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hepatitis c virus (hcv) is one of the major causes of chronic liver disease in the world. more importantly, many of those patients with chronic hepatitis c eventually develop cirrhosis and hepatocellular carcinoma. following infection with hcv, only about 15% of patients can clear the virus, while 6080% of patients develop persistent chronic infection [3, 4 ]. previous studies have revealed that the persistence of viral infection and chronic inflammation are dependent on the interaction among the virus, hepatocyte, and the host immune system [4, 5 ]. the viral infection and related hepatocyte injuries are known to suppress the immune system [6, 7 ]. although experimental evidence suggests that antigen - specific th1 immunity and proinflammatory cytokines play an important role in the hcv - related liver injury and clearance of viruses [810 ], the pathogenesis of chronic hcv infection has not been fully understood. interleukin-33 (il-33) is one of the newly described members in the il-1 family and can be produced by epithelial tissues and vascular endothelial cells. il-33 binds to its heterodimer receptors composed of il-1 receptor - related protein st2 and il-1 receptor accessory protein (il-1rap) and can activate the myd88 and nf-b - related signal pathway [11, 12 ]. st2 has transmembrane form of st2 (st2 or st2l) and soluble form of st2 (sst2). st2 is expressed on th2 and mast cells and functions as a mediator of il-33 bioactivities, while sst2 acts as a decoy receptor for il-33. biologically, il-33 induces th2 cell differentiation and activates mast cells, leading to th2 cytokine production and th2 response as well as pulmonary and mucosal th2 inflammation. furthermore, il-33 can antagonize the lps - induced mortality in a model of septic shock, and the levels of serum il-33 were elevated in sle and ra patients. recently, il-33 has been found to be an important factor of the pathogenesis of hiv infection and dengue virus infection [14, 15 ]. however, little is known on whether il-33 could participate in the pathogenic process of hcv infection. in the current study, we examined the concentrations of serum il-33 and sst2 in patients with chronic hepatitis c (chc), individuals with spontaneously resolved hcv infection (sr - hcv), and healthy controls (hc) to evaluate the potential role of il-33/st2 axis in the pathogenic process of chc. furthermore, we determined the concentrations of serum il-33 before and after antivirus therapies in patients with chc. we found that il-33 response appeared to be an important factor of the pathogenesis of chc, associated with the severity of liver injury in chc patients. a total of 154 patients with chc, 24 cases with sr - hcv, were recruited sequentially at the outpatient service of first hospital. another 20 gender-, age- and ethnic - matched hc were recruited, and they had no historical liver diseases and no evidence of hbv, hcv, and hdv infection. individuals with positive anti - hcv antibodies and serum hcv rna for at least six months were diagnosed with chc. individuals with sr - hcv were defined as those subjects who had prior hcv rna detection (hcv - ab) but lacked hcv rna detection for 12 weeks after enrollment in the absence of treatment. genotyping of hcv showed that 22 chc patients had genotype 2a, 45 had genotype 1b, and 14 had unclassified genotype. individuals with historical and current hepatitis b, d virus or hiv infection, autoimmune hepatitis, or metabolic liver disease, who had received immunosuppressive therapy or antiviral therapy during the past 12 months before entry, were excluded. fifty patients with chc were treated subcutaneously with 500 million units of a short - acting interferon (sinogen, jinan, china) every other day for 12 weeks. the concentrations of serum il-33, sst2, hcv - rna, alt, and ast were measured before and after treatment. individuals with 100-fold reduced serum virus load were defined as drug - responsive patients ; otherwise, individuals were defined as drug - nonresponsive patients. written informed consent was obtained from individual participants, and the study was approved by the first hospital ethical committee of jilin university. peripheral blood samples were obtained from individual subjects, and their sera were prepared and then stored at 80c till needed. the levels of serum il-33 were measured at the entry of individual participants, before and after treatment of patients with chc, and the concentrations of serum ifn-, tnf-, il-2, il-4, il-10, and il-6 in individual chc patients were also determined. the concentrations of serum il-33 and sst2 in individual patients and healthy controls were determined by elisa using human il-33 and sst2 elisa kits, according to the manufacturers ' instruction (roche diagnostics, lewes, uk). briefly, individual sera at 1 : 4 dilutions were subjected to elisa analysis, and the concentrations of serum il-33 and sst2 in individual samples were calculated, according to the standard curve established using the recombinant il-33 and sst2 provided. the concentrations of serum cytokine levels (ifn-, tnf-, il-2, il-4, il-10, and il-6) were determined by cytometric bead array (cba), according to the manufacturer 's protocol (bd biosciences, san joes, usa) with minor modification. briefly, 25 l of individual sera was used in duplicate for analysis, as described previously. the concentrations of serum cytokines were quantified using the cellquestpro and cba software (becton dickinson) on a facscalibur cytometry (bd biosciences). the concentrations of serum antibodies against hcv were detected by elisa ii (abbott laboratories, abbott park, usa). the levels of serum alt and ast were detected using a biochemistry automatic analyzer (roche diagnostics, branchburg, usa). the amounts of serum hcv rna were measured by quantitative pcr assay using a luciferase quantization detection kit, following the protocols (roche amplicor, basel, switzerland). the detection limit of viral rna was 300 copies / ml. the data are expressed as median and range unless specified. the differences between the groups were analyzed by wilcoxon - rank sum test and chi - square test using the spss 14.0 software. to determine the potential role of il-33 in the pathogenic process of chc, a total 154 patients with chc, 24 with sr - hcv, and 20 with hc were sequentially recruited. as expected, there was no significant difference in the distribution of age and gender among these groups of subjects, but the concentrations of serum alt and ast in patients with chc were significantly higher than those in the hc and those with sr - hcv (table 1). while high levels of virus rna were detected in chc patients, there was no detectable viremia in both hc and sr - hcv. in addition, the anti - hcv antibody was detected in chc patients and individuals with sr - hcv, but not in hc. analysis of serum cytokines indicated that there was no significant difference in the concentrations of serum il-33 between individuals with sr - hcv and hc, while the concentrations of serum il-33 in patients with chc were significantly higher than those in individuals with sr - hcv and hc (p 50 units / l) or ast (> 40 units / l) were significantly higher than those in chc patients with normal levels of alt (< 50 units / l) or ast (< 40 units / l), respectively (p < 0.001, p < 0.001, resp., figures 1(b) and 1(c)). the concentrations of serum il-33 in chc patients were correlated positively with the levels of serum alt and ast (r = 0.388, p < 0.001 ; r = 0.400, p < 0.001, resp., figures 1(d) and 1(e)). apparently, il-33 is a pathogenic factor, associated with the damage of the liver in chc patients. further analysis revealed that the levels of serum sst2 were significantly higher in chc and sr - chc patients than that in hc (p = 0.004, p = 0.041, resp., figure 2), but there was no significant correlation between the levels of serum il-33 and sst2 in those subjects (r = 0.050, p = 0.678). moreover, the concentrations of serum ifn- and il-6, but not tnf-, il-2, il-10, and il-4, in patients with chc, were significantly lower than those in individuals with sr - hcv (figures 3(a) and 3(b)). following treatment of chc patients with ifn for 12 weeks, 45 out of 50 chc patients displayed dramatically reduced levels of serum hcv virions. notably, treatment with ifn remarkably reduced il-33 responses in those patients because the levels of serum il-33 in patients with chc after treatment with ifn were significantly lower than those before treatment. however, treatment with ifn for 12 weeks did not change significantly in the levels of serum sst2 in chc patients (p = 0.641, figure 4(b)). il-33, through the receptor complex composed of st2 and il-1rap, can activate the map kinase and nf-b signal pathways and promote th2 response and cytokine production. indeed, intranasal administration of il-33 triggered an immediate allergic response in the airway, and endogenous il-33 contributes to airway inflammation. a recent study revealed that the levels of serum il-33 were elevated in sle and ra patients and correlated with the levels of serum esr and crp, two inflammation markers, indicating that il-33 may participate in the acute - phase response of sle. in addition, il-33 can protect against septic shock by enhancing neutrophils infiltration at the site of inflammation. other studies suggest that il-33 participates in the pathogenic process of acute hepatitis induced by con - a [25, 26 ], and il-33 overexpression is associated with the development of hbv / hcv - related liver fibrosis. to investigate the role of il-33 in the pathogenic process of chc, we determined the levels of serum il-33 and sst2 in 154 chc patients. we found that the levels of serum il-33 in chc patients were significantly higher than in those with sr - hcv and hc. furthermore, the levels of serum il-33 in chc patients with abnormal concentrations of alt or ast were significantly higher than in those with normal levels of alt and ast in this population. in addition, treatment with ifn to inhibit the replication of hcv dramatically decreased the levels of serum il-33 in chc patients. more importantly, the concentrations of serum il-33 were correlated positively with the levels of serum alt and ast in chc patients. given that abnormal levels of alt and ast are indicative of abnormal liver function and injuries, our data suggest that il-33 may be a pathogenic factor of the pathogenic process of chc in chinese patients. therefore, if the levels of serum il-33 are also correlated with pathogenic degrees of the liver in chc patients, the levels of serum il-33 may be used as a new biomarker for the diagnosis of liver damages in chc patients. furthermore, we found that the levels of serum sst2 were significantly higher in chc and sr - chc patients than those in hc, but were not correlated with the levels of il-33 in patients. treatment with ifn for 12 weeks did not significantly change the levels of serum sst2. a previous study has shown that the levels of serum sst2 in patients with acute liver failure are higher than in those with chronic liver failure and healthy controls. it is possible that high levels of serum sst2 are an early biomarker of liver injury, while high levels of serum il-33 may be associated with the development and progression of liver fibrosis and damage. we are interested in further examining the mechanisms underlying the action of il-33/sst2 axis in hcv - related liver injury. we found that the levels of serum ifn- and il-6 in chc patients were significantly lower than those in sr - hcv, supporting the notion that proinflammatory cytokines, such as ifn- and il-6, are not only important factors for the clearance of infected hcv, but also for liver injury [2931 ]. the lower levels of ifn- and il-6 in chc patients were unlikely to have come from the antagonization of il-33-induced th2 responses in those patients, because we failed to detect significant difference in the levels of serum il-4 and il-10 between those chc patients and sr - hcv and hc. given that il-6 is a critical factor of the functional development of th17 cells and that ifn- is an effector of th1 response, the lower levels of serum ifn- and il-6 in those chc patients indicated continual viral replication and pathogenic progression. although il-33 has been shown to promote ifn- production by invariant nkt and nk cells, il-33 may, through an unknown pathway, downregulate the functional development of hcv - related th1 response and inhibit ifn- production. our data indicate, for the first time, that the concentrations of serum il-33 are significantly higher in those with sr - hcv and hc and are significantly correlated with the levels of serum alt and ast, suggesting that il-33 may be a pathogenic factor of hcv - related liver injury in chc patients. we recognized that the current study has limitations, including no source for il-33 and no histopathological examination of liver tissues. although more detailed studies are necessary to determine the role and mechanisms of il-33 in regulating the pathogenic process of chc, our novel findings may provide new insights into understanding the pathogenesis of chc.	interleukin-33 (il-33) is associated with the development of th2 responses. this study examined the potential role of il-33 in the pathogenic process of chronic hepatitis c (chc) in chinese patients. the levels of serum il-33 and sst2 in 154 patients with chc, 24 with spontaneously resolved hcv (sr - hcv) infection and 20 healthy controls (hc), were analyzed by elisa. the concentrations of serum il-2, ifn-, tnf-, il-4, il-6, and il-10, hcv loads, alt, ast, and hcv - ab were measured. we found that the levels of serum il-33 in chc patients were significantly higher than those of sr - hcv and hc but decreased after treatment with interferon for 12 weeks. more importantly, the levels of serum il-33 were correlated with the concentrations of alt and ast in chc patients. the levels of serum sst2, as a decoy receptor of il-33, were significantly higher in chc and sr - chc patients than those in hc, and there was no correlation between the levels of serum sst2 and il-33. the concentrations of serum ifn- and il-6 in chc patients were significantly lower than those of sr - hcv. these data suggest that il-33 may be a pathogenic factor contributing to chc - related liver injury.
funding for travel or speaker honoraria : (1) istanbul ms days, novartis pharmaceuticals, speaker honoraria payment to mayo clinic. no personal compensation. (2) invited professor program, biogen - boston, speaker honoraria payment to mayo clinic. no personal compensation. other activities : grant review for the national multiple sclerosis society. research support, commercial entities : dr. orhun h. kantarci receives research support from the european regional development fund (fnusaicrc cz.1.05/1.1.00/02.0123), the national multiple sclerosis society, and has given a scientific presentation at a meeting supported by teva pharmaceuticals but has received no personal fees or personal compensation for this activity (all compensation for consulting activities paid directly to mayo clinic) nor has spoken about the specific medications involving this company. kejal kantarci (spouse) serves on the data safety monitoring board of takeda global research and development center, inc., and the data monitoring boards of pfizer inc. and janssen alzheimer immunotherapy and is funded by the nih (r01 ag040042 [pi ], r21 ns066147 [pi ], p50 ag44170/project 2 [pi ], p50 ag16574/project 1 [pi ], r01 ag11378 [co - i ], u19 ag10483 [co - i ] u01 ag042791 [co - i ]) and minnesota partnership for biotechnology and medical genomics (po03590201 [pi ]). kejal kantarci serves on the data safety monitoring board of takeda global research and development center, inc., and the data monitoring boards of pfizer inc. and janssen alzheimer immunotherapy and is funded by the nih (r01 ag040042 [pi ], r21 ns066147 [pi ], p50 ag44170/project 2 [pi ], p50 ag16574/project 1 [pi ], r01 ag11378 [co - i ], u19 ag10483 [co - i ] u01 ag042791 [co - i ]) and minnesota partnership for biotechnology and medical genomics (po03590201 [pi ]) kejal kantarci serves on the data safety monitoring board of takeda global research and development center, inc. janssen alzheimer immunotherapy and is funded by the nih (r01 ag040042 [pi ], r21 ns066147 [pi ], p50 ag44170/project 2 [pi ], p50 ag16574/project 1 [pi ], r01 ag11378 [co - i ], u19 ag10483 [co - i ] u01 ag042791 [co - i ]) and minnesota partnership for biotechnology and medical genomics (po03590201 [pi ]). research support, foundations and societies : 20062009 multiple sclerosis society for support in multiple sclerosis research unrelated to this publication ; 20082009 mayo foundation cr20 award for support in multiple sclerosis research unrelated to this publication ; 20092011 hilton foundation support in multiple sclerosis research unrelated to this publication.	before the genomics technology revolution allowed us to do genome - wide science, genetics research relied on our limited knowledge about a subject to generate hypothesis and candidate genes to study. despite the level of naivet, several associations with susceptibility to a complex disease such as multiple sclerosis (ms) were discovered. of these, hla - drb1 and il7r1 stand out as being confirmed and refined early by the genome - wide association studies (gwas) that followed.2 despite the expense and gargantuan efforts, these gwas have successfully led to the discovery of more than 100 additional genes, albeit with smaller effect sizes, that contribute to ms susceptibility.3 this list keeps growing, but it comes with no surprise that most of these genes identified the immune system as one large candidate for ms susceptibility.
this open, randomized controlled trial recruited diabetes hamilton registrants between june 2005 and november 2008. people aged 40 years with type 2 diabetes of at least 1 year duration and whose a1c was 7% were included. individuals who were pregnant, institutionalized, living with another study participant, or residing outside the city of hamilton were excluded. participants were recruited through diabetes hamilton and/or through television advertisements, direct - mail campaigns, flyers in pharmacies, and physician offices and public diabetes - information sessions. eligible consenting participants provided information regarding their demographics, diabetes - related behaviors, health status, medication use, quality of life, and a capillary blood sample for a1c measurement, which were all mailed to the project office. participants then were randomly allocated to either receive or not receive computer - generated, evidence - based recommendations targeting an a1c < 7% and other diabetes - related goals, such as blood pressure control, smoking cessation, and foot care, that were based on their measured a1c levels and other information that they provided ; participants were provided with the a1c result and an interpretation of its meaning, as well as the diabetes hamilton resources described above. the recommendations were designed to reinforce canadian evidence - based guidelines for the management of diabetes and were piloted in a small group of volunteers. interactions with participants were by mail or telephone only ; all written communication with participants was copied to their primary care physician. concealed random allocation in blocks of four stratified by a1c level (a1c 8.5 vs. < 8.5%) was conducted centrally using a computer - generated algorithm. all participants signed a written informed consent, and the study was approved by the local research ethics board. participants in the intervention group received tailored feedback comprising 1) up to 20 automatically generated recommendations for glycemic control and self - management on the basis of their diabetes questionnaire responses and a1c levels (supplementary appendix 1), 2) a list of local resources specific to their residence, and 3) a simple booklet to track personal diabetes - related information. feedback was sent at baseline, and a copy of these recommendations was resent after 3 months. six months after randomization, all participants submitted new study questionnaires and another blood sample to measure a1c, which triggered a new set of recommendations that was reinforced 3 months later. participants in the control group were sent their a1c results with an interpretation at baseline and 6 months. a sample of capillary blood was collected at home on the via post filter paper card (roche) using a lancing device and mailed to the project office for central assay. a1c levels were measured by immunoturbidimetry with the roche tina - quant assay on the hitachi 917. the assay system used in the laboratory was traceable to the diabetes control and complications assay result and had a precision < 4.4% at all levels (18,19). general health - related quality of life and utility was measured using the european quality of life5 dimension (eq-5d) scale, and the u.s. this instrument has demonstrated reliability and validity and measures quality of life on five domains, with a higher score indicating better quality of life (20). the visual analog scale (vas) for health status also was used, allowing individuals to rate their perceived health status on a scale of 0100, with 100 reflecting perfect health. the audit of diabetes dependent quality of life (addqol) scale was used to measure diabetes - specific quality of life. the addqol has established reliability and validity properties, and higher scores indicate a better quality of life (21,22). a composite score reflecting diabetes self - management behaviors was computed on the basis of information provided by participants. this score tracked 18 specific behaviors (e.g., glucose monitoring, being physically active, foot inspection) and was calculated as a count of the number of behaviors that the participant reported. a value of one was assigned for any of the 18 recommended behaviors that were reported to have been achieved ; otherwise, a value of zero was assigned. this score was calculated at baseline and at 6 and 12 months (supplementary appendix 2). sample size calculations showed that with an level of 0.05 and an sd of the change in a1c of 1.4%, at least 206 people had to be allocated to each group to detect a difference in a1c reduction of at least 0.5% between groups with 90% power (and a difference of at least 0.4% with 80% power). baseline characteristics were summarized as mean values, sds, counts, and percentages and compared using t tests or tests. the difference between the change in a1c level from baseline to 12 months in the intervention group and the change in the control group was compared using an independent - sample t test. t tests were used to compare the change from baseline to 12 months for the addqol, eq-5d, and the composite self - management scores. an exploratory linear regression was conducted to determine whether between - group differences in baseline a1c, education, ethnicity, age, sex, and diabetes duration confounded the effect of the intervention on the primary outcome. the proportions of individuals who reduced their a1c by 0.3, 0.5, or 0.7% at 12 months were calculated. odds ratios for these three proportions were estimated through logistic regression, adjusting for baseline a1c, education, ethnicity, age, sex, and diabetes duration. in a post hoc analysis to investigate whether there were changes in process behaviors (dependent variables), such as seeking primary care, oral medications, physical activity, glucose monitoring, and hospitalizations over time, a generalized estimating equation was used in which the presence or absence of the behavior was regressed onto the randomized group, time, and the group - by - time interaction (independent variables), using a logistic link. a nonsignificant interaction was taken to indicate that there was not substantial evidence that the two groups differed in the way their behaviors changed over time. in the event that the interaction was not significant, all statistical analyses were conducted using r statistical software (version 2.4) (23). a sample of capillary blood was collected at home on the via post filter paper card (roche) using a lancing device and mailed to the project office for central assay. a1c levels were measured by immunoturbidimetry with the roche tina - quant assay on the hitachi 917. the assay system used in the laboratory was traceable to the diabetes control and complications assay result and had a precision < 4.4% at all levels (18,19). general health - related quality of life and utility was measured using the european quality of life5 dimension (eq-5d) scale, and the u.s. this instrument has demonstrated reliability and validity and measures quality of life on five domains, with a higher score indicating better quality of life (20). the visual analog scale (vas) for health status also was used, allowing individuals to rate their perceived health status on a scale of 0100, with 100 reflecting perfect health. the audit of diabetes dependent quality of life (addqol) scale was used to measure diabetes - specific quality of life. the addqol has established reliability and validity properties, and higher scores indicate a better quality of life (21,22). a composite score reflecting diabetes self - management behaviors was computed on the basis of information provided by participants. this score tracked 18 specific behaviors (e.g., glucose monitoring, being physically active, foot inspection) and was calculated as a count of the number of behaviors that the participant reported. a value of one was assigned for any of the 18 recommended behaviors that were reported to have been achieved ; otherwise, a value of zero was assigned. this score was calculated at baseline and at 6 and 12 months (supplementary appendix 2). sample size calculations showed that with an level of 0.05 and an sd of the change in a1c of 1.4%, at least 206 people had to be allocated to each group to detect a difference in a1c reduction of at least 0.5% between groups with 90% power (and a difference of at least 0.4% with 80% power). baseline characteristics were summarized as mean values, sds, counts, and percentages and compared using t tests or tests. the difference between the change in a1c level from baseline to 12 months in the intervention group and the change in the control group was compared using an independent - sample t test. t tests were used to compare the change from baseline to 12 months for the addqol, eq-5d, and the composite self - management scores. an exploratory linear regression was conducted to determine whether between - group differences in baseline a1c, education, ethnicity, age, sex, and diabetes duration confounded the effect of the intervention on the primary outcome. the proportions of individuals who reduced their a1c by 0.3, 0.5, or 0.7% at 12 months were calculated. odds ratios for these three proportions were estimated through logistic regression, adjusting for baseline a1c, education, ethnicity, age, sex, and diabetes duration. in a post hoc analysis to investigate whether there were changes in process behaviors (dependent variables), such as seeking primary care, oral medications, physical activity, glucose monitoring, and hospitalizations over time, a generalized estimating equation was used in which the presence or absence of the behavior was regressed onto the randomized group, time, and the group - by - time interaction (independent variables), using a logistic link. a nonsignificant interaction was taken to indicate that there was not substantial evidence that the two groups differed in the way their behaviors changed over time. in the event that the interaction was not significant, all statistical analyses were conducted using r statistical software (version 2.4) (23). four hundred and sixty - five participants were recruited and randomly assigned for the trial, with 233 assigned to the intervention group and 232 to the control group. as shown in table 1, the mean sd age of participants was 62 11 years and 50% were female. at randomization, intervention - group participants were slightly heavier than control - group participants, with a bmi of 34 8 kg / m and 32 7 kg / m, respectively (p = 0.02), and intervention - group participants were slightly less likely to have had more than a high school education (p = 0.04). the mean sd baseline a1c levels in the intervention and control groups were similar at 7.85 0.88% and 7.81 0.83%, respectively (p = 0.61). baseline characteristics of study participants data are mean sd or n (%). as shown in table 2, by 12 months, the a1c had decreased by an absolute amount of 0.24% (95% ci 0.37 to 0.12 ; p < 0.001) and 0.15% (0.27 to 0.03 ; p = 0.01) in the intervention and control groups, respectively. however, as noted in table 2, the change in a1c did not differ significantly between the two groups (between - group difference 0.09% [95% ci 0.26 to 0.08 ] ; p = 0.3). the effect of the intervention was unchanged after adjusting for baseline a1c, age, education, bmi, and diabetes duration in a multiple regression analysis, and similar effects were seen within the two randomized strata of baseline a1c (< 8.5 vs. 8.5%). there also was no difference in the likelihood of achieving a 0.3, 0.5, or 0.7% reduction in a1c, either before or after adjusting for baseline a1c, education, ethnicity, age, sex, and diabetes duration (table 3). proportion of participants obtaining 0.3, 0.5, and 0.7% reductions in a1c and 95% cis data are counts (%), unless otherwise indicated. adjusted for baseline a1c, education, ethnicity, age, sex, and diabetes duration. the possibility that postrandomization changes in the behavior or management of participants could have attenuated the effect of the intervention was explored by examining between - group changes in 1) health care provider visits, 2) use of new oral medications and/or insulin, 3) self - reported physical activity, 4) frequency of glucose self - monitoring, or 5) hospitalization. regression analysis did not demonstrate differences between the two groups in the use of services, nor was there evidence of significant change in the use of services over time. both groups improved their eq-5d scores, vas scores, and addqol scores by the end of the study. however, the changes did not differ significantly between the two groups (table 4). likewise, a composite measure reflecting diabetes self - management behaviors improved by a similar amount in both groups during the study period (0.02 [95% ci 0.51 to 0.47 ] ; p = 0.95) (table 4). the incidence of clinical outcomes, including hospitalization, amputations, foot ulcer treatment, laser therapy, cataract surgery, heart attacks, strokes, and kidney failure were similar in both groups during the trial. baseline and 12-month results for quality of life and diabetes management behaviors data are means (95% ci). the eq-5d scale scores range from 1 (optimal health) to 0.11 (worse than being dead), which indicates poor quality of life. in the eq-5d vas, 0 reflects poor overall quality of life and 100 reflects optimal quality of life. addqol scores range from 9 to 3, with higher scores reflecting a better diabetes - related quality of life. composite score has a maximum score (optimal diabetes management behaviors), which is 17 at baseline and 18 at both 6 and 12 months. as shown in table 1, the mean sd age of participants was 62 11 years and 50% were female. at randomization, intervention - group participants were slightly heavier than control - group participants, with a bmi of 34 8 kg / m and 32 7 kg / m, respectively (p = 0.02), and intervention - group participants were slightly less likely to have had more than a high school education (p = 0.04). the mean sd baseline a1c levels in the intervention and control groups were similar at 7.85 0.88% and 7.81 0.83%, respectively (p = 0.61). baseline characteristics of study participants data are mean sd or n (%). as shown in table 2, by 12 months, the a1c had decreased by an absolute amount of 0.24% (95% ci 0.37 to 0.12 ; p < 0.001) and 0.15% (0.27 to 0.03 ; p = 0.01) in the intervention and control groups, respectively. however, as noted in table 2, the change in a1c did not differ significantly between the two groups (between - group difference 0.09% [95% ci 0.26 to 0.08 ] ; p = 0.3). the effect of the intervention was unchanged after adjusting for baseline a1c, age, education, bmi, and diabetes duration in a multiple regression analysis, and similar effects were seen within the two randomized strata of baseline a1c (< 8.5 vs. 8.5%). there also was no difference in the likelihood of achieving a 0.3, 0.5, or 0.7% reduction in a1c, either before or after adjusting for baseline a1c, education, ethnicity, age, sex, and diabetes duration (table 3). proportion of participants obtaining 0.3, 0.5, and 0.7% reductions in a1c and 95% cis data are counts (%), unless otherwise indicated. adjusted for baseline a1c, education, ethnicity, age, sex, and diabetes duration. the possibility that postrandomization changes in the behavior or management of participants could have attenuated the effect of the intervention was explored by examining between - group changes in 1) health care provider visits, 2) use of new oral medications and/or insulin, 3) self - reported physical activity, 4) frequency of glucose self - monitoring, or 5) hospitalization. regression analysis did not demonstrate differences between the two groups in the use of services, nor was there evidence of significant change in the use of services over time. both groups improved their eq-5d scores, vas scores, and addqol scores by the end of the study. however, the changes did not differ significantly between the two groups (table 4). likewise, a composite measure reflecting diabetes self - management behaviors improved by a similar amount in both groups during the study period (0.02 [95% ci 0.51 to 0.47 ] ; p = 0.95) (table 4). the incidence of clinical outcomes, including hospitalization, amputations, foot ulcer treatment, laser therapy, cataract surgery, heart attacks, strokes, and kidney failure were similar in both groups during the trial. baseline and 12-month results for quality of life and diabetes management behaviors data are means (95% ci). the eq-5d scale scores range from 1 (optimal health) to 0.11 (worse than being dead), which indicates poor quality of life. in the eq-5d vas, 0 reflects poor overall quality of life and 100 reflects optimal quality of life. addqol scores range from 9 to 3, with higher scores reflecting a better diabetes - related quality of life. composite score has a maximum score (optimal diabetes management behaviors), which is 17 at baseline and 18 at both 6 and 12 months. this trial tested the efficacy of tailored feedback on glycemic control for individuals with type 2 diabetes in the community. the findings from the study demonstrated an improvement in glycemic control in both the control and intervention groups but a nonsignificant difference between groups at 12 months. second, the study had a large sample size of 465 participants who were followed for 1 year. third, follow - up was very high, with a 96% completion rate of participants from the community setting. finally, the study was completed in the community, reflecting the relevance and reality of diabetes management in a nonclinical or hospital setting. as with any educational, behavioral - change intervention it is therefore possible that participants who did not receive tailored recommendations (control group), along with their doctor, initiated extra changes to improve diabetes control, such as medication initiation or modification. although such an effect was not observed in an exploratory analysis of postrandomization changes, unmeasured cointerventions may have been instituted. second, participants in both groups were voluntary registrants in a community - based diabetes program. any incremental benefit of the intervention studied in this trial may have been insufficient to surpass the effect of the community - based program alone. third, a1c testing and reporting was provided to the control group, which may not reflect a subject s usual or routine care. the receipt of an a1c level, in addition to the community program resources, such as newsletters and the inventory of community resources, may have led to the seeking of additional information, education, and support by control - group participants to improve glycemic control. finally, the intervention mainly comprised mail- and telephone - based interactions and did not include systematic, intensive, real - time interactions of a clinical or advisory nature by telephone, through a website, or face - to - face visits. the addition of such real - time interactions and coordination of care with health care providers has been demonstrated to have a greater effect on both a1c, quality of life, and rates of hypoglycemia (24,25). however, such an approach would have represented an individually focused, provider - driven intervention rather than a community - based, patient - driven intervention. the literature suggests that diabetes care can be improved by providing focused information and care reminders directly to patients (12). likewise, tailored feedback interventions geared toward patients directly in clinical or work settings were found to be effective when implemented with other interventions, such as telephone and face - to - face contact (1015). however, our study using printed tailored feedback via mail suggested that such an approach is insufficient to significantly change a1c or quality of life in individuals with diabetes in the community setting who are exposed to information and resources offered by a community - based support program. diabetes is a growing public health problem that requires evidence - based community approaches. in this study, tailored recommendations were provided to 233 individuals living with diabetes in the community, along with an inventory of community resources, tools to facilitate self - care, quarterly newsletters describing evidence - based information pertaining to diabetes, feedback support, and reminder prompts. this study showed that a quarterly community - based intervention providing computer - generated recommendations is insufficient to significantly change a1c levels. future research involving community - based approaches may include promoting community awareness of diabetes, promoting self - efficacy and self - management, promoting greater interaction between individuals and their diabetes care providers, and reducing barriers to adopting healthy lifestyles.	objectiveit is unknown whether computer - generated, patient - tailored feedback leads to improvements in glycemic control in people with type 2 diabetes.research design and methodswe recruited people with type 2 diabetes aged 40 years with a glycated hemoglobin (a1c) 7%, living in hamilton, canada, who were enrolled in a community - based program (diabetes hamilton) that provided regular evidence - based information and listings of community resources designed to facilitate diabetes self - management. after completing a questionnaire, participants were randomly allocated to either receive or not receive periodic computer - generated, evidence - based feedback on the basis of their questionnaire responses and designed to facilitate improved glycemic control and diabetes self - management. the primary outcome was a change in a1c after 1 year.resultsa total of 465 participants (50% women, mean age 62 years, and mean a1c 7.83%) were randomly assigned, and 12-month a1c values were available in 96% of all participants, at which time the a1c level had decreased by an absolute amount of 0.24 and 0.15% in the intervention and control groups, respectively. the difference in a1c reduction for the intervention versus control group was 0.09% (95% ci 0.08 to 0.26 ; p = 0.3). no between - group differences in measures of quality of life, diabetes self - management behaviors, or clinical outcomes were observed.conclusionsproviding computer - generated tailored feedback to registrants of a generic, community - based program that supports diabetes self - management does not lead to lower a1c levels or a better quality of life than participation in the community - based program (augmented by periodic a1c testing) alone.
study participants were drawn from the women 's health study (whs), a completed randomized, double - blinded, placebo - controlled trial of low - dose aspirin and vitamin e in the primary prevention of cardiovascular disease (cvd) and cancer in women (1113). whs participants were apparently healthy female health care professionals, aged 45 years, who were free of self - reported cvd and cancer at study entry (19921995). at the time of enrollment, women gave written informed consent and completed questionnaires on demographics, anthropometrics, medical history, and lifestyle factors. they were also asked to provide a baseline blood sample ; 28,345 women did so, and of these, 98.5% (n = 27,909) had nmr measurements. for this study, we excluded women missing other lipids (n = 33), those with self - reported baseline type 2 diabetes (n = 770), and those with baseline a1c 6.5% (n = 270), leaving 26,836 women for analysis. we also repeated the analyses after excluding 169 women with a1c 6.0 and 90% response rate to either telephone interview or supplemental questionnaire by women who self - reported diabetes. glucose screening rates in this population were similar to contemporaneous screening rates in the general population (18), with 68.2% of nondiabetic women having reported a screening fasting glucose performed in the prior 3 years. statistical analyses were performed using stata version 10.1 (stata, college station, tx). statistical comparisons were obtained from student t tests for continuous variables expressed as means, from wilcoxon rank - sum tests for variables expressed as medians, and tests for categorical variables. following guidelines from the department of health and human services (19), lipids and lipoproteins were divided into quintiles based on the distribution among women not taking hormone replacement. cox proportional hazard regression models were used to calculate the hazard ratios (hrs) and 95% cis according to these quintiles. the proportional hazard assumption was tested using schoenfeld residuals and the natural logarithm of follow - up time. some of the variables did not satisfy the proportionality assumption ; hence, we also divided the follow - up time into the first and second 6 years, finding no substantial differences within each 6-year period. stronger associations were noted for nmr lipoproteins with diabetes during the first 6 years compared with the second 6 years of follow - up, but the relative magnitude of associations was generally similar within each 6-year period ; therefore, we report the main results for the overall follow - up period unless otherwise specified. we initially considered two levels of adjustment for 1) age, race, and randomized treatment assignment (minimally adjusted ; model 1) ; and 2) covariates in model 1 plus smoking status, exercise, education, menopausal status, hormone use, blood pressure, bmi, family history of diabetes, a1c, and hscrp (nonlipid risk factors ; model 2). to determine the magnitude of association of nmr lipoproteins with diabetes independent of standard lipids, we additionally adjusted model 2 for triglycerides and hdl and ldl cholesterol and evaluated the association of nmr lipoproteins with diabetes using likelihood ratio tests. since lipoprotein particles are metabolically interrelated (7,20), nmr lipoproteins were also analyzed in a single model that included the nine nmr lipoprotein subclasses (two ldlnmr, one idlnmr, three hdlnmr, and three vldlnmr lipoprotein subclasses) in addition to the nonlipid risk factors, in order to estimate the independent associations of these correlated lipoproteins with diabetes. based on prior work from this cohort suggesting that nonfasting concentrations of certain lipids may be superior to fasting concentrations for risk prediction (21,22), we examined whether fasting status modified the association of nmr lipoproteins with diabetes. statistical tests for interaction between fasting status and lipoproteins in relation to diabetes were obtained using likelihood ratio tests. we repeated the analyses after excluding 169 women with a1c 6.0 and 90% response rate to either telephone interview or supplemental questionnaire by women who self - reported diabetes. glucose screening rates in this population were similar to contemporaneous screening rates in the general population (18), with 68.2% of nondiabetic women having reported a screening fasting glucose performed in the prior 3 years. statistical analyses were performed using stata version 10.1 (stata, college station, tx). statistical comparisons were obtained from student t tests for continuous variables expressed as means, from wilcoxon rank - sum tests for variables expressed as medians, and tests for categorical variables. following guidelines from the department of health and human services (19), lipids and lipoproteins were divided into quintiles based on the distribution among women not taking hormone replacement. cox proportional hazard regression models were used to calculate the hazard ratios (hrs) and 95% cis according to these quintiles. the proportional hazard assumption was tested using schoenfeld residuals and the natural logarithm of follow - up time. some of the variables did not satisfy the proportionality assumption ; hence, we also divided the follow - up time into the first and second 6 years, finding no substantial differences within each 6-year period. stronger associations were noted for nmr lipoproteins with diabetes during the first 6 years compared with the second 6 years of follow - up, but the relative magnitude of associations was generally similar within each 6-year period ; therefore, we report the main results for the overall follow - up period unless otherwise specified. we initially considered two levels of adjustment for 1) age, race, and randomized treatment assignment (minimally adjusted ; model 1) ; and 2) covariates in model 1 plus smoking status, exercise, education, menopausal status, hormone use, blood pressure, bmi, family history of diabetes, a1c, and hscrp (nonlipid risk factors ; model 2). to determine the magnitude of association of nmr lipoproteins with diabetes independent of standard lipids, we additionally adjusted model 2 for triglycerides and hdl and ldl cholesterol and evaluated the association of nmr lipoproteins with diabetes using likelihood ratio tests. since lipoprotein particles are metabolically interrelated (7,20), nmr lipoproteins were also analyzed in a single model that included the nine nmr lipoprotein subclasses (two ldlnmr, one idlnmr, three hdlnmr, and three vldlnmr lipoprotein subclasses) in addition to the nonlipid risk factors, in order to estimate the independent associations of these correlated lipoproteins with diabetes. based on prior work from this cohort suggesting that nonfasting concentrations of certain lipids may be superior to fasting concentrations for risk prediction (21,22), we examined whether fasting status modified the association of nmr lipoproteins with diabetes. statistical tests for interaction between fasting status and lipoproteins in relation to diabetes were obtained using likelihood ratio tests. we repeated the analyses after excluding 169 women with a1c 6.0 and 0.05). however, there were several borderline significant interactions noted for fasting status with each of small ldlnmr particles, ldlnmr size, and hdlnmr size in relation to diabetes (p for interaction 0.08, 0.06, and 0.05, respectively). moreover, while the p value for interaction was nonsignificant, nonfasting large vldlnmr particles carried much higher risk for diabetes than fasting measurements. hrs and 95% cis were adjusted similar to fig. 1 and stratified according to fasting (black circles) or nonfasting (gray diamonds) status. when we evaluated all nine nmr - measured lipoprotein particle concentrations in one model that also adjusted for nonlipid risk factors, we found that large and small ldlnmr, large and small hdlnmr, and large vldlnmr remained associated with diabetes. idlnmr and small vldlnmr particles were no longer significant (p = 0.38 and 0.62, respectively). medium hdlnmr and medium vldlnmr particles now showed inverse associations with diabetes. since nmr lipoproteins are correlated with standard lipids, in particular hdl cholesterol and triglycerides, we performed cox models that adjusted for triglycerides and hdl and ldl cholesterol in addition to the nonlipid (model 2) risk factors. although the associations were attenuated, smaller particle size for ldlnmr and hdlnmr remained significant (quintile 1 vs. 5, hr 1.79, [95% ci 1.372.33 ] and 2.39 [1.753.28 ], respectively ; p for trend 52 mg / dl). compared with the rest of the cohort, these women were more likely to be hypertensive and hormone users. we then examined the association of small ldlnmr with incident diabetes in these women after adjusting for nonlipid risk factors (including hypertension and hormone use). higher concentration of small ldlnmr particles was significantly associated (p for trend 0.003) with incident diabetes, despite that these women had normal levels of triglycerides and hdl cholesterol (fully adjusted hr for the top versus bottom quintile of small ldlnmr 3.95 [95% ci 1.639.55 ]). hrs for diabetes according to quintiles of ldl cholesterol and ldlnmrparticle concentration and size are shown in table 2. in fully adjusted models, neither total cholesterol (data not shown) nor ldl cholesterol was associated with diabetes (p for trend 0.53 and 0.64, respectively), but other ldl measures, such as ldlnmr particle concentration and size, were significantly associated with diabetes (p for trend 0.05). however, there were several borderline significant interactions noted for fasting status with each of small ldlnmr particles, ldlnmr size, and hdlnmr size in relation to diabetes (p for interaction 0.08, 0.06, and 0.05, respectively). moreover, while the p value for interaction was nonsignificant, nonfasting large vldlnmr particles carried much higher risk for diabetes than fasting measurements. hrs and 95% cis were adjusted similar to fig. 1 and stratified according to fasting (black circles) or nonfasting (gray diamonds) status. when we evaluated all nine nmr - measured lipoprotein particle concentrations in one model that also adjusted for nonlipid risk factors, we found that large and small ldlnmr, large and small hdlnmr, and large vldlnmr remained associated with diabetes. idlnmr and small vldlnmr particles were no longer significant (p = 0.38 and 0.62, respectively). since nmr lipoproteins are correlated with standard lipids, in particular hdl cholesterol and triglycerides, we performed cox models that adjusted for triglycerides and hdl and ldl cholesterol in addition to the nonlipid (model 2) risk factors. although the associations were attenuated, smaller particle size for ldlnmr and hdlnmr remained significant (quintile 1 vs. 5, hr 1.79, [95% ci 1.372.33 ] and 2.39 [1.753.28 ], respectively ; p for trend 52 mg / dl). compared with the rest of the cohort, we then examined the association of small ldlnmr with incident diabetes in these women after adjusting for nonlipid risk factors (including hypertension and hormone use). higher concentration of small ldlnmr particles was significantly associated (p for trend 0.003) with incident diabetes, despite that these women had normal levels of triglycerides and hdl cholesterol (fully adjusted hr for the top versus bottom quintile of small ldlnmr 3.95 [95% ci 1.639.55 ]). consistent with prior studies in individuals with insulin resistance, we found that in initially healthy women followed prospectively for incident clinical type 2 diabetes, both triglycerides and hdl cholesterol were independently associated with diabetes but not ldl or total cholesterol. furthermore, nmr - measured size and concentrations of ldl, hdl, and vldl particles were also associated with diabetes, independent of triglycerides, hdl cholesterol, and other factors. smaller average size of ldlnmr and hdlnmr particles, as well as the concentration of small ldlnmr and hdlnmr particles, was associated with increased risk, while the concentration of large ldlnmr and hdlnmr particles carried lower risk. ldlnmr, hdlnmr, and vldlnmr particle size remained associated with diabetes in models that already included standard lipids and nonlipid risk factors, adding incremental risk information beyond that obtained from established risk factors. a uniting feature of these lipoprotein alterations and their association with type 2 diabetes may be a state of insulin resistance. the associations we found in this study in relation to the nmr - measured lipoproteins have been previously linked to insulin resistance as measured by the euglycemic clamp (9). garvey. (9) demonstrated a progressive increase in insulin resistance associated with larger vldlnmr size, smaller ldlnmr size, and smaller hdlnmr size, all of which are consistent with our findings in relation to predicting incident type 2 diabetes. in 830 subjects with insulin resistance followed in the insulin resistance atherosclerosis study over a 5-year period, nmr - measured larger vldlnmr size and smaller hdlnmr particles were independently associated with increased risk of type 2 diabetes (14), while ldlnmr size and ldlnmr particles were not significant independent of other risk factors. factor analysis revealed a single factor that correlated with insulin resistance accounted for nearly half the variance in these lipoprotein measures (10). our study, which was conducted in a large population of healthy women, found independent associations for incident diabetes with baseline ldlnmr size and concentration, with larger ldlnmr particles associated with lower risk and smaller ldlnmr particles associated with higher risk. moreover, small ldlnmr imparted higher risk of diabetes even in women with normal triglyceride and hdl cholesterol levels. the inverse association of large ldlnmr particles with type 2 diabetes contrasts with the positive association noted previously in relation to incident cvd in this population of women (23). risk factors for type 2 diabetes may differ from those for cvd (24). for cvd risk, we reported that both small and large ldlnmr particles had similar increase in risk, which contrasts with the inverse association of large ldlnmr, and positive association of small ldlnmr, with risk of diabetes in the current study. for cvd events, nmr lipoprotein profiles in this cohort of women were comparable but not superior to standard lipids, as recently reported (23). this is in contrast to the current findings for type 2 diabetes, where nmr - measured lipoprotein classification by size provided additive and independent risk information to standard lipids and other risk factors. for hdl particles, the inverse association of hdlnmr size with risk of type 2 diabetes was also noted previously in relation to risk of cvd in this population of women (23). of the hdlnmr particles, only large particles were associated with lower risk of diabetes, to a magnitude similar to the association of hdl cholesterol with diabetes, while small particles carried higher risk. furthermore, adjusting for hdl cholesterol and other risk factors attenuated the association, but larger hdlnmr size remained associated with more than twofold increased risk. previous studies have found strong inverse relationships between insulin resistance and the large hdlnmr subclass as measured by nmr (9,10,14) or the corresponding hdl2 subclass as measured by ultracentrifugation (25). for vldl particles, large particles had a greater magnitude of association with diabetes compared with smaller particles, which we explain by large vldl carrying more triglycerides than small vldl and correlating more with the severity of insulin resistance (9). hepatic overproduction of large vldl particles is a key feature of the dyslipoproteinemia of insulin resistance and type 2 diabetes, with evidence for independent regulation of large and small vldl particles (26). in addition, our finding of similar lipoprotein associations with diabetes both early and late in follow - up suggests that these lipoprotein alternations may occur years before the onset of overt hyperglycemia and clinical diagnosis of diabetes, providing a potential opportunity for the early detection and prevention of type 2 diabetes and its complications. our study is largely limited to caucasian women, these data may not be generalizable to men or other patient groups. while our study found incremental predictive information for nmr lipoproteins, further studies should be performed in the appropriate patient settings to determine whether a strategy using nmr lipoprotein testing is cost - effective for prevention of type 2 diabetes and related metabolic disorders. undetected diabetes at study entry is unlikely to have biased our results, since we excluded women with baseline a1c levels 6.5% from our primary sample and found similar results when we excluded those with a1c 6.0%. in addition, similar results during the first 6 years of follow - up compared with the second 6 years. we conclude that the size and concentration of nmr - measured ldl, hdl, and vldl particles were associated with clinical type 2 diabetes, independent of other risk factors, particularly chemically measured hdl cholesterol and triglycerides. the associations of ldlnmr and hdlnmr particles with diabetes differed according to size, with larger particles carrying lower risk and smaller particles carrying higher risk. for vldlnmr ldlnmr, hdlnmr, and vldlnmr particle size remained significant in models that already included standard lipids and risk factors, adding incremental risk information beyond that obtained from established risk factors for type 2 diabetes.	objectivediabetic dyslipoproteinemia is characterized by low hdl cholesterol and high triglycerides. we examined the association of lipoprotein particle size and concentration measured by nuclear magnetic resonance (nmr) spectroscopy with clinical type 2 diabetes.research design and methodsthis was a prospective study of 26,836 initially healthy women followed for 13 years for incident type 2 diabetes (n = 1,687). baseline lipids were measured directly and lipoprotein size and concentration by nmr. cox regression models included nonlipid risk factors (age, race, smoking, exercise, education, menopause, blood pressure, bmi, family history, a1c, and c - reactive protein). nmr lipoproteins were also examined after further adjusting for standard lipids.resultsincident diabetes was significantly associated with baseline hdl cholesterol, triglycerides, and nmr - measured size and concentration of ldl, idl, hdl, and vldl particles. the associations of these particles differed substantially by size. small ldlnmr and small hdlnmr were positively associated with diabetes (quintile 5 vs. 1 [adjusted hazard ratios and 95% cis ], 4.04 [3.215.09 ] and 1.84 [1.542.19 ], respectively). by contrast, large ldlnmr and large hdlnmr were inversely associated (quintile 1 vs. 5, 2.50 [2.122.95 ] and 4.51 [3.685.52 ], respectively). for vldlnmr, large particles imparted higher risk than small particles (quintile 5 vs. 1, 3.11 [2.354.11 ] and 1.31 [1.101.55 ], respectively). lipoprotein particle size remained significant after adjusting for standard lipids and nonlipid factors.conclusionsin this prospective study of women, nmr lipoprotein size and concentrations were associated with incident type 2 diabetes and remained significant after adjustment for established risk factors, including hdl cholesterol and triglycerides.
the symposium is conducted annually by the angelman treatment and research institute, a part of the angelman syndrome foundation (www.angelman.org). the theme for this year 's symposium was the neuroscience of ube3a from genes to behavior. the program consisted of invited speakers and other presentations and was attended by approximately 80 scientists. it became clear during the conference that angelman syndrome research is providing a new vista into the understanding of neurodevelopmental disorders. here, we first review some basic aspects of angelman syndrome (as) and then thematically summarize the meeting presentations. in normal neurons, ube3a is transcriptionally inactivated (imprinted) on the paternally derived allele of chromosome 15 and is active only on the maternally derived allele. all other somatic cells have biallelic transcription. in 1997, loss of ube3a function was identified as the cause of as (kishino. the syndrome can occur by four different mutational mechanisms affecting the maternally derived chromosome 15 : intragenic mutation, chromosome microdeletion of the gene, paternal uniparental disomy (absence of maternal chromosome 15), and a defect in the imprinting center that controls ube3a transcription. the syndrome occurs in 1:1215,000 births and is characterized by severe to profound cognitive delay, seizures, severely limited speech, pro - social behaviors, tremulous limb movements, and an ataxic gait (williams. to understand the role of ube3a and potential therapies for as, an important goal is to identify both the substrates that ube3a targets for degradation and other ube3a - interacting proteins. moreover, the role of ube3a must also be considered in the context of how it and its substrates might be regulated by neural activity. with this in mind, michael e. greenberg, the symposium 's keynote speaker, provided an overview of how neuronal stimulation induces the transcription of hundreds of genes, including transcription factors and genes encoding synaptic proteins, as reviewed in greer and greenberg (2008). an increase in intracellular calcium appears to be the first step in this gene induction process. calcium influx, typically through l - type voltage - sensitive calcium channels and nmda receptors, can initiate synapse - to - nucleus signaling. calcium - mediated responses activate cytoplasmic proteins, including protein kinases, which translocate to the nucleus to phosphorylate and activate transcription factors. the camp - response element - binding protein (creb) was one of the first studied proteins involved in this type of induction. creb acts on genes with cre - response elements through a repressor complex spanning the promoter and upstream elements of these genes. calcium - mediated phosphorylation of creb increases its association with creb binding protein and other proteins in the basal transcription complex to promote gene transcription. this intricate process involves changes in histone acetylation and recruitment of the rnaii polymerase complex to promote gene transcription. the greenberg lab has suggested that ube3a is itself an activity - regulated gene (flavell. 2010) and thus protein substrates of ube3a would likely have similar activity - dependent expression. to identify new ube3a targets, the greenberg lab has taken advantage of a transgenic mouse with a hemagglutinin epitope - tagged version of ubiquitin (ha - ubiquitin) knocked into the hprt locus. by crossing these mice with either wild - type or ube3a - deficient mice, and comparing ubiquitination differences between the two crosses, the investigators were able to identify direct brain lysate proteins in as mice that had reduced ha - ubiquitin tagging. using this approach, they identified sacsin as an ube3a substrate (mutation in the sacsin gene causes autosomal recessive spastic ataxia of charlevoix saguenay). then, by comparing sequences between sacsin and another known ube3a substrate, hhr23a, they identified a ube3a - binding domain having loose sequence homology. using this domain, they identified arc (activity - regulated cytoskeletal protein) as a new ube3a target (greer. arc is known to regulate ampa receptor (ampar) trafficking and internalization at the synapse (bramham. accordingly, they found diminished surface counts of synaptic ampar receptors using array tomography (a technique in which ultra - thin sections of brain tissue are stained, imaged, and synapses visualized as a 3d reconstruction) and increased arc protein levels in direct brain lysate analysis of as mouse neurons (which lack ube3a). the greenberg lab hypothesized that the absence of ube3a could lead to accumulations of arc, which in turn could lead to increased ampar internalization. notably, the converse could occur in situations of ube3a excess, as in the 1% of individuals with autism arising from maternal duplications of the 15q11q13 region (christian. 1997), which spans the ube3a gene. increased ube3a gene expression has been demonstrated for these duplications, both the interstitial ones and the isodicentric marker chromosomes (herzing. interestingly, arc is known to be translationally inhibited by the fragile x protein (fmrp), and the simplistic picture emerges that dysregulation of arc protein homeostasis may be a commonality between as and fragile x mental retardation. it is not clear at this time whether inhibitors of the metabotropic glutamate 5 receptor, a therapeutic strategy being pursued in fragile x, could be of benefit in as. greenberg also reported on discovery of a new ube3a substrate involved with the ephb subfamily of receptor tyrosine kinases. eph receptors are known to be enriched at synapses and are important in regulating dendritic spine density. the ephb receptors interact with ephrin ligands and regulate dendritic development through small gtpases of the rho family (rho, rac, and cdc42) by activation of guanine nucleotide exchange factors (gefs) (murai and pasquale 2003). gefs serve as a switch to convert gtpases from an inactive gdp to an active gtp state. the gtp - bound forms can interact with downstream effectors that control cytoskeletal rearrangements. ephb - linked rho activation appears to inhibit dendritic spine outgrowth, while rac activation antagonizes this effect and promotes outgrowth. the greenberg lab determined that a newly identified ube3a substrate, named ephexin 5, acts as a gef for rho (margolis. consistent with ephexin5 being a ube3a substrate, ube3a - deficient neurons exhibit increased ephexin 5 levels. this raises the possibility that loss of ube3a function could lead to restricted synaptic development ; conversely, duplication of ube3a (as seen in some cases of autism) could result in diminished ephexin 5 levels with promotion of spine development. peter howley, a long - time investigator into the role of ube3a, reported on an unbiased proteomics approach to identify substrates and regulators of ube3a. with most proteomic studies, it is difficult to determine the physiologically relevant proteins among the large number of nonspecific interacting proteins dominating a mass spectral analysis. to overcome this problem, the howley lab is collaborating with wade harper 's lab to improve the identification of relevant interacting proteins and the analysis of interaction networks by using a newly established platform (comppass). comppass uses catalytically inactive forms of the three major isoforms of ube3a to identify high confidence interacting proteins (sowa. initial work with this approach used human embryonic kidney cells but is now being extended into human neural cells using a tet - regulatable human neuroblastoma sh - sy5y cell line to identify neural - specific substrates. the expectation is that this new approach will soon lead to the identification of novel ube3a substrates, providing additional therapeutic avenues in as. lawrence t. reiter reported on his work toward identifying substrates and interacting proteins for the drosophila ortholog of ube3a (dube3a). reiter used proteomic profiling in drosophila heads to show that dube3a may regulate punch (gtp cyclohydrolase i, gch1). gch1 catalyzes the first and rate - limiting step in tetrahydrobiopterin biosynthesis and thus is a key regulator of monoamine (dopamine / serotonin) synthesis in flies and humans. consequently, dube3a overexpression elevates tetrahydrobiopterin levels while diminished expression of dube3a has the opposite effect on both gch1 transcripts and dopamine levels. reiter proposed that this regulatory effect could be due to a transcriptional co - activation function of dube3a since an enzymatically inactive form of dube3a (dube3a - c / a) is also able to elevate dopamine levels through gch1. performing rapid genetic and functional assays in drosophila is an advantage of this model system, one that has already been used to demonstrate the importance of ube3a in dendritic structure (lu. 2009). using the more traditional, but still powerful, approach of yeast - two - hybrid screening and affinity proteomics, konstantin matentzoglu and interestingly, the herc2 gene is located in close proximity to ube3a and frequently found co - deleted with ube3a in individuals with as. the research shows that one of the rlds, the rcc1b domain of herc2, not only mediates binding to ube3a but also greatly enhances ube3a ligase activity. herc2 recruits ube3a into complexes and enhances auto - ubiquitination ; furthermore, substrate ubiquitination of ring1b appears to be activated in the same manner. the overlapping tissue distribution of ube3a and herc2 suggests a tantalizing possibility for functional interactions, although the physiological relevance of these potential interactions remains unknown. in addition to the importance of identifying substrates and interacting partners for ube3a, another important approach for guiding therapeutic approaches is to understand the neurological consequences of reduced ube3a expression. towards this end, ben philpot has used a mouse model of as to demonstrate that ube3a is required for neocortical plasticity in vitro as well as for experience - driven plasticity in vivo (yashiro. his work and that of others demonstrate deficits in as mouse for long - term potentiation and long - term depression, indicating that the loss of ube3a leads to a severe synaptic rigidity that likely underlies impaired learning in individuals with as. new data show that reduced ube3a causes a loss of synaptic connectivity across a variety of neuronal cell types and that this deficit in connectivity may be one of several factors limiting experience - driven synaptic learning in vivo. the philpot lab has previously shown that excitatory synapse development is blunted in ube3a - deficient mice by postnatal day 21. the philpot lab now finds that there are also severe deficits in inhibitory synapse development, but these deficits arise later in development. the deficits in inhibitory transmission appear to arise from decreased synaptic contacts made by inhibitory interneurons onto excitatory pyramidal neurons. it is unclear at this time whether the change in inhibition is primary or adaptive. however, he speculated that the loss of inhibition is maladaptive, as the mice have increased seizure susceptibility that is likely to arise from a severe loss of inhibition. these observations suggest that an imbalance between excitation and inhibition in the neocortex may contribute to epilepsy and neocortical dysfunction associated with as. although many factors are likely to contribute, the increases in arc expression observed in ube3a - deficient mice (noted above) could explain both the loss of functional synapses, due to the loss of ampars, and deficits in synaptic plasticity, due to aberrant ampar trafficking. indeed, it is now clear that arc expression must be tightly regulated for normal experience - driven plasticity in learning. along these lines, jason d. shepherd reported on recent data demonstrating that genetic deletion of arc prevents experience - dependent plasticity in the developing visual cortex (mccurry. arc knockout mice exhibited deficits in ocular dominance plasticity due to deficient depression of the deprived eye responses, similar to that observed in as model mice (yashiro. arc knockout mice also exhibit deficits in stimulus - specific response potentiation (srp), a relatively newly identified form of experience - dependent plasticity (frenkel. daily exposure to a horizontal grating stimulus of a single orientation selectively potentiates the amplitude of visual evoked potentials to that orientation, but not other orientations. these deficits occurred despite normal visual acuity and retinotopic organization, also similar to what has been observed in the as mouse model. thus, accumulating data suggest that having either too little or too much ube3a, and by extension arc, causes a severe impairment in synaptic plasticity. in addition to plasticity defects, the loss of ube3a can alter a number of other neuronal properties. indeed, hanoch kaphzan reported that the loss of ube3a alters intrinsic membrane properties in hippocampal area ca1 neurons. these changes may be caused, at least in part, by increased expression of the na / k - atpase alpha1 subunit, ankyrin - b and ankyrin - g, all three known to interact with the atpase transporter. the na / k - atpase transporter consists of alpha and beta subunits, and at least four genes encode for different isoforms of the alpha subunits. missense mutations in the alpha 2 gene (atp1a2) have been associated with familial hemiplegic migraine, a disorder in which some affected individuals can also have seizures and ataxia. because na / k - atpases play a seminal role in controlling neuronal excitability, it is tempting to speculate that the observed abnormalities could be related to the seizure liability observed in individuals with as. simone kehnle used established non - neural mammalian cell lines and rnai - mediated downregulation of ube3a to demonstrate that reduced ube3a levels result in severe cytotoxicity. he also found that ectopic ube3a expression is cytotoxic too, indicating that ube3a levels have to be delicately balanced in mammalian cells, contributing once again to the recurring theme that either the loss or overexpression of ube3a can lead to many similar defects. he then performed studies showing that, for cells depleted for ube3a, camkii overexpression can partially rescue the cytotoxic phenotype. camkii activity is known to be perturbed in as mouse neurons (weeber. hence, the cytotoxicity of ube3a depletion, as observed in non - neuronal cell lines, may at least in part involve the same pathways that are deregulated in the as mouse model. kehnle 's research with established cell lines may be of potential use to elucidate the pathways involved in as development. future advances in as will rely heavily on the development of new mouse models and their creative use. scott dindot has developed an ube3a - yfp reporter mouse (dindot. 2008) and is now using crosses of this mouse having the yfp reporter on either the maternal or paternal allele to examine the allelic expression patterns of ube3a during neurogenesis. current observations suggest that both paternal and maternal alleles express ube3a in the stem cell niches lining the lateral ventricles and in the dentate gyrus of the hippocampal formation. progenitor cells and immature neurons both express ube3a, while expression of ube3a in mature neurons is derived primarily from the maternal allele. notably, low levels of expression were detected from the paternal allele, demonstrating that the paternal allele remains functional and intact. collectively, these data suggest that imprinting of ube3a is specific to neuronal cells, as previously reported (yamasaki. 2003), but is not complete until the later stages of neuronal maturation. while most mouse models of as have involved selective deletion of ube3a (jiang. 1998 ; miura. 2002), 70% of individuals with as have a much larger deletion from 15q11 to q13. to create a more realistic model of the most common form of as, yong - hui jiang and his coworkers generated mutant mice with a 1.6 mb chromosomal deletion that spans ube3a to gabrb3, including deletion of atp10a. homozygous deletion mice show cleft palate and die in the perinatal period, but the heterozygotes survive and show parent - of - origin phenotypes (jiang. expression analysis of maternal and paternal deletion mice confirmed that the ube3a gene is exclusively maternally expressed in brain and that the atp10a and gabrb3 genes are biallelically expressed in all brain subregions studied. the maternal deletion mice showed many of the behavioral features seen in other ube3a - deficient mouse models. in addition, ultrasonic vocalization recordings in newborns revealed that maternal deletion pups emitted significantly more vocalizations than wild - type littermates, but the behavioral correlate to communication deficits observed in human as is unclear. this mouse model will be invaluable to future as research and will help to provide a biological basis for the more severe deficits in speech and epilepsy observed in as individuals with 15q11q13 deletions compared to those with ube3a mutations (lossie. marc lalande elegantly described how he and stormy chamberlain are using induced pluripotent stem (ips) cells to develop an innovative approach to study as. their lab has established two human as deletion and two normal control ips cell lines derived from fibroblasts (chamberlain. the ips cell lines express nanog and oct4, two markers that characterize the undifferentiated stem cell state. the ips cell lines can be differentiated into mature neurons, using a technique that mimics neural development in the embryo. during this process of in vitro neurogenesis, there is upregulation of pax6, a marker of neuronal precursors, at early stages, followed by expression of map2, synapsin i and iii tubulin in neurons. single - cell recordings from the ips - derived neurons demonstrate that these cells fire action potentials and exhibit spontaneous synaptic activity, hallmarks of mature neurons. a marked decrease in ube3a mrna and protein is observed in neurons derived from as ips cells, indicating that silencing of the paternal ube3a allele occurs in this in vitro platform, similar to what is likely to occur in vivo. as neurons derived from ips cell lines may thus provide important insights about the developmental timing and regulation of ube3a imprinting and for characterizing the functional abnormalities in human as neurons. the imprinting of ube3a presents unique treatment opportunities. one strategy for developing an as therapeutic is to activate the silenced, but intact, paternal ube3a allele. towards this end, barbara bailus presented an update on work with david segal in developing artificial transcription factors (atfs) that may serve such a purpose. by attaching an activation or repression domain to a zinc finger two approaches are being explored to activate the paternal copy of murine ube3a in mouse cells. the direct approach targets an activator atf to the promoter region of the paternal ube3a, while the indirect approach will use repressor atfs to silence the ube3a antisense transcript thought to silence the paternal copy of ube3a. while still preliminary, this approach represents a promising new front on the development of as therapeutics. clearly, a number of approaches to develop as therapeutics rely on methodologies that restore normal ube3a levels in individuals with as. to explore the possibility that this might be achievable with advances in gene therapy, ron mandel reviewed the use of recombinant adeno - associated viral vectors (raav) for adjusting gene expression in the brain. transduction efficiency has steadily increased since the first use of the raav vectors in 1996. to date, raav2 production methods have improved, the purity of the vector and the amount recovered through the production process has increased. in addition, with the advent of alternative aav capsid serotypes, vector titers have increased, and brain transduction has become more efficient. neurons that are transduced (glial cells do not become transduced) have generally robust and durable gene expression. the raav has a size constraint of approximately 5 kb of genomic code (including the promoter), the approximate size of the ube3a cdna, so packaging may be difficult. also, gene copy number varies widely in transduced neurons, and this may be problematic for ube3a since both diminished and increased cellular levels of ube3a appear to be harmful. while gene therapy may not present a readily viable option for restoring ube3a levels in the brain and treating as, the expectation is that advances in gene therapy may someday make this approach viable. there is no salient hypothesis as to why seizures are so prevalent in the syndrome, and the clinical treatment with anticonvulsants remains largely empirical but fairly effective. to date, no class of anticonvulsants has shown a clearly superior benefit. thus, an important future goal is to understand the pathophysiology underlying the severe and relatively intractable forms of epilepsy in as so that more rationally based anticonvulsants can be designed. due to the relative rarity of as, best treatment practices are often derived from extensive parent questionnaires. with this in mind, sarika peters provided an update of a web - based parent questionnaire project aimed at delineating the use of traditional and alternative treatments for challenging behaviors in as. more than half (53% of 333) the caregivers reported individuals with as using at least one alternative treatment. the most popular alternative treatments included melatonin, omega-3 fatty acids / fish oil, folic acid, and magnesium. however, the efficacy of these approaches still needs to be studied under rigorous control conditions, as was recently done by peters. (2010) for analysis of folic acid and betaine as a putative as therapeutic. in addition to using surveys to help determine best practices in as, surveys have also helped to highlight the remarkable strain on caregivers. frances ulman presented results from an online anonymous questionnaire survey investigating parenting experiences in mothers of a child with a developmental handicap. a total of 211 mothers completed the questionnaire and, of the sample, 30 had a child with as. mothers of a child with as demonstrated a unique profile of caregiving experiences, including devoting significantly greater time to caregiving and physical demands. thus, there is an unmet need to develop therapies that can alleviate the behavioral issues surrounding angelman syndrome, and great consideration needs to be given to the extreme challenges of being a caregiver. she reported on her efforts to develop a behavioral battery to assess memory, motor imitation, and motor performance skills in individuals with as. she studied 12 children, ages 3.6 to 12.7 years, and used structured and reproducible assessment protocols. her work demonstrates that there are a range of abilities among individuals with as and some individuals exhibit relatively well - developed motor performance skills and an ability to reproduce actions from memory. participants ' skill levels were tested several times and remained stable over the assessment period. this work provides a way to evaluate key learning skills in individuals with as and study the impact of different therapeutic approaches on their ability to learn. more details of the findings from the conference are available in the abstracts at the angelman syndrome foundation website (www.angelman.org). we are gaining a better understanding of the biological actions of ube3a as more neuroscientists investigate its role in neural plasticity and learning. newly discovered ube3a targets and interacting proteins are creating speculation about therapeutic, drugable targets, and this is an exciting change in the research landscape for as. while deficiency of ube3a in brain causes as, overexpression appears to be associated with intellectual impairments and developmental abnormalities in the autism spectrum. whether an increase in ube3a. continued research into the area of autism may thus bring better understanding about as, and continued research on ube3a 's role in as may likewise bring benefits to those with autism.	this report is a meeting summary of the 2010 angelman syndrome foundation 's scientific symposium on the neuroscience of ube3a. angelman syndrome is characterized by loss of speech, severe developmental delay, seizures, and ataxia. these core symptoms are caused by maternal allele disruptions of a single gene ube3a. ube3a encodes an e3 ubiquitin ligase that targets certain proteins for proteasomal degradation. this biology has led to the expectation that the identification of ube3a protein targets will lead to therapies for angelman syndrome. the recent discovery of ube3a substrates such as arc (activity - regulated cytoskeletal protein) provides new insight into the mechanisms underlying the synaptic function and plasticity deficits caused by the loss of ube3a. in addition to identifying ube3a substrates, there have also been recent advances in understanding ube3a 's integrated role in the neuronal repertoire of genes and protein interactions. a developmental picture is now emerging whereby ube3a gene dosage on chromosome 15 alters synaptic function, with deficiencies leading to angelman syndrome and overexpression associated with classic autism symptomatology.
high - resolution peripheral quantitative computed tomography (hr - pqct) is a novel imaging modality that noninvasively assesses trabecular and cortical bone microstructure at the distal radius and tibia [1, 2 ]. although areal bone mineral density (abmd), as assessed by dual - energy x - ray absorptiometry (dxa), remains an important predictor of fractures, dxa technology is nevertheless limited in its inability to measure the trabecular and cortical bone compartments separately, each of which contributes to bone strength. the ability to assess these compartments may help improve fracture prediction in men and women. until recently, assessment of trabecular and cortical microstructure has required a bone biopsy. with the development and subsequent validation of hr - pqct [1, 47 ], trabecular and cortical microstructure of peripheral bone sites can now be determined noninvasively and is thus more amenable for clinical study. indeed, there are a growing number of studies that have now used hr - pqct to evaluate relatively large cohorts of men and women. the findings from these studies have provided important advances to our understanding of the age - related changes in bone microarchitecture that occur, which may in turn help to explain the lower fracture incidence observed in aging men relative to aging women. bone histomorphometry from iliac crest bone biopsies provided invaluable two - dimensional information on bone microarchitecture, but the use of bone biopsies in clinical studies was limited due to its invasive nature and the inability to assess a section of bone more than once. furthermore, the three - dimensional (3d) aspect of bone microarchitecture could not be taken into account to fully understand its biomechanical properties. imaging modalities were thus developed that could assess, noninvasively, the 3d bone microarchitecture in vivo [1, 2 ] and this work led to the now commercially available 3d hr - pqct scanner (xtreme ct, scanco medical ag, switzerland ; voxel size 82 m) that can image the distal radius and distal tibia of human subjects. the imaging protocol provides not only an assessment of both trabecular and cortical bone microstructure parameters, which will be described in more detail below, but also total volumetric bmd (vbmd, g / cm) of the distal radius or tibia as well as separate measures for trabecular and cortical vbmd. in recent software updates, an assessment of cortical porosity has become available as well as an assessment of bone strength at the radius and tibia determined through microfinite element analysis (ipl v1.12, scanco medical ag) although similar parameters can be derived from the images independently from the built - in software. several additional trabecular and cortical microstructure parameters are provided through the commercial software ; however, these novel parameters have not been as well studied as those described below. imaging of the distal radius or tibia by hr - pqct involves a similar protocol with the acquisition of a 3d stack of 110 high - resolution ct slices at either the distal end of the radius (figure 1(a)) or tibia, with an isotropic voxel size and slice thickness of 82 m. the processing and analyses of these images has been validated [47 ]. figures 1(b) and 1(c) show representative cross - sectional scan slices and 3d images, respectively, that can be obtained from hr - pqct imaging. some trabecular microstructure parameters assessed using hr - pqct are measured directly while others are derived. the trabecular vbmd is determined directly, and then the trabecular bone volume / total volume (bv / tv) is derived, assuming a mineral density of fully mineralized bone of 1.2 g hydroxyapatite / cm. recognizing that individual trabeculae would not be resolved at their correct thickness (~100 m) due to partial volume effects, a thickness - independent structure extraction is employed whereby 3d ridges (the center points of the trabeculae) are detected in the gray - level images. trabecular number (tbn, 1/mm) is then taken as the inverse of the mean spacing of the ridges. then, in analogy with standard histomorphometry, trabecular thickness (tbth, mm) is calculated using the formula tbth = bv / tv tbn, and trabecular spacing or separation (tbsp, mm) is calculated using the formula tbsp = (1 bv / tv) tbn. validation studies show excellent correlation (r 0.96, p < 0.0001) for these parameters when compared with the gold - standard ex vivo microcomputed tomography (ct) technique. the cortex is segmented from the gray scale image with a gaussian filter and threshold. cortical vbmd (g / cm) bone cross - sectional area (csa, mm) and cortical cross - sectional area (ct csa, mm) are measured directly, and the periosteal circumference calculated from the contour. cortical thickness (ctth, mm) is then derived using the formula ctth = ct csa circumference. recent studies that have assessed both men and women using hr - pqct have led to important insights into the sex- and age - related differences in bone microarchitecture. greater bone strength in men than women is, in part, related to larger bone size of men. however, differences between the sexes with respect to both the trabecular and cortical compartments of bone may also contribute to their differences in bone strength and fracture risk. first reported on the bone microarchitecture by hr - pqct in both men and women. using the initial prototype of the hr - pqct scanner, the distal radius was imaged in 278 men and 324 women (ages of 2197 years), who represented an age - stratified, random sample of the rochester, mn community adults. relative to young women age 2029 years of age (n = 17), young men of a similar age range (n = 19) had higher bv / tv and greater tbth but had similar tbn and tbsp. young men also had greater bone csa, but similar ctth, when compared with young women. imaged both the distal radius and tibia in 146 healthy volunteers (53 men and 93 women), aged 2078 years. they found that young men (age 2029 years) had greater bv / tv and tbth at both the distal radius and tibia when compared with young women of the same age range. tbn and tbsp were not significantly different between the sexes at the radius but were at the tibia. findings were thus similar to what was observed by khosla. at the radius. the tibia was not imaged in the study by khosla., but findings by sode. suggested that the sex - related differences in bone microarchitecture may not be the same between the radius and tibia sites. sode. also noted substantial variations in trabecular parameters within a cross - section of either the distal radius or tibia and identified sex - specific subregional variations in trabecular microstructure. in a population - based study from canada involving 202 men and 442 women (ages 2099 years) where both the distal radius and tibia were measured, macdonald n = 28) had greater bone size, bv / tv and tbth at the distal radius when compared with young women of the same age range (n = 58), observations that were again similar to reports at the same site by khosla. and sode. in contrast, they observed that young men also had greater tbn and ctth than women. at the distal tibia, young men again had greater bone size, bv / tv, tbn and ctth than women. tbth at the tibia was also slightly higher in men but did not reach statistical significance. interestingly, in a study involving almost 300 adolescent boys and girls between the ages of 1520 years who had their distal tibia imaged by hr - pqct, sex - related differences were more apparent by the age of 1720 years, with boys in this age range having greater bone size, bv / tv and ctth relative to girls. however in this study, tbth was not significantly different between the sexes at any age, whereas tbn was higher, while tbsp was lower, in boys than girls at all ages. however, a cross - sectional study by wang., involving almost 130 boys and girls ages 518 years, found that bv / tv and tbth increased more in boys than girls over puberty at both the radius and tibia sites, while tbn remained similar for both boys and girls. similarly, in a cross - sectional study by kirmani. assessing the distal radius of 140 healthy boys and girls ages 621 years, bv / tv and tbth also increased more in boys than girls over puberty. tbn transiently increased in boys more than girls across age but then became similar again in both sexes at more mature ages. collectively, these studies confirm that there are clear bone microarchitectural differences apparent between young men and women, particularly bone size, bv / tv and tbth, with these differences developing over puberty and appearing to be established as early as late adolescence. these studies also suggest that sex - related differences for some parameters of bone microstructure may be site - specific. nevertheless, there was a lack of consistency among studies on findings for some parameters, but they may reflect imaging resolution issues or power limitations. to date, studies reporting on the age - related differences in bone microarchitecture in men and women have been cross - sectional in nature. the determination of true age - related changes in bone microarchitecture will require longitudinal studies. nevertheless, these cross - sectional studies do provide key information on differences in the trabecular and cortical compartments of bone between young and old and thus impart the most practical insights on structural changes that may be related to aging in men and women. in the study by khosla., cross - sectional decreases in bv / tv at the radius between the ages of 20 and 90 years were reported to be similar in men and women. however, in contrast to women, where tbn decreased and tbsp increased with age, there were little age - related changes in these parameters in men. on the other hand, tbth appeared to decrease to a greater extent in men than women (p < 0.01) (figure 2). these findings suggested that the mechanism for loss in bv / tv with aging in men and women may be different, with loss of trabeculae being the main factor in women but trabecular thinning being the mechanism for men. as decreases in tbn have been shown to have greater impact on impairing bone strength compared with decreases in tbth, it was suggested that these findings could explain the lower life - long fracture risk in men relative to women. macdonald. observed similar findings as khosla. at the distal radius. however, at the distal tibia, macdonald. found that tbth, tbn, and tbsp changed to a similar extent in both men and women with age. reported that age - related differences in men and women were more prominent at particular subregions of each bone. on the other hand, dalzell. failed to see any significant association between trabecular microstructure parameters and age, at either the distal radius or tibia, in their study involving 58 men and 74 women (ages 2079 years) from the uk. khosla. reported that, in both men and women, bone csa increased with aging, but while the percentage change was similar between men and women, older men continued to have greater bone size than older women. in contrast, ctth decreased to a lesser extent in men than women (38% versus 52%, resp. thus confers a greater biomechanical advantage for bones of elderly men compared with elderly women. in contrast to the trabecular compartment, where parameters showed cross - sectional age - related declines before and after age 50 years, the cortical parameters tended to remain stable until after age 50 years when they then started to decline. observed that cortical porosity increased to a lesser extent in men than women with advancing age. however, cortical porosity appeared to be greater in men than women at all age ranges except at the eighth decade. in a study involving 57 men and 94 women (age range 2078 years), burghardt. reported similar observations, with men having greater cortical porosity than women, especially before age 30. differences became attenuated in those over the age of 50 years due to a 3-fold increase in cortical porosity in women from age 20 to 60 years, with men having an increase in cortical porosity of only 60% over the same age range. based on observations from these cross - sectional studies, men appear to have more trabecular thinning than drop - out with increasing age, while women have both trabecular thinning and dropout. furthermore, men have greater bone size than women across age and suffer less cortical thinning than women with aging. although men have greater cortical porosity than women, it increases more in women than men with age. overall, these apparent age - related changes in trabecular and cortical microstructure in men would thus seem to confer less of an adverse effect on bone strength and, thereby, explain the lower fracture risk, in aging men when compared with aging women. to date, only the structure of the aging men 's bones (strambo) study has evaluated the relation between prevalent fractures in men with bone microarchitecture parameters from hr - pqct [21, 22 ]. in a subset of men from the strambo cohort, 185 men (mean age, 71 years) with a prevalent fracture and 185 men without fracture had both the distal radius and tibia imaged by hr - pqct and had assessments of bone strength by finite element analysis. at both bone sites, ctth, tbn, and tbsp bone strength parameters of stiffness and failure load also were 8 - 9% lower in fracture cases relative to controls (p < 0.01). in a larger cohort from strambo, involving 920 men over the age of 50 years who had hrpqct measurements at the distal radius and tibia, 98 men had a prevalent vertebral fracture, and 100 men had a prevalent peripheral fracture. again, almost all bone microarchitecture parameters from hrpqct scans were worse in men with either vertebral or peripheral fractures. following adjustment for abmd, there were no associations observed between bone microarchitecture parameters and peripheral fractures, whereas cortical vbmd and ct th at both the distal radius and tibia remained lower in men with vertebral fractures. whether the bone microarchitecture parameters based on the radius or tibia will be able to predict overall or site - specific fragility fractures in men prospectively remains to be determined. imaging studies using hr - pqct of bone microarchitecture among children at various stages of puberty also have provided further understanding on the pathogenesis of increased fractures in adolescence among both boys and girls although findings are again limited by their cross - sectional nature. in the study by kirmani., the proportion of load borne by cortical bone and the ratio of cortical to trabecular bone volume at the radius decreased transiently during mid - to - late puberty in both sexes, with apparent cortical porosity peaking during this time. these findings appeared to mirror the incidence of distal forearm fractures seen in boys and girls of this age group. regional deficits in cortical bone were thus suggested to underlie the adolescent peak in forearm fractures, a hypothesis that was also suggested by wang.. although studies in men imaged by hr - pqct are limited, current findings have provided novel insights into the sex- and age - related differences in trabecular and cortical microstructure that underlie the differences in bone strength and fracture risk observed in men relative to women. nevertheless, the cross - sectional nature of studies to date does limit the ability to make definitive conclusions, and longitudinal studies remain the ideal in order to best determine age - related changes in bone microarchitecture and their relation to fracture risk in both men and women.	the trabecular and cortical compartments of bone each contributes to bone strength. until recently, assessment of trabecular and cortical microstructure has required a bone biopsy. now, trabecular and cortical microstructure of peripheral bone sites can be determined noninvasively using high - resolution peripheral quantitative computed tomography (hr - pqct). studies that have used hr - pqct to evaluate cohorts of both men and women have provided novel insights into the changes in bone microarchitecture that occur with age between the sexes, which may help to explain the lower fracture incidence in older men relative to women. this review will highlight observations from these studies on both the sex- and age - related differences in trabecular and cortical microstructure that may underlie the differences in bone strength, and thereby fracture risk, between men and women.
the sequential deletion method and other biological and biochemical experiments are generally used to locate the functional domain of a protein. for example, a previous study (1) used the sequential deletion method accompanied by manual pcr primers design to generate the n - terminal truncated mutants of different lengths (figure 1), which in turn were used in further biological experiments to decipher the functional domain of the 5s rna - protein complex (5s rrnp). the 5s rrnp is believed to be formed by a co - translation event leading to the binding of the 5s rrna to the nascent ribosomal protein l5. lin. (1) used an in vitro translation system to investigate how and when 5s rrna triggers the formation of the eukaryotic 5s rrnp. the l5 and truncated l5 mutant mrnas were prepared on a large scale for their investigation and a great amount of time was needed to manually modify the in - frame pattern of atg start codon for conventional pcr and truncated mutant translation experiments. the illustration of the produced n - terminal truncated mutants. in order to save time and money needed in the traditional sequential deletion method, this web - based application system ntmg is proposed to automatically do the multiplex pcr assays design in order to generate the various n - terminal truncated mutants. given a protein cdna sequence, the ntmg will first find those atg - like codons that are suitable to act as the starting positions of truncated mutants. then, the ntmg will design the forward primers for all possible truncated mutants. finally, with all these primers, the ntmg will choose those primers that can be divided into the least number of groups such that each group constitutes a multiplex pcr assay. in this section, we describe the input to the ntmg, the methodology of the ntmg and the output of the ntmg. since the primer design and the multiplex pcr primer design are two important parts of the ntmg, some factors concerning the primer design such as the primer length and the melting temperature are input as parameters. there are also some factors that may affect the multiplex pcr amplification with multiple primers in the same tube (2,3). these factors include the cross - dimerization, the melting temperature, the products co - existence and others. figure 2 gives the input screen of the ntmg. on the top of the screen, the cdna sequence, the start codon address and the stop codon address are input. then comes four classes of parameters : primer criteria (47)these include the forward primer length (default 2030 bp), the reverse primer length (default 1428 bp) and the gc content (default 40 - 60%).temperature criteria (5,810)these include melting temperature (default 5160c), the melting temperature range for each group (default 5c), the molar concentration of monovalent cation (default 50 mmol / l), the molar concentration of mg (default 1.25 mmol / l) and the dntps concentration (default 0.02 mmol / l).complementary criteria (4,8,1012)these include the terminal repeated sequence (default 3 bp), the intra self - complementary sequence (default 3 bp), the specificity (default 65%), the cross - dimer distance (default 10 bp), the cross - dimer total similarity (default 50%) and the cross - dimer terminal similarity (default 3 bp).grouping criteriathese include the product length difference (default 80 bp) and the maximum number of primers in each group (default 16). figure 2.the input screen of the ntmg. primer criteria (47) these include the forward primer length (default 2030 bp), the reverse primer length (default 1428 bp) and the gc content (default 40 - 60%). temperature criteria (5,810) these include melting temperature (default 5160c), the melting temperature range for each group (default 5c), the molar concentration of monovalent cation (default 50 mmol / l), the molar concentration of mg (default 1.25 mmol / l) and the dntps concentration (default 0.02 mmol / l). complementary criteria (4,8,1012) these include the terminal repeated sequence (default 3 bp), the intra self - complementary sequence (default 3 bp), the specificity (default 65%), the cross - dimer distance (default 10 bp), the cross - dimer total similarity (default 50%) and the cross - dimer terminal similarity (default 3 bp). these include the product length difference (default 80 bp) and the maximum number of primers in each group (default 16). first, a cdna sequence is input, then the ntmg searches the sequence with in - frame criterion in order to find all the atg - like codons. an atg - like codon is a xtg, an axg or an atx with x representing a, t, c or g. for each atg - like codon, the ntmg generates candidate primers using the sliding window. the ntmg also modifies the atg - like codon into the atg codon in each candidate primer. hence, each candidate primer contains the atg start codon with in - frame criterion that can translate correctly to truncated mutants. after that, the ntmg applies the primary criteria and the secondary criteria to check if the candidate primers satisfy all the criteria. those candidate primers that pass the criteria checking are then divided into classes according to the melting temperature. each class contains primers whose melting temperatures are within the same range (default 5c). so the primers chosen from the same class may be used in the same pcr experiment. for candidate primers in the same class, the ntmg does the cross - dimer check and the co - existence check and builds the cross - dimer matrix and the co - existence matrix. these two matrices are then anded to produce a new matrix which acts as an adjacency matrix of a graph. previous studies (13,14) proposed the transformation of minimizing the number of primers into the finding of the maximum clique in a graph. next, each class is further divided into subclasses according to the positions of the atg - like codons. the proposed genetic algorithm is then applied for each class in order to choose a set of primers, one from each subclass and the objective is to divide these primers into as few groups as possible such that each group of primers can be put in a tube in the pcr experiment. that is, the genetic algorithm tries to find a good multiplex pcr assay design. a heuristic maximum clique algorithm is proposed to calculate the fitness of a chromosome in the genetic algorithm. this heuristic algorithm tries to find the maximum clique in the graph previously mentioned and the maximum clique corresponds to the minimum number of grouping. the ntmg is written in java using java 2 platform standard edition 5.0 development kit (j2sdk) and employs the java server page (jsp) on the apache tomcat server (http://tomcat.apache.org/). the ntmg outputs the number of primer groups, the number of forward primers, the number of reverse primers and the primers in each group (figure 4 shows a solution report). figure 2 gives the input screen of the ntmg. on the top of the screen, the cdna sequence, the start codon address and the stop codon address are input. then comes four classes of parameters : primer criteria (47)these include the forward primer length (default 2030 bp), the reverse primer length (default 1428 bp) and the gc content (default 40 - 60%).temperature criteria (5,810)these include melting temperature (default 5160c), the melting temperature range for each group (default 5c), the molar concentration of monovalent cation (default 50 mmol / l), the molar concentration of mg (default 1.25 mmol / l) and the dntps concentration (default 0.02 mmol / l).complementary criteria (4,8,1012)these include the terminal repeated sequence (default 3 bp), the intra self - complementary sequence (default 3 bp), the specificity (default 65%), the cross - dimer distance (default 10 bp), the cross - dimer total similarity (default 50%) and the cross - dimer terminal similarity (default 3 bp).grouping criteriathese include the product length difference (default 80 bp) and the maximum number of primers in each group (default 16). figure 2.the input screen of the ntmg. primer criteria (47) these include the forward primer length (default 2030 bp), the reverse primer length (default 1428 bp) and the gc content (default 40 - 60%). temperature criteria (5,810) these include melting temperature (default 5160c), the melting temperature range for each group (default 5c), the molar concentration of monovalent cation (default 50 mmol / l), the molar concentration of mg (default 1.25 mmol / l) and the dntps concentration (default 0.02 mmol / l). complementary criteria (4,8,1012) these include the terminal repeated sequence (default 3 bp), the intra self - complementary sequence (default 3 bp), the specificity (default 65%), the cross - dimer distance (default 10 bp), the cross - dimer total similarity (default 50%) and the cross - dimer terminal similarity (default 3 bp). these include the product length difference (default 80 bp) and the maximum number of primers in each group (default 16). the input screen of the ntmg. first, a cdna sequence is input, then the ntmg searches the sequence with in - frame criterion in order to find all the atg - like codons. an atg - like codon is a xtg, an axg or an atx with x representing a, t, c or g. for each atg - like codon, the ntmg generates candidate primers using the sliding window. the ntmg also modifies the atg - like codon into the atg codon in each candidate primer. hence, each candidate primer contains the atg start codon with in - frame criterion that can translate correctly to truncated mutants. after that, the ntmg applies the primary criteria and the secondary criteria to check if the candidate primers satisfy all the criteria. those candidate primers that pass the criteria checking are then divided into classes according to the melting temperature. each class contains primers whose melting temperatures are within the same range (default 5c). so the primers chosen from the same class may be used in the same pcr experiment. for candidate primers in the same class, the ntmg does the cross - dimer check and the co - existence check and builds the cross - dimer matrix and the co - existence matrix. these two matrices are then anded to produce a new matrix which acts as an adjacency matrix of a graph. previous studies (13,14) proposed the transformation of minimizing the number of primers into the finding of the maximum clique in a graph. next, each class is further divided into subclasses according to the positions of the atg - like codons. the proposed genetic algorithm is then applied for each class in order to choose a set of primers, one from each subclass and the objective is to divide these primers into as few groups as possible such that each group of primers can be put in a tube in the pcr experiment. that is, the genetic algorithm tries to find a good multiplex pcr assay design. a heuristic maximum clique algorithm is proposed to calculate the fitness of a chromosome in the genetic algorithm. this heuristic algorithm tries to find the maximum clique in the graph previously mentioned and the maximum clique corresponds to the minimum number of grouping. the ntmg is written in java using java 2 platform standard edition 5.0 development kit (j2sdk) and employs the java server page (jsp) on the apache tomcat server (http://tomcat.apache.org/). the ntmg outputs the number of primer groups, the number of forward primers, the number of reverse primers and the primers in each group (figure 4 shows a solution report). a web - based application system called the ntmg is provided for researches who need to apply the sequential deletion method to locate the functional domain of a protein. each group of primers can be put in a tube and all tubes can be accommodated in a single batch of the multiplex pcr amplification under the same condition. we conducted a wet laboratory experiment on the multiplex pcr assay design proposed by the ntmg on input hl5 cdna. the ntmg found 48 forward primers and one reverse primer and it divided them into 8 groups. in the wet pcr experiment, 44 pcr products had been found and the success rate is 91.7% (see supplementary data). hence, the ntmg is of practical use to researchers who need to apply the sequential deletion method. as a future work, we plan to develop the more general multiplex pcr assay design. given a set of pcr experiment requirements, we plan to develop a system that can automatically find the primers and try to divide the primers into as few groups as possible such that the primers in each group can be put in a tube and all tubes can be accommodated in a single batch of the multiplex pcr experiment.	the sequential deletion method is generally used to locate the functional domain of a protein. with this method, in order to find the various n - terminal truncated mutants, researchers have to investigate the atg - like codons, to design various multiplex polymerase chain reaction (pcr) forward primers and to do several pcr experiments. this web server (n - terminal truncated mutants generator for cdna) will automatically generate groups of forward pcr primers and the corresponding reverse pcr primers that can be used in a single batch of a multiplex pcr experiment to extract the various n - terminal truncated mutants. this saves much time and money for those who use the sequential deletion method in their research. this server is available at http://oblab.cs.nchu.edu.tw:8080/websdl/.
anti - glomerular basement membrane (gbm) antibody disease is a prototypical autoimmune disease characterized by pulmonary hemorrhage and crescentic necrotizing glomerular disease as a result of antibodies targeting the non - collagenous 1 domain of the 3 subunit of type 4 collagen [3(iv)nc1 ]. the disease course is usually monophasic, with initially severe pulmonary and renal involvement, but subsequent relapses are not commonly seen. in > 95% of cases, anti - gbm antibodies can be detected in the serum using commercially available elisas that use various forms of 3(iv) collagen as the target antigen [2, 3 ]. however, there have been increasing reports of atypical anti - gbm antibody disease characterized by isolated pulmonary disease with minimal renal involvement or without detectable anti - gbm antibodies [49 ]. proposed mechanisms to explain negative anti - gbm antibody elisa testing include low levels of pathogenic antibodies below the detectable limit of the assay, different igg subtypes (such as igg4) or non - igg antibodies that are less detectable by elisa, low antigen binding affinity or target antigens other than the usual epitopes in 3(iv)nc1 [6, 1013 ]. these atypical characteristics have also been suggested to result in fewer pathogenic antibodies and hence might explain cases of isolated pulmonary disease in the context of superimposed lung injury from hydrocarbons or smoking [1, 1214 ]. we report a case of anti - gbm antibody disease with a highly unique frequently relapsing disease course that varied between anti - gbm antibody positive flares with both pulmonary and renal involvement and anti - gbm antibody negative flares that were pulmonary limited. to our knowledge, this clinical pattern of anti - gbm antibody disease has not been previously described. by comparing elisa results with igg subtypes detected along the gbm in serial kidney biopsies, this case provides unique insight into the role of longitudinal changes in antibody characteristics associated with atypical variation in the clinical phenotype of anti - gbm antibody disease. a 41-year - old woman with normal baseline kidney function presented in december 2005 with pulmonary hemorrhage confirmed on ct scan and bronchoscopy, an elevated creatinine of 957 mol / l (egfr 5 ml / min/1.73 m) requiring dialysis, > 50 red blood cells (rbcs)/high power field (hpf) on urinalysis, negative anti - neutrophil cytoplasmic antibodies (ancas), and positive anti - gbm antibodies (see figure 1). a renal biopsy showed crescentic glomerulonephritis in which 60% of the 24 glomeruli contained active cellular or fibrocellular crescents, with strong linear capillary glomerular staining on direct immunofluorescence (if) that was igg2 dominant (see figure 2 and table 1). she was treated with daily plasmapheresis, steroids and monthly intravenous (iv) cyclophosphamide for 6 months. her hemoptysis resolved, and she recovered renal function but had residual chronic kidney disease with baseline creatinine of 170 mol / l. table 1.results of linear igg subtype staining along the gbm on serial kidney biopsies, graded on a scale of 0 to 4 +, performed using indirect immunofluorescencefirst biopsy (2005)second biopsy (2009)third biopsy (2011)igg11+/2 + 1 + 1+/2+igg23 + 3 + 1+/2+igg3000igg41+/2 + 1 + 1 + fig. (a) first renal biopsy (2005). a glomerulus with a large cellular crescent characteristic of active crescentic glomerulonephritis [periodic acid (b and c) second (2009) and third (2011) biopsies. both panels show glomeruli with segmental scarring (short arrows) or global sclerosis (long arrows) (pas stain, original magnification 200). results of linear igg subtype staining along the gbm on serial kidney biopsies, graded on a scale of 0 to 4 +, performed using indirect immunofluorescence serum creatinine, anti - gbm titers and disease presentations over time. (a) first renal biopsy (2005). a glomerulus with a large cellular crescent characteristic of active crescentic glomerulonephritis [periodic acid schiff (pas) stain, original magnification 400 ]. (b and c) second (2009) and third (2011) biopsies. both panels show glomeruli with segmental scarring (short arrows) or global sclerosis (long arrows) (pas stain, original magnification 200). the glomerulus shows strong linear capillary staining for igg (original magnification 200). in june 2009 she presented with diffuse alveolar hemorrhage confirmed on ct scan and bronchoscopy, creatinine near baseline at 201 mol / l with an egfr of 25 ml / min/1.73 m and negative anti - gbm antibody and anca testing. a kidney biopsy showed strong linear capillary igg staining on if that was igg2 dominant, but no active disease (24 glomeruli17 were globally sclerotic, 2 had segmental scarring and 5 were normal ; see figure 2 and table 1). she was treated with plasmapheresis, steroids and monthly iv cyclophosphamide followed by azathioprine maintenance therapy, with improvement in her hemoptysis. in 2011, while on low - dose azathioprine, she presented again with pulmonary hemorrhage confirmed on ct scan, creatinine near her baseline at 215 mol / l with an egfr of 21 ml / min/1.73 m and negative anti - gbm antibody testing. a third renal biopsy again showed linear igg staining that was igg1/2 codominant but with no active disease (48 glomeruli33 were globally sclerotic and the rest were normal or had segmental scarring ; see figure 2 and table 1). she was treated with plasmapheresis, steroids and oral cyclophosphamide, followed by azathioprine maintenance therapy, with resolution of her pulmonary disease. in december 2012, she presented with a final disease flare while on low - dose azathioprine. she had diffuse alveolar hemorrhage confirmed on ct scan and bronchoscopy, > 20 rbcs / hpf on urinalysis, creatinine increased to 677 mol / l requiring dialysis and positive anti - gbm antibodies (see figure 1). her hemoptysis resolved but she remained dialysis dependent. throughout her disease course she had no evidence of a lymphoproliferative disorder, but she was a lifelong smoker and had recurrent occupational exposures to paints and solvents. all testing for the presence of anti - gbm antibodies was by the quanta lite gbm elisa (inova diagnostics, san diego, ca, usa), which uses purified bovine 3(iv) as substrate and was performed according to the manufacturer 's protocol. we describe a unique case of anti - gbm antibody disease that is the first report of an atypical frequently relapsing disease course alternating between combined pulmonary and renal involvement and isolated pulmonary hemorrhage. renal involvement paralleled the presence or absence of detectable anti - gbm antibodies by conventional elisas but was independent of igg subtype or staining intensity on serial kidney biopsies. anti - gbm antibody disease most commonly presents as a monophasic illness that results in crescentic glomerular disease alone or in combination with pulmonary hemorrhage. while the risk of relapse was originally thought to be quite rare, there have been recent reports of classic pulmonary - renal disease with positive anti - gbm antibodies that were followed by a single pulmonary limited relapse without detectable anti - gbm antibodies [5, 7, 8 ]. these relapses were often triggered by inhalation injury from smoking or hydrocarbons, which has been proposed to cause local alveolar injury precipitating pulmonary disease but without sufficient systemic involvement to result in crescentic glomerulonephritis. this is similar to the current case, in which multiple frequently relapsing pulmonary limited flares without detectable anti - gbm antibodies were likely precipitated by ongoing exposure to cigarettes, paint fumes or solvents. however, to our knowledge this is the first report of pulmonary limited antibody - negative relapses followed by a second occurrence of combined pulmonary and renal disease with reappearance of detectable anti - gbm antibodies. this emphasizes that in rare instances anti - gbm antibody disease can result in multiple relapses associated with inhalational irritants, that organ involvement in the lungs and kidney can fluctuate over time and that anti - gbm antibodies can be variably detectable and parallel more severe renal involvement. this case offers novel insight into anti - gbm antibody characteristics that have been previously proposed to explain variation in disease severity and false - negative conventional elisas. although igg4 is the least common subtype seen on kidney biopsy, it has been associated with more mild renal involvement and is less able to bind anti - igg antisera during elisa preparation, resulting in false - negative results [1315 ]. low levels of pathogenic antibodies below the detectable limit of elisas have also been proposed to explain pulmonary limited disease [6, 8 ]. however, neither of these explain the variation in clinical disease or anti - gbm assay results seen in this case, given that igg1 and 2 were the dominant subtypes on serial kidney biopsies, and the stable intensity of linear igg staining across disease flares suggests sufficient antibody production to result in the same degree of gbm deposition irrespective of the presence or absence of renal involvement. because commercial elisas use various forms of 3(iv)nc1 as a substrate, they are less able to detect antibodies that either target atypical antigens or have lower binding affinity and are removed during the preparation process [9, 10 ]. more severe disease is known to be associated with increasing binding affinity to target antigens and with epitope spreading to atypical nc1 domains in non-3 chains, such that the majority of patients with severe disease have antibodies against two or more antigens in different chains [1, 14, 16, 17 ]. as such, we propose that this patient may have had multiple subpopulations of antibodies with different characteristics, such as antigen binding affinity or epitope specificity, whose pathogenicity varied over time and resulted in different clinical presentations. this is consistent with the initial and final severe disease flares that had both pulmonary and renal involvement and had detectable antibodies against 3(iv)nc1 of sufficient binding affinity to result in positive elisas. the intervening more mild pulmonary limited flares occurred in the context of igg anti - gbm antibodies, as demonstrated by linear gbm staining on kidney biopsies, but these had sufficiently different properties to result in negative elisas. these findings suggest that the population of pathogenic antibodies in anti - gbm antibody disease may not be static and instead may change over time, resulting in disease flares of different severity and organ involvement and inconsistent detection of circulating anti - gbm antibodies by conventional elisas. in summary, we demonstrated a case of highly atypical anti - gbm antibody disease with a frequently relapsing course in which disease flares alternated between combined lung and renal involvement and isolated pulmonary hemorrhage. the variable detection of anti - gbm antibodies by elisa paralleled disease severity and was independent of persistent linear igg staining along the gbm, suggesting a role for changing subpopulations of pathogenic autoantibodies as an explanation for variation in disease phenotype.	anti - glomerular basement membrane (gbm) antibody disease is a typically monophasic autoimmune disease with severe pulmonary and renal involvement. we report an atypical case of frequently relapsing anti - gbm antibody disease with both anti - gbm antibody positive flares with pulmonary and renal involvement, and anti - gbm antibody negative flares that were pulmonary limited with no histologic renal disease. this is the first report of alternating disease phenotype and anti - gbm antibody status over time. disease severity paralleled the detection of anti - gbm antibodies but was independent of igg subtype staining along the gbm. this case suggests a role for changing subpopulations of pathogenic antibodies as an explanation for variation in disease phenotype and anti - gbm antibody results.
nucleotides stored in the cells not only serve as substrates for nucleic acid biosynthesis but also participate in the energy metabolism and signal transduction. nitric oxide (no) is a gaseous free radical that is synthesized by nitric oxide synthases (noss). no plays important roles in the regulation of diverse physiological phenomena such as vascular and neuronal signal transduction, host defense, and cell death regulation. signal transduction by no primarily involves a nucleotide signal molecule, guanosine 3',5'-cyclic monophosphate (cgmp), generated by soluble guanylate cyclase (sgc) from guanosine triphosphate (gtp). cgmp thus formed binds to allosteric regulatory domains of target proteins, including protein kinases, ion channels, and phosphodiesterases, with various downstream biological consequences that allow cells to adapt to changes and stresses occurring under different environmental conditions and metabolic demands. excess production of no has been suggested to be a cause of diverse pathophysiological conditions, such as inflammation, neurodegenerative and cardiovascular diseases, and cancer. these detrimental effects of no are attributed to reactive nitrogen oxide species (rnos), including nitrogen dioxide (no2) and peroxynitrite (onoo), which are formed by the reaction of no with molecular oxygen and reactive oxygen species (ros) such as the superoxide anion radical o2. peroxynitrite is thought to act as a strong oxidizing and nitrating agent in different pathophysiological situations. such rnos include nitryl chloride (no2cl), which is formed from nitrite (no2) and hypochlorous acid (hocl), and no2, which is generated by oxidation of no with molecular oxygen or by catalysis of peroxidases such as myeloperoxidase (mpo) and eosinophil peroxidase using hydrogen peroxide (h2o2) and nitrite as substrates. when production of rnos exceeds the cellular antioxidant capacity, these molecules can cause nitrative and oxidative damage of nucleic acids, proteins, lipids, and carbohydrates by nitrosation, nitration, and oxidation reactions. rnos are known to have a strong potential to oxidize and nitrate nucleic acids at the level of their base structures, e.g., guanine and adenine. during the past several years, oxidized and nitrated guanine derivatives, including 8-oxoguanine and 8-nitroguanine, were identified in diverse cultured cells, in tissues and organs from humans with cancer or degenerative diseases, and in different organisms with viral pneumonia, cancer, and other inflammatory conditions. not only the mutagenic potential but also the redox - active property of 8-nitroguanine derivatives suggested that guanine nitration may have significant biological effects yet to be identified. in fact, we recently discovered a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro - cgmp), that was produced in cells expressing inducible nos (inos). 8-nitro - cgmp is an extremely potent signaling molecule in biological systems because of its dual nature in signal transduction, i.e., in the canonical no / cgmp pathway and in noncanonical electrophilic signaling. among the nitrated guanine derivatives studied, 8-nitro - cgmp possessed the strongest redox - active and electrophilic properties. because of its electrophilic behavior, 8-nitro - cgmp reacts effectively with highly nucleophilic sulfhydryl groups of certain cysteine (cys) residues and formed a protein - s - cgmp adduct via a unique posttranslational modification named s - guanylation. in addition, some particular biological effects, e.g., cell death induction, rather than mutagenic potential are now well recognized to be caused by 8-oxoguanine accumulated in the nucleotide pool in the cells. here, we will review current knowledge about endogenous nucleotides chemically modified via oxidative / nitrative stress with respect to their formation and biological significance. there is now ample evidence from a number of data indicating relatively frequent formation of 8-oxoguanine in various cells and tissues under oxidative stress. ros derived from both endogenous origins such as mitochondria, leukocytes (oxidative burst), peroxisomes (degradation of fatty acids) and cytochrome p450 system (mixed function oxidative system), as well as exogenous origins such as cigarette smoking, uv radiation, and ionizing radiation can contribute to the formation of 8-oxoguanine. epidemiological studies showed the increased formation of 8-oxoguanine as a risk factor for cancer, atherosclerosis, diabetes and neurodegenerative disorders. there are two pathways for the accumulation of 8-oxoguanine in dna or rna : one is a result of the incorporation of oxidized (deoxy)guanosine triphosphate (8-oxo - dgtp) generated in nucleotide pools while the other is a result of the direct oxidation of guanine in dna or rna. recent progress in studies of the sanitization of nucleotide pools, as well as dna repair, has revealed that the impact of oxidation of free nucleotides such as dgtp is unexpectedly large, in comparison with the direct oxidation of dna. similarly, in vitro and in vivo experiments have shown possible nitration of nucleic acids, more specifically guanine derivatives, that have been associated with various inflammatory conditions. yermilov. found that peroxynitrite reacted with the guanine base of nucleic acids in vitro to form 8-nitroguanine. our group was the first to report in vivo evidence of guanine nitration : we found marked guanine nitration in the lungs of influenza virus - infected mice and in the lungs of patients with idiopathic pulmonary fibrosis and lung cancer, with the nitration depending on production of no by inos. we also observed formation of 8-nitroguanosine in mice infected with bacteria such as salmonella typhimurium. in addition, hoki. detected 8-nitroguanine formation in malignant fibrous histiocytoma specimens from patients. it is also interesting that formation of 8-nitroguanine was recently suggested to be linked to diabetic retinopathy, which is a major cause of blindness. our chemical analyses using high - performance liquid chromatography - based electrochemical detection and tandem mass spectrometry (lc - ms / ms) revealed that, from among a series of 8-nitroguanine derivatives and related compounds, only a nitrated derivative of cgmp, 8-nitro - cgmp, was generated in significant amounts in cell culture models with different types of cells. for example, by using lc - ms / ms, we identified 8-nitro - cgmp in murine macrophage raw 264.7 cells that had been stimulated with interferon- and lipopolysaccharide to produce no via inos. as just mentioned, infection of murine macrophages with the gram - negative bacterium salmonella also facilitated formation of 8-nitro - cgmp, which was reported to be involved in host defense against infection. formation of 8-nitro - cgmp and 8-nitroguanine derivatives can be easily detected by means of conventional immunocytochemistry with the use of anti-8-nitro - cgmp monoclonal antibodies. it was intriguing that intracellular 8-nitro - cgmp formation and 8-nitroguanine formation had similar immunostaining profiles for time and location. this may suggest that a major nitrated guanine derivatives formed in the cells is likely to be 8-nitro - cgmp rather than other nitrated nucleotides and dna / rna. we recently precisely quantified the no - dependent formation of 8-nitro - cgmp in c6 glioma cells via lc - ms / ms. treatment of cultured rat c6 glial cells with the no donor s - nitroso - n - acetylpenicillamine (snap) led to a rapid and transient increase in cgmp, but the level of 8-nitro - cgmp gradually increased linearly up to a peak value comparable to that of cgmp at 24 h and declined thereafter. markedly high levels (reaching up to 100 m) of 8-nitro - cgmp were also evident in c6 cells that had been stimulated to express inos and produce excessive no. the amount of 8-nitro - cgmp generated was much higher than that of cgmp, whose production profile slightly preceded 8-nitro - cgmp formation in the activated inos - expressing cells. because of these unexpectedly large amounts of 8-nitro - cgmp, we suspected that gtp (a substrate of cgmp biosynthesis), rather than cgmp per se, may undergo guanine nitration. this idea was indeed supported by the fact that 8-nitroguanosine 5'-triphosphate (8-nitro - gtp), produced in a cell - free chemical reaction of gtp with peroxynitrite, served as an effective substrate for sgc. among the pathological effects associated with oxidative and nitrative stress, the mutagenic potential of ros and rnos is of great interest. rnos such as peroxynitrite that commonly generated during infection and inflammation nonselectively affect a host s cells and tissues. obviously, such host defense molecules are produced to kill invading pathogens, which then suffer oxidative stress because of the host s antimicrobial attack. it may therefore be logical to expect that mutagenesis of various microbial pathogens occurs during infections in biological systems as a result of host defense. evidence of this mutagenesis includes the finding that human leukocytes producing o2, but not leukocytes from patients with chronic granulomatous disease, were shown to be mutagenic for s. typhimurium ta100. our earlier study also confirmed that oxidative and nitrative stress induced by a high output of no and ros accelerated mutation of the rna virus. related to this in vivo rna virus mutation, our investigations also found that 8-nitroguanine formed by rnos in the viral genome led to an increased frequency of mutations in an rna virus (fig. 1). in addition, authentic 8-nitroguanosine added exogenously to an rna virus - infected cells caused a dose - dependent increase in the frequency of viral mutations, especially c to u transitions. an earlier study by wogan s group documented that a high no output induced mutations in an endogenous hypoxanthine - guanine phosphoribosyltransferase gene (hprt) in murine macrophages expressing inos. genetic analysis of the no - induced mutated gene indicated that the no - associated mutational spectrum was similar to that arising spontaneously, but small deletions and insertions were found in the no - induced mutants. the same group showed that mutagenicity was enhanced by no overproduction in vivo, as assessed by mutation of an exogenously expressed lacz gene in lacz - containing pur288 plasmid - transgenic mice. excess production of no by inos induced by inammatory cytokines, possibly through rnos (particularly peroxynitrite), caused dna damage and impaired dna repair in human cholangiocarcinoma cells, as assessed by the comet assay, which suggests an no - dependent development and progression of cholangiocarcinoma. it is important to note that guanine nitration appears to cause dna mutagenesis as well, with a mutation spectrum similar to that induced by peroxynitrite. thus, 8-nitroguanine in dna may be rapidly depurinated from dna in vitro, within 14 h under physiological conditions, the result being the formation of mutagenic abasic sites and release of free 8-nitroguanine. formation of 8-nitroguanine in dna may therefore facilitate g to t transversion via abasic site formation. in addition, suzuki. used photochemical synthesis to obtain an oligodeoxynucleotide containing a single 8-nitrodeoxyguanosine at a specific position and utilized the oligodeoxynucleotide as a template in primer extension reactions catalyzed by mammalian dna polymerases. this finding suggests that 8-nitrodeoxyguanosine in dna can mispair with adenine, thereby directly inducing a g to t transversion in mammalian cells. treatment of chinese hamster ovary as52 cells with 8-nitroguanosine significantly increased the mutation frequency of the xanthine - guanine phosphoribosyltranserase gene, with g to t transversion at the gene locus. concomitant increase of abasic sites in dna further supports the notion that depurination - dependent mutagenesis may be operative for 8-nitroguanosine - induced mutation. a similar mutation spectrum is observed for oxidative damaged nucleic acids caused by peroxynitrite and ros. specifically, 8-oxoguanine can pair with both cytosine and adenine during dna synthesis, and this base mismatching could contribute significantly to spontaneous mutations in genomic dna. direct oxidation in g : c pair can result in g to t transversion during replication, whereas 8-oxo - dgtp can be incorporated in dna opposite to the adenine, leading to the a to c transversion. for example, 8-oxoguanine dna glycosylase1 (ogg1) excises 8-oxoguanine from 8-oxo - g : c pairs in dna, thus initiating base excision repair. mutt homolog-1 (mth1) is an enzyme that hydrolyzes 8-oxo - dgtp to the monophosphate form, thus preventing incorporation of 8-oxo - dgtp into dna during replication. immunohistochemical studies demonstrated that 8-oxoguanine and 8-nitroguanine are also present in the cytosol of various cells and tissues suffering from oxidative / nitrative stress, which suggests that not only dna but also the nucleotide pool, rna, and other cytosolic compartments are modified chemically via oxidative / nitrative stress. for example, because certain enzymes such as mpo are unlikely to nitrate dna directly, nitrated nucleic acids may be formed in the nucleotide pool or in rna by reactive nitrogen species that are generated by such enzyme systems. as shown for certain oxidized and halogenated nucleosides, 8-nitroguanine and related nucleosides and nucleotides may be misincorporated into dna, which can result in mutations. similarly, 8-nitroguanine and related nucleosides and nucleotides, like oxidized and halogenated nucleosides, may be incorporated into rna and interfere with rna function and metabolism. the studies summarized here therefore clearly show that oxidative and nitrative dna damage, as evidenced by increased formation of 8-oxoguanine and 8-nitroguanine, can be induced under various inflammatory conditions. in fact, chronic inflammation induced by various biological, chemical, and physical factors has also been associated with an increased risk of human cancer at many locations. however, to establish a causal relationship between this type of dna damage and human cancer, additional studies that utilize a molecular epidemiological approach in a large human population are required. 8-nitro - cgmp has the potential to activate cgmp - dependent protein kinase (pkg) in vascular smooth muscle cells ; in addition, 8-nitroguanosine and its derivatives possess a significant redox activity. 8-nitro - cgmp had the highest redox activity among the 8-nitroguanosine derivatives we tested, with redox activity decreasing in the following order : 8-nitro - cgmp > 8-nitroguanosine > 8-nitroguanosine 5'-monophosphate (8-nitro - gmp) 8-nitro - gtp. 8-nitroguanine had only negligible redox activity. in the presence of certain oxidoreductases and electron donors such as nadph, 8-nitroguanosine derivatives were readily reduced to form their anion radicals, after which a single electron was transferred to molecular oxygen to form superoxide anion radical. because of their electrophilicity, nitro - nucleotides readily react with nucleophilic thiol compounds of low and high molecular weight to form 8-thioalkoxy - guanosine (8-rs - cgmp) adducts (fig. 2). this reaction seems to occur via a nucleophilic attack by the thiol group of a protein cys or gsh on c8 of 8-nitro - cgmp, the results being release of the nitro moiety and formation of the 8-rs - cgmp adduct (fig. 2). the second - order rate constant for the reaction of 8-nitro - cgmp with the sulfhydryl of gsh was determined to be 0.03 m s at ph 7.4 and 37c. this value is much smaller than values for other electrophiles such as 4-hydroxynonenal, 15-deoxy--prostaglandin j2, and nitrolinoleic and nitrooleic acids. those compounds have reaction rate constants with gsh of 1.3, 0.7, 355, and 183 m s, respectively, at ph 7.4 and 37c. this comparatively lower second - order rate constant may account for the stable nature of this novel compound in the cellular compartment where gsh is abundant (at ~ mm levels) and may be responsible for the fact that 8-nitro - cgmp causes very selective s - guanylation with sulfhydryls possessing high nucleophilicity, as determined, at least in part, by low pka values of sulfhydryls of the cys moiety. because of its electrophilicity, 8-nitro - cgmp may mediate electrophilic signaling by means of induction of s - guanylation of redox sensor proteins. among this class of proteins, kelch - like ech - associated protein 1 (keap1) was identified as a highly sensitive s - guanylation target. keap1 is a negative regulator of nf - e2-related factor 2 (nrf2), which is a transcription factor that regulates phase-2 detoxifying and antioxidant enzymes for electrophiles and ros. our chemical analyses revealed that keap1 expressed by various cultured cells was highly susceptible to s - guanylation induced by no - dependent 8-nitro - cgmp. in fact, we found that no and rnos could activate the keap1-nrf2 pathway in macrophages during bacterial infections and in rat c6 glial cells in culture after treatment with proinflammatory stimuli. that 8-nitro - cgmp may act as an endogenous electrophilic ligand and affect keap1 sulfhydryls via s - guanylation, which would lead to antioxidant signaling, is therefore highly plausible. cytoprotection and host defense conferred by 8-nitro - cgmp were clearly associated with increased expression of heme oxygenase 1 (ho-1) in cultured macrophages and in vivo during salmonella infection. ho-1 is an enzyme with various physiological roles including vasoregulation, cytoprotection, and anti - inflammatory effects. we also reported earlier that ho-1 expression induced by no contributed to cell survival in certain solid tumor models. we recently found, in rat c6 glial cells, that keap1 is the major target that is s - guanylated by no exposure and that its s - guanylated structure derives primarily from 8-rs - cgmp adducts. s - guanylated keap1 led to nrf2 activation and subsequent induction of antioxidant enzymes including ho-1, so 8-nitro - cgmp protected cells against the cytotoxic effects of hydrogen peroxide. proteomic analysis for endogenously modified keap1 with matrix - assisted laser desorption / ionization time - of - flight - ms / ms revealed that 8-nitro - cgmp s - guanylated the cys434 of keap1 (fig. 2). this finding is therefore the first compelling corroboration of the potential roles of 8-nitro - cgmp in the nrf2-dependent antioxidant response. recent studies have suggested that 8-oxoguanine formation not only can contribute to mutagenesis, but also may play an important role in the regulation of cell death. excessive formation of 8-oxoguanine was found to induce cell death via nuclear dna - dependent and mitochondrial dna - dependent mechanisms. in addition to ogg1, muty homolog (mutyh) excises adenine opposite 8-oxoguanine in template dna, with concomitant formation of single strand breaks (ssbs) during the base excision repair. the accumulation of ssbs in nuclear dna by the action of mutyh leads to poly - adp - ribose polymerase - dependent nuclear translocation of apoptosis - inducing factor, and triggers cell death. on the other hand, the accumulation of ssbs in mitochondrial dna, which is also dependent on mutyh, caused mitochondrial dysfunction and calcium ion release, thereby activating calpain, and finally triggers cell death. recent study also demonstrated that excessive production of 8-oxo - dgtp in the nucleotide pool is a major source of oxidized bases, which may in turn cause accumulation of 8-oxoguanine in dna, leading to activate the cell death pathway as mentioned above. taken together, 8-oxoguanine may function as a signaling molecule triggering the cell death, which appears to cause a completely opposite effect compared with the downstream cytoprotective effect of 8-nitro - cgmp signaling as described above (fig. 2). in this article, we overviewed available data on the physiology and pathophysiology of oxidized and nitrated guanine nucleotides, with emphasis on information on mutagenesis and cell signaling related to these nucleotides reported in the last decades. different rnos produced under various pathophysiological conditions may oxidize and nitrate guanine and its related nucleosides and nucleotides, which exist as part of dna or rna or in free form as an abundant component of the intracellular nucleotide pool. not only do 8-oxoguaine and 8-nitroguanine function biologically as an endogenous mutagen but it may also serve as a biomarker for ros- and rnos - induced nucleic acid damage. more important, the major nitrated guanine nucleotide product, 8-nitro - cgmp, may play a critical role in signal transduction during cellular responses to oxidative stress that are primarily mediated by no and ros. this concept was unambiguously confirmed by our recent observation that no - dependent formation of 8-nitro - cgmp is a potent contributor to activation of the antioxidant signaling pathway controlled by the keap1-nrf2 system via unique site - specific s - guanylation of keap1 in cells. also, structural evidence of keap1 s - guanylation at cys434 in vivo indicated that 8-nitro - cgmp, formed from the major nucleotide sensor gtp that exists in an intracellular pool, appears to act as a critical signaling molecule in the initial defense against nitrative and oxidative stress. of importance is that, while guanine oxidation to form 8-oxoguanine may activate the cell death pathway, either dependent or independent of the p53 regulation, guanine nitration involving 8-nitro - cgmp - mediated antioxidant responses exhibits a completely opposite cellular protective effect (fig therefore, the nucleotides in the intracellular pool seem to be sensing the oxidative and nitrative stress occurring in cells, as being differentially recognized based on the altered chemical structures of nucleotides (e.g., oxo- and nitro - moieties) modified by ros and rnos. in other words, oxidative and nitrative nucleotide modifications may not be simple chemical damages, which mostly lose their biological functions because of the altered nucleotide structures, but may be physiologically relevant phenomena, which allow the cells to evoke the versatile cell signaling for adaptive responses to the various chemical stress. further clarification of the signaling functions via oxidized and nitrated nucleotides may shed light on the chemical biology and mutation research and may support an emerging paradigm for oxidative stress - related chemical sensing and signaling of no and ros via cellular nucleotides functioning potentially as nitrative / oxidative stress sensors.	oxidized and nitrated nucleotides including 8-oxogunanine and 8-nitroguanine derivatives such as 8-nitroguanosine 3',5'-cyclic monophosphate were generated by reactive nitrogen oxides and reactive oxygen species in cultured cells and in tissues. 8-oxoguanine and 8-nitroguanine in dna and rna are potentially mutagenic, and the former also induces cell death. some derivative, 8-nitroguanosine 3',5'-cyclic monophosphate a major nitrated guanine nucleotide, was identified as a novel second messenger. surprisingly, the amount of 8-nitroguanosine 3',5'-cyclic monophosphate generated was found to be higher than that of guanosine 3',5'-cyclic monophosphate in cells expressing inducible nitric oxide synthase. more important, 8-nitroguanosine 3',5'-cyclic monophosphate is electrophilic and reacted efficiently with sulfhydryls of proteins to produce a novel posttranslational modification (named s - guanylation) via guanosine 3',5'-cyclic monophosphate adduction. for example, 8-nitroguanosine 3',5'-cyclic monophosphate - induced s - guanylation of kelch - like ech - associated protein 1 led to nf - e2-related factor activation and induction of antioxidant enzymes. 8-nitroguanosine 3',5'-cyclic monophosphate may thus protect cells against oxidative stress - related cytotoxicity. therefore, although chemically modified nucleotides produced via oxidative and nitrative stress are regarded simply as endogenous mutagens, the endogenous nucleotides stored in cells per se may serve functionally as a sensing mechanism for reactive nitrogen oxides and oxygen species to induce cellular adaptive responses to oxidative stress.
globally, noncommunicable diseases (ncds), mainly cancer, cardiovascular diseases (cvd), chronic obstructive pulmonary diseases (copd), and diabetes mellitus (dm), are the most important causes of deaths and disability - adjusted life years (dalys). as of 2010, 65% of total deaths in the world were attributable to ncds (34.5 million of 52.8 million deaths). according to the 2010 global burden of disease (gbd) study, ncds caused 54% of dalys worldwide. a recent analysis also showed that ncds accounted for more than half of total mortality in all regions except for sub - saharan africa. it is expected that ncds will be the major causes of death in sub - saharan africa by 2030. based on age - standardized mortality rates, mortality for most ncds is higher in low - income and middle - income countries than in high - income countries. studies on within - country inequalities in mortality indicated that socioeconomically disadvantaged groups had greater mortality rates than their counterparts for most ncds except for certain causes such as breast cancer and colon cancer. these results on within - country and between - country inequalities challenge a popular view that ncds are diseases of affluence and westernization. in fact, 80% of all ncd deaths in the world occur in low- and middle - income countries. many important international actions on ncds have been initiated through international agencies including the united nations (un) and the world health organization (who) during the past decade (table 1). the global strategy for ncd prevention and control was first adopted by the 53rd world health assembly (wha) in 2000 and asked the who director - general to continue giving priority to ncd prevention and control. the who made important steps toward controlling major ncd risk factors by adopting the framework convention on tobacco control, the global strategy on diet, physical activity and health, and the global strategy to reduce harmful use of alcohol in 2003, 2004, and 2010, respectively. based on the 2007 resolution calling for concrete action on ncds, the wha passed a resolution endorsing the action plan for the global strategy for the prevention and control of noncommunicable diseases in 2008. the action plan set out six objectives, actions to be implemented over the six - year period of 2008 to 2013, and performance indicators. subsequently the global noncommunicable disease network (ncdnet) supported by the who was created for who member states, the who itself, international partners, and other stakeholders to promote partnerships for the prevention and control of ncds. in 2010, the ncd alliance was founded by four international nongovernmental federations representing the four main ncds. the ncd alliance now represents over 2,000 civil society organizations in more than 170 countries and acts as a positive initiative for cooperation to combat the ncd epidemic by putting health at the center of all governmental policies. based on the 2010 un general assembly resolution calling for a high - level meeting on ncds, the ncd high - level summit was held in new york in september 2011. this summit was the second un general assembly meeting devoted to a global health topic, following the 2001 un general assembly special session dedicated to hiv / aids. in 2012, the wha endorsed a goal for the reduction of ncds : to reduce avoidable mortality from ncds by 25% by 2025 (the 25 by 25 goal). in the recent wha in may 2013, the 2013 - 2020 ncd global action plan and the global monitoring framework for ncds were endorsed. it has been also argued that ncds should be embedded in the post-2015 development agenda. this is because ncds are leading causes of death and disability and subsequently impair sustainable development through their impacts on the societal, economic, and environmental domains. according to a recent estimation, tobacco use annually costs 1% to 2% of the global gross domestic product (gdp). a recent study by the world economic forum and harvard university estimated that ncds will cost the world economy $ 47 trillion over the next 20 years, representing 75% of the global gdp and surpassing the cost of the global financial crisis. in a similar vein, the 2012 un conference on sustainable development, rio+20, acknowledged that the ncd burden constitutes " one of the major challenges for sustainable development in the 21st century ", emphasizing the relationship between the ncds and development. ncds accounted for 82% of the total deaths in 2008 in south korea (hereafter ' korea '). based on the recent gbd study, many ncds were included in the 10 leading health problems causing the burden of disease measured by years of life lost : stroke, ischemic heart disease, liver cancer, stomach cancer, lung cancer, liver cirrhosis, diabetes, and colorectal cancer. ncds including stroke, low back pain, dm, ischemic heart disease, liver cancer, other musculoskeletal disorders, stomach cancer, and neck pain were in the top 10 causes of dalys in 2010 in korea (table 2). in korean men, the burden of liver cancer, self - harm, road injury, lung cancer, and liver cirrhosis were relatively great while in women musculoskeletal disorders, neck pain, major depressive disorders, anxiety disorders, and migraine were important causes of dalys. using mortality and national health insurance claim data in korea, researchers also found that the four major ncds (cancer, cvd, dm, and copd) were important contributors to years of life lost and dalys. the importance of ncds has been recognized by the korean government, while it sometimes issues official documents on disease trends not backed by scientific evidence. for example, it was asserted in a white paper of the ministry of health and welfare that chronic disease mortality rates are rapidly increasing in korea. this, however, is misleading, considering that korea recorded a huge increase in life expectancy during the past several decades, mainly due to the reduction of ncd mortality (especially cvd mortality). the increasing importance of ncds in population health does not mean increases in ncd mortality. as indicated in a recent science paper, mortality from major ncds (cvd, copd, and even some cancers) has declined or shifted to older ages, but this occurred later and more slowly than decreases in mortality from infectious diseases. thus, the relative importance of ncds can be better explained by differences in the speed of mortality reduction between ncds and infectious diseases, rather than an absolute increase in ncd mortality. figure 1 presents trends in age - standardized mortality rates (with the world standard population being the referent), crude mortality rates, and numbers of all - cause and ncd deaths by age group in korea between 1983 and 2011. however, of all - cause mortality rates, the proportion of mortality rates from the four major ncds (cancer, cvd, dm, and copd) was 39.4% in 1983 but increased to 56.0% in 2011. although crude cancer mortality rates are increasing, crude ncd mortality rates have shown rather stable trends during the past decade. differences between age - standardized and crude mortality rates would be mainly due to population ageing. as seen in (c) and (d) of figure 1, similar numbers of all - cause deaths were recorded during the past three decades. however, the number of deaths from the four major ncds has increased, and this increase was mainly due to the increase in the numbers of ncd deaths among those aged 65 or over. figure 1 also shows that mortality from communicable diseases (infectious and parasitic diseases and acute respiratory infection) accounted for only a small part of total mortality over the period between 1983 and 2011. a detailed analysis indicated that mortality from infectious and parasitic diseases (international classification of diseases 10th revision [icd-10 ] code : a00-b99) substantially decreased during the period while mortality from acute respiratory infection (icd-10 code : j09-j22) among the elderly population recently increased (data not shown here). the time trends of the prevalence of ncds and their risk factors vary by the outcome analyzed. while body mass index showed increasing trends in both genders according to the global burden of metabolic risk factors of chronic diseases study, decreasing trends were found in systolic blood pressure and fasting blood glucose in korea. based on the cancer registry data in korea, the number of cancer incident cases and the crude incidence rate doubled between 2000 and 2010. a recent cause - specific analysis of cancer registry data also showed that increasing trends in incidence were found for thyroid cancer, colorectal cancer, prostatic cancer (for men), and breast cancer (for women). because of the lack of a nationwide active surveillance system for cvd, national health insurance claims data has been used to present time trends of the incidence of two major cvd, ischemic heart disease and stroke, in korea. these data showed increasing trends between the mid-1990s and mid-2000s, but decreasing trends afterward since the mid-2000s. no significant increasing trends were found for diabetes prevalence based on the data from the korea national health and nutrition examination survey (knhanes) between 1998 and 2009. however, according to a review of community surveys on diabetes prevalence, it was concluded that the prevalence of diabetes in korea has increased six- to seven - fold in the past 40 years. for copd, population - based spirometric surveys have been included as a component of the knhanes in 2001 and 2008. survey results show that the prevalence of copd among participants aged 40 or over was 17.2% in 2001 and 13.4% in 2008. the decreasing tendency between 2001 and 2008 is, however, thought to be due to different criteria for spirometry rather than an actual decrease in the prevalence of copd. socioeconomic inequalities in ncds and ncd risk factors have been demonstrated in government reports and epidemiological studies in korea. using census and mortality data, educational inequalities in mortality longitudinal follow - up data also showed mortality inequalities by income group for most ncds except for a few cancers (kidney cancer for men and non - hodgkin 's lymphoma for women). using cancer registry and health insurance data, socioeconomic inequalities in cancer incidence and cancer survival the ministry of health and welfare and the korea centers for disease control and prevention have published annual reports, ' korea health statistics ', based on the knhanes and have included age - standardized prevalences or means by income and urbanization levels in a wide range of ncd prevalences, ncd risk factors, and health care utilization rates related to ncds. recently, in a special issue of the journal of the korean medical association on health inequalities, korean researchers reviewed the prior literature and concluded that socioeconomic inequalities in ncd risk factors and health care utilization related to ncds are widespread in korea and the magnitude of the inequalities for some measures is even increasing. ncds are the main contributor to between - country and within - country inequalities in total mortality. a recent analysis examined cause - specific contributions to socioeconomic inequalities in total mortality in england, new zealand, korea, and japan and showed that four major ncds (cancer, cvd, copd, and dm) accounted for the major part of mortality inequalities by geographical deprivation index. in this international comparative study, the contribution of ncds was relatively smaller in korea than the contribution in the other three industrialized countries. the finding can be explained by the discrepancy in the size of the geographical unit in each country, the lower level of residential segregation in korea than other countries, and ill - defined causes in korean death certificate data. meanwhile, several korean studies using individual socioeconomic position indicators indicated that ncds are the main determinants of inequalities in mortality and life expectancy. for example, in an analysis using mortality linkage data of korean public servants aged 35 to 64, four major ncds accounted for 50.4% and 70.8% of the absolute inequalities in total mortality of men and women, respectively, as measured by slope indices of inequality. tobacco and alcohol use, salt intake and unhealthy diet, high blood pressure, and physical inactivity and obesity have been reported to be major determinants of ncds. the relative importance of these risk factors in explaining the ncd burden is debatable and thus findings on comparative risk assessment are helpful in understanding the role of ncd determinants. based on the recent comparative risk assessment of the global burden of disease and injury, high blood pressure, smoking, and alcohol use were the three leading risk factors for the global disease burden measured by dalys. in the same analysis, these three risk factors were also the leading determinants of dalys in asia pacific high income countries (japan, korea, brunei, and singapore). considering the population size of the four countries in this region (japan 128 million, korea 50 million, brunei 0.4 million, and singapore 5.2 million), this result primarily represents findings from japan and korea. comparative risk assessment results from japan, with which korea shares similarities regarding causes of death structure and risk factors, are thus informative. according to a japanese study estimating the impact of 16 risk factors on cause - specific mortality and life expectancy, tobacco smoking and high blood pressure were found to be the two major risk factors for adult mortality and shortened life expectancy. recently, the institute for health metrics and evaluation at the university of washington released gbd country profiles and now provides online access to country - specific results on the burden of disease and comparative risk assessment in terms of death, years of life lost, years lost due to disability, and dalys. according to the results of the comparative risk assessment, dietary risks (including salt intake), alcohol use, smoking, and high blood pressure were the leading risk factors of dalys in korea. gender - specific results indicated the relative importance of alcohol and smoking in korean men and high blood pressure in korean women (table 3). although upstream determinants such as poverty and financial resources, education, working conditions, housing, living environment, and social policies including health care policies play important roles in the creation and maintenance of health inequalities, major modifiable risk factors of ncds have been considered to be the major proximal determinants of socioeconomic inequalities in ncds. a prior korean study showed that behavioral risk factors accounted for a significant magnitude of absolute mortality inequalities. cigarette smoking explained about 34% to 42% of absolute inequalities in all - cause mortality by income group among men aged 30 - 64 and its explanatory ability was even greater for cvd mortality (47% to 68%). similar results regarding the role of major established cvd risk factors including smoking were reported in prospective studies from other countries. despite the continuing academic activities and policy development surrounding ncds, interest in recent international policy development on ncds has been sparse in both the academic community and policy makers of korea. for example, except for a small discussion section held by a non - governmental research organization, no meaningful public discussion regarding the 2011 un ncd summit occurred in 2010 and 2011. it is only recently that the korean society for preventive medicine held academic meetings on ncds in 2012 and 2013. this lack of interest in recent international actions on ncds is partly because policy makers and academic societies in korea tend to consider policies and activities discussed among international agencies (the un and who for ncds) as ones for underdeveloped countries. policy makers may think that the ncd problems in korea have already been addressed legislatively by the national health promotion act established in 1995 and that measures to control ncds are now partly established with support of national health insurance and other ncd screening programs at the community public health centers. there may also be a pessimistic view on ncd policies from non - governmental organizations of korea, in that fundamental approaches to controlling ncds can be expected to be discouraged in the ncd policy making processes because of lobbying from associated tobacco, alcohol, salt, and unhealthy food industries as well as pharmaceutical companies. for these reasons, the korean government may not take the ncd issue seriously, as it did not for the millennium development goals. nevertheless, the ncd policies of the international agencies may well allow the korean government to review its own policies on ncds and ncd risk factors and provide opportunities to develop more aggressive and population - wide policy measures. international discussions to include ncd targets in the post-2015 development agenda in the un would be a good platform for drawing attention to the links between ncds and social, economic, and environmental factors. many challenges lie ahead in the development of comprehensive national strategies to control ncds in korea (table 4). establishment of a rigorous and independent accountability mechanism has been suggested to be critical to ensure that the goal of ncd reduction is on track to be met. at the national level, because of the multisectoral nature of ncds, comprehensive and rigorous national strategies and a whole - of - government approach are urgently needed, and thus a presidential ncd commission would be appropriate in korea. a governmental unit for monitoring and evaluation should be established. development of a national health promotion fund (or the equivalent) has been suggested as the most sustainable funding mechanism for ncd prevention and control. national policies to strengthen the korean health promotion fund by substantially and regularly increasing the price of tobacco products and starting to impose surcharges on alcohol and unhealthy food products are needed in korea. based on the global monitoring framework for ncds, the goals and targets on ncds and ncd risk factors in the national health plan 2020 of korea should be reviewed and revised and new indicators may also be added. many governmental policies should be developed in korea to achieve a meaningful reduction in ncds and ncd risk factors and to meet the targets and indicators of the global monitoring framework (table 4). many reports have indicated population approaches such as taxation, regulation of marketing and sales, and public education and campaigns are effective measures for controlling ncds and ncd risk factors. such population - wide policies have been chosen as ' best buy ' interventions that are not only highly cost - effective but also cheap, feasible, and culturally acceptable to implement. considering the paucity of population - wide policies to control major ncd risk factors in korea including tobacco and alcohol, and the preponderance of high - risk approaches using screening mechanisms through the national health insurance and public health centers, rigorous population approaches should be further developed in korea. aggressive taxation of tobacco and comprehensive smoke - free policies should be implemented right away to further control the use of tobacco. taxation of alcohol and unhealthy food and drink should be initiated in korea by including these products as additional items for which the tax is allocated to the national health promotion fund. the global strategy to reduce the harmful use of alcohol. pointed out the lack of evidence supporting the effectiveness of the reliance on industry self - regulation and public - private partnerships in reducing ncd risk factors. they also concluded that public regulation and market intervention are the only evidence - based measures for controlling unhealthy commodities including tobacco, alcohol, and unhealthy food and drink. a comprehensive review of the health promotion act of korea and associated legislation is required to develop more aggressive policies to restrict the sales and marketing of unhealthy commodities. active and rigorous enforcement of the acts should be also conducted. in addition, public education and both media and non - mass media campaigns especially tailored toward disadvantaged populations need to be developed. socioeconomic inequalities in mortality, risk factor prevalence, and treatment of ncds have been identified. social determinants including poverty, working conditions, education, housing, and living environment contribute to the development of ncds and ncd inequalities. policies to address such social determinants of ncds should be developed in korea. policies and programs to improve early childhood development and working conditions are crucial to effectively address inequalities in ncds. the commission on social determinants of health recommended health equity in all policies, systems, and programs to ensure the reduction of health inequalities by equalizing distributions of power, money, and resources. national strategies with explicit consideration for health and health equity in all policies would help to effectively reduce ncds and ncd inequalities. the importance of the roles of civil society and the academic community should also be recognized. studies on the burden of ncds and the roles of risk factors (comparative risk assessment) should be conducted in korea with updated data from death certificates, hospitalization records, and community surveys as well as country - specific disability weighting. research on the social, economic, and environmental impacts of the ncd burden would be helpful in drawing attention from other sectors (e.g., the economic sector) to the issue. considering the paucity of detailed information on the activities of unhealthy commodity industries in korea, mechanisms for monitoring industrial activities related to tobacco, alcohol, and unhealthy food and drink should be developed, especially with the participation of civil society and the academic support of the research community. furthermore, active involvement by civil society in the development of ncd policies is necessary and research interest in population approaches to controlling ncds should be pursued. ncds are the main contributor to both the disease burden and health inequalities in korea. international discussions on ncd policies in the un and the who have provided a good opportunity to review governmental ncd policies and further develop rigorous and population - wide policy measures in korea. considering the paucity of aggressive population - wide policies to control major ncd risk factors in korea, rigorous population approaches such as taxation and regulation of unhealthy commodities as well as public	noncommunicable diseases (ncds) are the most important causes of premature mortality and disability - adjusted life years in korea. ncds are also the main contributor to socioeconomic inequalities in mortality and life expectancy. reduction of ncds and ncd inequalities would result in significant improvement in healthy life expectancy and health equity in korea. major ncd risk factors such as dietary risks (including salt intake), alcohol consumption, cigarette smoking, and high blood pressure were found to be the leading modifiable risk factors of disability - adjusted life years in korea, based on the 2010 global burden of disease study. several korean studies have shown that these risk factors play an important role in creating socioeconomic inequalities in ncd mortality and total mortality. current international discussions on ncd policies in the united nations and the world health organization would provide better opportunities for developing aggressive population - wide policy measures in korea. considering the paucity of population - wide policies to control major ncd risk factors in korea, rigorous population approaches such as taxation and regulation of unhealthy commodities as well as public education and mass campaigns should be further developed in korea.
to enable a broadscale screening and protein profiling for the potential identification of novel biomarker candidates associated with dili, we here set out an explorative affinity proteomics strategy. an initial nontargeted discovery screening that encompassed 4594 antibodies targeting 3450 unique proteins, representing approximately 17% of the human gene products, was combined with targeted profiling using a carefully designed dili array. the screening and verification strategy was based on the analysis of 1196 plasma and serum samples from 241 individuals divided across four independent cohorts (hv apap, hiv / tb, safet dili and hv heparin, see supplementary demographics tables and table s2) in combination with an antibody suspension bead array approach (fig. overview of the study design with information regarding the various cohorts (number of samples and collection time points) and the antibody arrays (number of antibodies and unique protein targets) used in the different stages. in the nontargeted discovery stage, a screening array of 4594 antibodies and a subset of the hv apap cohort (day 2, baseline) were used. the baseline samples served to identify potential dili susceptibility markers that could be included in the dili array. these were in combination with a selection of antibodies from previous inhouse protein profiling efforts and thorough literature mining, pathway analysis, gene and protein expression data and information on liverenriched expression of proteins from the human protein atlas project 10 used to generate the targeted dili array of 287 antibodies representing 251 proteins (table s1). in the targeted discovery stage, the dili array was used to profile two independent cohorts of longitudinal design, the complete hv apap cohort (355 samples, 14 cases, 15 controls, 13 time points) and the hiv / tb cohort (472 samples, 38 cases and 38 controls, 7 time points). in a first verification, the same cohorts were again profiled but with a smaller set of antibodies defined by the results from the targeted discovery stage and incorporating additional antibodies per protein, resulting in a set of 91 antibodies targeting 47 proteins (table s3). the safet dili cohort (88 samples, 26 cases, 62 controls) was included in verification 2, to replicate the initial findings. in verification 3, an even smaller set of 34 selected targets represented by 61 antibodies and more antibodies per protein target was used. finally, the hv heparin cohort (281 samples, 48 cases, 6 time points), was profiled in verification 4 and repeated. two proteins, cdh5 (cadherin 5, type 2) and fabp1 (fatty acidbinding protein 1), were identified to be elevated in dili cases. the longitudinal dependence of these markers was first investigated by profiling serum and plasma from the hv apap and hiv / tb cohort, covering a time frame of 14 days and 12 weeks respectively. in hv apap, cdh5 showed elevated median levels in dili cases already in the predose / baseline samples (day 13). although only significant at day 11, the trend of increased levels was sustained across the studied time frame including time points of dosing as well as during washout with the exception of day 6. the same trend with baselineelevated levels was observed in the hiv / tb cohort, with significant differences for week 2 and 4 as well as for week 8 and 12 (fig. alt and fabp1 displayed a treatmentdependent appearance with increasing levels in dili cases after initiation of the dosing in both the hv apap and the hiv / tb cohort (fig. 2a+c). in hv apap, a significant elevation in fabp1 was observed one day prior to alt (day 8 compared to day 9). the elevation for both remained significant during the continued dosing as well as after treatment had stopped. interestingly, towards the end of the study period, fabp1 levels started to decrease (compare day 9 with day 14) whereas alt remained unchanged. for the hiv / tb cohort, a significantly higher level of fabp1 was found for week 6 and onwards. the elevated levels were confirmed in the safet dili cohort with significant differences of 10 and 10 for cdh5 and fabp1 respectively (fig. furthermore, profiling of the heparin cohort showed an elevation in fabp1 levels across the studied time frame whereas cdh5 levels were relatively stable (fig. (a) alt, (b) cdh5 and (c) fabp1 in dili cases and controls of three independent cohorts measured with a standard clinical test (enzymatic activity assay) or with the antibody suspension bead array assay. in hv apap / tb, the treatment started at week 1 and continued across the analysed time frame. antibodies targeting cdh5 and fabp1 detected elevated levels of the proteins in dili cases in all the three cohorts. p 3 the upper limit of normal. direct comparison of the extent of an increase in alt between the healthy and patient cohorts may not be applicable. a slight increase in alt and ast level is more damaging or fatal in sick immunocompromised hiv and tb patients. in most cases, this rise is accompanied by clinical symptoms of liver toxicity and consequently treatment is discontinued before alt reaches the levels observed in the hv apap or safet cohort. however, for both the hv apap and hiv / tb cohorts the observed time course of dili was valuable. other limitations of the study are an imbalance in age and gender distributions as well as the slightly varying dili case definition used for the different cohorts, as the standardization in case definition has evolved during the course of the study. this exploratory study aimed to identify potential protein markers that could have use as dili biomarkers irrespective of the drug inducing the injury. the antibody suspension bead array assay in the employed format is a method for relative quantification. the format is convenient in the discovery stages of biomarker research, generating multiplex data for hundreds of proteins in hundreds of samples in one single assay. although it could be of interest to reveal potential drugrelated differences in the proteins across the four cohorts or within cohorts, the division of subjects into subgroups of agents would generate groups too small to be able to draw any conclusion. in addition, this would demand that the same dili criteria were used for all cohorts and would require absolute quantitative data., this biomarker discovery study has been based on affinity proteomics using human samples from both healthy volunteers and patients with dili caused by a wide spectrum of therapeutical agents. we decided here to utilise a very stringent approach by only focusing on those proteins that were differentiated in all four cohorts. fabp1 showed superior characteristics regarding tissue distribution and kinetics compared to alt but limited predictive value for the development of severe dili. both biomarker candidates have been taken up in the clinical biomarker qualification performed by the safet consortium. clinically applicable elisa assays have been validated according to the safet biomarker qualification strategy for largescale analysis of clinical samples. further analysis of cdh5 and fabp1 in comparison to and in combination with current standard assays in samples from large cohorts of healthy volunteers, patients with drug and none druginduced liver injury and with common disorders is currently ongoing to establish the performance characteristics and potential use for cdh5 and fabp1 as biomarkers. finally, the safety biomarker community together with regulators will need to determine the utility of these potential novel dili biomarkers in drug development and clinical practice. additional supporting information may be found at onlinelibrary.wiley.com/doi/10.1111/liv.13174/suppinfo click here for additional data file.	abstractbackground & aimsthe occurrence of druginduced liver injury (dili) is a major issue in all phases of drug development. to identify novel biomarker candidates associated with dili, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human dili cases and controls.methodsan initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. resulting candidate proteins together with proposed dili biomarker candidates generated a dili array of 251 proteins for subsequent target analysis and verifications. in total, 1196 samples from 241 individuals across four independent cohorts were profiled : healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, dili cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins.resultswe observed elevated levels of cadherin 5, type 2 (cdh5) and fatty acidbinding protein 1 (fabp1) in dili cases. in the two longitudinal cohorts, cdh5 was elevated already at baseline. fabp1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (alt). the elevations were verified in the dili cases treated with various drugs. in the heparin cohort, cdh5 was stable over time whereas fabp1 was elevated.conclusionsthese results suggest that cdh5 may have value as a susceptibility marker for dili. fabp1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to alt but likely with limited predictive value for the development of severe dili. further studies are needed to determine the clinical utility of the proposed markers.
anorectal melanoma is associated with an extremely poor prognosis regardless of the aggressiveness of surgical therapy ; it is commonly incurable at presentation, with many patients developing systemic metastasis within a year after diagnosis. however, reports have noted that long - term survival was seen only in patients who underwent an abdominoperineal resection instead of wide local excision. it is estimated that only 0.1 - 0.5% of all rectal tumors are leiomyosarcomas ; only 136 cases of rectal leiomyosarcoma had been recorded in a 1986 literature review. local excision carries an approximately 80 - 85% chance of recurrence. since 80% of these tumors are in the distal rectum, abdominoperineal excision has been the most frequently performed operation. the five - year survival rate in most series seems to be 20 - 25% after radical surgery. kaposi 's sarcoma is often asymptomatic and as such usually requires no treatment, but surgery is sometimes indicated to control bleeding or obstructive symptoms. proponents of laparoscopic abdominoperineal resection cite arguments in favor, that the extended lymph node dissection, the mobilization of the rectum and the mesorectum and stoma creation can be laparoscopically performed ; furthermore, the transperineal tumor removal is affected. nonetheless, local wound recurrence of tumor cells in the port sites of patients who have undergone curative laparoscopic procedures for cancer are of major concern. due to relative prevalence rates, other series therefore, the aim of this study was to assess the results of laparoscopic abdominoperineal resection for treatment of non - carcinomatous malignancies. all five patients were referred to our department for treatment of biopsy - proven neoplasms. in four cases preoperative staging included computerized axial tomography (cat) scan and anorectal ultrasound and in all cases failed to identify distant or nodal disease, respectively. all patients underwent conventional preoperative mechanical bowel preparation and received routine oral and parenteral antibiotic prophylaxis. operative steps include : 1) mobilization of the left colon ; 2) division of the inferior mesenteric vessels ; 3) division of the mesentery ; 4) total mesorectal excision ; 5) division of bowel at the sigmoid - descending junction ; 6) perineal dissection in the standard fashion with specimen removal ; and 7) end colostomy creation. the operative time, intraoperative findings, transfusion requirement, intra- and postoperative complications, length of hospitalization, and outcomes of surgery were recorded for each patient. a 75-year - old female patient presented to another surgeon with a primary tumor located 5 cm cephalad to the dentate line, on the posterior lateral aspect of the rectum. histopathologic evaluation revealed an invasive malignant melanoma with vascular involvement ; high mitotic activity (mean 6 mitoses per 107 high - power field [hpf ]) ; and a positive immunocytochemical profile of 100s - hmb 45/ 50. chest, abdominal and pelvic ct scans failed to reveal any local or distant metastasis. the melanoma was transanally removed. six months after the local excision the patient presented with a gelatinous 4 cm diameter mass, localized on the left postero - lateral aspect of the dentate line, extending to the anorectal ring. a pelvic and abdominal ct scan revealed a dense 2 cm ovoid mass on the left lateral wall of rectum, caudal to the levator ani, involving the puborectalis muscle. although the patient had enlarged lymph nodes in the left ischiorectal fossa at the level of the levator ani, there were no distant metastases noted. at that time the patient was referred to our department for evaluation of severe pain and rectal bleeding and underwent a laparoscopic abdominoperineal resection. pathologic assessment showed metastatic, multicentric melanoma of the rectum, involving 8 of 9 lymph nodes. four months after abdominoperineal resection, the ct scan found the liver extensively involved with metastatic lesions without port - site or local metastasis. an 87-year - old male patient presented to another surgeon with a malignant melanoma infiltrating the external anal sphincter and puborectalis muscle. a wide local excision of the lesion was performed 3 cm cephalad to the dentate line. the patient was followed up for 18 months, during which time no distant or local metastases were revealed. eighteen months later he was referred to our department with a deeply infiltrative 3 cm diameter anteriorly bound tumor at the level of the dentate line, involving the anterior rectal wall. after abdominoperineal resection, the pathologist reported a tumor free distal margin of 0.8 cm, and 3 of the 7 pericolic harvested lymph nodes had metastatic foci. postoperatively he developed a common bile duct obstruction due to stone impaction at the ampulla of vater, which was successfully removed by endoscopic retrograde cholangiopancreaticography. the patient had uneventful recovery and two months after surgery had no evidence of any distal, local or port - site recurrence. a 69-year - old patient presented to another surgeon with a broad - based polypoid lesion of 1.5 cm diameter in the anal canal removed by endoscopic snare. histologic studies showed a poorly differentiated primary tumor, with high mitotic activity which infiltrated the rectal wall ; immunohistochemical profile was positive for s100-hmb 45/50. the patient was followed up for two months, after which a biopsy revealed a local recurrence of malignant melanoma at the anterior dentate line. the patient was referred to our department for curative resection of an early recurrence. rectal ultrasonography and anoscopic evaluation revealed a 3 mm diameter anteriorly based lesion at the dentate line. because preoperative ct did not reveal any distant metastasis, the patient underwent laparoscopy with a view towards abdominoperineal resection. unfortunately, two 4 cm diameter right - sided hepatic metastasis were identified and confirmed by percutaneous fine needle biopsy. she died secondary to liver metastasis but without any port - site recurrence 46 days after surgery. a 68-year - old female patient who was receiving immunosuppressive agents for rheumatoid arthritis was referred to our department for a primary tumor presenting as a firm mass in the left wall of the anal canal. a graytan soft tissue weighing 42 grams within a diameter of 5.5 3.6 3.5 cm was removed by wide local excision, and histologic evaluation confirmed the diagnosis of leiomyosarcoma. the patient underwent laparoscopic abdominoperineal resection after which the pathologist described the lesion as a lymphosarcoma. two years later, the patient presented with a 2 cm diameter left obturator mass, with bilobar hepatic and bilateral pulmonary nodules. however, neither ct scan nor magnetic resonance imaging revealed any port - site recurrences. a 34-year - old white male patient presented to another surgeon with an advanced hiv infection. during his course, the patient had experienced both cutaneous and gastrointestinal kaposi 's sarcoma, cytomegalovirus gastritis, cryptosporidium, peripheral neuropathy, wasting, neutropenia and anemia. office evaluation revealed two 4 cm diameter kaposi 's lesions in the rectum, one 5 cm and one 8 cm cephalad to the dentate line. the patient was treated by chemotherapy and radiotherapy. however, one month later, he presented with obstructive symptoms, increased bleeding, and severe pain. proctoscopy revealed near - occlusion of the lumen due to the two rectal lesions. accordingly, it was elected to proceed with a laparoscopic loop ileostomy after which he was discharged home on postoperative day five in stable condition. the patient died five months after the procedure due to systemic cytomegalovirus infection and seizures. a 75-year - old female patient presented to another surgeon with a primary tumor located 5 cm cephalad to the dentate line, on the posterior lateral aspect of the rectum. histopathologic evaluation revealed an invasive malignant melanoma with vascular involvement ; high mitotic activity (mean 6 mitoses per 107 high - power field [hpf ]) ; and a positive immunocytochemical profile of 100s - hmb 45/ 50. chest, abdominal and pelvic ct scans failed to reveal any local or distant metastasis. the melanoma was transanally removed. six months after the local excision the patient presented with a gelatinous 4 cm diameter mass, localized on the left postero - lateral aspect of the dentate line, extending to the anorectal ring. a pelvic and abdominal ct scan revealed a dense 2 cm ovoid mass on the left lateral wall of rectum, caudal to the levator ani, involving the puborectalis muscle. although the patient had enlarged lymph nodes in the left ischiorectal fossa at the level of the levator ani, there were no distant metastases noted. at that time the patient was referred to our department for evaluation of severe pain and rectal bleeding and underwent a laparoscopic abdominoperineal resection. pathologic assessment showed metastatic, multicentric melanoma of the rectum, involving 8 of 9 lymph nodes. four months after abdominoperineal resection, the ct scan found the liver extensively involved with metastatic lesions without port - site or local metastasis. an 87-year - old male patient presented to another surgeon with a malignant melanoma infiltrating the external anal sphincter and puborectalis muscle. a wide local excision of the lesion was performed 3 cm cephalad to the dentate line. the patient was followed up for 18 months, during which time no distant or local metastases were revealed. eighteen months later he was referred to our department with a deeply infiltrative 3 cm diameter anteriorly bound tumor at the level of the dentate line, involving the anterior rectal wall. after abdominoperineal resection, the pathologist reported a tumor free distal margin of 0.8 cm, and 3 of the 7 pericolic harvested lymph nodes had metastatic foci. postoperatively he developed a common bile duct obstruction due to stone impaction at the ampulla of vater, which was successfully removed by endoscopic retrograde cholangiopancreaticography. the patient had uneventful recovery and two months after surgery had no evidence of any distal, local or port - site recurrence. a 69-year - old patient presented to another surgeon with a broad - based polypoid lesion of 1.5 cm diameter in the anal canal removed by endoscopic snare. histologic studies showed a poorly differentiated primary tumor, with high mitotic activity which infiltrated the rectal wall ; immunohistochemical profile was positive for s100-hmb 45/50. the patient was followed up for two months, after which a biopsy revealed a local recurrence of malignant melanoma at the anterior dentate line. the patient was referred to our department for curative resection of an early recurrence. rectal ultrasonography and anoscopic evaluation revealed a 3 mm diameter anteriorly based lesion at the dentate line. because preoperative ct did not reveal any distant metastasis, the patient underwent laparoscopy with a view towards abdominoperineal resection. unfortunately, two 4 cm diameter right - sided hepatic metastasis were identified and confirmed by percutaneous fine needle biopsy. she died secondary to liver metastasis but without any port - site recurrence 46 days after surgery. a 68-year - old female patient who was receiving immunosuppressive agents for rheumatoid arthritis was referred to our department for a primary tumor presenting as a firm mass in the left wall of the anal canal. a graytan soft tissue weighing 42 grams within a diameter of 5.5 3.6 3.5 cm was removed by wide local excision, and histologic evaluation confirmed the diagnosis of leiomyosarcoma. the patient underwent laparoscopic abdominoperineal resection after which the pathologist described the lesion as a lymphosarcoma. two years later, the patient presented with a 2 cm diameter left obturator mass, with bilobar hepatic and bilateral pulmonary nodules. however, neither ct scan nor magnetic resonance imaging revealed any port - site recurrences. a 34-year - old white male patient presented to another surgeon with an advanced hiv infection. during his course, the patient had experienced both cutaneous and gastrointestinal kaposi 's sarcoma, cytomegalovirus gastritis, cryptosporidium, peripheral neuropathy, wasting, neutropenia and anemia. office evaluation revealed two 4 cm diameter kaposi 's lesions in the rectum, one 5 cm and one 8 cm cephalad to the dentate line. the patient was treated by chemotherapy and radiotherapy. however, one month later, he presented with obstructive symptoms, increased bleeding, and severe pain. accordingly, it was elected to proceed with a laparoscopic loop ileostomy after which he was discharged home on postoperative day five in stable condition. the patient died five months after the procedure due to systemic cytomegalovirus infection and seizures. anal cancer is a relatively infrequent tumor, accounting for less than 2% of all large bowel cancer and less than 2000 cases annually in the united states. only 1.5% of all malignant melanomas develop in the anorectal region and only 460 cases have been reported in the medical literature. anorectal melanoma is associated with an extremely poor prognosis despite aggressive surgical therapy. in the literature the average survival rates vary from 9 months to 2.8 years for all patients with only a 10% five - year survival. (memorial sloan - kettering cancer center) published 85 cases ; seventy - five percent of patients had tumors greater than 1 cm with a mean survival of only 17 months. only six patients survived five years, and all of these six had undergone abdominoperineal resection. interestingly, two of the long - term survivors had mesenteric lymph nodes involved by tumor. several other authors have found that long - term survival was seen only in patients who underwent abdominoperineal resection and have strongly suggested this procedure for cure. a swedish group reported that only 2 of 33 patients treated for cure survived for five years. although one of the two survivors underwent local excision, they found that local recurrences occurred significantly more often after a local excision than after an abdominoperineal resection (50% vs. 27%, respectively). other researchers believe that radical surgery does not alter the natural history of a high rate of distant metastasis and, therefore, advocate sphincter - sparing surgery. siegel., in a review of 30 patients, found that the only two 5-year survivors had been treated with local excision. similarly, cooper. reported that two of six 5-year survivors had local excision. although melanoma has been successfully used to produce tumor cell lines with increased metastatic capacity from many other experimental tumors, none of the three patients in our small series developed port - site recurrences. leiomyosarcoma of the anorectum is rare, and wide local excision is the best treatment option. radiation or chemotherapy, alone or in combination, have not been found to be effective, although they may play a palliative role. as emphasized in a st. mark 's series, the treatment of choice for leiomyosarcoma of the rectum arising from sites other than the muscularis mucosa is abdominoperineal excision. in this current series, one patient who was treated with a previous wide local excision previously had local recurrence and finally underwent laparoscopic abdominoperineal resection. although most of these lesions are asymptomatic, complications can include bleeding, obstruction, intussusception and mesenteric cyst formation. treatment of anorectal kaposi 's sarcoma rarely entails surgery although neither the chemotherapy, radiotherapy, nor immunotheraphy is of proven benefit. because of the decreased immune function in hiv - infected patients, laparoscopy may be beneficial. these lesions described in this small series of case reports are infrequently occurring but highly aggressive. it, therefore, seems logical that if these patients need surgery, they be offered the least traumatic, least immune - system compromising type available. in addition, therapy which can expedite hospital discharge and minimize disability is desirable in patients with a limited life expectancy. issues germane to care of carcinomatous lesions, such as port - site recurrences, may not be as relevant in these other tumors.	objective : numerous concerns have been raised relative to the appropriateness of laparoscopic surgery for cure of rectal adenocarcinomas. however, because of their rarity, little information exists about the role of laparoscopy for other anorectal malignancies. we report the outcome of five patients who underwent laparoscopic surgery for other anorectal malignancies.methods:all patients who underwent laparoscopic surgery for treatment of non - carcinomatous anorectal malignancy were assessed by means of endoscopic, radiological and histopathologic diagnostic tests.results:two patients with anorectal melanoma and one with anal leiomyosarcoma underwent laparoscopic abdominoperineal resection. a laparoscopic loop ileostomy was performed for an hiv - positive patient with rectal kaposi 's sarcoma. another patient with anorectal melanoma had intraoperative identification of distant liver metastasis and therefore underwent diagnostic laparoscopy instead of an intended abdominoperineal resection. there were no intraoperative laparoscopic complications. during the follow - up period three patients who underwent abdominoperineal resection were alive, one of whom had rectal melanoma and developed liver metastasis without local recurrence. the two patients with distant liver metastasis and rectal kaposi 's sarcoma died 46 days and five months after surgery, respectively. there were no port - site or local recurrences.conclusion:laparoscopic abdominoperineal resection for non - carcinomatous anorectal malignancies is technically feasible and avoids many of the concerns associated with attempted curative laparoscopic resection of carcinoma.
secondary data analysis is analysis of data that was collected by someone else for another primary purpose. increasingly, generalist researchers start their careers conducting analyses of existing datasets, and some continue to make this the focus of their career. using secondary data enables one to conduct studies of high - impact research questions with dramatically less time and resources than required for most studies involving primary data collection. for fellows and junior faculty who need to demonstrate productivity by completing and publishing research in a timely manner, secondary data analysis can be a key foundation to successfully starting a research career. successful completion demonstrates content and methodological expertise, and may yield useful data for future grants. despite these attributes, conducting high quality secondary data research requires a distinct skill set and substantial effort. however, few frameworks are available to guide new investigators as they conduct secondary data analysies.13 in this article we describe key principles and skills needed to conduct successful analysis of secondary data and provide a brief description of high - value datasets and online resources. the primary target audience of the article is investigators with an interest but limited prior experience in secondary data analysis, as well as mentors of these investigators, who may find this article a useful reference and teaching tool. while we focus on analysis of large, publicly available datasets, many of the concepts we cover are applicable to secondary analysis of proprietary datasets. datasets we feature in this manuscript encompass a wide range of measures, and thus can be useful to evaluate not only one disease in isolation, but also its intersection with other clinical, demographic, and psychosocial characteristics of patients. many worthwhile studies simply can not be done in a reasonable timeframe and cost with primary data collection. for example, if you wanted to examine racial and ethnic differences in health services utilization over the last 10 years of life, you could enroll a diverse cohort of subjects with chronic illness and wait a decade (or longer) for them to die, or you could find a dataset that includes a diverse sample of decedents. even for less dramatic examples, primary data collection can be difficult without incurring substantial costs, including time and money scarce resources for junior researchers in particular. secondary datasets, in contrast, can provide access to large sample sizes, relevant measures, and longitudinal data, allowing junior investigators to formulate a generalizable answer to a high impact question. for those interested in conducting primary data collection, beginning with a secondary data analysis may provide a bird s eye view of epidemiologic trends that future primary data studies examine in greater detail. secondary data analyses, however, have disadvantages that are important to consider. in a study focused on primary data, you can tightly control the desired study population, specify the exact measures that you would like to assess, and examine causal relationships (e.g., through a randomized controlled design). in secondary data analyses, the study population and measures collected are often not exactly what you might have chosen to collect, and the observational nature of most secondary data makes it difficult to assess causality (although some quasi - experimental methods, such as instrumental variable or regression discontinuity analysis, can partially address this issue). while not unique to secondary data analysis, another disadvantage to publicly available datasets is the potential to be scooped, meaning that someone else publishes a similar study from the same data set before you do. on the other hand, intentional replication of a study in a different dataset can be important in that it either supports or refutes the generalizability of the original findings. if you do find that someone has published the same study using the same dataset, try to find a unique angle to your study that builds on their findings. the same basic research principles that apply to studies using primary data apply to secondary data analysis, including the development of a clear research question, study sample, appropriate measures, and a thoughtful analytic approach. for purposes of secondary data analysis, these principles can be conceived as a series of four key steps, described in table 1 and the sections below. table 2 provides a glossary of terms used in secondary analysis including dataset types and common sampling terminology. table 1a practical approach to successful research with large datasetsstepspractical advice(1) define your research topic and question(1) start with a thorough literature review(2) ensure that the research question has clinical or policy relevance and is based on sound a priori reasoning. a good question is what makes a study good, not a large sample size(3) be flexible to adapt your question to the strengths and limitations of the potential datasets(2) select a dataset(1) use a resource such as the society of general internal medicine s online compendium (www.sgim.org/go/datasets) (table 3)(2) to increase the novelty of your work, consider selecting a dataset that has not been widely used in your field or link datasets together to gain a fresh perspective(3) factor in complexity of the dataset(4) factor in dataset cost and time to acquire the actual dataset(5) consider selecting a dataset your mentor has used previously(3) get to know your dataset(1) learn the answers to the following questions:why does the database exist?who reports the data?what are the incentives for accurate reporting?how are the data audited, if at all?can you link your dataset to other large datasets?(2) read everything you can about the database(3) check to see if your measures have been validated against other sources(4) get a close feel for the data by analyzing it yourself or closely reviewing outputs if someone else is doing the programming(4) structure your analysis and presentation of findings in a way that is clinically meaningful(1) think carefully about the clinical implications of your findings(2) be cautious when interpreting statistical significance (i.e., p - values). large sample sizes can yield associations that are highly statistically significant but not clinically meaningful(3) consult with a statistician for complex datasets and analyses(4) think carefully about how you portray the data. a nice figure sometimes tells the story better than rows of datatable 2glossary of terms used in secondary dataset analysis researchtermmeaningtypes of datasets (not mutually exclusive)administrative or claims datadatasets generated from reimbursement claims, such as icd-9 codes used to bill for clinical encounters, or discharge data such as discharge diagnoseslongitudinal datadatasets that measure factors of interest within the same subjects over timeclinical registriesdatasets generated from registries of specific clinical conditions, such as regional cancer registries used to create the surveillance epidemiology and end results program (seer) datasetpopulation - based surveya target population is available and well - defined, and a systematic approach is used to select members of that population to take part in the study. for example, seer is a population - based survey because it aims to include data on all individuals with cancer cared for in the included regionsnationally representative surveysurvey sample that is designed to be representative of the target population on a national level. often uses a complex sampling scheme. the health and retirement study (hrs), for example, is nationally representative of community - dwelling adults over age 50panel surveya longitudinal survey in which data are collected in the same panel of subjects over time. as one panel is at the middle or end of its participation, a panel of new participants is enrolled. in the medical expenditures panel survey (meps), for example, individuals in the same household are surveyed several times over the course of 2 yearsstatistical sampling termsclusteringeven simple random samples can be prohibitively expensive for practical reasons such as geographic distance between selected subjects. identifying subjects within defined clusters, such as geographic regions or subjects treated by the same physicians, reduces cost and improves the feasibility of the study but may decrease the precision of the estimated variance (e.g., wider confidence intervals)complex survey designa survey design that is not a simple random selection of subjects. surveys that incorporate stratification, clustering and oversampling (with patient weights) are examples of complex data. statistical software is available that can account for complex survey designs and is often needed to generate accurate findingsoversamplingintentionally sampling a greater proportion of a subgroup, increasing the precision of estimates for that subgroup. for example, in the hrs, african - americans, latinos, and residents of florida are oversampled (see also survey weights)stratificationin stratification, the target population is divided into relatively homogeneous groups, and a pre - specified number of subjects is sampled from within each stratum. for example, in the national ambulatory medical care survey physicians are divided by specialty within each geographic area targeted for the survey, and a certain number of each type of physician is then identified to participate and provide data about their patientssurvey weightsweights are used to account for the unequal probability of subject selection due to purposeful over- or under - sampling of certain types of subjects and non - response bias. the survey weight is the inverse probability of being selected. by applying survey weights, the effects of over- and under - sampling of certain types of patients can be corrected such that the data are representative of the entire target population a practical approach to successful research with large datasets glossary of terms used in secondary dataset analysis research case a fellow in general medicine has a strong interest in studying palliative and end - of - life care. building on his interest in racial and ethnic disparities, he wants to examine disparities in use of health services at the end of life. he is leaning toward conducting a secondary data analysis and is not sure if he should begin with a more focused research question or a search for a dataset.investigators new to secondary data research are frequently challenged by the question which comes first, the question or the dataset ? in general, we advocate that researchers begin by defining their research topic or question. a good question is essential an uninteresting study with a huge sample size or extensively validated measures is still uninteresting. the answer to a research question should have implications for patient care or public policy. if possible, select a question that will be interesting regardless of the direction of the findings : positive or negative. also, determine a target audience who would find your work interesting and useful.it is often useful to start with a thorough literature review of the question or topic of interest. this effort both avoids duplicating others work and develops ways to build upon the literature. once the question is established, identify datasets that are the best fit, in terms of the patient population, sample size, and measures of the variables of interest (including predictors, outcomes, and potential confounders). once a candidate dataset has been identified, we recommend being flexible and adapting the research question to the strengths and limitations of the dataset, as long as the question remains interesting and specific and the methods to answer it are scientifically sound. some measures of interest may not have been ascertained directly, but data may be available to construct a suitable proxy. in some cases, you may find a dataset that initially looked promising lacks the necessary data (or data quality) to answer research questions in your area of interest reliably. in that case, you should be prepared to search for an alternative dataset.a specific research question is essential to good research. however, many researchers have a general area of interest but find it difficult to identify specific research questions without knowing the specific data available. in that case, combing research documentation for unexamined yet interesting measures in your area of interest can be fruitful. beginning with the dataset and no focused area of interest may lead to data dredging simply creating cross tabulations of unexplored variables in search of significant associations is bad science. yet, in our experience, many good studies have resulted from a researcher with a general topic area of interest finding a clinically meaningful yet underutilized measure and having the insight to frame a research question that uses that measure to answer a novel and clinically compelling question (see references for examples).48 dr. warren browner once exhorted, just because you were not smart enough to think of a research question in advance does nt mean it s not important ! case continued after a review of available datasets that fit his topic area of interest, the fellow decides to use data from the surveillance epidemiology and end results program linked to medicare claims (seer - medicare).the range and intricacy of large datasets can be daunting to a junior researcher. fortunately, several online compendia are available to guide researchers (table 3), including one recently developed by this manuscript s authors for the society of general internal medicine (sgim) (www.sgim.org/go/datasets). the sgim research dataset compendium was developed and is maintained by members of the sgim research committee. sgim compendium developers consulted with experts to identify and profile high - value datasets for generalist researchers. the compendium includes a description of and links to over 40 high - value datasets used for health services, clinical epidemiology, and medical education research. the sgim compendium provides detailed information of use in selecting a dataset, including sample sizes and characteristics, available measures and how data was measured, comments from expert users, links to the dataset, and example publications (see box for example). sgim members can request a one - time telephone consultation with an expert user of a large dataset (see details on the compendium website). table 3online compendia of secondary datasetscompendiumweb addressdescriptionsociety of general internal medicine (sgim) research dataset compendiumwww.sgim.org/go/datasetsdesigned to assist investigators conducting research on existing datasets, with a particular emphasis on health services research, clinical epidemiology, and research on medical education. includes information on strengths and weaknesses of datasets and the insights of experienced users about making best use of the datanational information center on health services research and health care technology (nichsr)http://www.nlm.nih.gov / nichsr / index.htmlthis group of sites provides links to a wide variety of data tools and statistics, including research datasets, data repositories, health statistics, survey instruments, and more. it is sponsored by the national library of medicineinter - university consortium for political and social research (icpsr)https://www.icpsr.umich.eduworld s largest archive of digital social science data, including many datasets with extensive information on health and health care. icpsr includes many sub - archives on specific topic areas, including minority health, international data, substance abuse, and mental health, and morepartners in information access for the public health workforcehttp://phpartners.org / health_stats.htmlprovides links to a variety of national, state, and local health and public health datasets. also provides links to sites providing a wide variety of health statistics, information on health information technology and standards, and other resources. sponsored by a collaboration of us government agencies, public health organizations, and health sciences librariescanadian research data centreshttp://www.statcan.gc.ca / rdc - cdr / index - eng.htmlinks to datasets available for analysis through canada s research data centres (rdc) programdirectory of health and human services data resources (us dept. of health and human services)http://aspe.hhs.gov / datacncl / datadir / index.shtmlthis site provides brief information and links to almost all datasets from national institutes of health (nih), centers for disease control and prevention (cdc), centers for medicare and medicaid services (cms), agency for healthcare research and quality (ahrq), food and drug administration (fda), and other agencies of the us department of health and human servicesnational center for health statistics (nchs)http://www.cdc.gov / nchs / index.htmthis site links to a variety of datasets from the national center for health statistics, several of which are profiled in table 4. these datasets are available for downloading at no costmedicare research data assistance center (resdac) ; and centers for medicare and medicaid services (cms) research, statistics, data & systemswww.resdac.umn.edu/available_cms_data.aspthese sites link to a variety of datasets from the centers for medicare and medicaid services (cms)veterans affairs (va) datawww.virec.research.va.gov/index.htma series of datasets using administrative and computerized clinical data to describe care provided in the va health care system, including information on outpatient visits, pharmacy data, inpatient data, cost data, and more. with some exceptions, use is generally restricted to researchers with va affiliations (this can include a co - investigator with a va affiliation)table 4examples of high value datasetscost, availability, and complexitydatasetdescriptionsample publicationsfree. readily available.. does not require special statistical techniques to address complex samplingsurveillance, epidemiology and end results program (seer) http://www.seer.cancer.gov/population-based multi - regional cancer registry database. can be linked to medicare claims and files (see medicare below)trends in breast - conserving surgery among asian americans and pacific islanders, 1992200012treatment and outcomes of gastric cancer among us - born and foreign - born asians and pacific islanders13free. readily available. requires statistical considerations to account for complex sampling design and use of survey weightsnational ambulatory medical care survey (namcs) & national hospital ambulatory care survey (nhamcs) http://www.cdc.gov/nchs/ahcd.htmnationally-representative serial cross - sectional surveys of outpatient and emergency department visits. can combine survey years to increase sample sizes (e.g., for uncommon conditions) or evaluate temporal trends. provides national estimatespreventive health examinations and preventive gynecological examinations in the us14the namcs and nhamcs are conducted annually. do not link to other datasetsprimary care physician office visits for depression by older americans15national health interview survey (nhis) http://www.cdc.gov/nchs/nhis.htmnationally-representative serial cross - sectional survey of individuals and families including information on health status, injuries, health insurance, access and utilization information. can combine survey years to look at rare conditionspsychological distress in long - term survivors of adult - onset cancer : results from a national survey16can be linked to national center for health statistics mortality data ; medicare enrollment and claims data ; social security benefit history data ; medical expenditure panel survey (meps) data ; and national immunization provider records check survey (niprcs) data from 19971999diabetes and cardiovascular disease among asian indians in the us17behavioral risk factor surveillance system (brfss) http://www.cdc.gov/brfss/serial cross - sectional nationally - representative survey of health risk behaviors, preventative health practices, and health care access. provides national and state estimates. since 2002, the selected metropolitan / micropolitan area risk trends (smart) project has also used brfss data to identify trends in selected metropolitan and micropolitan statistical areas (mmsas) with 500 or more respondents. does not link to other datasetsperceived discrimination in health care and use of preventive health services18use of recommended ambulatory care services : is the veterans affairs quality gap narrowing?19free or minimal cost. readily available accounting for complex sampling design and use of survey weights can be more complex when using multiple waves seek support from a statistician. or can restrict sample to single waves for ease of usenationwide inpatient sample (nis) http://www.hcup-us.ahrq.gov/databases.jspthe largest us database of inpatient hospital stays that incorporates data from all payers, containing data from approximately 20% of us community hospitals. sampling frame includes approximately 90% of discharges from us hospitalsfactors associated with patients who leave acute - care hospitals against medical advice20nis data is collected annually. for most states, the nis includes hospital identifiers that permit linkages to the american hospital association (aha) annual survey database and county identifiers that permit linkages to the area resource file (arf)impact of hospital volume on racial disparities in cardiovascular procedure mortality21national health and nutrition examination survey (nhanes) http://www.cdc.gov / nchs / nhanes.htmnationally- representative series of studies combining data from interviews, physical examination, and laboratory testsdemographic differences and trends of vitamin d insufficiency in the us population,1988 - 200422nhanes data are collected annually. can be linked to national death index (ndi) mortality data ; medicare enrollment and claims data ; social security benefit history data ; and medical expenditure panel survey (meps) data ; and dual energy x - ray absorptiometry (dxa) multiple imputation data files from 19992004association of hypertension, diabetes, dyslipidemia, and metabolic syndrome with obesity : findings from the national health and nutrition examination survey, 1999 to 200423the health and retirement study (hrs) http://hrsonline.isr.umich.edu/index.phpa nationally - representative longitudinal survey of adults older than 50 designed to assess health status, employment decisions, and economic security during retirementchronic conditions and mortality among the oldest old24hrs data is collected every 2 years. can be linked to social security administration data ; internal revenue service data ; medicare claims data (see medicare below) ; and minimum data set (mds) dataadvance directed and surrogate decision making before death25medical expenditure panel survey (meps) http://www.meps.ahrq.gov/mepsweb/serial nationally - representative panel survey of individuals, families, health care providers, and employers covering a variety of topics. meps data are collected annuallyloss of health insurance among non - elderly adults in medicaid26can be linked by request to the agency for healthcare research and quality to numerous datasets including the nhis, medicare data, and social security datainfluence of patient - provider communication on colorectal cancer screening27data costs are in the thousands to tens of thousands of dollars. requires an extensive application and time to acquire data is on the order of months at a minimum. require additional statistical considerations to account for complex sampling design, use of survey weights, or longitudinal analysis. complex database structure requires a higher degree of analytic and programming skill to create a study dataset efficiently.medicare claims data (alone), seer - medicare, and hrs - medicare http://www.resdac.org/medicare/data_available.aspclaims data on medicare beneficiaries including demographics and resource utilization in a wide variety of inpatient and outpatient settings. medicare claims data are collected continually and made available annually. can be linked to other medicare datasets that use the same unique identifier numbers for patients, providers, and institutions, for example, the medicare current beneficiary survey, the long - term care minimum data set, the american hospital association annual survey, and others. seer and the hrs offer linkages to medicare data as well (as described above)long - term outcomes and costs of ventricular assist devices among medicare beneficiaries28association between the medicare modernization act of 2003 and patient wait times and travel distance for chemotherapy29medicare current beneficiary survey (mcbs) http://www.cms.gov/mcbs/panel survey of a nationally - representative sample of medicare beneficiaries including health status, health care use, health insurance, socioeconomic and demographic characteristics, and health expenditures. can be linked to other medicare datacost - related medication nonadherence and spending on basic needs following implementation of medicare part d30medicare beneficiaries and free prescription drug samples : a national survey31dataset complexity, cost, and time to acquire the data and obtain institutional review board (irb) approval are critical considerations for junior researchers, who are new to secondary analysis, have few financial resources, and limited time to demonstrate productivity. table 4 illustrates the complexity and cost of large datasets across a range of high value datasets used by generalist researchers. dataset complexity increases by number of subjects, file structure (e.g., single versus multiple records per individual), and complexity of the survey design. many publicly available datasets are free, and others can cost tens of thousands of dollars to obtain. some datasets can be downloaded from the web, others require multiple layers of permission and security, and in some cases data must be analyzed in a central data processing center. if the project requires linking new data to an existing database, this linkage will add to the time needed to complete the project and probably require enhanced data security. one advantage of most secondary studies using publicly available datasets is the rapid time to irb approval. many publicly available large datasets contain de - identified data and are therefore eligible for expedited review or exempt status. if you can download the dataset from the web, it is probably exempt, but your local irb must make this determination.linking datasets can be a powerful method for examining an issue by providing multiple perspectives of patient experience. many datasets, including seer, for example, can be linked to the area resource file to examine regional variation in practice patterns. however, linking datasets together increases the complexity and cost of data management. a new researcher might consider first conducting a study only on the initial database, and then conducting their next study using the linked database. for some new investigators, this approach can progressively advance programming skills and build confidence while demonstrating productivity. online compendia of secondary datasets examples of high value datasets case continued the fellow s primary mentor encourages him to closely examine the accuracy of the primary predictor for his study race and ethnicity as reported in seer - medicare. the fellow has a breakthrough when he finds an entire issue of the journal medical care dedicated to seer - medicare, including a whole chapter on the accuracy of coding of sociodemographic factors.9 in an analysis of primary data you select the patients to be studied and choose the study measures. this process gives you a close familiarity with study subjects, and how and what data were collected, that is invaluable in assessing the validity of their measures, the potential bias in measuring associations between predictors and outcome variables (internal validity), and the generalizability of their findings to target populations (external validity). the importance of this familiarity with the strengths and weaknesses of the dataset can not be overemphasized. secondary data research requires considerable effort to obtain the same level of familiarity with the data. practically, this objective requires scouring online documentation and technical survey manuals, searching pubmed for validation studies, and closely reading previous studies using your dataset, to answer the following types of questions : who collected the data, and for what purpose ? do your measures capture what you think they capture?we strongly recommend taking advantage of help offered by the dataset managers, typically described on the dataset s website. for example, the research data assistance center (resdac) is a dedicated resource for researchers using data from the centers for medicare and medicaid services (cms).assessing the validity of your measures is one of the central challenges of large dataset research. for large survey datasets, a good first step in assessing the validity of your measures is to read the questions as they were asked in the survey. others, unfortunately, were collected in a way that makes the measure meaningless, problematic, or open to a range of interpretations. these ambiguities can occur in how the question was asked or in how the data were recorded into response categories.another essential step is to search the online documentation and published literature for previous validation studies. a pubmed search using the dataset name or measure name / type and the publication type validation studies is a good starting point. the key question for a validity study relates to how and why the question was asked and data were collected (e.g., self - report, chart abstraction, physical measurements, billing claims) in relationship to a gold standard. for example, if you are using claims data you should recognize that the primary purpose of those data was not for research, but for reimbursement. consequently, claims data are limited by the scope of services that are reimbursable and the accuracy of coding by clinicians completing encounter forms for billing or by coders in the claims departments of hospitals and clinics. some clinical measures can be assessed by asking subjects if they have the condition of interest, such as self reported diagnosis of hypertension. self - reported data may be adequate for some research questions (e.g., does a diagnosis of hypertension lead people to exercise more ?), but inadequate for others (e.g., the prevalence of hypertension among people with diabetes). even measured data, such as blood pressure, have limitations in that methods of measurement for a study may differ from methods used to diagnose a disorder in the clinician s office. in the national health and nutrition examination survey, for example, subject s blood pressure is based on the average of several measures in a single visit. this differs from the standard clinical practice of measuring blood pressure at separate office visits before diagnosing hypertension. rarely do available measures capture exactly what you are trying to study. in our experience measures in existing datasets are often good enough to answer the research question, with proper interpretation to account for what the measures actually assesses and how they differ from the underlying constructs.finally, we suggest paying close attention to the completeness of measures, and evaluating whether missing data are random or non - random (the latter might result in bias, whereas the former is generally acceptable). statistical approaches to missing data are beyond the scope of this paper, and most statisticians can help you address this problem appropriately. however, pay close attention to skip patterns ; some data are missing simply because the survey item is only asked of a subset for which it applies. for example, in the health and retirement study the question about need for assistance with toileting is only asked of subjects who respond that they have difficulty using the toilet. if you were unaware of this skip pattern and attempted to study assistance with toileting, you would be distressed to find over three - quarters of respondents had missing responses for this question (because they reported no difficulty using the toilet).fellows and other trainees usually do their own computer programming. although this may be daunting, we encourage this practice so fellows can get a close feel for the data and become more skilled in statistical analysis. datasets, however, range in complexity (table 4). in our experience, fellows who have completed introductory training in sas, stata, spss, or other similar statistical software have been highly successful analyzing datasets of moderate complexity without the on - going assistance of a statistical programmer. however, if you do have a programmer who will do much of the coding, be closely involved and review all data cleaning and statistical output as if you had programmed it yourself. close attention can reveal all sorts of patterns, problems, and opportunities with the data that are obscured by focusing only on the final outputs prepared by a statistical programmer. programmers and statisticians are not clinicians ; they will often not recognize when the values of variables or patterns of missingness do nt make sense. if estimates seem implausible or do not match previously published estimates, then the analytic plan, statistical code, and measures should be carefully rechecked.keep in mind that the perfect may be the enemy of the good. no one expects perfect measures (this is also true for primary data collection). the closer you are to the data, the more you see the warts dont be discouraged by this. the measures need to pass the sniff test, in other words have clinical validity based primarily on judgement that they make sense clinically or scientifically, but also supported where possible by validation procedures, reference to auditing procedures, or in other studies that have independently validated the measures of interest. case continued the fellow finds that blacks are less likely to receive chemotherapy in the last 2 weeks of life (blacks 4%, whites 6%, p < 0.001). he debates the meaning of this statistically significant 2% absolute difference.often, the main challenge for investigators who are new to secondary data analysis is carefully structuring the analysis and presentation of findings in a way that tells a meaningful story. based on what you ve found, what is the story that you want your target audience to understand ? when appropriate, it can be useful to conduct carefully planned sensitivity analysis to evaluate the robustness of your primary findings. a sensitivity analysis assesses the effect of variation in assumptions on the outcome of interest. for example, if 10% of subjects did not answer a yes or no question, you could conduct sensitivity analyses to estimate the effects of excluding missing responses, or categorizing them as all yes or all no. because large datasets may contain multiple measures of interests, co - variates, and outcomes, a frequent temptation is to present huge tables with multiple rows and columns. these tables can be challenging to sort through, and the clinical importance of the story resulting from the analysis can be lost. in our experience, a thoughtful figure often captures the take - home message in a way that is more interpretable and memorable to readers than rows of data tables.you should keep careful track of subjects you decide to exclude from the analysis and why. the best way to keep track is to construct a flow diagram from the original denominator to the final sample.dont confuse statistical significance with clinical importance in large datasets. due to large sample sizes one concern that frequently arises at this stage in large database research is the acceptability of exploratory analyses, or the practice of examining associations between multiple factors of interest. on the one hand, exploratory analyses risk finding a significant association by chance alone from testing multiple associations (a false - positive result). on the other hand, the critical issue is not a statistical one, but rather whether the issue is important.10 exploratory analyses are acceptable if done in a thoughtful way that serves an a priori hypothesis, but not if merely data dredging looking for associations.we recommend consulting with a statistician when using data from a complex survey design (see table 2) or developing a conceptually advanced study design, for example, using longitudinal data, multilevel modeling with clustered data, or surivival analysis. the value of input (even if informal) from a statistician or other advisor with substantial methodological expertise can not be overstated. case a fellow in general medicine has a strong interest in studying palliative and end - of - life care. building on his interest in racial and ethnic disparities, he wants to examine disparities in use of health services at the end of life. he is leaning toward conducting a secondary data analysis and is not sure if he should begin with a more focused research question or a search for a dataset.investigators new to secondary data research are frequently challenged by the question which comes first, the question or the dataset ? in general, we advocate that researchers begin by defining their research topic or question. a good question is essential an uninteresting study with a huge sample size or extensively validated measures is still uninteresting. the answer to a research question should have implications for patient care or public policy. if possible, select a question that will be interesting regardless of the direction of the findings : positive or negative. also, determine a target audience who would find your work interesting and useful.it is often useful to start with a thorough literature review of the question or topic of interest. this effort both avoids duplicating others work and develops ways to build upon the literature. once the question is established, identify datasets that are the best fit, in terms of the patient population, sample size, and measures of the variables of interest (including predictors, outcomes, and potential confounders). once a candidate dataset has been identified, we recommend being flexible and adapting the research question to the strengths and limitations of the dataset, as long as the question remains interesting and specific and the methods to answer it are scientifically sound. some measures of interest may not have been ascertained directly, but data may be available to construct a suitable proxy. in some cases, you may find a dataset that initially looked promising lacks the necessary data (or data quality) to answer research questions in your area of interest reliably. in that case, you should be prepared to search for an alternative dataset.a specific research question is essential to good research. however, many researchers have a general area of interest but find it difficult to identify specific research questions without knowing the specific data available. in that case, combing research documentation for unexamined yet interesting measures in your area of interest can be fruitful. beginning with the dataset and no focused area of interest may lead to data dredging simply creating cross tabulations of unexplored variables in search of significant associations is bad science. yet, in our experience, many good studies have resulted from a researcher with a general topic area of interest finding a clinically meaningful yet underutilized measure and having the insight to frame a research question that uses that measure to answer a novel and clinically compelling question (see references for examples).48 dr. warren browner once exhorted, just because you were not smart enough to think of a research question in advance does nt mean it s not important ! case continued after a review of available datasets that fit his topic area of interest, the fellow decides to use data from the surveillance epidemiology and end results program linked to medicare claims (seer - medicare).the range and intricacy of large datasets can be daunting to a junior researcher. fortunately, several online compendia are available to guide researchers (table 3), including one recently developed by this manuscript s authors for the society of general internal medicine (sgim) (www.sgim.org/go/datasets). the sgim research dataset compendium was developed and is maintained by members of the sgim research committee. sgim compendium developers consulted with experts to identify and profile high - value datasets for generalist researchers. the compendium includes a description of and links to over 40 high - value datasets used for health services, clinical epidemiology, and medical education research. the sgim compendium provides detailed information of use in selecting a dataset, including sample sizes and characteristics, available measures and how data was measured, comments from expert users, links to the dataset, and example publications (see box for example). sgim members can request a one - time telephone consultation with an expert user of a large dataset (see details on the compendium website). table 3online compendia of secondary datasetscompendiumweb addressdescriptionsociety of general internal medicine (sgim) research dataset compendiumwww.sgim.org/go/datasetsdesigned to assist investigators conducting research on existing datasets, with a particular emphasis on health services research, clinical epidemiology, and research on medical education. includes information on strengths and weaknesses of datasets and the insights of experienced users about making best use of the datanational information center on health services research and health care technology (nichsr)http://www.nlm.nih.gov / nichsr / index.htmlthis group of sites provides links to a wide variety of data tools and statistics, including research datasets, data repositories, health statistics, survey instruments, and more. it is sponsored by the national library of medicineinter - university consortium for political and social research (icpsr)https://www.icpsr.umich.eduworld s largest archive of digital social science data, including many datasets with extensive information on health and health care. icpsr includes many sub - archives on specific topic areas, including minority health, international data, substance abuse, and mental health, and morepartners in information access for the public health workforcehttp://phpartners.org / health_stats.htmlprovides links to a variety of national, state, and local health and public health datasets. also provides links to sites providing a wide variety of health statistics, information on health information technology and standards, and other resources. sponsored by a collaboration of us government agencies, public health organizations, and health sciences librariescanadian research data centreshttp://www.statcan.gc.ca / rdc - cdr / index - eng.htmlinks to datasets available for analysis through canada s research data centres (rdc) programdirectory of health and human services data resources (us dept. of health and human services)http://aspe.hhs.gov / datacncl / datadir / index.shtmlthis site provides brief information and links to almost all datasets from national institutes of health (nih), centers for disease control and prevention (cdc), centers for medicare and medicaid services (cms), agency for healthcare research and quality (ahrq), food and drug administration (fda), and other agencies of the us department of health and human servicesnational center for health statistics (nchs)http://www.cdc.gov / nchs / index.htmthis site links to a variety of datasets from the national center for health statistics, several of which are profiled in table 4. these datasets are available for downloading at no costmedicare research data assistance center (resdac) ; and centers for medicare and medicaid services (cms) research, statistics, data & systemswww.resdac.umn.edu/available_cms_data.aspthese sites link to a variety of datasets from the centers for medicare and medicaid services (cms)veterans affairs (va) datawww.virec.research.va.gov/index.htma series of datasets using administrative and computerized clinical data to describe care provided in the va health care system, including information on outpatient visits, pharmacy data, inpatient data, cost data, and more. with some exceptions, use is generally restricted to researchers with va affiliations (this can include a co - investigator with a va affiliation)table 4examples of high value datasetscost, availability, and complexitydatasetdescriptionsample publicationsfree. readily available. population - based survey with cross - sectional design. does not require special statistical techniques to address complex samplingsurveillance, epidemiology and end results program (seer) http://www.seer.cancer.gov/population-based multi - regional cancer registry database. can be linked to medicare claims and files (see medicare below)trends in breast - conserving surgery among asian americans and pacific islanders, 1992200012treatment and outcomes of gastric cancer among us - born and foreign - born asians and pacific islanders13free. readily available. requires statistical considerations to account for complex sampling design and use of survey weightsnational ambulatory medical care survey (namcs) & national hospital ambulatory care survey (nhamcs) http://www.cdc.gov/nchs/ahcd.htmnationally-representative serial cross - sectional surveys of outpatient and emergency department visits. can combine survey years to increase sample sizes (e.g., for uncommon conditions) or evaluate temporal trends. provides national estimatespreventive health examinations and preventive gynecological examinations in the us14the namcs and nhamcs are conducted annually. do not link to other datasetsprimary care physician office visits for depression by older americans15national health interview survey (nhis) http://www.cdc.gov/nchs/nhis.htmnationally-representative serial cross - sectional survey of individuals and families including information on health status, injuries, health insurance, access and utilization information. can combine survey years to look at rare conditionspsychological distress in long - term survivors of adult - onset cancer : results from a national survey16can be linked to national center for health statistics mortality data ; medicare enrollment and claims data ; social security benefit history data ; medical expenditure panel survey (meps) data ; and national immunization provider records check survey (niprcs) data from 19971999diabetes and cardiovascular disease among asian indians in the us17behavioral risk factor surveillance system (brfss) http://www.cdc.gov/brfss/serial cross - sectional nationally - representative survey of health risk behaviors, preventative health practices, and health care access. provides national and state estimates. since 2002, the selected metropolitan / micropolitan area risk trends (smart) project has also used brfss data to identify trends in selected metropolitan and micropolitan statistical areas (mmsas) with 500 or more respondents. does not link to other datasetsperceived discrimination in health care and use of preventive health services18use of recommended ambulatory care services : is the veterans affairs quality gap narrowing?19free or minimal cost. readily available accounting for complex sampling design and use of survey weights can be more complex when using multiple waves seek support from a statistician. or can restrict sample to single waves for ease of usenationwide inpatient sample (nis) http://www.hcup-us.ahrq.gov/databases.jspthe largest us database of inpatient hospital stays that incorporates data from all payers, containing data from approximately 20% of us community hospitals. sampling frame includes approximately 90% of discharges from us hospitalsfactors associated with patients who leave acute - care hospitals against medical advice20nis data is collected annually. for most states, the nis includes hospital identifiers that permit linkages to the american hospital association (aha) annual survey database and county identifiers that permit linkages to the area resource file (arf)impact of hospital volume on racial disparities in cardiovascular procedure mortality21national health and nutrition examination survey (nhanes) http://www.cdc.gov / nchs / nhanes.htmnationally- representative series of studies combining data from interviews, physical examination, and laboratory testsdemographic differences and trends of vitamin d insufficiency in the us population,1988 - 200422nhanes data are collected annually. can be linked to national death index (ndi) mortality data ; medicare enrollment and claims data ; social security benefit history data ; and medical expenditure panel survey (meps) data ; and dual energy x - ray absorptiometry (dxa) multiple imputation data files from 19992004association of hypertension, diabetes, dyslipidemia, and metabolic syndrome with obesity : findings from the national health and nutrition examination survey, 1999 to 200423the health and retirement study (hrs) http://hrsonline.isr.umich.edu/index.phpa nationally - representative longitudinal survey of adults older than 50 designed to assess health status, employment decisions, and economic security during retirementchronic conditions and mortality among the oldest old24hrs data is collected every 2 years. can be linked to social security administration data ; internal revenue service data ; medicare claims data (see medicare below) ; and minimum data set (mds) dataadvance directed and surrogate decision making before death25medical expenditure panel survey (meps) http://www.meps.ahrq.gov/mepsweb/serial nationally - representative panel survey of individuals, families, health care providers, and employers covering a variety of topics. meps data are collected annuallyloss of health insurance among non - elderly adults in medicaid26can be linked by request to the agency for healthcare research and quality to numerous datasets including the nhis, medicare data, and social security datainfluence of patient - provider communication on colorectal cancer screening27data costs are in the thousands to tens of thousands of dollars. requires an extensive application and time to acquire data is on the order of months at a minimum require additional statistical considerations to account for complex sampling design, use of survey weights, or longitudinal analysis. complex database structure requires a higher degree of analytic and programming skill to create a study dataset efficiently.medicare claims data (alone), seer - medicare, and hrs - medicare http://www.resdac.org/medicare/data_available.aspclaims data on medicare beneficiaries including demographics and resource utilization in a wide variety of inpatient and outpatient settings. medicare claims data are collected continually and made available annually. can be linked to other medicare datasets that use the same unique identifier numbers for patients, providers, and institutions, for example, the medicare current beneficiary survey, the long - term care minimum data set, the american hospital association annual survey, and others. seer and the hrs offer linkages to medicare data as well (as described above)long - term outcomes and costs of ventricular assist devices among medicare beneficiaries28association between the medicare modernization act of 2003 and patient wait times and travel distance for chemotherapy29medicare current beneficiary survey (mcbs) http://www.cms.gov/mcbs/panel survey of a nationally - representative sample of medicare beneficiaries including health status, health care use, health insurance, socioeconomic and demographic characteristics, and health expenditures. can be linked to other medicare datacost - related medication nonadherence and spending on basic needs following implementation of medicare part d30medicare beneficiaries and free prescription drug samples : a national survey31dataset complexity, cost, and time to acquire the data and obtain institutional review board (irb) approval are critical considerations for junior researchers, who are new to secondary analysis, have few financial resources, and limited time to demonstrate productivity. table 4 illustrates the complexity and cost of large datasets across a range of high value datasets used by generalist researchers. dataset complexity increases by number of subjects, file structure (e.g., single versus multiple records per individual), and complexity of the survey design. many publicly available datasets are free, and others can cost tens of thousands of dollars to obtain. some datasets can be downloaded from the web, others require multiple layers of permission and security, and in some cases data must be analyzed in a central data processing center. if the project requires linking new data to an existing database, this linkage will add to the time needed to complete the project and probably require enhanced data security. one advantage of most secondary studies using publicly available datasets is the rapid time to irb approval. many publicly available large datasets contain de - identified data and are therefore eligible for expedited review or exempt status. if you can download the dataset from the web, it is probably exempt, but your local irb must make this determination.linking datasets can be a powerful method for examining an issue by providing multiple perspectives of patient experience. many datasets, including seer, for example, can be linked to the area resource file to examine regional variation in practice patterns. however, linking datasets together increases the complexity and cost of data management. a new researcher might consider first conducting a study only on the initial database, and then conducting their next study using the linked database. for some new investigators, this approach can progressively advance programming skills and build confidence while demonstrating productivity. case continued the fellow s primary mentor encourages him to closely examine the accuracy of the primary predictor for his study race and ethnicity as reported in seer - medicare. the fellow has a breakthrough when he finds an entire issue of the journal medical care dedicated to seer - medicare, including a whole chapter on the accuracy of coding of sociodemographic factors.9 in an analysis of primary data you select the patients to be studied and choose the study measures. this process gives you a close familiarity with study subjects, and how and what data were collected, that is invaluable in assessing the validity of their measures, the potential bias in measuring associations between predictors and outcome variables (internal validity), and the generalizability of their findings to target populations (external validity). the importance of this familiarity with the strengths and weaknesses of the dataset can not be overemphasized. secondary data research requires considerable effort to obtain the same level of familiarity with the data. therefore, knowing your data in detail is critical. practically, this objective requires scouring online documentation and technical survey manuals, searching pubmed for validation studies, and closely reading previous studies using your dataset, to answer the following types of questions : who collected the data, and for what purpose ? do your measures capture what you think they capture?we strongly recommend taking advantage of help offered by the dataset managers, typically described on the dataset s website. for example, the research data assistance center (resdac) is a dedicated resource for researchers using data from the centers for medicare and medicaid services (cms).assessing the validity of your measures is one of the central challenges of large dataset research. for large survey datasets, a good first step in assessing the validity of your measures is to read the questions as they were asked in the survey. others, unfortunately, were collected in a way that makes the measure meaningless, problematic, or open to a range of interpretations. these ambiguities can occur in how the question was asked or in how the data were recorded into response categories.another essential step is to search the online documentation and published literature for previous validation studies. a pubmed search using the dataset name or measure name / type and the publication type validation studies is a good starting point. the key question for a validity study relates to how and why the question was asked and data were collected (e.g., self - report, chart abstraction, physical measurements, billing claims) in relationship to a gold standard. for example, if you are using claims data you should recognize that the primary purpose of those data was not for research, but for reimbursement. consequently, claims data are limited by the scope of services that are reimbursable and the accuracy of coding by clinicians completing encounter forms for billing or by coders in the claims departments of hospitals and clinics. some clinical measures can be assessed by asking subjects if they have the condition of interest, such as self reported diagnosis of hypertension. self - reported data may be adequate for some research questions (e.g., does a diagnosis of hypertension lead people to exercise more ?), but inadequate for others (e.g., the prevalence of hypertension among people with diabetes). even measured data, such as blood pressure, have limitations in that methods of measurement for a study may differ from methods used to diagnose a disorder in the clinician s office. in the national health and nutrition examination survey, for example, subject s blood pressure is based on the average of several measures in a single visit. this differs from the standard clinical practice of measuring blood pressure at separate office visits before diagnosing hypertension. rarely do available measures capture exactly what you are trying to study. in our experience measures in existing datasets are often good enough to answer the research question, with proper interpretation to account for what the measures actually assesses and how they differ from the underlying constructs.finally, we suggest paying close attention to the completeness of measures, and evaluating whether missing data are random or non - random (the latter might result in bias, whereas the former is generally acceptable). statistical approaches to missing data are beyond the scope of this paper, and most statisticians can help you address this problem appropriately. skip patterns ; some data are missing simply because the survey item is only asked of a subset for which it applies. for example, in the health and retirement study the question about need for assistance with toileting is only asked of subjects who respond that they have difficulty using the toilet. if you were unaware of this skip pattern and attempted to study assistance with toileting, you would be distressed to find over three - quarters of respondents had missing responses for this question (because they reported no difficulty using the toilet).fellows and other trainees usually do their own computer programming. although this may be daunting, we encourage this practice so fellows can get a close feel for the data and become more skilled in statistical analysis. datasets, however, range in complexity (table 4). in our experience, fellows who have completed introductory training in sas, stata, spss, or other similar statistical software have been highly successful analyzing datasets of moderate complexity without the on - going assistance of a statistical programmer. however, if you do have a programmer who will do much of the coding, be closely involved and review all data cleaning and statistical output as if you had programmed it yourself. close attention can reveal all sorts of patterns, problems, and opportunities with the data that are obscured by focusing only on the final outputs prepared by a statistical programmer. programmers and statisticians are not clinicians ; they will often not recognize when the values of variables or patterns of missingness do nt make sense. if estimates seem implausible or do not match previously published estimates, then the analytic plan, statistical code, and measures should be carefully rechecked.keep in mind that the perfect may be the enemy of the good. no one expects perfect measures (this is also true for primary data collection). the closer you are to the data, the more you see the warts dont be discouraged by this. the measures need to pass the sniff test, in other words have clinical validity based primarily on judgement that they make sense clinically or scientifically, but also supported where possible by validation procedures, reference to auditing procedures, or in other studies that have independently validated the measures of interest. case continued the fellow finds that blacks are less likely to receive chemotherapy in the last 2 weeks of life (blacks 4%, whites 6%, p < 0.001). he debates the meaning of this statistically significant 2% absolute difference.often, the main challenge for investigators who are new to secondary data analysis is carefully structuring the analysis and presentation of findings in a way that tells a meaningful story. based on what you ve found, what is the story that you want your target audience to understand ? when appropriate, it can be useful to conduct carefully planned sensitivity analysis to evaluate the robustness of your primary findings. a sensitivity analysis assesses the effect of variation in assumptions on the outcome of interest. for example, if 10% of subjects did not answer a yes or no question, you could conduct sensitivity analyses to estimate the effects of excluding missing responses, or categorizing them as all yes or all no. because large datasets may contain multiple measures of interests, co - variates, and outcomes, a frequent temptation is to present huge tables with multiple rows and columns. these tables can be challenging to sort through, and the clinical importance of the story resulting from the analysis can be lost. in our experience, a thoughtful figure often captures the take - home message in a way that is more interpretable and memorable to readers than rows of data tables.you should keep careful track of subjects you decide to exclude from the analysis and why. the best way to keep track is to construct a flow diagram from the original denominator to the final sample.dont confuse statistical significance with clinical importance in large datasets. due to large sample sizes, associations may be statistically significant but not clinically meaningful. one concern that frequently arises at this stage in large database research is the acceptability of exploratory analyses, or the practice of examining associations between multiple factors of interest. on the one hand, exploratory analyses risk finding a significant association by chance alone from testing multiple associations (a false - positive result). on the other hand, the critical issue is not a statistical one, but rather whether the issue is important.10 exploratory analyses are acceptable if done in a thoughtful way that serves an a priori hypothesis, but not if merely data dredging looking for associations.we recommend consulting with a statistician when using data from a complex survey design (see table 2) or developing a conceptually advanced study design, for example, using longitudinal data, multilevel modeling with clustered data, or surivival analysis. the value of input (even if informal) from a statistician or other advisor with substantial methodological expertise can not be overstated. case conclusion two years after he began the project the fellow completes the analysis and publishes the paper in a peer - reviewed journal.11a 2-year timeline from inception to publication is typical for large database research. academic potential is commonly assessed by the ability to see a study through to publication in a peer - reviewed journal. this timeline allows a fellow who began a secondary analysis at the start of a 2-year training program to search for a job with an article under review or in press.in conclusion, secondary dataset research has tremendous advantages, including the ability to assess outcomes that would be difficult or impossible to study using primary data collection, such as those involving exceptionally long follow - up times or rare outcomes. for junior investigators, the potential for a shorter time to publication may help secure a job or career development funding. some of the time saved by not collecting data yourself, however, needs to be spent becoming familiar with the dataset in intimate detail. ultimately, the same factors that apply to successful primary data analysis apply to secondary data analysis, including the development of a clear research question, study sample, appropriate measures, and a thoughtful analytic approach.	abstractsecondary analyses of large datasets provide a mechanism for researchers to address high impact questions that would otherwise be prohibitively expensive and time - consuming to study. this paper presents a guide to assist investigators interested in conducting secondary data analysis, including advice on the process of successful secondary data analysis as well as a brief summary of high - value datasets and online resources for researchers, including the sgim dataset compendium (www.sgim.org/go/datasets). the same basic research principles that apply to primary data analysis apply to secondary data analysis, including the development of a clear and clinically relevant research question, study sample, appropriate measures, and a thoughtful analytic approach. a real - world case description illustrates key steps : (1) define your research topic and question ; (2) select a dataset ; (3) get to know your dataset ; and (4) structure your analysis and presentation of findings in a way that is clinically meaningful. secondary dataset analysis is a well - established methodology. secondary analysis is particularly valuable for junior investigators, who have limited time and resources to demonstrate expertise and productivity.
the genus listeria is composed of 17 species, including two that are recognized pathogens. of these, l. monocytogenes is considered as the only human pathogen causing listeriosis, a highly lethal opportunistic infection caused by ingestion of contaminated food. the second pathogenic species is l. ivanovii, which mostly responsible for abortions, stillbirths and neonatal septicaemias in sheep and cattle. however, rare cases of human infection by this species have been published. to date, eight human l. ivanovii infections have been reported, including four cases of bacteraemia and one case each of mesenteric adenitis, uterine discharge and stillbirth (table 1). here we report a rare case of l. ivanovii vascular infection in a 78-year - old man. we applied real - time genomics to compare the genetic content of this strain to those of other l. ivanovii strains available in public databases. on 29 november 2014 a 78-year - old man was hospitalized in timone hospital, marseille, france, for suspected infectious endocarditis. the patient had had a mechanical bentall prosthesis inserted in 2010 as well as a mitral valve regurgitation repaired in 1998. he also had a dissection of the descending thoracic aorta. over the past 10 days transesophageal echocardiography revealed moderate leakages of the mitral and mechanical aortic valves but no vegetation. however, because endocarditis was suspected, empirical intravenous amoxicillin (12 g / d) and gentamicin (3 mg / kg / d) was initiated after blood was collected for culture. on 4 december, as a result of a persistent inflammatory syndrome (c - reactive protein level 264 mg / l), a new aortic paraprosthetic leakage was detected by transesophageal echocardiography. a thoracic computed tomographic scan revealed increased size of the previously known descending aorta dissection. blood cultures and systematic serology assays (aspergillus sp., bartonella sp., brucella sp., legionella pneumophila, mycoplasma pneumoniae, q fever) were negative. antibiotics were changed to intravenous imipenem (3 g / d) and vancomycin (2 g / d). on 24 december liposomal amphotericin b (3 mg / kg / d) was added to treat a persistent biological inflammatory syndrome. on 30 december the patient underwent a right posterolateral thoracotomy for resection of a mediastinal abscess. culture of peroperative specimens were positive for a listeria isolate that was identified as l. ivanovii using 16s rrna sequencing (99.9% identity with genbank accession no. the antibiotic therapy was changed again to intravenous amoxicillin (12 g / d) and gentamicin (3 mg / kg / d). however, on 8 january 2015 the patient developed respiratory distress and was transferred to the intensive care unit, where atrial fibrillation and pleural effusion were diagnosed. on 14 january the patient underwent pleural drainage, right axillofemoral bypass, reimplantation of the subclavian artery on the common carotid artery, removal of the thoracic endoprosthesis and closure of the aortic stump. listeria ivanovii strain g770 was deposited in the csur collection (wdcm 875) under reference p1995. genomic dna of listeria ivanovii was extracted using an ez - one automate (qiagen, hilden, germany). genomic dna was quantified by a qubit assay with the high sensitivity kit (life technologies, carlsbad, ca, usa) to 10.8 ng/l. genomic dna (1 ng) was used to prepare the paired - end library with the nextera xt dna sample prep kit (illumina, san diego, ca, usa). the tagmentation step fragmented and tagged the dna. then limited - cycle pcr amplification (12 cycles) after purification on ampure xp beads (beckman coulter, fullerton, ca, usa), the library was sequenced on the miseq sequencer (illumina) in a single 39-hour run in 2 250 bp. a total of 455 380 paired - end reads was obtained for this project, with a 871k / mm cluster density with a cluster passing quality control filters of 80.5%. the reads obtained after sequencing were assembled using the a5 assembler. then a finishing step was performed with the mauve aligner software and clc bioserver. open reading frames (orfs) were predicted by prodigal software (http://prodigal.ornl.gov/) with default parameters. functional annotation was done by comparison of orf sequences to the genbank and clusters of orthologous groups (cogs) database using blastp. trnas and rrnas were detected using trnascan - se v.1.21 and rnammer 1.2, respectively. the absence of plasmids was verified both by searching the gene annotation for any plasmid - related gene and by mapping all contigs against previously published listeria plasmid sequences. to identify the most closely related l. ivanovii genomes, we performed a phylogenetic analysis (fig. 1) by comparing the sequences of the hlya gene using the neighbour - joining method and mega 6 software. subsequently we compared the genomic content of strain g770 and those of the most closely related l. ivanovii strains available in genbank. the genome sequence similarity between studied strains was evaluated with ggdc software (http://ggdc.dsmz.de). the analysis of virulence factors was performed by searching the genes already identified as responsible for virulence in l. ivanovii (six genes of the lipi-1 cluster, internalin genes, genes of stress survival islet 1 (ss1)) and by screening every gene that was specific to strain g770 for a putative role in virulence. the crisprfinder algorithm was used to identify putative crispr (clustered regularly interspaced short palindromic repeat) loci. the genome sequence from l. the draft genome sequence of l. ivanovii strain g770 consisted of 17 scaffolds after assembly and finishing. no plasmid was detected. the chromosome size, g+c content and number of genes were 2 965 602 bases, 37.10% and 2946 genes, respectively. among these genes, 2850 were protein - coding genes and 96 were rnas (21 rrnas, 74 trnas, one tmrna). a total of 2433 genes were assigned to cogs (82, 25%). of these, 2393 genes were assigned a putative function (81.2%), and 553 (18.8%) were annotated as hypothetical proteins. phylogenetically, two lineages were identified among l. ivanovii strains, including one that was made of l. ivanovii subsp. ivanovii strains, including strain g770, and a second made of l. ivanovii subsp. the percentages of nucleotide sequence similarity between strain g770 and l. ivanovii strains for the hlya gene ranged from 94.35% with the two strains of subspecies londoniensis to 99.87% with the other three strains of subspecies ivanovii. for 16s rrna, the percentages of nucleotide similarity were 99.87 and 100% between strain g770 and the strains of the subspecies londoniensis and ivanovii, respectively. londoniensis strains (supplementary table s1). as detailed in table 2, 21 were absent from all other compared genomes l. ivanovii strains (table 3). in addition, strain g770 had 12, 11 and 13 missing genes with regard to subspecies londoniensis (table 2). supplementary table s2 lists the missing in strain g770. among the 21 strain g770specific genes, five encoded proteins similar to type i restriction - modification genes of l. monocytogenes and one was mostly similar to vanz, a component of the vancomycin - resistant operon in staphylococcus pseudintermedius (table 3). strain g770 also had five specific genes encoding metabolic enzymes such as helicases, transferases and acetyl coenzyme a synthase. finally, the strain exhibited six genes encoding hypothetical proteins and one encoding a membrane protein (table 3). as other l. ivanovii strains, strain g770 had a complete lipi-1 virulence cluster. however, although the sequences from the five lipi-1 genes prfa, llo, mpl, plcc and plcb of all compared genomes were identical, strain g770 differed from other strains in the acta gene sequence (supplementary table s3). nevertheless, the annotated domains of the acta protein were similar to those of acta proteins from other strains of the subspecies ivanovii (supplementary fig. strain g770, like all other compared l. ivanovii strains, exhibited 16 internalin or internalin - like genes including inla, inlb and inlc (supplementary table s4). in addition, we identified in strain g770 five genes related to the ssi-1 (supplementary table s5). s2), whereas the other three subspecies ivanovii strains exhibited three crispr regions each and the subspecies londoniensis strains wslc30151 and wslc30167 had two and three crisprs, respectively. to date, only eight cases of human infections caused by l. ivanovii have been reported in the literature. of these, two were diagnosed in patients over 60 years, and most occurred in immunocompromised patients (two pregnant women, and one case each of aids, drug abuse, hepatic lymphoma and immunosuppression ; table 1). in the remaining two cases of human l. ivanovii infection, no underlying condition was described (table 1). to our knowledge, we herein present the first case of l. ivanovii vascular infection. in an effort to determine whether this unusual presentation was due to an increased virulence of strain g770, we compared the genome of strain g770 to those of other l. ivanovii strains. in listeria species, ivanovii, the lipi-1 cluster is made of six genes, including a pore - forming toxin (listeriolysin o) and two phospholipases (plcc and plcb) which cooperate to lyse the membrane of the phagocytic vacuole ; an actin - polymerization surface protein (acta) that is responsible for intracellular bacterial motility and spread ; a metalloprotease (mpl) that is involved in the maturation of proplcb ; and a transcriptional activator (prfa) that controls the expression of lipi-1 genes. studies have shown that mutations in the prfa gene inhibit bacterial cell - to - cell spread and reduce bacterial virulence in l. monocytogenes. mutations in different parts of acta, notably the n - terminal region, cause several unusual motility and actin polymerization phenotypes in l. monocytogenes. strain g770 possessed all six genes of the lipi-1 cluster but varied in hly and acta when compared to other strains from l. ivanovii subsp. therefore, the observed differences in cluster lipi-1 or acta do not support an increased virulence of strain g770. among other known virulence factors of listeria species, internalins such inla, inlb and inlc are essential for host invasion and cell - to - cell spread. however, strain g770 did not exhibit differences in internalin content (supplementary table s4) compared to other l. ivanovii strains. in addition, strain g770 also possessed the stress survival islet 1 (supplementary table s5), known in l. monocytogenes to be linked to tolerance towards acidic, salt, bile and gastric stress. again, we found no difference in ssi-1 content among studied strains. finally, we detected crisprs in strain g770 (supplementary fig. the presence of crispr regions confers to listeria species an adaptive immunity that protects them against invading bacteriophages and plasmids. however, no relationship between the number of crisprs and virulence has been reported for these bacteria. the best matches for 12 of these genes were found in both listeria seeligeri and l. monocytogenes. only one specific gene (scaffold1.1_178) was not found in the genus listeria, thus confirming the highly conserved nature of genomes in this genus. of these additional genes, six may have roles in virulence or resistance of strain g770, including five genes similar to a type i restriction - modification system of listeria monocytogenes. this is the first time that a type i restriction - modification system is found in l. ivanovii. in addition, type i restriction - modification systems play roles in various cellular processes including host defence and virulence, and they even control the speed of evolution of the organism. one study showed that bacteria that have acquired a competent restriction - modification system acquire new properties especially during colonization of new habitats. therefore, we assume that the type i restriction - modification system identified in strain g770 may have conferred to it an increased virulence. the presence of this transposon confers resistance to glycopeptides in enterococcus faecium. in the absence of the other components of the tn1546 transposon however, no case of resistance to vancomycin has been reported for strains of listeria spp. originating from humans, food or ivanovii strain g770, which caused a deadly aortic infection, enabled identification of specific characteristics among other l. ivanovii strains, including a type i restriction - modification system that may have conferred to it an increased virulence.	we sequenced the genome of listeria ivanovii strain g770, which caused a deadly infection of the thoracic aortic prosthesis of a 78-year - old man. the 2.9 mb genome exhibited 21 specific genes among l. ivanovii strains, including five genes encoding a type i restriction modification system and one glycopeptide resistance gene.
single coronary artery (sca) is a rare congenital anomaly of the coronary arteries arising from a single coronary ostium1). the clinical significance of sca is not clear, and it is generally considered to be a benign entity. there are some case reports of sca in adults, but there are a few such reported cases in children. we report a case of a 13-year - old girl with right coronary artery arising from the left circumflex artery who presented with chest pain. a previously healthy 13-year - old girl visited our emergency department due to left - sided chest pain that had occurred suddenly 10 hours ago. the pain was continuous pressing in nature without any radiation, and it improved on bending forward but worsened with deep inspiration. she had no specific past medical history such as kawasaki disease, asthma, and no family history of heart or lung disease. she did not appear ill ; and her vital signs were as follows : blood pressure was 133/79 mmhg, heart rate was 117 beats / min, and temperature was 37.6. her weight was 40 kg. the physical examination did not reveal any other specific findings, especially her lung sound was normal electrocardiogram (ecg) showed sinus tachycardia with a heart rate of 114 beats / min without st segment changes (fig. however, her cardiac enzyme levels were slightly elevated ; n - terminal prohormone brain natriuretic peptide was 257.6 pg / ml, troponin i was 2.138 ng / ml, and creatine kinase - myoglobin (ck - mb) was 13.02 ng / ml. the next day, she again complained of severe chest pain. her ecg was taken again, and it showed marked st elevation in the v4-v6 left precordial leads and inferior leads and st depression in avr lead, which was suggestive of myocardial ischemia (fig. 2). also, her cardiac enzyme levels were elevated ; troponin i was 4.310 ng / ml, ck - mb was 10.78 ng / ml. although the chest pain regressed spontaneously, further evaluation was performed. we performed coronary angiography ; left coronary artery (lca) was bifurcated normally into left anterior descending (lad) and left circumflex (lcx) coronary arteries, and it showed the absence of normally originating right coronary artery (rca). an aberrant branch was extended from the distal lcx, which crossed the crux and continued to the right atrioventricular groove, covering the territory of the rca (fig. lad and its branches (septal perforator branch, diagonal branch) also had no stenotic lesion. we also performed cardiac computed tomography and confirmed the absence of the rca and the presence of a well - developed lcx coronary artery covering the right side of the heart (fig. she was discharged without any medications ; however, a regular follow - up was recommended. an isolated sca is defined when only one coronary artery arises from the aorta by a single coronary ostium, supplying the entire heart1). sca is a very rarely encountered disease entity, and its reported incidence in adults is only from 0.024% to 0.066%1,2). also, it is occasionally associated with other cardiac anomalies such as transposition of the great vessels, a coronary arteriovenous fistula, or a bicuspid aortic valve3). a sca can result in the development of cardiac ischemia, cardiomyopathy, and congestive heart failure4). myocardial ischemia may occur if there is only insignificant or absent coronary artery narrowing. based on lipton classifications1), the first division was made between the right - type (r) and left - type (l) according to the site of origin of sca. next, the artery was designated as group i, ii, or iii depending on its anatomical course group ii anomalies arise from the proximal part of the normal rca or lca, and cross the base of the heart before assuming the normal position of the inherent coronary artery. group iii describes the anomaly where the lad and lcx arise separately from the proximal part of the normal rca. according to this classification1), our patient was classified as l - i type, which is the presence of a markedly dominant lcx artery but the absence of a rca. there would be some published reports of an anomalous rca arising from the lad5,6), but the reported cases of an anomalous rca originating from the lcx are relatively less7,8,9,10). but, there are only a few cases in the pediatric and adolescent age group ; these anomalies are rarely suspected or identified during life and are usually first recognized at autopsy, largely because there is insufficient clinical suspicion as well as the difficulties implicit in routine examination or clinical testing for these malformations12). mostly, sca has a benign course, and it is known that sca does not interfere with coronary perfusion. but some reports stated that patients with sca could develop significant ischemic heart disease13), and the patients with sca who usually present with myocardial infarction have stenosis of the coronary artery. especially, the r ii - iii type and l ii - iii type anomalies, having a coronary artery branch following a dangerous course, may predispose to fatal myocardial ischemia. but there were some reports suggesting that the incidence of coronary artery bypass graft in patients with l - i type of anomaly such as the rca originating from the lcx was about 33%9). sca was incidentally detected on angiography while we were evaluating the patient for chest pain. our patient had abnormal ecg showing st - elevation in inferior leads and left precordial leads with reciprocal change in avr and elevated cardiac enzyme levels suggesting myocardial ischemia. the cause of myocardial ischemia was uncertain, because there was no stenosis and the result of treadmill test was negative. certain mechanisms could be considered, such as the coronary steal phenomenon due to the abnormal vessel or microvascular damage, and slow controlled ischemia caused by long travel distance of abnormal coronary artery7). there is a report of a case similar to our patient, in which there was no stenosis, but the authors thought that the patient had myocardial ischemia due to an abnormally slow coronary flow in the rca, because there was abnormally slow angiographic filling in the rca territory7). hence, they treated the patient with calcium channel blockers like diltiazem and nitrates, and the chest pain resolved after taking medications. the cause of chest pain can be considered to be coronary artery spasm - induced myocardial ischemia10). since we did not perform coronary artery spasm test such as the ergonovine test, the diagnosis of coronary artery spasm could not be definitely excluded. we advised her to avoid heavy exercise and to develop healthy eating habits to prevent atherosclerosis. also, we explained to the patient that she might need to take medications and to undergo a regular follow - up of symptoms. if a patient complains of chest pain due to sca, but does not exhibit significant stenotic lesions in the sca on coronary angiography like our patient, other tests should be considered. we have to consider performing stress tests such as single photon emission computed tomography, treadmill test whether or not myocardial and strategies for clinical identification of coronary artery anomalies are as follows : if clinical symptom such as exertional syncope or chest pain appears, physicians have to consider noninvasive examination like transthoracic or transesophageal echocardiography. and then, sonographer can not find coronary arteries from their usual coronary sinuses, we may suggest further examination such as coronary arteriography or computed tomography. we believe that this is the first report of a pediatric case of sca with rca originating from the lcx artery in korean population. therefore, although it is very rare, we can consider the diagnosis of coronary artery anomaly in pediatric patients with an unusual presentation of myocardial ischemia.	the presence of a single coronary artery is a rare congenital anomaly ; such patients often present with severe myocardial ischemia. we experienced the case of a 13-year - old girl with the right coronary artery originating from the left circumflex artery. she visited our emergency department owing to severe chest pain ; her cardiac enzyme levels were elevated, but her initial electrocardiogram (ecg) was normal. echocardiography showed normal anatomy and normal regional wall motion. when she presented with recurrent chest pain on admission, the ecg showed significant st - segment elevation in the left precordial leads and inferior leads with st - segment depression in avr lead, suggesting myocardial ischemia, and her cardiac enzyme levels were also elevated. we performed coronary angiography that showed a single right coronary artery originating from the left circumflex artery without stenosis. we confirmed the presence of a single coronary artery using coronary computed tomography. in addition, the treadmill test that was performed showed normal results. she was discharged from the hospital without any medications but with a recommendation of a regular follow - up.
hypertension (ht) is a common cardiovascular disease globally and it causes significant morbidity and mortality. ht affects about one - fifth of adult nigerians.[14 ] it is a risk factor for erectile dysfunction (ed) in men. many studies have reported a higher prevalence of ed in hypertensive induviduals compared with normotensive individuals.[57 ] ed is defined as the persistent inability to achieve and/or maintained penile erection sufficient for sexual intercourse. microalbuminuria (ma) is an important predictor of cardiovascular risk and complications in hypertensives.[810 ] ma has been described as a marker of generalized vascular damage. ht plays an important role in endothelial damage which may affect the nitric oxide production and/or release leading to ed.[1113 ] there are few nigerian studies on male ed and ht. the primary objective was to determine the prevalence of ma and ed in newly diagnosed hypertensive nigerians. in addition, we also evaluated the relations between ed and ma and some other cardiovascular risk factors in essential ht. the study was conducted at the cardiology clinic of a tertiary referral hospital in nigeria. the study was done in newly diagnosed hypertensive patients who were yet to commence antihypertensive drugs. the study protocol was approved by the ethics and research committee of the study center. all the participants gave informed consent and the procedures followed were consistent with institutional guidelines. individuals with diabetes mellitus, renal disease, endocrine disorder, heart failure, liver disease, prostatic enlargement, psychiatric illness, obesity, and proteinuria were excluded from the study. a standardized protocol was followed, in which systolic blood pressure (sbp) and diastolic blood pressures (dbp) were measured on the left arm after the participants had been seated for at least 5 min. three measurements were done at least 5 min apart and the mean value was used for the study. ht was defined as sbp 140 mmhg and/or dbp 90 mmhg, or use of antihypertensive drugs. general as well as specific examination of the external genitals for local abnormalities and digital rectal examination for prostatic enlargement were conducted. venous blood samples were collected and analyzed for fasting plasma glucose (fpg), serum total cholesterol (tc), and serum triglyceride (tg). resting 12-lead electrocardiogram (ecg) of all the patients and controls were recorded using schiller at-1 machine at a sensitivity of 10 mm / mv and a paper speed of 25 mm / sec. ecg left ventricular hypertrophy (lvh) was diagnosed based on standard ecg criteria.[1416 ] ed was evaluated using a standardized questionnaire of the international index of erectile function (iief).[17195 ] we used an inform - then - probe approach, whereby the participants were generously assured that their ed (if present) was not an uncommon condition. the iief has been proved to be a reliable and widely used test to define sexual function. ed is graded as severe (6 - 10 points), moderate (11 - 16 points), mild (17 - 25 points), and none (26 - 30 points). ma was determined using the micra test strips (boehringer mannheim, mannheim, germany). this dipstick has been found to be a fast, accurate, and relatively, the cheapest way to screen patients for the presence of ma. there are four color blocks on the test strip corresponding to negative (or 0), 20, 50, and 100 mg / l of albumin. the test was done on three consecutive first morning - voided urine samples collected at 3 weeks intervals. ma was considered to be present when two of the three urine samples tested produce a reaction color corresponding to 20 mg / l or more. statistical analyses were done using the student 's t - test and chi?square to compare means and proportions respectively. statistical analyses were done using the student 's t - test and chi?square to compare means and proportions respectively. the mean age of the patients and the controls was 53.8 5.6 and 51.2 7.1 respectively. ed was found in 32.1% of hypertensive patients and 16% of normotensive controls and the difference was statistically significant (< 0.001). among the hypertensive patients, there was a significant difference between the mean age of those with ed and those without ed (54.2 8.5 vs. 49.2 6.8, p = 0.02). the prevalence of ma was significantly higher in patients with ed (65.4%) than in those without ed (23.6%) (p < 0.001). hypertensive patients with ed were significantly more likely to have ecg lvh than their counterparts without ed (p < 0.001). the other characteristics and features of hypertensive patients with ed compared with hypertensive patients without ed are shown in table 2. when hypertensive patients with ed are divided into two subsets based on the presence or otherwise of ma, and then compared, the results obtained are shown in table 3 : the means of sbp (p = 0.04), dbp (p = 0.02), ecg lvh (p < 0.001), serum tc (p = 0.1), serum ldl - c (p = 0.001) were significantly higher in those with ma than their counterparts without ma. multiple regression analysis showed that ed was independently associated with ma, ecg lvh, age, dbp, and cigarette smoking. characteristics of hypertensive patients and the controls characteristics of hypertensive patients with and without erectile dysfunction characteristics of hypertensive patients with erectile dysfunction with and without microalbuminuria our study demonstrated a higher prevalence of ed in hypertensive patients than in normotensive individuals (32.1% vs. 16.0%). for instance, aranda. reported a prevalence of ed among hypertensive patients of 46% in spain where patients with diabetes mellitus were included in the study. in this study, thus, the difference in prevalence may be due to study methodology including the selection of patients. hypertensive patients with ed have significantly higher cardiovascular risk factors when compared to their counterparts without ed and to the normotensive controls. ma was found in 37% of the hypertensive patients as against 6.7% in the controls. this finding is higher than that of akinsola. who reported 17.4% prevalence of ma in ht. the difference may be attributed to patient selection as we recruited hypertensive patients who had never been treated previously for the study. treatment of hbp with antihypertensive medications, particularly angiotensin - converting - enzyme (ace) inhibitors, has been found to improve ma. in our study, ma, a cardiovascular risk factor and marker of endothelial damage, is also significantly more common in hypertensives with ed than in hypertensives without ed. that is, patients with ed are more likely to have endothelial damage and dysfunction than their counterparts without ed. the study also revealed that ed may also be related to cardiac damage as significantly more hypertensive patients with ed have ecg lvh. ed is often a sentinel manifestation of damage to the vascular endothelium.[2830 ] the main link between ed and cardiovascular disease is the vascular endothelium, which has a fundamental role in the regulation of circulation. thus, once the ability to generate nitric oxide has been compromised, ed may ensue. the study shows that mal and ed might represent different variables with a common pathological process such as vascular damage. this hypothesis is consistent with the finding of pedrinelli. who reported that ma was strongly associated with c - reactive protein (crp) which is an evidence of subclinical inflammation. crp is also a cardiovascular risk factor and predicts cardiovascular prognosis independent of conventional risk factors. thus, vascular damage with possible atherosclerosis and the resultant inadequate blood flow to the erectile tissue might cause ed. there are conflicting reports on the role of cigarette smoking in the etio - pathogenesis of ed. moreira. showed a 2.5-fold increased risk of ed among smokers in contrast to the study by doumas. and the massachusetts male aging study where smoking was found not to be associated with ed. this notwithstanding, long - term cigarette smoking is a major risk factor for vasculogenic ed because of its effects on the vascular endothelium. it also causes nicotine - induced vasoconstriction of the cavernous smooth muscle. in our study, patients with ed were significantly more likely to be cigarette smokers than those without ed. the effect of alcohol on penile erection depends on the volume and the duration of consumption. although small quantities of alcohol may improve erection and enhance libido, large amounts result in central sedation, depression of libido, and ed. however, one of the limitations of the study is that the alcohol ingestion was not quantified. the study reveals that both ed and ma are common in adult male nigerians with ht. it also points out that male ed in hypertensive patients is associated with ma and the subsets with either or both have increased risk of cardiovascular disease. cigarette smoking, a major risk factor for atherosclerosis and cardiovascular disease, is also associated with ed. with a somewhat common pathological process of vascular damage between ed and ma, it could be expected that antihypertensive drugs, particularly ace - inhibitors and angiotensin - receptor blockers, that improve ma might have some impact on ed. this hypothesis is yet to be proven and may become a focus of research in the future.	background : microalbuminuria has been described as a marker of generalized vascular damage.aims:the aim of the present study was to determine the prevalence of erectile dysfunction (ed) and microalbuminuria in adult male nigerians with newly diagnosed hypertension. we also evaluated the relations between ed and microalbuminuria, electrocardiographic left ventricular hypertrophy, serum lipids, and cigarette smoking.materials and methods : a total of 81 male adult nigerians with newly diagnosed hypertension were recruited into the study. there were also 75 age- and sex - matched healthy normotensive controls. ed was evaluated using a standardized questionnaire of the international index of erectile function and microalbuminuria was determined using the micra test strips (boehringer manneheim gmbh, mannheim, germany).results : eighty - one hypertensive patients and 75 normotensive controls were studied. mean age of the patients and the controls was 53.8 5.6 and 51.2 7.1 respectively. ed was found in 32.1% of the hypertensive patients and 16% of normotensive controls (p < 0.001). the prevalence of microalbuminuria was significantly higher in patients with ed than in those without it (65.4% vs. 23.6%, p < 0.0001).conclusion : the study shows that ed and microalbuminuria are common in male adult nigerians with hypertension. it also demonstrates that male ed is associated with an increased risk of cardiovascular disease.
even though the quantity of brain tissue accounts for only 23% of the total body weight, it requires 2025% of the total oxygen supply. because of the limited oxygen and glucose reserves, brain tissue is susceptible to hypoxic - ischemic injury young and middle - aged patients are a high - risk population for traumatic brain injury, and cognitive impairment is one of the most common symptoms of patients with moderate and severe brain injuries. further, hypoxic - ischemic brain injury may seriously affect quality of life and prognosis. at present, hyperbaric oxygen (hbo) is a historical therapeutic option in the treatment of various types of brain damage. hbo functions as follows in the treatment of brain injuries : (1) by correcting local brain tissue hypoxia, the oxygen partial pressure increases nearly 20-fold and strengthens the ability of oxygen to infiltrate local brain tissue ; (2) hbo can improve the oxygen supply and blood supply in the brainstem reticular system and induce the excitability of brain cells, which plays a role in the promotion of consciousness ; (3) by eliminating cerebral edema and protecting brain cells ; and (4) by promoting the recovery of neural functionality and reducing sequelae [14 ]. all procedures were in accordance with the chinese capital medical university animal welfare guidelines and approved by the institutional animal care and use committee of the chinese capital medical university. seventy clean and healthy male sprague dawley (sd) rats (3 months old ; 275 + 25 g) were purchased from the laboratory animal center of the academy of military medical sciences (beijing, china). the rearing conditions were as follows : temperature, 211c ; light / dark cycle,12 h/12 h ; illumination time, 19:007:00 ; sufficient moisture and food were provided daily pre - and post - operatively ; and there was an 8-hour fast before surgery. the sd rats were randomly divided into control (n=10) and intervention (n=60) groups. randomized sd rats in the intervention groups had hbo treatment for 1, 3, and 5 days and 1, 2, and 4 weeks (n=10 in each group), while routine breeding resumed in the control group. described by dixon, the models were established by ecci6.3. abdominal anesthesia with 10% chloral hydrate (0.3 ml/100 g) was prepared for bilateral frontal craniotomy surgery. craniotomies were performed 3 mm from the center line between the anterior fontanelle and lambdoid suture. the rats in the intervention group were put into a small high - pressured oxygen cabin for animals (hong yuan gy3200,yantai, china).the cabin was washed with pure oxygen for 10 min, then a uniform pressurization was initiated to achieve a 0.2 mpa cabin pressure in 10 min and maintained for 30 min. hbo therapy was performed four times during the day, each 30 min apart for 7 days continuously. the water temperature was controlled at 281c melan was added to the water to shield the platform underwater, and to reduce the influence of positioning. (1) navigation experiment : the platform was put in 1 quadrant 2 cm beneath the surface. when heading towards the wall, the rats were randomly put into the other three quadrants without a platform in daily experiments. if the rat could not locate the hidden platform in 60 s, the rat would be guided to the platform and remain for 10 s continuously for 3 days. four sessions of training were necessary every day for each rat with intervals of 1520 min. (2) space exploration experiment : the day after the navigation experiment, the rats were put into the water from the contralateral quadrant of platform one. the percentage of time spent locating the quadrant with the platform in the total time would be recorded in 60 s. the water maze baseline was measured in all rats 1 day before modeling. the percentage of residence time during escape latency, in the target quadrant, and the total time were recorded for the water maze test 8 weeks after controlled cortical impact (cci). after the behavioral test, the rats were sacrificed and the brain tissues were removed. the brain tissues were placed on ice ; the hippocampuses were isolated and frozen in liquid nitrogen. three photographs magnified 200 times were randomly selected from each slice and from each group. brain tissues should fill the entire field of vision when taking photos to ensure the background light is consistent in each photo. image - pro plus 6.0 software was used to select the same degree of tan as a uniform standard to assign positive results in all photos, and the integrated optical density (iod) was analyzed and concluded in all photos. count data are denoted by absolute values and percentages, while measurement data are recorded as the mean standard deviation. the results of the water maze test were determined with repetitive measurement deviation analysis, and the measurement data were compared with single factor analysis of variance by the least significant difference (lsd) test post hoc. single factor variance analysis was applied in analysis of time spent in the targeted quadrant in the spatial probe test between each group, and the lsd test was applied in comparisons between each group. all procedures were in accordance with the chinese capital medical university animal welfare guidelines and approved by the institutional animal care and use committee of the chinese capital medical university. seventy clean and healthy male sprague dawley (sd) rats (3 months old ; 275 + 25 g) were purchased from the laboratory animal center of the academy of military medical sciences (beijing, china). the rearing conditions were as follows : temperature, 211c ; light / dark cycle,12 h/12 h ; illumination time, 19:007:00 ; sufficient moisture and food were provided daily pre - and post - operatively ; and there was an 8-hour fast before surgery. the sd rats were randomly divided into control (n=10) and intervention (n=60) groups. randomized sd rats in the intervention groups had hbo treatment for 1, 3, and 5 days and 1, 2, and 4 weeks (n=10 in each group), while routine breeding resumed in the control group. described by dixon, the models were established by ecci6.3. abdominal anesthesia with 10% chloral hydrate (0.3 ml/100 g) was prepared for bilateral frontal craniotomy surgery. craniotomies were performed 3 mm from the center line between the anterior fontanelle and lambdoid suture. the rats in the intervention group were put into a small high - pressured oxygen cabin for animals (hong yuan gy3200,yantai, china).the cabin was washed with pure oxygen for 10 min, then a uniform pressurization was initiated to achieve a 0.2 mpa cabin pressure in 10 min and maintained for 30 min. hbo therapy was performed four times during the day, each 30 min apart for 7 days continuously. the water temperature was controlled at 281c melan was added to the water to shield the platform underwater, and to reduce the influence of positioning. (1) navigation experiment : the platform was put in 1 quadrant 2 cm beneath the surface. when heading towards the wall, the rats were randomly put into the other three quadrants without a platform in daily experiments. if the rat could not locate the hidden platform in 60 s, the rat would be guided to the platform and remain for 10 s continuously for 3 days. four sessions of training were necessary every day for each rat with intervals of 1520 min. (2) space exploration experiment : the day after the navigation experiment, the rats were put into the water from the contralateral quadrant of platform one. the percentage of time spent locating the quadrant with the platform in the total time would be recorded in 60 s. the water maze baseline was measured in all rats 1 day before modeling. the percentage of residence time during escape latency, in the target quadrant, and the total time were recorded for the water maze test 8 weeks after controlled cortical impact (cci). after the behavioral test, the rats were sacrificed and the brain tissues were removed. the brain tissues were placed on ice ; the hippocampuses were isolated and frozen in liquid nitrogen. three photographs magnified 200 times were randomly selected from each slice and from each group. brain tissues should fill the entire field of vision when taking photos to ensure the background light is consistent in each photo. image - pro plus 6.0 software was used to select the same degree of tan as a uniform standard to assign positive results in all photos, and the integrated optical density (iod) was analyzed and concluded in all photos. count data are denoted by absolute values and percentages, while measurement data are recorded as the mean standard deviation. the results of the water maze test were determined with repetitive measurement deviation analysis, and the measurement data were compared with single factor analysis of variance by the least significant difference (lsd) test post hoc. single factor variance analysis was applied in analysis of time spent in the targeted quadrant in the spatial probe test between each group, and the lsd test was applied in comparisons between each group. spatial learning ability was reflected by changes of escape latency in the water maze test. eight weeks later, no statistical differences (p>0.05) existed in the 3- and 5-day groups when compared with baseline. the other groups were statistically different based on auto - comparison (p0.05) existed in the 5-day and 1-week groups when compared with baseline. the other groups were statistically different based on auto - comparison (p0.05) was observed in the level of expression of gap-43 and syn in the 1-day group compared with the control group. when comparing the remaining groups and the control group, there were statistically significant differences (p 2 weeks after hypoxic - ischemic brain injury, the improvement in cognitive function was less than that observed with earlier treatment, while the effect on neural remodeling was still evident.	backgroundhyperbaric oxygen (hbo) is a historical therapeutic option in the treatment of various types of brain damage. at present, clinical treatment of hypoxic - ischemic injury is giving priority to cognitive training. the effects of hbo on cognitive dysfunction were observed in a controlled cortical impact (cci) rat model.material/methodsseventy male sd rats were randomly divided into control (n=10) and intervention (n=60) groups. all rats underwent baseline water maze testing 1 day before modeling, and were retested 8 weeks after modeling. the percentage of residence time during escape latency in the target quadrant and the total time were recorded. data were analyzed by spss 16.0 software. p0.05) existed in spatial learning ability in the 3-day and 5-day groups when compared with baseline. the other groups were statistically different by auto - comparison (p0.05) in spatial memory existed in the 5-day and 1-week groups when compared with baseline, while a significant difference was noted in the other groups by self - comparison (p0.05) was noted in the level of expression of growth - associated protein-43 (gap-43) and synaptophysin (syn) in the 1-day group compared with the control group. the remaining groups and the control group were statistically different (p<0.05), while the level of expression of gap-43 and syn in the 5-day, 1-week, and 2-week groups was significantly different compared with that in the control group (p<0.01).conclusionsif hbo therapy was provided 57 days after craniocerebral trauma, there was apparent improvement in cognitive function and neuroplasticity.
solitary fibrous tumors (sfts) are rare mesenchymal neoplasms of fibroblastic origin, most commonly found in the pleura. in fact, they are ubiquitous neoplasms, and non - serosal extrathoracic locations have been reported during the last 2 decades, including intra - abdominal cases. given the rarity of these tumors, misdiagnosis is frequent, and information regarding prognosis and treatment is lacking. herein, we report the first case of sft developing in the round ligament of the liver. in august 2010, a 46-year - old caucasian man presented with a 12-month history of abdominal pain. he had no specific medical history, personal or familial. the initial ultrasonography (us) scan, performed 1 year earlier, was interpreted as normal. on self - examination a ct scan showed a solid and inhomogeneous mass measuring 182 66 mm, located under the abdominal wall, causing displacement of the left and right hepatic lobes but distinct from them (fig. iodine injection showed heterogeneous contrast uptake, with a fat interface between the tumor and intra- and retroperitoneal elements (fig. histological examination showed a proliferation of nonatypical round to spindle - shaped cells without mitoses, admixed with collagen. immunohistochemistry (ihc) showed no expression of epithelial, smooth muscle or schwann cell markers by tumor cells but solely expressed cd34. a diagnosis of gastrointestinal stromal tumor (gist) was ruled out by ihc (no expression of cd117 or dog1) and molecular biology (no kit or pdgfra mutation), and the diagnosis of desmoid tumor was made. second - line treatment based on imatinib (400 mg daily) was started. despite 3 months of treatment, the initial biopsy was reexamined and regarded as suspicious for low - grade sarcoma or sft. mri was performed : on t2-weighted sequences, the tumor had a higher signal than the muscle (fig. 2a) ; gadolinium injection was associated with gradual and heterogeneous contrast enhancement on t1-weighted sequences, evoking fibrosis (fig. 2b), but diffusion mri showed important cellularity that was highly suspicious for a malignant process (fig. 2c). laparotomy found a huge and well - limited tumor that, unexpectedly, was appended to the round ligament of the liver (fig. the tumor was free from any other intra - abdominal contact, especially from the liver parenchyma, and was easily removed without any further dissection. the tumor measured 210 70 mm and weighed 1,990 g. after formalin fixation, it showed a fleshy appearance with gray - white color on cut section and was centered on and appended to the round ligament (fig. unlike the microbiopsy, the histological examination of the surgical specimen showed many more characteristic areas of small nonatypical spindle cells scattered in a fibrous stroma without any clear architecture (fig. 3e). ihc analysis confirmed strong expression of cd34 by tumor cells and, above all, highlighted very characteristic ramified vessels, so - called 3f). there was no expression of epithelial (pancytokeratin, ema), smooth muscle (-smooth actin), and schwann cell (ps100) markers. mitotic count was very low, less than 1 mitosis per 10 high - power fields (hpf). a regular clinical and radiological monitoring was set up. at the 10-month follow - up, the patient was alive with excellent ps, without any signs or symptoms of relapse. in the past 2 decades, sfts have been identified in the abdominopelvic cavity [2, 3 ]. regarding the liver, fewer than 30 cases have been reported in the english literature [4, 5, 6 ]. to our knowledge, our case represents the first case of sft arising from the round ligament of the liver. the round ligament, or ligamentum teres, is a degenerative string of tissue stemming from the umbilical vein involution and that anatomically divides the left lobe of the liver from the medial and lateral sections. most of them are metastases of gastrointestinal cancers (in particular, mucinous appendiceal neoplasm), peritoneal mesothelioma, and more rarely of breast cancer and hepatocarcinoma [8, 9 ]. cases of primary mesenchymal tumors have also been reported, including sarcomas (malignant fibrous histiocytoma, leiomyosarcoma), and benign tumors such as lipomas, lymphangiomas, stromal tumors and pecomas. sfts of the round ligament of the liver have not been reported so far. given they often manifest as large, asymptomatic, slow - growing tumors, and their clinical presentation has no specificity (pain, abdominal mass). in our case, it is likely that the tumor had been evolving slowly for several years before the onset of abdominal pain, and that the us scan was falsely negative because of similar echogenicity between the tumor and the perihepatic tissues. these tumors demonstrate remarkable radiological heterogeneity, with variable degrees of enhancement, necrosis, or hemorrhage. ct is the initial investigative modality of choice for the detection of sfts, and is better than a us scan, as exemplified in our case. typically, the tumor appears as a well - circumscribed hypervascular mass that may displace or exert pressure effects on neighboring organs. at mri, it typically displays intermediate signal intensity on t1-weighted images, heterogeneous low - signal intensity with flow voids on t2-weighted images, and intense enhancement after administration of gadolinium. in our case, these features existed, but diffusion mri was falsely alarming. on presurgical imaging, the large size of the tumor did not allow for the definition of its precise origin or the prediction of a simple surgical removal. the origin in the round ligament was obvious during the laparotomy only ; however, a postoperative re - reading of the initial ct and mri scans showed that the tumor had infiltrated the umbilical fissure. the positive diagnosis of sfts is histological, while keeping in mind the frequent difficulty to precisely characterize a mesenchymal neoplasm on small biopsies, as illustrated by our case. analysis shows a proliferation of rounded to fusiform cells arranged without any well - defined architecture (so - called appearance can be very different from one area to another depending on the relative proportion of cells and stroma. ihc shows strong expression of cd34 by tumor cells and importantly helps to rule out numerous differential diagnoses, namely smooth muscle tumors, desmoid tumors, tumors of peripheral nerves, gist, spindle cell sarcomas, notably dedifferentiated liposarcomas and synovial sarcomas. as with our patient 's tumor, molecular biology can also help to rule out some differential diagnoses, mainly gists. regarding their prognosis, extrapleural sfts have a classically more indolent behavior than pleural sfts and are most always defined as benign. however, their clinical outcome is unpredictable, and some of them can display histological aggressiveness criteria (size > 5 cm, mitotic count > 4 per 10 hpf, high cellularity, necrosis, hemorrhage, cytological atypia and infiltrative growth pattern) or present local invasiveness or local or metastatic relapses [3, 13 ]. like in pleural locations, the presence of aggressive features does not always forecast an unfavorable clinical outcome, and half of cases with a malignant lesion (defined by the presence of high cellularity and/or high mitotic count and/or pleomorphism) are cured by simple excision. for many authors, the complete resection of the tumor is the most important prognostic factor. in our case, and thanks to its fortunate origin in the round ligament, the tumor was easily and completely removed, and the only pejorative criterion was its large size. surgical excision is the treatment of choice for sfts, with a 5-year survival rate close to 100% after complete excision. systemic chemotherapy and radiotherapy have been tried, but with variable success rates. in unresectable sfts, antiangiogenic therapy is associated with promising results. as the evolution of extrapleural sfts is unpredictable this is the first report of sft arising from the round ligament of the liver described in the literature. even though rare, this diagnosis should be kept in mind to allow early diagnosis and complete surgical resection, which provide the best chance for recovery.	solitary fibrous tumors (sfts) are mesenchymal neoplasms of fibroblastic origin, most commonly found in the pleura. numerous extrathoracic locations have been reported during the last 2 decades. herein, we report the first case of an sft in the round ligament of the liver. a 46-year - old caucasian man presented with a 12-month history of abdominal pain. an ultrasonography - guided microbiopsy first revealed a desmoid tumor. after failure of first- and second - line medical treatments (celecoxib and tamoxifen, then imatinib), histological reexamination was suspicious for a low - grade sarcoma. mri was also suspicious for a malignant process. hence, surgery was decided. laparotomy found a huge and well - limited tumor that, unexpectedly, was appended to the round ligament of the liver and free from any other intra - abdominal contact. the tumor was easily removed. excision was monobloc and macroscopically complete. histological analysis diagnosed an sft arising from the round ligament of the liver. no adjuvant treatment was given. ten months after surgery, the patient is alive without any signs or symptoms of relapse. this is the first report of sft arising from the round ligament of the liver. it illustrates the difficulty in diagnosing such tumors. whilst diagnosis of sft is rare, it should be kept in mind to allow early diagnosis and complete surgical resection, which provide the best chance for recovery.
plasmacytoid urothelial carcinoma is a rare malignant neoplasm in the urinary bladder recognized by recent who classification of urothelial carcinoma. it is an aggressive variant associated with poor prognosis that presents at an advanced clinical stage. limited data is available about the pathological, immunohistochemical characteristic as well as clinical behavior of this rare variant. morphological distinction from other malignant neoplasms with plasmacytoid phenotype is critical for its clinical management. treatment remains a challenge because of late presentation of the disease and presence of metatstasis at the time of initial work up.[27 ] we present here a case of 54-year - old male patient who was referred to our hospital for diagnostic work - up of bladder cancer. ct scan of abdomen and pelvis showed thickened urinary bladder wall with irregularity of the mucosa involving entire bladder. radical cystectomy was done which on gross examination showed a grey white firm to hard, ill - circumscribed ulcerative tumor involving almost the entire bladder measuring 2 cms in thickness. microscopic examination showed a high - grade tumor composed of discohesive plasmacytoid cells replacing the lamina propria [figures 1 and 2 ]. the tumor cells were seen extending from the mucosal aspect and invaded through the detrusor muscle to invade the perivesical fat and the serosa [figure 3 ]. the tumor cells were seen extending along the adventitia of both the ureters even up to the surgical cut margins. on immunohistochemistry the tumor cells were positive for ck, ck 7 [figure 4 ] and cd 138 ; while they were negative for lca, cd20, and light chains. plasmacytoid urothelial carcinoma : the tumor cells are seen undermining the normal urothelium with retraction spaces around them. immunohistochemical staining with antibody to ck7 marks the tumor cells as well the overlying urothelium thereby confirming their epithelial origin. the patient is on regular follow - up and is doing fine for last 6 months without any evidence of recurrence or metastasis elsewhere in the body. recent years have witnessed the illustrations and documentation of several morphological variants of urothelial carcinomas, which have significance from therapeutic and prognostic perspective. the plasmacytoid variant is an unusual variant that has been recognized in the latest who classification of urothelial carcinomas. earliest description of this variant came from the case reported by sahin, in 1991 and since then, not more than 40 cases have been reported,[26 ] there is very limited data available about the pathological, immunohistochemical characteristic as well as clinical behavior of this rare variant. most common presenting symptom is hematuria.[48 ] histopathologically, plasmacytoid urothelial carcinoma is characterized by plasmacytoid tumor cells arranged in cords and single - file pattern, small nests, solid sheet like growth or diffuse discohesive pattern - less architecture. the nuclei vary from hyperchromatic to vesicular with evenly distributed chromatin and variability in the prominence of nucleoli. prominent retraction artifact around each tumor cell may also be seen. in the present case the plasmacytoid urothelial carcinoma can coexist with non - invasive papillary urothelial carcinoma, urothelial carcinoma in situ or invasive high - grade urothelial carcinoma. however, in our case there were no areas of either papillary or in situ urothelial malignancy. plasmacytoid appearance of this tumor can lead to diagnostic dilemma, especially in smaller biopsies. differential diagnosis ranges from benign conditions like chronic cystitis with prominent plasma cell infiltrate to malignant tumors like plasmacytoma and signet ring carcinoma. in our case some cells also had focal signet ring - like appearance which led to confusion with signet ring cell carcinoma. however, the nuclei were eccentric rather then being peripherally compressed as in true signet ring cell carcinoma. metastatic carcinoma from breast or stomach, malignant melanoma and rhabdomyosarcoma can have striking plasmacytoid morphology and can cause considerable diagnostic difficulties. in such situations, it is imperative to employ a panel of antibodies for confirmation of diagnosis. immunohistochemical studies have shown that plasmacytoid urothelial carcinoma cells to be positive for ck-7, ck-20, ck, ae1/ae3, ema and cd-138, but negative for lca, s 100, hmb 45,, and cd 79-, as was seen in our case also. because of late presentation, biopsy usually reveals a high - grade tumor infiltrating the lamina propria and muscularis, with high potential of metastasis. some reports have shown good response to neoadjuvant chemotherapy, but this aspect needs further evaluation. our patient also had high - stage disease (pt3) and underwent radical cystectomy with ileal conduit. on final histopathology, the tumor cells were seen extending along the ureteric adventitia till the surgical resection margins. prognosis is extremely poor with an aggressive behavior of this variant. in the case series by nigwekar, no patient with greater than 1-year another report by lopez - beltran, showed that within 2 years of follow - up, all patients either died of cancer or had metastasis. in our case the patient is free of disease since last 6 months after surgery. in conclusion, plasmacytoid variant of urothelial carcinoma is a rare and important variant form diagnostic, therapeutic and prognostic point of view. it can be mistaken for other pathologies of bladder having plasmacytoid features and should be differentiated from them because of the difference in the therapeutic approach.	plasmacytoid urothelial carcinoma is an uncommon and aggressive variant of urothelial carcinoma associated with late presentation and poor prognosis. we discuss here the first reported case from india of a 54-year - old male who presented with hematuria. cystoscopy showed edematous and ulcerated mucosa throughout the bladder. a transurethral biopsy revealed urothelial carcinoma with plasmacytoid appearance. he underwent a radial cystectomy which on histopathology showed plasmacytoid urothelial carcinoma of the bladder of high stage with involvement up to bladder serosa and adventitial walls of the ureter. the diagnostic dilemmas of this unusual variant of urothelial malignancy and its clinical impact are discussed.
fabry disease (fd ; omim 301500) is a rare x - linked lysosomal storage disorder caused by the deficiency of the lysosomal enzyme -galactosidase a (-gal a ; ec 3.2.1.22) which is encoded by the gla gene (omim 300644, refseq nm_000169.2). phenotypic expression of fd is highly variable, ranging from a milder phenotype with cardiac and/or renal abnormalities, to classic fd, featuring, in addition to cardiac vascular degeneration, chronic pain, angiokeratoma, and kidney manifestations usually leading to renal failure. although fd is x - linked, heterozygous females may develop mild to severe clinical manifestations. the gla gene transcribes two alternatively spliced mrnas of which the most abundant (to which we refer as the -gal a transcript) encodes the lysosomal -gal a. the minor mrna (to which we refer as the glains57) accounts for about 5% of total gla transcripts. glains57 retains a 57-nucleotide - long cryptic exon from intron 4 and is predicted to code for a shorter protein of unknown function. (g.9273c > t or ivs4 + 861c > t) and nm_000169.2:c.639 + 919g > a (g.9331g > a or ivs4 + 919g > a) gla deep intronic mutations has been reported to cause fd. the c.639 + 861c > t mutation was associated with the classic fd phenotype in an italian family. the c.639 + 919g > a mutation is predominantly associated with late - onset fd and is highly prevalent in taiwanese newborns (1 in 1,600 males ; approximately 8286% of positives). the therapeutic skipping of specific exons (exon skipping) can be obtained by antisense oligonucleotides (aons) annealing with splice junctions or exonic splicing enhancers (eses). aons have been used against several genes associated with disease to restore correct splicing or the open reading frame through selective removal of the mutated exon. this strategy has been used in the treatment of duchenne muscular dystrophy (dmd). aons have also been successfully applied to the in vitro correction of aberrant splicing events caused by mutations which activated cryptic splice sites in several genetic diseases, such as leber congenital amaurosis, propionic and methylmalonic acidemia, ataxia telangiectasia, congenital disorders of glycosylation, niemann - pick disease type c, neurofibromatosis type 1 and 2, megalencephalic leukoencephalopathy with subcortical cysts type 1 or pelizaeus - merzbacher disease (reviewed in refs. it is vital that antisense molecules, when delivered to the cells, accumulate in the nucleoplasmic compartment where mrna is synthesized and spliced. to improve localization efficacy, the antisense sequences can be inserted into one of the nuclear u small nuclear rnas (usnrna). the advantage of using antisense - rnas (asrnas) as therapeutic agents is that they are very specific and not immunogenic ; as yet no undesired activity for asrnas has been reported. the therapeutic benefits of aons are time - limited, and repeated administration is necessary. the use of asrnas offers a major advantage as they can be delivered in vivo within expression cassettes as in conventional gene replacement therapies. it is possible to transduce or transfect target cells using viral or nonviral delivery systems, leading to the production of asrnas exploiting endogenous transcription. at present, two classes of usnrnas have been successfully modified to modulate splicing : u1 snrna and u7 snrna. u1 snrna recognizes the 5 ' splice site and mediates the first step of spliceosome assembly, while u7 snrna has a role in the processing of the 3 ' end of histone mrna. u1snrna promoter has been shown to be useful for the stable expression of antisense molecules and other therapeutic rnas. eight nucleotides at the single - stranded 5 ' terminus of u1 can be replaced by unrelated sequences with up to 50 nucleotides, without affecting either stability or the ability to assemble into snrnp particles. the u1snrna and u7snrna expression cassettes are small (about 600 bp), they work efficiently both in in vitro systems and in animals and can be delivered as part of lentiviral or aav vectors. we focus on the development of antisense u1snrnas (u1asrnas), designed to restore -gal a levels in the c.639 + 861c > t and c.639 + 919g > a gla deep intronic mutations. three different antisense constructs were tested for exon skipping activity in cos-1 cells transfected with gla minigenes. u1asrnas constructs were designed to target both the 3 ' and the 5 ' splice site of the gla cryptic exon and differed by one nucleotide corresponding to the position complementary to the nucleotide mutated in the gla pre - mrna. our results indicate that a single u1asrna molecule can be used to correct different mutations. we and other authors have shown that the gla gene transcribes two alternatively spliced mrnas : the major gla mrna -gal a transcript and the minor transcript glains57. gla deep intronic mutations c.639 + 861c > t and c.639 + 919g > a cause fd by increasing the inclusion of the cryptic exon to around 85 or 70% of the total transcripts. to replicate the splicing process of the cryptic exon, we constructed a minigene in which the entire 1,718-bp - long sequence of gla intron 4 was inserted into gla cdna between exons 4 and 5 (figure 1a). this wild - type splicing reporter minigene, which we named wt minigene, carries a sv40 early region polyadenylation site and expresses the gla mrna under the transcriptional control of the cmv promoter (figure 1a). upon transfection in cos-1 cells, the wt minigene produced a shorter, more abundant mrna, and a longer mrna, as assessed by semiquantitative reverse transcriptase - pcr (pcr) and by real - time pcr (figure 2a and table 1). we confirmed both amplicons by direct sequencing : the shorter band corresponded to the -gal a mrna, while the longer to the glains57 mrna harboring the inclusion of the 57 nt cryptic exon, as expected. we subsequently constructed the splicing reporter minigenes for the c.639 + 861c > t and c.639 + 919g > a intronic mutations which we named c.639 + 861c > t minigene and c.639 + 919g > a minigene, respectively. when transfected into cos-1 cells, the glains57/total glamrnas ratio was 40% for wt minigene, as revealed by quantitative real - time pcr. this ratio markedly increased for mutated minigenes : 95% for the c.639 + 861c > t minigene and 88% for the c.639 + 919g > a minigene (table 1). such data confirm that c.639 + 861c > t and c.639 + 919g > a mutations unbalance the expression of the gla mrnas causing an increment in the production of the gla alternatively spliced mrna. our real - time pcr probes are specific for the sequence of human gla transcripts and did not yield any amplification when tested on nontransfected cos-1 cells (data not shown). therefore, our quantitative data are not affected by the endogenous gla expression of cos-1 cells. in order to induce skipping of the cryptic exon, we designed and produced four antisense constructs, in the backbone of the u1 snrna, and named them u1-glawt, u1-gla3 t, u1-gla5a, and u1-glascramble. nucleotides from position 310 at the 5-end of u1 snrna, required for the recognition of the 5 splice site, were substituted with 48-nt - long antisense sequences complementary to both cryptic 3 ' and 5 ' splice sites (figure 1a, b). the use of double - target u1asrnas was dictated by indications in the literature demonstrating that efficient exon skipping is obtained with antisense u7 snrnas and antisense u1snrnas targeting at the same time two distinct splicing - regulating regions. the chimeric u1-antisense sequences were cloned under the control of the strong rna polymerase ii u1 snrna gene promoter and termination sequences and subsequently inserted into the paav2.1-cmv - egfp plasmid. construct u1-glawt produces a u1asrna complementary to the cryptic splice sites of the wild - type gla gene (figure 1b), while constructs u1-gla3 t and u1-gla5a differ from u1-glawt each by one nucleotide, being perfectly complementary to the 3 ' splice site of mutation c.639 + 861c > t and to the 5 ' splice site of mutation c.639 + 919g > a, respectively. as a negative control, we also produced the u1-glascramble construct that bears a 23-nt - long sequence (figure 1b) with no known target in mammalian cells. u1-gla5a, u1-gla3 t, and u1-glawt antisense constructs did not induce any significant variation on the gla mrna expression level when cotransfected with the wild - type minigene (figure 2a). cotransfection analysis of u1-gla3 t and u1-glawt partially restored the expression of the -gal a mrna in the presence of the c.639 + 861c > t mutation (figure 2b) and u1-gla5a and u1-glawt corrected, albeit not completely, the c.639 + 919g > a mutation (figure 2c). these results indicate that the u1-glawt construct induces skipping of the cryptic exon in both c.639 + 861c > t and c.639 + 919g > a gla mutations with a reduction of the glains57 mrna of 66% for the c.639 + 861c > t and 20% for the c.639 + 919g > a, relative to cells transfected with the minigene alone (figure 2). the control u1-glascramble did not cause any considerable alteration to the expression profile of any minigene, as expected (figure 2). because the different antisense constructs considerably extend the length of the u1 snrna (by 40 nucleotides), we checked the integrity of the u1 antisense rnas and compared their levels in cotransfected cos-1 cells. we performed semiquantitative rt - pcr analyses with a primer annealing to the 5 ' variable tail and a primer annealing to a common portion of the u1 snrna (figure 2d). a specific amplification product of the expected size (around 100 bp) was obtained exclusively in the cells transfected with one of the u1asrna constructs, confirming that they were produced in their entire length at comparable levels (figure 2). in order to assess the efficiency of exon skipping, we performed quantitative analyses of mrna products from the gla minigenes by absolute real - time pcr. to determine the absolute quantities of the two products, we used pcd - gla vector and the oligonucleotide glamut to generate the standard curves. we analyzed total rna isolated from the transfected cos-1 cells with gla minigenes alone or in combination with u1asrnas. relative to the level of expression of -gal a mrna in cells transfected with the wt minigene (100%), cells transfected with the c.639 + 861c > t minigene expressed 46.3% less the -gal a mrna and 1323.9% more the glains57 mrna, while cells transfected with the c.639 + 919g > a minigene expressed 57.1% less the -gal a mrna and 633% more the glains57 mrna (table 1). administration of the u1-gla3 t to the c.639 + 861c > t minigene enhanced the expression of the -gal a mrna by 150.7% and reduced the expression of the glains57 mrna by 66.8% (table 1). administration of the u1-gla5a to the c.639 + 919g > a minigene enhanced the expression of the -gal a mrna by 166.7% and reduced the expression of the glains57 mrna by 41.6% (table 1). the cotransfection of the c.639 + 861c > t or the c.639 + 919g > a minigenes with the u1-glawt enhanced the expression of the -gal a mrna by 141.8 and 166.7%, respectively ; while it reduced the expression of the glains57 mrna by 64.8 and 37.2%, respectively (table 1). cotransfection of both minigenes with the u1-gla scramble had no influence on the transcripts levels, as expected (table 1). the results show that the u1-glawt construct is as efficient as the mutation - specific u1asrnas, indicating that one mismatch in the 48-nt long antisense sequence is well tolerated. these data could have important implications as the same antisense molecule could be used to correct different mutations, without the need for personalized therapeutic molecules. a reduced amount of -gal a protein was detectable in cos-1 cells transfected with c.639 + 861c > t (figure 3a) or c.639 + 919g > a (figure 3b) minigenes, with respect to cells transfected with wild - type minigene. these results were consistent with what we observed at the level of gla transcripts. the c.639 + 861c > t mutation reduced the expression of -gal a protein to a greater extent than the c.639 + 919g > a (figure 3). cotransfection of u1-gla3 t with the c.639 + 861c > t minigene as well as of u1-gla5a with the c.639 + 919g > a minigene caused a significant increase in the -gal a protein detected by western blot (figure 3). an increase in -gal a was also present when we cotransfected c.639 + 861c > t and c.639 + 919g > a minigenes with the u1-glawt (figure 3). as expected, no differences in -gal a protein levels were detected when we cotransfected minigenes with the control u1-glascramble (figure 3). the -gal a basal activity of cos-1 cells was 103 6.9 nmol / mg prot / hour (average of five independent replicates) and this amount was subtracted from the -gal a activities measured in the transfected cells. the -gal a enzyme activity of cos-1 cells transfected with wild - type minigene with respect to untransfected cells rose nearly 800% (816.2 62.2 nmol / mg prot / hour, average of three independent replicates after subtraction of cos-1 basal -gal a activity). residual -gal a activity was 5% of the wild - type for the c.639 + 861c > t mutation (figure 4b) and 55% of the wild - type for the c.639 + 919g > a mutation (figure 4c). in particular, u1-gla3 t and u1-glawt efficiently recovered enzyme activity in the mutation c.639 + 861c > t to levels equal to the wild - type (figure 4b). the u1-glascramble control did not perturb -gal a enzyme activity which resulted to be as low as in cells transfected with mutant minigenes alone (figure 4). when cotransfected with the wild - type minigene, none of the u1asrnas significantly influenced -gal a enzyme activity (figure 4a). we and other authors have shown that the gla gene transcribes two alternatively spliced mrnas : the major gla mrna -gal a transcript and the minor transcript glains57. gla deep intronic mutations c.639 + 861c > t and c.639 + 919g > a cause fd by increasing the inclusion of the cryptic exon to around 85 or 70% of the total transcripts. to replicate the splicing process of the cryptic exon, we constructed a minigene in which the entire 1,718-bp - long sequence of gla intron 4 was inserted into gla cdna between exons 4 and 5 (figure 1a). this wild - type splicing reporter minigene, which we named wt minigene, carries a sv40 early region polyadenylation site and expresses the gla mrna under the transcriptional control of the cmv promoter (figure 1a). upon transfection in cos-1 cells, the wt minigene produced a shorter, more abundant mrna, and a longer mrna, as assessed by semiquantitative reverse transcriptase - pcr (pcr) and by real - time pcr (figure 2a and table 1). we confirmed both amplicons by direct sequencing : the shorter band corresponded to the -gal a mrna, while the longer to the glains57 mrna harboring the inclusion of the 57 nt cryptic exon, as expected. we subsequently constructed the splicing reporter minigenes for the c.639 + 861c > t and c.639 + 919g > a intronic mutations which we named c.639 + 861c > t minigene and c.639 + 919g > a minigene, respectively. when transfected into cos-1 cells, the glains57/total glamrnas ratio was 40% for wt minigene, as revealed by quantitative real - time pcr. this ratio markedly increased for mutated minigenes : 95% for the c.639 + 861c > t minigene and 88% for the c.639 + 919g > a minigene (table 1). such data confirm that c.639 + 861c > t and c.639 + 919g > a mutations unbalance the expression of the gla mrnas causing an increment in the production of the gla alternatively spliced mrna. our real - time pcr probes are specific for the sequence of human gla transcripts and did not yield any amplification when tested on nontransfected cos-1 cells (data not shown). therefore, our quantitative data are not affected by the endogenous gla expression of cos-1 cells. in order to induce skipping of the cryptic exon, we designed and produced four antisense constructs, in the backbone of the u1 snrna, and named them u1-glawt, u1-gla3 t, u1-gla5a, and u1-glascramble. nucleotides from position 310 at the 5-end of u1 snrna, required for the recognition of the 5 splice site, were substituted with 48-nt - long antisense sequences complementary to both cryptic 3 ' and 5 ' splice sites (figure 1a, b). the use of double - target u1asrnas was dictated by indications in the literature demonstrating that efficient exon skipping is obtained with antisense u7 snrnas and antisense u1snrnas targeting at the same time two distinct splicing - regulating regions. the chimeric u1-antisense sequences were cloned under the control of the strong rna polymerase ii u1 snrna gene promoter and termination sequences and subsequently inserted into the paav2.1-cmv - egfp plasmid. construct u1-glawt produces a u1asrna complementary to the cryptic splice sites of the wild - type gla gene (figure 1b), while constructs u1-gla3 t and u1-gla5a differ from u1-glawt each by one nucleotide, being perfectly complementary to the 3 ' splice site of mutation c.639 + 861c > t and to the 5 ' splice site of mutation c.639 + 919g > a, respectively. as a negative control, we also produced the u1-glascramble construct that bears a 23-nt - long sequence (figure 1b) with no known target in mammalian cells. u1-gla5a, u1-gla3 t, and u1-glawt antisense constructs did not induce any significant variation on the gla mrna expression level when cotransfected with the wild - type minigene (figure 2a). cotransfection analysis of u1-gla3 t and u1-glawt partially restored the expression of the -gal a mrna in the presence of the c.639 + 861c > t mutation (figure 2b) and u1-gla5a and u1-glawt corrected, albeit not completely, the c.639 + 919g > a mutation (figure 2c). these results indicate that the u1-glawt construct induces skipping of the cryptic exon in both c.639 + 861c > t and c.639 + 919g > a gla mutations with a reduction of the glains57 mrna of 66% for the c.639 + 861c > t and 20% for the c.639 + 919g > a, relative to cells transfected with the minigene alone (figure 2). the control u1-glascramble did not cause any considerable alteration to the expression profile of any minigene, as expected (figure 2). because the different antisense constructs considerably extend the length of the u1 snrna (by 40 nucleotides), we checked the integrity of the u1 antisense rnas and compared their levels in cotransfected cos-1 cells. we performed semiquantitative rt - pcr analyses with a primer annealing to the 5 ' variable tail and a primer annealing to a common portion of the u1 snrna (figure 2d). a specific amplification product of the expected size (around 100 bp) was obtained exclusively in the cells transfected with one of the u1asrna constructs, confirming that they were produced in their entire length at comparable levels (figure 2). in order to assess the efficiency of exon skipping, we performed quantitative analyses of mrna products from the gla minigenes by absolute real - time pcr. to determine the absolute quantities of the two products, we used pcd - gla vector and the oligonucleotide glamut to generate the standard curves. we analyzed total rna isolated from the transfected cos-1 cells with gla minigenes alone or in combination with u1asrnas. relative to the level of expression of -gal a mrna in cells transfected with the wt minigene (100%), cells transfected with the c.639 + 861c > t minigene expressed 46.3% less the -gal a mrna and 1323.9% more the glains57 mrna, while cells transfected with the c.639 + 919g > a minigene expressed 57.1% less the -gal a mrna and 633% more the glains57 mrna (table 1). administration of the u1-gla3 t to the c.639 + 861c > t minigene enhanced the expression of the -gal a mrna by 150.7% and reduced the expression of the glains57 mrna by 66.8% (table 1). administration of the u1-gla5a to the c.639 + 919g > a minigene enhanced the expression of the -gal a mrna by 166.7% and reduced the expression of the glains57 mrna by 41.6% (table 1). the cotransfection of the c.639 + 861c > t or the c.639 + 919g > a minigenes with the u1-glawt enhanced the expression of the -gal a mrna by 141.8 and 166.7%, respectively ; while it reduced the expression of the glains57 mrna by 64.8 and 37.2%, respectively (table 1). cotransfection of both minigenes with the u1-gla scramble had no influence on the transcripts levels, as expected (table 1). the results show that the u1-glawt construct is as efficient as the mutation - specific u1asrnas, indicating that one mismatch in the 48-nt long antisense sequence is well tolerated. these data could have important implications as the same antisense molecule could be used to correct different mutations, without the need for personalized therapeutic molecules. a reduced amount of -gal a protein was detectable in cos-1 cells transfected with c.639 + 861c > t (figure 3a) or c.639 + 919g > a (figure 3b) minigenes, with respect to cells transfected with wild - type minigene. the c.639 + 861c > t mutation reduced the expression of -gal a protein to a greater extent than the c.639 + 919g > a (figure 3). cotransfection of u1-gla3 t with the c.639 + 861c > t minigene as well as of u1-gla5a with the c.639 + 919g > a minigene caused a significant increase in the -gal a protein detected by western blot (figure 3). an increase in -gal a was also present when we cotransfected c.639 + 861c > t and c.639 + 919g > a minigenes with the u1-glawt (figure 3). as expected, no differences in -gal a protein levels were detected when we cotransfected minigenes with the control u1-glascramble (figure 3). the -gal a basal activity of cos-1 cells was 103 6.9 nmol / mg prot / hour (average of five independent replicates) and this amount was subtracted from the -gal a activities measured in the transfected cells. the -gal a enzyme activity of cos-1 cells transfected with wild - type minigene with respect to untransfected cells rose nearly 800% (816.2 62.2 nmol / mg prot / hour, average of three independent replicates after subtraction of cos-1 basal -gal a activity). residual -gal a activity was 5% of the wild - type for the c.639 + 861c > t mutation (figure 4b) and 55% of the wild - type for the c.639 + 919g > a mutation (figure 4c). in particular, u1-gla3 t and u1-glawt efficiently recovered enzyme activity in the mutation c.639 + 861c > t to levels equal to the wild - type (figure 4b). the u1-glascramble control did not perturb -gal a enzyme activity which resulted to be as low as in cells transfected with mutant minigenes alone (figure 4). when cotransfected with the wild - type minigene, none of the u1asrnas significantly influenced -gal a enzyme activity (figure 4a). since 2001, enzyme replacement therapy (ert) has been the only available specific treatment for fd. ert involves regular intravenous infusions of one of the two recombinant enzyme formulations available in europe : agalsidase alfa (replagal ; shire, cambridge, ma), produced using cultured human skin fibroblasts and agalsidase beta (fabrazyme ; genzyme, cambridge, ma), produced by expressing human -galactosidase cdna in chinese hamster ovary (cho) cells. published reports strongly support the overall clinical benefit of ert, but benefits on renal and nervous system function are less obvious. some patients may develop a specific antibody response to the exogenous protein and, overall, therapy is very expensive. pharmacological chaperone therapy (pct) can offer an alternative approach. for some -gal a missense mutations the supplementation of pharmacological chaperone 1-deoxygalactonojirimycin (dgj or at1001), an imminosugar with a structure resembling the -galactose of globotriaosylceramide (gb3), improves endogenous -gal a activity. pharmacological chaperone therapy is currently being tested in clinical trials. a significant number of gla disease - causing mutations are missense variants, which cause the newly synthesized lysosomal protein to be misfolded, but still catalytically competent. the misfolded enzyme forms are unable to undergo appropriate trafficking to the lysosomal compartment and are prematurely degraded in the endoplasmic reticulum. the active - site - specific chaperone dgj stabilizes the conformation of the mutant proteins assisting protein folding and preventing premature degradation by endoplasmic - reticulum - associated protein degradation (erad). dgj could provide additional advantages in terms of cost, whose use, however, would be limited to patients with specifically responsive gla missense mutations. an attractive alternative approach aimed to correct rna defects in human diseases is based on the exon skipping (es) strategy. an interesting way to pursue es is based on the use of antisense modified u snrnas (such as u1 or u7) which can be delivered by adeno - associated viral vectors (aav). es based on virally - vectored modified u1 snrnas offers the possibility of providing a long - term therapy which would only require a single administration in a patient 's lifetime, by promoting stable genomic integration with viral delivery particles. modified u1 snrnas have been shown to produce effective es in several diseases. here, we have sought to evaluate u1-based es for the correction of c.639 + 861c > t and c.639 + 919g > a mutations in fd. we constructed a set of specific u1asrnas and built gla minigenes to express two deep intronic mutations, c.639 + 861c > t and c.639 + 919g > a. a different repertoire of splicing factors and other rna - binding proteins is present in different cell types. additionally, it has been suggested that nucleosome density and histone modifications, which could also be cell - specific, may play a role in splice site recognition and regulation. nonetheless, cell - based splicing of minigenes, a simplified system, is used extensively for identifying or confirming the in vivo relevance of cis- and trans - acting factors in the regulation of particular splicing patterns. although bioinformatic tools might be used to predict the effect of a nucleotide change on splicing, experimental verification by minigenes is still required for diagnostic purposes, for revealing disease mechanisms and for monitoring therapeutic interventions. the minigene - based analysis represents a complementary approach to reverse transcription - pcr analyses of patients ' rna, for the identification of pathogenic splicing. the exon carrying the mutation under study is cloned along with its flanking intronic sequence. the resulting construct, in its wild - type and mutant sequence version, is transfected in established cell lines, and the vector splicing pattern is analyzed. ideally, the wild - type minigene results in correct exon inclusion, while the mutant construct results in aberrant transcripts. the splicing pattern observed when transfecting minigenes in different cell lines recapitulates the effect that a mutation has on the splicing of the different isoforms in patients ' primary cells. however, in minigene studies, it is not unusual to observe a different ratio of correct vs. aberrantly spliced transcripts, with respect to patients ' cells and from cell type to cell type. this might be likely due to the cell type - specific repertoires of splicing factors and rna - binding proteins and to the fact that the minigene is a simplified system compared to the genomic dna. in this paper, we report the construction of a gla wild - type minigene and two mutant gla minigenes, harboring the mutations c.639 + 861c > t and c639 + 919g > a. upon transfection into cos-1 cells, the wild - type gla minigene generated gla transcripts of which 60% were -gal a mrna and 40% were glains57, as assessed by real - time pcr. our real - time pcr probes are specific for the human gla sequence and did not yield any amplification when tested on non - transfected cos-1 cells (data not shown). therefore, our real - time pcr data are not affected by the endogenous expression of the cos-1 gla gene. the difference in the percentage of glains57 can be explained by the simplified nature of reporter minigenes systems, compared to the endogenous gene and the fact that cos-1 cells contain a different repertoire of splicing factors. however, our data are in line with the observations of ishii and colleagues that showed small amounts of glains57 transcripts (t minigene gave rise to a higher number of glains57 transcripts (ca. ten - fold increase) and a lower number of -gal a mrna (ca. similarly, the c.639 + 919g > a minigene caused a two - fold higher expression of the glains57 transcript and a five - fold decrease of -gal a mrna, when compared to the wild - type minigene. importantly, the c.639 + 861c > t minigene led to a higher expression of the glains57 transcript and a lower residual -gal a activity than the c.639 + 919g > a minigene. such results confirm what has been observed in fabry patients, because the c.639 + 861c > t mutation is associated with a heavily -gal a enzyme deficiency in males and a more severe phenotype than the c.639 + 919g > a mutation. by testing u1asrnas against the minigene systems the u1-gla5 t was able to restore full -gal a enzyme activity and protein for the c.639 + 861c > t mutation and a similar effect was obtained with u1-gla3a for the c.639 + 919g > a mutation. the administration of u1-gla3 t or u1-glawt to the c.639 + 861c > t minigene, and of u1-gla5a or u1-glawt to the c.639 + 919g > a minigene, significantly reduced the expression of the glains57 mrna. results indicate that u1-gla5a, u1-gla3 t, and u1-glawt antisense constructs are able to prevent the inclusion of the cryptic exon in the mutated minigenes with variable efficiencies, depending on the targeted sequence. the u1-glawt construct is able to prevent the inclusion of the cryptic exon in both the c.639 + 861c > t and c.639 + 919g > a mutations, indicating that one mismatch in the 48-nt long antisense sequence is well tolerated, as expected. in fact, u1-glawt would bind with a g : u wobble base pair in the 3'ss to the c.639 + 861 t mutant pre - mrna, compared to a g : c base pair present in the same position in the binding to the wild - type pre - mrna. this translates in a difference in standard free energy of only about 1.5 kcal / mol. however, u1-glawt would bind with a a / c mismatch the 5'ss of the c.639 + 919a mutant pre - mrna, compared to a g : c base pair present in the same position in the binding to the wild - type. this translates into a difference in standard free energy of about 6 kcal / mol. hence, a single mismatch probably is not likely to abrogate the binding of the 48-nt long sequence to the target (which in the wild - type conditions is of about g = 80 kcal / mol). these data have important implications because a universal u1-based es strategy could be used to correct different mutations affecting this specific alternative splicing process of the gla gene. the accumulation of globotriaosylceramide (gb3) and related glycolipids in fd is progressive and occurs in fluids and tissues including vascular endothelium, kidney, heart, connective tissue, and peripheral nerves. fd males who have little or no functional -gal a enzyme activity (a mutation is quite common in the asian population. on the other hand, in particular, the c.639 + 919g > a mutation was found with a very high prevalence among newborns in taiwan (1 in 1,600 males ; approximately 8286% of patients carrying a gla mutation). if a specific antisense rna - based drug to correct this mutation was made available, it could benefit a high number of fabry patients. a u1asrna - based therapy would act on the pre - mrna endogenously produced in the affected cells, thus maintaining the physiological gla gene transcription regulation. since the c.639 + 919g > a mutation does not completely impair the -gal a protein production leading to residual enzyme activity, we can hypothesize that a u1-based therapeutic approach would be well tolerated in patients without the development of an anti--gal a immune response as occurring with ert. anyhow, further experiments focused on patients ' derived cell lines or animal models will be necessary to confirm such speculations. our study provides evidence that splicing correction through a u1-vectored antisense rna could be developed to restore -gal a production and enzyme activity in the c.639 + 861c > t and c.639 + 919g > a gla mutations. it will now be possible to proceed towards preclinical trials for this therapeutic approach, delivering the antisenseu1-producing cassette to patients ' cells and animal models via viral vectors. we constructed wild - type and mutated gla minigenes for the expression of c.639 + 861c > t and c.639 + 919g > a mutations by integration of the full - length sequence of the human gla intron 4 within the wild - type human gla cdna. we engineered the wild - type human gla cdna sequence that had previously been cloned in the pcd - x vector in our laboratory. the entire gla intron 4 was amplified from human wild - type dna or from a male patient 's dna harboring the c.639 + 861c > t gla mutation. we used oligonucleotides cdnacap4f and cdnacap5r (table 2) that anneal in exon 4 and exon 5, respectively. two gla cdna fragments corresponding to exons 14 and exons 57 were amplified from pcd - gla plasmid with primer pairs 5xbagla + cdnacap4r and cdnacap5f + 3ecogla. the obtained pcr fragments were mixed in separate mixtures with wild - type or c.639 + 861c > t gla intron 4 and used as templates for a pcr reaction with external primers 5xbagla and 3ecogla to produce the minigene cassette. all the pcr amplifications were performed with high fidelity platinum pfx dna polymerase (thermo fisher scientific, waltham, ma). the pcd - gla plasmid and the minigene cassettes were cut with endonucleases xbai and ecori (new england biolabs, ma) that each recognize a unique restriction site. the digested fragments were linked using solution i of the dna ligation kit ver.2.1 (takara bio, kusatsu, japan) and cloned by transforming the e. coli solopack gold cells (agilent technologies, santa clara, ca). endofree plasmid maxi kit (qiagen, hilden, germany) was used to purify the plasmids, which were called wt minigene (with the wild - type gla intron4) and c.639 + 861c > t minigene (with the c.639 + 861c > t mutation). to introduce the asian gla c.639 + 919g > a mutation, for which patient - derived genomic dna was not available, we used general procedures for site directed mutagenesis. the wt minigene was amplified by pcr with specific mutated oligonucleotide primers gla919a - fw and gla919a - rev, in combination with primers 3ecogla and 5xbagla. the obtained pcr fragments were mixed and used as templates for a pcr reaction with external oligonucleotides 5xbagla and 3ecogla to produce the minigene cassette. the pcd - gla plasmid and the minigene cassette were cut with endonucleases xbai and ecori (new england biolabs). the final mutated construct was obtained by ligation and cloning and was verified by direct sequencing, as previously described. we obtained plasmids u1-gla3 t, u1-gla5a, u1-glawt and u1-glascramble by inverse pcr of the u1 snrna genomic sequence, as described previously, using the oligonucleotides (eurofins mwg operon company, ebersberg, germany) reported in table 2. 100 pmol of u1-gla3cfor, u1-gla3tfor, u1-gla5grev, u1-gla5arev, u1-scramblerev primers were phosphorylated in a final volume of 100 l by using t4 polynucleotide kinase (fermentas, thermo fisher scientific), following the manufacturer 's protocols. after incubation of 30 minutes at 37 c, the reaction was stopped by the addition of 1 l of edta 0.5 m ph 8.0. oligonucleotides were purified by the adding 100 l of chloroform and centrifuged for 2 minutes at 16,000 g. the u1-gla3 t, u1-gla5a, u1-glawt, and u1-scramble fragments were obtained via two distinct inverse pcr amplifications, performed on the pbsu1 plasmid containing the u1 snrna gene. the following phosphorylated and non - phosphorylated primer couples were used for the first pcr amplification reaction : u1-gla3cfor and u1-casdown - revnhei ; u1-gla3tfor and u1-casdown - revnhei ; u1-gla5grev and u1-casup - fornhei ; u1-gla5arev and u1-casup - fornhei ; u1-scramblerev and u1-casup - fornhei, u1-univ - nhei and u1-casdown - revnhei. all the pcr amplifications were performed in a final volume of 50 l in a reaction mixture containing 1 of cloned pfu dna polymerase reaction buffer, 200 mol / l dntpmix, 0.2 mol / l of each primer, 2u of cloned pfu dna polymerase taq (agilent technology, santa clara, ca). dna was denatured for 1 minute at 95 c, followed by 30 cycles of amplification with cycling conditions of 95 c for 1 minute, 65 c for 40 seconds, extension at 72 c for 45 seconds, and extra final extension at 72 c for 7 minutes. amplification products sizes are 215 bp for u1-gla3c, u1-gla3 t, u1-univ fragments and 419 bp for u1-gla5 g, u1-gla5a and u1-scramble fragments. after pcr reactions all the products were extracted from a 1% agarose gel in tris - acetic acid - edta (tae) 1x (sigma - adrich, st. louis, mo) using the qiaquick gel extraction kit according to the manufacturer 's protocol (qiagen, hilden, germany), and the single fragments were ligated in order to obtain the whole u1-gla3 t, u1-gla5a, u1-glawt, and u1-scramble fragments. ligation was performed in a final volume of 20 l at 16 c for 3 hours, according to the manufacturer 's protocol (t4 dna ligase, new england biolabs, neb, ipswich, ma). the whole fragments (634 bp) obtained from the ligation reaction were used as templates for a second pcr reaction, using the external u1-casup - fornhei and u1-casdown - revnhei primers. the reaction was performed in a final volume of 50 l with a single series of 30 cycles, preceded by a 1 minute long predenaturation step, according to the above - mentioned protocol. after amplification and purification, the resulting fragments were cloned in the forward orientation in the nhei site of a paav2.1-cmv - egfp plasmid : the total amount of pcr products and 10 g of paav2.1-cmv - egfp2 vector were digested with nhei restriction enzyme (new england biolabs, neb) for 3 hours at 37 c in a final volume of 50 l. in order to prevent its potential recircularization, the restricted paav-2.1-cmv - egfp vector was dephosphorylated by treatment with calf intestine alkaline phosphatase (ciap, fermentas, thermo fisher scientific) at 37 c for 30 minutes. the ligation reaction was performed at 16 c for 3 hours in a final volume of 20 l by using t4 dna ligase (new england biolabs, neb). we confirmed the exact sequence and the orientation of the inserts within the paav-2.1cmv - egfp vector by automated dna sequencing (bmr genomics, padova), using the aav rev primer (table 2). we transiently overexpressed wild type and mutant plasmids into african green monkey kidney cells (cos-1). cos-1 cells were cultured in dulbecco 's modified eagles - hams f10 medium (1:1 vol / vol) with fetal bovine serum (10%) and antibiotics (penicillin - streptomycin 1) and grown in a humidified incubator, at 5% co2 and 37 c. transfection of cos-1 cells was mediated by lipofectamine ltx (life technologies, carlsbad, ca) using a plasmid dna / lipofectamine ratio of 1 g : 3 l. cos-1 cells were plated onto 6-multi well plates (7.5 10 cells / well). for each transfection an amount of 2.5 g for reporter minigenes (size about 6.0 kb) and 1.25 g for u1asrna constructs (size about 6.0 kb) were used, to obtain a final molar ratio of 2:1. after 4 hours of incubation, the medium was replaced with fresh growth medium and cells were incubated for 48 hours before harvesting by trypsinization. after trypsinization, cells were washed once with phosphate buffered saline (pbs) 1x and resuspended in water supplemented with a protease inhibitor (sigma - aldrich). pellets were stored at 20 c for protein analysis or at 80 c for rna analysis. rna isolation, retrotranscription, and reverse transcriptase - pcr (rt - pcr). total rna was isolated from transfected cos-1 cells using the rneasy mini kit (qiagen). rna integrity and concentration were checked both by 1% agarose gel and nanodrop nd-1000 spectrophotometer (nanodrop technologies, wilmington). total rna (200 ng) was reverse transcribed with random hexamers using taqman reverse transcriptase kit (applied biosystems by life technologies) as previously described. we performed rt - pcr analysis using 2 l of retrotranscribed products as template and primers rna3f and rna5r that anneal on the gla exons 3 and 5 respectively. this primer couple includes the region that differs between the two alternative gla transcripts and produces an amplicon of 406 bp corresponding to the wild - type gla mrna and an amplicon of 463 bp from the glains57 transcript. pcr reactions were prepared in 25 l of final volume in a reaction mixture containing 1x pcr buffer, 1.5 mmol / l mgcl2, 250 mol / l each dntp, 15 pmol of each primer, 1 u of taq dna polymerase (applied biosystems by life technologies). after a primary denaturation for 5 minutes at 95 c, amplification was carried out for 30 cycles of 30 seconds at 95 c, 30 seconds at 56 c and 2 minutes at 72 c, with a final extension of 7 minutes at 72 c. to verify the transcription of u1asrnas from the vectors transfected in cos-1 cells, and confirm their integrity, we adapted the previously reported protocol by denti. rt - pcrs were carried out on retrotranscribed total rna (2 l corresponding to 10 ng of rna) with primer u1 + 130, which anneals on the body of the u1 mrna molecule, in combination with a primer specific for the engineered antisense modified u1 tail. primer pu1-gla was used to amplify the transcript from u1-gla3 t, u1-wt and u1-gla5a and primer pu1-scramble for the amplification of the u1-glascramble rna. pcr reactions were performed as described above for rt - pcr changing the annealing temperature to 54 c. we measured the gla mrna transcripts using a quantitative real - time rt - pcr method based on taqman technology and a fast real - time pcr system (applied biosystems by life technologies). probes, primers and protocols used were previously reported. for the detection of the gla mrna that encodes -gal a : probe 631/666 nt, we used forward primer 579/602 nt and reverse primer 688/668 nt. for the detection of the glains57 mrna, we used probe 670/701 nt, forward primer 642/668 nt and reverse primer 738/718 nt. for the absolute quantification of gla mrna which encodes the lysosomal -gal a, we used the wild - type pcd - gla plasmid while for the glains57 mrna, the following oligonucleotide was designed on the cdna carrying the intronic insertion of 57 nt (1347 bp) and generated by roche, mannheim, germany : oligonucleotide glamut 5-ctccacactatttggaagtatttgttgacttgttaccatgtctccccactaaagtcccaattatacagaaatccgacagtactgcaatcactggcga-3 (642/738 nt). the pcd - gla plasmid and glamut oligonucleotide were serially diluted 10-fold from a starting quantity of 10 ag for the wild - type pcd - gla and 10 ag for the glamut oligonucleotide and used as standard curves. the absolute values of gla gene mrna products were expressed as copy / mg of total rna (mean sd). transfected cos-1 cells were harvested by trypsin and cell pellets were frozen at 80 c until assayed. cell pellets were lysed in sterile distilled water containing complete protease inhibitor cocktail (bd biosciences, erembodegem, belgium) by sonication (10 seconds). the protein concentration for each lysate was measured using the micro bca protein assay kit (pierce, rockford, il). total protein (30 g) from each lysate was loaded onto homemade 12.5% polyacrylamide gels containing 0.1% sds and transferred onto nitrocellulose membranes (bio - rad, hercules, ca) using the mini trans - blot system (bio - rad). immunoblots were then probed with a 1:200 dilution of rabbit polyclonal antibody for -gal a kindly provided by genzyme corporation (genzyme, italy) and a 1:2,500 dilution of rabbit polyclonal antibody for cyclophilin a (millipore, billerica, ma). the blots were then incubated with secondary anti - rabbit igg (whole molecule) conjugated to alkaline phosphatase (sigma ; milano, italy), and signals were revealed using the ap conjugate substrate kit (bio - rad). -gal a enzyme assay. -gal a enzyme assays were performed in triplicate by fluorogenic method with the following modifications : 1 mg / ml cell lysates (diluted in 10 l of heat inactivated 0.2% bsa+0.02% na azide) were added to 20 l substrate (4-mu - a - d - galactopyranoside 5 n - acetyl - d - galactosamine and 0.02% na azide) ; stop solution : 200 l of 0.5 m na - hco3-na2co3 buffer, ph 10.7, 0.025% triton x-100. the micro bca protein assay kit (pierce) was used to set up the starting protein concentrations used in each enzyme assay performed in black 96-well microplates. fluorescence was read on a spectra max m2 microplate reader (molecular devices, toronto, canada). statistical analysis. we evaluated data for qpcr for statistical significance using the student 's t - test (graphpad software, san diego, ca), expressed as mean sd (n = 3). a p value of < 0.05 was considered significant for data acquired in density units. gene variants are given in the text by following the human genome variation society (hgvs) recommendations (http://www.hgvs.org/mutnomen/). exon numbering refers to the gla refseq nm_000169.2 (gla ; gene i d 2717).	fabry disease is a rare x - linked lysosomal storage disorder caused by deficiency of the -galactosidase a (-gal a) enzyme, which is encoded by the gla gene. gla transcription in humans produces a major mrna encoding -gal a and a minor mrna of unknown function, which retains a 57-nucleotide - long cryptic exon between exons 4 and 5, bearing a premature termination codon. nm_000169.2:c.639 + 861c > t and nm_000169.2:c.639 + 919g > a gla deep intronic mutations have been described to cause fabry disease by inducing overexpression of the alternatively spliced mrna, along with a dramatic decrease in the major one. here, we built a wild - type gla minigene and two minigenes that carry mutations c.639 + 861c > t and c.639 + 919g > a. once transfected into cells, the minigenes recapitulate the molecular patterns observed in patients, at the mrna, protein, and enzymatic level. we constructed a set of specific double - target u1asrnas to correct c.639 + 861c > t and c.639 + 919g > a gla mutations. efficacy of u1asrnas in inducing the skipping of the cryptic exon was evaluated upon their transient co - transfection with the minigenes in cos-1 cells, by real - time polymerase chain reaction (pcr), western blot analysis, and -gal a enzyme assay. we identified a set of u1asrnas that efficiently restored -gal a enzyme activity and the correct splicing pathways in reporter minigenes. we also identified a unique u1asrna correcting both mutations as efficently as the mutation - specific u1asrnas. our study proves that an exon skipping - based approach recovering -gal a activity in the c.639 + 861c > t and c.639 + 919g > a gla mutations is active.
the mitochondrial genomes of angiosperms exhibit a diverse array of features that contribute to increased genomic complexity, including numerous genes and introns, a large amount of intergenic and repetitive dna, and abundant cytidine - to - uridine (c - to - u) rna editing (knoop 2012 ; mower. for example, the 783 kb mitogenome of liriodendron tulipifera has 64 genes, 25 introns, and > 750 edit sites (richardson. 2013), whereas the 6.7 mb mitogenome of silene noctiflora has only 32 genes, 18 introns, and 20 sites of rna editing in this gene (rice. 2013) as exemplified by the 28 edit sites in melianthus nad1, nearly all of these sites have been converted to thymidines in the corresponding positions of geraniaceae nad1 genes. the correlated loss of introns and edit sites from geraniaceae nad1 is consistent with retroprocessing activity. despite these changes, the geraniaceae nad1 gene is probably functional : it is full length and free of internal stop codons, and rna editing of the few remaining sites improves sequence conservation to homologous genes from non - geraniaceae species. in addition to the presence of a putatively functional nad1 gene, most geraniaceae species contain another partial nad1 sequence at a distinct genomic position (fig. 1). unlike the putatively functional nad1 copies, however, which lack the nad1i728 intron and the associated matr gene, these partial nad1 sequences include the matr gene and parts of the nad1i728 intron (fig. the flanking intron sequences show clear signs of degradation based on the numerous nucleotide substitutions, insertions, and deletions that disrupt the predicted secondary structure (fig. 2b). in contrast, the matr sequence is presumably functional because it is full length, free of internal stop codons, and transcribed based on detectable reads in the rnaseq library. the rnaseq reads also revealed 25 positions that are edited in at least one of the seven geraniales matr sequences, of which eight positions (32, 43, 326, 1679, 1700, 1720, 1756, and 1844) are edited in most species (fig. 3). these data indicate that matr functions as a freestanding gene in most geraniaceae species. (a) the diagram depicts the degree of degradation of nad1i728 in selected geraniaceae species. (b) the secondary structure of the functional cis - spliced nad1i728 intron of m. villosus (in black) was determined by a manual comparison with existing group ii introns models (michel and ferat 1995 ; qin and pyle 1998 ; toor,. the intron folds into a typical group ii intron secondary structure, with a central core and six branched domains (i vi). colored intron regions represent changes in the secondary structure of the degraded introns in g. maderense (in green), e. moschatum (in magenta), h. biloba (in orange), and m. emarginata (in blue) : numbers, positive numbers, and negative numbers show sequence replacements, insertions, and deletions, respectively. the position of the maturase matr gene in domain iv is indicated by a bold black arrow. exon binding sites (ebs) within domain i of the intron sequence (capital letters) and intron binding sites (ibs) in the adjacent exon sequence (small letters) are highlighted by a thin black arrow. (a) the diagram depicts the degree of degradation of nad1i728 in selected geraniaceae species. (b) the secondary structure of the functional cis - spliced nad1i728 intron of m. villosus (in black) was determined by a manual comparison with existing group ii introns models (michel and ferat 1995 ; qin and pyle 1998 ; toor,. the intron folds into a typical group ii intron secondary structure, with a central core and six branched domains (i vi). colored intron regions represent changes in the secondary structure of the degraded introns in g. maderense (in green), e. moschatum (in magenta), h. biloba (in orange), and m. emarginata (in blue) : numbers, positive numbers, and negative numbers show sequence replacements, insertions, and deletions, respectively. the position of the maturase matr gene in domain iv is indicated by a bold black arrow. exon binding sites (ebs) within domain i of the intron sequence (capital letters) and intron binding sites (ibs) in the adjacent exon sequence (small letters) are highlighted by a thin black arrow. because of the deep illumina sequencing performed here, the matr gene was easily detectable in the draft mitochondrial assemblies of most geraniaceae species. in contrast, matr was not detected in the mitochondrial assemblies of p. x hortorum and p. citronellum (fig., a homolog was recovered in the assemblies of the nuclear genome and transcriptome (fig. 4a). in both pelargonium species, these nuclear matr sequences (annotated as nmatr) were split into two distinct genes termed nmatrt, encoding the subdomains ii to iv of the reverse transcriptase (rt) domain, and nmatrx, encoding subdomains v to vii of the rt domain and the entire maturase (x) domain. a glutaredoxin (grx) gene was identified upstream of nmatrx and a copper / zinc superoxide dismutase (cuznsod) gene was identified downstream of nmatrt, demonstrating that both nmatr genes are located in the nuclear genome. furthermore, both nmatr genes are transcribed, and the nuclear intron residing in the 5 utr of nmatrx is properly spliced, indicating that both nmatr genes are functionally expressed nuclear genes. in contrast, two additional copies of nmatrt (found in a tail - to - tail arrangement on a different scaffold) had no transcriptional activity and may not be functional. (a) the mitochondrial matr gene in the nad1 intron nad1i728 in m. villosus is shown at top, and homologous matr sequences in contigs from the pelargonium nuclear genome are shown below. grey shading shows conserved reverse transcriptase (rt) and maturase (x) domains. the thick blue bar following the first potential start codon of the nmatr sequences defines the location of the acquired mitochondrial targeting sequence ; targeting prediction scores for mitoprot, targetp, and predotar are shown in blue in parentheses below the target sequence. (b) excerpt of a phylogenetic tree of the mitochondrial matr gene of angiosperms (thin black branches) and the nuclear nmatr paralogs of pelargonium (thick red branches). (a) the mitochondrial matr gene in the nad1 intron nad1i728 in m. villosus is shown at top, and homologous matr sequences in contigs from the pelargonium nuclear genome are shown below. start and stop codons are indicated by the arrow and bar, respectively. grey shading shows conserved reverse transcriptase (rt) and maturase (x) domains. the thick blue bar following the first potential start codon of the nmatr sequences defines the location of the acquired mitochondrial targeting sequence ; targeting prediction scores for mitoprot, targetp, and predotar are shown in blue in parentheses below the target sequence. (b) excerpt of a phylogenetic tree of the mitochondrial matr gene of angiosperms (thin black branches) and the nuclear nmatr paralogs of pelargonium (thick red branches). the full phylogenetic tree is presented in supplementary figure s1, supplementary material online. to infer the origin of the pelargonium nmatr genes, phylogenetic analysis was used to determine their relationship to mitochondrial matr sequences from a diversity of other angiosperms (fig. the two nmatr sequences group together with 100% bootstrap support, and they cluster within the geraniaceae clade of mitochondrial matr genes with strong (95%) bootstrap support. because p. citronellum and p. x hortorum span the full taxonomic diversity of pelargonium (weng. 2012), this phylogenetic result indicates that the nmatr genes were probably derived by intracellular transfer of a mitochondrial matr sequence into the nuclear genome of the common ancestor of pelargonium. the extreme sequence divergence for the pelargonium nmatr sequences probably results from the elevated substitution rates known to affect pelargonium mitochondrial genes (parkinson. 2005 ; mower. 2007) and the generally higher rates of nuclear substitution relative to typical mitochondrial genomes (wolfe. 1987 ; drouin. 2008). similar to the mitochondrial matr sequences, the nuclear nmatr genes are more conserved within the domain regions relative to the rest of the protein sequence (supplementary fig.s2, supplementary material online). this pattern exists in both halves of the split gene, nmatrt and nmatrx, and indicates that the gene remained functional after the transfer into the nuclear genome. many of the edited positions in the geraniaceae mitochondrial matr transcripts have been converted to a thymidine in the pelargonium nmatrt and nmatrx genes (fig. 3), suggesting that transfer to the nucleus involved a partially edited rna intermediate. because c - to - u rna editing is not known to exist for nuclear genes we predict that the lack of conversion of some edit sites does not inhibit activity of the two nmatr gene products. the assemblies of the nad1 gene from representative geraniaceae species revealed an unusual pattern of evolution, including (1) the first demonstrated loss of a trans - configured intron (nad1i669) from a functional gene, (2) the loss of the fourth nad1 intron (nad1i728) that hosts the matr gene, (3) the establishment of matr as a freestanding gene within the mitogenome, and (4) the first reported transfer of matr to the nuclear genome. all five nad introns in trans configurations have been lost from several mistletoe mitogenomes (petersen. 2015), but in these cases the intron losses were due to loss of the nad genes themselves. pcr results suggested that nad1i728 was lost from two species in malpighiales (wurdack and davis 2009), but this intron is not trans - spliced in malpighiales (qiu and palmer 2004 ; rivarola., the absence of nad1i669 in geraniaceae represents the first reported loss of a trans - configured intron from any genome. thus, although trans splicing appears to be a strong barrier to intron loss in eukaryotes, this result demonstrates that it is not an absolute barrier. multiple genomic changes are necessary to explain the loss of introns from nad1 and the origin of a freestanding matr gene in geraniaceae, and one possible evolutionary scenario is shown in figure 5. the shared loss of most, but not all, introns and edit sites in geraniaceae could be attributed to incomplete retroprocessing, in which a segment of the nad1 gene was gene converted by a transcript that was only partially edited and spliced. most other examples of retroprocessing also involve the removal of a subset of introns and edit sites (ran. a second coexisting locus, lacking the nad1i728 intron and almost all edit sites, may have been created by a retroduplication event, involving the genomic integration of a partially spliced and edited transcript into a new genomic locus, eliminating the nad1i728 intron and additional edit sites from one of the loci. we then propose that the nad1 locus lacking the nad1i728 intron became the functional component of the gene. at the other locus, once the matr gene was established as a freestanding gene, the splicing of nad1i728 became unnecessary, and the intron sequence underwent lineage - specific degradation from its 5 and 3 ends. finally, ongoing gene conversion, which has been documented for homologous sequences in plant mitogenomes (hao and palmer 2009 ; hao. 2010a), could explain the 100% sequence identity of the nad1 exon sequences shared between the two loci. more generally, the newly translocated matr gene described here provides a rare glimpse into the mechanism of gene translocation. the effects of retroprocessing, retroduplication, gene conversion, and lineage - specific gene degradation, as described in the text, are shown. the effects of retroprocessing, retroduplication, gene conversion, and lineage - specific gene degradation, as described in the text, are shown. the mitochondrial - to - nuclear transfer of maturase genes has occurred multiple times during land plant evolution (mohr and lambowitz 2003 ; guo and mower 2013), but until now, there was no evidence for a nuclear transfer of matr, the sole maturase remaining in seed plant mitogenomes. in viscum scurruloideum, matr and nearly all introns are absent from the mitogenome, but surveys of genome sequencing data did not identify a homolog in the nucleus suggesting that matr and all of its splicing targets have been lost from this species (skippington., pcr results suggest that matr was lost from the mitogenome, but nothing is known about a potential nuclear transfer (wurdack and davis 2009). the transfer of matr to the nucleus in pelargonium was likely facilitated by several of the unusual characteristics of this genus. first, the heavy reduction in rna editing in this genus (parkinson. 2005) raises the possibility of either a dna - mediated or an rna - mediated transfer event (henze and martin 2001), whereas the presence of editing sites in mitochondrial genes of most other species precludes functional transfers occurring via dna. a similar case has been suggested for the mitochondrial rpl5 gene in grasses, in which an ancestral retroprocessing event may have facilitated multiple instances of functional dna - mediated gene transfer into the nuclear genome (ong 2006). second, if a transfer event occurs, the very high substitution rate in pelargonium mitogenomes (parkinson. 2005) would quickly degrade the mitochondrial copy, thus making the transferred nuclear copy essential. from a functional perspective, the retention of a matr gene in geraniaceae after loss of its host nad1i728 intron implies that matr has functions in the mitochondrion beyond the splicing of its host intron. one possibility is that matr facilitates the removal of other mitochondrial group ii introns, and thus is still required for one or more introns remaining in geraniaceae (supplementary table s1, supplementary material online). a recent review of mitochondrial splicing factors suggested an association of matr to other introns (brown. 2014) consistent with additional splicing assignments, and the plastid maturase matk was shown in vivo to have binding activity to several chloroplast introns (zoschke. it is also possible that matr performs other essential transcriptional functions, such as rna processing or stabilization. genetic manipulation of the plant mitochondrial genome is not yet possible in plants, precluding any direct assessment of matr function. in contrast, because nuclear transformation is possible in many plants, including pelargonium (colling. 2012), the nuclear location of matr in pelargonium raises the possibility of genetic and transcriptional manipulation of this gene. furthermore, the split nature of these nmatr genes enables the independent assessment of the functions of the rt and x domains. source of plant materials and procedures for nucleic acid extraction and illumina sequencing were described previously (weng. draft mitochondrial genomes were assembled with velvet 1.1.06 (zerbino and birney 2008) using a combination of kmer (5191) and expected coverage (20500) values as previously described (grewe. the assembly with the longest average length of identifiable mitochondrial sequences (based on blastn searches with known mitochondrial protein - coding genes from related species as query sequences) was selected for further processing (supplementary table s2, supplementary material online). mitochondrial gene and intron content was identified by inspection of the blastn search results (supplementary table s1, supplementary material online). for some species, the presence of two nad1 gene sequences resulted in a failure to assemble complete loci. these regions were manually corrected by aligning and inspecting individual sequence reads that cover the respective regions. the assembled matr and nad1 sequences from this study were deposited in genbank (accession numbers kx824067kx824107). a draft nuclear genome for p. citronellum was assembled using velvet with kmer (41) and expected coverage (20) values that were reduced (relative to the mitochondrial assembly parameters) in order to preferentially assemble the lower - depth nuclear genome. a blastn search identified matr homologs on three contigs, with sizes of 19,729 bp (containing nmatrx), 5,204 bp (containing nmatrt), and 16,625 bp (containing pseudo - nmatrt). in plant cells, the mitogenome is typically present at a substantially higher number of copies compared with the nuclear genome (lamppa and bendich 1984 ; draper and hays 2000). thus, the much lower depth of coverage for these three contigs (relative to the identified mitochondrial contigs) provides reliable evidence that these contigs are nuclear rather than mitochondrial. additional nuclear genes were detected in these three contigs by querying them against the non - redundant protein database using blastx, and some of the genes contain nuclear spliceosomal introns with the canonical gt and ag bases at their 5 and 3 splice sites, providing additional support that these contigs are from the nuclear genome. mitochondrial targeting signals were predicted with the programs mitoprot ii (claros and vincens 1996), targetp 1.1 (emanuelsson. 2000), and predotar v1.03 (small. 2004). the three contig sequences were deposited in genbank (accession numbers kx824108kx824110). rnaseq reads were mapped onto the matr and nad1 sequences and the nmatr nuclear contigs using bowtie2 2.2.6 (langmead and salzberg 2012). mapping results were manually inspected to identify exon intron splicing junctions. for the mitochondrial genes, edit sites were identified by searching for c - t mismatches detectable in at least 10% of the mapped rna reads. phylogenetic analysis of matr included sequences from 39 angiosperms (supplementary table s3, supplementary material online). 2011) and trimmed using gblocks (castresana 2000) in codon mode with relaxed parameters (b2 = 20 ; b4 = 5 ; b5 = 20). maximum likelihood trees were constructed with the gtr + g substitution model in raxml (stamatakis 2006). supplementary figures s1s2 and tables s1s3 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).	the mitochondrial nad1 gene of seed plants has a complex structure, including four introns in cis or trans configurations and a maturase gene (matr) hosted within the final intron. in the geranium family (geraniaceae), however, sequencing of representative species revealed that three of the four introns, including one in a trans configuration and another that hosts matr, were lost from the nad1 gene in their common ancestor. despite the loss of the host intron, matr has been retained as a freestanding gene in most genera of the family, indicating that this maturase has additional functions beyond the splicing of its host intron. in the common ancestor of pelargonium, matr was transferred to the nuclear genome, where it was split into two unlinked genes that encode either its reverse transcriptase or maturase domain. both nuclear genes are transcribed and contain predicted mitochondrial targeting signals, suggesting that they express functional proteins that are imported into mitochondria. the nuclear localization and split domain structure of matr in the pelargonium nuclear genome offers a unique opportunity to assess the function of these two domains using transgenic approaches.
the history of microbiological research has been dominated by investigations of the agents of human infectious disease. motivated by the desire to culture, characterize, and understand the pathogenicity mechanisms of these organisms, several centuries of microbiological research culminated in a broad range of antimicrobial therapies, vaccines, and immunizations. in more recent years, similar analytical methodologies have been applied to facilitate exploitation of bacteria for industrial applications. two related branches of microbiology environmental microbiology, and the study of intestinal commensals (a branch of the first in purest terms)lagged behind until relatively recently. from the mid 1990s, a range of techniques allowed environmental microbiologists to indentify soil microorganisms in situ, without resorting to culture, based upon ribosomal small subunit rna gene probes. a natural extension of this approach was to sequence large numbers of cloned ribosomal rna gene amplicons, yielding catalogs of all the organisms (the microbiota) present in complex samples. latterly the field of metagenomics has provided technical approaches to sequence large fractions of the entire microbial dna present in an ecological system. coupled with the application of molecular tools for studying commensal bacteria, many of which were originally developed for studying pathogens, there is now an exciting nexus between technologies and research foci whereby commensal bacteria may be studied in the context of intestinal ecosystems. this review will summarize what is known about the effect of introducing probiotic bacteria on the composition and activities of the microbiota, with an emphasis on recent studies using culture - independent methods. the likely mechanisms whereby commensals exert their influence are discussed, and directions for future research are outlined. the notion that certain intestinal microorganisms might benefit the host derives historically from suggestions by metchnikoff and others that putrefying bacteria that contribute to toxification and aging could be deliberately replaced by fermentative organisms [1, 2 ]. in this context, some of the fermentative bacteria, probiotic bacteria are live microorganisms which when administered in adequate amount confer a health benefit on the host [3, 4 ]. many microorganisms that are considered probiotic have been traditionally used to preserve food products by fermentation, and are present in the food in varying numbers, along with their fermentation end products and other metabolites. thus another operational definition of the term probiotic requires the organism in question to be consumed in adequate amounts to confer a benefit. the host benefits that have been attributed to consumption of probiotic microorganisms are diverse (reviewed in [3, 68 ] ; some major examples are listed in table 1), and have been substantiated to different degrees. probiotic bacteria are now included in a wide range of consumer formulations including yoghurts, drinks, capsules, and dietary supplements, and they represent a significant element in the modern functional foods market. organisms used as probiotic agents are frequently members of the genera lactobacillus or bifidobacterium, but escherichia coli, bacillus subtilis, saccharomyces boulardii, and enterococcus faecium are also employed, among others. thus, an organism employed as a probiotic agent may not necessarily be part of what is considered the normal microbiota. tannock distinguishes between allochthonous and autochthonous species [10, 11 ]. autochthonous means bacteria both present and replicating in situ in the human gi tract, as distinct from transiently passing through (allochthonous). bacteria administered as probiotic agents are not necessarily autochthonous to the consuming animal, and indeed some lactobacillus species may only be autochthonous for certain human individual subjects, and possibly not the majority of subjects (see below). rate of growth of allochthonous lactobacilli may be a critically limiting step preventing their establishment. with regard to developing probiotic strains for exploitation, it may prove easier to identify beneficial traits in species that are autochthonous to the human consumer, as consumer acceptance is likely to be easier if the probiotic ingredient in a functional food (a food product with benefit to the consumer over and above inherent nutrition) was first cultured from humans. related to the consumption of probiotic agents a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improves host health [30, 31 ]. it follows from this definition that the bacteria capable of metabolizing prebiotics should be restricted to a small number of beneficial species or strains (reviewed in). in practice, prebiotic compounds must also be refractory to host digestive processes, and the combined catabolic activities of bacteria higher up in the gastrointestinal tract, so that prebiotic compounds are often oligosaccharides towards which probiotic bacteria produce specific hydrolases. prebiotics are commonly found in, or extracted from, plant material including fruits, cereal, and vegetables, but are also present in human milk and colostrum. the best characterized prebiotics include inulin, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, and lactulose (reviewed in). unravelling the health benefits of prebiotics is a challenging task, because these compounds have parallel direct effects on the host, and potentially on multiple members of the microbiota. for example, -glucans are unbranched polysaccharides with (1 - 4) and (1 - 3)-linked -d glucopyranosyl units, that are recognized as important dietary ingredients (reviewed in). -glucans are components of plant cell walls, and are abundant in the endosperm of cereals such as barley and oatmeal. consumption of -glucans has attendant health benefits that are recognized by health and regulatory bodies in several jurisdictions including the us. these benefits include lowering of blood cholesterol and lipoprotein, lowering of postprandial glucose and insulin responses, and enhancement of antitumor monoclonal antibodies. supplementation of mammalian diet with -glucan, or modification by prehydrolysis of in vitro bacterial growth medium, leads to increased numbers and proportions of lactobacilli or bifidobacteria [3941 ]. barley supplementation of rat diet led to an increase in lactobacillus numbers, a decrease in bacteroides and coliforms, and an increase in the production of butyrate. butyrate is an important energy source, signalling metabolite, proliferation stimulus for normal colonic epithelial cells, and anti - proliferative signal for neoplastic colonocytes [42, 43 ], suggesting a potential direct benefit from dietary ingredients or prebiotics that promote growth of clostridia. until recently, the composition of the microbiota was examined by relatively insensitive techniques. culturing the bacteria was unrepresentative, because a large proportion of the bacteria do not grow on standard laboratory media. analysis by temperature gradient gel electrophoresis provided one of the earliest insights into the uncharted complexity of the microbiota. using denaturing gradient gel electrophoresis (dgge) of 16s rrna gene amplicons, the same group later showed that the colonic mucosal microbiota and faecal microbiota were different, and the colonic mucosal microbiota was likely dependent on host factors. meanwhile fluorescent hybridization of probes for 16s rrna genes was being applied to determine species identities, numbers, and proportions of intestinal bacteria [47, 48 ], exemplified by the studies of dore, blaut and colleagues [49, 50 ]. these analyses highlighted extensive inter - individual variation at phylotypes level (among northern europeans), and some correlations of microbiota with age, gender, and geographic origin but which varied between countries. our current understanding of the intestinal microbiota (reviewed in [51, 52 ]) has been significantly shaped by culture - independent methods, in particular the sequencing of 16s rrna gene amplicons, either from clone libraries or direct pyrosequencing of the pcr product. a consensus appears to be emerging in the literature of somewhere between 800 and 1000 bacterial phylotypes being present in the healthy human intestine ; the evidence for which will be selectively presented here. a relatively small - scale investigation by benno and colleagues in 2003 revealed an unexpectedly high number of novel phylotypes in 240 partially sequenced 16s rrna gene amplicons clones derived from six elderly individuals. in a pioneering study, relman and colleagues applied the 16s rrna molecular inventory - based approach, at a much larger scale than previously published, on samples from both colonic sites and faeces ; strikingly, the majority of the sequences derived corresponded to uncultivated species and novel microorganisms. the human stomach, previously considered sterile except for infections with helicobacter pylori, was revealed by 16s rrna gene library sequencing to be well populated by bacteria, based on detection of 128 bacterial phylotypes from 23 gastric endoscopy samples. gill and colleagues sequenced not just 16s rrna genes, but also randomly cloned bacterial dna gill. showed by this metagenomic approach that the bacteria in the gut significantly expand the metabolic capabilities of the human gut. by generating two to three 454 pyrosequencing runs per mouse cecum, gordon and colleagues showed the existence of an obesity - associated gut microbiome with increased capacity for energy harvest. significantly, this balance of the microbiota was borne out when investigating obese humans, showing a seminal link between human obesity and changes in the microbiota. furthermore, the complexity of the microbiota in humans and 59 other mammalian species was shown to be linked to phylogeny (of the mammal) and the composition of the diet. analysis of the metabolic capability likely conferred by the microbial metagenome recently revealed 237 gene families commonly enriched in adult - type and 136 families in infant - type microbiomes. thus, any consideration of the effect of probiotics on the intestinal metagenome should ultimately include analysis of the downstream effects upon the host of impacting on this metabolic capability. a more fundamental consideration is that the genera whose members are among the most commonly employed probiotics bifidobacterium and lactobacillus are not present in the human gastrointestine at the high levels traditionally expected based on culture - based approaches, being represented by 20 phylotypes (ca. 2%) and 36 phylotypes (ca. 3.6%), respectively. thus if probiotic bacteria impart health benefits to the host under natural conditions, that is, in individuals who have normal nonmanipulated numbers of probiotic bacteria, they accomplish this despite being at much lower numbers than are achieved by consumption of probiotic products. there have been relatively few studies which have rigorously characterized the effect upon the whole microbiota of administering probiotic cultures, and until recently, all such studies applied targeted analysis of specific groups of bacteria. in one of the earliest investigations, tannock and colleagues observed transient and modest fluctuations in lactobacillus and bifidobacterium numbers following consumption of a probiotic l. rhamnosus strain dr20. lactobacilli and enterococci were detected more frequently (among 10 subjects) and in higher numbers during consumption. interestingly, the presence of stable populations of lactobacilli before the administration period precluded long - term colonization by the administered probiotic strain. most subjects ceased shedding the probiotic strain in faeces soon after its consumption stopped, but the l. rhamnosus strain remained detectable in faeces of one subject over 2 months after the test period. these data suggest inter - host variables such as bacterium - host or bacterium - diet interactions. probiotics and prebiotics are commonly applied in companion animals and production animals [63, 64 ], and there have been some studies of effects upon the microbiota. administration of a cocktail containing lactobacilli, bifidobacteria, enterococci, and pediococci improved weight gain in broiler chickens, which was associated with an increase in numbers of bifidobacterium spp., administration of a probiotic enterococcus faecium strain reduced e. faecalis numbers in the intestines of weanling piglets, but total numbers of e. faecium remained unchanged, suggesting that the administered strain had displaced part of a fixed number of niche sites occupied by the same species. many investigations have been published describing the effects of probiotic bacteria on human pathogens (reviewed in), some of which are normal components of production animal microbiota. enterobacteriaceae numbers were reduced when a cocktail of two lactobacillus strains was administered to pigs, and a five - strain probiotic combination reduced salmonella enterica serovar typhimurium shedding in pigs. although data from small animal models for human probiotic strains must be interpreted with caution, it was interesting to note from a recent study that administration of l. casei and l. plantarum affected the diversity of murine intestinal lactobacilli, but not the overall bacterial community structure. there was an increase in the number of lactobacilli related to the acidophilus complex in the inoculated mice. these animal models provide an opportunity for determining the effect of probiotic administration on the entire microbiota but must ultimately be repeated in humans if that species is the desired host. studies in humans are currently few in number, and are often focused in nature. for example, consumption of a commercial probiotic yoghurt reduced clostridium difficile - related diarrhoea in hospitalized patients, but effects on the broader microbiota were not studied. alterations in the fecal microbiota have been reported in irritable bowel syndrome (ibs) [72, 73 ]. however, administration of a multispecies probiotic supplementation which alleviated ibs had negligible effect upon the composition of microbiota as measured by quantitative pcr with group - specific primers. however, this approach may have missed changes in microbial composition within these groups. a follow - up study reported stabilization of the microbiota over time, which was related to amelioration of symptomatology that was absent from the placebo control group. alterations of the human intestinal microbiota have also been reported in inflammatory bowel disease (ibd) [7678 ]. given the clinical impetus to find simple non - medicinal solutions to ibd and ibs, one can anticipate renewed vigor in studies of probiotic bacteria as agents for microbiota modulation in these subjects. probiotics also appear to be efficacious as adjunct therapy for infectious diarrhea, with a recent meta - analysis revealing reduction in risk and duration of diarrhea. most of the 23 studies included in this analysis were descriptive rather than investigative of the microbiological aspects, and future determination of the effects on the microbiota wrought by probiotic intervention will be very informative. as recently as 2006, the effect of probiotic administration in humans was still being followed by bacteriological culture, but as concluded by the authors of one such study, there was a clear case for culture - independent molecular methods to be applied instead. community profiling by dgge showed that lactulose increased the levels of bifidobacterium adolescentis in subjects consuming the prebiotic lactulose, whereas the probiotic yeast s. boulardii did not cause any significant universal changes in dgge profiles. figure 1 shows a schematic overview of the potential mechanisms whereby probiotic micro - organisms might influence the intestinal microbiota. consumption of probiotic cultures may modulate the microbiota or change its metabolic properties by competition for nutritional substrates. gordon and colleagues have used transcriptional microarrays to show that introducing a probiotic into the mouse gut changes the way the endogenous microbiota metabolize the diet. when germ - free mice that had been monoassociated with bacteroides thetaiotaomicron were challenged with bifidobacterium animalis or lactobacillus casei, both interventions caused shifts in the gene expression pattern of the b. thetaiotaomicron genome. these differentially expressed gene sets (i.e., in response to the two probiotics) did not overlap, emphasizing that different probiotics elicit different responses. however, many of the genes in b. thetaiotaomicron whose expression was altered by presence of either probiotic strain were related to expansion of the carbohydrate metabolizing capability of b. thetaiotaomicron. thus, one of the ways in which probiotics can impact upon the composition of the microbiota is apparently by competing with them for substrate availability, and by altering the dynamics of carbohydrate utilization by individual microbiota components. this competition is probably not restricted to the intestine, since recent evidence indicates that oral bifidobacterium strains (b. adolescentis) reduce vitamin k concentration, and may thus compete with porphyromonas gingivalis in the oral cavity. the application of metabolic profiling methods to animal models has suggested another indirect way in which probiotic bacteria might impact on the microbiota, namely, by production of a significantly different microenvironment due to a diverse range of metabolic pathway outcomes. in a recent study using germ - free mice colonized by human baby microbiota and exposed to two lactobacillus strains, nicholson and colleagues observed microbiome modification, measured by selected culture regimes. this was accompanied by changes in cecal concentrations of short - chain fatty acids, and marked changes in fecal levels of diverse metabolites including choline, acetate, ethanol, a range of putative n - acetylated metabolites (nams), unconjugated bile acids (bas), and tauro - conjugated bile acids. while a natural focus of these studies is the effect of these metabolites upon the host, it is likely that such gross changes in metabolic profile also impact upon intestinal microbiota composition. as noted in table 1, some probiotic bacteria also produce vitamins, enhanced availability of which may modulate the microbiota. in addition, exopolysaccharide produced by probiotics including lactic acid bacteria could act as a growth substrate for selected components of the microbiota (see figure 1). it has been shown in several recent studies that they can modulate numbers of single model organisms in experimental systems. for example, probiotic l. salivarius strains inhibit the growth of h. pylori in vitro in a strain - dependent manner, by mechanisms involving lactic acid secretion, and another as yet uncharacterized mechanism (k. a. ryan and p. w. o'toole, unpublished). intestinal l. salivarius strains are distinguished by production of a broad - spectrum bacteriocin abp118, but this is not likely to contribute to antagonism to gram - negative bacteria like h. pylori. however, production of this bacteriocin abp118 was identified as the mechanism whereby l. salivarius ucc118 eliminated listeria monocytogenes infection in a murine model, providing the first definitive mechanism for anti - infective activity of a probiotic bacterium in vivo. interestingly, both the wild - type strain ucc118 and a bacteriocin - negative derivative were equally able to suppress salmonella typhimurium infection in the mouse model, suggesting that broader antimicrobial effects on the gram - negative components of the microbiota may occur. from an opposite perspective, production of a bacteriocin - like substance by vaginal enterococci has been linked to reduction in levels of commensal lactobacilli that is linked to vaginosis. natural competition between commensals and opportunistic pathogens may therefore be mediated by mechanisms such as bacteriocin production, that can be exploited for using probiotics to modulate the microbiota. competitive exclusion (see figure 1), whereby adherent probiotic species occlude access of members of the microbiota to the epithelium [89, 90 ], represents another way of modulating the microbiota, although strong evidence for this occurring in vivo is lacking. the most subtle effects wrought by probiotics on the microbiota are potentially those that operate by indirect mechanisms involving the host. improvement of the intestinal epithelium barrier function might theoretically, for example, impact on efficiency of invasion of pathogens, severity of subclinical tissue damage, and release rates of host - derived micronutrients (see figure 1), that could translate into impacts on the microbiota. in an analogous manner, pathological changes in intestinal epithelium might also favor growth of certain members of the microbiota, if inflamed or damaged epithelial cells differentially affect the microbiota. it is well established that some probiotics can suppress inflammation by inhibiting proinflammatory cytokine production [9294 ], and although the molecular basis for this is not currently understood for probiotics, mechanisms and molecules have recently been identified in commensals and pathogens [14, 95 ]. reduction in gut inflammation by probiotics furthermore, some probiotic bacteria have been reported to stimulate the innate immune system both in animal models and in elderly subjects [96, 97 ], by an unknown mechanism. administration of probiotic bacteria could thus bolster innate immune activity against transient pathogens, or non - commensal elements in the microbiota, leading to subtle changes in long - term overall composition. however, more studies are required to substantiate the mechanisms in the probiotic - host interactions, and to investigate if they do in fact impact on the microbiota. there has been a rapid recent accumulation of sequence - based information on the composition of the gut microbiota. however, for pragmatic reasons of sample collection facility, this is largely based on fecal analysis, and the microbiota of the colon and small intestine will be different from feces. studies of the small intestine are particularly warranted because probiotics are proportionally more numerous there, and may exert significant biological activity at this site. there is adequate information in the literature to support the hypothesis that administration of probiotic cultures in high doses to human subjects will impact on the intestinal microbiota. a comprehensive intervention study, supporting this hypothesis by deep compositional and functional metagenomics approaches, and supplemented by metabolomics, is not currently available (june 2008). in this hypothetical study, mechanisms whereby changes in the microbiota that were achieved could be inferred to a degree by global transcriptional analysis, but definitive linkages between bacterial gene products and effects upon the microbiota could be impossible to establish because of the regulatory issues surrounding human trials with genetically modified organisms. as noted above, proof of principle may be established in animal models, but ultimately these studies must be validated in human subjects. there remains the intriguing question of the role, if any, of the relatively small numbers of potentially probiotic organisms as part of the microbiota in ostensibly healthy individuals. do these organisms contribute to maintenance of health or avoidance of disease ? is the level of candidate probiotic organisms in the microbiota critical, and does its importance vary with age ? as noted above, there is reasonable evidence that changes in the microbiota accompany disease states like ibd and ibs, conditions whose prevalence increases with aging. there are attractive hygiene - related hypotheses suggesting that depletion of probiotic commensal microbiota in early life may be responsible for the dramatic rise in diseases involving immune dysregulation. the challenge now is to rigorously tackle the interplay of diet, microbiota, and host factors in tractable experiments that will elucidate the key elements in determining outcomes of this interplay, and allow its manipulation.	probiotics have a range of proposed health benefits for the consumer, which may include modulating the levels of beneficial elements in the microbiota. recent investigations using molecular approaches have revealed a human intestinal microbiota comprising over 1000 phylotypes. mechanisms whereby probiotics impact on the intestinal microbiota include competition for substrates, direct antagonism by inhibitory substances, competitive exclusion, and potentially host - mediated effects such as improved barrier function and altered immune response. we now have the microbial inventories and genetic blueprints to begin tackling intestinal microbial ecology at an unprecedented level of detail, aided by the understanding that dietary components may be utilized differentially by individual phylotypes. controlled intervention studies in humans, utilizing latest molecular technologies, are required to consolidate evidence for bacterial species that impact on the microbiota. mechanistic insights should be provided by metabolomics and other analytical techniques for small molecules. rigorous characterization of interactions between the diet, microbiota, and probiotic bacteria will provide new opportunities for modulating the microbiota towards improving human health.
in this paper, we will try to highlight the importance of various investigations and their crucial role in identifying whether the defect is structural or functional. subsequently, although all ophthalmic signs resolved, she complained of decreased vision in her left eye. initial ophthalmic injury was detected by optical coherence tomography scan showing a neurosensory detachment of the fovea. on later examination, although the optical coherence tomography scan showed no structural damage, electrodiagnostic tests showed a functional defect at the fovea. a 24-year - old female presented to the eye department complaining of significantly blurred vision in the left eye and bilateral subconjunctival hemorrhages immediately after a bungee jump from the top of a crane. there was associated nausea, headache, neck pain, and a feeling of tightness in the chest. the patient had a history of epilepsy during childhood and was symptom - free since the age of 11 years. on general examination, she was alert with a score of 15 on the glasgow coma scale. neurological examination was normal. computed tomography of the head and neck did not reveal any abnormalities. unaided visual acuity of the right eye was 0.00 logmar, and the left eye was 1.08 logmar, not improving with pinhole. anterior segment examination revealed normal pupillary reactions to light and accommodation without a relative afferent pupillary defect. there were a few round deep retinal hemorrhages in the left eye temporal to the optic disc along with a dull foveal reflex (figure 1). optical coherent tomography (oct) of both maculae showed a localized left subfoveal neurosensory detachment (figure 2). fundus fluorescein angiography (ffa) was undertaken, which showed the presence of hemorrhages in the left macula, without hyperfluorescence, leakage, or pooling. a week later, the unaided vision of the right eye remained stable at 0.00 logmar, while the unaided vision of the left eye had improved to 0.00 logmar, but was subjectively blurred. left macular hemorrhages had resolved and oct images showed normal foveal contour with no signs of neurosensory detachment (figure 3). one month later, the patient was still complaining of left eye vision being blurred with no obvious corresponding clinical, biomacroscopically, or oct imaging findings. three and a half months later, subjective left eye blurred vision remained even though oct mapping was normal in both eyes, and unaided vision was 0.00 logmar bilaterally. pattern electroretinogram (perg) was borderline for the right eye, and slightly subnormal for the left eye indicating a degree of macular dysfunction. with the increased popularity of bungee jumping as a leisure activity over recent years, there have been reports of associated ocular complications.13 most of these ocular complications were subconjunctival and benign, but they were also serious given that patients have been found with severe retinal hemorrhages.24 most authors agree that the sudden increase in intrathoracic and intravenous blood pressure leads to the development of these retinal hemorrhages,5 even though the actual mechanism of this pressure increase is not clear. chan suggests that a sudden change in the hydrostatic pressure in the ciliary and retinal circulations that occurs in relation to the wide change of gravitational forces during the dive may be the most important etiological factor.4 this view is supported by other authors who agree that the most likely cause of these retinal hemorrhages is a sudden rise of intravascular venous pressure in the upper portion of the body during the deceleration phase of the jump in a fashion similar to purtscher and valsalva retinopathy.3,6 however, innocenti and bell suggested that breath - holding and tensing of the abdominal muscles are the main causes of the sudden rise in intrathoracic and intravenous pressure, which are thought to cause the retinal damage.5 habib suggested that as the intravenous pressure increases, there is a spontaneous rupture of superficial capillaries resulting in a hemorrhagic detachment of the internal limiting membrane in the foveal region.7 the gravitational force (g - force) on a body is its acceleration relative to the freefall. the term g - force is technically incorrect, as it is a measure of acceleration, not force. g - forces, when multiplied by mass, are associated with a certain type of mechanical force.8 although the human body can survive high positive and negative gs, a rapid deceleration in short duration of more than 3 gs (negative) is enough to cause ocular injuries.9 retinal and subconjunctival hemorrhages are the most common ocular complications, as a rapid -3 g deceleration will increase the intravenous pressure to 100 mmhg or more. this is the same mechanism that produces the retinal hemorrhages in the shaken baby syndrome. it has been calculated that the rapid gs during the shaken baby syndrome can go up to 9 gs, resulting in hemorrhages in different anatomical layers of the retina.8 however, there are species that can handle the rapid gs without any problem, such as the woodpecker. the woodpecker is able to use his chisel - tipped bill to hammer wood at the rate of 16 times a second, or nearly 1000 pecking blows per minute. the suddenness of this movement in which the woodpecker s head is brought to a halt during each peck results in a stress equivalent of 1000 times the g - force. his highly adapted eyes10 have an avascular retina and pectin, which play a role in maintaining an effective cushion. this pectin can be briefly filled with blood, which can elevate his intraocular pressure which, in effect, maintains a firm pressure on the crystalline lens and retina to prevent any retinal damage from a higher g - force. human orbits contain enough quantity of orbital soft tissue (retrobulbar fat pads), tight fibrous compartments, and ligaments allowing for a freer, but controlled, globe movement within the orbit. without a structure containing pectin and acting as a vascular cushion (as is the case in the woodpecker s orbits), a bungee jumper (or infant in shaken baby syndrome) is subject to excessive acceleration deceleration forces, and he or she would, obviously, develop ocular complications. optic nerves are firmly attached to sclera and to the orbital apex, so violent translational movements of the globe can potentially generate severe traction forces at these sites. the mechanical effect of the g - force on vitreous movement also contributes to retinal injuries by avulsing at the anterior segment of the vitreous base, causing oral retinal tears, retinal detachments, vitreous hemorrhages, posterior vitreous detachment, and posterior macular holes. furthermore, there is another pathophysiological cause of the ocular injuries due to bungee jumping, which is the valsalva phenomenon s effect on the eye. the patient s normal erg a test of retinal photoreceptors and a test of inner nuclear layer function showed no evidence of diffuse retinal dysfunction ; however, the abnormal result of the perg test a test of retinal ganglion cell function is consistent with a degree of macular dysfunction, particularly in the left eye of our patient. we could not comment on our patient s multifocal ergs as she was unable to perform this test and we had to abandon the procedure. our case report highlights the importance of tests such as oct and perg in the diagnosis of posttraumatic macular cone dysfunction. we were able to point out a plausible cause that might be able to explain the patient s complaint of slightly blurred central vision in the left eye. the posttraumatic macular neurosensory detachment resolved, and the visual acuities were -0.00 logmar, bilaterally. indeed, the oct showed no structural damage, but the electrodiagnostic tests showed a functional defect at the fovea. ultimately, bungee jumpers need to make sure that stringent safety guidelines are implemented by the bungee jump provider.	aimin this paper, we will try to highlight the importance of various investigations and their crucial role in identifying whether the defect is structural or functional.case historya 24-year - old woman presented with ocular complications after bungee jumping. subsequently, although all ophthalmic signs resolved, she complained of decreased vision in her left eye.conclusioninitial ophthalmic injury was detected by optical coherence tomography scan showing a neurosensory detachment of the fovea. this was not initially detected on slit - lamp examination or fluorescein angiography. on later examination, although the optical coherence tomography scan showed no structural damage, electrodiagnostic tests showed a functional defect at the fovea.
acute - phase response is a set of immediate host inflammatory reactions that counteract challenges, such as tissue injury, infection, and trauma. acute - phase proteins, including c - reactive protein (crp) and serum amyloid a (saa), are primarily produced by hepatocytes and chiefly induced by the proinflammatory cytokine interleukin-6 (il6). in the setting of an acute - phase reaction, crp and saa levels can increase up to 100- to 1000-fold for a brief period and typically return to baseline levels within 2 wk. an elevated crp level has been reported to predict the incidence of cardiovascular events and vascular mortality among apparently healthy individuals and among patients with established cardiovascular disease (cvd) [25 ]. the large jupiter trial prospectively confirmed that crp levels aid in tailoring statin treatments for primary prevention in patients with elevated crp but normal low - density lipoprotein (ldl) cholesterol levels. family and twin studies have reported additive genetic factors accounting for 27%40% of the variance in crp levels [79 ]. recent genome - wide association studies (gwas) have identified multiple loci influencing crp levels, including crp [6, 10, 11 ]. saa is secreted by hepatocytes, macrophages, vascular smooth muscle cells, and endothelial cells. saa promotes the chemotaxis of monocytes and neutrophils and plays critical roles in a wide range of functions including cholesterol transport, high - density lipoprotein (hdl) metabolism, and host defense [13, 14 ]. furthermore, saa induces, promotes, or influences susceptibility to several chronic diseases such as atherosclerosis and its clinical complications [1518 ]. twin studies have suggested a substantial genetic contribution of baseline saa levels, with heritability estimates of 49%67%. gwas have reported that the chromosome region at 11p15.5-p13, which includes the saa family, accounts for most of the explained variance in circulating saa levels. the saa1 single nucleotide polymorphism (snp) rs12218 was found to be associated with multiple atherosclerotic cvds and related risk factors [2023 ]. mediation analysis suggested a suppression effect of soluble e - selectin (se - selectin) on the association between abo genotypes and triglyceride to hdl cholesterol ratios. thus, using pathway and mediation analysis is crucial to further elucidating the genetic determinants of inflammatory marker levels, which may be one of the reasons for the missing heritability in gwas. therefore, we tested the clinical and biomarker correlates of crp and saa levels and the statistical association of crp and saa locus variants with the circulating levels of metabolic and inflammatory biomarkers by using mediation analysis in a taiwanese sample population. this study was approved by the institutional review board of taipei tzu chi hospital, buddhist tzu chi medical foundation (irb number : 02-xd38 - 089). a total of 599 han chinese subjects (315 men and 284 women with mean ages of 46.1 10.0 and 46.8 9.9 y, resp.) were recruited during routine health examinations between october 2003 and september 2005 at chang gung memorial hospital. the subjects responded to a questionnaire on their medical history and lifestyle characteristics and underwent a physical examination that involved measurement of height, weight, waist and hip circumference, and blood pressure (bp) in the sitting position after 15 min of rest. exclusion criteria included an age younger than 18 y, crp levels of above 10 mg / l, a history of myocardial infarction, stroke, or transient ischemic attack, cancer, and current renal or liver disease. the clinical characteristics and biometrics of the study population nine snps around the crp and saa1 loci were selected for this study (see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/5830361). genotyping was performed using pcr, followed by restriction enzyme digestion or using taqman snp genotyping assays obtained from applied biosystems (abi, foster city, ca, usa). the laboratory examinations and assays were performed as described previously [27, 28 ]. most markers, including serum crp, saa, soluble intercellular adhesive molecule (sicam1), soluble vascular cell adhesive molecule (svcam1), se - selectin, adiponectin, matrix metalloproteinase-9 (mmp-9), and plasma monocyte chemotactic protein-1 (mcp-1), were measured using a sandwich enzyme - linked immunosorbent assay (elisa) developed in - house. all in - house kits showed good correlation with commercially available elisa kits. circulating serum resistin, lipocalin-2, mmp-2 and plasma mmp-1, soluble p - selectin (sp - selectin), soluble tumor necrosis factor receptor ii (stnfrii), and interleukin-6 (il6) were measured using commercially available elisa kits from r&d (minneapolis, mn, usa). the chi - square test was used for testing to compare categorical variables of diabetes mellitus and smoking. the clinical characteristics that were continuous variables are expressed as means sds and were tested using a two - sided t - test or analysis of variance (anova). pearson correlation coefficients (r) were calculated to determine the association between crp or saa levels and clinical and biochemical factors with the adjustment of age and sex. furthermore, a general linear model was applied to capture the major effect of each polymorphism on clinical and biochemical variables with bmi, age, gender, and smoking status as confounding covariates. we also used dominant models for numeric association test after recoding our snps from categorical variables to continuous variables, such as 0, 1 of a particular allele. a value of p < 0.05 using two - sided tests was considered statistically significant. all the above calculations were performed with standard statistical spss 12 software (spss, chicago, il, usa). golden helix svs win32 7.3.1 software was used to analyze the deviation from the hardy - weinberg equilibrium and to estimate the linkage disequilibrium between polymorphisms. values of hdl cholesterol, ldl cholesterol, total cholesterol, triglyceride, crp, saa, sicam1, svcam1, se - selectin, sp - selectin, mmp-1, mmp-2, mmp-9, mcp-1, stnfrii, and il6 were logarithmically transformed prior to statistical analysis to adhere to a normality assumption. the bonferroni method was used to correct for multiple comparisons where applicable. to explore the mediating effects of saa levels on the relationship between the saa1 genotypes / haplotypes and crp levels, and vice versa for the crp genotypes / haplotypes, a conceptual model was hypothesized for the test, and four criteria were suggested for evaluating the mediating and suppression effects. for example, in criterion one, independent variable (saa1 genotypes / haplotypes) must predict the mediator (saa levels). in criterion two, the mediator must predict the dependent variable when adjusting for independent variable (crp level). the mediation effect was calculated as the product of the two regression coefficients from criterion one and criterion two, which reflected the intermediate pathways from independent variable to mediator and in turn to dependent variable. the regression coefficient relating independent variable to dependent variable adjusting for the mediator was expressed as direct effect. for criterion three, the total effect, expressed as the effect of independent variable on dependent variable, can be obtained by summation of direct and mediation (indirect) effects. in criterion four, the mediation effect must be significant using the procedure outlined by sobel [26, 29 ]. a suppression effect may be indicated in a situation when the direct effect is larger than the total effect. in this situation, the direct and indirect effects often have fairly similar magnitudes and opposite signs, which may entirely or partially cancel each other out and result in zero or a nonzero but insignificant total effect. the coefficients and standard errors from the model above were further used to conduct a sobel test for mediation. the sobel test was performed using a tool for mediation tests (http://www.quantpsy.org/sobel/sobel.htm), in which the null hypothesis h0 : = 0 is tested. the test statistic s, which is approximately distributed as z (2), is obtained by dividing the estimated mediation effect () by the standard error () in (1). the reported p values are drawn from the unit normal distribution under the assumption of a z value of the hypothesis that the mediated effect equals zero in the population. 1.96 are the critical values of the test ratio which contain the central 95% of the unit normal distribution:(1)2=22+22(2)z=sqrt22+22,where sqrt is square root. a summary of demographic features, clinical profiles, and biomarker levels for the health examination participants is provided in table 1. no significant deviation from the hardy - weinberg equilibrium was detected for the studied polymorphisms. all of the studied polymorphisms in the same chromosomal region were in strong pairwise linkage disequilibrium (supplementary tables 2 and 3). circulating crp levels were positively associated with approximately all anthropometric and metabolic traits, except for the quicki index and hdl cholesterol level, in which a negative association was observed, and the ldl cholesterol and urinary acr levels, in which no significant association was observed (table 2). by contrast, saa levels had a similar but less significant trend of the associations, having no significant association with waist - hip ratios, egfr, fasting plasma glucose, hdl cholesterol levels, and urinary acr. regarding inflammatory markers and adipokine levels, crp levels were positively associated with circulating saa, fibrinogen, se - selectin, stnfrii, il6, leptin, and lipocalin-2 levels and negatively associated with adiponectin levels, whereas saa levels were positively associated with circulating crp, fibrinogen, il6, and leptin levels. after adjusting for clinical covariates, significant association of three saa1 polymorphisms with saa levels was observed using an additive inheritance model (table 3). using a dominant inheritance model, a minor allele of rs4638289 was found to be associated with a higher saa level (p = 7.48 10), whereas minor alleles of rs11024591 and rs7131332 were found to be associated with a lower saa level (p = 1.28 10 and p = 1.93 10, resp.). using haplotype analysis, two haplotypes (gatt and aaac) inferred from four snps were found to be associated with saa levels (p = 4.15 10 and p = 1.20 10, resp.) the association between crp genotypes / haplotypes and crp levels has been reported previously. in this study, we excluded subjects aged younger than 18 y or had crp levels above 10 mg / l, and the results were similar to those of previous reports, with the exception of no significant association of the rs1800947 genotypes with crp levels (table 3). association of crp and saa1 genotypes / haplotypes with various clinical parameters and biomarker levels is shown in supplementary tables 47. after further adjusting for saa levels, a significant association of crp levels with rs4638289, rs7131332, and rs11024591 was observed in the additive inheritance model (p = 2.48 10, p = 0.040, and p = 0.016, resp.) and with rs4638289 in dominant inheritance model (p = 2.41 10). adjustment of circulating saa and crp levels revealed that the saa1 haplotype aaac was associated with lower crp levels (p = 1.91 10) and that the crp haplotype aagcg was associated with lower saa levels (p = 0.046). subgroup analysis of saa quartiles revealed at least a trend of lower crp levels with the minor allele of the rs4638289 genotypes in each saa quartile (p = 0.008, p = 0.002, p = 0.023, and p = 0.275, separately). by contrast, a significant increase in the minor allele frequencies of the rs4638289 genotypes was observed in the higher saa quartiles (p = 9.39 10), which illustrated no significant association between crp levels and rs4638289 genotypes when all of the subjects were pooled (p = 0.733) (figure 1). saa1 and crp genotypes / haplotypes were analyzed (table 5). in brief, the saa1 genotypes / haplotypes were significantly associated with saa levels (criterion 1), which in turn had significant positive effects on crp levels (criterion 2). the total effect of saa1 genotypes / haplotypes on crp levels was 0.002, 0.006, 0.006, and 0.054 with all p values nonsignificant (criterion 3). sobel tests for mediation of the results of the corresponding crp levels showed z = 9, 6, 6.67, and 8.02 (all p < 10) (criterion 4). moreover, the direct effects () of the saa1 genotypes / haplotypes on crp levels were greater than their total effects (+) and had similar magnitudes as mediation effects but opposite signs (), demonstrating a suppression effect in this model (figure 2). the suppression effects of crp levels on the association between the crp haplotype aagcg and saa levels, of the criteria, were also observed. this investigation involved analyzing the association of crp / saa1 snps and crp / saa levels with various clinical parameters and biomarker levels. as predicted, both crp and saa levels correlated with multiple metabolic phenotypes and inflammatory marker levels, suggesting their crucial roles in atherosclerotic processes. similar to the results of previous studies, our data showed a strongly significant association between crp / saa1 snps and their respective circulating levels. by adjusting their individual circulating levels further, we also found saa and crp variants to be significantly associated with circulating crp and saa levels, respectively. mediation analysis revealed that saa levels have a suppression effect on the association between saa1 genotypes / haplotypes and crp levels, and crp levels have a suppression effect on the association between crp haplotypes and saa levels. these results suggested the relevance of saa1/crp variants as the genetic determinants of both crp and saa levels. reported two gene loci, 11p15.5 and 1p31, to have a considerable impact on saa levels, which compose approximately 20% of the total estimated heritability. in this study, we confirmed a highly significant association of rs4638289 with saa levels in a taiwanese sample population. previous studies have shown that the saa1 snp rs12218 was associated with multiple atherosclerotic cvds and related risk factors [2023 ]. our data showed no evidence of the association of the rs12218 genotypes with saa levels and various other metabolic or inflammatory phenotypes (table 3 and supplementary table 5). additional studies may be necessary to elucidate further the role of this snp in different ethnic populations. compelling experimental and clinical evidence suggests a crucial role of inflammation in the initiation and progression of atherosclerosis. an abundance of epidemiological data has linked circulating inflammatory biomarkers with the risk of atherosclerotic cvds and their adverse outcomes. among the wide array of inflammatory biomarkers that have been studied, high - sensitivity crp has received the most attention for its use in screening and risk reclassification and as a predictor of clinical response to satin therapy. our study revealed a significant association of crp and saa levels with approximately all of the studied atherosclerosis - related traits, including glucose metabolism, renal function, levels of various adipokines, and inflammatory markers, as well as adiposity and bp status. although most of the correlations were not strong, one possibility is that the study participants are relatively healthy without manifested systemic diseases. further study in patient populations, such as those with atherosclerotic cardiovascular diseases, will help us to understand whether crp / saa levels have a stronger association with various atherosclerotic risk factors in disease populations. these results further support the multifaceted perspective of the crp and saa levels affecting the pathogenesis of atherosclerosis. recent gwas studies have shown multiple genetic determinants of crp and saa levels ; however, the probability of snps contributing much to the current approach of risk assessment, based on conventional risk factors, is low. our data provide the first evidence suggesting the importance of crp and saa1 variants as genetic determinants of both crp and saa levels. showed that gene - gene interaction between rs4638289 and genetic variants of saa regulators influences cvd. shah. revealed the association of interleukin-6 receptor (il6r) genotypes with both il6 and crp levels. thus, a paradigm shift would involve using a gene - centric approach to investigating an entire pathway rather than assessing isolated mutations for providing more useful information on complex traits that involve a high number of genes and are subject to environmental regulation of gene expression and cellular phenotypes. suppression effect and possible mechanisms. mediation hypotheses suggest a suppression effect if the statistical removal of a mediating effect enhances the relationship between the independent and dependent variables. we previously reported that se - selectin levels have a suppression effect on the association between abo blood group genotypes and triglyceride to hdl cholesterol ratios ; this emphasizes the importance of the relationship between abo blood groups and atherogenesis. in our recent study, adiponectin levels also acted as suppressors for the association between cdh13 snps and metabolic syndrome and metabolic phenotypes. the data in our current study suggested that saa and crp levels act as suppressors of the association between their respective gene variants and circulating levels of other inflammatory biomarkers. the association between saa rs4638289 genotypes and crp levels became more significant in subgroup analysis with different quartiles of saa levels. lower crp levels were observed in each saa quartile in subjects with the minor allele a of the rs4638289 genotypes, whereas higher aa+at genotype frequencies were observed with increased saa quartiles (which are associated with higher crp levels) ; this may partially explain why no significant difference was observed between rs4638289 genotypes and crp levels when all of the studied subjects were pooled for analysis without adjusting the saa levels. micrornas have emerged as key gene regulators, including trans- or cisregulation, for diverse biological pathways in various vascular and metabolic diseases. online resources for microrna target prediction may be helpful in searching for possible candidates linked between crp and saa loci. it is also possible that the association of the saa polymorphism with crp levels may be due to linkage disequilibrium with another polymorphism that is linked to crp regulation. the proinflammatory cytokine il6 is a critical mediator that induced both crp and saa expression in hepatocytes. significant associations of circulating il6 levels with crp and saa levels were noted in this investigation ; however, our preliminary data revealed that the association of crp / saa genotypes / haplotypes with crp and saa levels was not affected with adjustment of il6 levels (data not shown). thus, further investigation involving multiple candidate targets will be necessary in the future to establish the molecular basis of the suppression effect. recent gwas have identified hundreds of genetic variants associated with complex human diseases and traits. however, complex inheritance can assume numerous forms and gwas have only partially explained heritability. the missing heritability may be due to multiple factors, including a high number of variants with smaller effects yet to be found, rare variants with larger effects, which are poorly detected by available genotyping arrays, structural variants poorly captured by existing arrays, low power for detecting gene - gene and gene - environment interactions, and sequence - independent epigenetic effects including noncoding micrornas [41, 42 ]. our recent investigations revealed that mediation analysis of suppression effects may elucidate several previously unidentified associations between genetic variants in one gene and other closely associated metabolic or inflammatory phenotypes, which may in turn partially explain the missing heritability in complex diseases. our study has several limitations, one of which is the relatively modest number of studied subjects. furthermore, only 9 of the crp and saa1 snps were analyzed, and this incomplete genotyping may not represent all of the haplotypes in crp and saa1. another limitation of this study is its cross - sectional design, which could draw only limited inference regarding the relationships between exposure and outcome. finally, the examined subjects were ethnically chinese ; hence, caution should be exercised when extrapolating our results to other ethnic groups. our data revealed a significant association of crp and saa1 variants with both crp and saa levels, which are highly correlated with multiple atherosclerosis - related traits. these results provide further evidence of the role of suppression effects in biological science and may partially explain the missing heritability in genetic association studies. further analysis of the interrelationship between entire pathways of inflammatory gene variants and circulating levels may further elucidate the pathogenesis of atherosclerotic cvds.	to test the statistical association of the crp and saa1 locus variants with their corresponding circulating levels and metabolic and inflammatory biomarker levels by using mediation analysis, a sample population of 599 taiwanese subjects was enrolled and five crp and four saa1 variants were genotyped. correlation analysis revealed that c - reactive protein (crp) and serum amyloid a (saa) levels were significantly associated with multiple metabolic phenotypes and inflammatory marker levels. our data further revealed a significant association of crp and saa1 variants with both crp and saa levels. mediation analysis revealed that saa levels suppressed the association between saa1 genotypes / haplotypes and crp levels and that crp levels suppressed the association between crp haplotypes and saa levels. in conclusion, genetic variants at the crp and saa1 loci independently affect both crp and saa levels, and their respective circulating levels act as suppressors. these results provided further evidence of the role of the suppression effect in biological science and may partially explain the missing heritability in genetic association studies.
urticaria represents a transient edema within the dermis ; the individual urticarial lesions characteristically clear within hours. it is a vascular reaction of the skin, triggered by many known as well as unknown factors that result in liberation of vasoactive substances such as histamine, prostaglandins, and kinins. most lesions of urticaria are small, less than 10 mm, but individual large lesions of 610 cm may occur the so - called giant urticaria. clinically, urticaria is classified into the acute (duration six weeks) types. episodic urticaria, which occurs intermittently but recurrently over months or years, is also recognized. acute urticaria in children can be treated symptomatically needing no further investigations, while chronic urticaria requires a detailed search for trigger factors. chronic urticaria is relatively less frequent in children compared to adults, with only occasional documentation of autoimmune urticaria. the etiology of chronic urticaria (cu) in childhood remains incompletely understood because of limited data available on children. the objective of this retrospective study is to examine some of the possible etiology of urticaria and its response to specific treatment in children, after evaluation of available data. a retrospective hospital data - based study study was conducted in children who had attended the out - patient departments of dermatology and pediatrics from june 2005 to may 2010. 80 patients suffering from urticaria (excluding physical urticaria) between the ages of 1 and 14 years were examined. detailed history of these children included food allergies, drug intake, personal and family history of atopy, insect bites, infections, and infestations. the initial evaluation consisted of a thorough history, detailed physical examination, and basic laboratory tests such as complete blood count, stool microscopy, and urine examination, to rule out a focus of infection. specific tests including thyroid function tests, thyroid anti peroxidase antibodies, serum ige levels, asst, x - ray of paranasal sinuses, and investigations for hepatitis a virus were done after baseline investigations. 80 children, comprised 49 males and 31 females with majority (44, 55%) of children under the age group of eight and eleven years [table 1 ]. chronic urticaria was observed in 56.25% of these children and the remaining had acute urticaria. a majority of parents gave a preceding history of infections and the second most common observation of parents was aggravation of urticaria after food [table 2 ]. during detailed history taking, the various food materials found to be aggravating the urticaria were peanuts, eggs, cheese, and chinese food in that order. drugs aggravating urticaria were mefanamic acid, ibuprofen, nimesulide, ofloxacin, ampicillin, and cotrimoxazole. a complete blood count showed the hemoglobin to be less than 10 gm% in eight children. demographic features of patients detailed histories obtained microscopic examination of the urine showed pus cells in 16 and ova / cysts were documented in the stool examination of 10 children. the autologous serum skin test was positive in 17 and thyroid dysfunction was documented in 12 patients. out of the 12 patients four had hyperthyroidism and the remaining eight had hypothyroidism, two among the eight were positive for thyroid antiperoxidase antibodies. raised serum ige levels were present in 15. a majority of the children were treated with antihistamines ; a definite association of suspected underlying cause with specific treatment was found in 16 (20%). out of 16, five were treated for worms (albendazole), three received treatment for upper respiratory tract infection (two were given amoxicillin and clavulinic acid, one was given azithromycin), two each had received specific treatment for urinary tract infection (nitrofurantoin), hypothyroidism (thyroxine), and removal of an offending drug, and one each was treated for hepatitis a virus and dental caries. the episode is short - lived and simply a nuisance, but for a few it may be a sign of a serious disorder. in contrast to the ease of its diagnosis, the etiological factors are often difficult to determine. the natural history of urticaria in children is distinct from that in adults. urticaria is a common disease that can persist or recur over long periods of time, spanning years, causing distress, and at times can be life - threatening. as a cause is often not found, it presents the physicians with a diagnostic and therapeutic challenge. due to the scarcity of data regarding the etiological factors of urticaria in children, this retrospective data - based study was undertaken in an effort to determine various etiological factors of childhood urticaria. urticaria in childhood is common ; according to different studies, 15 20% of children suffer from at least one episode of urticaria by adolescence.[47 ] in our study 56.25% had chronic urticaria ; another study from a tertiary referral center from india reported that 80% children had cu out of 44 children. in this study, a definite association to an underlying cause was present in 37.5% of the patients, although suspicion was entertained in 66.25% of the patients, in the remaining there was no response seen on offering specific treatment. a review of 94 children with cu revealed that 58% became symptom - free for six months or more, whereas the cause in this study was determined in only 16%. in another report of 226 children (age : 1 14 years) with cu, only 21% were determined to have a causal factor. only a few studies have been conducted on the etiological factors of urticaria in children and few of these studies in children showed that in more than 70% of the cases, the exact cause could not be identified. in our study, asst was positive in 17 of the 80 (21.25%) children ; another study of asst in children showed a positive reaction in 35.29% of the patients. in western literature, a positive asst was reported in 25 45% of adult patients with chronic idiopathic urticaria. a positive test is suggestive, but not the diagnostic of an autoimmune basis for a patient 's urticaria. confirmation is needed by in vitro testing of the patient 's serum for anti - fcr1 or anti - ige autoantibodies. an autologous serum skin test is a fairly good baseline test for diagnosing suspected cases of autoimmune urticaria, particularly where a basophil histamine releasing test is not available. it has good sensitivity and an even better specificity for detecting autoantibodies, even in children. management of urticaria in children requires a detailed structured history, a complete review of systems, baseline investigations, and other specific in vivo and in vitro tests. various studies suggest that in a majority of pediatric patients, the history of a preceding infection, particularly respiratory infection, is obtained.[571314 ] in our study also, a majority (20% out of 32.25%) of the patients had infections as the underlying cause [table 4 ]. details of patients showing response to specific treatment exhaustive work up with extensive laboratory, technical diagnostics, challenge tests and skin prick testing should be reserved for individual cases following detailed history. a well designed structured study is still in need to find the etiological factors of urticaria in children.	background : urticaria is a common dermatological manifestation in adults with relatively infrequent occurrence of chronic urticaria (cu) in childhood. the etiology of urticaria in childhood remains incompletely understood because of limited data on children.objective:we carried out this retrospective data - based study to determine different etiological factors and response to treatment in pediatric patients presenting with urticaria.materials and methods : eighty children (m : f 49 : 31) between the ages of 1 and 14 years, who presented with urticaria excluding patients of physical urticaria attending the outpatient department of pediatrics and dermatology were included in the study. patients were evaluated after taking a detailed history, doing a thorough physical examination, and basic laboratory investigations. specific in vivo and in vitro tests were performed after the initial evaluation and consent of patients.results:out of eighty patients, 35 (43.75%) presented with acute urticaria and 45 (56.25%) gave a history of chronic urticaria.cu. an underlying cause was suspected in 53 cases (66.25%), although a definite association with response to a specific treatment was correlated in 30 (37.5%). infection was the most common underlying causative factor. underlying thyroid dysfunction was observed in 12 patients, of whom two had thyroid anti peroxidase antibodies. autologous serum skin test was positive in 17 patients.conclusions:in the present study infections were the most common etiological factor for urticaria in children.
tsetse flies are large biting and blood - feeding flies of great economic, veterinary, and medical importance, due to their ability to transmit african animal and human trypanosomoses. tsetse - transmitted trypanosomoses occur in 38 sub - saharan african countries with averages of 15,000 human cases and one million cattle deaths reported yearly, exposing over 70 million people and 160 million cattle to the risk of infection in the region. these vectors are distributed over wide range of habitats covering about 10 million square kilometers of potential grazing and farming lands in sub - saharan africa. tsetse abundance and feeding behaviours determine the degree of vector - host contact and may have a serious impact on the risk of pathogen transmission. the degree of contacts between vectors and vertebrate hosts is an important determinant of their vectoral capacity and is determined by the vector feeding patterns on its hosts. as vectors of human and animal trypanosomoses, the epidemiology of these diseases is determined largely by their abundance, density, and feeding behaviours. the importance of vector feeding patterns in the epidemiology of diseases has been recognized for quite a long time. however, majority of the traditional approaches used earlier were serologically based and included haemagglutination crystallization, agglutination reactions, passive haemagglutination test, immunohistochemical methods [4, 5 ], precipitin test [6, 7 ], and enzyme linked immunosorbent assay [810 ]. despite the level of successes achieved by these traditional approaches, limitations ranging between high percentage of false positives, low sensitivity, species cross infectivity, the need for producing specific antibodies to several species, and the inability to discover unpredicted hosts the development of molecular based techniques for the identification of vertebrate host blood meals provides more convenient approaches and has already been employed in the detection of host blood meals in several arthropod vectors including tsetse flies, ticks [13, 14 ], triatomine bugs, and mosquitoes [16, 17 ]. in order to understand the epidemiology of trypanosomosis in the gashaka - gumti national park in nigeria, we trapped and studied the feeding patterns of tsetse flies using the mitochondrial cytochrome b pcr and xenomonitored trypanosomes in tsetse flies using its pcr. the study was carried out in gashaka - gumti national park which is located in taraba state, northeastern nigeria, between latitudes 655 and 617n and longitudes 1113 and 1121e. it is the largest national park in nigeria, covering 6402 km and consisting of both savannah grassland and montane forest vegetation. altitude ranges from 457 meters (1,499 ft) to 2419 meters (7,936 ft). it is an important water catchment area for the benue river with abundant river flow even during the markedly dry season. enclaves for local fulani pastoralists exist within the park 's boundary allowing for farming and grazing. the reserve also contains a wide range of wild fauna of over 103 different species. we conducted a cross - sectional study in four randomly selected villages (burto, gindin dutse, goje, and serti) located at least 510 km apart around the gashaka community taking cognisance of their relationship with the gashaka - gumti national park which is a tourist centre. we identified forested and riverine areas where 6 traps were mounted 100 meters apart two times weekly for four months in each of the four villages. tsetse flies were trapped by the use of biconical traps as described by charllier and laveissiere. six traps were mounted 100 meters apart along streams twice every week between july and october, 2014. traps were emptied every 24 hours and the flies were identified using morphological characteristics as described by murray.. following identification, a proportion of the collected tsetse flies were dissected using dissection microscope to check for vector stages of trypanosomes on the field while the most engorged were selected for blood meal identification. flies that were positive following dissection and the engorged were all preserved at 80c until needed for pcr. blood meal dna extraction was done using the modified salt procedure earlier described by norris.. tsetse abdomen was detached from the thorax and head and homogenized in 100 l of extraction buffer containing 0.1 m nacl, 0.2 m sucrose, 0.1 m tris - hcl, 0.05 m edta, ph 9.1, and 0.5% sodium dodecyl sulfate (sds) and incubated at 65c for one hour. trypanosome dna was extracted using genejet genomic dna extraction kit (thermo scientific, germany). midguts of positive tsetse flies were pooled together in twos and homogenized and 200 l of the homogenate was lysed by adding 400 l of lyses solution and 20 l of proteinase k as recommended by the manufacturer. extracted dna we conducted multiplex pcr with four forward primers and a universal reverse primer to identify mitochondrial cytochrome b of vertebrate hosts in blood meals of tsetse flies (table 1). cycling conditions were as follows : denaturation at 98c for 10 seconds to activate the phusion flash ii dna polymerase, followed by 36 cycles at 98c for 1 second, annealing for 50 seconds at temperatures of 58.5c, 59.5c, 62.0c, and 62.5c for cattle, dog, pig, and human primers, respectively. extension was at 72c for 40 seconds and final extension at 72c for 5 minutes according to the manufacturers ' instructions. all amplified products were analyzed by electrophoresis in a 2% agarose gel and uv illumination after ethidium bromide staining. its-1 pcr was used to identify the species of trypanosomes in the tsetse flies using primer sets with the sequences 5-ccggaagttcaccgatattg-3 (forward) and 5-ttgctgcgttcttcaacgaa-3 (reverse) designed by. pcr conditions were as follows : an initial denaturation step for 10 seconds at 98c to activate the phusion flash ii dna polymerase, four cycles of amplification with 1-second denaturation at 98c, 5-second hybridization at 58c, and 15-second elongation steps at 72c ; eight cycles of amplification with 1-second denaturation at 98c, 5-second hybridization at 56c, and 15-second elongation steps at 72c ; 23 cycles of amplification with 1-second denaturation at 98c, 5-second hybridization at 54c, and 15-second elongation steps at 72c ; and a final extension step of 5 minutes at 72c as described by the manufacturer. infection rates were calculated by dividing the number of infected tsetse flies by the total number of flies we dissected and expressed as percentages. average tsetse catch per day was determined by summing the daily catch per week and dividing by the number of days traps were mounted for the week while average catch per trap was determined by summing daily catch and dividing by the number of traps. we employ the chi square () and fishers exact test where appropriate to determine variations in infection rates and the distribution of trypanosoma species. the analysis of variance (anova) was also employed to determine variations in the distribution of mammalian blood in tsetse blood meals and values of p < 0.05 were considered significant. a total of 631 tsetse flies were captured with average trap and daily catches of 4.39 and 26.34, respectively. we dissected 531 (84.2%) of the flies to check for the vector stages of trypanosomes while the remaining 100 (15.8%) were analyzed for vertebrate sources of tsetse blood meals. overall tsetse infection rate was 5.08% while those in relation to study sites were 6.84%, 4.65%, 3.03%, and 4.96% for burto, gindin dutse (g / dutse), goje, and serti, respectively (table 2). of the 2 species of tsetse flies captured, 408 (64.7%) were glossina palpalis while 223 (35.3%) were g. tachinoides. tsetse infection rates of 5.5% (19/347) and 4.3% (8/184) the 27 tsetse flies positive for trypanosomes using light microscopy were distributed into 12 pools of two flies each and a pool of three flies yielding 13 pools which were subjected to the its pcr. we identified trypanosoma brucei in 10, trypanosoma congolense savannah in 3, and trypanosoma congolense forest in 2 of the pools. two of the pools revealed mixed infections of trypanosoma brucei and trypanosoma congolense (table 4). of the 100 engorged tsetse flies subjected to the mitochondrial cytochrome b pcr to identify sources of tsetse blood meals, 45, 17, 6, and 32 fed on cattle, dog, man, and pig, respectively (table 5). in addition, 24% of the flies fed on at least 2 vertebrate hosts, 17% fed on at least 3 vertebrate hosts, and 1%, 5%, 22%, and 31% of the flies fed on man, dog, pig, and cattle, respectively (table 5). the distribution of dual feeding among the 24 tsetse flies that fed on at least 2 vertebrate hosts was 15%, 7%, 20%, and 24% for flies that fed on cattle - dog, cattle - man, cattle - pig, and dog - pig, respectively. of the 17 tsetse flies that fed on at least 3 vertebrate hosts, 27%, 5%, and 2% fed on cattle - dog - pig, cattle - man - pig, and dog - man - pig, respectively (figure 1). the species of tsetse flies we reported in our study were earlier reported in the same region by karshima. and in other parts of nigeria [24, 25 ] and were among the first eleven species of tsetse flies reported in nigeria. glossina palpalis was predominantly higher than g. tachinoides in line with earlier reports [25, 27, 28 ]. glossina palpalis usually has preference for thick forested areas with high temperatures and humidity for efficient breeding. considering the site we studied which has similar kind of vegetation we presume that the vegetation may be a reason for the predominance of this species of tsetse fly. we observed an overall average daily tsetse catch per trap of 4.39 which falls within the range of 0.618.1 reported by other workers [2931 ]. we expected a higher average daily catch considering the game reserve terrain in which we conducted our study and reports that confirm tsetse abundance during the rainy season. we attributed our size of catch to the consecutive rainfall we observed during the trapping as tsetse activities are grossly reduced during rain and cold weather. the location of gashaka - gumti national park in serti and its close proximity to burto may explain the higher tsetse catch we recorded in these two areas considering the fact that the park may serve as suitable habitat for tsetse flies and provide sources of blood meals for these flies. this may also explain the higher infection rates in these two areas especially with the expected increased tsetse animal contact with over 103 wildlife species and livestock grazing in the area. the mixed infections of t. brucei and t. congolense observed may be due to tsetse acquiring the infection from animals carrying the mixed infection in nature, from two different animal sources during feeding or even from the pools we made in our study. tsetse flies that were positive for trypanosomes by the use of light microscopy were pooled together before being subjected to pcr to increase the quantity of dna in the samples and thus increase the chances of pcr detection. the 2 species of trypanosomes identified in tsetse flies are not reported for the first time in this region, suggesting that they are endemic. of the trypanosoma species identified t. brucei was most prevalent among tsetse flies indicating that they may be the commonest species infective to the domestic animals and wildlife species in the region as earlier documented [23, 32, 33 ]. majority of trypanosome infections were observed among glossina palpalis contrary to earlier reports by desta.. this variation may not be unconnected with factors such as differences in tsetse feeding frequencies, feeding patterns, and vectoral capacity. the feeding pattern of tsetse vector is an important instrument in understanding the epidemiology of human and animal trypanosomoses. in our study, we detached engorged tsetse abdomens from the thorax and head before pcr to reduce contamination of blood meal dna by tsetse dna. selection of only engorged tsetse flies for the identification of host blood meals was to ensure the availability of host dna in the blood meals since tsetse digestion of blood meals which usually occurs few days after ingestion may denature host dna and impair its detection. we also included only four species of vertebrate hosts in our assay because they were the commonest animals in the region where this work was conducted. tsetse feeding preference for cattle based on cattle size has been reported by torr.. of all the vertebrate hosts included in our assay, cattle were the largest and this may explain why majority of the tsetse flies fed on them. the influence of host availability on tsetse feeding habits has also been documented. this may also be a probable reason for the large proportion of pig blood identified by our study. hunting dogs would have contributed to the number of tsetse flies that fed on dogs while activities like hunting, fishing, farming, and visit to rivers and streams would have exposed humans to the tsetse bites amounting to the number of tsetse that fed on humans. of particular concern are the dual and multiple feeding patterns involving more than one species of vertebrate hosts because this may be of great epidemiological importance in the area of spreading tsetse - borne infections between different vertebrate hosts including man in the region. considering the proximity of this game reserve with the fonten sleeping sickness focus of the republic of cameroon, there is the risk of trans - boundary tsetse flies acquiring trypanosoma brucei gambiense and spreading the infection among humans and animal reservoirs in the region. tsetse flies fed predominantly on cattle and less frequently on humans and also showed mixed feeding habits. the risk of tsetse flies transmitting tsetse - borne infection between humans and other vertebrate hosts exists in the region.	in order to understand the epidemiology of trypanosomoses in gashaka - gumti national park, nigeria, we determined the density, infection rates, and feeding patterns of tsetse flies using biconical traps, its, and mitochondrial cytochrome b pcrs. a total of 631 tsetse flies were captured, of which 531 (84.2%) and 100 (15.8%) were analyzed for trypanosomes and blood meals, respectively. tsetse distribution varied significantly (p < 0.05) across study sites with average trap and daily catches of 4.39 and 26.34, respectively. overall tsetse infection rate was 5.08% and ranged between 3.03% and 6.84% across study sites. we identified 10 t. brucei, 3 t. congolense savannah, 2 t. congolense forest, and 2 mixed infections among the 13 pools made from the 27 flies positive for trypanosomes with light microscopy. the distribution of vertebrate blood meals in tsetse flies varied significantly (p < 0.05) and ranged between 6.0% and 45% across hosts. we also observed dual feeding patterns involving at least 2 hosts in 24% and multiple feeding involving at least 3 hosts in 17% of the flies. we observed predominance of g. palpalis which also recorded higher infection rate. t. brucei was more prevalent among tsetse flies. tsetse flies fed predominantly on cattle and less frequently on humans and also showed mixed feeding habits.
membrane depolarization elicited outward current through inward rectifier k channels exhibits profound relaxation. consequently, with identical k concentrations on both sides of the membrane the steady - state outward macroscopic current at positive membrane voltages is much smaller than the inward current at the corresponding negative voltages, a feature called initially anomalous rectification and subsequently inward rectification (katz, 1949 ; noble, 1965). the first clue to a possible mechanism came from the work by armstrong and binstock (1965), who showed that intracellular tea blocks squid voltage activated k channels in a strongly voltage - dependent manner, rendering them inwardly rectifying. 20 y later, mg was identified as an endogenous voltage - dependent channel blocker causing inward rectification (matsuda., 1987 ; vandenberg, 1987). however, the voltage dependence of channel block by mg alone is too weak to account for the strong inward rectification observed in intact cells. furthermore, significant rectification remains in the absence of mg. thus arose the concept of intrinsic (voltage - dependent) channel gating to explain mg - independent current relaxation and rectification (e.g., ishihara. little progress was made in the search for additional endogenous blockers until certain polyamines were found to block the channels in a strongly voltage - dependent manner (ficker., 1994 ; lopatin. still, variable residual inward rectification remains after the inside of a membrane patch is perfused with solutions nominally devoid of polyamines and mg (aleksandrov. this finding underlies the hypothesis that inward rectification results from intrinsic channel gating modulated by the binding of mg and polyamines to a putative channel - gating machinery, rather than from voltage - dependent channel block by these intracellular cations. we found, however, that the residual rectification independent of mg and polyamines is related to hepes in recording solutions (guo and lu, 2000a). voltage jump induced, time - dependent relaxation is not exclusively associated with outward currents, since hyperpolarization - induced inward currents also exhibit relaxation (kubo., 1993). (1999) showed that hyperpolarization reduces the open probability of irk1, and argued that the reduction in channel open probability results from both channel block by extracellular divalent cations and channel gating. (1999) found that the protein segments that form the ion conduction pore underlie the putative channel gating. (2001) reported that gating of irk1 at negative voltages is significantly perturbed when ester carbonyls replace the amide carbonyls of the two glycine residues within the signature sequence that forms the ion selectivity filter. also, shieh (2000) showed that in low k solutions in the absence of extracellular divalent cations, hyperpolarization induces a significant inward current relaxation which is likened to the c - type inactivation of voltage - activated shaker k channels (hoshi., 1991 ; lopez - barneo., 1993 ; yellen., whether the macroscopic conductance of irk1 has any significant intrinsic voltage dependence, we present here a systematic experimental investigation of the causes underlying voltage jump induced current relaxations. irk1 cdna (kubo., 1993) was cloned into the pgem - hess plasmid. oocytes harvested from xenopus laevis (xenopus one) were incubated in a solution containing nacl, 82.5 mm ; kcl, 2.5 mm ; mgcl2, 1.0 mm ; hepes (ph 7.6), 5.0 mm ; and collagenase, 24 mg / ml. it was then rinsed thoroughly and stored in a solution containing nacl, 96 mm ; kcl, 2.5 mm ; cacl2, 1.8 mm ; mgcl2, 1.0 mm ; hepes (ph 7.6), 5 mm ; and gentamicin, 50 g / ml. defolliculated oocytes were selected and injected with rna at least 2 and 16 h, respectively, after collagenase treatment. irk1 currents were recorded from inside - out membrane patches of xenopus oocytes (injected with irk1 crna) with an axopatch 200b amplifier (axon instruments, inc.), filtered at 5 khz, and sampled at 25 khz using an analogue - to - digital converter (digidata 1200 ; axon instruments, inc.) interfaced with a personal computer. pclamp6 software (axon instruments, inc.) was used to control the amplifier and acquire the data. during current recording, the voltage across the membrane patch was first hyperpolarized from the 0 mv holding potential to 100 mv and then stepped to various test voltages, or stepped directly from the holding potential. the duration of the voltage test pulse was 100 ms, which is comparable to those used in the studies where the mg- and polyamine - independent rectification was initially observed (aleksandrov. background leak current correction was performed as described previously (lu and mackinnon, 1994 ; guo and lu, 2000a). to effectively perfuse the patch, the tip of the patch pipette (3 m) was immersed in a stream of intracellular solution exiting one of ten glass capillaries (i d = 0.2 mm) mounted in parallel. all recording solutions contained 100 mm k contributed by kcl, k2edta, k2hpo4, kh2po4, and koh. the phosphate - buffered intracellular solution contained (mm) : 5 k2edta (unless specified otherwise), 10 k2hpo4 + kh2po4 in a ratio yielding the desired ph, and sufficient kcl to bring total k concentration to 100 mm (guo and lu, 2000a). to adjust ph to 8.0 and above a small amount of koh 3, contained (mm) : 100 k (cl + oh), 5 egta and 10 hepes, ph 7.2 (adjusted with koh). the hepes - buffered extracellular solution contained (mm) : 100 k (cl + oh), 0.3 cacl2, 1.0 mgcl2, and 10 hepes, ph 7.6 (adjusted with koh). in the phosphate - buffered extracellular solution, hepes was replaced by an equal concentration of k2hpo4 + kh2po4 in a ratio yielding ph 7.6. (a d) currents were recorded from the same inside - out patch with the voltage pulse protocol shown in fig. (e) normalized i - v curves constructed from the currents determined at the end of each test voltage - pulse. the i - v curves a d correspond to the currents from a d, respectively. irk1 cdna (kubo., 1993) was cloned into the pgem - hess plasmid. oocytes harvested from xenopus laevis (xenopus one) were incubated in a solution containing nacl, 82.5 mm ; kcl, 2.5 mm ; mgcl2, 1.0 mm ; hepes (ph 7.6), 5.0 mm ; and collagenase, 24 mg / ml. it was then rinsed thoroughly and stored in a solution containing nacl, 96 mm ; kcl, 2.5 mm ; cacl2, 1.8 mm ; mgcl2, 1.0 mm ; hepes (ph 7.6), 5 mm ; and gentamicin, 50 g / ml. defolliculated oocytes were selected and injected with rna at least 2 and 16 h, respectively, after collagenase treatment. irk1 currents were recorded from inside - out membrane patches of xenopus oocytes (injected with irk1 crna) with an axopatch 200b amplifier (axon instruments, inc.), filtered at 5 khz, and sampled at 25 khz using an analogue - to - digital converter (digidata 1200 ; axon instruments, inc.) interfaced with a personal computer. pclamp6 software (axon instruments, inc.) was used to control the amplifier and acquire the data. during current recording, the voltage across the membrane patch was first hyperpolarized from the 0 mv holding potential to 100 mv and then stepped to various test voltages, or stepped directly from the holding potential. the duration of the voltage test pulse was 100 ms, which is comparable to those used in the studies where the mg- and polyamine - independent rectification was initially observed (aleksandrov. background leak current correction was performed as described previously (lu and mackinnon, 1994 ; guo and lu, 2000a). to effectively perfuse the patch, the tip of the patch pipette (3 m) was immersed in a stream of intracellular solution exiting one of ten glass capillaries (i d = 0.2 mm) mounted in parallel. all recording solutions contained 100 mm k contributed by kcl, k2edta, k2hpo4, kh2po4, and koh. the phosphate - buffered intracellular solution contained (mm) : 5 k2edta (unless specified otherwise), 10 k2hpo4 + kh2po4 in a ratio yielding the desired ph, and sufficient kcl to bring total k concentration to 100 mm (guo and lu, 2000a). to adjust ph to 8.0 and above a small amount of koh 3, contained (mm) : 100 k (cl + oh), 5 egta and 10 hepes, ph 7.2 (adjusted with koh). the hepes - buffered extracellular solution contained (mm) : 100 k (cl + oh), 0.3 cacl2, 1.0 mgcl2, and 10 hepes, ph 7.6 (adjusted with koh). in the phosphate - buffered extracellular solution, hepes was replaced by an equal concentration of k2hpo4 + kh2po4 in a ratio yielding ph 7.6. (a d) currents were recorded from the same inside - out patch with the voltage pulse protocol shown in fig. (e) normalized i - v curves constructed from the currents determined at the end of each test voltage - pulse. the i - v curves a d correspond to the currents from a d, respectively. blocks irk1 channels with varying potency depending on the commercial sources (guo and lu, 2000a). as shown there and in fig. 1 a, the blocking kinetics are slow even with 10 mm hepes present. usually, hepes is synthesized by reacting hydroxyethylpiperazine (hep) with bromoethanesulfonate (good., 1966). b shows the current records in the presence of 3 m hep ; the i - v curves determined at the end of the voltage pulses in the presence of 10 mm hepes and 3 m hep are shown in fig. 1, d and e, respectively. hepes and hep block the channels in a qualitatively comparable manner, although hep is much more potent. the blocking activity of hep must come from the piperazine ring since piperazine itself is even more potent (fig. 1 2 shows the fraction of unblocked currents in the presence of three representative concentrations of hepes, hep, or piperazine. analyzing the data in fig. 2 with the woodhull (1973) equation gives an apparent kd(0 mv) = 2.47 m and z (valence) = 1.02 for hepes, kd(0 mv) = 2.97 mm and z = 1.09 for hep, and kd(0 mv) = 0.27 mm and z = 1.08 for piperazine. therefore, the apparent channel block by hepes can be accounted for by a trace amount of hep, which is well below the limit of impurity specified by the supplier (0.5%). comparisons of the effects of intracellular hepes, hep, and piperazine on irk1 currents (structures shown at top). (a c) currents were recorded with 10 mm hepes, 3 m hep and 0.3 m piperazine, respectively, from the same inside - out patch with the voltage pulse protocol shown below. in all cases the intracellular solutions contained 100 mm k, 5 mm edta, and 10 mm phosphate (ph 7.6) besides the tested chemicals, whereas the extracellular solution contained 100 mm k, 0.3 mm ca, 1 mm mg, 10 mm phosphate (ph 7.6). c, which were constructed from the currents determined at the end of each test voltage - pulse. current at each voltage is normalized (except for its signs) to that at 100 mv. each data point represents the mean (sem) of currents recorded from 57 patches. the fraction of unblocked currents in the presence of three representative concentrations of hepes (a), hep (b), or piperazine (c) is plotted against membrane voltage. the theoretical curves are fits of the woodhull equation, which give kd(0 mv) = 2.47 0.02 m (mean sem, n = 6) and z = 1.02 0.02 for hepes, kd(0 mv) = 2.97 0.16 mm (n = 6) and z = 1.09 0.03 for hep, and kd(0 mv) = 0.27 0.04 mm (n = 8) and z = 1.08 0.02 for piperazine. to our surprise, we found in the experiment shown below that hepes is not the sole source of contaminating blockers in commonly used intracellular solutions. 3 shows that irk1 channels exhibit inward rectification in the presence of an intracellular solution like that used by aleksandrov. (1996), which contained kcl, egta and hepes (ph 7.2). the rectification was reduced only modestly when hepes (10 mm) was replaced by phosphate (10 mm), but reduce significantly further when we also substituted edta (5 mm) for egta (5 mm), although the i - v curve did not approach linearity until solution ph was raised from 7.2 to 7.6 [the final composition is the one we used previously (guo and lu, 2000a) ]. these findings show that the persisting rectification after removal of endogenous blockers (aleksandrov., 1996) intracellular egta, hepes (each up to 10 mm), and ph 10,000-fold more potent. therefore, the blocking effect associated with edta can be accounted for by trace of contaminating ethylenediamine used to synthesize edta. all subsequent data were recorded with intracellular solutions containing 5 mm edta and 10 mm phosphate (without hepes). (a and c) currents with 0.1 mm and 30 mm edta normalized to that with 5 mm edta are plotted against membrane voltage, respectively. (b and d) the fraction of unblocked currents in the presence of mg or ethylenediamine (ed), respectively, is plotted against membrane voltage. all curves are fits of the equation i / io = 1/(1 + [blocker]/kd), where kd = kd(0 mv)e. for a and b, the two nearly superimposed curves for each data set are fits either all data points (continuous curves) or all but the rightmost three data points (dashed curves). for c or d, the dashed curve through each dataset is a fit to all but the rightmost three data points. comparisons of the effects on irk1 currents of intracellular edta and ethylenediamine (structures shown at top). (a and b) currents were recorded from the same membrane patch with, respectively, 30 mm edta and 0.1 m ethylenediamine. (c and d) normalized i - v curves ; each data point represents the mean (sem ; n = 5) of currents. to determine the optimal intracellular ph we examined how ph affects irk1 currents (fig. 9 a). lowering ph inhibited irk1 currents minimally between 8.5 and 7.5 but dramatically between 7.5 and 6.5. 10 c, which shows that channel inhibition has voltage - dependent and -independent components. interestingly, much of the voltage - dependent component of proton inhibition vanished when acidic d172 (located in the inner pore) was replaced by neutral asparagine (figs. effects of intracellular ph on the currents of wild - type and d172n mutant irk1 channels. currents were recorded at various intracellular ph, with extracellular ph = 7.6 throughout. for each channel type effects of intracellular ph on the i - v curves of wild - type and mutant irk1 channels. (a and b) i - v curves of wild - type and d172n mutant irk1 channels at various intracellular ph, determined from the current records, as shown (and including those) in fig. 9, except for those at ph 7.6, which were taken from those as shown in fig. 4 a. (c and d) currents through irk1 and d172n channels, normalized to those at ph 8.5, are plotted against membrane voltage. all data points are mean sem (n = 5). as a practical matter, very small membrane patches must be used to achieve adequate removal of endogenous blockers by perfusion. to illustrate this, fig. 11 a shows three consecutive sets of current records from a small membrane patch, taken at 1-min intervals after the start of perfusion. any endogenous blockers present in that excised patch were apparently effectively removed within the first minute. in contrast, for a much larger patch (from a separate oocyte injected with much less rna), removal of endogenous blockers was very slow and incomplete even after prolonged perfusion (fig. 11 b). variable degree and rate of removal of endogenous blockers by perfusion. current traces shown in a and b were recorded from two separate patches excised from two oocytes injected with different amounts of crna (higher in a than in b). the recordings were made at the indicated times following the start of perfusion. under certain commonly used experimental conditions membrane hyperpolarization also induces relaxation of inward irk1 currents. the currents in fig. 12 a (and all those above) were recorded with 0.3 mm ca and 1.0 mm mg present in the extracellular solution buffered with phosphate. the voltage dependence of the ratio of currents at the end and the beginning of voltage pulses (iend / ibgn) is shown in fig. (1999) that extracellular divalent cations block irk1 in a voltage - dependent manner. the extracellular solution contained 0.3 mm ca and 1 mm mg in a, but 5 mm edta and no added ca or mg in b. (c) the ratio of currents at the end and the beginning of voltage pulses (iend / ibgn) is plotted against membrane voltage. the data corresponding to a and b are labeled by letters a and b, respectively. all data points are mean sem (n = 5). furthermore, shieh (2000) observed relaxation of inward current even in the nominal absence of extracellular divalent cations. the relaxation was prominent only at low concentrations of k. since solutions in the quoted study were hepes buffered, we wondered whether the current relaxation was due to the use of extracellular hepes. confirming the results of shieh (2000), we found that the inward current after strong membrane hyperpolarization exhibits little or no relaxation when the extracellular solution contained 100 mm k, 10 mm hepes, and 5 mm edta, but no added divalent cations (fig. 13 c). also confirming shieh 's finding, lowering the k concentration to 20 mm revealed prominent inward current relaxation (fig. 13 b). however, this relaxation vanished when we replaced hepes with phosphate (fig the voltage dependence of the ratio of currents at the end and the beginning of voltage pulses (iend / ibgn) is shown in fig. 13 e. furthermore, we examined whether channel block associated with extracellular hepes could also be caused by residual contaminants. 14, like hepes, hep and piperazine block the channels in the presence of 20 mm but not 100 mm extracellular k. therefore, channel block associated with extracellular hepes can also be accounted for by a trace amount of contaminating hep. 12, with 5 mm edta and no added divalent cations present in the extracellular solution. extracellular solutions were buffered with 10 mm hepes (a and b) or phosphate (c and d), and contained either 100 mm k (a and c) or 20 mm k (b and d). (e) the ratio of currents at the end and the beginning of voltage pulses (iend / ibgn) is plotted against membrane voltage. the extracellular solution contained 3 m hep (a and b) or 0.3 m piperazine (c and d), and either 100 mm k (a and c) or 20 mm k (b and d). (e) the ratio of currents at the end and the beginning of voltage pulses (iend / ibgn) is plotted against membrane voltage. in the present study, we systematically investigated the causes underlying the reported irk1 current relaxations after step changes of membrane voltage, and found that none of them is intrinsic to the channels. in other words, within the usual voltage range the macroscopic irk1 conductance has no significant intrinsic voltage dependence that causes either inward rectification or inactivation. the reported apparent voltage dependence of macroscopic irk1 currents is caused by traces of contaminants in routinely used chemicals, such as hepes and metal ion chelators among which (the latter) edta has the least effect. specifically, block associated with both intracellular and extracellular hepes can be accounted for by residual hep (500 part per million (ppm) in weight), and that associated with intracellular edta by residual ethylenediamine (1 ppm). these contamination levels, estimated from the relative specific inhibitory activity of compared chemicals (figs. 2 and 8), are well below the limits (5,000 ppm) specified by the supplier. intracellular divalent chelators must be used both to suppress endogenous ca - activated cl currents and to minimize channel block by contaminating divalent cations. however, a divalent cation chelator such as edta has two opposing effects : (a) it reduces free divalent cation concentration and thereby relieves channel block by these ions, and (b) it contains residual ethylenediamine that blocks the channels. consequently, the current is a biphasic function of edta concentration with a maximum at an intermediate concentration of edta (fig. 6), which may vary somewhat depending on the extent of contamination in a given lot of edta and on the level of divalent cation concentration in the solution. under the present solution conditions, the maximal current corresponding to 5 mm edta where there is little time - dependent relaxation of outward current during a 100-ms test - pulse to positive voltage (even up to 100 mv ; fig. 5), which is sufficiently long for many common studies. in the solution with 8), which practically causes no channel block, given that the kd(0 mv) = 1.4 mm and z = 2 (guo and lu, 2000b). therefore, at this low concentration of edta the channels are blocked primarily by contaminating metal ions (fig. the common divalent cations, ca and mg, block inward rectifiers with the same mechanism, although the latter binds with somewhat higher affinity (matsuda, 1993 ; matsuda and cruz, 1993). examining block by intracellular ca is technically more difficult due to the presence of ca - activated cl currents in oocytes ; thus, we estimated the total concentration of contaminating divalent cations in the equivalent of mg. to do so, we first determined that kd (0 mv) = 17 m and z = 1.1 from the fit of the woodhull equation to the mg - inhibition curve (fig. then, based on these values, we found, from the same analysis of current inhibition in 0.1 mm edta, that the extent of channel block is equivalent to that caused by 68 nm free mg (fig. 7 a). this free mg concentration requires 20 m total mg in a 0.1 mm edta solution while the edta - mg stability constant is 3.5 10 m. theoretically, 20 m mg in a 30 mm edta solution results in 0.2 nm free mg, which would practically cause no block. thus, at this high edta concentration the channels are primarily blocked by contaminating ethylenediamine (0.1 m ; fig. 8). with these estimates, we calculated for each edta concentration the current at 80 mv, using the following equation, (1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{i}{i_{o}}=\frac{1}{1+\displaystyle\frac { \left \left[mg^{2+}\right ] \right _ { free}}{^{mg}k_{d } \left e^{-\displaystyle\frac{^{mg}zfv}{rt}}}+\displaystyle\frac { \left \left[ed\right ] \right } { ^{ed}k_{d } \left \left(0\;mv\right) \right e^{-\displaystyle\frac{^{ed}zfv}{rt}}}}{\mathrm{,}}\end{equation}\end{document}where quantities f, v, r, and t have their usual meaning. since the current at 80 mv does not significantly deviate from the fit of the woodhull equation (fig. 7, c and d), 10, a and c, shows that inhibition by protons has both voltage - dependent and -independent components. several residues underlying voltage - independent inhibition by intracellular protons have been identified in various inward rectifiers (e.g., fakler., 1996 ; if d172 in the pore of irk1 acts as a surface charge and if protonation of d172 is voltage dependent, protonation of d172 (d to d) may reduce the single channel conductance and thereby render the channels inwardly rectifying. for the following reasons, this may not, however, be the primary cause underlying the observed voltage - dependent channel inhibition at low ph. replacing d172 with neutral asparagine does not reduce the single channel conductance (oishi., furthermore, it renders neither the single channel i - v curve nor the macroscopic i - v curve inwardly rectifying (oishi., 1998 ; 10 b). on the other hand, voltage - dependent inhibition of irk1 by intracellular protons probably primarily reflects protonation of amine groups in the residual endogenous and contaminating exogenous organic blockers and/or edta. protonation of these blockers enhances their affinity for irk1 (guo and lu, 2000b), whereas protonation of edta reduces its affinity for trace divalent cations, thus causing further channel block. consistent with this reasoning, mutant d172n channels, whose affinity for intracellular blocking cations is dramatically reduced (lopatin., 1994 ; ficker., 1994 ; fakler., 1995 ; yang., 1995), 10 d). in any case, for practical purposes one can obtain essentially uninhibited irk1 currents at intracellular ph 7.6 or higher. the affinity of irk1 channels for some endogenous blockers is exceedingly high, so that even trace amounts of endogenous blocker may cause significant inward rectification. for example, the kd(0 mv) for the binding of fully protonated spermine is 10 m, whereas the effective valence of channel block by spermine is 5 (guo and lu, 2000a, b). the calculated kd(100 mv) for spermine binding is therefore 10 m, which is almost certainly below the concentration of spermine remaining in an exhaustively perfused membrane patch. therefore, although spermine is a permeant blocker whose effect can be somewhat relieved by membrane depolarization, at 100 mv in the steady - state most channels will be blocked by spermine at concentrations as low as 1 nm (typical oocyte concentrations are submillimolar ; osborne., 1989). assuming the rate constant for spermine binding at 100 mv is diffusion limited and as high as estimated for quaternary ammoniums (1010 m s ; guo and lu, 2001), the predicted current reduction caused by 1 nm spermine is 110% at the end of a 100-ms voltage pulse to 100 mv (full steady - state inhibition would require many seconds). consequently, to limit the extent of channel block by spermine to at most a few percent during a 100-ms pulse, spermine concentration may need to be reduced to 1 nm. even if the precise values of kd and kon (at 100 mv) for spermine are unknown, the above exercise illustrates the practical challenge posed by the need to lower spermine concentration to a level that will leave channel currents essentially unaffected. not surprisingly, in cases where endogenous blockers can not be adequately removed despite exhaustive perfusion, significant voltage - dependent channel inhibition persists (fig. the problem of residual high - affinity inhibitors can be dramatically relieved, or even practically eliminated, by lowering channel affinity for intracellular cations. for example, a linear i - v curve is readily obtained in irk1 channels containing the d172n mutation (figs. 9 and 10 ; guo and lu, 2000a), which significantly lowers their affinity for intracellular spermine (e.g., yang., 1995). also as expected, we obtained satisfactory removal of endogenous blockers only with very small patches (fig. 11). as stated in materials and methods, to more effectively perfuse the patch we positioned the tip of the patch pipette in a rapid stream of the intracellular solution instead of perfusing the entire recording chamber. we also kept oocytes away from the recorded patch, since they are known to release substantial amounts of polyamines (ficker. 12), that some inward current relaxation results from channel block by divalent cations in the extracellular solution. furthermore, shieh (2000) showed that, in the absence of extracellular divalent cations but with hepes present, lowering k concentration reveals profound current relaxation after strong membrane hyperpolarization. based on this finding, the author suggested that the current relaxation resembles c - type inactivation of voltage - activated shaker k channels, which is similarly protected by k. however, we found that this k - sensitive inward current relaxation can be also accounted for by residual hep in the hepes used to buffer extracellular ph (figs. 13 and 14). on the basis of these findings, we argue that the k - sensitive current relaxation is also not an intrinsic gating property of these channels. the dramatic channel block induced by lowering k on both sides of the membrane probably results from both a reduced competition of extracellular k with extracellular blocking ions and a reduced knock off effect of the blocking ions by intracellular k (armstrong and binstock, 1965 ; armstrong, 1971 ; yellen, 1984 ; mackinnon and miller, 1988 ; neyton and miller, 1988a, b ; spassova and lu, 1998). in summary, at the macroscopic level irk1 channels inherently have practically ohmic characteristics although in principle, the i - v curve may exhibit slight outward rectification in the complete absence of any endogenous or exogenous blockers. in intact cells, the observed inward rectification of the i - v curve results from voltage - dependent block by intracellular cations such as mg and polyamines (matsuda., 1987 ; vandenberg, 1987 ; ficker., 1994 ; lopatin., 1994 ; fakler., 1995). however, in excised membrane patches, perfused with solutions nominally devoid of mg and polyamines, the relaxation of both inward and outward irk1 currents induced by voltage jumps and the resulting nonlinearity of the i - v curve is a reflection, not of intrinsic gating properties of irk1 channels, but of the unusually high affinity of irk1 for cations. because of the extraordinarily high affinity for cations, traces of usually insignificant contaminants in commonly used organic ph buffers and metal ion chelators become highly significant and problematic in the study of irk1 channels. despite this, practically uninhibited (inward and outward) irk1 currents and therefore linear i - v curves can be obtained, provided that the recorded membrane patch is adequately perfused and that both intracellular and extracellular solutions contain 100 mm kcl, 5 mm edta, and 10 mm phosphate at ph 7.6 or above.	in intact cells the depolarization - induced outward irk1 currents undergo profound relaxation so that the steady - state macroscopic i - v curve exhibits strong inward rectification. a modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of mg2 + and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. furthermore, irk1 exhibits significant extracellular k+-sensitive relaxation of its inward current, a feature that has been likened to the c - type inactivation observed in the voltage - activated shaker k+ channels. we found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in hepes and ethylenediamine in edta. therefore, inherently, irk1 channels are essentially ohmic at the macroscopic level, and the voltage jump induced current relaxations do not reflect irk1 gating but the unusually high affinity of its pore for cations. furthermore, our study helps define the optimal experimental conditions for studying irk1.
	the generation of genetic mutants in caenorhabditis elegans has long relied on the selection of mutations in large - scale screens. directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. here, we adapt the crispr / cas9 system to generate mutations at specific sites in the c. elegans genome.
cervical intraepithelial neoplasia (cin) is a premalignant disease, often leading to invasive cervical cancer. it is widely known that cin is caused by an infection with high - risk human papillomavirus (hpv) types [3, 4 ]. interestingly, most hpv infections are transient and do not lead to cin. genetic, immunologic, and socioeconomic factors play a role in the natural history of hpv infection and development of cervical intraepithelial neoplasia. lifestyle factors, such as cigarette smoking, are well known to be a risk factor for hpv persistence and development of cervical intraepithelial neoplasia, the precursor lesion of cervical cancer. compared to women who have never smoked, current cigarette smokers have a significantly increased risk of high - grade cervical intraepithelial neoplasia and cervical cancer. host factors such as the immune systems and genetic factors also seem to be important factors in hpv persistence and subsequent cervical cancer carcinogenesis [8, 9 ]. the transporter associated with antigen processing (tap) belongs to the superfamily of atp - binding cassette (abc) transporter that is essential for peptide loading onto class i major histocompatibility complex (mhc - i) molecules [10, 11 ]. transporter associated with antigen processing is composed of two integral membrane proteins, tap1 and tap2, which have one hydrophobic region and one atp - binding region each. tap 1 functions by providing a supply of candidate peptides to the mhc i molecules within the peptide loading complex and by transporting antigen peptides from the cytoplasm into the endoplasmic reticulum (er) [12, 13 ]. mhc - i molecules play a major role in the immune response against viral infections and transformed cells by presenting peptide antigens to cytotoxic t lymphocytes (ctl) [14, 15 ]. the loss of mhc class i expression is observed commonly in various tumours and is possibly the mechanism underlying the ability of neoplastic cells to evade the immune system. regarding cervical cancer, mhc - i antigen has been reported to be downregulated in hpv-16 and -18 positive cervical malignancies. tap facilitates the detection of hpv by mhc - i molecules and contributes to successful detection and eradication of hpv despite various immunoevasion mechanisms of the virus. therefore tap gene polymorphisms have been investigated in women with cervical cancer precursor lesions and in women with cervical cancer [1922 ]. einstein. ascertained a reduced risk for high - grade cin at the presence of tap1 i333v and tap1 d637 g. in the present study, we evaluated the association between five common tap gene polymorphisms in tap1 and tap2 : tap1 1341 (rs1057141), tap1 2254 (rs1135216), tap2 1135 (rs1800454), tap2 1693 (rs2228396), and tap2 1993 (rs241447) and the risk of cin in a total number of 822 women. women, who were referred to the department 's outpatient clinic for genital dysplasia between 2004 and 2009 because of a cytological result atypical cells of undetermined significance (asc - ucs) or higher, were asked to participate at the present study. in total, 616 caucasian women with histologically proven cin 1 - 3, treated at the department of gynaecology and gynaecological oncology, medical university of vienna, comprehensive cancer centre, vienna, austria, were included in this study. the examination in our outpatient clinic comprised ecto- and endocervical cytology, human papilloma virus (hpv) dna testing (hybrid capture 2-test, digene corporation, gaithersburg, md, usa), application of 3% acetic acid, colposcopy, and colposcopically guided biopsy. after the women agreed to participate at the study by signing informed consent, they were asked about their smoking status (yes including previous smokers versus no), and the clinical examination was performed. the patients, in whom no cin 1 - 3 was diagnosed after colposcopically guided biopsy, were excluded of further analysis (n = 60 patients). as a control group, we included 206 consecutive caucasian women, who presented at the department 's gynaecologic outpatient clinic between 2004 and 2009 with vaginal infection, pelvic pain or similar symptoms, and without a history of conization or histologically proven cin 1 - 3. patients, who agreed to participate, signed informed consent and had in addition to their particular examination, ecto- and endocervical cytology and one cervical smear to collect the dna samples. patients with known hiv, hepatitis b or c infection, or a history of gynaecologic malignancy were excluded from the study. the collected pap smears and histological samples were analyzed by a board certified pathologist specialized in gynaecological oncology. the institutional review board at the medical university of vienna approved this study (reference : ek number 017/2004). dna was extracted from patients ' cervical smear using qiaamp dna blood mini kit (qiagen, germany) according to the instruction of manufacturer. dna was amplified with polymerase chain reaction (pcr) and dna pyrosequencing was performed according to established protocols as described previously. after researching dbsnp, the snp database from national center for biotechnology information, and pubmed, the five most common and clinically relevant tap gene polymorphisms (i.e., tap 1341, tap 2254, tap 1135, tap 1693, and tap 1993) were chosen for investigation. primers used for the amplification of the fragments including the polymorphic sites, the length of the pcr products, and the sequencing primers were shown in table 1. pcr was carried out in a total volume of 25 l including 25 ng total dna, 5 pmol of each sense and antisense primers and 12.5 l jumpstart redtaq readymix reaction mix (sigma, missouri, usa), which contains 20 mm tris - hcl, ph 8.3, 100 mm kcl, 4 mm mgcl2, 0.002% gelatin, 0.4 mm each dntp, inert dye, stabilizers, 0.06 unit/l taq dna polymerase, jumpstart taq antibody. the reaction was performed on a perkin - elmer geneamp pcr system 9600 with 40 cycles at 94c for 30 seconds, at the corresponding annealing temperature (see table 1) for 30 seconds and 72c for 30 seconds. the reaction was preceded by a primary denaturation step at 94c for 1 minute and incubated at 72c for 7 min at last. 25 l pcr product was used for pyrosequencing according to the instruction of the manufactory. hardy - weinberg equilibrium was tested by chi - square tests comparing observed and expected genotype frequencies. differences in gene polymorphism frequencies and smoking between women with cin and controls were assessed by chi - square tests ; results are given as p value and odds ratio (or) (95% confidence interval (95% ci)). in a multivariate logistic regression model, we assessed the independent association between tap gene polymorphisms, smoking, and risk of cin. differences in gene polymorphism frequencies between women with cin 1 and cin 2/3 were assessed by chi - square tests ; results are given as p value and odds ratio (or) (95% confidence interval (95% ci)). haplotypes can only be estimated and then probability values between 0 and 1 are assigned, expressing the likelihood for each haplotype to be present in one allele of a patient. these probabilities sum up to 2 in each patient (for the two alleles) and were included as continuous covariates (one for each haplotype) in the logistic regression model. a logistic regression model was estimated to evaluate whether the presence of one or more copies of each particular haplotype in a patient is associated with a higher or lower risk of cin, compared to a patient with two copies of the wild - type haplotype. women with cin presented with the following histological results : cin i n = 206/616 (33.4%), cin ii n = 205/616 (33.3%), and cin iii n = 205/616 (33.3%). sixty - one of 422 (14.5%) women with cin were tested negative and 361 of 422 (85.5%) positive for high - risk hpv infection. of note, patients with cin (58.8%) were smoking significantly more often than controls (35.8%) (p = 0.0001) (table 2). the tap snps 1341, 2254, 1135, 1693, and 1993 were genotyped in 616 patients with cin and 206 controls. the genotype frequencies for all the studied snp 's were in hardy - weinberg equilibrium in both controls and patients with cin. distribution of genotypes for the five snp 's did not differ significantly between patients with cin and controls (table 3). in a multivariate model, smoking, but none of the investigated five snps, was independently associated with risk of cin (table 4). moreover, in 616 patients with cin the risk of high - grade cin was investigated. no significant association was found between the investigated tap genotypes and risk for high - grade cin (p = 0.08 for tap 1341 ; p = 0.3 for tap 2254 ; p = 0.8 for tap 1135 ; p = 0.3 for tap 1693 ; p = 0.2 for tap 1993) (table 3). the combined effect of the five tap gene polymorphisms on the risk for cervical intraepithelial neoplasia was investigated by haplotype analysis (table 5). haplotype analysis revealed that women with haplotype mut - wt - wt - wt - wt (tap polymorphisms t1135-t1341-t1693-t1993-t2254) had a significantly lower risk for cin, compared to women with the haplotype wt - wt - wt - wt - wt (p = 0.006 ; or 0.5 [0.350.84 ]). the aim of the present study was to evaluate the association between five common tap gene polymorphisms and risk for cin. the haplotype combination mut - wt - wt - wt - wt (tap polymorphisms t1135-t1341-t1693-t1993-t2254) was associated with a reduced risk for the presence of cervical intraepithelial neoplasia (or 0.5 (0.40.8)). this finding seems biologically plausible, as tap facilitates the detection of hpv by mhc - i molecules and contributes to successful detection and eradication of hpv despite various immunoevasion mechanisms of the virus. although the biological implication of the observed haplotype combination is not fully elucidated, this haplotype combination might facilitate the detection of hpv and subsequently reduce the risk for cin development. our observation is in line with studies investigating the role of tap gene polymorphisms and risk for high - grade cin and cervical cancer [7, 13 ]. einstein. ascertained a reduced risk for high - grade cin at the presence of tap1 i333v (p = 0.02) and tap1 d637 g (p = 0.01). in women with cervical cancer, a tap2 gene polymorphism has been associated with a reduced risk for cervical cancer. of note, this study was performed in an indian cohort although no population diversities for these gene polymorphisms are described. interestingly, we were not able to ascertain any association between five common tap polymorphisms and risk of cin in a relatively large number of women. as we did not aim to evaluate the exact biological mechanisms exerted by the five tap gene polymorphisms or their haplotype combinations, we did not investigate mhc - i serum levels and we did not obtain tissue of the patients. moreover, patients in the present study represent a highly selected group of women, as all patients were of caucasian origin and have been recruited at a single tertiary care center in vienna, austria. in a multivariate model, smoking, but none of the investigated five snps, was independently associated with risk of cin. it is well known that women smoking cigarettes are at higher risk for development of cin. due to these facts patients should be advised to quit smoking to reduce the risk for high - grade cin development and increase spontaneous regression. in the present study the protective effect of a tap1 and tap2 haplotype combination for the presence of cin has been observed in a relatively large cohort of caucasian women. although its further implication has to be elucidated, this haplotype combination could be used to identify women, less susceptible for development of cin following an infection with high - risk human papillomavirus.	background. transporter associated with antigen processing (tap) is responsible for peptide loading onto class i major histocompatibility complex (mhc - i) molecules. tap seems to facilitate the detection of hpv by mhc - i molecules and contributes to successful eradication of hpv. tap polymorphisms could have an important impact on the course of hpv infection. objective. the aim of this study is to evaluate the association between five tap gene polymorphisms and the risk of cin. methods. this case - control study investigated five common tap polymorphisms in tap1 (1341 and 2254) and tap2 (1135, 1693, and 1993) in 616 women with cin and 206 controls. associations between gene polymorphisms and risk of cin were analysed by univariate and multivariable models. the combined effect of the five tap gene polymorphisms on the risk for cin was investigated by haplotype analysis. results. no significant difference in genotype distribution of the five tap polymorphisms was observed in women with cin and controls. haplotype analysis revealed that women with haplotype mut - wt - wt - wt - wt (tap polymorphisms t1135-t1341-t1693-t1993-t2254) had a significantly lower risk for cin, compared to women with the haplotype wt - wt - wt - wt - wt (p = 0.006 ; or 0.5 [0.350.84 ]). conclusion. identification of this haplotype combination could be used to identify women, less susceptible for development of cin following hpv infection.
it is transmitted by infected female anopheline mosquitoes when they feed on humans for their oviposition and is responsible for millions of deaths every year around the world (1). malaria as on today is among the major infectious diseases causing considerable deaths and can be assumed as prime etiological factor of slowed economic growth as a result of low production associated with mortality. majority of malaria infections are caused by plasmodium falciparum and present with high fever and convulsions, which may lead to coma and death. in addition to the parasite, thick density of vector mosquitoes manifolds the burden (24). malaria infections are common during pregnancy posing substantial risks to the mother, fetus and the newborn, which include severe anemia, placental parasitemia and intra - uterine growth retardation (5). these factors contribute to low birth weight and stillbirth, which are principal causes of infant mortality in the africa (6). primigravid, in particular, and secundigravid women are at higher risk for placental malaria than women with multiple prior pregnancies (7). the pregnant women experience more mosquito bites as compared to non pregnant women, which may be due to increased body surface and specific odors secretions during pregnancy (8, 9). pregnant women are highly susceptible to malaria as compared to the adults, and both frequency and severity of disease are higher in pregnant women due to depressed cellular immunity during pregnancy (10, 11). until the early 20th century the problem of malaria in pregnancy was not described adequately however in the last few decades, many comprehensive studies have highlighted various issues of malaria in pregnancy and its effect on maternal and infant health (12). despite comprehensive malaria control programme, control measures specifically aimed at malaria in pregnancy the epidemiological data are scanty in order to develop effective policies to address this problem. in the present article, spectrum of malaria manifestation in pregnancy, we have used data from relevant articles published between 19802010, which were searched on google and pubmed using malaria, pregnancy and related key words. a characteristic infection of plasmodium falciparum includes a process involving the accumulation of parasitized red blood corpuscles (rbc) in various organs and organ systems. pregnant women have a large number of rbc accumulated in the intervillous spaces of placenta. many studies have been carried out to explain the preference of malaria parasite proliferation in the placenta and accumulation of brown malarial pigment was found in almost all cases of infected placenta examined (13,14). the placental infection occurs by cytoadherence - sequestration mediated by adhesive interactions between ligands of parasite present on infected rbc s surface and host molecules (glycosaminoglycans chondroitin sulphate - a and hyaluronic acid) present on endothelium. malaria infection during pregnancy may cause hepato - splenomegaly and megaloblastic changes in the bone marrow. further during the labour and course of pregnancy falciparum malaria may cause pyrexia and heart attack to the mother, while intra uterine growth retardation (iugr) and intra uterine death (iud) of the developing fetus (10, 12). placental infection acquired during the high transmission season may persist for a long time in the placenta itself. the extent of clogging incurred due to infection is directly proportion to the severity of infection (12). severe parasitization of placenta causes congenital anemia, premature delivery and stillbirth in the newborns which is primarily be due to the decrease in maternal blood output and exchanges between mother and fetus (15). several epidemiological studies have been carried out but still a concrete strategy to decrease malaria in pregnant women is awaited. two prospective hospital - based studies conducted in chandigarh and gujarat, india demonstrated that severity of clinical illness was significantly higher in pregnant patients for both plasmodium vivax and p. falciparum (1620). the pregnant woman affected by malaria experience maternal anemia, higher maternal death, intrauterine fetal death and severe peripartum complications like abortion, stillbirth, premature labour, low birth weight (lbw) (table 1). the studies carried out in different parts of africa have shown that > 25% of pregnant women have malaria infection at the time of delivery (10, 12). malaria infections in most parts of asia are very symptomatic (12) however in more endemic areas particularly in north eastern states, orissa and some parts of west bengal, where asymptomatic infections are common. plasmodium falciparum infections may not result in fever and therefore remain undetected and untreated during pregnancy (21). meta - analyses of intervention trials suggest that successful prevention of these infections reduces the risk of severe maternal anemia by 38%, low birth weight by 43% and perinatal mortality by 27% among pregnant women (12). the studies conducted in tribal villages in central india on pregnant women and infants revealed that 55 % pregnant women and 44 % infants investigated had malaria at some time during the study period (22, 23). a hospital based study in tanzania showed that out of 413 women 91% slept under bed nets, still 43% were having fever. malaria parasites were found in 8% of the placenta samples and parasite density was > 2,100 parasites/l. this investigation has revealed that despite of using insecticide treated bed nets ; malaria remains a problem in pregnancy. the delivery of intermittent preventive treatment in pregnancy could be useful at all levels of control implementation to attain maximum coverage at community level (5). several investigations have shown that p. falciparum is responsible for the majority of malaria incidences (> 75%) whereas the rest are contributed by p. vivax (16, 19, 20, 24). the infections due to p. vivax are generally ignored as compared to the p. falciparum in term of prevention and intermittent treatment. however, various studies have demonstrated that p. vivax malaria was associated with mild anemia and increased risk of low birth weight, which may be more pronounced in multigravidae than in primigravidae (25). the p. vivax infection, have not been found responsible for miscarriage, stillbirth and reduced duration of pregnancy in many investigations (20, 25). a report from thai - burmese border showed that the risk of severe malaria is reduced to > 25% in both p. falciparum and p. vivax mixed infection as compared to p. falciparum alone (26). in the other studies, the multiplicity of the infection in placenta was associated with occurrence of low birth weight babies with some parasite genotypes were able to persist over several weeks (27). the infection multiplicity decreased significantly with an increasing number of pregnancies, and infection with multiple p. the women infected with four or more strains have > 2 times chance to be anemic than women harboring fewer strains (28). the severity and timing of obtaining the infection plays a significant role in the intrauterine growth retardation (igur) and preterm delivery of pregnant women. a study carried out at malawi has suggested that women who have parasitemia or clinical episode of malaria in the antenatal period were more than three times likely to deliver an iugr infant than other women (29). similarly the preterm delivery was found associated with cord parasitaemia (29). malaria parasites infecting the pregnant women have distinct antigenic and adhesive properties than infecting the non pregnant women and other (30). further the primigravidae mothers are likely to be more parasitaemic than multigravidae because antibodies are present in higher concentration in multigravidae (12, 24, 30). many studies have showed that the young primigravidae and multi - gravidae have greater risk of acquiring malaria and its adverse effects than older primigravidae and multigravidae (3135). therefore, in addition to the parity level immunity, which comes through consecutive pregnancies, the immunity associated with the age of mother also plays considerable role in reducing the malaria infection during pregnancy. the risk of malaria infection in the first trimester as compared to the second trimester is very little, which may be due to changes in splenic function early in pregnancy (12, 36). however, the susceptibility in the first trimester should increase to explain the peak malaria prevalence in the second trimester. some studies have demonstrated the risk in the early trimester but the results are not consistent every time (3739). recent study in mozambique suggests that one - fifth of pregnant women with malaria symptoms are in their first trimester (12). little information is available about the infection after delivery and the picture is still unclear, however some studies propose the increase in new and recurrent infections during first trimester (3739). in contrast, a few studies have shown a rapid clearance of peripheral parasitemia within two days post delivery (40). therefore the chances of malaria infection depend upon the level of immunity present in the women at the time of becoming pregnant. studies have proved that peripheral malaria may enhance the risk of placental malaria up to five times (41). however the chance of placental infection due to the peripheral malaria infection occurred in the beginning of pregnancy is much higher as compared to the malaria infection occurred toward end of pregnancy (3). this can be concluded due to the limited immunity at the beginning of pregnancy (12). the prevention and control programmes in the areas of stable malaria transmission ensure the use of antimalarials and other intervention measures, still the malaria problem remains uncoverable. several studies have revealed the importance of prophylaxis for malarial infection during pregnancy (4143). however low prevention coverage, lack of personnel protective measures including insecticide treated bed nets (itbn s), socio economic factors and resistance to antimalarials have posed a great challenge to the control programmes (18, 44, 45). world health organization (who) in malaria risk nations and national vector borne disease control programme (nvbdcp) in india have clear guidelines towards prevention and treatment of malaria infection but these remain largely in the papers only due to several region specific problems namely, difficult terrain to reach the remote locations, resource scarcity, intentional underreporting of the cases due to various reasons, poverty and insurgency etc. there are information lacunae regarding the true burden of malaria infection which is very important for proper understanding and control of this serious health problem. correct measurement on the direct or indirect measurement of malaria - associated maternal morbidity and mortality is an essential component to quantify the exact malaria burden in pregnancy. there are only a few systematic studies carried out in asia and america which are insufficient to clarify the exact situation of malaria burden in pregnancy in these continents (12). the effective antenatal coverage is very inadequate and pregnant women mostly take care in the middle of second trimester or in the third trimester of pregnancy. the evaluation of the effect of anti - malarial drug used for control purpose is difficult as there is widespread use of incomplete treatment course, leading to frequent recrudescent malaria infections with repeated production of gametocytes after infective mosquito bite (12, 36, 46). mefloquine and quinine have been found unable to produce satisfactory responses for the treatment of highly drug - resistant p. falciparum malaria in pregnancy, when used as single agents (47). consequently, options for the treatment of pregnant woman are limited because of the unknown effects of antimalarials on the fetus (46). socio - behavioural issues are also needed to be addressed in developing countries such as india, where people have strong religious and cultural values (48). social and cultural barriers to the pregnant women limit the efficacy of any antimalarial intervention and success of control programmes. a study targeted on pregnant women from low socioeconomic groups in central india showed that > 80 % did not accept chemoprophylaxis due to objections by family members (49). therefore proper understanding of socio - cultural factors is important in tackling the problem of malaria among pregnant women. a characteristic infection of plasmodium falciparum includes a process involving the accumulation of parasitized red blood corpuscles (rbc) in various organs and organ systems. pregnant women have a large number of rbc accumulated in the intervillous spaces of placenta. many studies have been carried out to explain the preference of malaria parasite proliferation in the placenta and accumulation of brown malarial pigment was found in almost all cases of infected placenta examined (13,14). the placental infection occurs by cytoadherence - sequestration mediated by adhesive interactions between ligands of parasite present on infected rbc s surface and host molecules (glycosaminoglycans chondroitin sulphate - a and hyaluronic acid) present on endothelium. malaria infection during pregnancy may cause hepato - splenomegaly and megaloblastic changes in the bone marrow. further during the labour and course of pregnancy falciparum malaria may cause pyrexia and heart attack to the mother, while intra uterine growth retardation (iugr) and intra uterine death (iud) of the developing fetus (10, 12). placental infection acquired during the high transmission season may persist for a long time in the placenta itself. the extent of clogging incurred due to infection is directly proportion to the severity of infection (12). severe parasitization of placenta causes congenital anemia, premature delivery and stillbirth in the newborns which is primarily be due to the decrease in maternal blood output and exchanges between mother and fetus (15). several epidemiological studies have been carried out but still a concrete strategy to decrease malaria in pregnant women is awaited. two prospective hospital - based studies conducted in chandigarh and gujarat, india demonstrated that severity of clinical illness was significantly higher in pregnant patients for both plasmodium vivax and p. falciparum (1620). the pregnant woman affected by malaria experience maternal anemia, higher maternal death, intrauterine fetal death and severe peripartum complications like abortion, stillbirth, premature labour, low birth weight (lbw) (table 1). the studies carried out in different parts of africa have shown that > 25% of pregnant women have malaria infection at the time of delivery (10, 12). malaria infections in most parts of asia are very symptomatic (12) however in more endemic areas particularly in north eastern states, orissa and some parts of west bengal, where asymptomatic infections are common. plasmodium falciparum infections may not result in fever and therefore remain undetected and untreated during pregnancy (21). meta - analyses of intervention trials suggest that successful prevention of these infections reduces the risk of severe maternal anemia by 38%, low birth weight by 43% and perinatal mortality by 27% among pregnant women (12). the studies conducted in tribal villages in central india on pregnant women and infants revealed that 55 % pregnant women and 44 % infants investigated had malaria at some time during the study period (22, 23). a hospital based study in tanzania showed that out of 413 women 91% slept under bed nets, still 43% were having fever. malaria parasites were found in 8% of the placenta samples and parasite density was > 2,100 parasites/l. this investigation has revealed that despite of using insecticide treated bed nets ; malaria remains a problem in pregnancy. the delivery of intermittent preventive treatment in pregnancy could be useful at all levels of control implementation to attain maximum coverage at community level (5). several investigations have shown that p. falciparum is responsible for the majority of malaria incidences (> 75%) whereas the rest are contributed by p. vivax (16, 19, 20, 24). the infections due to p. vivax are generally ignored as compared to the p. falciparum in term of prevention and intermittent treatment. however, various studies have demonstrated that p. vivax malaria was associated with mild anemia and increased risk of low birth weight, which may be more pronounced in multigravidae than in primigravidae (25). the p. vivax infection, have not been found responsible for miscarriage, stillbirth and reduced duration of pregnancy in many investigations (20, 25). a report from thai - burmese border showed that the risk of severe malaria is reduced to > 25% in both p. falciparum and p. vivax mixed infection as compared to p. falciparum alone (26). in the other studies, the multiplicity of the infection in placenta was associated with occurrence of low birth weight babies with some parasite genotypes were able to persist over several weeks (27). the infection multiplicity decreased significantly with an increasing number of pregnancies, and infection with multiple p. the women infected with four or more strains have > 2 times chance to be anemic than women harboring fewer strains (28). the severity and timing of obtaining the infection plays a significant role in the intrauterine growth retardation (igur) and preterm delivery of pregnant women. a study carried out at malawi has suggested that women who have parasitemia or clinical episode of malaria in the antenatal period were more than three times likely to deliver an iugr infant than other women (29). similarly the preterm delivery was found associated with cord parasitaemia (29). malaria parasites infecting the pregnant women have distinct antigenic and adhesive properties than infecting the non pregnant women and other (30). further the primigravidae mothers are likely to be more parasitaemic than multigravidae because antibodies are present in higher concentration in multigravidae (12, 24, 30). many studies have showed that the young primigravidae and multi - gravidae have greater risk of acquiring malaria and its adverse effects than older primigravidae and multigravidae (3135). therefore, in addition to the parity level immunity, which comes through consecutive pregnancies, the immunity associated with the age of mother also plays considerable role in reducing the malaria infection during pregnancy. the risk of malaria infection in the first trimester as compared to the second trimester is very little, which may be due to changes in splenic function early in pregnancy (12, 36). however, the susceptibility in the first trimester should increase to explain the peak malaria prevalence in the second trimester. some studies have demonstrated the risk in the early trimester but the results are not consistent every time (3739). recent study in mozambique suggests that one - fifth of pregnant women with malaria symptoms are in their first trimester (12). little information is available about the infection after delivery and the picture is still unclear, however some studies propose the increase in new and recurrent infections during first trimester (3739). in contrast, a few studies have shown a rapid clearance of peripheral parasitemia within two days post delivery (40). therefore the chances of malaria infection depend upon the level of immunity present in the women at the time of becoming pregnant. studies have proved that peripheral malaria may enhance the risk of placental malaria up to five times (41). however the chance of placental infection due to the peripheral malaria infection occurred in the beginning of pregnancy is much higher as compared to the malaria infection occurred toward end of pregnancy (3). this can be concluded due to the limited immunity at the beginning of pregnancy (12). the prevention and control programmes in the areas of stable malaria transmission ensure the use of antimalarials and other intervention measures, still the malaria problem remains uncoverable. several studies have revealed the importance of prophylaxis for malarial infection during pregnancy (4143). however low prevention coverage, lack of personnel protective measures including insecticide treated bed nets (itbn s), socio economic factors and resistance to antimalarials have posed a great challenge to the control programmes (18, 44, 45). world health organization (who) in malaria risk nations and national vector borne disease control programme (nvbdcp) in india have clear guidelines towards prevention and treatment of malaria infection but these remain largely in the papers only due to several region specific problems namely, difficult terrain to reach the remote locations, resource scarcity, intentional underreporting of the cases due to various reasons, poverty and insurgency etc. there are information lacunae regarding the true burden of malaria infection which is very important for proper understanding and control of this serious health problem. correct measurement on the direct or indirect measurement of malaria - associated maternal morbidity and mortality is an essential component to quantify the exact malaria burden in pregnancy. there are only a few systematic studies carried out in asia and america which are insufficient to clarify the exact situation of malaria burden in pregnancy in these continents (12). the effective antenatal coverage is very inadequate and pregnant women mostly take care in the middle of second trimester or in the third trimester of pregnancy. the evaluation of the effect of anti - malarial drug used for control purpose is difficult as there is widespread use of incomplete treatment course, leading to frequent recrudescent malaria infections with repeated production of gametocytes after infective mosquito bite (12, 36, 46). mefloquine and quinine have been found unable to produce satisfactory responses for the treatment of highly drug - resistant p. falciparum malaria in pregnancy, when used as single agents (47). consequently, options for the treatment of pregnant woman are limited because of the unknown effects of antimalarials on the fetus (46). socio - behavioural issues are also needed to be addressed in developing countries such as india, where people have strong religious and cultural values (48). social and cultural barriers to the pregnant women limit the efficacy of any antimalarial intervention and success of control programmes. a study targeted on pregnant women from low socioeconomic groups in central india showed that > 80 % did not accept chemoprophylaxis due to objections by family members (49). therefore proper understanding of socio - cultural factors is important in tackling the problem of malaria among pregnant women. the above issue illustrates the need for a greater degree of co - ordination and participation by the community with local organization and govt. focus should be given on the reduction of parasite load by developing first rate surveillance in prevention and treatment programmes. pregnant women must be tested for parasite infection on regular basis using rapid diagnostic kits on the spot itself and serious cases should be hospitalized without any delay. the community awareness can play a backbone role in malaria eradication as only personnel protective measures and malaria surveillance alone can not assist effectively to fight with malaria. better estimates and reporting of malaria maternal morbidity and mortality in pregnancy are also needed. global risk maps will allow better estimation of potential impact and successful control of malaria in pregnancy. there is urgent need to stick the prevention guidelines and to provide medical service to the people living in remote areas in order to prevent the malaria related adverse effects and mortality upto considerable extent. the antimalarials which have been found effective and tested no side effects in pregnancy should be used in limit so as to check the resistance spread. community based awareness and education programmes are essential to educate women about the benefits and risk associated with taking recommended chemoprophylaxis during the pregnancy. 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.	malaria remains a complex problem during the pregnancy, which threatens > 35 millions pregnant women every year. malaria pathogenesis in pregnancy results in accumulation of infected rbcs in the intervillous spaces causing severe alterations leading to the reduced materno - foetal exchanges. in this article we have revisited the current evidences of clinical implications and overall burden of malaria in pregnancy. many adverse aftermaths including, low birth weight, intrauterine growth retardation, preterm delivery, stillbirth and anemia were found associated with malaria in pregnant women. despite of worldwide comprehensive control programmes for malaria in pregnancy, the disease control has been a daunting task everywhere. socio cultural, economical, lack of awareness and various logistic problems compound the disease in developing countries. thorough evidence based information and estimates, education and awareness and strengthening of prevention programmes are needed urgently to achieve success in malaria control in pregnancy.
large - scale programs to provide anti retroviral therapy (art) have expanded to combat the epidemic of hiv / aids in india which is third among the populations of hiv - positive cases in the world. however, the expansion art programs have also brought challenges. successful implementation of an art program hinges on a lifelong commitment by patients to adhere diligently to daily medication dosing schedules and scheduled visits to the art center. a systematic review assessing the rates of follow - up in patients undergoing art across sub - saharan africa reported approximately 25% of patients lost to follow - up 1 year after initiation of art and the rate of lost to follow - up was 40% after 2 years. patients lost to follow - up (i.e. discontinuing treatment) are at high risk of more opportunistic infections, illness, and death. they also often reflect patients with advanced disease who, untreated, can further spread infection. however, despite the critical importance of lost to follow - up as a public health issue to combat the epidemic of hiv / aids, it is surprising that it has received very little attention. there have been studies to determine the rates of follow - up and the reasons for lost to follow - up in lower income countries across the world which shows the seriousness of the issue. nevertheless, in case of india, reliable data on rates of lost to follow - up or the reasons for lost to follow - up is unavailable. the current study is carried out to study the rate of drug adherence and loss to follow - up (lfu) and factors affecting drug adherence among people living with hiv / aids (plwha) attending art centre of a tertiary care government hospital in western india. the study was carried out after taking permission from the local institutional ethics committee and gujarat state aids control society. shree sayaji general hospital (ssgh) is the largest tertiary care government hospital in central gujarat and third largest hospital in western india. all plwha registered at art centre in this ssgh, from april 01, 2010, to march 31, 2011, were enrolled in the study. a retrospective chart review of all consecutive patients initiating art at the primary art center in vadodara was done. data from the medical chart of the patients were collected using a standardized data extraction form based on the national aids control organization (naco) treatment card which broadly includes patient demographics, previous and current medical history, 1 day of treatment, and date of the last visit for collection of medications. as per naco treatment card entry point has been recorded as the mechanism by which client was referred to art center whether the client was referred from integrated counselling and testing center, revised national tuberculosis control programme / tuberculosis, outpatient department of the hospital, pptct, sti clinic, outreach workers, nongovernmental organization, or by private practitioner. data were entered and analyzed using epi info software version 6.0 (centers for disease control and prevention (cdc) in atlanta, georgia (usa).). according to the current treatment scheme patients undergoing art, patients not showing up at the clinic to collect their medications for three consecutive months were classified as lost to follow - up. category b : 8095% drug adherence where 311 pills missed a month and category c : < 80% drug adherence where more than 11 pills missed in a month. factors affecting drug adherence among plwha were studied by putting the variables in multiple logistic regression model in which p < 0.05 is considered to be statistically significant. out of 755 plwha registered at art center, 534 (70.7%) subjects were actively undergoing art, 61 (8%) were transferred out, 68 (9%) died, and 92 (17%) were lfu. the average duration of lfu patients on art was 2 months before they became lfu. the sociodemographic profile of 534 plwha actively undergoing art is as shown in table 1. nearly, 69% were married and 23% were illiterate. majority (67%) of them half of the plwha were from rural areas and a half from the urban areas. sociodemographic profile of people living with hiv / aids attending antiretroviral treatment center as shown in figure 1, the drug adherence rate among plwha was 306 (57.3%) plwha were in category a followed by 158 (29.6%) plwha were in category b while 70 (13.1%) plwha were in category c. drug adherence rate among people living with hiv / aids attending art center table 2 shows the multivariate analysis related to factors affecting drug adherence rate among plwha using logistic regression model, in which address, age, employment status, entry point, marital status, place of hiv testing, and mode of transmission were not found to be associated with drug adherence. however, education status of the participant showed independent and significant association with drug adherence. those who were literate were 1.8 times (odds ratio = 1.8, 95% confidence interval : 1.82.50, p < 0.05) adherent to art as compared to illiterate. based on hiv sentinel surveillance 20082009, it is estimated that india has an adult hiv prevalence of 0.31% (0.25% among women and 0.36% among men). in gujarat, estimated hiv prevalence is 0.37% in total while 0.30% among females and 0.44% among males. india 's national art programme was launched on april 01, 2004 under the naco, as an initiative of the ministry of health and family welfare of the government of india and the introduction of antiretroviral therapy (art) at free of cost at all art centers has also been initiated. however, now, the question arises of lifelong commitment by plwha to adhere diligently to daily medication dosing schedules and scheduled visits to the art center. a total of 755 patients were registered during a period of 1 year, out of which 70% were actively undergoing art, 17% were lfu, 8% died, and 9% transferred out. similar baseline characteristics were also observed by sharma. in a tertiary care art centre in north india. while a systematic review assessing the rates of follow - up in patients undergoing art across sub - saharan africa also reported approximately 25% lfu within 1 year of initiation of art. in the current study, the drug adherence rate (category a) among plwha attending the art centre of tertiary care hospital in gujarat was found to be 57.3%. whereas peltzer. in south africa and bijal. in private clinics of mumbai found as high as 70.8% and 73% drug adherence rate, respectively. however, in many studies cost has been well - documented barrier to drug adherence because of high cost of art in private sectors giving a load to the out of pocket expenditure to plwha. in india, few studies have identified factors associated with adherence to art. in this study, we have tried to find out the sociodemographic factors associated with drug adherence. literacy status of the patient was found to be significantly associated with drug adherence with aor of 1.8, which is comparable to studies conducted by kleeberger. and wasti. contradictory to this, sarna. in pune and new delhi ; and achappa. in south india found literacy significantly associated with drug adherence in bivariate analysis but not in multivariate analysis. being a cross - sectional study, this research has its own limitations. since, it was not our main objective, so we have tried to find relation only between drug adherence and sociodemographic characteristics, but there are many other factors associated with drug adherence which needs to be explored. this study observed 57.3% drug adherence rate and 17.22% lfu among plwha attending art centre in tertiary care government hospital in western india. as government programs in india expand access to free art for plwha, the development of validated low - cost interventions that optimize adherence is quite essential now.	introduction : national aids control organization (naco) has expanded the reach of anti retroviral therapy (art) to combat the epidemic of hiv / aids in india which has one of the largest populations of people living with hiv / aids (plwha) in the world. one of the major challenges related to art is a lifelong commitment by patients to adhere diligently to daily medication dosing schedules and scheduled visits to the art center. hence, the current study is carried out to assess the drug adherence rate and loss to follow - up (lfu) among plwha attending art centre of a tertiary care hospital in western india.materials and methods : the current cross - sectional study was carried out using medical records of all patients registered at art center, shree sayaji general hospital, vadodara after taking ethical clearance from local irb. lfu was classified according to naco guidelines. data were collected using a standardized data extraction form as per naco treatment card. data entry and analysis were performed using epi info software.results:of 755 plwha registered at art center, 534 (70.7%) subjects were alive on art, 61 (8%) were transferred out, 68 (9%) died, and 92 (17%) were lfu. nearly, 57% plwha have drug adherence rate of more than 95%. education status of the participant showed independent and significant association with drug adherence.conclusion:this study showed 57.3% were adherent to art among plwha, whereas 17.22% were lost to follow - up. hence, there is a need to emphasize on increasing drug adherence rate and on outreach activities to combat lfu.
levetiracetam is an anti - epileptic drug used for the treatment of partial and generalized seizures. we report the case of a 69-year - old woman with previously normal renal function who developed acute granulomatous interstitial nephritis (gin) requiring dialysis following levetiracetam consumption. a 69-year - old woman with a background of early stage chronic lymphocytic leukaemia (cll) was noted to have episodes of phrase repetition and instances of memory loss over 1 year. a magnetic resonance imaging showed mild deep white matter small vessel ischaemic changes with a small old infarct of the right caudate nucleus. six weeks later, she developed generalized erythematous macular rash (figure 1a) and was admitted to hospital for wet dressings. a skin biopsy confirmed a lichenoid drug reaction and serological markers of vasculitis were negative. at this time, the patient had normal renal function as assessed by a serum creatinine of 49 mmol / l (0.55 mg / dl) and the absence of pyuria, haematuria (by urinary microscopy) or albuminuria on a spot urine assessment. carbamazepine was discontinued and the patient was commenced on levetiracetam 500 mg twice a day and discharged home. (a) rash representative of that affecting the entire body after commencement of carbamazepine. (b) renal biopsy [haematoxylin and eosin (h&e) stain 250 ] showing diffuse active non - caseating granulomatous tubulointerstitial nephritis. (c) h&e stain (400) demonstrating areas of gin with lymphohistiocytic infiltrate and giant cells. fourteen days following commencement of levetiracetam, she presented with a 2-day history of severe oral mucositis and feeling generally unwell. on examination, she was afebrile and her blood pressure was 120/65 mmhg. urinalysis demonstrated + 1 protein only. initial laboratory evaluation revealed a serum urea of 37.1 mmol / l (103 mg / dl), creatinine of 393 mmol / l (4.44 mg / dl) and an elevated leucocyte count of 26.3 10/l (26.3 10/l), which were predominantly lymphocytes. urine microscopy showed > 100 10 leucocytes per litre and < 10 10 erythrocytes per litre. the urinary albumin : creatinine ratio was 47.6 mg / mmol (421.7 mg / g). a serological vasculitic screen was negative in particular, the anti - nuclear anibody, extractable nuclear antibodies, double - stranded dna antibody, anti - neutrophil cytoplasmic antibody and rheumatoid factor were all negative. computed tomography (ct) of the urinary tract showed large kidneys (left measuring 13.5 cm and right 12.4 cm) with no hydronephrosis or hydroureter. fluid overload, worsening azotaemia and metabolic acidosis required treatment with the commencement of haemodialysis via temporary venous catheter. the renal biopsy contained cortex and medulla with eight glomeruli, two of which were globally sclerosed. there was a diffuse active inflammation of the tubulointerstitium with non - necrotizing lymphohistiocytic giant cell granulomas as well as associated tubule destruction (figure 1b and c). ziehl neelsen, auramine and periodic acid - schiff stains were negative for mycobacterium and fungi. there was no evidence of glomerulonephritis by routine light microscopic, immunofluorescence or electron microscopic examination. she was initially treated with intravenous methylprednisolone daily (500 mg for 3 days) and the levetiracetam was replaced by sodium valproate 500 mg twice a day. other more common causes of granulomatous interstitial disease were considered including tuberculosis, which was excluded with a negative urine culture and polymerase chain reaction (pcr). sarcoidosis was also excluded with a ct scan of the chest and abdomen that did not reveal hilar lymphadenopathy or other features suggestive of sarcoidosis and a normal serum angiotensin - converting enzyme 35 the oral mucositis was secondary to herpes simplex virus as demonstrated on pcr of a swab of an oral lesion and was treated with acyclovir (dose adjusted for renal impairment, 250 mg intravenously for 3 days). g / l (540 mg / dl), igm < 10 mg / l (10 mg / dl), iga = 480 mg / l (48 mg / dl) ] secondary to cll and was given intravenous gammaglobulin (24 g daily over 3 days). she had three haemodialysis treatments over 6 days and rapidly recovered renal function with increasing urine output and improved biochemistry (figure 2). she had a course of oral prednisone commencing at 50 mg daily (1 mg / kg / day) that was withdrawn gradually over 3 months. her renal function normalized and her current serum creatinine is 70 mmol / l (0.80 mg / dl). renal toxicity secondary to levetiracetam has not previously been reported in adults although there is a single paediatric case report. the most common side effects of levetiracetam in adults are somnolence, asthenia and dizziness. it has high oral bioavailability and is excreted predominantly in the urine (93% after 48 h). adjustment of the dose is required in renal impairment as its elimination is directly dependent on creatinine clearance. although tubulointerstitial nephritis is relatively common, gin is rare, accounting for 1% of diagnoses in native renal biopsies [4, 5 ]. the most common causes of gin are sarcoidosis, drugs and infection, particularly tuberculosis. drugs implicated in cases of gin are varied and include non - steroidal anti - inflammatory drugs, phenytoin, nitrofurantoin and vancomycin. gin has been seen as a consequence of cll with one case reporting leukaemic cells infiltrating the renal tissue with surrounding t cells and granuloma formation. this patient had renal failure requiring haemodialysis and a diagnosis of gin confirmed by renal biopsy. the outcome of patients with gin is generally favourable but some patients with an insidious presentation may progress to end - stage renal failure. steroids have been used at varying dosages but due to the infrequency of the disease, there are no defined treatment guidelines.	granulomatous interstitial nephritis (gin) is an uncommon cause of renal failure, which may be caused by drugs. levetiracetam is an increasingly used anti - epileptic medication that is not known to cause renal toxicity in adults. to our knowledge, levetiracetam has not previously been reported as a cause of gin. we report the case of a 69-year - old woman who developed haemodialysis - requiring acute renal failure after commencement of treatment with levetiracetam, which was shown to be gin by renal biopsy. she made a complete recovery with cessation of levetiracetam and treatment with steroids.
" man is a creature composed of countless millions of cells : a microbe is composed of only one, yet throughout the ages the two have been in ceaseless conflict " septic shock remains a common cause of death in intensive care units worldwide and presents the clinician with a variety of management problems. the surviving sepsis campaign has gone far in collating the considerable wealth of information currently available about this devastating condition and provides excellent guidelines, which are essential reading for consultant and trainee alike. however, new insights into the management of these patients continue to accumulate, and the last few months have been no exception. one of the basic tenets of treating septic shock is the provision of cardiovascular support through the use of catecholamines, although there is much interest in other agents as addressed in the study by russell and colleagues in the new england journal of medicine. they examined the use of the pituitary - derived peptide hormone vasopressin in patients with septic shock through a multi - centred, randomised trial involving 778 patients given low - dose vasopressin (0.01 to 0.03 u / min) in addition to noradrenaline (norepinephrine) compared with noradrenaline alone. no overall differences were found between the two groups in terms of either the primary endpoint of 28-day mortality or the various secondary endpoints. interestingly, the vasopressin - treated group had a 28-day mortality of 35% compared with 39% of the group treated with noradrenaline alone, which is clearly much less than one would expect. the authors do suggest that this may reflect an improvement in the overall care of patients with septic shock, but equally it could reflect the selection criteria used : 6,229 patients were assessed for eligibility but more than 5,000 patients were not enrolled, a significant proportion of whom had cardiac disease. the authors themselves do concede that the baseline mean arterial pressures observed (72 to 73 mmhg) were somewhat higher than expected and therefore the trial probably reflects the use of vasopressin as a catecholamine - sparing drug rather than being an evaluation of vasopressin as an agent in shock unresponsive to catecholamines. subgroup analysis on those " thought to be more severe " (at least 15 g noradrenaline per minute), on whom vasopressin might be deemed to have a greater effect, failed to show any decrease in mortality. it does show that, in this carefully selected patient cohort, vasopressin use is safe but does not confer any additional benefit over catecholamine use. as with many studies, we await the next randomised controlled trial ! the differences between everyday clinical practice and the restrictive environs of the randomised controlled trial are further highlighted in a retrospective observational study by wheeler and colleagues on the use of drotrecogin alfa (activated ; drotaa). this is an interesting study that compares drotaa use in five teaching institutions with that reported in the recombinant human activated protein c worldwide evaluation in severe sepsis (prowess) trial and demonstrates that in the group observed, which reflects current practice, patients who received drotaa were younger, had more severe illness, increased comorbidities and received treatment later than those enrolled in the prowess study. indeed, nearly 50% of those treated would have been ineligible for the prowess trial principally through delayed onset of treatment, with 33.9% of patients receiving drotaa later than 2 days after the onset of severe sepsis. what is clear is that those patients receiving early treatment (day 0) fared similarly in terms of mortality to those in the prowess study despite the disparity in the groups. those treated later did not ; for example, patients treated at day 0 had a 33% mortality, whereas those treated at day 2 or later had a 52% mortality. these results probably strengthen the case for drotaa use when applied appropriately and yet again emphasises the need for the early recognition and treatment of sepsis. the evolution of the treatment of our patients with septic shock requires continuing to search for improvements in outcome through alternative agents. relatively recently, the anti - inflammatory role of the vagus nerve has been explored in an animal model of endotoxaemia and shock, the so - called ' cholinergic anti - inflammatory pathway ', and is a mechanism for the neural inhibition of inflammation through regulation of the inflammatory response. a recent study by hofer and colleagues in critical care medicine addresses this through assessing the role of pharmacological cholinesterase inhibition on survival in experimental sepsis. a murine model of sepsis animals were then treated with intraperitoneal injections of nicotine, physostigmine or lipopolysaccharide - free 0.9% normal saline. animals treated with intraperitoneal injections of nicotine (400 g / kg), physostigmine (80 g / kg) and neostigmine (80 g / kg) demonstrated improved survival, and treatment with physostigmine significantly reduced lethality as efficiently as direct stimulation of the cholinergic anti - inflammatory pathway with nicotine. downregulation of the binding activity of nuclear factor-b was observed, together with a significant decrease in the concentrations of the circulating pro - inflammatory cytokines tumour necrosis factor-, interleukin-1 and interleukin-6 as well as a decrease in pulmonary neutrophil invasion. there was no observed difference in survival between the groups treated with neostigmine and physostigmine ; interestingly, as in most things, septic delay of treatment beyond 6 hours negated any positive effects on survival. the last 5 years has seen a small collection of studies that have demonstrated a potential role for cholinesterase inhibitors and nicotine in the management of sepsis and improved outcomes ; we await further studies with interest. barnato and colleagues present a different slant on sepsis, performing a retrospective study on the racial variation of patients with sepsis admitted to hospitals within six us states. this study demonstrates that the incidence of severe sepsis in blacks is significantly higher than in whites or hispanics. the authors have made considerable efforts to negate the confounding effects of poverty and geography, although they admit that unmeasured differences in behaviour may be relevant to their results. however, they do raise the hypothesis that biological differences in susceptibility may also have a role. no doubt, with the rising tide of genetic studies, further information is not too far away. finally we return to the surviving sepsis campaign guidelines, which ' recommend the protocolized resuscitation of a patient with sepsis - induced shock... this protocol should be initiated as soon as hypoperfusion is recognized and should not be delayed pending icu admission '. the studies highlighted here all reinforce the need for the prompt recognition and early treatment of sepsis. adherence to these guidelines may have more of an impact on the outcome of our patients than any new, as yet unproven, treatments. drotaa = drotrecogin alfa (activated) ; prowess = recombinant human activated protein c worldwide evaluation in severe sepsis.	the choice of inotropic agent, particularly in catecholamine - resistant septic shock, remains an area of debate. here we discuss a recent trial examining the use of vasopressin in a carefully controlled trial setting. yet more data on the use of drotrecogin alfa (activated) in septic shock are described, as are novel but as yet experimental approaches to the treatment of sepsis. finally, it is important not to forget to read the latest surviving sepsis guidelines.
aging is currently perceived as an ongoing physiological process that intertwines the patho - biological mechanisms of a diseased state, influencing the development of a clinically evident morbidity. the field of cardiovascular disease is thus oriented to consider the aging process as the determinant of a priori structural and functional alterations of cardiac and vascular substrates that are further exposed to superimposed pathogenic noxae. the interaction between age - associated structural and functional changes and the actual biological mechanisms of a disease along with a plethora of other risk factors will define threshold, severity, and prognosis of cardiovascular disease occurrence in older persons. in the cardiovascular clinical practice, the commonest conditions encountered in the elderly are progressive heart failure, arrhythmias, and degeneration of heart valve apparatus. conduction disorders in this population carries a considerable high morbidity and mortality requiring pacemakers or defibrillators implantation in the majority of the cases. nodal dysfunction leading to chronotropic insufficiency, or increased susceptibility to reentry phenomena triggering ventricular or supraventricular arrhythmias characterize the clinical picture of these patients. degeneration of the conduction system and nodal pacemaker is thought to begin after the seventh decade of life, and ion channel alterations, along with beta adrenergic receptor down regulation and signaling impairment, have been reported as physiological substrates for tachyarrhythmia or tachyarrhythmia in the elderly. a reduction to less than 10% of cardiac pacemaker cells has also been reported in respect to young adults, and calcium, potassium and sodium handling systems have been shown to be defective leading to prolonged action potential and repolarization time with further increased susceptibility to reentrant arrhythmias. at the cellular level, a so called electrical remodeling including post translational modification of sarcoplasmic reticulum ca - atpase (serca-2), sarcoplasmic reticulum ca - release channel (ryr2) and phospholamban changes,, coupled with impairment in gap junction function and energy generation at mitochondrial level, has been claimed to constitute an electrophysiological substrate for arrhytmogenicity in the elderly. however, the generation of specific zones of myocardial refractoriness, or areas characterized by heterogeneity in the impulse propagation and conduction anisotropy suggests the role of different mechanisms, other than the described intracellular alterations, in the determinism of arrhytmogenicity in the elderly. myocyte loss and compensatory hypertrophy together with interstitial focal fibrosis induce the appearance of specific zones of functional conduction block or slowing eventually generating and stabilizing reentry circuits. the description of specific ectopic foci, intracardiac pathways or reentrant circuits often target of specific therapeutic interventions further substantiate this point and progressively led to individuate other co - responsible for cardiac arrhythmias in the aged population. in this context, in spite of the interest addressed by the literature to the aged cardiomyocyte as the main pathological responsible of age - related conduction disturbances, there are several evidences pointing at changes in the structure and function of the connectival extracellular matrix (ecm) as an important actor. at the biophysical level, cardiac ecm exhibits a peculiar degree of anisotropy, which is responsible for the elastic and compliant properties of the ventricle and for the structural properties of heart valves. however, ecm components and their arrangement are also the main determinants of the conductive properties of the specialized electrical conduction system. moreover alterations of ecm function in the elderly might additionally exert a detrimental effect on the normal function of the conduction system and on overall ventricular function and cardiac performance. thus, this review will focus on changes of ecm components in the aged myocardium and on their relevance in conduction disorders appearance. keeping an eye on the clinical side, it will explore the potential implications of ecm changes in the clinical management and on the therapeutic strategies potentially deriving from the scientific knowledge currently acquired on ecm. prevalence of cardiac arrhythmias increases over time during aging, carrying significantly higher morbidity and mortality in the elderly. in particular, the commonest arrhythmic conditions encountered in the elderly regard atrial fibrillation and ventricular tachyarrhythmia, but major ventricular arrhythmic events are the main responsible for sudden cardiac death (scd) in older population, greatly impacting health care resource utilization. the most recent epidemiological analyses are also remarking a striking incidence of atrial fibrillation or ventricular dysrrhytmias both malignant and benign independently on an underlying cardiac structural disease. on the other side, atrio - ventricular block and asystole are also increasingly frequent with aging and account for up to 20% of sudden cardiac death. in this regard, generation and conduction of the electrical impulse has been reported to be defective in the elderly, generating increased need for pacemaker devices implantation in the clinical management of this population. the function of the sinoatrial node (san) deteriorates with age with an increase in the nodal conduction time and a decrease in the intrinsic heart rate. collectively, those alterations translate at the clinical side in the so - called sick sinus syndrome, whose manifestations include bradycardia, sinus arrest, and sinus exit block. beside sinus node dysfunction, neurally mediated syndromes, acquired atrioventricular block, fascicular blocks, or (supra) ventricular tachyarrhythmias are the commonest indication for pacemaker implantation. additionally, considering the hemodynamic changes occurring with aging, which are basically constituted by a reduction of ventricular compliance and an increased contribution of atrial contraction to ventricular filling, dual chamber pacemakers maintaining synchrony between atria and ventricles are advantageous in older adults. during the aging process, the described structural and functional changes occurring in the left ventricle are interlaced with malfunction of the conduction system, which in turn results in non - efficient and non - synchronous activation of both ventricles, fostering a vicious circle eventually worsening the detrimental effects on cardiac performance. therefore, the use of biventricular leads in attempt to resynchronize ventricles activity is becoming a routine practice, usually coupled with a defibrillator system in order to protect the patient from malignant ventricular arrhythmias. also, with the aim to overcome issues of chronotropic incompetence and weakened response to adrenergic stimulation, the use of rate - responsive ventricular pacing has been proven effective in improving the quality of life in older patients compared to fixed - rate devices. multiple factors may influence age - related arrhythmic events and scd, including structural and electrical changes in the heart at the microscopic level. aging results in increased fibrosis, reduced cellular coupling in the cardiac muscle, as well as retarded activation and slowed velocity of the specialized conduction system throughout both the ventricle and the his - purkinje system. age - related alterations in anisotropic conduction velocity with a preferentially reduced transverse conduction provide a substrate for reentrant arrhythmias and exert a pro - arrhythmic effect by decreasing the threshold for ventricular fibrillation., this phenomenon is associated to reorientation of myofibrillar and myocardial sheet structures, as occurring during aging and contributes to myocardial wall thickening. propagation of the electrical impulse is well orchestrated within the heart and relies on a complex interplay between excitability, cell - to - cell coupling, and architecture of myocardial tissue. myocardial interstitium is emerging as playing a pivotal role in this context : collagen fibers constitute the main component of extracellular matrix cardiac architecture and, in association with connexin-43 (cx43), determine and modulate cell - to - cell coupling in ventricular myocardium. under normal physiological conditions, myocardial collagen between cardiomyocytes is organized in a delicate network constituting less than 1% of total tissue volume. the peculiar distribution of type i collagen confers the particular conduction anisotropy and modulate electrical cell - to - cell coupling by determining the distance among myocytes. during aging, a 200% increase in collagen content, together with a 50% decrease in cx43 expression, has been reported. increased collagen content, together with the enhanced interstitial and reactive fibrosis seen during aging, mechanistically leads to separation of the cardiomyocytes, with subsequent reduction of gap junction plaques and impairment in cell coupling. as a result of these changes, an increase in the vulnerability to tachyarrhythmias occurs, as the augmented anisotropic ratio and heterogeneity of conduction dramatically impair the conduction velocity, predisposing to reentrant arrhythmias. the clinical reflex of this condition lies in the increased risk for fatal arrhythmias, that also partially accounts for the reported high incidence of scd in patients with age - related remodeled hearts. the relation between myocardial interstitium and cx43 has been stressed by studies in which long - term inhibition of the renin - angiotensin - aldosterone system (raas) of aging mice to blunt excessive intramyocardial fibrosis preserved the normal cx43 expression and reduced arrhythmia vulnerability., interestingly, if treatment is administered in a model of age - associated ventricular hypertrophy, in which gap junction downregulation and fibrosis formation is well established, it is possible to obtain a reversal of cx43 expression but not to rescue the fibrosis process. to clarify this mechanism, a recent work by jansen,. demonstrated that cx43 decrease in aged hearts precedes the process of interstium remodeling, and that antifibrotic treatment determines primarily an increase in gap junction expression, just as if cx43 could have a permissive action towards collagen deposition. explanation to this phenomenon is thought to lie in the fact that reduced cx43 levels may alter cardiomyocyte - fibroblast and fibroblast - fibroblast communication, leading to increased fibroblast proliferation and/or activity. in this regard, jansen and colleagues demonstrated that gap junction remodeling, i.e., cx43 age - related downregulation, induces an increase in fibroblast activation rather than proliferation. in their study on aged mice (cx43 vs. cx43) following transverse aortic constriction, the authors demonstrated that the expression of discoidin domain receptor 2, a fibroblast marker, remained unchanged, while the expression of procollagen peptide (type i and iii) and of collagen type i significantly increased in the cx43-impaired group. this study also demonstrates how both gap junction negative remodeling and collagen deposition (fibrosis) are required for slowing electrical conduction. the exact mechanism underlying the cx43 mediated activation of cardiac fibroblast still needs to be unraveled, but a recent work by bowers,. demonstrated that communication between cardiomyocytes and fibroblasts via cx43 channel exerts a potent influence on cytockine production. in support of these findings, pedrotty,. underlined the role of paracrine fibroblast activity in influencing myocyte electrophysiology favoring conductance slowing. however, it is interesting to notice that the most recent literature is increasingly pointing at the role of cardiac ecm as a culprit of age - related cardiac dysfunction. in this context, the proliferative and secretory activity of fibroblasts and myofibroblasts mediating ecm deposition and intramyocardial fibrosis have been recently elucidated, and four different patterns of fibrosis, compact, patchy, interstitial and diffuse have been described. areas of patchy fibrosis with collagen bundles separating cardiac cells carry an important arrhythmogenic potential altering source sink relationships between myocytes and therefore favoring reentry phenomena or triggering ectopic electric activity. several reports describe a shift in the ratio between collagen type i and iii at the ventricular side and the accumulation of exuberant quantity of collagen seems to occur in association with fibronectin deposition, contributing to interstitial fibrosis and dramatically affecting conduction anisotropy. stein,. while remarking the link between increased collagen deposition and reduced expression of cx43 and of nav1.5 sodium channel, observed a difference in the fibrosis distribution between the right ventricle (rv) and left ventricle (lv). areas of fibrosis were found in the entire thickness of the rv wall, while only in the endocardium and mid - myocardium in the lv. the subepicardic zone of the ventricle, the region form where ventricles are normally stimulated, was found to be affected by fibrosis degeneration only in the rv and this might partially explain the increased vulnerability to arrhythmias of this side of the heart in the elderly. along with collagen and fibronectin, also accumulation of 1 and 5 integrin have been reported, and an imbalance in the metalloproteinases (mmps) and their specific tissue inhibitors (timps) has been claimed to be at the root of the profibrotic shift occurring in aged ventricles. specifically, bonnema,. found a decrease in the mmp-9/timp-1, mmp-9/timp-4 and mmp-2/timp-4 ratios during aging suggesting that a reduced ecm degradative ability might be at the basis of interstitial fibrosis. additionally, a recent clinical study has shown that serum markers of ecm degradation positively correlate with malignant ventricular arrhythmias. in particular, procollagen type i carboxyterminal peptide (picp) and procollagen type iii aminoterminal peptide (piiinp), representative markers of collagen i and iii synthesis, and mmp9/timp-1 ratio were found to be associated to tachyarrhytmic episodes. beside intraventricular conduction, impulse generation at the level of san and atrioventricular node (avn) is thought to be defective in the elderly. at the microscopic level, a recent autoptic study on human tissue showed fatty infiltration of the san, with observable signs of calcification and general inflammation, but no amyloid accumulation was detected. again, the recent literature is revealing the importance of changes in the structural component of san in aging - related nodal dysfunction. a study of yanni,. demonstrated that an interstitium remodeling occurs also in the san of aged animals beside the known nodal enlargement and nodal cells hypertrophy. the authors correlated nodal dysfunction to both cellular abnormalities concerning reduced expression of nav1.5 sodium channel and to san connective tissue reorganization during the aging process. interestingly, they did not report an increase in fibroblast number, but modifications in ecm component of san. decreased protein collagen density and mrna expression was reported and, unexpectedly, the ratio between the stiff collagen i, which is abnormally high in diseased heart, and the more elastic collagen iii, did not change with age in san. conversely, elastin, the other main structural component of cardiac ecm, was decreased in expression, but the ratio (at the mrna level) between collagen i and elastin did not vary significantly within nodal region or with age. those changes were substantiated by alteration in the balance between fibrotic modulators and degrading enzymes (cardiac mmps). transforming growth factor 1 (tgf-1) and tumor necrosis factor (tnf) were significantly upregulated in aged animals at nodal level, while mmp-2 showed an age - dependent decrease. this picture converges toward a pro - fibrotic activity, but this biological movement does not translate into an actual increase of structural matrix proteins levels in the san of old hearts. however, these data find a correlate in a recent clinical study demonstrating an association between mmp-9 and the risk of developing atrial fibrillation in a cohort of aged patients. interestingly, age - dependent changes in other ecm components seen at the ventricular muscle side during aging could not be detected at the san level. no significant changes at the mrna level of fibronectin 1 (adhesive protein), decorin (anti - fibrotic proteoglycan), connective tissue growth factor (ctgf, a cysteine - rich protein induced by tgf-1 and shown to trigger many cellular processes underlying fibrosis), and integrins 1, 5, and 1 were reported. however, the finding of changes in the expression of mmp-1, mmp-9, and mmp-13 further underpins the involvement of ecm in this context. tgf1 : transforming growth factor 1 ; tnf : tumor necrosis factor ; mmp-2 : metalloproteinases-2. prevalence of cardiac arrhythmias increases over time during aging, carrying significantly higher morbidity and mortality in the elderly. beside atrial fibrillation, ventricular tachyarrhythmias and major ventricular arrhythmic events are the main responsible for scd in older population, greatly impacting health care management. defective impulse generation and conduction and ecm disarray with augmented intramyocardial fibrosis during aging are considered the main biological responsible of these disturbances. complex cellular interplay and paracrine biological signaling underlie this phenomenon and targeting fibrosis generation and its pathological characteristics might be a promising therapeutical approach for age - related arrhythmic disease. the knowledge obtained over the electrophysiological significance of intramyocardial fibrosis distribution fueled an interesting piece of research concentrating on the spatial precise resolution of fibrotic strands and bundles within the ventricle by means of mri - based imaging techniques. gadolinium enhanced mri combined with computer - aided image processing could describe patterns and amount of fibrosis at the submillimeter scale, potentially allowing for tailored ablation interventions or device implantations. another important clinical translational remark that could be inferred by the mentioned paracrine interaction between cardiac myocytes and fibroblasts regards the fact that pharmachological stabilization of cell - to - cell coupling could exert a positive role not only on the immediate electrical homeostasis, but also on the prevention of collagen deposition and intramyocardial fibrosis. cellular interaction via gap junction has been shown to be associated to a paracrine signaling activation which in turns reflects in to enhanced matrix production and deposition by fibroblasts. compounds able to optimize cell electrical coupling might therefore induce a regulation of ecm production. alternatively, targeting directly the fibrosis process could represent a valid approach, and, despite both ace inhibitors and aldosterone antagonists have been shown to reduce fibrosis and decrease sudden death in aged decompensated patients, exploration of novel biological therapeutical approaches might deserve further investigations in this context. inhibitors of at-1 receptors for angiotensin have been shown to be a promising option, and inhibition of collagen synthesis through blockers of geranylgeranyl transferase and farnesyl transferase, or antagonist of prenyl chain biosynthesis as statins, is under investigation. however, targeting the complex paracrine signaling established in the delicate network between cellular and extracellular component of the heart might potentially enlighten new avenues in the treatment of age - related disease. despite the interest lavished in the current research on the cellular cardiac component, ecm plays an active and pivotal role during the aging process, influencing several aspects of cardiac biology and conditioning myocardial structural properties and function. conduction disturbances are frequent among the elderly and carry significant morbidity and mortality representing a clinical and economical burden. from this analysis of the literature deepening knowledge on ecm age - associated alterations might be important in the development of novel therapeutical approaches in the widespread panorama of age - related disease.	cardiovascular aging is a physiological process gradually leading to structural degeneration and functional loss of all the cardiac and vascular components. conduction system is also deeply influenced by the aging process with relevant reflexes in the clinical side. age - related arrhythmias carry significant morbidity and mortality and represent a clinical and economical burden. an important and unjustly unrecognized actor in the pathophysiology of aging is represented by the extracellular matrix (ecm) that not only structurally supports the heart determining its mechanical and functional properties, but also sends a biological signaling regulating cellular function and maintaining tissue homeostasis. at the biophysical level, cardiac ecm exhibits a peculiar degree of anisotropy, which is among the main determinants of the conductive properties of the specialized electrical conduction system. age - associated alterations of cardiac ecm are therefore able to profoundly affect the function of the conduction system with striking impact on the patient clinical conditions. this review will focus on the ecm changes that occur during aging in the heart conduction system and on their translation to the clinical scenario. potential diagnostic and therapeutical perspectives arising from the knowledge on ecm age - associated alterations are further discussed.
a number of studies over the last 15 years have provided converging evidence showing that dyslexic readers of languages with orthographies more regular than english suffer from a pervasive and persistent reading speed deficit, but much less from the reading accuracy problem which is characteristic for dyslexic children learning to read english (e.g., dutch : van den bos., 1998 ; yap and van der leij, 1993 ; german : wimmer, 1993 ; italian : zoccolotti., 1999 ; spanish : gonzalez and valle, 2000 ; norwegian : lundberg and hoien, 1990 ; greek : porpodas, 1999). the reading accuracy advantage of dyslexic children in regular orthographies was substantiated in direct english and german dyslexia comparisons which used similar words in the two orthographies (landerl., 1997 ; to illustrate, the english dyslexic children (11-year - olds) studied by landerl. had a problem with the word character. some refused to read it, and others produced misreadings ranging from chancellor and calendar to nonwords such as tschraekter. their german peers produced few misreadings for charakter (all nonwords close to the target), but their reading time was between 2 and 3 times longer than normal. with respect to regularity, it should be noted that german like many other alphabetic orthographies is more regular in the reading (grapheme - to - phoneme) direction than in the writing (phoneme - to - grapheme) direction. this asymmetry has the effect that, in a substantial number of cases, accurate but slow reading is accompanied by incorrect, but phonetically acceptable spellings. this profile, in terms of dual - route theorizing, suggests that fully specified memory representations of the letter sequences of words (i.e., orthographic lexicon entries) are necessary for correct spellings, but not for correct readings. a recent cognitive analysis by bergmann and wimmer (2008) based on both orthographic and phonological lexical decisions localized the source of the reading speed problem of german dyslexic readers in both the lexical and the sublexical route of the well - known dual - route model of visual word processing (coltheart. the lexical route leads to phonology via orthographic whole - word recognition units which get instantiated by familiar letter strings and provide direct access to whole - word phonology and meaning. the sublexical route of the dual - route model leads to word phonology via serial conversion of graphemes into phonemes. in an orthographic lexical decision task (i.e., is xxx correctly written ?), bergmann and wimmer found that dyslexic readers exhibited major difficulty with the orthographic distinction between words and pseudohomophones (e.g., taxi and taksi), indicating that their orthographic word lexicon contained fewer fully specified orthographic word recognition units. however, even when such recognition units were available and used, the speed of access to word phonology was markedly impaired. moreover, the sublexical route was found to be even more speed impaired than the lexical route. a hallmark of impaired functioning of the sublexical route is the dramatic increase of reading onset time with each additional letter of a word or nonword (marinus and de jong, 2010, this issue ; moll., 2005 ; spinelli., 2005 ; ziegler., 2003 ; based on a review of cognitive deficits associated with dyslexia in regular orthographies, bergmann and wimmer hypothesized that the underlying problem of slow functioning of both the lexical and the sublexical route resides in slow access to phonology, that is, in slow access from orthographic to phonological word representations (lexical route) and in slow access from graphemes to phonemes (sublexical route). recently, the manifestation of slow lexical and sublexical route functioning in dyslexic readers was also examined in a study of eye - movements (hawelka., 2010). the present study extended the work by bergmann and wimmer (2008) by using their phonological lexical decision task for measuring brain activity in german dyslexic readers (adolescents and adults). the task requires evaluation of whether letter strings sound like an existing word, and it presents familiar strings of existing words, unfamiliar strings of the very same words (i.e., pseudohomophones) and unfamiliar strings of nonwords. examples are taxi, taksi and tazi. in terms of the dual - route model, processing of familiar strings should primarily depend on the lexical route, and processing of the unfamiliar strings should involve the sublexical route. a methodological advantage is that both the familiar and the unfamiliar letter string of an existing phonological word result in the same yes response. we expected that abnormalities of the brain response in specific regions will specify the rather broad dual - route explanation of the speed impairment of our dyslexic readers. for expectations, the fmri results of a preceding study from our group are important (kronbichler., 2007), because this study used the present phonological lexical decision task, and the nonimpaired participants of kronbichler. identified a left hemisphere reading network consisting of occipitotemporal (ot), parietal and frontal regions which were engaged by both the lexical and the sublexical routes, but with increased demands posed by the sublexical route. importantly, the left ot region corresponded closely to the visual word form area (vwfa) of cohen. a straightforward expectation is that the present dyslexic readers may exhibit activation abnormalities in one or several of the mentioned regions engaged by efficient lexical and sublexical route processes in nonimpaired readers. given that our dyslexic readers suffer primarily from impaired reading speed, a main candidate region for abnormality is the vwfa in the left ot cortex, which was originally conceptualized as brain region recruited by highly efficient letter string processing in competent readers (cohen. underactivation of the left ot cortex is a common finding as shown in a review by mccandliss and noble (2003) and in a quantitative meta - analysis of imaging findings by our group (richlan., 2009). however, the majority of imaging studies with dyslexic participants found underactivation of left temporoparietal regions (i.e., posterior aspect of the superior temporal gyrus / sulcus, supramarginal gyrus) as the main brain signature of dyslexia (see richlan., the dysfunction of left posterior language regions is linked to the phonological deficit explanation of dyslexia (mccandliss and noble ; pugh., 2000 ; sandak., twenty german - speaking dyslexic readers (19 males, 1 female) were added to the sample of nonimpaired readers of the kronbichler. however, for matching purposes, and because of partial data loss in one case, the present sample of nonimpaired readers (i.e., the control group) included the data from only 19 (17 males, 2 females) of the original 24 participants of kronbichler. the dyslexic group consisted of 12 adolescents (age range : 1517 years) and 8 young adults (age range : 1834 years) and the control group consisted of 11 adolescents and 8 adults in the same age range. the adolescents were recruited from a longitudinal study and were invited to participate based on a marked reading fluency deficit on previous assessments. the adult dyslexic participants were university students who volunteered to take part in the study. they reported a childhood history of reading and/or spelling problems and still felt that their reading speed and spelling were not adequate. however, only few participants of the adult group had received a formal dyslexia diagnosis. all participants were right - handed and had normal or corrected - to - normal vision. final group assignment relied on a reading fluency test which is under development in our lab. this test presents a list of sentences (all of simple content) for 1 min with the instruction to mark as many sentences as possible as true (i.e., making sense) or false. the format of this test corresponds to the reading fluency subtest of the woodcock - johnson iii (wj iii) test of cognitive abilities (cog ; woodcock., 2001). participants were included in the dyslexic sample when their reading speed score was below the 10th percentile. a score above the 15th percentile qualified for the control group. these thresholds were chosen based on preliminary norm samples of about 300 university students for the adult participants and about 200 adolescents for the younger participants. table 1 shows that the mean score of the dyslexic group on the sentence reading test was only about half of the score of the controls, and that this mean corresponds to a reading quotient of about 2 sds below norm. the reading quotient was scaled like the iq score (m = 100 and sd = 15). the adult dyslexic subgroup tended to score somewhat higher than the adolescent dyslexic subgroup (means of 13.9 and 10.0 sentences, respectively, pooled sd = 2.5) and this was also the case for the nonimpaired subgroups (means of 24.9 and 21.3 sentences, respectively, pooled sd = 4.1). the close to perfect sentence accuracy in table 1 of the dyslexic group speaks against the possibility of the low sentence reading scores reflecting a deficit in vocabulary or knowledge required for evaluating the sentences. table 1 further shows reading speed (syllables per second) and accuracy scores for reading aloud a short text consisting of 137 words. unfortunately, data are missing for three nonimpaired and one dyslexic reader on this test. dyslexic readers similar to their slow performance on the sentence processing test read with only about half of the speed of the controls. furthermore, the scores for sentence processing and reading aloud were substantially associated : r(35) =.87 for the combined groups and also within groups (dyslexic readers :.72, nonimpaired readers :.68). table 1 further shows that the present dyslexic readers were not only slow readers but also poor spellers. for spelling assessment, a standardized test (kersting and althoff, 2004) one may wonder how there can be massive spelling problems in an orthography which is characterized as regular. the answer is that german, like other orthographies, is more regular in the reading direction than in the writing direction, with the effect evident in table 1 from the comparison of the reading and spelling accuracy that reading accuracy tends to be perfect and spelling accuracy tends to be comparatively low. a further inclusion criterion for the dyslexic sample was a nonverbal iq score in the normal range. this score was based on two subtests (object assembly and block design) of the german adaptation (tewes, 1991) of the wechsler adult intelligence scale - revised (wais - r). averaged over the two subtests, a mean standard score of higher than 7 was required, which would correspond to a performance iq of higher than 85. the subtests of the wais - r are standardized with a mean of 10 and a standard deviation of 3. in addition to the performance scale subtests, two subtests (vocabulary and similarities) of the verbal scale were also presented. the means in table 1 show that the dyslexic participants tended to score above average on all four subtests. only for the vocabulary subtest, their mean performance was reliably lower than that of the controls. the mean estimated iq (based on the 4 subtests) in table 1 shows that the dyslexic readers, similar to controls, tended to score above average. the lower section of table 1 shows measures from earlier assessments of the longitudinal participants (i.e., 12 dyslexics, 11 controls). before the beginning of systematic reading instruction in grade 1 there is no reading preparation involving letters in kindergarten a rapid automatized naming (ran) task, modelled after denckla and rudel (1976), was administered, which required quick naming of a sequence of pictured objects. furthermore, a peg moving task, modelled after annett (1985), required participants to quickly move 10 pegs from one line of holes in a frame into the holes of the line closer to the child (for details, see mayringer and wimmer, 2002). at the end of grade 1, the longitudinal participants were instructed to read aloud a list of 10 words and a list of 10 nonwords quickly and accurately. in grade 3, the mean syllables per minute scores in table 1 show that the dyslexic participants exhibited slow ran performance and marked early reading fluency impairments. however, even at the end of grade 1, reading accuracy was rather high with about 80% correct. importantly, table 1 shows that on the peg moving task, the later dyslexic readers performed faster than the controls. this speaks against a general speed impairment as cause of their slow ran performance and of their slow reading. on a visual coherent motion detection task, which was part of a cognitive assessment in grade 3 (for details this speaks against a visual magnocellular deficit as cause of the reading speed problem of the present dyslexic sample. the 180 stimuli consisted of 60 orthographically familiar forms of german nouns, 60 orthographically unfamiliar forms of the same words (i.e., pseudohomophones) and 60 nonwords. examples for the three item types are taxi taksi tazi or chaos kaos kuse. quantitative information about item characteristics is provided in table 2 of kronbichler. the familiar forms consisted of 49 letters and began with a consonant (in upper case following german spelling convention for nouns). the familiar forms and the pseudohomophones did not differ in number of letters, syllables, bigram frequency, or in number of orthographic neighbours (i.e., words of the same length differing by one letter). the mean frequency of 86 occurrences per million according to the celex database (baayen., 1993) indicates that the majority of words was of moderate to high frequency. nonwords were generated in such a way that they could not be distinguished from pseudohomophones by superficial characteristics such as absence of vowel letters or length. to examine differences in the bold response to the three item types, an event - related design was used. each item was displayed for 1600 msec with an inter - stimulus interval of 2100 msec during which a fixation cross was shown. this stimulus onset asynchrony of 3700 msec is not a multiple of the tr of 2000 msec (see below) which enhances the efficiency of the design by sampling the haemodynamic response at different time - points. the 180 stimuli were presented in two pseudo - randomized lists, and each list was divided into two runs of 90 items, each composed of 30 items per stimulus type. in addition, 10 null - events of 3700 msec duration with a fixation cross were included in each run to improve evaluation of stimulus related activation relative to baseline. the order of the 90 stimuli and of the 10 null events within each run was determined by a genetic algorithm (wager and nichols, 2003) which selects the most efficient sequence for testing stimulus contrasts. a critical feature for creating the two pseudo - randomized lists was the sequencing of the familiar and the unfamiliar forms of the same phonological word. when in list one the familiar form was presented in the first run, which was the case for half of the words, then this order was reversed in list two. item order was varied between participants so that the familiar form was equally often presented before and after the unfamiliar form of the same word. participants were familiarized with the phonological lexical decision task (i.e., does it sound like an existing word ?) and with the response mode outside the scanner. participants responded with the index finger (yes) and middle finger (no) of their right hand. 190 functional images sensitive to blood oxygenation level dependent (bold) contrast were acquired with a t2 weighted echo - planar imaging (epi) sequence (te echo time, 40 msec, tr repetition time, 2000 msec, fa flip angle, 86, 21 slices with a thickness of 6 mm, 220 mm fov field of view, with a 64 64 matrix resulting in 3.44 3.44 mm in plane resolution). additionally, a low (3.5 3.5 6 mm) and a high resolution (1 1 1.3 mm) structural scan were acquired from each participant with t1 weighted mprage sequences. a philips 1.5 tesla intera scanner (philips medical system, best, the netherlands) was used for mr imaging. functional images were realigned, unwarped and slice time corrected. then the functional images were first co - registered to the low resolution structural image and, subsequently, the functional and the structural images were coregistered to the high resolution structural image. this two - step procedure was found to obtain higher coregistration accuracy for previous data sets from this scanner than directly coregistering functional and high resolution structural images. the high resolution structural image was normalized to the montreal neurological institute (mni) t1 template image, and the resulting parameters were used for normalisation of the functional images, which were re - sampled to isotropic 3 mm voxels and smoothed with a 9 mm full width at half maximum (fwhm) gaussian kernel. we also characterized brain activation in regions of interest (rois) based on results of the voxel - based analysis. for roi analyses, parameter estimates of stimulus effects versus regions were defined as spheres of 5 mm radius centered on peak coordinates from group comparisons (see results section). voxel - based analysis was performed in a two stage mixed effects model. in the subject - specific first level model, each stimulus type was modelled by a canonical haemodynamic response function and its temporal derivative. the functional data in these first level models were high pass filtered with a cut - off of 128 sec, and corrected for autocorrelation by an ar(1) model (friston., 2002). the parameter estimates reflecting signal change for each item type versus fixation baseline (which consisted of the interstimulus interval and the null events) were calculated in the context of a general linear model (glm) (see henson, 2004). these comparisons were thresholded at p <.005, uncorrected, in conjunction with a cluster size threshold of at least 10 voxels. furthermore, these thresholds allow comparison with other recent fmri studies which also used uncorrected thresholds (booth., 2004 ; schulz., 2008 ; temple., 2000). we also provide information about which of the liberally identified regions survive a more conservative false discovery rate (fdr) corrected threshold (genovese., 2002). first, brain activity against baseline (averaged across the three item types, p <.001, uncorrected) was computed separately for each group. second, reliable activations of each group were combined, so that the mask contained all voxels which were activated in at least one of the two groups. table 2 shows accuracy and latency data for the in - scanner phonological lexical decision task. even for these more difficult item types, their accuracy was about 85% correct. only for nonwords was the group difference reliable. table 2 further shows that dyslexic readers exhibited markedly prolonged latencies for correct decisions on all item types and that these group differences were larger for pseudohomophones and nonwords than for words. the main effects of group and item type were reliable, f(1, 35) = 13.80, p <.01, and f(2, 70) = 190.08, p <.001, respectively. the group by item type interaction was also reliable, f(2, 70) = 9.63, p <.01. there was no main effect of age, f(1, 35) < 1, n. s., and none of the interactions involving age were reliable, fs(2, 70) < 1, n. s. a possible concern is that the group by item type interaction on latencies may reflect an over - additivity effect resulting from overall higher latencies of the dyslexic group. (1999), we standardized the item type latencies of each individual (i.e., item type latency minus average of the three latencies divided by standard deviation of these latencies). for these transformed scores, the group by item type interaction was no longer reliable, f(2, 70) < 1, n. s. in summary, the unfamiliar letter strings (pseudohomophones and nonwords) which required sublexical route processes led to more decision errors in dyslexics than in controls, although accuracy was still high for the dyslexic group. for words, dyslexic readers, similar to their slow performance on the reading tests, exhibited substantially prolonged decision latencies for all item types. importantly, for both accuracy and latency of phonological lexical decision, dyslexics profited at least as much as nonimpaired readers when presented with familiar compared to unfamiliar letter strings of existing words (e.g., taxi vs taksi). this finding is suggestive for reliance on the lexical route for familiar letter strings, and on the sublexical route for unfamiliar letter strings. because of the similar in - scanner performance of adolescents and adults, age was not used as a separate factor in the analyses of the fmri results. for nonimpaired readers, the renders of fig. 1 (first section) show that word items activated large bilateral occipital regions which were accompanied by bilateral frontal and parietal regions, including the motor cortex. the unfamiliar letter strings of pseudohomophones compared to words led to an activation increase in three left hemisphere regions (ot, inferior parietal, inferior frontal). nonwords compared to pseudohomophones led to a further increase in occipital regions and in large left frontal regions reaching from the inferior frontal gyrus (ifg) to precentral regions. in response to words, dyslexic readers activated largely the same regions as the nonimpaired readers, but the extent of the left parietal and left frontal activations was larger, and there were additional activations in several right hemisphere regions. word contrast identified a larger number of regions than in nonimpaired readers : bilateral middle occipital, bilateral parietal, bilateral frontal and insula regions. the largest regions with increased activity to pseudohomophones compared to words were localized in left inferior frontal regions and in the supplementary motor area (sma). importantly, the left ot region identified by the pseudohomophone word contrast for nonimpaired readers was not identified for dyslexic readers. pseudohomophone contrast in dyslexic readers identified a left precentral region and the left putamen, but did not identify the large left inferior frontal region found in nonimpaired readers. the results of the group comparisons are shown in fig. 2 and in table 3. for each of the three item types, dyslexic readers exhibited underactivation with an identical peak voxel in a left ot region. the extent of this underactivation increased from words to pseudohomophones and from pseudohomophones to nonwords in the posterior and lateral directions. in response to nonwords, but not in response to pseudohomophones, this left ot underactivation was accompanied by underactivation in a left inferior parietal and a left inferior frontal opercular region. these few regions with underactivation in dyslexic readers stood in contrast to a substantial number of regions with overactivation. for each item type, dyslexic readers exhibited overactivation in a large medial occipital region, a left postcentral region and in the left caudate. in response to words, there was additional overactivation in bilateral frontal and cingulum regions. in response to pseudohomophones and nonwords, a substantial number of regions with overactivation in frontal and subcortical regions were observed. for pseudohomophones, the largest anterior overactivations were in the left primary motor cortex and in the left cingulum. for nonwords, again large overactivations were identified in the left primary motor cortex and in the left cingulum and also in the left putamen. word difference of the nonimpaired readers in the left ot and they failed to exhibit the nonword pseudohomophone difference of the nonimpaired readers in left inferior frontal and precentral regions and in the left lingual gyrus., we also searched for brain regions showing the inverse item type effects, that is, higher activity to words compared to pseudohomophones and higher activity to pseudohomophones compared to nonwords. (2008) found that dyslexic readers exhibited an activation pattern inverse to that of the nonimpaired readers (i.e., more activity to high compared to low frequency words). in the present study, however, no such region was identified for the word the occipital roi was centered around the peak of the large medial occipital region, where dyslexic readers exhibited higher activity compared to nonimpaired readers in response to all three item types. the anterior one of the two left ot rois (y = 48) exhibited reduced activity in dyslexic readers in response to all three item types, and the posterior one (y = 60) was selected to illustrate the posterior extent of this group difference in the case of pseudohomophones and nonwords. 3 illustrate the substantial size of these opposite dyslexic abnormalities, that is, overactivation in occipital and underactivation in ot regions. furthermore, in the occipital roi our dyslexic readers exhibited increased activity to nonwords compared to words. this was not the case for the ot rois, where dyslexic readers failed to exhibit the activation pattern of the nonimpaired readers (i.e., words < pseudohomophones = nonwords). 3 also includes brain activity for two superior temporal gyrus regions and an inferior parietal region. the middle superior temporal roi is centered around the maximum of dyslexic underactivation found in the letter - sound integration task of blau. (2009) and the posterior temporal roi is based a maximum of dyslexic underactivation in our quantitative meta - analysis (richlan., 2009). the inferior parietal roi is based on the present voxel - based finding of dyslexic underactivation in response to nonwords. in the middle superior temporal roi there was generally little activity compared to baseline, but, interestingly, a tendency towards higher activity for dyslexic readers. in the posterior superior temporal roi, activation levels were increased in dyslexic readers and the group difference was of borderline reliability (p =.07). for the inferior parietal roi, the underactivation of the dyslexic readers was not limited to nonwords, but was also present for pseudohomophones and words. the one in the left ifg, opercular part, was identified by reduced activity in dyslexic readers compared to nonimpaired readers in response to nonwords, but the means show that dyslexic underactivation was not limited to nonwords, as there was a tendency in this direction also for pseudohomophones and words. the left motor cortex roi was identified by higher activity in dyslexic readers compared to nonimpaired readers in response to pseudohomophones and nonwords, but fig. we also examined correlations between individual reading speed scores outside the scanner (averaged over silent sentence evaluation and reading a text aloud) and individual brain activity estimates for the rois (averaged over the three item types). there was a positive correlation of (35) =.56, p <.001, between reading speed and combined left ot activation (i.e., the higher the speed score, the higher the activation) and a negative association between reading speed and right occipital (calcarine) activation, (35) =.57, p <.001, as well as between reading speed and left motor cortex activation, (35) =.51, p <.01. within each group, associations were lower and none was reliable. the effect of age on brain activity was examined for the rois by analyses of variance (anovas) with age (adolescents vs adults) and reading skill group (dyslexic vs nonimpaired) as between - subjects factors and item type as within - subjects factor. corresponding to the absence of an age effect on response latencies for the in - scanner task, none of the main effects of age was reliable, fs(1, 35) < 1, all n. s., and, with a single exception, none of the interactions involving age was reliable, fs < 3.2, all n. s. the only exception was the age by group interaction for the left motor cortex roi, f(2, 70) = 6.4, p <.05, which resulted from the higher brain activity of the dyslexic readers compared to the controls in the adolescent subsample, p < 01. the dyslexic participants of the present study exhibited the behavioral manifestation of dyslexia in regular orthographies, that is, they suffered from a severe impairment of reading speed but not of reading accuracy. this pattern is expected from the asymmetric regularity of grapheme phoneme correspondence in german, that is, high in the reading (grapheme - to - phoneme) direction and low in the spelling (phoneme - to - grapheme) direction. in terms of dual - route processes, it indicates that dyslexic readers suffered from a poor orthographic lexicon, that is, they possessed memory representations containing all letters for only a reduced number of words. this reduced size of the orthographic lexicon may contribute to the reading speed problem, because it requires reliance on serial grapheme phoneme processing for words, for which nonimpaired readers can rely on fast whole - word recognition based on orthographic word recognition units. for the subsample of 12 adolescents coming from the longitudinal study, we additionally showed that accuracy for fast reading of a list of nonwords was about 90% correct even at the end of grade 3. in contrast to english - based dyslexia findings, this early accuracy of sublexical reading most plausibly is due to the regular grapheme phoneme relations of german and to reliance on a synthetic phonics teaching approach (for details, see wimmer., 2000). the early acquisition of an accurately functioning sublexical reading route is important, as it can be seen as precondition of long - term storage of the letter - sequences of correctly decoded words (share, 1995). a specific difficulty of dyslexic readers for such orthographic learning was found in training studies (reitsma, 1983 ; thaler., in contrast to these high levels of early reading accuracy, reading speed was impaired over all three longitudinal assessments, and this reading speed impairment was preceded by poor performance in a ran task. importantly, this early ran impairment was not accompanied by a slow speed on a peg moving test which was part of the precursor assessment. the performance of the dyslexic participants on the in - scanner phonological lexical decision task (i.e., does xxx sound like an existing word ?) corresponded to their reading performance outside the scanner. similar to their generally high reading accuracy, importantly, similar to the nonimpaired readers, the dyslexic sample exhibited more accurate and faster yes responses to familiar strings such as taxi compared to pseudohomophones such as taksi. actually, they profited at least as much from orthographic familiarity as nonimpaired readers. this marked orthographic familiarity effect suggests that dyslexic readers relied on the efficient lexical route to word phonology (via orthographic word recognition units) for a substantial number of the familiar letter strings. however, their shorter decision latencies in response to familiar compared to unfamiliar strings were still substantially prolonged compared to the latencies of the nonimpaired readers. following bergmann and wimmer (2008), this speed impairment may emerge from two sources : one is absence of fully specified orthographic word recognition units for some words, so that dyslexic readers may have had to rely on the slower sublexical route to reach the yes response for these items. bergmann and wimmer showed that dyslexic readers exhibited prolonged phonological lexical decision latencies even when availability of orthographic whole - word recognition units could be inferred from their orthographic lexical decisions. importantly, prolonged decision latencies on the in - scanner task were not limited to words, but were also observed for pseudohomophones and nonwords. actually, in absolute terms the speed deficit was larger for the unfamiliar letter strings. this suggests an at least similar efficiency problem of the sublexical route as of the lexical route. of relevance for interpreting the reading speed impairment in terms of the dual - route model is the finding that dyslexic readers exhibited underactivation in a left ot brain region in response to all three item types. the center of this region was identical at around x = 45, y = 48, z = 15 (mni coordinates) for all three item types. this center in the ot cortex is slightly anterior to the classical vwfa of cohen. (2002) at around x = 43, y = 54, z = 12. our quantitative meta - analysis (richlan., 2009) identified a maximum of dyslexic underactivation in the fusiform gyrus at x = 46, y = 50, z = 16. the extent of the left ot underactivation was relatively small for words and was enlarged in the posterior and lateral direction for pseudohomophones and nonwords. this left ot underactivation stood in contrast to dyslexic overactivation in a large medial occipital region. therefore, it can be excluded that the left ot underactivation is a down - stream consequence of a posterior occipital dysfunction. dyslexic overactivation in an occipital region (lingual gyrus) was also found in our quantitative meta - analysis. besides underactivation of the vwfa, dyslexic readers completely failed to exhibit the modulation of the vwfa shown by the nonimpaired readers, that is, increased activity to unfamiliar letter strings of pseudohomophones and nonwords compared to familiar letter strings of words. the failure of dyslexic readers to exhibit this modulation can not be attributed to a lack of familiarity with the letter strings of words, because the effect of familiarity on response time (in absolute terms) was stronger for dyslexic than nonimpaired readers. the absence of orthographic familiarity effects on left ot activation of the present poor readers is not an isolated finding, but was also found in two other fmri studies which used the present procedure (bruno., 2008 ; van der mark., 2009). this study measured brain activity in response to reading aloud words of high or low frequency and found that dyslexic readers exhibited underactivation of the left ot cortex and failed to exhibit any modulation in response to word frequency. one may be concerned that the presently found underactivation of the vwfa in the left ot cortex and the absence of a familiarity effect in this region may simply reflect a delay in the acquisition of reading skill, which may be overcome with further reading development. however, we note that the present dyslexic readers were adolescents and adults who typically show little further gains in reading skill. (2007) compared dyslexic and younger nonimpaired readers matched for reading ability, and, consistent with the present study, found underactivation in the left ot / fusiform gyrus, together with left inferior parietal and right ot underactivation. in previous studies (kronbichler., 2007, 2009 ; schurz., 2010) with nonimpaired readers, we have found similar vwfa activation patterns as in the present study (i.e., words < pseudohomophones = nonwords). this was interpreted as recruitment of the vwfa by both lexical and sublexical coding processes. specifically, we proposed that the lower activity in response to the familiar letter strings reflects efficient assimilation of whole - word strings by often used orthographic word recognition units, whereas the higher activity in response to unfamiliar letter strings reflects coding into grapheme sequences. in this perspective, the present dyslexic result pattern reduced activity to all three items types, absence of orthographic familiarity related modulation suggests that the vwfa was not recruited in dyslexic readers by lexical and sublexical orthographic coding processes. this failure to recruit the vwfa for lexical and sublexical route processes can be seen as a serious abnormality in the neural organization of reading processes. this interpretation is suggested by evidence showing that the vwfa is critically involved in highly efficient processing of letter string information (cohen., 2002 ; dehaene., 2005). in direct support of this function, disruption or deafferentation of the vwfa was found to result in letter - by - letter reading in formerly fluent readers (cohen., 2004 ; gaillard., 2006 ; a different, but also critically important function of left ot regions in visual word processing was proposed by price and devlin (2003). these authors presented evidence for the position that the left ot cortex functions as an efficient interface which channels visual or tactile information to brain regions engaged by language and knowledge representation (see also devlin., 2006). as noted in the introduction, reviews of dyslexic brain activation abnormalities summarize the largely english - based evidence as speaking for a primary dysfunction of left temporoparietal language regions (e.g., pugh., 2000 ;, 2004, mccandliss and noble, 2003 ; shaywitz., 2007). indeed, our quantitative meta - analysis of functional imaging research identified several maxima of dyslexic underactivation in left posterior temporal regions (richlan., 2009). the mentioned reviews suggest that left temporoparietal regions are engaged primarily by the sublexical phonological reading route which is known to be specifically error - prone for (english) dyslexic readers. recently, further support for a left superior temporal dysfunction was supplied by fmri studies which used a letter - sound integration paradigm with dutch adult dyslexic readers (blau., 2009). the dyslexic readers although knowing all relevant letter - sound associations failed to exhibit modulation of left superior temporal areas in response to incongruent letter - sound pairs. in a more general perspective, underactivation of posterior temporal language areas in response to reading - related tasks is linked to the phonological deficit explanation of dyslexia, which assumes that underspecified phonological word representations give rise to a phonemic awareness deficit which hinders the extraction of grapheme phoneme associations on which sublexical reading is dependent (e.g., snowling, 2000). the present findings stand in marked contrast to the position that sublexical reading engages a left temporoparietal reading system, and that dyslexic readers suffer from a primary dysfunction of these regions. in the whole - brain analysis, neither nonimpaired nor dyslexic readers exhibited reliable activation in left superior temporal regions in response to pseudohomophones and nonwords, despite a liberal statistical threshold. (2009) as exhibiting dyslexic abnormalities in response to letter - sound matching, did not show reliable activation in the present study. actually, we found the opposite from what is expected from a dysfunction of left superior temporal regions, because our dyslexic readers exhibited a tendency towards higher activity in the left posterior superior temporal gyrus. a rather obvious explanation of the present absence of left temporal activation in both nonimpaired and dyslexic readers is that our activation task was based on silent reading. however, one may note that the instruction required a judgement based on sound (i.e., does xxx sound like a real word ?) and presented pseudohomophones and nonwords (e.g., taksi and tazi) which can only be reliably distinguished by sublexical reading processes. of course, the present negative finding on a dysfunction of the left temporoparietal can not rule out that such a dysfunction would have become apparent if we had relied on a more demanding phonological task, for example, judging whether two visual words do or do not rhyme (see richlan., 2009, for a list of activation task). furthermore, there is evidence that the left supramarginal gyrus plays an important role in the early phase of learning to read (e.g., church., 2008). from this finding one may infer that a left temporoparietal dysfunction may have been identified with a younger group of german dyslexic readers. consistent with an important role of the left parietal cortex in reading, our nonimpaired readers exhibited high activity in a left inferior parietal region, specifically in response to pseudohomophones and nonwords, and dyslexic readers exhibited reduced activity in this region. for interpretation, one may note that this inferior parietal cluster at x = 51, y = 45, z = 54 is quite distant from the left posterior superior temporal regions which are considered as core regions of phonological reading processes. furthermore, the activation pattern shown by nonimpaired readers, that is, high activity in the inferior parietal cortex but no substantial activity in the posterior superior temporal area, speaks against the possibility that the inferior parietal activation reflects phonological processing (i.e., hearing of phonemes or assembled pronunciations). following milner and goodale (1995), one may hypothesize that the left inferior parietal cortex in silent reading serves as attention guiding interface between visual - orthographic coding in ot regions and productive phonological processes in left ifg regions. however, one may note that the presently identified region with dyslexic underactivation in response to nonwords is part of an extended left parietal region which was activated by all item types in dyslexic and nonimpaired readers. the only brain region where dyslexic readers, in addition to left ot and left inferior parietal regions, exhibited underactivation was identified in the left ifg, opercular part. different from the left ot region which exhibited underactivation in response to all three item types, the left inferior frontal together with the left inferior parietal region were identified by the voxel - based analysis only for nonwords. this suggests that a dysfunction of these regions became apparent only for the most difficult item types for which sublexical processing did not find a phonological lexicon entry. however, the roi analysis found reliable underactivation in the ifg not only for nonwords but also for pseudohomophones and a tendency was also apparent for words. left ifg underactivation was also found in our meta - analysis (richlan., 2009) where it was high - lighted as a new finding which is overlooked in narrative reviews of imaging studies. these reviews summarily speak of dyslexic overactivation in a left frontal reading system in order to compensate for underactivation in the left temporoparietal reading system (e.g., pugh. the present study differs from this pattern by finding left ifg underactivation without underactivation in left tp regions. the underactivation in the left ifg in response to pseudohomophones and nonwords is suggestive of a dysfunction in the efficient access to sublexical phonological segments. the mentioned reviews summarily interpret the left frontal overactivation in dyslexic readers as reflection of compensatory silent articulatory processes in visual word processing. although we did find the opposite of left frontal overactivation in the left ifg, there is specific support for this interpretation in our results. we did find dyslexic overactivation in pre- and post - central regions and in the primary motor cortex. these findings, together with overactivation in the caudate and putamen, speak for reliance on silent articulatory processes. a predominance of dyslexic overactivation was also found in our preceding study which measured brain activity in response to sentence verification (kronbichler., 2006) and in another german - based study which measured dyslexic brain activity in response to rhyme judgements of nonwords (grnling., 2004). this predominance of overactivation in german - based dyslexia studies stands in contrast to a predominance of underactivation in a substantial number of english - based dyslexia studies (richlan., 2009) and may be related to effortful sublexical route processes based on the reliable grapheme phoneme relations of german. these orthography - related differences in dyslexic brain dysfunctions challenge current neurocognitive accounts of dyslexia, which assume that all dyslexic readers irrespective of the particular writing system of their language have the same underlying brain dysfunction (for a discussion, see hadzibeganovic., 2010, this issue). the behavioral in - scanner data suggested that the present german dyslexic readers, similar to nonimpaired controls, relied on lexical route processes (orthographic word recognition, direct access to word phonology) for familiar letter strings of words, and on sublexical route processes (grapheme phoneme conversion) for unfamiliar letter strings of pseudohomophones and nonwords. the imaging results were suggestive for a different neural organization of reading processes in dyslexic readers. specifically, dyslexic readers, in response to lexical route processes, exhibited under - activation in a left ot region corresponding to the vwfa, presumably engaged by visual - orthographic whole word recognition. this region was also insensitive to the increased visual - orthographic processing demands of the sublexical route. reduced engagement in response to sublexical route processes was also found in a left inferior parietal region, presumably engaged by attentional processes, and in a left inferior frontal region, presumably engaged by phonological processes. in contrast, to this reduced engagement of the nonimpaired reading network, our dyslexic readers exhibited increased engagement of visual occipital regions and of regions presumably engaged by silent articulatory processes (premotor / motor and subcortical caudate and putamen). different from largely english - based imaging finding, no dyslexic abnormalities were found in left posterior temporal regions.	this study examined functional brain abnormalities in dyslexic german readers who due to the regularity of german in the reading direction do not exhibit the reading accuracy problem of english dyslexic readers, but suffer primarily from a reading speed problem. the in - scanner task required phonological lexical decisions (i.e., does xxx sound like an existing word ?) and presented familiar and unfamiliar letter strings of existing phonological words (e.g., taxi - taksi) together with nonwords (e.g., tazi). dyslexic readers exhibited the same response latency pattern (words < pseudohomophones < nonwords) as nonimpaired readers, but latencies to all item types were much prolonged. the imaging results were suggestive for a different neural organization of reading processes in dyslexic readers. specifically, dyslexic readers, in response to lexical route processes, exhibited underactivation in a left ventral occipitotemporal (ot) region which presumably is engaged by visual - orthographic whole word recognition. this region was also insensitive to the increased visual - orthographic processing demands of the sublexical route. reduced engagement in response to sublexical route processes was also found in a left inferior parietal region, presumably engaged by attentional processes, and in a left inferior frontal region, presumably engaged by phonological processes. in contrast to this reduced engagement of the optimal left hemisphere reading network (ventral ot, inferior parietal, inferior frontal), our dyslexic readers exhibited increased engagement of visual occipital regions and of regions presumably engaged by silent articulatory processes (premotor / motor cortex and subcortical caudate and putamen).
community acquired pneumonia (cap) is one of the main concerns of health services which only causes to 1.7 million cases of hospitalization annually in the united states (1, 2). it is one of the most common infectious diseases and important causes of mortality and morbidity in worldwide (3). streptococcus pneumonia, hemophilic influenza, and moraxella catarrhalis are typical bacterial pathogens in approximately 85% of cap cases (4). the presentation of cap includes fever, cough, and pleuritic chest pain. beside the physical examination, chest x - ray (cxr), as the simplest diagnostic tool, is suggested to exclude conditions mimic cap (1). however, cxr findings may be negative in patients if cap presents at early stages of the disease. moreover, noticing to high costs and the need for more irradiation, the routine usage of computed tomography (ct) scan in diagnosis of pneumonia in emergency departments is not recommended (5). ultrasonography (cus) has been recently applied in detection of pulmonary diseases such as pulmonary edema, pulmonary embolisms, pericardial and pleural effusion, and pneumothorax (6, 7). however therefore, the aim of this study was evaluating diagnostic accuracy of cxr and cus in findings of pneumonia. study design and setting the present study was conducted to evaluate diagnostic accuracy of cus and cxr in detection of pneumonia. it was performed in the emergency department (ed) of imam reza medical research and training hospital, tabriz, east azerbaijan, iran, through february to april 2014. the local ethical committee of tabriz university of medical sciences approved the protocol of the study and written consent was obtained from all patients after describing the aims and methods of the study. the consecutive samples of suspected cap patients were underwent cus, cxr, and ct scan. presence of fever, cough, pleuritic pain, sputum production, and dyspnea were considered as signs and symptoms of cap (8). patients with asthma, acute coronary syndrome, septic shock, pulmonary embolism, chronic obstructive pulmonary disease (copd), cardiac and pleural disorders were excluded. after clinical examinations, patients with clinical suspicion of cap underwent cxr, cus, and chest ct scan. clinical examinations and cus were done with bedside machine available in the ed (gh healthcare ; logiq 200, pro series ; korea) with a convex 3.5-mhz transducer by a trained emergency medicine specialist. then, patient referred to radiology unit to perform cxr and chest ct scan (as a gold standard) with toshiba asteion 16 slices scanner with considering one - millimeter distance between image slices. the radiography and ct scan findings were interpreted by a radiologist blinded to clinical examinations. diagnostic accuracy of cus and cxr were assessed by calculating the sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios. diagnostic accuracy of chest ultrasonography and radiography in detection of pneumonia and plural effusion (95% confidence interval) we studied imaging findings of 30 patients with clinical suspicion of community - acquired pneumonia (93.3% male, mean age 63.818.3 years). ct scan findings showed 29 (96.7%) cases of pneumonia, while cus revealed the diagnosis of pneumonia for all 30 cases (1 case of false positive). cxr also showed 28 (93.3%) pneumonia cases (2 false negative and 1 false positive) (table1). the sensitivity of cus in pneumonia detection was calculated 100.0% (95% ci : 85.4 - 100.0), but its specificity was not calculable because of being positive of all subjects. however, sensitivity and specificity of cxr in pneumonia detection were 93.1% (95% ci : 75.8 - 98.8) and 0.0% (95% ci : 0.0 - 94.5), respectively. based on ct scan findings, cus detected 12 (40%) subjects (1 false negative), while cxr could only detect 8 (26.7%) patients (4 false positive and 4 false negative). sensitivity and specificity of cus in detection of pleural effusion were 92.3% (95% ci : 62.1 - 99.6) and 100.0% (95% ci : 77.1 - 100.0), respectively. sensitivity and specificity of cxr were also 66.7% (95% ci : 35.4 - 88.7) and 77.8% (95% ci : 51.9 - 92.6), respectively (table 1). findings of the present study demonstrated that diagnostic accuracy of cus in detection of pneumonia and plural effusion were higher than cxr. several studies stated that cus is a simple, fast, and effective diagnostic tool for detection of pulmonary diseases. for example, in a study done by mathis and colleagues, authors recommended that ultrasonography is the simplest and most sensitive imaging tool for measurement of pleural fluid (9). bsrilleni and colleagues concluded that ultrasonography is a useful and reliable method in diagnosing the pulmonary diseases (10). in another study conducted by lichtenstein., it was stated that cus is a fast and cost effective method in detecting pulmonary diseases, without radiation exposure (11). neesse and colleagues evaluated the results of cxr in pulmonary diseases and concluded that ultrasonography is a rapid tool in detecting the pulmonary diseases, leads to accurate diagnosis in 68% of cases (12). these findings are along with advanced trauma life support (atls), represented that ultrasonography can be used in detection of abdominal free fluid, organ trauma, and pneumothorax. one of the most important solving is the exact and long - term training of ed specialists regarding performing and interpreting of ultrasonography finding. the small sample size was the limitation of this study and consequently specificity of cus was not calculable. in addition, the small sample size caused that no true negative case was seen by cxr ; therefore, the power of calculated specificity of cxr was week. the cus operator was not blind to the clinical presentation of patients, while the radiologist did not aware from the clinical presentation of patients and less diagnostic accuracy of cxr might be derived from that. finding of the present study demonstrates that diagnostic accuracy of cus in detection of pneumonia and plural effusion were higher than cxr. ali taghizadieh, alireza ala, and akbar nadi performed the data collection, literature review, and drafting of the manuscript. farzad rahmani undertook the major parts of the study design and performed the statistical analysis.	introduction : chest x - ray (cxr) is the simplest diagnostic tool of community acquired pneumonia (cap), but it has some limitation. therefore, the aim of this study is comparing the diagnostic accuracy of cxr and chest ultrasonography (cus) in detection of cap. methods : in the present study, a consecutive sample of suspected patients with cap was underwent cus, cxr, and chest computed tomography (ct) scan. diagnostic accuracy of cus and cxr was assessed by calculating the sensitivity, specificity, predictive values, and likelihood ratios using spss 20 statistical software. results:30 patients with cap were enrolled (93.3% male with mean age of 63.8 18.3 years). sensitivity of cus and cxr in detection of cap were 100.0% (95% cl : 85.4 - 100.0) and 93.1% (95% cl : 75.8 - 98.8), respectively. specificity of cxr was 0.0 (95% cl : 0.0 - 94.5), while the cus specificity was not calculable. conclusion : findings of the present study demonstrated on the higher diagnostic accuracy of cus versus cxr in detection of pneumonia.
metaplastic breast cancer (mpbc) is an extremely rare breast cancer subtype constituting 0.25% to 1% of all breast cancers. histopathologically, mpbc is characterized by a heterogeneous phenotype, most commonly with epithelial and mesenchymal components. mpbc is typically associated with rapid metastasis and poor survival compared to other breast cancer subtypes. most mpbcs are negative for estrogen receptor (er), progesterone receptor (pr), and her2/neu, and are managed as triple - negative breast cancer. it has been suggested that the poor prognosis of mpbc could be due to its stem cell - like characteristics. phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit (pik3ca) mutations and loss of the phosphatase and tensin gene (pten) have been identified in this group, but their prognostic and therapeutic significance is unclear. due to its rarity, tumor protein 53 (tp53) mutation and overexpression are present in 20% to 40% of breast cancers, but the incidence and significance of this mutation in mpbc are unknown. herein, we report the clinical course of a 59-year - old african american woman with mpbc presenting with pik3ca and tp53 mutations, who was treated using a molecularly matched therapy. a 59-year - old african american woman presented with a left - sided breast mass. the biopsy revealed a triple - negative, poorly differentiated, high - grade invasive ductal carcinoma with necrosis. clinical staging indicated that the lesion was a t2 stage, n1 or 3, and m0 with biopsy - proven axillary metastasis and suspicion of internal mammary nodal involvement. the patient received standard neoadjuvant therapy with weekly paclitaxel (12 cycles) and 5-fluorouracil, adriamycin, and cyclophosphamide (fac) (2 cycles). subsequently, the patient declined further chemotherapy and underwent a left modified radical mastectomy followed by chest wall radiation. the surgical pathological report indicated an invasive matrix - producing (metaplastic) carcinoma with patchy necrosis and 10% er positivity, pr and her2/neu negative, and stage pt2 n1 with 2 of 25 axillary lymph nodes presenting metastatic carcinoma. after radiation, the patient started a course of anastrozole, which was later switched to exemestane due to arthralgia. a year after surgery, the patient presented with pulmonary metastases and metastatic lymph nodes in the mediastinum and left internal mammary region (figure 1) the patient 's tumor, including the left breast primary and the left axillary lymph node metastases were profiled separately. genomic dna polymerase chain reaction - based sequencing, using a next - generation sequencing (ngs) platform, was performed to screen for the frequently reported point mutations in a 46-gene panel on the primary and metastatic tissues. this 46-gene panel was validated using an ngs platform for the detection of frequently reported point mutations in human malignancies in the clinical laboratory improvement amendments - certified molecular diagnostics laboratory at the university of texas md anderson center. a minimum of 250coverage is required at a given base for the interpretation of a wild - type or variant call. although the ngs platform is capable of achieving a much higher analytical sensitivity, for clinical purposes, we determined the effective lower limit of detection of this assay (analytical sensitivity) for single - nucleotide variations to be in the range of 5% (one mutant allele in the background of 19 wild - type alleles) to 10% (one mutant allele in the background of nine wild - type alleles) by taking into consideration the depth of coverage at a given base and the ability to confirm low - level mutations using independent conventional platforms. to avoid false negatives, we required that the tumor nuclei represented 20% of the nuclei in the tested sample. a pik3ca gene mutation was detected in codon 545, exon 10 (gag to aag ; p.glu545lys) and another mutation was found in thetp53 gene in codon 286, exon 8 (gaa to aaa ; p.glu286lys). the patient was given a combination therapy of intravenous temsirolimus, 25 mg weekly, plus intravenous bevacizumab, 15 mg / kg, and liposomal doxorubicin, 20 mg / m, once on day 1 every 21 days. repeat positron emission tomography / computed tomography (pet / ct) after two cycles of therapy showed a decrease in the standard uptake value from 12.3 to 3.9 in one intrathoracic lymph node and a decrease from 13.1 to 7.0 in another lesion as well as the complete resolution of the disease in most of the sites on a pet scan, with corresponding anatomic improvement observed on a ct scan (figures 1,2,3). pet / ct or ct - chest / abdomen / pelvis was performed every 2 months to evaluate the response. after seven cycles, the patient requested a month break from chemotherapy related to grade 2 - 3 fatigue. the patient resumed her therapy with reasonable tolerance and continued with clinical benefit and radiological response. after 14 cycles of therapy, a plateau of response was reached and the patient developed grade 4 dyspnea. the rarity, heterogeneity, and clinically aggressive nature of mpbc have made this disease a therapeutic challenge. since these tumors are largely chemoresistant, the identification of novel targeted therapies is necessary to treat patients with this malignancy. early identification of targetable molecular aberrations, during the course of the disease, might provide therapeutic benefits, particularly in the absence of other options. in this patient, the combination of liposomal doxorubicin, bevacizumab, and temsirolimus was selected based on the strong scientific rationale that the pik3ca mutation is found in approximately 14% to 21% of breast cancer patients referred to early phase clinical trials. a phase i trial using this combination resulted in a 42% partial response (pr)/complete response (cr) rate in mpbc (5 out of 12 patients). moreover, 39% of patients with a pik3ca mutation and/or a pten mutation / loss (11 out of 28 patients) achieved a pr / cr. previous studies have demonstrated that therapies using mammalian target of rapamycin (mtor) inhibitors show a response rate of 35% to 39% in patients presenting with activating mutations of the phosphatidylinositide 3-kinase / akt / mtor (pi3k / akt / mtor) pathway when compared to patients who did not have a documented pik3ca mutation (response rate, 6%-10%). in addition to blocking the mtor pathway, temsirolimus decreases hypoxia - inducible factor (hif-) levels. it has been shown that resistance to doxorubicin - based therapy is mediated through hif-1. thus, hif-1 inhibition by temsirolimus might increase the sensitivity to doxorubicin. in addition to this putative synergy, the p53 tumor suppressor gene plays an important role in angiogenesis, apoptosis, and tumor stability. tp53 mutations are found in approximately 53% of patients referred to early phase clinical trials. intriguingly, tp53 patients present a longer progression - free survival when treated with bevacizumab in combination with other drugs compared to patients treated without bevacizumab (10.97 months vs. 4.37 months). mtor inhibitors can reduce vascular endothelial growth factor (vegf) levels, increasing the antiangiogenic effects of bevacizumab. the fact that mpbc commonly displays vegf and hif- aberrations strengthens the rationale for using this combination. a clinical trial combining these three agents for mpbc is ongoing and may help us to further define the effect of this combination. in summary, identifying the underlying molecular aberrations in each patient as early as possible is crucial to select the most rational matched treatment regimen. interestingly, in our case study, the same molecular aberrations were identified in both the primary and metastatic specimens. in addition to pik3ca mutations, other mutations and gene amplifications have been reported in mpbc. leibl and moinfar reported epidermal growth factor receptor amplification in 14 out of 20 patients with mpbc. the case described in this study is unique in ways other than its specific molecular aberrations. later analysis of the surgical sample identified a matrix - producing (metaplastic) carcinoma with patchy necrosis and 10% er positivity, although er was not detected initially. another important observation in this patient was the complete resolution of fluorodeoxyglucose (fdg) uptake in most lesions after treatment. using mtor inhibitors can lead to decreased metabolism in cancer cells due to their predominant cytostatic properties. fdg uptake, which is reflective of metabolic activity, may be a useful biomarker to monitor the response to mtor inhibitor treatments. additionally, pet / ct might be a better imaging modality than ct alone to evaluate the tumor response in patients treated with mtor inhibitors. in conclusion, mpbc is a heterogeneous breast cancer with no current standard therapy. patients may benefit from being offered molecular profiling early during the course of the disease followed by a therapy guided accordingly. liposomal doxorubicin+bevacizumab+temsirolimus is one combination that has a strong underlying scientific rationale, and could be used a valuable therapeutic strategy for patients with mpbc.	metaplastic breast cancer (mpbc) is an extremely rare breast cancer subtype, characterized by a heterogeneous phenotype. mpbc aggressive biology is attributed to its stem cell - like characteristics. since these tumors are largely chemoresistant, novel targeted therapies should be explored. herein, we report the clinical course of a 59-year - old african american woman with mpbc with a pik3ca mutation in codon 545, exon 10 (gag to aag ; p.glu545lys) and a tp53 mutation in codon 286, exon 8 (gaa to aaa ; p.glu286lys). the same mutations were observed in the primary and secondary sites. the patient was treated with a molecularly matched therapy using a combined antiangiogenic and mammalian target of rapamycin kinase inhibitor strategy that included liposomal doxorubicin, bevacizumab, and temsirolimus. partial remission was achieved. in this report, the scientific rationale underlying the activity of this combination was explored. in conclusion, patients may benefit from being offered molecular profiling early during the course of the disease to receive a therapy guided accordingly.
epstein - barr virus (ebv) and cytomegalovirus (cmv) are members of the herpesvirus family. co - infection with these two viruses occurs occasionally in children, but co - reactivation of these viruses has not been reported. ebv is a double - stranded dna herpesvirus that infects and persists in > 90% of humans worldwide. ebv infection frequently causes self - limiting infectious mononucleosis ; latent infection is preferentially sustained in b cells over the lifetime of the host. cmv is also a common viral infection in humans, which occurs in 40100% of humans worldwide. primary cmv infection is followed by either chronic infection or viral latency from which the virus may be reactivated. cmv infection of the gastrointestinal tract occurs mainly in immunocompromised individuals, including those with inflammatory bowel disease (ibd) and those who have received transplants. we herein report, to our knowledge, the first case of an immunodeficient patient with severe hemorrhagic colitis associated with the reactivation of both ebv and cmv, and whose endoscopic findings mimicked ibd. a 56-year - old man was diagnosed with severe aplastic anemia (saa), showing pancytopenia and severe bone marrow hypocellularity ; therefore, he was treated with antithymocyte globulin (atg) and cyclosporine (csp). after 2 years of atg / csp therapy, he suddenly started passing bloody diarrhea and developed a high fever. blood tests revealed the following results : hemoglobin level 4.2 g / dl, platelet count 3.7 10 cells / l, and white blood cell count 3,230 cells / l. the patient 's c - reactive protein level was 4.7 mg / dl and serum cmv antigen c7-hrp was positive in 2/50,000 cells. we initially thought the colitis to be associated with cmv infection because of the endoscopic features and positive result for serum cmv antigen in spite of negative results obtained using immunohistochemical analysis. therefore, the patient was treated with ganciclovir for 2 weeks ; however, his symptoms did not resolve. however, his symptoms persisted for the next 2 weeks. therefore, assuming a potential misdiagnosis, a second colonoscopy was performed, which showed multiple deep ulcerations (fig. 2b), and in situ hybridization revealed ebv - encoded small rna-1 (eber-1)-positive cells (fig. 2c). additionally, serum c7-hrp was positive in 33/50,000 cells, and both blood and colon tissues were positive for ebv dna, which was detected using pcr analysis. we finally diagnosed the patient with colitis associated with the reactivation of cmv and ebv. after cessation of csp and treatment with gamma globulin and ganciclovir, his symptoms resolved. a subsequent blood sample was negative for ebv dna, and colon cells were negative for eber-1, but positive for ebv dna. interestingly, 6 months after the first remission, the patient had a recurrence of severe bloody diarrhea after an increase in the csp dose. both blood and colon tissues were positive for ebv dna and a colon biopsy showed eber-1-positive cells ; however, we did not detect cmv reactivation. after a second round of treatment with gamma globulin and ganciclovir and cessation of csp, his symptoms resolved. the patient has had no abdominal symptoms for 1.5 years with the scheduled antiviral treatments and after cessation of csp. to our knowledge, this is the first reported case of severe colitis associated with reactivation of both ebv and cmv in a patient with saa after atg / csp therapy. ebv primary infected b cells, cmv was designed to enter monocytes besides lymphocytes, whereas both of them also infected epithelial cells. reactivation of ebv or cmv after atg / csp therapy has been reported in patients with saa [1, 2 ]. after atg / csp therapy in patients with saa, ebv reactivation occurred in 87% and cmv reactivation in 33%. however, reactivation of both these viruses has not been reported, and the clinical features are not well known. in this case, we have several reasons to associate ebv with the severe colitis : the serum samples from the patient were positive for ebv dna and the colonic tissue showed the presence of eber-1-positive cells on in situ hybridization. in contrast, at the time of colitis remission, neither ebv dna in the blood nor eber-1-positive cells in the colon were detected. interestingly, the patient had two colitis attacks with identical clinical symptoms and ebv results. therefore, colitis likely reflects ebv reactivation, which is indicated by the presence of ebv dna in the blood and eber-1-positive cells in the colon. there is only one report of ebv colitis after atg therapy, however ebv and cmv colitis after atg therapy have never been reported. in the case of this patient, it was necessary to distinguish severe colitis from other ebv - associated diseases, including chronic active ebv infection, ebv - associated t / natural killer cell lymphoproliferative disorder (ebv - t / nk - lpd) and ebv - associated lymphomagenesis. this case did not satisfy chronic active ebv infection guidelines for diagnosis of lymphomagenesis [6, 7 ]. ebv - t / nk - lpd has 4 categories : a1, a2, a3 and b. ohshima. reported that ebv - t / nk - lpd could develop into category a1 (smoldering), a2 (chronic) or a3 (leukemia / lymphoma). our patient was successfully treated twice with antiviral drugs only and after cessation of csp treatment and without the use of chemotherapy such as rituximab. in addition, he had no recurrence of symptoms for 1.5 years after treatment. therefore, this case was considered category a1, i.e. smoldering ebv - t / nk - lpd. however, careful follow - up is necessary as colitis is not a well - known symptom of ebv - t / nk - lpd. there have been some reports of ebv / cmv infection in patients with ibd [9, 10, 11 ]. those studies used pcr analysis to show the presence of ebv in gastrointestinal tissue from patients with ibd. however, these reports never used immunohistochemical analysis or tested blood samples to detect the presence of ebv dna. weinberg. reported the difficulty of differentiating between ebv - associated diarrhea and ibd. besides, the mechanisms of ulcer formation and the differences in endoscopic features caused by ebv are not well understood. in the future, more case studies are necessary to differentiate ebv - associated intestinal diseases from ibd in patients with ebv infections. in conclusion, to our knowledge, this is the first reported case of a patient with saa who developed severe hemorrhagic colitis owing to the reactivation of both ebv and cmv.	epstein - barr virus (ebv) and cytomegalovirus (cmv) are members of the herpesvirus family and common causes of viral infection in humans. cmv infection of the gastrointestinal tract occurs mainly in immunocompromised individuals, on the other hand ebv infection and reactivation involving the gastrointestinal tract is very rare. a 56-year - old man was diagnosed with severe aplastic anemia and treated with antithymocyte globulin (atg) and cyclosporine (csp). after 2 years of atg / csp therapy, he suddenly started passing bloody diarrhea and developed a high fever despite csp treatment. endoscopic features included severe edema and multiple superficial ulcers ; the patient was initially diagnosed with severe colitis resembling inflammatory bowel disease (ibd). however, his symptoms did not resolve with steroid treatment. immunohistochemical analysis of samples obtained from a second colonoscopy showed cells positive for cmv, and in situ hybridization revealed ebv - encoded small rna-1-positive cells. additionally, the patient 's serum was positive for c7-hrp, and both blood and colon tissues were positive for ebv dna, which was detected using pcr analysis. we finally diagnosed the patient with colitis associated with reactivation of both cmv and ebv. the patient remains diarrhea - free after 1.5 years with scheduled globulin treatment and after cessation of immunosuppressive drug therapy. to our knowledge, this is the first reported case of an immunodeficient patient with severe hemorrhagic colitis that was associated with reactivation of both ebv and cmv, and whose endoscopic findings mimicked ibd.
alzheimer 's disease (ad) is a neurodegenerative disorder that will affect 15 million people in usa alone in the next ten years [1, 2 ]. the most common form of the disease is the late onset form (load) that affects people older than 65. load is caused by a complex interaction of risk factors including age, genetics, and environmental factors, such as level of education, diet, and physical activity [37 ]. the accumulation of a, a neurotoxic product of amyloid precursor protein (app) cleavage, is central to ad pathogenesis [810 ]. this accumulation causes synaptic dysfunction and eventually neuronal death [9, 10 ]. therefore proteins that affect app metabolism and synaptic function are likely to be important in ad pathogenesis. the neurotrophin receptors (trka, trkb, and trkc) are important in neuronal development and synaptic function [11, 12 ]. levels of trka, trkb, and trkc, but not p75, are downregulated in ad brain samples. trk downregulation has been proposed as a biomarker of ad progression since trk mrna levels correlate with the degree of cognitive impairment. further evidence for a role of trkb in ad is the fact that trkb can modulate app levels and proteolysis. expression of the longest trkb isoform, full - length trkb (trkb fl), can increase app promoter transcription and promotes accumulation of sapp- [1417 ]. conversely, a has been found to reduce trkb fl / bdnf levels and to impair trkb - mediated signaling [1821 ]. interestingly, knockdown of another splice variant of trkb, truncated trkb (trkb t) in a mouse model of down syndrome rescued neuronal death. conversely, mice overexpressing trkb t display synaptic dysfunction and long - term potentiation defects. the gene encoding trkb, ntrk2, is located on chromosome 9, specifically 9q22. this region has been genetically linked to ad [24, 25 ]. despite the experimental evidence functionally linking trkb signaling to app metabolism and synaptic function, case - control and genomewide association studies of ntrk2 single nucleotide polymorphisms (snps) found no significant association with ad [2530 ]. one family - based study did observe genetic association of ntrk2 haplotypes with ad. three major splice variants of trkb are expressed in neurons, trkb fl, trkb shc, and trkb t. we hypothesized that these different trkb isoforms differentially affect app metabolism and could play a role in the pathogenesis of ad. the three trkb splicing isoforms we investigated share an extracellular bdnf binding domain and differ in their cytoplasmic - domain (figure 1). two splice variants encode full - length receptors, trkb full - length (fl), that contain a tyrosine kinase domain, an shc - binding domain and a plc--binding domain in the intracellular portion [32, 33 ]. two isoforms encode shorter receptors, trkb shc, that contain only an shc - binding domain, and the remaining isoform is a truncated receptor, trkb t, that does not have any known intracellular functional domain (figure 1). we used a previously described cell - based functional screen to identify putative app metabolism regulators. we found that ntrk2 knockdown altered both aicd - mediated luciferase activity and app full - length levels. to characterize the role of trkb fl and truncated isoforms we knocked down and overexpressed the isoforms in an sh - sy5y neuroblastoma cell line overexpressing app as a fusion protein with the yeast transcription factor gal-4. we then measured app fl levels and proteolytic products using western blots and luciferase assays. specifically, overexpression of trkb fl increases aicd - gal4-mediated luciferase activity. while overexpression of trkb t does not alter the luciferase activity and trkb shc decreases the luciferase activity compared to control. we determined that the tyrosine kinase and plc- functional domains contribute to the observed trkb fl - mediated effects. we also found that the shc - binding site contributed to the observed trkb - shc - mediated effects. bdnf stimulation of the exogenously expressed trkb receptors amplified the app metabolism effects and cotransfection of the trkb - truncated isoforms with trkb fl altered the effects on app metabolism. four shrna - containing plasmids specific for ntrk2 were obtained from the psm2 retroviral library of the drexel rnai resource center purchased from open biosystems. the constructs i d numbers are ntrk2.1 : 1920 ; ntrk2.2 : 2295 ; ntrk2.3 : 29734 ; ntrk2.4 : 30795. we also used app (i d 39147) and luciferase targeting shrna (rhs1705) as positive controls and a scrambled shrna sequence (nonsilencing, ns, rhs1707) as a negative control. the trkb full - length and truncated gfp fusion constructs and the gfp - f control overexpression plasmid were kindly donated by dr. eero castren (university of helsinki, finland) and were previously described [36, 37 ]. site - directed mutagenesis (stratagene, quikchange mutagenesis kit) was utilized to generate point mutants on the trkb full - length receptor functional domains. mutagenesis was carried out according to manufacturer 's instructions and the primers employed are reported in table 1 (the bolded sequences represent the mutations / insertion). therefore trkb fl k571 m indicates the tyrosine kinase dead receptor since it is mutated on the atp - binding site ; trkb fl y515f indicates the receptor mutated on the shc - binding site ; trkb fl y816f indicates the receptor mutated on the plc--binding site. note that in some literature the trkb mutants are referred to with the numbering of the amino acidic sequence of trka, the ngf receptor, that has functional sites in common with trkb and therefore are referred to as k560 m, y490f and y785f respectively [32, 33 ]. trkb shc indicates the other human truncated isoform (isoform d and e, ncbi gene nm_0010180642 and nm_0010180662). trkb shc was obtained by insertion of the exon 19 followed by a stop codon after the shc - binding site on the trkb fl constructs. after obtaining the trkb shc isoform by insertion, the shc - binding site was mutated on that isoforms using the same primer sequence employed for the trkb fl mutant on the shc - binding site (trkb y515f). one clone per construct was transformed in e. coli (dh5- competent cells, invitrogen). transformed bacteria were selected on 100 g / ml ampicillin lb - agar plates and liquid cultures were grown overnight at 37c. bacterial cultures were miniprepped (miniprep kit, quiagen) and used for transfection after dna quantification. sh - sy5y cells stably transfected with uas - luciferase and app - gal4 described before were maintained in dmem (gibco) supplemented with 10% fbs, penicillin streptomycin and 200 g / ml g418 (gibco). to assess the effects of trkb knockdown or overexpression on aicd - gal4 mediated luciferase we used the following transfection protocol previously described. briefly, one day before transfection cells were plated in 96-wells plates at approximately 4050% confluency. the day of transfection media was removed from the cells and replaced with transfection media : 100 l of serum free dmem media containing 2 g / well arrest - in (open biosystems) and 0.2 g / well plasmid dna. cells were also transfected with shrna targeting app, luciferase and a control shrna that contains a scrambled sequence that does not target any human gene. in addition a mock transfection, containing only arrest - in was performed to control for selection effectiveness. 6 replicate wells per shrna constructs and mock control transfection were set up for each independent experiment. the transfection media was left on the cells for 8 hours and then replaced with complete media. 48 hours after transfection, transfected cells were selected with 4 g / ml puromycin (sigma) in 10% fbs dmem with 200 g / ml g418. the media was changed every 48 hours and cell death was monitored and compared to the mock - transfected control. once all the cells in the mock control wells were dead, surviving cells in the shrna transfected wells were split and transferred to another 96-well plate and a 24-well plate. cell lysates were collected from 6080% confluent 96-wells 1113 days after transfection in 100 l glo lysis buffer per well (promega). lysates were used immediately after collection or frozen prior to performing steady glo luciferase assays (promega). shrna - mediated knockdown effectiveness was monitored by comparing the luciferase signal of the nonsilencing control shrna with the app targeting shrna. after assessing successful knock - down, luciferase data for the experimental shrna targeting ntrk2 was collected and analyzed. in parallel, 24-well plates and 12-well plates were seeded with the same cells that had been assayed for luciferase signal and collected for western blot analysis. the same transfection procedure was followed for the overexpression experiments, but lysates were collected 48 hours after transfection and transfection efficiency was monitored by fluorescence microscopy, no antibiotic selection was performed in this case. conditioned media was collected from the cells (48 hours after transfection) in eppendorf tubes and centrifuged at 14,000 rpm for 10 minutes at 4c (beckman coulter, microfuge 22r). the resulting supernatant was collected and 142 l were mixed with 33 l of 4x reducing loading buffer (invitrogen) supplemented with 0.4% -mercaptoethanol (sigma). whole cell lysates were collected (48 hours after transfection) by lysing the cells with ice - cold radio immuno - precipitation (ripa) buffer (150 mm nacl, 1% np40, 0.5% doc, 1% sds, 50 mm tris, ph 8.0) supplemented with halt cocktail of protease and phosphatase inhibitors (thermoscientific). cell lysates were sonicated in an ice - cold water bath sonicator for 6 minutes then centrifuged 20 minutes at 4c at 14,000 rpm. the resulting supernatants were collected and protein concentration measured with a bca protein concentration kit (pierce) according to manufacturer 's instructions. western blot samples were prepared at a final concentration of 1 - 2 g/l in 4x reducing loading buffer (invitrogen) and heated at 70c for 10 minutes. 1525 g of total protein / well were separated on 412% tris - glycine midi gels (invitrogen) in mes - sds running buffer (invitrogen) and run at 190 mvolts for 45 minutes. the separated proteins were transferred to pvdf fl membranes (millipore) in a semi - dry transfer apparatus (aa hoefer te77x) for 3 hours at 125 milli amp per gel. membranes were blocked one hour at room temperature using licor blocking buffer then probed overnight with primary antibodies diluted in licor blocking buffer at 4 or 25c. membranes were then washed for 5 minutes 4 times with 0.1% tween (sigma) in pbs. after washing, membranes were incubated in the dark with the appropriate secondary antibody irdye (licor) diluted in licor blocking buffer for one hour. membranes were scanned on an odyssey infrared scanner (licor) at appropriate intensities and images acquired. band intensities were quantified with the provided in - built software (licor) and always normalized to the actin loading control. when conditioned media was analyzed the signals were normalized to the protein concentration of the corresponding lysates. detection of trkb - gfp tagged constructs utilized mouse anti - gfp antibody (1 : 1000, living colors, clontech) ; detection of app full - length and c - terminal fragments utilized a8717 rabbit antibody (1 : 2000, rb, sigma) ; detection of sapp 22c11 utilized mouse antibody (1 : 1000, millipore) ; detection of sapp- 6e10 utilized mouse antibody (1 : 1000, covance) ; detection of actin a5441 utilized mouse antibody (1 : 15,000, sigma). the secondary antibodies : irdye700 anti - mouse antibody (1 : 15,000) and irdye800 anti - rabbit antibody (1 : 15,000) were obtained from by licor. we applied our functional screening method to all the genes in the linkage region on chromosome 9 that displays a high likelihood of disease score for ad. this screening is conducted in sh - sy5y cells stably transfected with a luciferase reporter driven by the yeast uas promoter and app fused to gal4. when app is cleaved by the secretases, the aicd - gal4 domain is released and can activate the transcription of the luciferase reporter. since changes in aicd - mediated luciferase activity can occur through a variety of mechanisms affecting app, this is an effective and general way of identifying regulators of app metabolism. we targeted ntrk2 with 4 different shrna constructs (see supplementary figures 1 and 2 in the supplementary material available online at doi : 10.4061/2011/729382). three shrnas targeted all the trkb isoforms (ntrk2.1 - 3) and one (ntrk2.4) targeted all the isoforms except the trkb t. we also transfected a nonsilencing scrambled shrna (ns) that does not target any human gene as a negative control and a shrna targeting app as a positive control. of the four transfected constructs ntrk2.1 - 3 decreased aicd - mediated luciferase to the same extent of the app targeting shrna compared to the ns shrna (figure 2(a)). the fourth construct, ntrk2.4 targeting all trkb isoforms except trkb t, consistently caused cell death (data not shown). this result suggests that ntrk2 can affect app metabolism and that the isoforms have different roles since downregulation of all the isoforms except trkb t was lethal. therefore we investigated the effect of the single isoforms in the same experimental model. we transiently transfected individual trkb isoform overexpression constructs in the cells and measured aicd - mediated luciferase activity. we found that there was no difference in aicd - mediated luciferase activity between trkb t and the gfp - control, while trkb fl significantly increased luciferase activity (p =.01) and trkb shc significantly decreased it (p =.01) (figure 2(b)). moreover, we show that there is a difference between the isoforms trkb shc and trkb t, even though both isoforms lack the tyrosine kinase domain. trkb t did not alter aicd - mediated luciferase activity compared to the gfp - f control, while trkb shc decreased it and trkb fl increased it. we hypothesized that the intracellular domains of the trkb shc and trkb fl are responsible for the effects observed. to determine which domain was responsible for this effect we generated a mutant of the trkb shc isoform that can not bind shc (y515f). we transfected this mutant and the other trkb wild - type isoforms, into sh - sy5y - app - gal4 cells and measured aicd - mediated luciferase activity. we observed that trkb shc y515f (shc - binding site mutant) does not significantly alter luciferase activity compared to trkb t but significantly increased it compared to the trkb shc wild - type isoform (p <.001) (figure 3(a)). therefore, disrupting the shc - binding site on the trkb shc isoform impairs its ability to decrease aicd - mediated luciferase activity. we mutated the shc - binding site (y515f) to generate a mutant that can not bind shc. then we mutated the atp - binding site (k571 m) to generate a trkb fl tyrosine kinase inactive receptor k571 m [32, 33 ]. we also generated a double mutant that is both tyrosine kinase inactive and does not bind shc (trkb y515f / k571 m). we then transfected these trkb fl mutant constructs in sh - sy5y - app - gal4 cells. we measured the aicd - mediated luciferase activity and compared it to trkb fl wild - type (figure 3(b)). the trkb y515f mutant (preventing shc - binding) did not significantly alter aicd - mediated luciferase activity compared to trkb fl (figure 3(b)). the trkb fl k571 m (tyrosine kinase inactive) significantly decreased luciferase activity compared to trkb fl (p =.0006). trkb fl y816f, (preventing plc--binding) also significantly decreased luciferase activity compared to trkb fl (p =.0002). the double mutant trkb y515f / k571 m (preventing shc - binding and tyrosine kinase inactive) significantly decreased luciferase compared to trkb fl (p =.002) but did not differ from the tyrosine kinase inactive trkb k571 m (figure 2(a)). in summary, trkb fl overexpression increases aicd - gal4 mediated luciferase activity two - fold compared to controls trkb t (figure 2(b)). the tyrosine kinase inactive mutant receptor, trkb k571 m, the plc--binding site mutant, and the trkb shc isoform mutated on the shc - binding site also cause a 6070% decrease in aicd - mediated luciferase activity compared to the trkb fl (figures 3(a) and 3(b)). the trkb shc wild - type isoform causes an aicd - mediated luciferase decrease of about 90% (figure 2(b)). the effects we observe on aicd - mediated luciferase activity can occur through many different mechanisms : decreased app transcription, increased app degradation, decreased app cleavage, destabilization of aicd, and trafficking that affects app localization. the most immediate way of decreasing aicd levels is to decrease app levels. to determine if ntrk2 knockdown decreased app levels, we tested if app levels were altered. we transfected the ntrk2 targeting shrna, a ctrl shrna, and an app targeting shrna as a positive control. as an additional control we used shrna targeting the luciferase gene : this construct accounts for overexpression of shrna that have to be processed by the endogenous rnai machinery. we found that knockdown of all the trkb splice variants cause a significant decrease in app fl levels (p <.05) (figure 4(b)) and we concluded that decreased app levels might be at least partially responsible for the observed reduction in luciferase activity. based on the previous knockdown results, we then hypothesized that overexpression of trkb fl causes increased aicd - mediated luciferase activity by increasing app fl levels. we transfected the trkb isoforms in the cells, performed western blot analysis and quantified app fl levels in cell lysates. overexpression of trkb fl significantly increased app fl levels compared to trkb t (p =.03) and trkb shc (p =.008) (figure 5(a)). there was no difference in app levels between trkb t- and trkb shc - transfected cells (figure 5(a)). we then verified that aicd - gal4 levels in trkb fl - transfected cells correlated to the observed increase in luciferase activity. aicd - gal4 is the intracellular domain of app that is, generated by -secretase cleavage, translocates to the nucleus and activates transcription. we found that, as expected, trkb fl displayed increased aicd - gal4 levels compared to trkb t, but this difference was not statistically significant. compared to trkb t, trkb shc overexpression resulted in a decrease in aicd - gal4 levels, as expected, but this difference was not statistically significant (figure 5(b)). interestingly, we consistently observed trkb fl lower levels compared to trkb t and trkb shc in our western blot analysis (figure 5(a)). to assess changes in app proteolysis we measured app c - terminal fragments (ctfs) and sapp levels upon trkb transfection. c83 and c99 are generated by the cleavage of app by -secretase and -secretase, respectively. in our cell line we measure c83-gal4 and c99-gal4 levels since app overexpressed is a fusion protein with gal4. these fragments are the precursors of aicd, that is, released in the cytoplasm by -secretase cleavage [40, 41 ]. while c83 and c99 are membrane - bound fragments of app, the soluble n - terminal fragment of app, sapp, generated by /-secretase cleavage is released in the extracellular environment. in sh - sy5y cells, therefore, the majority of the luciferase signal observed is due to aicd - gal4 generated from -secretase cleavage of c83-gal4. if the aicd - gal4 levels are increased, as measured by luciferase and western blot, then the levels of its precursor c83-gal4 should also be increased. we then tested the hypothesis that c83-gal4 and sapp levels are increased by trkb fl overexpression and decreased by trkb shc. to aid detection of c83-gal4 we treated cells with the -secretase inhibitor l-685,485. we did not detect a difference in c83-gal4 levels among the cells transfected with the different trkb isoforms (figure 5(c)). surprisingly, trkb fl decreased sapp levels compared to trkb t (p =.01). trkb shc showed a nonsignificant difference in sapp levels compared to trkb t (figure 5(d)). all three trkb isoforms studied here are capable of binding bdnf. moreover, it has been previously shown that trkb fl bdnf - mediated intra - cellular signaling can alter app metabolism [1417 ]. we hypothesized that application of exogenous bdnf would stimulate the trkb fl - mediated effects on app fl and proteolytic products levels. we then tested this hypothesis applying bdnf to cells transfected with trkb isoforms and measured the levels of app fl by western blot. we found that short term (10 minutes) bdnf application increases app fl levels in cells transfected with the trkb t or trkb shc isoforms and to a greater degree in cells that had been transfected with trkb fl (figure 6). twenty - four hour bdnf treatment of trkb fl transfected cells did not further increase app fl levels compared to short - term bdnf treatment. it has been previously shown that trkb t has a dominant negative effect on trkb fl. we hypothesized that cotransfection of trkb t with trkb fl would eliminate the trkb fl effects on app metabolism observed when we transfect trkb fl alone. moreover we hypothesized that cotransfection of the trkb shc with trkb fl would also have dominant negative effect on trkb fl. finally we hypothesized that cotransfection of trkb fl with trkb y515f or trkb y816f would not significantly alter the effects seen on app since they seem to be primarily mediated by the tyrosine kinase domain and not by the shc - binding domain. for this reason we also hypothesized that cotransfection of trkb fl with trkb k571 m (trkb fl / k571 m) would have the same effect as the cotransfection of trkb fl and trkb t (trkb fl / t). consistent with our hypothesis, trkb fl / t cotransfection did not increase app fl levels, nor did cotransfection of trkb fl / k571 m, the tyrosine kinase inactive mutant (figure 7). also, as expected, there was very little difference between the app fl levels in cells transfected with trkb fl / y515f, trkb fl / y816f, and trkb fl / fl. surprisingly, cotransfection of trkb fl / shc increased app fl levels compared to trkb fl / t cotransfection but not compared to trkb fl / fl (figure 7). we also hypothesized that bdnf treatment of the cotransfected cells would affect the transfected isoforms mediated effects on app. surprisingly bdnf treatment did not significantly alter these effects of the cotransfected trkb receptors. in summary, both truncated isoforms were able to decrease app fl levels compared to trkb fl / fl transfection ; trkb t to a greater extent than trkb shc. the tyrosine kinase inactive receptor decreased app fl levels to the same extent of trkb fl / t cotransfection while trkb fl / y515f and trkb fl / y816f cotransfection did not alter app fl levels compared to trkb fl / fl. we investigated the role of the trkb isoforms on app metabolism in sh - sy5y cells overexpressing an app - gal4 fusion protein that can transactivate a luciferase reporter gene. this system monitors changes in app metabolism that are reflected in altered aicd - mediated transcription of the luciferase gene. we found that knockdown of all trkb isoforms in sh - sy5y - app - gal4 cells caused a decrease in aicd - mediated luciferase activity. this decrease is probably due to a decrease in app levels observed in cells with ntrk2 knock - down. we hypothesize that decreased app levels in this system are mainly due to increased app degradation caused by altered trafficking in absence of trkb. transcriptional downregulation of app might be partially responsible for the decreased signal observed in the western blot but that is only possible for the endogenous app. because the endogenous app gene is under the physiologic transcriptional regulation, while the app - gal4 overexpressed is under cmv promoter regulation. concomitant knockdown of the trkb fl and shc isoforms lead to cell death, and this is consistent with the finding that trkb t is one of the causes of neuronal death in a mouse model of trisomy 21. to discriminate between the effects of the different isoforms, we overexpressed one isoform at a time and measured the resulting aicd - mediated luciferase activity. as a control we employed a gfp expression vector (gfp - f) that includes a farnesylation sequence that targets gfp to the cell membrane this is a better control for a membrane - bound receptor than a cytoplasmatic gfp. trkb fl increased luciferase activity while no difference was observed between trkb t and gfp - f control transfected cells. we hypothesize that the decrease in aicd - mediated luciferase activity induced by trkb shc might be mediated by binding of shc adaptor proteins. binding of adaptor proteins to trkb and possibly to app, might decrease the endocytosis of app decreasing its -secretase cleavage. the luciferase assay described here has been found to be particularly sensitive in detecting decreased -secretase processing and that can be the cause of the decrease in luciferase activity that we observe, at least with cotransfection of the trkb shc isoform. our data demonstrates differential effects of the trkb isoforms on aicd - mediated transcription showing that trkb shc behaves differently from both trkb fl and trkb t. it has been previously demonstrated that bdnf application does not improve the cognitive function in a trisomy 21 mouse model because trkb t is upregulated [43, 46 ]. therefore, a better understanding of the individual trkb isoforms and their signaling role will improve the therapeutic potential of bdnf or bdnf agonists. experimentally, we found that the detected protein levels of trkb fl were much lower than trkb t and trkb shc levels. we can exclude effects due to plasmid copy number in the cells since we used equimolar amounts of plasmid dna that account for differences in plasmid size. we can also exclude differences in transcription levels due to plasmid promoters since the trkb shc vector was generated by mutagenesis of the trkb fl vector. the difference in expression levels of the trkb isoforms is highly reproducible suggesting that there might be a tight regulation of trkb fl expression levels. trkb fl is stored in intracellular vesicles that rapidly fuse to the cell membrane upon bdnf stimulation of the cells. this causes a fast bdnf - mediated phosphorylation of the receptor and initiates intracellular signaling. after this spike of activity trkb / bdnf complexes high trkb fl expression levels increase malignancy in neuroblastomas reinforcing the idea that regulatory mechanisms of trkb expression and signaling are necessary to maintain homeostasis [49, 50 ]. trkb fl expression is also decreased by chronic bdnf stimulation of h4 neuroblastoma cells while trkb t levels remain constant. we therefore hypothesize that in our model system, trkb fl levels are controlled by mechanisms that can not be overcome by trkb fl overexpression and that bdnf expressed by the cell line might be one of the causes of this downregulation. to determine which trkb functional domain and signaling pathway was mediating the trkb mediate effects, we overexpressed the mutant trkb isoforms and monitored aicd - mediated luciferase activity. the observed trkb fl - mediated increase in luciferase activity was suppressed by either inactivating the tyrosine kinase activity (k571 m) or mutating the plc--binding site (y816f). we hypothesize that the plc- effect is due to lack of plc- activation which produces dag (diacyl glycerol), an activator of pkc, a protein that mediates adam10 activation. the fact that there is a difference between the trkb k571 m mutant and the trkb y816f plc--binding site mutant suggests both of these functional domains and their associated pathways can regulate app metabolism. the shc - binding site on the trkb fl receptor did not seem to be involved in mediating increased aicd - mediated luciferase activity since the trkb y515f mutant did not differ from the trkb fl isoform in increasing aicd - mediated luciferase activity. also the aicd - mediated luciferase signal in cells transfected with the double mutant trkb k571m / y515f did not differ from the cells transfected with the trkb k571 m mutant suggesting that there is no additive effect in eliminating both signaling pathways. in fact, the binding of shc might occur more efficiently when the site is phosphorylated so that when phosphorylation is prevented the small change in luciferase signal is not detectable in our experimental system. supporting this hypothesis is a nonsignificant decrease of aicd - mediated activity caused by the trkb y515f mutant compared to trkb fl. we observe a significant effect when the trkb shc isoform is mutated to eliminate shc - binding. this mutant induces the same luciferase activity signal of the trkb t. this finding suggests that binding of shc to the trkb shc isoform might mediate signaling pathways independently of phosphorylation. importantly, we demonstrate that there is a difference in signal transduction between the two truncated trkb isoforms and that they act on app - mediated transcription. moreover, we identify the shc - binding domain as responsible for the difference in signaling mechanism between trkb t and shc. mutation of the binding site for shc adaptor proteins on the truncated trkb shc isoform increases aicd - mediated luciferase signal, while the same shc site on the trkb fl is not responsible for the observed changes in luciferase activity. this contrasting result suggests that interaction between the same proteins and specific trkb isoforms mediates different signaling pathways. the cell line used, sh - sy5y, expresses basal levels of trkb receptors and bdnf, the endogenously expressed bdnf can promote dimerization and activation of the overexpressed receptors. also, bdnf independent activation of trkb fl receptors has been previously demonstrated and we hypothesize that both bdnf independent and dependent activation coexist in our experimental system. endogenous trkb receptors might also be upregulated or downregulated in response to exogenous trkb expression. to assess the effect of bdnf - dependent activation we added exogenous bdnf to the transfected cells. bdnf is hypothesized to activate the receptors by mediating their dimerization. in our experimental system bdnf treatment did not significantly alter the effects of the trkb isoform overexpression on app fl levels. the close proximity of the overexpressed receptors on the membrane probably allows dimerization and activation of the receptors independently from bdnf so that even when bdnf is added to the system any additional effect on trkb activation is not detectable. we mentioned above that sh - sy5y cells express basal levels of the trkb receptors. to investigate the role of trkb isoforms interaction on trkb fl - mediated signaling, we coexpressed exogenous trkb fl with truncated isoforms and mutated variants. cotransfection of the trkb fl with the truncated t and shc isoforms or the tyrosine kinase inactive mutant abrogated the increase in app fl levels induced by trkb fl. interestingly trkb fl / shc cotransfection had higher app fl levels than trkb fl / t cotransfection. this points to a possible difference between the two trkb truncated isoforms in the regulation of the trkb fl catalytic receptor. the fact that in the cotransfection experiments trkb fl / shc showed increased app fl levels compared to trkb fl / t also suggests that shc - binding to this isoform might occur more efficiently when trkb fl and trkb shc interact, maybe causing phosphorylation on the shc - binding site. cotransfection of trkb fl / y515f had similar effects on app fl to trkb fl / fl but was less effective in inducing an increase in app fl levels. trkb fl / y816f cotransfection was indistinguishable from trkb fl single transfection suggesting that plc- signaling is not involved in determining app fl levels. bdnf treatment of the cotransfected cells seemed to accentuate the effect of trkb fl on app fl levels. for example, it increased app fl in cells cotransfected with trkb fl / t but not in cells cotransfected with trkb fl / k571 m. on the contrary, trkb fl / y515f cotransfection seemed to cause lower app fl levels when bdnf was applied. it is intriguing to think that when all trkb isoforms are expressed in the cells, as should be the case in our model, bdnf promotes homodimerization versus heterodimerization. the issue of preferential homo- versus heterointeraction of trkb isoforms has not been investigated so far and it would be important to address. this work demonstrates that truncated trkb isoforms affect app processing and transcriptional signaling differently than full - length trkb. not only do the truncated isoforms have a different effects when transfected alone, they were also able to modify the trkb fl effects when co - transected with it. these findings point to the possible roles of the trkb isoforms in the pathogenesis of ad. in fact all the isoforms are present on neurons and other cell types of the cns. the proportion of trkb fl to trkb t and trkb shc is then important in determining the effect on trkb signal transduction and app metabolism. since all the isoforms bind bdnf in the extracellular domain, a therapeutic approach that uses bdnf biomimetic drugs in fact, expression of truncated isoforms could scavenge the drugs, decrease the benefit of engaging trkb fl triggered pathways, and also inhibit the trkb fl effects. depending on the relative amounts of the trkb receptors on the cells, bdnf - mimetic drugs could cause an overall worsening of the conditions by, for example, increasing the inflammatory response. it will be important in the future to dissect the contributions of the trkb isoforms to bdnf - dependent and -independent signaling pathways in the context of ad to better understand which isoforms and pathways are beneficial and which ones are detrimental.	we report that ntrk2, the gene encoding for the trkb receptor, can regulate app metabolism, specifically aicd levels. using the human neuroblastoma cell line sh - sy5y, we characterized the effect of three trkb isoforms (fl, shc, t) on app metabolism by knockdown and overexpression. we found that trkb fl increases aicd - mediated transcription and app levels while it decreases sapp levels. these effects were mainly mediated by the tyrosine kinase activity of the receptor and partially by the plc-- and shc - binding sites. the trkb t truncated isoform did not have significant effects on app metabolism when transfected by itself, while the trkb shc decreased aicd - mediated transcription. trkb t abolished trkb fl effects on app metabolism when cotransfected with it while trkb shc cotransfected with trkb fl still showed increased app levels. in conclusion, we demonstrated that trkb isoforms have differential effects on app metabolism.
it has been documented that pregnancy is a unique immunological state with both immune response and tolerance against fetus antigens (1). previous studies demonstrated that the imbalanced immune responses against fetus antigens can result in abnormality in pregnancy. cytokines as important immune molecules, play crucial roles in regulation of immune responses against microbes and foreign antigens including graft and fetus antigens (2). interleukin-10 (il-10), as a main anti - inflammatory cytokine, significantly participates in regulation of immune responses against self and foreign antigens in the case of tolerance to these antigens (3 - 5). therefore, alteration in expression of this cytokine during pregnancy may lead to several pathologic conditions such as preterm labor (ptl) (6, 7). ptl is the common disorder in the pregnancy and is defined as birth before the 37 week of gestation (8). previous studies revealed that ptl can be associated with inflammation : hence, regarding the important roles played by il-10 in the suppression of inflammation, it appears that this cytokine can play key roles in the pathogenesis of ptl. therefore, the main aim of this review article is to present the status and function of il-10 in the pathogenesis of ptl. ptl is a birth that occurs before the 37 week of gestation and as described in the previous section the complication is a common disorder in the pregnancy occurring in approximately 10% of deliveries (9). in the normal human fetus, several organ systems mature between 34 and 37 weeks, and the fetus reaches adequate maturity by the end of this period (9, 10). the preterm birth is associated with several complications leading to mortality and morbidity such as neurological, respiratory, gastrointestinal and metabolic problems, hematologic disorders, and infection (10). based on the fact that the etiology and the main responsible mechanisms that lead to preterm birth are unknown, several hypotheses are in process worldwide. il-10 is categorized as an anti - inflammatory cytokine and is produced by several cells including activated macrophages, t regulatory and th2 lymphocytes and so on (11). the main function of this cytokine is to suppress th1, th2 and b lymphocytes, nk cells, macrophages, and dendritic cells inflammatory functions (12, 13). 1q31 - 1q32 is the location of the il-10 gene (approximately 5.2 kb), which consists of five exons (14), and encodes a protein of 178 amino acids called pro - il-10 (13). pro - il-10 activation is done by cleavage of a signal peptide of 18 amino acids (13). it has been demonstrated that il-10r consists of and chains and is categorized as a type ii cytokine receptor (15). several intracellular signaling pathways will be activated following il-10/il-10r and chain interaction by phosphorylation of jak1 (janus kinase-1) and tyk2 (tyrosine kinase-2), respectively, as intracellular protein kinases (16). in a positive feedback, activated jak1 and tyk2 phosphorylate y446 and y496 positions of il-10r chain (17). the phosphorylation is a place for binding and phosphorylation of signal transducer and activator of transcription 3 (stat3) tyrosine residues (18). homodimerized stat3 is translocated into the nucleus and recognizes stat - binding element (sbe) regions at the promoters of several genes including il-10, anti - apoptotic, cell - cycle - progression, suppressor of cytokine signaling 3 (socs3) genes, and so on (19). several other pathways such as phosphoinositide-3 kinase (pi3k) (20) and mitogen - activated protein kinase (p38/mapk) (21) are also regulated by il-10/il-10r interaction. based on the aforementioned information, it appears that il-10 may significantly participate in the outcome of pregnancy. for instance, it is documented that il-10 suppresses the production and function of pro - inflammatory cytokines including il-12, ifn-, il-1, and so on (22), and numerous studies have reported its expression at the maternal - fetal interface (23 - 25). it was also demonstrated that, under effects of progesterone, il-10 serum levels are elevated, which results in suppression and activation of th1 and th2-type cytokines, respectively (i.e., il-10 and il-4). robertson also showed that il-10 mrna and protein is up - regulated in gestational tissues in normal pregnancy. interestingly, they have concluded that up - regulation of il-10 can be considered as a critical factor for resistance to preterm labor (26). it has been demonstrated that lps could induce preterm labor in some cases (27). experimental studies have shown that exogenous il-10 inhibits lps - induced preterm labor (28, 29). a study by gotsch revealed that in 4157 days (third phase) after gestation, the concentration of il-10 was increased by high levels of 17-estradiol (30). up - regulation of il-10 and 17-estradiol leads to the suppression of dendritic cells (dcs) and consequently inhibits the stimulation of t lymphocytes by dcs (30), which is associated with a normal pregnancy. additionally, another study demonstrated that decreased number of peripheral blood mononuclear leukocytes that produce il-10, leads to recurrent preterm births during the second trimester (31). it has also been documented that preterm delivery during mid - trimester is associated with unchanged levels of il-10 in amniotic fluid, where the concentration of il-10 needs to be increased (32). our previous study also revealed that serum levels of il-10 were not differed in preterm in comparison to term neonates (33). the results have been confirmed by gotsch who reported that il-10 is expressed in high concentrations in the amniotic fluid of normal term women (30). the investigators also showed that spontaneous parturition in either term or preterm gestation is associated with elevated concentrations of il-10 in amniotic fluid (30). increased amniotic fluid concentrations of il-10 during intra - amniotic infection / inflammation have also been reported by gotsch and colleagues (30). moreover, another research demonstrated that preterm neonates compared to term neonates, produce higher and lower inflammatory and anti - inflammatory cytokines in response to specific bacteria, respectively (34). therefore, it may result in uncontrolled inflammatory response, which is associated with preterm labor (34). researchers have evaluated the expression profiles of il-10 and cyclo - oxygenase-2 (cox-2), as an enzyme for producing prostanoids (prostaglandins, prostacyclin, and thromboxane), which are the potential inducers of delivery (35). they have reported that il-10 significantly regulates the expression of cox-2 and consequently prostaglandins, hence, the authors concluded that il-10 plays important roles in countering inflammation that is produced in preterm labor (35). although, the aforementioned studies have demonstrated that the il-10 levels were lower in preterm vs. term delivery, some investigations reported that il-10 levels were high in preterm delivery associated with infection. for example, it was shown that the cord blood levels of il-10 were significantly increased in infected versus non - infected mothers (36). another study demonstrated that the cord blood il-10 levels are increased during intrauterine infection (37). based on these results, it may be concluded that up - regulation of il-10 in the infected preterm delivery is a normal response of the immune system to regulate the infection - dependent inflammation. for example, it has been demonstrated that il-10 levels are positively associated with elevated odds ratio of placental - mediated preterm birth (38). it may be related to the infections that have not been examined in the evaluated preterm neonates. according to the data presented here, it appears that il-10 plays significant roles in the induction of an appropriate pregnancy because its expression is up - regulated in the normal pregnancy, while its production is disrupted during preterm labor. for example, it has been established that surfactant protein a (sp - a) is produced in the fetus to provide signals for the onset of parturition (39). interestingly, the study revealed that sp - a suppresses preterm delivery via tlr2-dependent il-10 production (39). indoleamine 2,3-dioxygenase (ido) is an enzyme for catabolism of tryptophan, which is crucial for t lymphocyte activation and proliferation (40). ido regulate maternal immune responses against fetus alloantigens via suppression of maternal t lymphocytes and also up - regulation of anti - inflammatory cytokines such as il-10 (40). additionally, it has been evidenced that tolerogenic dcs plays key roles in the induction of maternal immune tolerance to fetus alloantigens (41). interestingly, il-10 is a potential factor that stimulates the production of tolerogenic dcs (42), hence, it can induce a successful pregnancy. previous investigations identified that regulatory t lymphocytes also significantly participate in induction of maternal immune tolerance to fetus alloantigens and subsequently a successful pregnancy (43). il-10 is not only produced by regulatory t lymphocytes, but also leads to the development of these cells (43). il-10 binding to its corresponded receptor (il-10r) leads to activation of the il-10/jak1/stat3 cascade and subsequently phosphorylation of stat3. the phosphorylation leads to the production of stat3 homodimer (stat3/stat3) and its translocation to the nucleus can trigger the expression of the target gene (see the previous section), which participates in the induction of a successful pregnancy (4446). as mentioned in the figure, sp - a, 17-esteradiol, ido vitamin d3, and il-10 genetic variations can alter expression of il-10. il-10 inhibits preterm delivery using several mechanisms including development of t regulatory lymphocytes and tolerogenic dcs, activation of the jak1/stat3 pathway, down - regulation of cox-2, and also inhibition of lps - induced preterm delivery interestingly, genetic research revealed that the genetic variations in the il-10 gene are also associated with preterm delivery. it has also been evidenced that the polymorphisms within il-10 gene are associated with its expression during pregnancy (47, 48). for instance, it was shown that il-10 (rs1800896) polymorphism is associated with gram - negative infections in preterm labor (49). the relation between the polymorphisms within the promoter region of il-10 and cervical insufficiency have been reported by warren and colleagues (50). another study also revealed that il-10 (-1082a) polymorphism is significantly associated with genitourinary infections and/or inflammation (51). another study has shown that il10 (-1082)g plays crucial roles in less than 29 weeks extreme preterm delivery (52). a study on australian population revealed that il-10 82a/-819t/-592a haplotype is associated with preterm delivery (53). interestingly the study demonstrated that this haplotype is more prevalent in women with preterm premature rupture of membranes (53). contrastly, one study was unable to find a relation between il-10 polymorphisms and preterm delivery (54). another study was also unable to find an association between the polymorphisms within il-10 gene (il10 -1082 g > a, il10 -819 c > t, and il10 -592 c > a) and spontaneous preterm delivery (55). a study on austrian population demonstrated that il-10 -1082 g / a single nucleotide polymorphism was not associated with preterm delivery (56). no association between this polymorphism and preterm delivery was also demonstrated by brazilian researchers (57). it appears that further studies are essential to complete our knowledge regarding the roles of genetic variations in the induction of preterm delivery. ptl is a birth that occurs before the 37 week of gestation and as described in the previous section the complication is a common disorder in the pregnancy occurring in approximately 10% of deliveries (9). in the normal human fetus, several organ systems mature between 34 and 37 weeks, and the fetus reaches adequate maturity by the end of this period (9, 10). the preterm birth is associated with several complications leading to mortality and morbidity such as neurological, respiratory, gastrointestinal and metabolic problems, hematologic disorders, and infection (10). based on the fact that the etiology and the main responsible mechanisms that lead to preterm birth are unknown, several hypotheses are in process worldwide. il-10 is categorized as an anti - inflammatory cytokine and is produced by several cells including activated macrophages, t regulatory and th2 lymphocytes and so on (11). the main function of this cytokine is to suppress th1, th2 and b lymphocytes, nk cells, macrophages, and dendritic cells inflammatory functions (12, 13). 1q31 - 1q32 is the location of the il-10 gene (approximately 5.2 kb), which consists of five exons (14), and encodes a protein of 178 amino acids called pro - il-10 (13). pro - il-10 activation is done by cleavage of a signal peptide of 18 amino acids (13). it has been demonstrated that il-10r consists of and chains and is categorized as a type ii cytokine receptor (15). several intracellular signaling pathways will be activated following il-10/il-10r and chain interaction by phosphorylation of jak1 (janus kinase-1) and tyk2 (tyrosine kinase-2), respectively, as intracellular protein kinases (16). in a positive feedback, activated jak1 and tyk2 phosphorylate y446 and y496 positions of il-10r chain (17). the phosphorylation is a place for binding and phosphorylation of signal transducer and activator of transcription 3 (stat3) tyrosine residues (18). homodimerized stat3 is translocated into the nucleus and recognizes stat - binding element (sbe) regions at the promoters of several genes including il-10, anti - apoptotic, cell - cycle - progression, suppressor of cytokine signaling 3 (socs3) genes, and so on (19). several other pathways such as phosphoinositide-3 kinase (pi3k) (20) and mitogen - activated protein kinase (p38/mapk) (21) are also regulated by il-10/il-10r interaction. based on the aforementioned information, it appears that il-10 may significantly participate in the outcome of pregnancy. for instance, it is documented that il-10 suppresses the production and function of pro - inflammatory cytokines including il-12, ifn-, il-1, and so on (22), and numerous studies have reported its expression at the maternal - fetal interface (23 - 25). it was also demonstrated that, under effects of progesterone, il-10 serum levels are elevated, which results in suppression and activation of th1 and th2-type cytokines, respectively (i.e., il-10 and il-4). robertson also showed that il-10 mrna and protein is up - regulated in gestational tissues in normal pregnancy. interestingly, they have concluded that up - regulation of il-10 can be considered as a critical factor for resistance to preterm labor (26). it has been demonstrated that lps could induce preterm labor in some cases (27). experimental studies have shown that exogenous il-10 inhibits lps - induced preterm labor (28, 29). a study by gotsch revealed that in 4157 days (third phase) after gestation, the concentration of il-10 was increased by high levels of 17-estradiol (30). up - regulation of il-10 and 17-estradiol leads to the suppression of dendritic cells (dcs) and consequently inhibits the stimulation of t lymphocytes by dcs (30), which is associated with a normal pregnancy. additionally, another study demonstrated that decreased number of peripheral blood mononuclear leukocytes that produce il-10, leads to recurrent preterm births during the second trimester (31). it has also been documented that preterm delivery during mid - trimester is associated with unchanged levels of il-10 in amniotic fluid, where the concentration of il-10 needs to be increased (32). our previous study also revealed that serum levels of il-10 were not differed in preterm in comparison to term neonates (33). the results have been confirmed by gotsch who reported that il-10 is expressed in high concentrations in the amniotic fluid of normal term women (30). the investigators also showed that spontaneous parturition in either term or preterm gestation is associated with elevated concentrations of il-10 in amniotic fluid (30). increased amniotic fluid concentrations of il-10 during intra - amniotic infection / inflammation have also been reported by gotsch and colleagues (30). moreover, another research demonstrated that preterm neonates compared to term neonates, produce higher and lower inflammatory and anti - inflammatory cytokines in response to specific bacteria, respectively (34). therefore, it may result in uncontrolled inflammatory response, which is associated with preterm labor (34). researchers have evaluated the expression profiles of il-10 and cyclo - oxygenase-2 (cox-2), as an enzyme for producing prostanoids (prostaglandins, prostacyclin, and thromboxane), which are the potential inducers of delivery (35). they have reported that il-10 significantly regulates the expression of cox-2 and consequently prostaglandins, hence, the authors concluded that il-10 plays important roles in countering inflammation that is produced in preterm labor (35). although, the aforementioned studies have demonstrated that the il-10 levels were lower in preterm vs. term delivery, some investigations reported that il-10 levels were high in preterm delivery associated with infection. for example, it was shown that the cord blood levels of il-10 were significantly increased in infected versus non - infected mothers (36). another study demonstrated that the cord blood il-10 levels are increased during intrauterine infection (37). based on these results, it may be concluded that up - regulation of il-10 in the infected preterm delivery is a normal response of the immune system to regulate the infection - dependent inflammation. for example, it has been demonstrated that il-10 levels are positively associated with elevated odds ratio of placental - mediated preterm birth (38). it may be related to the infections that have not been examined in the evaluated preterm neonates. according to the data presented here, it appears that il-10 plays significant roles in the induction of an appropriate pregnancy because its expression is up - regulated in the normal pregnancy, while its production is disrupted during preterm labor. for example, it has been established that surfactant protein a (sp - a) is produced in the fetus to provide signals for the onset of parturition (39). interestingly, the study revealed that sp - a suppresses preterm delivery via tlr2-dependent il-10 production (39). indoleamine 2,3-dioxygenase (ido) is an enzyme for catabolism of tryptophan, which is crucial for t lymphocyte activation and proliferation (40). ido regulate maternal immune responses against fetus alloantigens via suppression of maternal t lymphocytes and also up - regulation of anti - inflammatory cytokines such as il-10 (40). additionally, it has been evidenced that tolerogenic dcs plays key roles in the induction of maternal immune tolerance to fetus alloantigens (41). interestingly, il-10 is a potential factor that stimulates the production of tolerogenic dcs (42), hence, it can induce a successful pregnancy. previous investigations identified that regulatory t lymphocytes also significantly participate in induction of maternal immune tolerance to fetus alloantigens and subsequently a successful pregnancy (43). il-10 is not only produced by regulatory t lymphocytes, but also leads to the development of these cells (43). il-10 binding to its corresponded receptor (il-10r) leads to activation of the il-10/jak1/stat3 cascade and subsequently phosphorylation of stat3. the phosphorylation leads to the production of stat3 homodimer (stat3/stat3) and its translocation to the nucleus can trigger the expression of the target gene (see the previous section), which participates in the induction of a successful pregnancy (4446). as mentioned in the figure, sp - a, 17-esteradiol, ido vitamin d3, and il-10 genetic variations can alter expression of il-10. il-10 inhibits preterm delivery using several mechanisms including development of t regulatory lymphocytes and tolerogenic dcs, activation of the jak1/stat3 pathway, down - regulation of cox-2, and also inhibition of lps - induced preterm delivery interestingly, genetic research revealed that the genetic variations in the il-10 gene are also associated with preterm delivery. it has also been evidenced that the polymorphisms within il-10 gene are associated with its expression during pregnancy (47, 48). for instance, it was shown that il-10 (rs1800896) polymorphism is associated with gram - negative infections in preterm labor (49). the relation between the polymorphisms within the promoter region of il-10 and cervical insufficiency have been reported by warren and colleagues (50). another study also revealed that il-10 (-1082a) polymorphism is significantly associated with genitourinary infections and/or inflammation (51). another study has shown that il10 (-1082)g plays crucial roles in less than 29 weeks extreme preterm delivery (52). a study on australian population revealed that il-10 82a/-819t/-592a haplotype is associated with preterm delivery (53). interestingly the study demonstrated that this haplotype is more prevalent in women with preterm premature rupture of membranes (53). contrastly, one study was unable to find a relation between il-10 polymorphisms and preterm delivery (54). another study was also unable to find an association between the polymorphisms within il-10 gene (il10 -1082 g > a, il10 -819 c > t, and il10 -592 c > a) and spontaneous preterm delivery (55). a study on austrian population demonstrated that il-10 -1082 g / a single nucleotide polymorphism was not associated with preterm delivery (56). no association between this polymorphism and preterm delivery was also demonstrated by brazilian researchers (57). it appears that further studies are essential to complete our knowledge regarding the roles of genetic variations in the induction of preterm delivery. due to the aforementioned results, it appears that il-10 plays key roles in the induction of a normal delivery via development of tolerogenic dcs, t regulatory lymphocytes, and up - regulation of stat3 target genes. up - regulation of this anti - inflammatory cytokine leads to suppression of nk cells and t lymphocytes against their fetus alloantigens. the factors that alter the expression of il-10, including infections and genetic variations within il-10 gene, could determine the outcome of pregnancy. additionally, it has also been demonstrated that preterm delivery is associated with several short and long - term health problems. it may be hypothesized that alteration in expression of il-10 is also associated with the incidence of the complications. moreover, il-10 can decline the pathologic effects of inflammation that is prevalent in the preterm delivery. therefore, it appears that regulation of il-10 expression during normal pregnancy is cautiously regulated.	imbalanced immune responses against fetus alloantigens can lead to abnormality in pregnancy. interleukin-10 (il-10) plays key roles in regulation of immune responses against self and foreign antigens to induce tolerance to these antigens. therefore, alteration in expression of il-10 during pregnancy may result in several pathologic conditions such as preterm labor. il-10 leads to a normal pregnancy via several molecular mechanisms including development of tolerogenic dendritic cells, t regulatory lymphocytes and activation of the jak1/stat3 pathway in the target cells. this review has collected recent data regarding the status of il-10 expression during term and preterm deliveries and also its molecular mechanisms that lead to a normal pregnancy.
chemotherapy - induced nausea and vomiting (cinv) is one of the most unpleasant side effects for patients receiving chemotherapy. it can have a significant effect on a patient 's qualify of lifeand prevent continuation of chemotherapy. the incidence and severity of cinv are affected by diverse factors, including the specific chemotherapeutic agents, the dosage of the agents, the schedule and route of administration of the agents, and individual patient variability [1 - 3 ]. cisplatin is one of the most effective chemotherapeutic agents available for treatment of many solid tumors. however, it is also highly emetogenic, resulting in poor compliance with chemotherapy. therefore, control of cinv by selection of a relevant antiemetic regimen is as important as the efficacy of the chemotherapy regimen. development of newer antiemetic agents, such as serotonin (5-ht3) receptor antagonists and neurokinin-1 (nk-1) receptor antagonists, has resulted in substantially reduced incidence and risk of cinv in patients receiving chemotherapy. in particular, a triple combination regimen consisting of a nk-1 antagonist, a 5-ht3 antagonist, and dexamethasone is recommended by key clinical guidelines groups, including the national comprehensive cancer network, the european society of medical oncology, and the american society of clinical oncology, for prevention of acute and delayed emesis in patients receiving highly emetic intravenous chemotherapy such as cisplatin [4 - 6 ]. the excellent efficacy of this triple - drug regimen with various 5-ht3 antagonists, including ondansetron, granisetron, and palonosetron, has been reported [7 - 9 ]. however, the most effective 5-ht3 antagonist for this combination has not yet been identified., tokyo, japan), has been used widely in asian countries for prevention of cinv. in several clinical trials, it showed equivalent efficacy and a similar safety profile when compared with ondansetron and granisetron [10 - 13 ]. however, there is currently no information with regard to whether ramosetron is as effective as other 5-ht3 receptor antagonists for the triple combination regimen. in this study, we evaluated the clinical efficacy and tolerability of a combination regimen comprising ramosetron, aprepitant (nk-1 antagonist), and dexamethasone (rad) for prevention of cinv in chemotherapy - nave patients with solid cancers. this study was a multicenter, open - label, prospective, phase ii clinical trial conducted for investigation of the effects of rad on prevention of cinv. chemotherapy - nave patients between the ages of 15 and 75 years with any solid cancer who were scheduled to receive single day chemotherapy with 50 mg / m or more of cisplatin were eligible. patients from five hospitals of hallym university medical center and keimyung university dongsan hospital in korea were enrolled in this study. all patients had an eastern cooperative oncology group performance status of 0 - 2, adequate renal function (serum creatinine level<2.5 mg / dl or calculated creatinine clearance50 ml / min), adequate hepatic function (serum total bilirubin level<2 mg / dl, aspartate aminotransferase / alanine aminotransferase level of<3 times the upper normal limit, and alkaline phosphatase level<5 times the upper normal limit), and adequate marrow function (absolute neutrophil count1,500/l and platelets100,000/l). the primary exclusion criteria were as follows : receipt of medication (antiemetics, steroids, and benzodiazepines, etc.) that might affect study results within one week before the start of chemotherapy ; symptomatic brain metastasis ; gastro - intestinal obstruction or other disease that could provoke nausea and vomiting ; administration of radiotherapy to the brain, abdomen, or pelvis within two weeks before the start of chemotherapy ; and known allergy or severe side effects to the study drugs. all patients provided written informed consent, and the study protocol was approved by the institutional review board of each institution and was registered with clinicaltrials.gov (identifier, nct01046461). on day 1, all eligible patients received intravenous administration of 0.6 mg ramosetron 30 minutes before administration of chemotherapy, 125 mg aprepitant orally 1 hour before administration of chemotherapy, and 12 mg dexamethasone orally 30 minutes before administration of chemotherapy. for the next two days, the patients received 80 mg aprepitant and 8 mg dexamethasone orally in the morning. rescue antiemetics were administered at any time during the study period for vomiting or severe nausea at the request of the patients or as recommended by the attending physicians. the primary efficacy endpoint was complete response (cr), which was defined as no vomiting, including retching, and no administration of rescue anti - emetic treatment, to the rad regimen from the start of chemotherapy (0 hour) until - day 5 (defined as the overall phase). the overall phase was classified into an acute phase (0 - 24 hours) and a delayed phase (24 - 120 hours). the secondary endpoints were cr in the acute phase and delayed phase, absolute cr (defined as cr plus no nausea), and severity of nauseain both phases. the severity of nausea was determined using the visual analog scale of the multinational association supportive care cancer antiemesis tool. tolerability was assessed on the basis of clinical and laboratory adverse events that occurred after the start of treatment and within 14 days after treatment ended and were evaluated according to the common toxicity criteria for adverse events (ctcae) v.3.0. during the overall phase, patients were asked to record daily episodes of vomiting or retching, the degree of nausea, and the use of rescue medication in a diary. the cr rate of high dose cisplatin - induced overall phase cinv is known to be approximately 55% (p0) for patients receiving ramosetron and dexamethasone. assuming that the addition of aprepitant to ramosetron and dexamethasone improves the cr rate by up to 75% (p1), the sample size should be 37 according to the " exact single - stage phase ii designs " procedure (5% -error and 80% power). considering a possible dropout rate of 5%, a target sample size of 39 descriptive examination of demographic data and patients ' characteristics was performed and the percentage of patients achieving cr was calculated. this study was a multicenter, open - label, prospective, phase ii clinical trial conducted for investigation of the effects of rad on prevention of cinv. chemotherapy - nave patients between the ages of 15 and 75 years with any solid cancer who were scheduled to receive single day chemotherapy with 50 mg / m or more of cisplatin were eligible. patients from five hospitals of hallym university medical center and keimyung university dongsan hospital in korea were enrolled in this study. all patients had an eastern cooperative oncology group performance status of 0 - 2, adequate renal function (serum creatinine level<2.5 mg / dl or calculated creatinine clearance50 ml / min), adequate hepatic function (serum total bilirubin level<2 mg / dl, aspartate aminotransferase / alanine aminotransferase level of<3 times the upper normal limit, and alkaline phosphatase level<5 times the upper normal limit), and adequate marrow function (absolute neutrophil count1,500/l and platelets100,000/l). the primary exclusion criteria were as follows : receipt of medication (antiemetics, steroids, and benzodiazepines, etc.) that might affect study results within one week before the start of chemotherapy ; symptomatic brain metastasis ; gastro - intestinal obstruction or other disease that could provoke nausea and vomiting ; administration of radiotherapy to the brain, abdomen, or pelvis within two weeks before the start of chemotherapy ; and known allergy or severe side effects to the study drugs. all patients provided written informed consent, and the study protocol was approved by the institutional review board of each institution and was registered with clinicaltrials.gov (identifier, nct01046461). on day 1, all eligible patients received intravenous administration of 0.6 mg ramosetron 30 minutes before administration of chemotherapy, 125 mg aprepitant orally 1 hour before administration of chemotherapy, and 12 mg dexamethasone orally 30 minutes before administration of chemotherapy. for the next two days, the patients received 80 mg aprepitant and 8 mg dexamethasone orally in the morning. rescue antiemetics were administered at any time during the study period for vomiting or severe nausea at the request of the patients or as recommended by the attending physicians. the primary efficacy endpoint was complete response (cr), which was defined as no vomiting, including retching, and no administration of rescue anti - emetic treatment, to the rad regimen from the start of chemotherapy (0 hour) until - day 5 (defined as the overall phase). the overall phase was classified into an acute phase (0 - 24 hours) and a delayed phase (24 - 120 hours). the secondary endpoints were cr in the acute phase and delayed phase, absolute cr (defined as cr plus no nausea), and severity of nauseain both phases. the severity of nausea was determined using the visual analog scale of the multinational association supportive care cancer antiemesis tool. tolerability was assessed on the basis of clinical and laboratory adverse events that occurred after the start of treatment and within 14 days after treatment ended and were evaluated according to the common toxicity criteria for adverse events (ctcae) v.3.0. during the overall phase, patients were asked to record daily episodes of vomiting or retching, the degree of nausea, and the use of rescue medication in a diary. the cr rate of high dose cisplatin - induced overall phase cinv is known to be approximately 55% (p0) for patients receiving ramosetron and dexamethasone. assuming that the addition of aprepitant to ramosetron and dexamethasone improves the cr rate by up to 75% (p1), the sample size should be 37 according to the " exact single - stage phase ii designs " procedure (5% -error and 80% power). considering a possible dropout rate of 5%, a target sample size of 39 would be needed. descriptive examination of demographic data and patients ' characteristics was performed and the percentage of patients achieving cr was calculated. a total of 41 patients were enrolled in this study between november 2010 and february 2012. the most common primary tumor site was the lung (49%), followed by the stomach (15%) and genitourinary tract (10%). all patients received cisplatin and other chemotherapeutic agents, including pemetrexed, taxanes, and gemcitabine. the median dose of a single administration of cisplatin was 70 mg / m of the body surface area (range, 50 to 75 mg / m). one patient was not a chemotherapy - nave patient and the other received a low dose of ramosetron. the cr rate was 92.3% in the overall phase, 94.9% in the acute phase, and 92.3% in the delayed phase (table 2). the absolute cr rate was 46.2% in the overall phase (74.4% in the acute phase and 51.3% in the delayed phase). median nausea scores during the overall, acute, and delayed phases were 2 (interquartile range [iqr ], 0 - 4), 0 (iqr, 0 - 1), and 0 (iqr, 0 - 4), respectively. mild nausea (score of 1 - 3 on the visual analogue scale) was observed in 10% of patients in the acute phase and in 13% of patients in the delayed phase (table 3). moderate - to - severe nausea (score of 4 - 10 on the visual analogue scale) was observed in 15% and 36% of patients in the acute and delayed phases, respectively. the most common grade 3/4 hematologic toxicity was neutropenia, with an incidence of 12%, followed by thrombocytopenia (10%). one patient was not a chemotherapy - nave patient and the other received a low dose of ramosetron. the cr rate was 92.3% in the overall phase, 94.9% in the acute phase, and 92.3% in the delayed phase (table 2). the absolute cr rate was 46.2% in the overall phase (74.4% in the acute phase and 51.3% in the delayed phase). median nausea scores during the overall, acute, and delayed phases were 2 (interquartile range [iqr ], 0 - 4), 0 (iqr, 0 - 1), and 0 (iqr, 0 - 4), respectively. mild nausea (score of 1 - 3 on the visual analogue scale) was observed in 10% of patients in the acute phase and in 13% of patients in the delayed phase (table 3). moderate - to - severe nausea (score of 4 - 10 on the visual analogue scale) was observed in 15% and 36% of patients in the acute and delayed phases, respectively. the most common grade 3/4 hematologic toxicity was neutropenia, with an incidence of 12%, followed by thrombocytopenia (10%). in the current study, the rad regimen showed significant efficacy for prevention ofcisplatin - induced cinv in chemonave cancer patients. ninety - two percent of patients did not experience vomiting episodes, nor did they receive rescue medication for cinv during the overall phase. the cr rates of a two - drug regimen with ramosetron plus dexamethasone for prevention of cisplatin - induced cinv were reported to range from 68% to 85% in the acute periodand from 58% to 75% in the delayed period. in their study with ondansetron, hesketh. reported that cr rates for a triple - drug regimen of aprepitant, dexamethasone, and the 5-ht3 antagonist were 89%, 75%, and 73% in acute, delayed, and overall phases, respectively. longo. conducted a study evaluating a three - drug regimen with palonosetron for prevention of cinv in patients receiving highly emetogenic chemotherapy. the cr rates in the acute, delayed, and overall periods were 98%, 73%, and 70%, respectively. in another triple regimen study with palonosetron, herrington. reported cr rates in the acute, delayed, and overall phases of 96%, 93%, and 93%, respectively, which was comparable to our results. in our trial, the cr rate was 95% in the acute period. only two patients experienced episodes of vomiting during the first 24 hours after initiation of chemotherapy : one was a 58-year - old male with bladder cancer receiving cisplatin plus gemcitabine chemotherapy and the other was a 54-year - old male with small cell lung cancer receiving cisplatin plus irinotecan. however, in the trial conducted by herrington., the proportion of patients receiving cisplatin - based chemotherapy was 55%, which was different from that observed in our study. however, in this trial, the proportion of female patients considered vulnerable to cinv was relatively low when compared with other studies. it is a tetrahydrobenzimidazole derivative, which is structurally different from ondansetron and granisetron, with more potent 5-ht3 receptor antagonizing effects than the reference compounds used in animal experiments. in addition, because its half - life is known to be longer than that of ondansetron and granisetron, it can be administered once per day. its known side effects include an elevation of hepatic transaminases, headache, diarrhea, and febrile sensation with a frequency of less than 1%. in this study although two patients (5% of cases) experienced grade 3 diarrhea, it could be considered a side effect of chemotherapeutic agents. most adverse events were also acceptable toxicities which could be related to chemotherapy and were similar in comparison with other studies [11 - 13 ]. aprepitant, the first nk-1 receptor antagonist to be developed, prevents binding of substance p to the nk-1 receptor forimprovement of cinv. although the rad regimen, ramosetron combined with aprepitant and dexamethasone, has been widely used in clinical practice in asian countries for prevention of cinv, there are not yet any published data on this triple - drug combination. despite advances in control of vomiting by development of effective antiemetic agents, many patients still suffer from chemotherapy - induced nausea. in particular, delayed nausea tends to show resistance to treatment. in our study, the absolute cr rate, defined as cr plus no nausea, was 46% during the overall period. while 15% of patients experienced moderate - to - severe nausea during the acute period, 36% developed moderate - to - severe nausea during the delayed period. this result was similar to those reported byother studies. in the trial conducted by hesketh., 48% of patients had no nausea in the overall phase, and 9% and 25% of patients were reported to have significant nausea in the acute and delayed phases, respectively. in a recent study, roscoe. reported that delayed nausea could be improved by addition of dexamethasone on days 2 and 3, but not by the 5-ht3 receptor antagonist. the patient sample size was relatively small, and the proportion of male patients was high. the effect of individual variable factors, such as alcohol consumption, chemotherapeutic agents administered in combination, and the impact of the cisplatin dose was not considered. however, to the best of our knowledge, this is the first prospectively conducted study on the efficacy of a three - drug regimen with ramosetron for prevention of cinv. the results of this study showed that the rad combination regimen is a very safe and effective antiemetic therapy for prevention of cinv in patients receiving highly emetogenic chemotherapy, although chemotherapy - induced nausea is still not completely overcome by use of this regimen. based on the results of this study, we are currently conducting a prospective multicenter, randomized, single - blind phase iii trial for comparison of rad with ondansetron, aprepitant, and dexamethasone (nct01536691).	purposecombination therapy with aprepitant, serotonin receptor antagonist, and steroids improves the complete response rate of both acute and delayed chemotherapy - induced nausea and vomiting (cinv). however, it is not known whether ramosetron is suitable for administration in combination with aprepitant. therefore, we conducted a multicenter, open - label, prospective, phase ii study in order to assess the efficacy and tolerability of combination therapy with ramosetron, aprepitant, and dexamethasone (rad) for prevention of cisplatin - based cinv in chemotherapy - nave patients with solid cancers.materials and methodsforty - one patients with various solid cancers (31 male and 10 female ; median age, 59 years) who received treatment with highly emetogenic chemotherapy (median cisplatin dose, 70 mg / m2 ; range 50 to 75 mg / m2) were enrolled in this study. oral aprepitant (125 mg on day 1 ; 80 mg on days 2 and 3), intravenous ramosetron (0.6 mg on day 1), and oral dexamethasone (12 mg on day 1 ; 8 mg on days 2 - 4) were administered for prevention of cinv.resultsthe complete response (no emesisand retching and no rescue medication) rate was 94.9% in the acute period (24 hours post - chemotherapy), 92.3% in the delayed period (24 - 120 hours post - chemotherapy), and 92.3% in the overall period (0 - 120 hours). the absolute complete response (complete response plus no nausea) rate was 74.4% in the acute period, 51.3% in the delayed period, and 46.2% in the overall period. there were no grade 3 or 4 toxicities related to these antiemetic combinations.conclusionrad regimen is a safe and effective antiemetic treatment for prevention of cinv in patients receiving highly emetogenic chemotherapy.
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors.	abstractanalytical methods that enable visualization of nanomaterials derived from solution selfassembly processes in organic solvents are highly desirable. herein, we demonstrate the use of stimulated emission depletion microscopy (sted) and single molecule localization microscopy (smlm) to map living crystallizationdriven block copolymer (bcp) selfassembly in organic media at the subdiffraction scale. four different dyes were successfully used for singlecolour superresolution imaging of the bcp nanostructures allowing micelle length distributions to be determined in situ. dualcolour smlm imaging was used to measure and compare the rate of addition of red fluorescent bcp to the termini of green fluorescent seed micelles to generate block comicelles. although wellestablished for aqueous systems, the results highlight the potential of superresolution microscopy techniques for the interrogation of selfassembly processes in organic media.
body weight and body mass index (bmi) are independent risk factors for mortality in chronic obstructive pulmonary disease (copd) patients [1, 2 ]. we researched the relationships between possible nutritional parameters (haemoglobin levels, serum albumin, blood urea nitrogen, creatinine, cholesterol, triglycerides, creatine kinase a myocyte - derived enzyme) and lung functions in copd. spirometric, laboratory, and demographic data of 450 consecutive patients (348 men and 102 women), aged 39 to 84 years, were analysed retrospectively. all the patients had copd, according to the gold consensus report and chronic airflow limitation, defined as measured forced expiratory volume in one second (fev1)/forced vital capacity (fvc)70%). all statistical analyses were performed with spss version 10.0 (spss inc, chicago, il). data were analysed using independent - samples t test, and pearson s correlation test, and spearman rank correlation test if the population was non - parametric. if correlation coefficient was less then 0.3, the results were considered as non - significant. the study was approved by the ethical committee of the school of medicine, cumhuriyet university (2005 - 2/2). the mean age years of 102 females (22.7%) and 348 males (77.3%) were 62.9 0.8 and 61.4 0.5 respectively (p>0.05). a non - copd group, consisted of 103 males (77.4%) and 30 females, was gender- and age - matched. demographic, spirometric, and biochemical parameters of two groups were presented in table 1. the patients who had airway obstruction had significantly greater pack - years of smoking, blood urea nitrogen and serum creatinine levels but lower bmi. when compared to female patients with copd, males had significantly greater pack - years of smoking, haemoglobin levels, more severe airway obstruction but lower bmi (table 2). the prevalence of a bmi less than 20 kg.m was 9.6% among the 450 outpatients studied, 13.9% in severe copd (fev1<50% of reference) and 4.0% in mild to moderate copd. there was a negative correlation between bmi and pack - years of smoking in copd (r : 0.24, p<0.01) but not in non - copd group. the decreasing effect of smoking on bmi was valid only in copd group (odds ratio : 1.41, 95% confidence intervals : 1.22 - 1.63). for the relationships between bmi and pulmonary functions, the correlation coefficients were 0.21 for fvc, 0.26 for fev1, and 0.32 for fev1/fvc in copd group. in non - smokers, bmi was significantly lower in men independent of age and pulmonary functions (table 3). in the entire copd group (table 4) and in non - smokers (table 5), bmi was not related any of pulmonary functions. multiple linear regression analysis revealed that serum albumin level was the best predictor of fev1 (beta = 12.307, p<0.001). creatine kinase levels showed no correlation any of the pulmonary function parameters (data not shown). table 1 showed that the patients with copd had significantly greater pack - years of smoking but lower bmi. this finding suggests that the lower bmi is due to the loss of lung functions. the higher correlation coefficients between bmi and lung functions than those between bmi and smoking also support this idea. table 2 demonstrated that males had more severe loss of pulmonary parenchyma because they had greater load of smoking than females. schols. reported that serum albumin levels are positively associated with exercise performance, katsura. found that low serum albumin can be a risk factor for poor outcome in copd. this finding suggests that systemic catabolic process in copd affects serum albumin concentration more than bmi. additionally, table 3 showed that bmi was significantly lower in male than in female in non - smokers subset. males were more susceptible to weight loss and protein degradation despite the anabolic effects of testosterone. another explanation is the different aetiology of copd. in our study, females had a history of tobacco use of less than 5.5 pack - years. however, the minimum requirement is 10 pack - years for the development of copd in most clinical trials.. demonstrated that biomass fuel exposure could cause chronic airway obstruction in our region. possibly copd due to biomass exposure can lead to different systemic effects than tobacco - induced copd. the effect of malnutrition on pulmonary function is mediated in part by its effect on respiratory muscles. arora and rochester showed that nutritional depletion reduced respiratory muscle strength in patients without lung disease. on the other hand, skeletal muscle weakness is frequently observed in patients with copd, and is associated with wasting of extremity fat - free mass. depletion of fat - free mass, indicating loss of muscle mass, plays a pivotal role in exercise tolerance. in this study, we assessed serum creatine kinase levels to evaluate myocyte dysfunction. however, we did not find a correlation between this muscle - derived parameter and pulmonary functions because serum creatine kinase is not a specific marker for malnutrition. in conclusion, nutritional depletion is associated with impaired pulmonary functions in copd. when compared to controls, rising levels of bun and serum creatinine suggest a protein catabolism in patients with copd. serum albumin concentration is more accurate parameter than bmi for assessing pulmonary dysfunction due to catabolic process in copd.	the aim of this study was to reveal the relationships between nutritional parameters and pulmonary functions in patients with chronic obstructive pulmonary disease (copd) in both sexes. spirometric, laboratory, and demographic data of 450 consecutive patients were analysed retrospectively. males had significantly greater pack - years of smoking, more severe airway obstruction, and lower body mass index (bmi). in non - smokers, bmi was significantly lower in men independent of age and pulmonary functions. creatine kinase levels showed no correlation with any pulmonary function parameters. serum albumin levels correlated better than bmi with pulmonary functions. in conclusion, females with copd were maintained weight better than men.
therefore, the main challenge for commercial plasma collectors in the us is to apply procedures that will safeguard the quality and safety of the product. in many other countries, however, nonremunerated plasma donation is the usual practice. this means that specific interventions not based on monetary incentives are required to recruit plasma donors. to our knowledge, in the scientific literature only a few studies have reported the impact of interventions on plasma donor recruitment [25 ]. each of these interventions mainly provided information about the plasma donation procedure and was successful in recruiting a significant number of new plasma donors. nonetheless, it was recently shown that one - on - one conversation was the best approach to motivate whole blood donors to become plasma donors especially if the conversation was donor - oriented, that is, focusing on donor 's needs and welfare. this latter observation is in agreement with the results reported by ferguson., showing that committed blood donors are more willing to donate blood when exposed to a benevolence message. moreover, their results were consistent with those of weyant who showed that when helping costs are high (e.g., giving plasma), beliefs in personal benefits are more important for promoting action. given the increasing importance for blood agencies to recruit new plasma donors without remuneration, the present research focused on (a) whether two interventions outperformed a control condition regarding the recruitment of new plasma donors, (b) whether any of the two interventions engendered a greater number of donations compared to a control condition, and (c) the moderating effects of gender, age, and donor status on the findings. the population targeted by this study was whole blood donors aged 18 to 70 years, who donated at one of the mobile blood drives organized by hma - qubec, the blood establishment in quebec. to be included in the study, donors had to be living near the fixed donation center in quebec city, where apheresis plasma collections are performed. thus, donors were included if they resided in the metropolitan area or if they gave blood at a mobile clinic within a driving distance of 45 minutes from the apheresis center. a total of 3,514 donors registered at one of the 33 different mobile blood drives held within the specified geographical area between february 14 and june 7, 2012. donors were excluded if they self - reported plasma donation before (n = 41) ; were first time female donor (n = 333) ; or had blood types other than o+, b+, ab+, and ab (n = 1, 753) (see figure 1). thus 1,387 donors were eligible for the study. given the variability in size of the blood drives, at each site the number of donors to be recruited was predetermined and varied between 12 and 60. recruitment of the donors was done at the time of registration, at a pace adapted to the flow of donors, and this is in order to avoid overloading the research nurses. for this latter reason, 396 the other 991 eligible donors were asked at registration if they agreed to participate in a project aimed at better understanding the motivation of blood donors towards plasma donation. if yes, they were given one of three folders that were placed in a prerandomized order. this was the randomization method applied to allocate blood donors to one of the two interventions or the control condition. subsequently, 37 donors were temporarily deferred from giving blood at the time of reviewing their screening questionnaire with the nurse, 25 donors had a history of apheresis according to the donor information system (progesa, mak system), and five registered twice during the study ; thus, these 67 individuals were excluded from the study. in sum the number of blood donors in each condition was 303 (information only by nurse) ; 310 (information plus self - positive image by nurse) ; and 311 (control). this study, realized under the legal mandate of hma - qubec, was conducted to evaluate possible changes in operational recruitment techniques that would have been applied to the donors in any case, except for randomization. notwithstanding this observation, all standard american psychological association (apa) ethical procedures were applied. participants in the control condition were given a folder containing the following : a leaflet explaining the plasma donation procedure (a paragraph of 95 words and a photograph showing someone giving plasma) and a registration form for a first plasma donation. participants in the information only by nurse condition received the same documents as those in the control condition but were told that the research nurse would provide further explanations at a later time during the donation process. thus, upon completion of the donor eligibility questionnaire for blood donation, the trained research nurse briefly reviewed (in less than two minutes) the content of the leaflet describing the plasma donation and the procedure itself, including when, where, and how donors could give plasma. participants in the information plus self - positive image by nurse condition also reviewed the documents at the time they met the nurse, but the leaflet contained an additional paragraph (101 words) about feelings of self - positive image expressed by plasma donors. for both experimental conditions, five nurses were previously trained to deliver the messages on sites of mobile blood drives and to respond to any questions blood donors might have regarding plasma donation. at random, they met blood donors of both experimental conditions on each site. the review of the leaflet by nurses was done within the usual screening procedure for eligibility to give blood and did not require more than two minutes. finally, before leaving the site of the blood drive, all donors of the three conditions could insert in an envelope their signed registration form providing consent to be phoned by the staff of the plasma donation center to set up an appointment for a first plasma donation, no earlier than 56 days after their whole blood donation. thus, for each blood donor, whether s / he gave a first lifetime plasma donation and the number of plasma donations during the six - month period following the index donation were determined. this information was extracted from the donor information system (progesa, mak system). third, the omnibus tests for the effect of condition (experimental versus control) on the proportion of new plasma donors and the mean number of plasma donations over the study period of 6 months. fourth, planned analyses were undertaken that compared the effect of each intervention on donation behavior, compared with the control condition. finally, tests for modification of intervention effects by gender, age, and donor status were conducted via moderated regression analysis. the final sample (n = 924) consisted of 378 women (40.9%) and 546 men (59.1%). participants were predominantly repeat donors (87.8%) and had a mean age of 41.5 years (sd = 14.9). overall, 141 (15.3%) donors gave plasma at least once during the 6-month follow - up period. in total to check on the success of randomization of participants, the three conditions (control, information only by nurse, and information plus self - positive image by nurse) were compared on sociodemographic variables (i.e., age, gender, and donor status) at the time of randomization. no difference was observed on any of the variables (gender, (2, n = 924) = 1.31, p =.518 ; age, (4, n = 924) = 2.58, p =.63 ; and donor status, (2, n = 924) = 2.83, p =.24), suggesting randomization was successful. because correlations between these variables and donation (spearman 's coefficient : rho,) were low (at 6 months : gender, =.02 ; age, =.06 ; and donor status, =.10), we did not control for these variables in subsequent analyses. the use of a dichotomized measure refers to the number of individuals who gave plasma at least once. the intention - to - treat analysis (genmod procedure, binomial distribution : 1 = gave plasma ; 0 = did not give plasma) showed a main effect on the proportion of blood donors in the experimental conditions who gave plasma at least once at 6 months compared to the control condition ((1, n = 924) = 31.97, p <.001). for the frequency of plasma donation (genmod procedure, poisson distribution), the analysis showed a main effect for the experimental versus control conditions at the 6-month follow - up, ((1, n = 924) = 5.04, p <.05). contrast analyses indicated that information only by nurse ((1) = 25.15, p <.001 ; 19.8%) and information plus self - positive image by nurse ((1) = 25.01, p <.001 ; 19.7%) showed significant greater proportions of donors who gave plasma at least once compared to control (6.4%). the two experimental conditions did not differ ((1) = 0.00, p =.969). table 1 presents the mean frequency of plasma donations for each condition at 6-month follow - up. as expected, the control condition exhibited the lowest mean donation (m =.135). at the 6-month follow - up, the information only by nurse ((1, n = 924) = 25.22, p <.001, d = 0.26) and information plus self - positive image by nurse conditions ((1, n = 924) = 52.56, p <.001 ; d = 0.32) showed significantly greater frequency of plasma donations compared to the control condition. moreover, the information plus self - positive image by nurse significantly outperformed the information only by nurse ((1, n = 924) = 5.04, p <.05, d = 0.13). as recommended by aiken and west, a series of three - step hierarchical regressions were used to test whether gender, age, and donor status moderated the effect of each intervention at the 6-month follow - up. behavior was regressed on the respective condition at step 1, the three moderator variables were entered on the second step, and the three condition moderator interaction terms entered the equation on the third step. concerning the proportion of whole blood donors who gave plasma at least once, only donor status (p <.05) emerged as a moderator for one of the interventions : information plus self - positive image by nurse. this latter intervention was significant only among repeat whole blood donors (b = 1.36, se =.28, p <.001). concerning the mean number of plasma donations, few moderation effects were observed. first, gender, age, and donor status (all ps <.01) moderated the effect of the information plus self - positive image by nurse intervention. more precisely, this latter intervention was more efficient among men (b = 0.81, se =.21, p <.001), although it remained significant among women (b = 1.82, se =.33, p < 001), donors aged 35 year and more (3549 years : b = 1.23, se =.26, p <.001 ; 50 years and more : b = 1.81, se =.38, p <.001), and repeat donors (b = 1.24, se =.18, p <.001). finally, it was observed that age (p <.01) moderated the effect of the information only by nurse intervention. this intervention was more efficient among donors aged 50 years and more (b = 1.78, se =.38, p <.001) ; it was not significant among the younger groups. the present study was quite successful and both interventions performed evenly in recruiting new plasma donors although the information plus self - positive image by nurse intervention outperformed the information only by nurse on the frequency of plasma donations. first, the effect of both interventions on the frequency of plasma donation was moderated by age, being significant only among older aged groups. second, the effect of the information plus self - positive image by nurse intervention on both outcomes (proportion of new plasma donors and frequency of donation) was moderated by donor status, the effect being significant only among repeat donors. finally, the information plus self - positive image by nurse intervention was more effective to increase the frequency of plasma donation among men, although it was also significant among women. it is quite interesting to note that only providing basic face - to - face information concerning the plasma donation procedure was sufficient to initiate this new behavior among a substantial proportion of blood donors. this observation runs contrary to the current trend observed in many public health policies to promote the adoption environmental change approaches instead of individuals change strategies. in the present study, our results suggest that providing information is a behavioral change technique that has the potential to change behavior of a significant proportion of blood donors. first, most blood donors were informed for the first time, face - to - face by a nurse, about the nature of this new behavior and given its similarity with blood donation, they likely evaluated having the capacity to adopt it. second, the adoption of this new behavior was likely well aligned with the prevailing underlying motivations, for instance to help others. third, there was a real opportunity to act, with the apheresis center being located within a driving distance of 45 minutes. in summary, in the present case, these above explanations would fit perfectly the com - b system, a framework for understanding behavior. according to this framework, when individuals have the capacity, are motivated, and have the opportunity to act, there is a high probability that they will take action. notwithstanding all of these possible explanations, the present study showed that providing face - to - face information changed the behavior of blood donors. the message referring to self - positive image did not have a significant effect above providing information only by a nurse on the recruitment of new plasma donors. however, our self - positive image message resulted in a higher number of donations in the group exposed to the information plus self - positive image by nurse intervention compared to information only by nurse. this suggests that reference to feelings of self - positive image such as feeling good and being proud and that giving plasma is a rewarding personal experience is a motivational approach to favor a higher frequency of plasma donation. this explanation would be supported by the results of ferguson and colleagues showing that donors exposed to a benevolent message (i.e., reading a leaflet) highlighting the personally rewarding nature of helping and of self - worth associated with donation significantly increased the willingness to give blood. our findings are also aligned with the observation reported by bagot. suggesting that messages that emphasize the donor 's needs and welfare are more effective to motivate whole blood donors to become plasma donors. first, donors in the spi condition were described the plasma donation by a nurse as a rewarding experience and told that those who give plasma are proud of doing this, feel good about themselves, and hold personal positive affect (spi). according to the theory of self - fulfilling prophecy, a positive expectation about the adoption of a given behavior (e.g., giving plasma) may affect a person 's behavior toward this action in a manner that causes those expectations to be fulfilled. therefore, once these new plasma donors were convinced that giving plasma really was a rewarding experience, they took very real actions in consequence and made more frequent donations than those who only received information. this theory would predict that an intrinsic source of motivation better support the adoption and maintenance of a given behavior. thus, the information provided by nurses can be viewed as a source of external motivation. however, donors in the self - positive image condition may have retained the idea that doing the behavior makes you feel good and, in turn, this makes you do it again. this would then be an internal or intrinsic source of motivation that favors giving plasma again. the three moderators analyzed, that is, age, gender, and donor status, carried some effect on the effectiveness of our interventions to initiate plasma donation among blood plasma donors. overall, these moderating effects indicate that interventions to promote starting giving plasma among blood donors are more likely to be successful among a population of men aged 35 years and more and who have some experience with blood donation. for instance, men have a higher circulating blood volume, which enhances the total quantity of plasma that can be collected on the long term compared to women. individuals aged 35 years and more might also be able to follow a rigorous collection schedule compared to younger individuals. finally, blood donors who have some experience with the procedure of blood donation likely have a better understanding of the ability required for giving plasma. in summary, this would provide support for policies targeting the recruitment of plasma donors only among those who have given whole blood at least once in their life, such as the case in australia. a few limitations of this study should be noted. first, our study was conducted on a sample of french speaking donors in quebec, canada, who might differ from other whole blood donors from different countries. second, five nurses were trained for the present project. it is likely that the quality of the intervention varied from one nurse to another and it is not possible to know if the intervention was always delivered as planned. providing information on how, where, and when to give plasma is enough to convince a significant proportion of whole blood donors to start giving plasma. moreover, a message highlighting self - positive image of giving plasma appears to enhance the frequency of donation. blood agencies can therefore introduce this simple low cost promotional approach to recruit new plasma donors, especially among men, aged 35 years and more and who have some experience with giving blood.	this study tested the efficacy of interventions to recruit new plasma donors among whole blood donors. a sample of 924 donors was randomized to one of three conditions : control ; information only by nurse ; and information plus self - positive image message by nurse (spi). participants in the control condition only received a leaflet describing the plasma donation procedure. in the two experimental conditions the leaflet was explained face - to - face by a nurse. the dependent variables were the proportion of new plasma donors and the number of donations at six months. overall, 141 (15.3%) new plasma donors were recruited at six months. there were higher proportions of new plasma donors in the two experimental conditions compared to the control condition (p <.001) ; the two experimental conditions did not differ. also, compared to the control condition, those in the experimental conditions (all ps <.001) gave plasma more often (information only by nurse : d =.26 ; spi : d =.32) ; the spi intervention significantly outperformed (p <.05) the information only by nurse condition. the results suggest that references to feelings of spi such as feeling good and being proud and that giving plasma is a rewarding personal experience favor a higher frequency of plasma donation.
ductal carcinoma is the most common exocrine tumor of the pancreas and is subdivided into adenocarcinoma and squamous cell carcinoma. adenocarcinoma, the most common subtype, comprises 80% of all malignant tumors of the pancreas,1,2 while primary squamous cell carcinoma is an extremely rare subtype of ductal carcinoma in the pancreas because the cell line does not exist in a normal pancreas.3,4 consequently, if the presence of squamous cell carcinoma is confirmed in a biopsy, the possibility of metastatic disease must be considered first, and an extensive search for the primary lesion should be undertaken.5,6 recently, we confirmed a case of synchronous esophageal cancer with pancreatic metastasis and hepatocellular carcinoma (hcc). a 58-year - old male with a 1-month history of abdominal bloating and indigestion visited our hospital for esophagogastroduodenoscopy. this detected a 34-cm erythematous lesion with an irregular nodular surface extending between 38 and 41 cm from the incisors in the 9 to 1 o, clock position (fig. it was shown as an iodine - void area with a well - defined boundary. a biopsy revealed squamous cell carcinoma, and he was admitted for further evaluation and management of this esophageal cancer. he was an alcoholic who had been drinking one bottle of soju daily for the past 30 years and had a 30-pack - year smoking history. he was a hepatitis b carrier without regular follow - up. at admission, the patient 's vitals were stable and he was alert mentally. the peripheral blood tests were normal, with hemoglobin 13.6 g / dl, white blood cell count 7,330/mm, platelets 238,000/mm, as were the blood chemistry and electrolytes. additional studies for hepatitis b showed hepatitis b e antigen (-) and anti - hepatitis b e antibody (+), and no virus was detected with hepatitis b virus real - time polymerase chain reaction. the results for tumor markers were as follows : carcinoembryonic antigen 2.39 ng / ml (reference range, < 4.1), ca19 - 9 2 u / ml (reference range, < 37), and alpha - fetoprotein (afp) 8.58 ng / ml (reference range, < 4.0). abdominal computed tomography (ct) showed a poorly demarcated and poorly enhancing mass at the pancreatic tail (fig. magnetic resonance imaging (mri) showed that the pancreatic mass appeared poorly demarcated and hyperintense on t2-weighted image and hypointense on t1-weighted image. so we were highly suspicious that the pancreatic mass was a primary adenocarcinoma rather than metastasis, because of tumor character and extremely low incidence of pancreatic metastasis from esophageal cancer. ct and mri showed 1.2 cm - sized single nodule in the liver (fig. mri showed that the hepatic mass was hypointense on t1-weighted image and hyperintense on t2-weighted image, and poorly enhanced on dynamic enhancing study. however, serum afp was elevated (8.52 ng / ml) and we had to undergo ultrasound - guided liver biopsy. the hepatic mass was confirmed as a primary hcc based on the loss of ductular reaction and cd34 positivity (fig. the patient underwent surgery for the esophagus and pancreas lesions and radiofrequency ablation for the hepatic cancer. histologically, the partial esophagectomy and distal pancreatectomy specimens showed squamous cell carcinoma and local lymph node invasion and the patient was diagnosed with stage iv esophageal cancer with pancreatic metastasis (fig. the tumor invaded to submucosal layer with lymphatic invasion and metastasis to 3 of 31 regional lymph nodes. in the pancreas, the size was 54.54 cm with clean resection margins but there was lymphatic invasion involving 2 of 20 regional lymph nodes. the patient had received 5-fluorouracil / cisplatin combination therapy six times for 4 months after the surgery. he has been followed up without any evidence of recurrence on abdomen ct and gastroscopy until this month. epithelial tumors of the pancreas are divided into exocrine tumors, which are derived from ductal and acinar cells, and endocrine tumors, which are derived from islet cells.1,2 most malignant tumors in the pancreas are primary, and approximately 80% are adenocarcinomas. the rest include metastatic lesions, squamous cell carcinoma, small cell carcinoma, and acinar cell carcinoma.7 primary squamous cell carcinoma of the pancreas is extremely rare, accounting for only 0.7% of 1,300 pancreatic cancer in a 1992 report from japan because the normal pancreas does not contain squamous epithelium.8 typically, primary squamous cell carcinoma of the pancreas is confirmed only after other primary sources have been excluded using diagnostic tests like ct of the head and chest, gastrointestinal endoscopy, a pelvic exam with a pap smear, and a thorough examination of the skin. once there is no evidence of a primary lesion elsewhere, the diagnosis of primary pancreatic cancer can be made.5,6 isolated pancreatic metastases from nonpancreatic primary tumors are also very rare, accounting for about 2% of all pancreatic tumors. autopsy studies of cancer patients with pancreatic metastasis have shown that the lungs and liver are the most common sites of primary lesions, followed by the colon and kidney. primary esophageal cancer compromises only 0% to 4.8% of pancreatic metastasis, and there has been no reported case in south korea.8 - 10 generally, it is difficult to distinguish a solitary pancreatic metastasis from a primary cancer and a double or triple primary pancreatic cancer is difficult to diagnose in almost all cases. a tissue diagnosis can be made before surgery with a fine needle aspiration biopsy, guided by ultrasonography, ct, or another modality. in the case of a pancreatic mass, resectability should be considered ahead of a preoperative biopsy. in our case, both primary pancreatic cancer and pancreatic metastasis from the esophageal cancer or hcc were possible. so we decided surgical resection because the tumors were thought to be triple primary cancer arising from liver, esophagus, and pancreas. after the operation, the pancreatic mass was unexpectedly confirmed as metastasis from the esophageal cancer. in the treatment of cancer patients with an isolated distant organ metastasis and no widespread disease, resection of the metastasis is beneficial for some types of tumor. hiotis.11 reported that the resection of pancreatic metastatic lesions in 16 patients with different primary tumors led to a longer disease - free interval and improved overall survival. in addition, the surgical resection of solitary metastatic lesions from colorectal cancer to the pancreas increases patient survival.10 furthermore, the resection of isolated extrahepatic recurrences of hcc prolonged the survival in selected patients.12 a review of the surgical outcomes of reported cases, including our patient, indicated that the surgical mortality rate in pancreatic surgery is less than 5%.13 in our case, surgical resection was reasonable for treatment and obtaining a definite diagnosis, as well as for improving patient survival, although there are few guidelines for managing cases such as ours. however, these previous reports are based on metastatic lesions that developed after the diagnosis of well - controlled primary tumors. there are some limitations to applying these reported cases to our case, in which a primary lesion and an isolated metastatic lesion were found simultaneously and the metastatic lesion was considered as primary tumor on imaging studies. further observations of the prognosis are required in cases diagnosed with synchronous double primary cancer with an isolated metastatic lesion. we reported this case because isolated pancreatic metastasis of esophageal cancer is rare, and careful treatment decisions are required. moreover, established guidelines for treating double primary cancer with an isolated metastatic lesion are needed.	solitary pancreatic metastasis of esophageal cancer is extremely rare. we report the case of a 58-year - old male admitted with esophageal cancer. additional asymptomatic solitary hepatic and pancreatic masses were observed in the staging work - up for esophageal cancer. the hepatic mass was confirmed as a primary hepatocellular carcinoma with an ultrasound - guided needle biopsy. an esophagectomy with a distal pancreatectomy and radiofrequency ablation for hepatocellular carcinoma were performed. histologically, the pancreatic mass was confirmed to be a metastasis from the esophageal cancer. the patient has been followed up with chemotherapy.
first generation antipsychotics along with amisulpride, risperidone, paliperidone, and zotepine have been consistently reported to be associated with the high rates of hyperprolactinemia and termed as prolactin - raising, whereas clozapine, olanzapine, quetiapine, ziprasidone, and aripiprazole, which have a more favorable profile, have been termed as prolactin - sparing. switching over to aripiprazole or adjunctive aripiprazole, has been advocated for the optimal management of antipsychotic - induced hyperprolactinemia. adjunctive treatment with aripiprazole has been shown to normalize prolactin levels without affecting improvements in the psychotic symptoms that had already been achieved with the previous antipsychotic treatment. however, a literature search shows the case reports of paradoxical hyperprolactinemic symptoms such as galactorrhea associated with aripiprazole use. saraf. in a case report of a female, who developed hyperprolactinemia, while on aripiprazole, postulated that aripiprazole could have dopamine antagonistic properties at higher doses as the partial d2 agonistic activity could be dose - related. recent research has also shown that the beneficial effect of aripiprazole on other antipsychotic - induced hyperprolactinemia is seen at lower doses and plateaus off at higher doses. however, here, we present the case of a female with the delusional disorder who developed symptomatic hyperprolactinemia even while on a low dose (10 mg / day) of aripiprazole. a 36-year - old married female was presented to the psychiatry outpatient services with 2 years of delusion of infidelity and significant sociooccupational dysfunction in the absence of any impairment in sleep or appetite. vitals were stable, body mass index (bmi) was calculated to be 26 and systemic examinations revealed no abnormality. baseline prolactin level was 14 ng / ml. in view of the high bmi and poor insight, she was started on aripiprazole. it was expected that the favorable side effect profile of aripiprazole with relatively less weight gain and extrapyramidal symptoms would ensure compliance. aripiprazole was started at an initial dose of 5 mg at night and was increased to 10 mg and later 15 mg after 4 days each and 15 mg was continued as maintenance. however, 3 weeks later, she was presented with galactorrhea and a missed menstrual period. the possibility of aripiprazole - induced hyperprolactinemia was considered and aripiprazole was stopped. as she was otherwise asymptomatic, no other medication was started and she was sent home. at follow - up, 1-month later, galactorrhea had subsided, she had menstruated, and repeat prolactin levels were 18 ng / ml. her psychotic symptoms continued to be in remission, and she returned home medication - free. aripiprazole was restarted as previously, but was maintained at 10 mg owing to the previous history of hyperprolactinemia. she reported back within a month with the complaints of galactorrhea and amenorrhea at which time her psychotic symptoms were in complete remission. serum prolactin levels were repeated and found to be elevated (84 ng / ml). serum prolactin levels were normalized (19 ng / ml) and hyperprolactinemia symptoms were subsided within 1-month. peptides, steroids, and neurotransmitters regulate the synthesis and release of the hormone prolactin from the lactotrophs of the anterior pituitary. dopamine acts as a tonic inhibitor of prolactin secretion through the tuberoinfundibular and the tuberohypophysial dopaminergic systems. the binding of dopamine to the d2 receptors on the membrane of the lactotroph cells inhibits the prolactin gene transcription, synthesis and release of prolactin, and lactotroph proliferation. on the other hand, d2 receptor blockade by antipsychotics counteracts the tonic inhibitory effect of dopamine on prolactin secretion, thus elevating serum prolactin levels. the degree of hyperprolactinemia caused correlates with the penetrability of the blood - brain barrier and the strength of the dopamine blockade. the quicker the drug dissociation from the receptor, the lesser is the increase in plasma prolactin. the functional activity of a partial agonist at the specific receptor depends on the presence or absence of other full agonists and antagonists in the surrounding milieu. they bind to the receptor to produce the physiological response seen, when the receptor is activated. however, if a full agonist is simultaneously present, the partial agonist shows functional antagonist activity. it competes with the full agonist for receptor occupancy and when bound, the response is lesser than that seen with the full agonist alone. aripiprazole has a lower intrinsic activity at the d2 receptor than dopamine, allowing it to act as both, a functional agonist and antagonist, depending on the surrounding levels of dopamine. in the presence of a prolactin - raising antipsychotic, adjunctive aripiprazole competes with it to possibly act as an agonist in the tuberoinfundibular pathway, thus bringing down the elevated prolactin levels. however, as a stand - alone treatment, the functional activity of aripiprazole in this pathway would be dependent solely on the dopamine levels. hence, it is possible that in the absence of a competing d2 antagonist and the presence of dopamine (the natural agonist), aripiprazole could act as a functional antagonist at lower doses also, and thus, elevate prolactin levels.	hyperprolactinemia is a common adverse effect of antipsychotic medication. switching over to aripiprazole or adjunctive aripiprazole has been advocated for optimal management of antipsychotic - induced hyperprolactinemia. adjunctive treatment with aripiprazole has been shown to normalize prolactin levels without affecting already achieved improvements in psychotic symptoms. however, here, we present the case of a 36 year old female with delusional disorder who developed symptomatic hyperprolactinemia while on aripiprazole treatment. dopamine acts as a tonic inhibitor of prolactin secretion through the tubero - infundibular dopaminergic system. aripiprazole being a partial agonist has a lower intrinsic activity at the d2 receptor than dopamine, allowing it to act as both, a functional agonist and antagonist, depending on the surrounding levels of dopamine. hence, in the absence of a competing d2 antagonist and the presence of dopamine (the natural agonist), aripiprazole could act as a functional antagonist and thus elevate prolactin levels.
the functions of the brain and other body system organs are strongly dependent on proper cardiac performance. our daily life appropriately emphasizes the strong connections between the heart, emotional reactions and social functions. therefore, the appearance of normal emotional reactions such as shock, fear, anger, sadness, and grief in response to the development of heart disease is not unexpected.1 three decades ago, epidemiologists began to report strong associations between depression and cardiovascular disease, morbidity and mortality ; though, this association was the subject of a lot of criticism.2 during the last decade, many large - scale well - designed controlled studies have identified depression as an important risk factor for both first myocardial infarction (mi) and cardiovascular mortality.35 all clinical grades of depression even minor depression have been found to be associated with significantly increased risk of subsequent cardiovascular morbidity and mortality in patients with known coronary heart disease (chd).614 the prevalence of major depression disorder (mdd) is estimated to be between 15% and 23% among patients with known chd,91315 though it is frequently underdiagnosed and undertreated.1617 nevertheless, clinical depression has been recognized as an independent risk factor for the incidence of chd for several decades (after the onset of clinical depression).18 the possible explanation of this association is the enhanced platelet activity in depressed patients. in the sertraline anti - depressant heart attack randomized trial (sadhart) platelet substudy, the treatment with sertraline (ssri) was associated with substantial reduction of platelet / endothelial activation, despite co - administration of widespread antiplatelet regimen including aspirin and clopidogrel.19 in this study, our aim was to determine the frequency of clinical depression among patients who were admitted to coronary care unit (ccu) with acute coronary syndrome (acs) in this part of the world with a different social and cultural environment. the objectives of this study were to : (1) determine the frequency of clinical depression among patients admitted with acs to the ccu at king fahd hospital of the university ; (2) find any association between depression and the traditional risk factors for coronary artery disease (cad) such as age, sex, diabetes mellitus, hypertension, smoking, dyslipidemia, and strong family history of coronary artery disease. this was a prospective study conducted in king fahd hospital of the university (450-bed teaching hospital), al khobar, eastern province, kingdom of saudi arabia (ksa), between july 2003 and february 2004. to qualify for inclusion, males and females were required to have a definite history of chest pain strongly suggestive of unstable angina (ua), acute non - st segment elevation myocardial infarction (nstemi) or st segment elevation myocardial infarction (stemi), the three components of acute coronary syndrome. the diagnosis of unstable angina (ua) mainly depended on a convincing history of episodes of chest pain occurring at rest and lasting at least 10 minutes and leading to hospitalization, or a history of ua in a patient with a known coronary artery disease and a documented history of a prior myocardial infarction. the patient should have undergone a prior revascularization procedure, or had documented coronary artery stenosis greater than 75% in one of the major epicardial coronary arteries. the diagnosis of nstemi and stemi depended mainly on a convincing prolonged chest pain lasting for more than 20 minutes, associated with electrocardiographic changes (st segment depression, t waves inversion, st segment elevation and q waves), and specific cardiac enzymes elevation, e.g. creatine phosphokinase isoenzyme mb (cpk - mb), greater than the upper limit of normal and troponin i or troponin t more than two times the upper limit of normal. the exclusion criteria included all of the following conditions : patients with an obvious language barrier, patients with abnormal cognitive functions, those with acs of non - atherosclerotic etiology (e.g. anemia or cocaine use), klipp class iii or iv status, those with persistent and clinically significant laboratory abnormalities such as severely impaired liver function test, thyroid function test and renal function test ; patients with other significant non - cardiac disease such as chronic renal failure, chronic hepatic failure, and hypothyroidism, alcohol and substance abuse or dependence in the past six months, and finally a history of psychosis, bipolar disorder, organic brain syndrome (obs), or dementia. one hundred and two patients admitted consecutively to the ccu with acute coronary syndrome (acs) who met our inclusion criteria completed the self - rated beck depression inventory (bdi) in their native language.24 the bdi which includes 21 multiple - choice questions (mcqs) was completed after the presenting symptoms were alleviated, 4 -7 days after the onset of acs. bdi has been an internationally accepted practical questionnaire since 1961, and has been validated.2122 the opinions of expertise in the fields of psychiatry, cardiology and community medicine were also obtained for the purpose of validation of bdi in ksa. the reliability of patient responses was calculated using chronbach = 0.82 which indicating high reliability. the study sample was divided according to patient ethnic background as follows : saudi arabs, non - saudi arabs, patients from the indian subcontinent (indians, pakistanis, bangladeshi and sri lankans) and others mainly asians. arabic, hindi, urdu and english were initially used and the inventory was subsequently translated into english. our patients were classified into two groups : group a included those patients with subclinical depression or with no depression scoring 0 - 20 points on bdi score scale, and group b for those scoring 21 points and above. the data were entered into a personal computer (pc) using statistical package for social science version 10 (spss). t - test and mann whitney were used to compare between bdi total score and each of the different variables as appropriate. analysis of variance (anova) or kruskal - wallis were used appropriately to ascertain any differences among nationalities, age and bdi mean total score. the levene test was used as the criteria for choosing between parametric and non - parametric tests. the chi - square test was used to determine any association between risk factors and the total scores after categorizing them into two groups : sub - clinical and clinical depression. logistic regression analysis was implemented to find the effect of different risk factors and bdi total score after clinical categorization. to qualify for inclusion, males and females were required to have a definite history of chest pain strongly suggestive of unstable angina (ua), acute non - st segment elevation myocardial infarction (nstemi) or st segment elevation myocardial infarction (stemi), the three components of acute coronary syndrome. the diagnosis of unstable angina (ua) mainly depended on a convincing history of episodes of chest pain occurring at rest and lasting at least 10 minutes and leading to hospitalization, or a history of ua in a patient with a known coronary artery disease and a documented history of a prior myocardial infarction. the patient should have undergone a prior revascularization procedure, or had documented coronary artery stenosis greater than 75% in one of the major epicardial coronary arteries. the diagnosis of nstemi and stemi depended mainly on a convincing prolonged chest pain lasting for more than 20 minutes, associated with electrocardiographic changes (st segment depression, t waves inversion, st segment elevation and q waves), and specific cardiac enzymes elevation, e.g. creatine phosphokinase isoenzyme mb (cpk - mb), greater than the upper limit of normal and troponin i or troponin t more than two times the upper limit of normal. the exclusion criteria included all of the following conditions : patients with an obvious language barrier, patients with abnormal cognitive functions, those with acs of non - atherosclerotic etiology (e.g. anemia or cocaine use), klipp class iii or iv status, those with persistent and clinically significant laboratory abnormalities such as severely impaired liver function test, thyroid function test and renal function test ; patients with other significant non - cardiac disease such as chronic renal failure, chronic hepatic failure, and hypothyroidism, alcohol and substance abuse or dependence in the past six months, and finally a history of psychosis, bipolar disorder, organic brain syndrome (obs), or dementia. one hundred and two patients admitted consecutively to the ccu with acute coronary syndrome (acs) who met our inclusion criteria completed the self - rated beck depression inventory (bdi) in their native language.24 the bdi which includes 21 multiple - choice questions (mcqs) was completed after the presenting symptoms were alleviated, 4 -7 days after the onset of acs. bdi has been an internationally accepted practical questionnaire since 1961, and has been validated.2122 the opinions of expertise in the fields of psychiatry, cardiology and community medicine were also obtained for the purpose of validation of bdi in ksa. the reliability of patient responses was calculated using chronbach = 0.82 which indicating high reliability. the study sample was divided according to patient ethnic background as follows : saudi arabs, non - saudi arabs, patients from the indian subcontinent (indians, pakistanis, bangladeshi and sri lankans) and others mainly asians. arabic, hindi, urdu and english were initially used and the inventory was subsequently translated into english. our patients were classified into two groups : group a included those patients with subclinical depression or with no depression scoring 0 - 20 points on bdi score scale, and group b for those scoring 21 points and above. the data were entered into a personal computer (pc) using statistical package for social science version 10 (spss). t - test and mann whitney were used to compare between bdi total score and each of the different variables as appropriate. analysis of variance (anova) or kruskal - wallis were used appropriately to ascertain any differences among nationalities, age and bdi mean total score. the levene test was used as the criteria for choosing between parametric and non - parametric tests. the chi - square test was used to determine any association between risk factors and the total scores after categorizing them into two groups : sub - clinical and clinical depression. logistic regression analysis was implemented to find the effect of different risk factors and bdi total score after clinical categorization. one hundred and two consecutive patients were able to complete bdi, 4 - 7 days after the onset of acs. 92 were males (90.2%) ; and the remainder females, with a mean age of 52.14 12.14 years ; 51.43 10.57 years for males, and 58.60 21.71 years for females (p=0.076). twenty - nine patients (28.43%) were admitted with unstable angina, 20 (19.61%) with non - st segment elevation myocardial infarction (nstemi), and 53 (51.96%) with st segment elevation myocardial infarction (stemi). there were 38 (37.3%) saudis, 26 (25.5%) non - saudi arabs, 32 (31.4%) from the indian subcontinent and 6 (5.9%) other patients who were mainly asians. the age difference between saudi patients and those from the indian subcontinent was statistically significant (p<0.0001). fifty - one (50%) were diabetics, 51 (50%) hypertensive, 65 (63.72%) smokers, 51 (50%) dyslipidemic, 20 (19.61%) had a strong family history of coronary artery disease ; 17 (16.66%) were obese (bmi 0f 30 kg / m and above according to the who classification), and 16 (15.68%) had a previous history of heart failure. general characteristics of the study patients table 2 shows that the highest mean bdi score was found among the patients from the indian subcontinent, 16.81 12.70 as compared to saudis and non - saudi arabs with a borderline significance (p=0.057). the mean bdi scores were not significantly different among patients presenting with the 3 different components of acute coronary syndrome (12.24 7.50 for patients presenting with unstable angina, 14.508.42 for patients with nstemi and 12.66 12.07 for patients with stemi). mean bdi scores among different nationality subgroups with acs table 3 shows that evidence of clinical depression was found in 20.6% of all patients, 13.2% of the saudi patients, 19.2% of the non - saudi arabs, 34.4% of the patients from the indian subcontinent and none of the other patients. the difference between the saudi patients and those from the indian subcontinent was statistically significant (p=0.035). prevalence of clinical depressions among patients with acs table 4 illustrates the possible association between traditional risk factors for cad and depression. diabetes mellitus was found to be associated with lower mean bdi score (p=0.018). the presence of hypertension, dyslipidemia, strong family history of coronary artery disease and obesity did not affect the bdi scores. however, smoking was the only risk factor associated with a significantly higher mean bdi score, 15.48 10.85 as compared to 8.38 7.17 in non - smoker (p=0.01). patients with a previous history of chf had a lower mean bdi score than patients without a previous history of chf (p=0.043). nevertheless, the frequency of clinical depression among smokers was significantly higher than in non - smokers, 26 out of 65 patients(40%) compared with 6 out of 37 patients(16.2%) respectively, p=0.02. risk factors as possible predictors of depression after controlling for the effect of other traditional risk factors (diabetes mellitus, hypertension, and strong family history of cad) in a logistic regression model, smoking and dyslipidemia remained the most important significant predictors of clinical depression (p=0.0072 and p=0.00342, respectively). after reviewing the literature, it appeared that depression is unquestionably associated with cad and it is hard not to think of this association in terms of depression as a cause of cad.2 it appeared also that depression is an independent risk factor for cad, even several decades after the first episode,3418 and is an independent risk factor for increased post acs morbidity and mortality.269101423 it is important to remember, however, that what has been demonstrated is an association and not causality.2 unfortunately, post - myocardial infarction depression often goes unrecognized.17 in our study, approximately one in five patients admitted with acs (20.6%) suffered from symptoms of moderate to severe clinical depression. this agreed quite strongly with other studies (15 - 23%).21017 the frequency of clinical depression among patients from the indian subcontinent was significantly higher than in saudis and non - saudi arabs. furthermore, though patients from the indian subcontinent had a significantly lower mean age compared to saudi patients, they had a high frequency rate of clinical depression. patients from the indian subcontinent were three and half times more prone to developing clinical depression as compared to saudi patients. the probable factors responsible for this high frequency are : the lack of family and social support, the relatively low socio - economic status, unsatisfactory accommodation and living conditions, work pressures, and the significant differences in the cultural and social backgrounds.715 there is a large body of evidence to suggest that depression may be associated with several pathophysiologic mechanisms including hypothalamic - pituitary axis hyperactivity, autonomic nervous system dysfunction, and increased platelet activity which may explain the increased risk of cad in a depressed population.2024 those pathophysiologic mechanisms, especially increased platelet/ endothelial activity, may be considered as active players in the pathogenesis of coronary atherosclerosis and the early occurrence of cad in immigrant workers from the indian subcontinent. however, this hypothesis needs confirmation in a well designed prospective study focusing on immigrant patients from the indian subcontinent suffering from acs. smoking was the only risk factor for cad which was strongly associated with depression in our study (p=0.01). this finding was similar to those reported by carney, and glassman.1025 after controlling for the effect of other traditional risk factors, such as diabetes mellitus, hypertension, and strong family history of cad in a logistic regression model, smoking and dyslipidemia remained the most important significant predictors of depression (p=0.0072 and p=0.0342, respectively). our findings are supported by those of kaplan who reported that the effect of smoking on the degree of carotid atherosclerosis in 1100 middle - aged men was 3.4 times greater in depressed than in non - depressed men ; and the effect of ldl cholesterol level on atherosclerosis was nearly doubled. however, the studies which focused on the association between traditional risk factors and depression failed to document such an association.27 nevertheless, in those studies in which a significant association has been found, depression remained an independent predictor of cardiac morbidity and mortality after controlling those risk factors.3 our findings may suggest that clinical depression, smoking and dyslipidemia represent a potentially serious confounding cause for cad, and the post - acs higher morbidity and mortality.2 however, we are still far short of proof that depression has a causal role in the etiology and pathogenesis of cad. there remain some unsolved questions concerning the biological mechanisms relating depression to cad, the causes and the nature of depression preceding the first or recurrent cardiac events and the power of anti - depressive therapeutic programs to reduce the morbidity and mortality of cad in depressed patients.28 the sadhart trial opened the door to the hope that treating depression with selective serotonin reuptake inhibitors may effectively reduce morbidity and mortality in depressed post - acs patients.19 depression is unquestionably associated with cad, and is an independent risk factor for post - acs morbidity and mortality. this is a preliminary prospective study intended to determine the frequency of clinical depression among patients suffering from acs. the frequency of clinical depression among our patients was 20.6% and was very high among patients from the indian subcontinent (34.4%). therefore, we are in accord with the opinion that depression, smoking and dyslipidemia may together be serious confounders of cad. however, the causative relationship between depression and cad was beyond the scope of this study. thus, further controlled studies are needed to elucidate this important problem in this part of the world.	background : three decades ago, epidemiologists began to report a strong association between depression and cardiovascular disease - morbidity and mortality, and in the last decade, many large - scale studies have identified depression as an important risk factor for coronary artery disease (cad) and its morbidity and mortality.objectives:to determine the frequency of clinical depression among patients admitted with acute coronary syndrome (acs) to the coronary care unit (ccu) at king fahd hospital of the university (kfhu), and to find out if there is any relationship between depression and the traditional risk factors for cad.methods:one hundred and two patients admitted consecutively with acs completed the self - rated beck depression inventory (bdi) in their native language. the patients were classified into two groups : group a comprising patients with no symptoms of depression and patients with subclinical or borderline depression, scored less than 21 points on bdi score scale ; and group b composed of those who scored 21 points and above. various statistical tests were used whenever appropriate.results:one hundred and two patients completed the bdi. ninety - two (90.2%) were males, with a mean age of 52.1412.14 years. of these, 37.3% were saudis, 25.5% non - saudi arabs, 31.4% from the indian subcontinent and 5.9% were other asians. patients from the indian subcontinent were significantly younger than the saudis (p<0.0001). the evidence of clinical depression was found in 20.6% of all patients, 13.2% of saudi patients, 19.2% of non - saudi arabs and 34.4% of those from the indian subcontinent. there was a significant difference in the frequency of clinical depression between saudi patients and the indians (p=0.035). smoking and dyslipidemia were the only strong predictors of clinical depression in our study.conclusion:depression is unquestionably associated with cad. its frequency in our patients with acs was 20.6%, and the highest frequency was recorded among patients from the indian subcontinent (34.4%). smoking and dyslipidemia were the strongest independent risk factors for depression.
after obtaining clearance from the institutional board of studies and informed consent, horizontal strabismus patients qualifying for symmetrical recession or resection surgeries on the horizontal rectii were included. anesthesia utilized was either standard peribulbar block or general anesthesia (propofol and nitrous oxide). one eye was randomized (using an unbiased coin) to standard paralimbal strabismus surgery (spss) while the other automatically qualified for miss. after separating the lids with universal eye speculum, a 5 - 0 silk traction suture (johnson and johnson ltd. aurangabad ; nw 5079) was passed through the superficial sclera near the limbus at 12 and 6 oclock to expose the quadrant of interest. four points were marked on the conjunctiva : two at the upper and lower ends of the rectus muscle insertion and two at the posterior limits of the planned para - muscular conjunctival incisions [fig. the two points on each side of the muscle insertion were then incised with a bard - parker blade (# 11) after raising a fold of conjunctiva by lifting the conjunctiva between forceps, so as to obtain two incisions parallel to the upper and lower edge of the horizontal rectus muscles, with their anterior limits at the upper and lower ends of the muscle insertion [fig. the radial length of these incisions was 1 mm less than the planned recession / resection if the latter was 5 mm, and two mm less if > 5 mm. using blunt wescott scissors, careful dissection was carried out to identify the muscle edge, under which a muscle hook was then passed [fig. further dissection then helped delineate the muscle and the adjacent sclera up to the site of recession. a similar conjunctival incision was made at the superior border of the muscle, aided by the muscle hook already placed below the muscle. subsequently, the strap of conjunctiva, overlying the muscle tendon, between the two conjunctival incisions, was meticulously dissected free of the muscle up to the insertion to facilitate subsequent surgery in that area [fig. this provided us access to the upper and lower edge of the muscle right up to the insertion, as also the sclera / muscle till the point where recession / resection was to be carried out. the planned site of recession on the sclera, or the site of resection on the muscle, were marked [fig. (a - i) figures showing surgical steps for miss (recession) (see text) for recessions : vicryl 6 - 0 (johnson and johnson ltd. aurangabad nw 2670) locking bites were taken from the muscle margins from near the insertion [fig. if the parallel conjunctival cut edges appeared to be in good apposition, no sutures were applied ; else, a single central vicryl 8 - 0 suture approximated the incisions. good apposition was considered wherein the edges were no more than 2 mm apart at a point of maximum separation, after gently smoothening the conjunctival surface [fig. 1i ]. in case of any complications, like excessive hemorrhage, or difficulty in suturing the muscles, were encountered, it was planned to prolong the conjunctival incisions towards the limbus and join them, effectively converting them to resemble the limbal approach ; in such an event, the miss approach would be considered to have failed. for resection : after a similar conjunctival approach as for recessions, two vicryl 6 - 0 sutures were applied at the edges of the extra - ocular muscle (eom) at the point of desired resection and passed through the insertion of the eom. the muscle between the suture bites was excised ; tying the sutures brought the point of resection to the insertion, effecting a resection. we recorded the duration of surgery (in minutes) from the start of the conjunctival incision to the completion of surgery. post - operatively graded scoring of inflammation (o to 3 : nil, mild, moderate, severe) was done by an observer masked to the randomization ; on day 1, at 2 - 3 weeks, and 6 weeks : specifically redness, congestion (these two were compared to standard photographs), chemosis, foreign body sensation (fbs), drop intolerance were scored ; and each score combined to yield a total inflammatory score (tis ; with a possible range from 0 to 15). visible scarring (from one meter) and final alignment were noted at 6 weeks. we included 14 parallel designed surgeries on 10 patient ; 10 were bilateral recessions, while 4 of subjects participated a second time (3 months later) undergoing planned second bilateral recestions on account of large angles not amenable to single strabismus - surgical correction. the mean duration of surgery in the spss was 29.6 (sd 5.37) minutes as compared to 40.4 (sd 7.98) minutes in the miss approach, with a mean difference of 10.8 minutes (95% ci for difference : 2.67 to 18.92 minutes.), which was significant (wilcoxon test ; p = 0.013). on the first post - operative day, there were significant differences (p < 0.05) in the median inflammatory scores for fbs and tis (wilcoxon test, table 2). at 2 - 3 weeks, in addition, significant differences for redness and congestion. by 6 weeks, significant differences were restricted to redness and tis. at final follow - up at 6 weeks, significant scarring was observed in all the eyes, which underwent spss while it was present in 9 miss eyes ; the difference was not statistically significant (chi - square, fisher 's exact test ; p = 0.09). there was no change in visual acuity postoperatively in any of the patients during any time in follow - up. amount of strabismus, target surgery, final alignment along with status of fusion, and stereopsis at 6 weeks postoperatively are also shown in table 1. demographic details of the patients along with post operative functional outcomes median inflammatory scores and p value during post - operative follow - up we compared post - operative comfort and cosmesis, by miss and spss, in patients undergoing symmetrical strabismus surgeries in a parallel design, randomizing one of two eyes, since that may be a superior model to compare inflammatory outcomes. on the first post - operative day, there was no significant difference between the two eyes in terms redness, congestion, chemosis, and drop intolerance ; only fbs (p value < 0.01) was significantly less in the miss eyes. this could be on account of the small incisions and posterior placement of conjunctival sutures (if any) in the latter group. at 2 - 3 weeks follow - up, scores for redness, congestion, and foreign body sensation were significantly less in the miss eyes [fig. 2 ]. while at 6 weeks, we did not find any significant difference, except for redness and total inflammatory score [table 2 ]. comparative photograph at 2 - 3 weeks postoperatively, of the same patient operated through miss and spss in his introductory paper, mojon utilized the miss approach and compared them with a historical group, which underwent a limbal approach in cases undergoing horizontal rectii surgeries. although his main outcome measures (fusion, alignment, and vision) were different from ours, mojon did report significantly less lid swelling (0% vs. 21%) in the miss group on the first post - operative day. on final analysis at 6 months, no significant difference was found. in another study on exotropes, pellanda and mojon they found significantly less conjunctival swelling and injection on the first post - operative day (test p < 0.001). they needed to convert 3/104 muscles in their miss group. in a similar analysis in esotropia, conjunctival swelling and injection were significantly less pronounced (p value < 0.001) in the miss group. we believe that a comparison of grades of inflammation by the mann - whitney test would have been superior, although the results are unlikely to have been different. although we found 9 of 14 cases (64.3%) of miss showing a significant scar at 6 weeks compared to all 14 cases (100%) of spss, the results did not reach statistical significance (fisher 's exact ; p value : 0.09). mojon has also commented in his comparative study that minimal cicatrization was seen along the incision line, which did not hinder free movement of the conjunctiva over the sclera. on occasion (sic), no scar was seen biomicroscopically. only larger studies with more statistical power may provide clear answers to this question. in our study, miss took significantly longer than spss ; but, it is likely that this would decrease as experience with miss accumulated. although comparison of alignment by its very nature can not be compared in a study like ours, we are reporting post - operative alignment outcomes [table 1 ], since that would be of interest to our readers. as is evident, 11 of 14 surgeries achieved correction to within 10 pd of target angle ; a success rate of 78.5%. as highlighted by kushner, the miss approach interferes less with the peri - limbal episcleral vessels, which should decrease the chances of anterior segment ischemia. although miss may appear to be superior to the paralimbal approach, it may not be much different to the parks fornix approach where too conjunctival incisions are far removed from the limbus. we are acutely aware of the small number of cases in our study and suggest that more studies with a larger sample size would yield better evidence to either support or refute our findings. since all our patients were young adults, the results should be interpreted preferable for this age group. the strength of our study is in its parallel design, a format superior in our opinion to compare inflammation scores. our small study shows that a miss approach is more comfortable and offers better cosmesis (in terms of redness) in the short - term (2 - 3 weeks) as compared to the spss ; this difference equates by 4 - 6 weeks.	introduction : minimal access surgery is common in all fields of medicine. we compared a new minimally invasive strabismus surgery (miss) approach with a standard paralimbal strabismus surgery (spss) approach in terms of post - operative course.materials and methods : this parallel design study was done on 28 eyes of 14 patients, in which one eye was randomized to miss and the other to spss. miss was performed by giving two conjunctival incisions parallel to the horizontal rectus muscles ; performing recession or resection below the conjunctival strip so obtained. we compared post - operative redness, congestion, chemosis, foreign body sensation (fbs), and drop intolerance (di) on a graded scale of 0 to 3 on post - operative day 1, at 2 - 3 weeks, and 6 weeks. in addition, all scores were added to obtain a total inflammatory score (tis).statistical analysis : inflammatory scores were analyzed using wilcoxon 's signed rank test.results:on the first post - operative day, only fbs (p = 0.01) and tis (p = 0.04) showed significant difference favoring miss. at 2 - 3 weeks, redness (p = 0.04), congestion (p = 0.04), fbs (p = 0.02), and tis (p = 0.04) were significantly less in miss eye. at 6 weeks, only redness (p = 0.04) and tis (p = 0.05) were significantly less.conclusion:miss is more comfortable in the immediate post - operative period and provides better cosmesis in the intermediate period.
chronic bronchitis is estimated to affect between 3.7% and 6.8% of the population in europe (ball and make 1998), and prevalence increases with age (mcguire 2001). patients with chronic bronchitis are predisposed to recurrent attacks of bronchial inflammation termed acute exacerbations of chronic bronchitis (aecb)characterized by increased cough, worsening dyspnea, and changes in sputum purulence and volume (anthonisen 1987). infectious agents are estimated to account for around 80% of these episodes, with the remaining 20% attributed to noninfectious causes such as inadequate medical treatment, congestive heart failure, pulmonary embolism, etc (sethi 2000). patients with recurrent exacerbations are exposed to frequent courses of antimicrobials with a possible selection of antimicrobial resistance among common bacterial pathogens. antibacterial therapy for aecb is aimed at relieving symptoms, preventing loss of pulmonary function that may lead to hospitalization, speeding recovery, and prolonging the time to the next exacerbation. fluoroquinolones are widely used antibiotics for the treatment of aecb due to their excellent pharmacokinetic / dynamic properties, high antimicrobial activity, and low incidence of side - effects (blasi 2003). we will review the available data on prulifloxacin efficacy and tolerability in the treatment of aecb.the review is based on a pubmed literature search, using as keyword prulifloxacin, for original articles and reviews published in english from january 1990 to april 2006. thirty - five articles were retrieved, 15 articles on urinary tract infections or strictly preclinical studies were discarded, and 20 papers were analysed. prulifloxacin (6-fluoro-1-methyl-7-[4-[(5-methyl-2-oxo-1, 3-dioxol-4-yl)methyl]-1-piperazinyl]-4-oxo-1h, 4h-[1, 3]thiazeto[3, 2-a]quinoline-3-carboxylic acid), the prodrug of ulifloxacin, is a broad - spectrum oral fluoroquinolone antibacterial agent. prulifloxacin is absorbed mainly from the upper small intestine and then metabolized to ulifloxacin in the liver by an -esterase (paraoxonase) (first pass or presystemic metabolism) (tougou 1998). table 1 shows pharmacokinetic characteristics of prulifloxacin (picollo 2003 ; matera 2006).ulifloxacin concentrations in serum and lung have been recently evaluated (concia 2005). in this open label study 27 a single dose of prulifloxacin 600 mg was administered and concentrations evaluated at 2, 4, 6, 12, or 24 hours preoperatively. ulifloxacin concentrations in plasma and lung tissue were determined by reversed - phase high - performance liquid chromatography. the results reported show lung tissue levels higher than plasma levels, however values appear to be widely dispersed. at time 2, 4, 6, 12, and 24-hour the values ranges (lung tissue after correction for blood contamination) the mean lung / plasma concentration ratio was 6.9 0.6 (standard error) reflecting the wide dispersion of concentration values. good intracellular penetration in macrophages and human polimorphonuclear cells has been reported (ozaki 1996). ulifloxacin strengthens the phagocytic and microbicidal activities of the peritoneal macrophages against klebsiella pneumoniae (tullio 2000). when intracellularly concentrated, ulifloxacin can kill the bacteria directly or make them more susceptible to the phagocyte bactericidal effect (tullio 1999) moreover, ulifloxacin seems to modulate human polymorphonuclear (pmn) s in vitro synthesis of proinflammatory cytokines such as interleukin (il)-8, il 1, and tumor necrosis factor- (tnf) (reato 2004). in healthy volunteers co - administration of theophylline and prulifloxacin induces an increase of 15% theophylline area under the curve (auc) and t and a 15% reduction of clearance (fattore 1998). likewise the other fluoroquinolones, cation - containing antacid, and iron preparations reduce absorption of prulifloxacin, these drugs should be administered 3 hours before or 2 hours after prulifloxacin (keam 2004 ; prats 2006). in healthy volunteers co - administration of theophylline and prulifloxacin induces an increase of 15% theophylline area under the curve (auc) and t and a 15% reduction of clearance (fattore 1998). likewise the other fluoroquinolones, cation - containing antacid, and iron preparations reduce absorption of prulifloxacin, these drugs should be administered 3 hours before or 2 hours after prulifloxacin (keam 2004 ; prats 2006). ulifloxacin, the active metabolite of prulifloxacin, shows a wide spectrum of activity against gram - positive and gram - negative bacteria. in this review we will address the activity against the main respiratory pathogen involved in exacerbations of chronic bronchitis. as stated in a recent paper by roveta and colleagues (2005), due to the absence of a defined breakpoint for prulifloxacin / ulifloxacin all the data are referred to ciprofloxacin breakpoint. the activity against streptococcus pneumoniae seems to vary considerably between studies. in three of these studies the ulifloxacin activity against s. pneumoniae resulted in fairly low minimum inhibitory concentration (mic) values ranging from 0.12 to > 4 g / ml, with a mic required to inhibit the growth of 90% of organisms (mic90) value of > 4 g / ml (ozaki 1991 ; yoshida and mitsuhashi 1993 ; prats 2002). in their study, prats and colleagues showed a mic50 of ulifloxacin of 1 g / ml against the penicillin - susceptible strains and 2 g / ml for those presenting moderate and high resistance ; the mic90 was 4 g / ml for the penicillin - sensitive and intermediate strains and 2 g / ml for the highly resistant strains (prats 2002). another study, performed on 36 italian strains, showed a better activity with mic values ranging from 0.015 to 2 g / ml and a mic90 value of 1 g / ml (montanari 2001). ulifloxacin in vitro activity against strains of methicillin - susceptible staphylococcus aureus is fairly good with a reported mic90 value of 50% of the predicted value), no comorbidities and 35%<50% of the predicted value) and/or significant comorbidity (eg, cardiac disease, diabetes, hepatic / renal insufficiency) and/or frequent exacerbations (4/year). patients considered at the highest risk for treatment failure are included in the third group and often demonstrate very severe impairment of lung function (fev1 < 35% of the predicted value) and/or multiple risk factors (including significant comorbidity, chronic corticosteroid therapy) and frequent exacerbations (4/year). this kind of patient stratification is also related to the bacterial flora associated with the exacerbations. thus, in patients with mild to moderate chronic bronchitis, h. influenzae and s. pneumoniae are the most commonly isolated bacteria during aecb, while staphylococcus aureus and gram - negative bacteria, including pseudomonas aeruginosa and enterobacteriaceae species, are predominantly isolated from patients with a severe degree of airflow obstruction (fev1 < 35% of the predicted value) (eller 1998 ; miravitlles 1999). notwithstanding the paucity of published clinical data, the antimicrobial spectrum and the results of grassi and colleagues (2002) study seem to indicate the possible role of prulifloxacin in the treatment of exacerbations of outpatients with moderate to severe copd, which are generally caused by gram - negative bacteria (mainly haemophilus influenzae), enterobacteriaceae, and pseudomonas spp. prulifloxacin is a new fluoroquinolone with indications in the treatment of urinary tract infection and acute exacerbations of chronic bronchitis. the antibacterial spectrum is similar to that of ciprofloxacin with clear advantages in terms of antipseudomonal in vitro activity. only few data on pharmacokinetic and pharmacodynamic characteristics, including bronchial and lung tissues, have been published. the available data indicate a fairly good penetration into the lung tissue with high intracellular concentrations in phagocytes and pmns, with an interesting immunomodulatory activity. the only published aecb treatment study shows a clinical and microbiological activity comparable with ciprofloxacin. more data are clearly required to better evaluate the role of this new fluoroquinolone in the panorama of antibiotics.	exacerbations of chronic bronchitis (aecb) are a major cause of morbidity and mortality in patients with chronic obstructive pulmonary disease (copd), and their impact on public health is increasing. the new fluoroquinolones have an excellent spectrum providing cover for the most important respiratory pathogens, including atypical and typical pathogens. not surprisingly, different guidelines have inserted these agents among the drugs of choice in the empirical therapy of aecb. the pharmacokinetic and dynamic properties of the new fluoroquinolones have a significant impact on their clinical and bacteriological efficacy. they cause a concentration - dependent killing with a sustained post - antibiotic effect. this review discusses the most recent data on the new fluoroquinolone prulifloxacin and critically analyses its activity and safety in the management of aecb.
periodontitis is an inflammatory disease of the periodontium which elicits an immune response resulting in loss of supporting structures of the teeth. it is independent of various ethnic groups and may be present in children to the elderly. the usual modes of treatment for periodontitis include informing the patient about the disease, oral hygiene instructions, scaling and root planing (srp), and if indicated periodontal surgery and in some cases administration of systemic and local chemotherapeutic agents. however, the final success rate of treatment depends on the status and maintenance of oral hygiene. it is well known that mechanical therapy alone provides an excellent clinical response in most patients but in certain patients increasing pocket depth (pd) and complicating anatomic factors limit the effectiveness of srp. many better - known antimicrobials and biomaterials have been used in the past, but in recent times certain less well - known compounds have significantly shown the potential to augment results of periodontal therapy. hyaluronic acid is a high molecular weight polysaccharide (glycosaminoglycan) and plays a vital role in the functioning of the extracellular matrix, including those of mineralized and nonmineralized periodontal tissues. it is a critical component of the extracellular matrix and contributes significantly to tissue hydrodynamics, cell migration, and proliferation. the use of hyaluronic acid in the treatment of inflammatory process has been well established in medical areas to treat radioepithelitis, osteoarthritis of the knee, rheumatoid arthritis, cataract surgery, etc., its wide use in the treatment of inflammatory conditions of the knee and temporomandibular joint has led to its application in the field of periodontics. hyaluronate has shown anti - inflammatory, anti - edematous, and antibacterial effects for the treatment of gingivitis and periodontitis. it has also been studied as a metabolite or diagnostic marker of inflammation in the gingival crevicular fluid as well as a significant factor in growth, development, and repair of tissues. its free radical scavenging and protein exclusion properties offer protection to cells and extracellular matrix. coq10 is a compound found naturally in mitochondria and plays an important role in electron transport chain process. however, its role as an antioxidant has been well established, so it is essential for the health of all tissues and organs. it is also helpful in the regeneration of other antioxidants, stimulation of cell growth, and inhibition of cell death. although coq10 is synthesized in the body, situations may arise in which the body 's synthetic capacity is insufficient to meet requirements for its production. this situation is seen in tissues that are metabolically active such as heart, immune system, gingiva, gastric mucosa, and can lead to dysfunctioning of these tissues. coq10 deficiency is frequently associated with periodontal diseases and its administration to periodontal tissues may control advanced periodontitis. topical administration to the gingiva as a sole treatment may decrease gcf flow and probing depths and improve clinical gingival attachment. the results have been quite controversial according to some studies showing statistically significant improved results while others showing no additional benefit of these molecules. this might be due to a difference in frequency and method of administration of the drugs. an attempt has been made through this study to evaluate the efficacy of administrating hyaluronic acid and coq10 as an adjunct to srp and find out if there is any beneficial role of both the molecules in treating chronic periodontitis. the subjects for this randomized, controlled study were selected from the outpatient department of periodontics, himachal institute of dental sciences college and hospital, paonta sahib (himachal pradesh). patients were selected for the study according to the following criteria : inclusion criteria for the study were patients between age group 25 and 55 years, patients in good systemic health ; chronic generalized periodontitis with 5 mm periodontal pocket, adequate attached gingiva, the diseased sites should bleed on probing, and patients who were able to follow verbal or written oral hygiene instructions. the exclusion criteria included patients having a history of oral prophylaxis in past 6 months, patients taking antibiotics in the past 3 months, patients with a history of systemic disease (cardiovascular diseases, diabetes, blood disorders, hepatitis, and renal diseases), and pregnant and lactating mothers. manchester, usa) [figure 1 ] or ubiquinone is essentially a vitamin or vitamin - like substance. it is supplied as a pack of gel, contains a mixture of coq10 and vegetable glycerine base in a ratio of 1:9. the gel should preferably be used within 48 months from the date of manufacture and stored in a dry area away from sources of light and heat. coq10 is found in small amounts in a wide variety of foods and is synthesized in all tissues. it is known as coenzyme because of its unique ability to participate in chemical reactions but remain at steady state levels in the cell and plays a central role in energy metabolism. test drugs 0.8% hyaluronic acid (gengigel) and coenzyme q10 gel (perio q). hyaluronic acid (gengigel, ricerfarma, milan, italy) [figure 1 ] is a mucopolysaccharide, which is present naturally in all living organisms. its functions include cellular and extracellular interactions, interactions with growth factors and regulation of the osmotic pressure, and tissue lubrication. the patients were divided into three groups ' viz., a, b, and c. if the outcome was numbered 1 or 2, the patient was allotted group a, if the outcome was 3 or 4 then the patient was allotted group b and if the outcome was 5 or 6 then the patient was treated in group c. blinding was also ensured as one investigator administered the drugs during subsequent visits while evaluation of results was done by a separate investigator who was unaware of the intervention given to the test groups. a groove on occlusal stents was made corresponding to the area of highest pd with a tapering low - speed bur. these grooves help in maintaining the same position and angulations during measurement recording. using the groove as a guide, the periodontal probe was inserted into the pocket and clinical measurements were obtained at the baseline and after 5 weeks, i.e., 6 weeks [figures 2 and 3 ]. (a) unc-15 probe measuring probing depth and clinical attachment level from fixed reference point on the acrylic stent at baseline ; (b) insertion of coenzyme q10 (perio q gel) at baseline, 1, 2 week ; and (c) reduction of pocket depth seen at 6 week (a) unc-15 probe measuring probing depth and clinical attachment level from fixed reference point on the acrylic stent, (b) insertion of hyaluronic acid (gengigel), and (c) pocket reduction seen at 6 week group a : treated by srp followed by placement of perio q gel at baseline, 1 and 2 weekgroup b : treated by srp followed by placement of gengigel at baseline, 1 and 2 weekgroup c : treated by srp alone at baseline. group a : treated by srp followed by placement of perio q gel at baseline, 1 and 2 week group b : treated by srp followed by placement of gengigel at baseline, 1 and 2 week group c : treated by srp alone at baseline. plaque index (pi), eastman interdental bleeding index (eibi), gingival color change index (gcci), pd, and clinical attachment level (cal). experimental sites received perio q gel in group a, gengigel in group b, and no drug was placed in group c following srp. the gels were placed in the pockets using a wide gauge needle which was inserted till the base of the pocket. the following parameters were assessed in the study : pi at baseline, 1, 2, and 6 weekeibi at baseline, 1, 2, and 6 weekgcci at baseline, 1, 2, and 6 weekpd at baseline and 6 weekcal at baseline and 6 week. pi at baseline, 1, 2, and 6 week eibi at baseline, 1, 2, and 6 week gcci at baseline, 1, 2, and 6 week pd at baseline and 6 week cal at baseline and 6 week. mean values and standard deviation were calculated for different parameters in group a, b, and c at baseline, 1, 2, and 6 week. wallis test was applied to determine if there was any significant difference between the parameters of the three groups. chi - square test was used to calculate the test of significance for eastman interdental bleeding index as it involved qualitative data, i.e., presence or absence of bleeding from the involved sites. mean values and standard deviation were calculated for different parameters in group a, b, and c at baseline, 1, 2, and 6 week. wallis test was applied to determine if there was any significant difference between the parameters of the three groups. chi - square test was used to calculate the test of significance for eastman interdental bleeding index as it involved qualitative data, i.e., presence or absence of bleeding from the involved sites. of these, forty sites were treated by srp + perio q gel, forty sites with srp + gengigel, and the remaining forty sites by srp alone. the following observations were made for different parameters in the three test groups. at baseline in group a, b, and c the mean pi was 1.35 0.53, 1.43 0.64, and 1.35 0.62, respectively [table 1 and figure 4 ]. at 1 week in group a, b, and c the mean pi was 0.70 0.52, 0.78 0.53, and 0.85 0.48, respectively (p < 0.05) [table 2 and figure 5 ], at 2 week in group a, b, and c the mean pi was 0.82 0.59, 0.82 0.59, and 0.87 0.65, respectively (p < 0.05), [table 3 and figure 6 ] and at 6 week in group a, b, and c the mean pi was 0.72 0.60, 0.92 0.57, and 0.82 0.75(p <.05) [table 4 and figure 7 ], respectively. on intragroup comparison, the results were statistically significant between baseline and 6 week in all the three groups [tables 57 and figures 810 ]. however, on intergroup comparison, no significant difference was noted at different time intervals. clinical parameters at baseline clinical parameters at baseline between group a, b, and c clinical parameters at week 1 clinical parameters at week 1 between groups a, b, and c clinical parameters at week 2 clinical parameters at week 2 between groups a, b, and c clinical parameters at week 6 clinical parameters at week 6 between groups a, b, and c evaluation of change in clinical parameters at different time intervals in group a (coenzyme q10) evaluation of change in clinical parameters at different time intervals in group b (hyaluronic acid) evaluation of change in clinical parameters between different time intervals in group c (control) comparison of change in clinical parameters between different time intervals in group a comparison of change in clinical parameters between different time intervals in group b comparison of change in clinical parameters between different time intervals in group c at baseline, the mean gcci was 1.52 0.69, 1.54 0.67, and 1.67 0.89, respectively [table 1 and figure 4 ]. at 1 week, the mean gcci was 0.20 0.46, 0.22 0.42, and 0.44 0.63, respectively [table 2 and figure 5 ]. at 2 week the mean gcci was 0.09 0.25, 0.37 0.17, 0.22 0.46 respectively [table 3 and figure 6 ], and at 6 week, the mean gcci was 0.15 0.43, 0.15 0.36, and 0.15 0.48, respectively [table 4 and figure 7 ]. on intragroup comparison, there was statistically significant decrease in gcci between baseline and 6 week [tables 57 and figures 810 ]. regardless of this, the results were nonsignificant when intergroup comparisons were made at different time intervals. at baseline, the percentage of eibi in all the three groups was 100% [table 1 and figure 4 ]. this reduced to 22.5% in coq10 group, to 35% in ha group, and to 32.5% in control group at 1 week [table 2 and figure 5 ]. at 2 week in coq10 group, only 17.5% of cases showed positive bleeding, while in ha group 22.5% showed positive bleeding and in control group 35% of cases showed positive bleeding [table 3 and figure 6 ]. at 6 week in coq10 and ha group only 12.5% of patients were positive for bleeding while in control group 20% cases reported positive [table 4 and figure 7 ]. these results were statistically significant when compared to baseline [tables 57 and figures 810 ]. on intergroup comparison, the results showed lesser values in both the test sites as compared to control group ; however, these results were statistically not significant. evaluating the pd at baseline shows the mean pd in group a, b, and c as 5.53 0.59, 5.80 0.79, and 5.60 0.87 [table 1 and figure 4 ]. following 6 week, the values of pd were as 3.20 0.69, 3.52 0.85, and 3.37 1.00 [table 4 and figure 7 ]. all these values were clinically significant from the baseline [tables 57 and figures 810 ]. however, no adjunctive beneficial effects of coq10 and ha was observed at 6 week in comparison to srp group. the relative cal between group a, b, and c at baseline shows mean of cal as 4.53 1.26, 4.83 1.24, and 4.50 1.01 [table 1 and figure 4 ]. the values at 6 week were 2.87 1.09, 3.15 1.37, and 2.75 1.40 [table 4 and figure 7 ]. on intragroup comparison, statistically significant difference was observed in all the groups between baseline and 6 week [tables 57 and figures 810 ]. on intergroup comparison, no statistically significant difference was found in between the three groups. at baseline in group a, b, and c the mean pi was 1.35 0.53, 1.43 0.64, and 1.35 0.62, respectively [table 1 and figure 4 ]. at 1 week in group a, b, and c the mean pi was 0.70 0.52, 0.78 0.53, and 0.85 0.48, respectively (p < 0.05) [table 2 and figure 5 ], at 2 week in group a, b, and c the mean pi was 0.82 0.59, 0.82 0.59, and 0.87 0.65, respectively (p < 0.05), [table 3 and figure 6 ] and at 6 week in group a, b, and c the mean pi was 0.72 0.60, 0.92 0.57, and 0.82 0.75(p <.05) [table 4 and figure 7 ], respectively. on intragroup comparison, the results were statistically significant between baseline and 6 week in all the three groups [tables 57 and figures 810 ]. however, on intergroup comparison, no significant difference was noted at different time intervals. clinical parameters at baseline clinical parameters at baseline between group a, b, and c clinical parameters at week 1 clinical parameters at week 1 between groups a, b, and c clinical parameters at week 2 clinical parameters at week 2 between groups a, b, and c clinical parameters at week 6 clinical parameters at week 6 between groups a, b, and c evaluation of change in clinical parameters at different time intervals in group a (coenzyme q10) evaluation of change in clinical parameters at different time intervals in group b (hyaluronic acid) evaluation of change in clinical parameters between different time intervals in group c (control) comparison of change in clinical parameters between different time intervals in group a comparison of change in clinical parameters between different time intervals in group b comparison of change in clinical parameters between different time intervals in group c at baseline, the mean gcci was 1.52 0.69, 1.54 0.67, and 1.67 0.89, respectively [table 1 and figure 4 ]. at 1 week, the mean gcci was 0.20 0.46, 0.22 0.42, and 0.44 0.63, respectively [table 2 and figure 5 ]. at 2 week the mean gcci was 0.09 0.25, 0.37 0.17, 0.22 0.46 respectively [table 3 and figure 6 ], and at 6 week, the mean gcci was 0.15 0.43, 0.15 0.36, and 0.15 0.48, respectively [table 4 and figure 7 ]. on intragroup comparison, there was statistically significant decrease in gcci between baseline and 6 week [tables 57 and figures 810 ]. regardless of this, the results were nonsignificant when intergroup comparisons were made at different time intervals. at baseline, the percentage of eibi in all the three groups was 100% [table 1 and figure 4 ]. this reduced to 22.5% in coq10 group, to 35% in ha group, and to 32.5% in control group at 1 week [table 2 and figure 5 ]. at 2 week in coq10 group, only 17.5% of cases showed positive bleeding, while in ha group 22.5% showed positive bleeding and in control group 35% of cases showed positive bleeding [table 3 and figure 6 ]. at 6 week in coq10 and ha group only 12.5% of patients were positive for bleeding while in control group 20% cases reported positive [table 4 and figure 7 ]. these results were statistically significant when compared to baseline [tables 57 and figures 810 ]. on intergroup comparison, the results showed lesser values in both the test sites as compared to control group ; however, these results were statistically not significant. evaluating the pd at baseline shows the mean pd in group a, b, and c as 5.53 0.59, 5.80 0.79, and 5.60 0.87 [table 1 and figure 4 ]. following 6 week, the values of pd were as 3.20 0.69, 3.52 0.85, and 3.37 1.00 [table 4 and figure 7 ]. all these values were clinically significant from the baseline [tables 57 and figures 810 ]. however, no adjunctive beneficial effects of coq10 and ha was observed at 6 week in comparison to srp group. the relative cal between group a, b, and c at baseline shows mean of cal as 4.53 1.26, 4.83 1.24, and 4.50 1.01 [table 1 and figure 4 ]. the values at 6 week were 2.87 1.09, 3.15 1.37, and 2.75 1.40 [table 4 and figure 7 ]. on intragroup comparison, statistically significant difference was observed in all the groups between baseline and 6 week [tables 57 and figures 810 ]. on intergroup comparison, no statistically significant difference was found in between the three groups. periodontal diseases are considered infections of the periodontium because of bacterial etiology, an immune response, and tissue destruction. the inflammatory and immune responses to the bacteria and viruses that colonize the periodontal and associated tissues involve the systemic circulation and ultimately the peripheral system of the body. this creates a complex bidirectional series of host - microbial interaction involving cellular and humoral factors and networks of cytokines, chemokines, and growth factors. the majority of periodontal tissue destruction is caused by an inappropriate host response to periopathogens and their product which includes overproduction of free radicals and reactive oxygen species, matrix metalloproteinases during the inflammatory process causing collagen and periodontal cell breakdown. immunomodulators like coq10 has been beneficial in many chronic diseases such as congestive heart failure, diabetes, hypertension, copd, and compromised immune states, whereas hyaluronic acid has been beneficial in diseases such as rheumatoid arthritis, osteoarthritis, and radioepithelitis. the pharmacology of these drugs suggested that they may show positive treatment outcomes in periodontitis. the present study was carried with the objective to carry out a comparative evaluation of coq10-based gel and 0.8% hyaluronic acid gel in the treatment of chronic periodontitis. the results obtained in the present study indicate that both the immunomodulators, i.e., coq10 and hyaluronic acid markedly improve the benefits of srp and are helpful in treating gingivitis. the clinical parameters, i.e., pi, gcci, eibi, pd, and cal showed a statistically significant difference in results between baseline and 6 week in the three test groups. on comparison, it has been observed that coq10 shows superior improvement in gingival parameters as compared to hyaluronic acid and control group though the results were statistically nonsignificant. as it plays crucial role in the generation of atp and cellular respiration which helps in regeneration of cells, it acts as a scavenger for free radicals and reactive oxygen species, regenerates other antioxidants, and inhibits cell death. finally, it has been known to mediate nfkb1-dependent pro - inflammatory cytokine tumor necrosis factor - alpha (tnf-). these all effects contribute to resolve inflammation and promote wound healing. on the other hand, hyaluronic acid being anti - edematous and acts as anti - inflammatory by draining prostaglandins, metalloproteinases, and other bioactive molecules. the significant improvement in the bleeding index can be addressed to mechanical debridement and properties of coq10 gel acting as anti - inflammatory and antioxidant which suppresses periodontal inflammation. the effect of coq10 on the nfkb1-dependent pro - inflammatory cytokine tnf- was studied and was suggested that coq10 exerts anti - inflammatory properties via nfb1-dependent gene expression. aforementioned is a common pathway for periodontal inflammation, suggesting that this could be the possible mechanism for gingival inflammation besides being scavenger for free radicals and reactive oxygen species. hyaluronic acid is known to enhance formation of extracellular connective tissue matrix, promoting wound healing, facilitating cell migration, and differentiation during tissue formation and repair leading to noninflamed and healthy periodontal tissue that is less susceptible to bleeding. more recently, hyaluronic acid has been shown to be the main ligand of cd44 receptor involved in the initial binding of leukocytes to endothelial cells activated by the inflammatory process. this same receptor is involved in the interaction between gingival fibroblasts and t, b lymphocytes and can speed up the gingival immune response. these findings suggest that both the test drugs are capable of treating gingivitis. in patients with periodontitis, no major additional advantage was seen when compared with the control group in coq10 group which was in agreement with an earlier study done by hans. similar findings were observed in hyaluronic acid group and were in accordance with the study done by xu. and gontiya and galgali. in coq10 group, the nonsignificant results may be due unfavorable thixotropic properties of gel, unknown substantivity, and unknown bioavailability of the gel and as it was neither a sustained release nor a controlled release formulation, it may have had a short washout period than that desired for optimal results. in the hyaluronic acid group, intergroup nonsignificant results may be due to no antibacterial effect of ha although it has shown a bacteriostatic effect on aggregatibacter actinomycetemcomitans and porphyromonas gingivalis in vitro, but periodontal environment is much more complex in vivo than an in vitro model can mimic. the other reason could be that the gel was placed with the help of a syringe and it was uncertain that the gel reached the base of the pocket. ha gel is also not yet known to bind and penetrate the periodontal tissues, whereas it has been shown to be bioadhesive and retentive in nasal mucosa and ocular tissue respectively. this difference can be due to differences in treatment protocols, observation intervals, disease severity, and measurements. coq10 and hyaluronic acid when used as an adjunct to srp, both proved to be effective in treating chronic periodontitis. the gingival parameters (eibi) were better in both the test drugs as compared to srp, but the results were nonsignificant. thus, further studies are encouraged to establish the role of the tested immunomodulators in periodontitis.	background : the anti - inflammatory and immune enhancing effects of coenzyme q10 (coq10) and hyaluronic acid are well established in medical literature. the present study was undertaken to evaluate their role in chronic periodontitis.materials and methods : one hundred twenty sites in 24 patients with clinically confirmed periodontitis were included in the study. a split - mouth design was used for intrasulcular application of coq10 as adjunct to scaling and root planing (srp), 0.8% hyaluronic acid as adjunct to srp and srp alone. clinical parameters such as plaque index (pi), gingival color change index (gcci), eastman interdental bleeding index (eibi), pocket depth (pd), and clinical attachment level (cal) were recorded. all the clinical parameters pi, eibi, gcci, pd, and cal were recorded at baseline before srp. only pi, eibi, and gcci were recorded at 1st and 2nd week. twenty - one days post 2nd week, i.e., 6th week all the clinical parameters were recorded again.results:intragroup analysis of all the clinical parameters showed clinical significant results between baseline and 6th week. however, on intergroup analysis, the results were not significant.conclusion:the local application of coq10 and hyaluronic acid gel in conjunction with srp may have a beneficial effect on periodontal health in patients with chronic periodontitis.
the world health organization defines suicide act as the injury with varying degrees of lethal intent and that suicide may be defined as a suicidal act with fatal outcome. deliberate self - harm is a major issue in the health care all over the world. many factors including biological, socio - cultural, and personality traits can modify this complex behavior. suicide is a significant problem in india also with a reported rate of 10.8 per 100,000 population. however it may be considerable under estimate due to underreporting and false reporting of many of the cases of suicides in india. however, some of the important psychosocial variables such as life events or stressors, social support, coping strategies, and quality of life have not yet been assessed in relation to deliberate self - harm in india. life change could act as a stressor causing physiological arousal and enhanced susceptibility for illness. suicide victims have experienced more changes in living conditions, work problems, and object losses than normal controls. a review of indian studies on stressors in suicide shows maladjustment with significant family members and domestic strife as the most important causes, followed by physical factors and mental illness. however, most of the indian studies have not used a proper scale to assess life events and many of them were descriptive and retrospective studies. a body of research in recent years has focused on the role of social support in maintaining emotional well - being and moderating the effects of life events. there is evidence that social network among suicide attempters are weaker than in nonsuicidal individuals. life events can alter the social support system in terms of size, frequency of interaction and stability, and such changes may be associated with suicidal behaviors. coping behavior, or the things people do to reduce the stress, has been a variable that has recently become the focus of research. coping behavior is operationally defined as the responses to external life stress that serve to prevent, avoid, reduce or control stress and emotional distress. reported a negative correlation with minimization, replacement, mapping, and reversal, and a positive correlation with suppression, blame and substitution in suicide victims. quality of life is another factor to assess with regard to suicide risk and a focus recent research in suicidology. it has been increasingly recognized in recent years that people who attempt suicide have certain individual predispositions, part of which is given by personality traits, in particular, impulsive, aggressive, and violent proneness. studies from india and west show identifiable personality disorder in suicide attempters. considering the paucity of such work from the indian context the present study was conducted to analyze and compare the type and severity of life events, coping strategies and social support, and quality of life of suicide attempters and matched normal controls and to identify the risk factors leading to suicide attempt. the sample comprised 50 suicide attempters qualifying the criteria for suicide attempt as defined by who admitted to different departments of a general hospital. patients below the age of 18 years and those whose physical condition did not allow detailed evaluation were excluded from study. wherever possible, relatives, friends, and other possible sources of information such as spouse and colleagues were also interviewed for eliciting further information. age, sex, and marital status - matched healthy controls from the community formed the comparison group. these subjects were initially screened by ghq-12 version to exclude the presence of common mental disorders. those who scored (cut - off score 2/3 mode) were excluded from the control group. a specially designed proforma was used for documenting socio - demographic variables, illness variables, and details of the current suicide attempt. presumptive stressful life events scale the scale consists of 51 life events commonly experienced by the normal indian adult population. scale items were further classified into (a) desirable, undesirable or ambiguous and (b) personal or impersonal (not dependent on the individual action). life event data collected from each patient were compared with life event data about the patient given by his relative and was found to be satisfactory (0.8). social support questionnaire the scale was specially developed by poling items from social support scale of asha and the social support scale of nehra, kulhara, and verma by item analysis. the positive statements were intermingled with negative statements to reduce the likelihood of response set occurring. aecom coping style scale this is a 95-item scale with a four - possibility spectrum ranging from never to very often. the scale measures eight basic coping styles that may be used for reducing stress and coping with life problems. these coping styles are (1) suppression, (2) help seeking, (3) replacement, (4) blame, (5) substitution, (6) mapping, (7) reversal, and (8) minimization. the internal validity of the scale was found to have an value of between 0.58 and 0.79 with a mean value of 0.70. the scale has been shown to have good discriminant validity, sound content validity, and good test -- retest reliability at several international whoqol centers. for comparison of quantitative variables we used a paired t - test or wilcoxon signed rank test applied depending on whether the data were normally distributed or not. the sample comprised 50 suicide attempters qualifying the criteria for suicide attempt as defined by who admitted to different departments of a general hospital. patients below the age of 18 years and those whose physical condition did not allow detailed evaluation were excluded from study. wherever possible, relatives, friends, and other possible sources of information such as spouse and colleagues were also interviewed for eliciting further information. age, sex, and marital status - matched healthy controls from the community formed the comparison group. these subjects were initially screened by ghq-12 version to exclude the presence of common mental disorders. those who scored (cut - off score 2/3 mode) were excluded from the control group. a specially designed proforma was used for documenting socio - demographic variables, illness variables, and details of the current suicide attempt. presumptive stressful life events scale the scale consists of 51 life events commonly experienced by the normal indian adult population. scale items were further classified into (a) desirable, undesirable or ambiguous and (b) personal or impersonal (not dependent on the individual action). life event data collected from each patient were compared with life event data about the patient given by his relative and was found to be satisfactory (0.8). social support questionnaire the scale was specially developed by poling items from social support scale of asha and the social support scale of nehra, kulhara, and verma by item analysis. the positive statements were intermingled with negative statements to reduce the likelihood of response set occurring. aecom coping style scale this is a 95-item scale with a four - possibility spectrum ranging from never to very often. the scale measures eight basic coping styles that may be used for reducing stress and coping with life problems. these coping styles are (1) suppression, (2) help seeking, (3) replacement, (4) blame, (5) substitution, (6) mapping, (7) reversal, and (8) minimization. the internal validity of the scale was found to have an value of between 0.58 and 0.79 with a mean value of 0.70. the scale has been shown to have good discriminant validity, sound content validity, and good test -- retest reliability at several international whoqol centers. a specially designed proforma was used for documenting socio - demographic variables, illness variables, and details of the current suicide attempt. presumptive stressful life events scale the scale consists of 51 life events commonly experienced by the normal indian adult population. scale items were further classified into (a) desirable, undesirable or ambiguous and (b) personal or impersonal (not dependent on the individual action). life event data collected from each patient were compared with life event data about the patient given by his relative and was found to be satisfactory (0.8). social support questionnaire the scale was specially developed by poling items from social support scale of asha and the social support scale of nehra, kulhara, and verma by item analysis. the positive statements were intermingled with negative statements to reduce the likelihood of response set occurring. this scale has approximately the same number of items from each area. the retest reliability obtained for this scale was 0.89. aecom coping style scale this is a 95-item scale with a four - possibility spectrum ranging from never to very often. the scale measures eight basic coping styles that may be used for reducing stress and coping with life problems. these coping styles are (1) suppression, (2) help seeking, (3) replacement, (4) blame, (5) substitution, (6) mapping, (7) reversal, and (8) minimization. the internal validity of the scale was found to have an value of between 0.58 and 0.79 with a mean value of 0.70. the questionnaire had both predictive validity and discriminative validity. who qol - bref contains 26 items with four domains 1. the scale has been shown to have good discriminant validity, sound content validity, and good test -- retest reliability at several international whoqol centers. for comparison of quantitative variables we used a paired t - test or wilcoxon signed rank test applied depending on whether the data were normally distributed or not. the sample comprised 50 suicide attempters and 50 controls matched on age, sex, and marital status. the mean age of attempters versus control was 30.8213.56 vs. 31.5413.1 (p=0.787) and the male female ratio was male attempters 22 (44%) vs. male control 22 (44%) and female attempters 28 (56%) vs. female control 28 (56%) (p=1.0). in both groups sample characteristics comparison of mean scores of different types of life events in attempters versus controls showed significantly higher total life events, and undesirable and personal life events in attempters [table 2 ]. comparison of different types of life events comparison of social support variables between attempters and controls showed that the confiding relationship was significantly less (35 (70%) vs. 49 (98%), pearson chi - square p=0.000) often present and loneliness was significantly more frequent (14 (28%) vs. 3 (6%), pearson chi - square p = 0.003) in attempters. comparison of various items from the social support scale showed significantly lower scores in attempters, except for religion [table 3 ]. comparison of variables in social support scale comparison of different types of coping behavior between attempters and controls showed that scores for minimization, replacement, and mapping were significantly higher in controls [table 4 ]. comparison of coping pattern between attempters and controls the mean scores of all the four domains of qol (physical health and well - being, psychological health and well - being, social relations and environment) were significantly lower in the attempters [table 5 ]. comparison of qol between attempters and controls all factors which were significant in one to one comparison were entered into a stepwise conditioned regression analysis. the final result showed that the lifetime score of desirable life events, longer education, and good social support were protective factors against suicide [table 6 ]. the present study attempted to differentiate suicide attempters from healthy controls based on their profile of life events, social support, coping strategies, psychiatric diagnosis, and quality of life. attempters had accumulation of life events especially unpleasant and personal events, lower social support, poor coping styles, and poor quality of life. life events and other psychosocial stressors are commonly associated with suicidal behavior when attempters were compared to the general population and nonsuicidal psychiatric patients osvath. reported recent life events in 80% of suicides ; job problems (28%), family discord (23%), somatic illness (22%), financial problems (18%), unemployment (16%), separation (14%), death (13%), and illness in a family member. in the present study psychosocial stressors like financial loss (34% vs. 14%), family conflict (30% vs 6%), marital conflict (18% vs 05), broken engagement, and love failure (12% vs. 2%) and major personal illness (10% vs 2%) were significantly higher in attempters than controls. hagnell and rorsman found more objective losses and humiliating experience in the week before death among suicide victims than people dying from natural causes and more changes in living condition, work problems, and objects losses in the final year. maladjustment with significant family members and domestic strife has been cited as the most important causes of attempted suicide in many indian studies the present study also figures out interpersonal problems such as the common life events experienced by attempters. coping skills are important protective factors against suicide. in the present study healthy coping behaviors such as minimization (ability to de - emphasize the burden of stressful events), replacement (ability to overcome stressful events by engaging in alternative behaviors), and mapping (ability to collect information for planning and to seek out alternative solutions to problems) were higher in controls. reported negative correlation of healthy coping mechanisms such as mapping, minimization, and replacement and positive correlation of coping styles of suppression (avoiding the problem or situation) with suicide risk. some other coping behaviors such as reversal, substitution and help seeking, which have been reported to be excessive in suicide attempters, were not found in this study. excessive use of substitution in attempters is harmful as it may predispose the individual to suicidal behavior reflecting the destructive nature inherent in excessive dependence on the environment. social support is provided by networks comprising family, relatives, friends, neighbors, and coworkers, especially when the interaction is positive. the personal networks may provide social support that helps to maintain emotional well - being and buffer the effect of adverse life events, or it can have a direct, independent effect on mental health irrespective of presence or absence of stressful life events. in the present study, confiding relationship, support from reliable attachment, friends, teachers, parental figures, elders, and other sources were significantly lower and loneliness was higher in attempters. there is evidence from comparative studies that social support systems are undermined among suicide attempters compared with nonsuicidal individuals. a psychological autopsy study by vijayakumar and rajkumar from india also showed low religiosity in suicide victims. social and family factors, negative life events, and medical illness may interact with psychiatric and personality disorders, genetic variables, biological factors, and psychosocial stressors and ultimately act as predisposing and precipitating or contributing factors to suicidal behavior. morano and cisler reported an influence of recent loss on serious suicide attempts, especially when paired with a perceived lack of family support and hopelessness, which provides evidence for a stress vulnerability model of suicide behavior. since this is relatively a new area, only few studies have looked into this aspect in suicide attempters. the score on all the four domains namely physical health and well - being, psychological health and well - being, social relations and environment were significantly lower in attempters in this study. the association was somewhat stronger in the first decade than in the second decade. throughout the entire follow - up, subjects who reported dissatisfaction at baseline and again 6 years later showed a high risk of suicide compared to those who repeatedly reported dissatisfaction. therefore it is beyond dispute that in a significant number of attempted suicides there are only minor psychiatric problems in the background but ratios mentioned in the literature seem to be exaggerated. the relationship between suicidal behavior and psychiatric diagnosis has always been a matter of debate pertaining to the indian context with low rate of psychiatric morbidity. western literature reports that about 90% of all those who attempt suicide suffer from a psychiatric disorder. in a series of studies from the indian context, the predominant psychiatric problem was adjustment disorder closely followed by major depression and alcohol abuse / dependence. moreover several of these attempts were of impulsive type and for 10% of the sample the diagnosis was emotionally unstable personality disorder. in a study from india on suicide attempters, han. identified personality disorder in 45.9% of his patients who attempted suicide. in the present study there were quite a few number of alcoholic / drug abusers who attempted suicide (14%). a chronic alcoholic in the course of his illness is more likely to face variety of stressors, interpersonal difficulties of weakening of social support all of which could push the person to suicide. it needs to be mentioned that social drinking is not a way of life in india. pondichery (which has a high rate of alcohol consumption) also has the highest suicide rate in (58%) in india. wasserman found that the suicide rate came down by 34% in 1984 - 1988 following strict restriction in the sale of alcohol in former ussr. hence there is an urgent need to address this issue at the societal and individual level. policies and programs should be initiated for reducing the alcohol availability and consumption and at the individual level there should be better availability and follow - up strategies for the treatment of alcoholics and their families. stepwise regression analysis shows that desirable life events, good education, and good social support are protective factors against suicide. desirable life events by virtue of its positive nature may prevent the individual from attempting suicide. good educational achievement may also help the individual to appraise the situation and to seek alternate solutions. adequate education is also a prerequisite for problem solving skills and to deal adequately with stressful situations. though lower education has not been directly cited as a risk factor, lower socio - economic status has been repeatedly shown as risk factor for suicide. moreover lower education may also invite more adverse life events because of related consequences such as unemployment, poverty, lower social economic status, etc. lower education and subsequent poor social status can also indirectly reduce the social support vulnerable individuals. good social support has always been cited as a protective factor against suicide. in an integrative path model analysis of the relationship between several variables and suicidal ideations found a significant relationship between social support and suicidal ideation. another one is the selection of a biased control group which was purposefully done to match the psycho - socio - demographic characteristics with the study group in order to reduce the confounding variables as much as possible. it seems that the quality of individual life events experienced by attempters and controls is unique. however one to comparison of these events requires higher frequency of events, which can be fulfilled with only larger sample size. other variables pertaining to suicidal behavior such as personality profile, proneness to violent behavior, and impulsivity should also be considered to differentiate suicidal individuals from controls. in the context of the present study, probably studies with long - term follow up would throw more light on suicidal tendency in individuals with lower social support, poor coping skills, poor qol, and excessive life stressors. an interventional study design may provide more information on the role of enhancing social support, improving coping styles and qol, and exposure to better life experiences in reducing the suicidal tendency. moreover, only qualitative individual case studies can provide in - depth exploration of multitude of factors operating in this complex behavioral problem. another one is the selection of a biased control group which was purposefully done to match the psycho - socio - demographic characteristics with the study group in order to reduce the confounding variables as much as possible. it seems that the quality of individual life events experienced by attempters and controls is unique. however one to comparison of these events requires higher frequency of events, which can be fulfilled with only larger sample size. other variables pertaining to suicidal behavior such as personality profile, proneness to violent behavior, and impulsivity should also be considered to differentiate suicidal individuals from controls. in the context of the present study, the following few suggestions seem to be relevant in planning for future research. probably studies with long - term follow up would throw more light on suicidal tendency in individuals with lower social support, poor coping skills, poor qol, and excessive life stressors. an interventional study design may provide more information on the role of enhancing social support, improving coping styles and qol, and exposure to better life experiences in reducing the suicidal tendency. moreover, only qualitative individual case studies can provide in - depth exploration of multitude of factors operating in this complex behavioral problem. this study concludes that suicide attempters experienced significantly more life events especially untoward events whereas the control group experienced more desirable and impersonal life events. social support, positive coping behaviors, and qol desirable life events, good education, and good social support were found to be protective against suicide. it is the complex interplay of various interrelated factors and the resultant buffering effect, which is protecting the individual against suicide. the present finding suggests that enhancing the social support, training individuals to adapt good coping skills, exposing the individuals to positive life experiences, promotion of good physical and psychological health and healthy environment are the most effective preventive strategies against individuals attempting suicide.	background : though deliberate self - harm encompasses a wide variety of medical and social disciplines some of the important psychosocial variable such as life events, social support, coping strategies, and quality of life have not yet been explored in depth in india.aims:the aim was to analyze and compare the type and severity of life events, coping strategies, social support, and quality of life of suicide attempters versus matched normal controls, and to identify the risk factors leading to suicide.materials and methods : a total of 50 consecutive suicide attempters were compared with same number of age, sex, and martial status matched healthy controls using presumptive stressful life events scale, social support questionnaire, aecom coping style scale, and who qol-bref.results:attempters experienced significantly more life events especially untoward events whereas the control group experienced more desirable and impersonal life events. social support, positive coping, and of qol were significantly lower in attempters. among all risk factors desirable life events, good education, and good social support were protective against suicide.conclusion:suicide attempters were differentiated from healthy controls based on more stressful life events, lower social support, less healthy coping, and poor qol. positive life events, good education, and good social support were protective factors against suicide. however, it is difficult to pinpoint a single factor responsible for suicidal behavior. it is the complex interplay of various interrelated factors and the resultant buffering effect, which is protecting the individual against deliberate self - harm.
if untreated, it can lead to pain and discomfort and finally loss of teeth. the disease is not self - limiting and without adequate intervention, the process can continue until the tooth is destroyed. the term caries denotes both the disease process and its consequences, that is, the damage caused by the disease process. the world health organization 's report on oral health in 2003 and global oral data bank of who confirm the international distribution of dental caries and stated that by the age of 12 only 15 to 30% of the population were caries - free with a global dmft of 1.74 [24 ]. the global distribution of dental caries presents a varied picture ; countries with low caries prevalence are experiencing an unprecedented increase in caries prevalence and severity of dental caries. on the other hand, in developed countries a reduction of dental caries incidence and improvement of gingival health care this decline in dental caries was mainly due to appropriate use of fluorides and preventive oral health care measures. dental caries has a multifactorial aetiology in which there is interplay of three principal factors : the host (saliva and teeth), the microflora (plaque), and the substrate (diet) and a fourth factor : time. there is no single test that takes into consideration all these factors and can accurately predict an individual 's susceptibility to caries. the risk of dental caries can be evaluated by analysing and integrating several causative factors such as fluoride, microbial plaque, diet, bacterial and salivary activity, and social and life style related behavioural factors. excessive body weight in children is a major public health problem. according to national family health survey (nfhs), obesity has reached epidemic proportions in india, affecting 5% of the country 's population. india is following a trend of other developing countries that are steadily becoming more obese [6, 7 ]. obesity status in children is measured by assessment of body mass index (bmi) corresponding to gender and age. consumption of soft drinks and fast foods together with less activity and exercise contributed to the increasing number of overweight people worldwide. high sugar intake, for example, sugar containing snacks and soft drinks, is reported to be more common among overweight and obese children / adolescents than those with normal weight. thus, the eating pattern among overweight or obese children may be a common risk factor in overweight children and dental caries. given that the strong evidence supporting the relation between dental caries with indiscriminate dietary intake has been linked to the development of obesity at a young age, a link between dental caries and weight is biologically possible. the role of sugar (and other fermentable carbohydrates such as highly refined flour) as a risk factor in the initiation and progression of dental caries is overwhelming. sugar acts as a favoured substrate for the cariogenic bacteria that reside in dental plaque, particularly the mutans streptococci, and the acid by - products of this metabolic process induce demineralization of the enamel surface. whether this initial demineralization proceeds to clinically detectable caries or whether the lesion is remineralized by plaque minerals depends on a number of factors, of which the amount and frequency of further sugars consumption are of utmost importance. another risk factor for development of caries is the existence of bacterial plaque on the teeth. caries can be reduced by mechanical removal of plaque from tooth surfaces ; however, most children do not remove it effectively which means the deficiency of maintenance of good oral hygiene. several studies have shown that, in countries where proper oral hygiene is followed, caries prevalence has decreased despite increases in sugar consumption, thus marking the importance of oral hygiene in caries etiology [11, 12 ]. dental caries is a multifactorial disorder and it is difficult to assess all the associated risk factors simultaneously. there have been no studies documented in literature in this part of india assessing the prevalence of dental caries in relation to body mass index, daily sugar intake, and oral hygiene. so an attempt was made to assess the prevalence of dental caries in relation to body mass index, daily sugar intake, and oral hygiene status in 12-year - old school children in mathura city. cross - sectional study was designed to evaluate the daily sugar intake, oral hygiene status, and body mass index and to correlate each of them with the prevalence of dental caries among 12-year - old school children in mathura city. sample size was determined by the formula based on the study population : (1)n=4pqne2(n1)+4 pq, where p = prevalence 27% (prevalence of dental caries obtained from previous study), q = (1 p) = 100 27 = 73, e = permissible error in estimation of prevalence 10%, n = study population 5000 (department of education mathura), and n = sample size. the estimated sample size for the study based on the prevalence of the dental caries came out to be 860.10% of sample size ; that is, 86 was included in pilot study, but to avoid any error slightly higher sample of 100 was taken for study. this study was planned to be conducted in high schools of mathura city in 12-year - old children. there are 141 primary schools and 37 high schools as per the record in the district education departments of mathura. out of 37 high schools, children do not have any specific habit such as tobacco chewing and smoking, but they were very fond of having sugar candies. level of fluoride ion concentration in drinking water in mathura city is in optimum range and subjects maintained their oral hygiene by using fluoridated toothpaste. out of 16 government aided high schools, 2 schools were randomly selected to obtain the sample size of 100 study subjects having similar socioeconomic and cultural background. subjects who were willing to participate, have completed 12 years of age, and were continuously residing in mathura city right from their birth were included in the study whereas subjects who were suffering from any acute or chronic diseases and were under medication, were below 12 years of age and above 13 years of age, and did not obtain parental consent were excluded from the study. body weight of study subjects was measured using standardized digital weighing machine. the fractional weight below 500 grams and above 500 grams was rounded to the nearest whole number. measurement of weight and height was taken without shoes and with their school dress. from the above data, bmi was calculated and plotted on cdc - bmi for age growth charts / curves for boys and girls to obtain a percentile ranking and subjects healthy weight : 5th percentile to less than 85th percentile. at risk of overweight : 85th to less than the 95th percentile. healthy weight : 5th percentile to less than 85th percentile. at risk of overweight : 85th to less than the 95th percentile. dental caries status was collected using dentition status of who criteria mentioned in basic oral health survey methodology (1997) and from the above data dmft / dmft was calculated. oral hygiene of study subjects was determined using oral hygiene index - simplified (ohi - s) by greene and vermilion. this index is based upon two parameters : debris and calculus and it has been validated by other authors in 12-year - old children of different geographic region. data regarding the daily sugar intake was recorded using 24-hour recall diet frequency chart and the subjects were grouped into excellent, good, and watch out zone based upon sugar sweet score (see table 1). all examinations and data collection were done by a single examiner and proforma was filled by a recording assistant after standardization. the examination of study subjects was carried out in their school premises using natural light, ordinary chair, plain mouth mirror and cpi probe for dental caries, and explorer no. 5 (shepard 's hook) for ohi - s. presterilized armamentarium was used to carry out the examinations. the data obtained was analysed using spss version 11.5 for windows. mean and standard deviations were calculated for each clinical parameter. differences between means were tested with one - way anova followed by post hoc tukey 's test. independent effects of bmi, oral hygiene status, and daily sugar intake on caries prevalence were tested using linear multiple regression analysis. significance for all statistical tests was predetermined at a probability (p) value of 0.05 or less. an epidemiological survey conducted showed that the study population consisted of 100 school children, out of which 50 (50%) were males and 50 (50%) were females. table 2 shows sex - wise distribution of various characteristics of study population collected by survey. one - way anova was applied to determine the association between mean dmft and bmi categories of study population (underweight, health weight, at risk of overweight and overweight) but no significant association was found (f = 1.145, p = 0.335, n.s.) but when it was applied to determine the association between mean dmft and bmi categories of study population (underweight, health weight, at risk of overweight and overweight), significant association was found (f = 7.783, p = 0.000, s). similarly, one - way anova was applied to determine the association between mean dmft / dmft and daily sugar intake categories of study population (excellent, good, and watch out) ; no significant associations were found (f = 1.348, p = 0.265, n.s., and f = 0.489, p = 0.615, n.s.), respectively. when one - way anova was applied to determine the association between mean dmft / dmft and oral hygiene status of study population (good, fair, and poor), no significant associations were found (f = 2.563, p = 0.082, n.s., and f = 1.051, p = 0.354, n.s.). multiple linear regression analysis was done to determine the independent effects of bmi, oral hygiene status, and daily sugar intake on caries prevalence. it was found that oral hygiene status had a significant effect on caries prevalence (or = 5.061, p = 0.004, s). however, body mass index and daily sugar intake had no significant effect on caries prevalence (table 3). the main objective of the present study was to determine the prevalence of dental caries in relation to body mass index, daily sugar intake, and oral hygiene status of 12-year - old school children of the mathura city. our study found a low caries prevalence (27%) with a mean dmft of 0.37 and mean dmft of 0.12, respectively, when compared with the global dmft for 12-year - olds. (2005) who reported 27% prevalence of dental caries with a mean dmft of 0.5. our study found no statistically significant association between dmft and bmi (f = 1.145, p = 0.335, n.s.). similarly, tramini. found no significant association between dmft and bmi. this finding is consistent with the results from the prospective study by pinto., where no correlation between dental decay and bmi was detected in a multiple regression analysis. even found an inverse association between bmi and caries experience : overweight children were less likely to have caries experience than normal weight children aged 611 years. after having performed a systematic review of obesity and dental caries, kantovitz. concluded that only one study with high level of evidence showed direct association between obesity and dental caries. showed that, overall, there was a significant relationship between childhood obesity and dental caries. however, this relationship was not significant for newly industrialized countries similar to present study conducted in mathura, india. this might be attributed to the fact that both obesity and dental caries are multifactorial in aetiology and various genetic and environmental factors have an impact on them. ludwig., in a longitudinal study, found that the increasing prevalence of obesity in children was linked to the consumption of sugar - sweetened drinks. however, our study found no significant association between dental caries (dmft / dmft) and daily sugar intake. even with increased consumption or high intake of sugar there was decrease in dental caries. this might be attributed to the widespread exposure to fluoride not only through drinking water but also through toothpaste, professional applications, and through fluoride 's presence in processed foods and drinks. this result is consistent with the findings of systematic review by burt and pai which concluded that the relationship between sugar consumption is much weaker in modern age of fluoride exposure. another study by loveren concluded that if good oral hygiene is maintained and fluoride is supplied frequently, teeth will remain intact even if the carbohydrate - containing food is frequently eaten. local oral factors such as retention around the teeth and salivary functions may be factors strongly modifying caries activity. poor oral hygiene leads to accumulation of dental plaque which has an important role in the aetiology of dental caries. the overall oral hygiene status among study population was recorded as fair in 65% and good in 31% and only 4% of the study population showed poor oral hygiene status. there was significant difference between oral hygiene status of males and females (p = 0.037). the ohi - s and its components showed a high mean value for males as compared to females. the probable reason for lower mean scores of ohi - s and its components in females was perhaps the increased grooming habits of girls in this age group. even though oral hygiene status of majority of the study population was between fair and good, 27% of the study subjects were affected by caries in the present study but no statistically significant difference was seen between dmft / dmft and oral hygiene status (dmft, p = 0.082 ; dmft, p = 0.354). however, multiple linear regression analysis found that oral hygiene status had a significant effect on caries prevalence (or = 5.061, p = 0.004, s). oral hygiene status had an intricate relationship with caries prevalence whereas body mass index and daily sugar intake did not reveal any significant association in 12-year - old school children of mathura city. the relationship between dental caries and obesity should be further explored by longitudinal studies as they both have common risk determinants.	aim. to correlate the prevalence of dental caries to body mass index, daily sugar intake, and oral hygiene status of 12-year - old school children of mathura city. material and methods. the study design was cross - sectional and included 100 school children aged 12 years (n = 50 boys and n = 50 girls) who were randomly selected from two schools based upon inclusion and exclusion criteria. body weight / height was recorded and bmi was calculated and plotted on cdc - bmi for age growth charts / curves for boys and girls to obtain percentile ranking. dental caries was recorded using who criteria. oral hygiene status of the study subjects was assessed using oral hygiene index - simplified. data regarding the daily sugar intake was recorded using 24-hour recall diet frequency chart. the data obtained was analysed using spss version 11.5 for windows. result. only 27 subjects were affected by caries. the mean dmft / dmft was 0.37 0.79 and 0.12 0.60, respectively. statistical analysis by means of a logistic regression model revealed that only oral hygiene status had a significant effect on caries prevalence (or = 5.061, p = 0.004), whereas daily sugar intake and body mass index had no significant effect. conclusion. from the analysis, it was concluded that oral hygiene status had a significant effect on caries prevalence of 12-year - old school children of mathura city.
endoscopic submucosal dissection (esd) was introduced for treating early gastric cancer with a minimal risk of regional lymph node and distant metastases 1. esd is currently performed worldwide because neoplasms can be resected en bloc, and a detailed pathological assessment of resected specimens can be performed 1 2. however, positive horizontal margins in the resected specimens are sometimes encountered after esd 1 2 3 4 5 6 7 8 9. nevertheless, appropriate treatment strategies for differentiated - type gastric cancers with positive horizontal or indeterminable margins after esd have not yet been established (fig. 1) 10. the aim of this study was to estimate the probability of positive horizontal or indeterminable margins after esd and evaluate the current empirical treatments for the patients with positive horizontal or indeterminable margins after esd. we performed a multicenter survey of the treatment strategies for early gastric cancer with positive horizontal or indeterminable margins after esd. data from 14 hospitals (cancer institute hospital, toranomon hospital, kitasato university east hospital, ntt medical center tokyo, st. luke s international hospital, tokai university school of medicine, kudanzaka hospital, koritsu showa hospital, tokyo metropolitan bokuto hospital, juntendo university school of medicine, tokyo women s medical university yachiyo medical center, keio university school of medicine, foundation of detection of early gastric carcinoma, and sanraku hospital) that participated in the 30th endoscopic gastric mucosal resection (egmr) conference were collected. questionnaires of the strategies used for early gastric cancer with positive horizontal or indeterminable margins after endoscopic submucosal dissection (esd). egmr conference is a well - known research conference focusing on endoscopic therapy for early gastric cancer, which has been held biannually since 2000 in tokyo, japan and many endoscopists from more than 20 medical centers usually attend the conference. these institutions covered medical centers where the majority of endoscopists with expertise in esd were working in tokyo. it was expected that these doctors behaviors would reflect current empirical therapeutic strategies after esd in japan. replies to our questionnaires and approval by the institutional review board (irb) for the study were obtained from 14 institutions. the proportion of patients with positive horizontal or indeterminable margins and the proportion of patients who were followed up without early interventions were calculated for each institution. two - sided or 1-sided 95 % confidence intervals (cis) of those proportions were also calculated. meta - analyses for proportions were performed to calculate pooled estimates of the above mentioned 2 proportions. the pooled proportions of positive horizontal or indeterminable margins and those of the patients who were followed up without early intervention were calculated by a logistic - normal random - effects model 11. for calculating pooled estimates, subgroup analyses of high- and non - high - volume centers a high - volume center was defined as an institution with more than 100 esd cases per year. all analyses were performed by stata version 14.1 (statacorp, college station, tx, usa). a total of 11,796 differentiated - type gastric adenocarcinomas that met the absolute or expanded indication for esd in the japanese gastric cancer treatment guidelines 2010 (tumors clinically diagnosed as t1a and either no ulcer findings regardless of size or positive ulcer findings in tumors sized 3 cm in diameter) 10 were treated with esd from september 2002 to may 2014. positive horizontal or indeterminable margins were observed in 235 resected specimens pathologically (2.0 %). six cases in which the description in the questionnaire was incomplete were excluded from the study, and 229 cases were enrolled. the treatment strategies were evaluated in 229 cases with positive horizontal or indeterminable margins using obtained questionnaires. the number of total esd cases and that of early gastric cancer with horizontal or indeterminable margins after esd in each institution are presented in table s1. esd, endoscopic submucosal dissection ; hm1, positive horizontal or indeterminable margins q2. the number of the cases in which the tumors were diagnosed as t1a and either no ulcer findings regardless of size or positive ulcer findings in tumors sized 3 cm in diameter, and positive horizontal or indeterminable margins were demonstrated in the esd - specimens. q3. strategies for q2 cases early repeat esd (re - esd) (esd within 30 days after initial esd) early coagulation (coagulation within 30 days after initial esd) early gastrectomy (additional gastrectomy without follow - up) follow - up without early treatments pooled estimates of positive or indeterminable margins in 14 institutions are demonstrated in fig. 2. the proportion of patients with horizontal or indeterminable margins ranged from 0.6 % to 11 %. the pooled estimate of positive horizontal or indeterminable margins was 2 % (95 % ci : 1 % 3 %). the pooled estimates of positive margins in high- and non - high - volume centers were 1 % (95 % ci : 1 % 2 %) and 2 % (95 % ci : 1 % 4 %), respectively. pooled estimates of these subgroups were marginally heterogeneous (p = 0.191). pooled estimates of positive or indeterminable margins in high and non - high volume centers. the treatment strategies used for early gastric cancer with positive horizontal or indeterminable margins after esd are presented in table s1 and fig. a total of 98 patients with early gastric cancer were treated within 30 days of esd treatment initiation : repeat esd (re - esd), n = 14 (14 %), 4 institutions ; coagulation, n = 55 (56 %), 6 institutions ; and surgical resection, n = 29 (30 %), 7 institutions. early re - esd was completed safely in all 14 cases without any complications such as perforation or post - treatment bleeding. residual cancer was demonstrated in the re - esd specimens in 6 cases (43 %). recurrence after coagulation was observed in 5 cases (9 %) in which coagulation was performed additionally. local residual cancers were observed in 13 of 29 surgically resected specimens (45 %). treatment strategy for early gastric cancer with positive horizontal or indeterminable margins after endoscopic submucosal dissection (esd). pooled estimates of follow - up rate without early intervention in 14 institutions the proportion of patients who were followed up without early intervention in those with horizontal or indeterminable margins ranged from 30 % to 100 %. the pooled estimate was 68 % (95 % ci : 50 % 83 %). the pooled estimates of high- and non - high - volume centers were 65 % (95 % ci : 38 % 85 %) and 72 % (95 % ci : 44 % 89 %), respectively. pooled estimates of these subgroups were not heterogeneous (p = 0.692). pooled estimates of follow - up rate without early intervention in high and non - high volume centers. results of follow - up cases without early intervention are demonstrated in table s2. in total, 131 cases were followed up without additional early treatments after initial esd ; 27 cases (21 %) recurred locally during the median follow - up period of 6 months (range, 1 the strategies used for the 27 recurrent cases more than 30 days after initial esd were as follows (table s3) : re - esd, n = 13, 6 institutions ; coagulation, n = 4, 3 institutions ; surgical resection, n = 7, 5 institutions ; and further observation or death due to other diseases, n = 3, 2 institutions. late coagulation was performed safely in all 4 cases without any complications such as perforation or post - treatment bleeding. however, recurrence occurred in 3 coagulation cases (75 %), in which additional coagulation was performed. lymph node or distant metastases were not found in the resected specimens from the seven surgically - treated patients. additional treatments were not performed and observation was done in three locally recurrent cases due to the patient s decision and/or comorbid cardiopulmonary diseases. esd, endoscopic submucosal dissection results of late re - esd for recurrent cases are demonstrated in table 1. the median size of the residual cancers and that of the resected specimens were 12 mm (range, 6 41 mm) and 40 mm (range, 20 59 mm), respectively. the median procedural time was 133 min (range, 70 353 min). the rate of en bloc resection was 92 % (12/13) in the late re - esd group. recurrence after late re - esd occurred in the piecemeal resected case, in which a third esd was performed and there was no recurrence during the follow - up period of 13 months after the third esd. needle - type knives (dual knife / flush knife / flex knife), hook knives, and insulated tip (it)-type knives (it knife / it knife 2) were preferred, and scissor - type grasping knives (sb knife / clutch cutter) were not selected during re - esd procedure. this is the first report of a multicenter survey of treatment strategies for early gastric cancer with positive horizontal or indeterminable margins after esd. the pooled estimates of positive margins in high - volume centers (1 %) were less than those in non - high - volume centers (2 %) in our study. although the heterogeneous test between above 2 groups was not statistically significant (p = 0.191), this test lacks power. kakushima reported that there were 3 types of lesions that resulted in positive margins after resection (lesions with a flat spreading area, lesions with an unexpected nearby lesion, and lesions with lateral extension beneath non - cancerous mucosa) and tumor diameter, recurrent - type cancer, submucosal cancer, and undifferentiated - type cancer were factors significantly related to margin - positive resection 3. therefore, early gastric cancers with these factors may need to be treated in high - volume centers. a first, and difficult, decision is whether the cases are treated promptly or followed up. in the current study, early treatment (early re - esd, coagulation, or surgery) was performed in 98 cases (43 %), and 131 cases (57 %) were followed up without additional early treatments. in addition, pooled estimates of the follow - up rate without early intervention in high- and non - high - volume centers were not heterogeneous (p = 0.692). recently, a cancer - positive lateral margin length 6 mm in the esd specimens has been reported as an independent risk factor for local recurrence after esd 5 6. before deciding whether the cases with positive horizontal or indeterminable margins are treated promptly or followed up, the length of cancer - positive lateral margins may need to be evaluated in the esd specimens. a second difficult decision is how to treat the cases with positive horizontal or indeterminable margins at an early date. re - esd, coagulation, or surgery were used in additional treatments within 30 days in the enrolled cases. with time, submucosal fibrosis becomes severe and it can be considered difficult to resect the residual cancers endoscopically.. that may be the reason why coagulation was used to treat more than half of the enrolled cases. however, a specimen is not obtained and the state of residual cancers can not be determined., specimens can be obtained in re - esd or surgery. in the early re - esd group, esd, the re - esd specimens could be evaluated pathologically and intramucosal residual cancer was demonstrated in 6 cases (43 %). however, local residual cancers were observed in 13 of 29 surgically resected specimens (45 %) and lymph node metastasis was not demonstrated in any cases. the third choice is how to manage the later recurrent cases after follow - up. application of coagulation to the later recurrent lesions may be easy and convenient as in the cases treated at an early date. however, the recurrence rate was high in late coagulation cases (75 %) and coagulation may not be sufficient for the treatment of later recurrent lesions. as in the cases treated at an early date, lymph node or distant metastases were not found in the cases treated surgically at a later stage and local resection may be sufficient for the treatment of these lesions. en bloc resection rate was high (92 %) and the recurrent case after late esd was treated using a third esd. in addition, there were no complications in the late re - esd group. because locally recurrent cancers were within the mucosal layer and esd is less invasive than gastrectomy, esd may be considered a more suitable additional treatment for locally recurrent lesions, as suggested in previous reports 7 8 9. first, the probability of positive horizontal or indeterminable margin should be collected not from the questionnaire, but from official reports such as published literatures. second, the selection criteria were insufficient because institutions that responded to our questionnaire were not selected randomly and limited in tokyo, japan. third, the detailed follow - up results after esd and additional treatments were not asked in our questionnaire. therefore, the effectiveness of each strategy for early gastric cancer with positive horizontal or indeterminable margins after esd could not be evaluated. in conclusion, the rate of positive margins after esd tended to be lower in high volume centers. there was insufficient consensus regarding the treatment strategies used for early gastric cancer with positive horizontal or indeterminable margins after esd.	background and study aims positive horizontal margins in resected specimens are sometimes encountered after endoscopic submucosal dissection (esd) for early gastric cancers, and appropriate treatment strategies for these cases are not established. the aim of this study was to evaluate current empirical treatments for patients with positive horizontal or indeterminable margins after esd. patients and methods we performed a multicenter survey and data from 14 hospitals were collected. the pooled proportions of positive horizontal or indeterminable margins and those of patients followed up without early intervention were calculated using a logistic - normal random - effects model. for calculating pooled estimates, subgroup analyses of high- and non - high - volume centers were conducted. results a total of 11,796 esd cases were enrolled and 229 patients (2 %) had positive horizontal or indeterminable margins. ninety - eight cases were treated within 30 days of esd and 131 cases were followed up without early treatments. pooled estimates of positive margins in high- and non - high - volume centers were 1 % (95 % ci : 1 % 2 %) and 2 % (95 % ci : 1 % 4 %), respectively, and were not heterogeneous (p = 0.191). the proportion of patients followed up without early intervention ranged from 30 % to 100 %. the pooled estimate was 68 % (95 % ci : 50 % 83 %). the pooled estimates of high- and non - high - volume centers were 65 % (95 % ci : 38 % 85 %) and 72 % (95 % ci : 44 % 89 %), respectively, and were not heterogeneous (p = 0.692). conclusion there was insufficient consensus regarding treatment strategies used for early gastric cancer with positive horizontal or indeterminable margins after esd. further studies are required to establish a consensus.
hormonal disorders of female reproductive system is comprised of a number of problems resulting from aberrant dysfunction of hypo - thalamic - pituitary - ovarian axis. proper evaluation of these disorders involves a multidimensional diagnostic approach, with a pivotal contribution from clinical laboratories (1). measurement of prolactin and thyroid hormones, especially thyroid stimulating hormone (tsh), has been considered an important component of infertility work up in women (2). several articles have highlighted the association of hyperthyroidism or hypothyroidism with menstrual disturbance, anovulatory cycles, decreased fecundity and increased morbidity during pregnancy (3, 4, 5). the increased prevalence of upper normal limit of serum tsh and raised anti - thyroperoxidase antibody titer indicate relatively more frequent occurrence of compensated thyroid function in infertile women than normal women of reproductive age. this necessitates considering such cases a subgroup of women in which all aspects of pituitary - thyroid axis should be thoroughly investtigated than merely do with tsh testing (6). despite normal tsh and free thyroxin (ft4) concentrations treating such thyroid dysfunction with low dose thyroxin slightly increases ft4 levels leading to inhibition of tsh secretion within normal range, resulting in subjective improvement in health status, normalization of menstrual abnormalities and restoration of normal fertility (7). hyperprolactinemia adversely affects the fertility potential by impairing pulsatile secretion of gnrh and hence interfering with ovulation (3, 8). this disorder has been implicated in menstrual and ovulatory dysfunctions like amenorrhea, oligomenorrhea, anovulation, inadequate corpus luteal phase and galactorrhea (9, 10). pituitary hormones such as tsh, prolactin or growth hormone may act synergistically with fsh and lh to enhance the entry of non - growing follicles into the growth phase (7). morphological changes observed in the follicles in hypothyroidism can be a consequence of higher prolactin production that may block both secretion and action of gonadotropins (11). adequate thyroid supplementation restores prolactin levels as well and normalizes ovulatory function (12). even in the absence of hyperprolactinemia, hypothyroidism itself may contribute to infertility since thyroid hormones may be necessary for the maximum production of both estradiol and progesterone (13). in areas with endemic goiter, the prevalence of thyroid dysfunction among the infertile females in delhi and its suburban areas, which is considered as a non - endemic zone for iodine deficiency, had not been studied prior to this research. the aims of the study were to find the prevalence of thyroid disorders in female infertility after exclusion of tubal factor and male factor infertility in delhi and suburbs from a hospital - based study and to investigate the impact of the thyroid status on serum prolactin, fsh and lh of the third day of menstrual cycle. the cases consisted 160 female subjects who were suffering from primary infertility and had been referred the department of biochemistry of maulana azad medical college, new delhi for hormonal evaluations. the inclusion criteria for the selection of cases were diagnosis of primary infertility, age between 20 - 40 years and duration of marriage more than one year. the exclusion criteria that were adopted during case selection were male factor infertility and amongst the female factors were tubal factor, any congenital anomaly of the urogenital tract, or any obvious organic lesion. any history of thyroid disease or previous thyroid surgery or being on thyroid medications were also amounted to exclusion for the study. the protocol for infertility work up in the women included : a detailed medical history, a gynecological examination, a premenstrual endo - metrial sampling, an ultrasonography, a hormonal profile (tsh, ft4, ft3, prolactin, fsh and lh), screening for infectious diseases and whenever indicated, hysterosalpingography and/or laparoscopy. eighty healthy fertile female employees of lok nayak hospital, new delhi with similar age range and socioeconomic status were enrolled as controls. five milliliters of fasting venous sample obtained in the morning of day three of menstrual cycle for serum biochemical analysis. all the hormones were estimated using electrochemilu - miniscence kits of tsh, ft3, ft4, prolactin, lh and fsh (roche diagnostics ; mannheim, germany) and estimated on elecsys 2010 (roche healthcare, basel, switzerland). assay reliability was determined by the use of commercially derived control sera of low and high concentrations. the normal range of serum prolactin and tsh were 2 - 25ng / ml and 0.5 - 4.7miu / l respectively. women with serum prolactin levels > 100ng / ml were advised to undergo ct scan or mri to rule out any pituitary pathology. as per the serum tsh profile the cases, as well as the controls, were divided into three groups : i euthyroidism was present when the value of tsh was within the normal range. patients with subclinical hyperthyroid as well as those with hypothyroidism were not included in the study. statistical analysis was done by using spss software, version 12 (spss inc, illinois, usa) through chi - square test and mann whitney u test calculations. spearman 's correlation was used to look for association between different variables in the study group. most of the control (86%) and infertile women (87%) were euthyroid. the prevalence of hyperthyroidism in the cases and the controls were 5% and 9%, respectively. hypothyroidism was seen in 8% of the infertile subjects whereas in the control group it was found to be 5%. the crude prevalence of hypothy - roidism was slightly higher when compared to hyperthyroidism in the infertile group. hormonal status in the whole study groups (values are expressed in mean sd) p<0.001 in comparing the distributions (mann - whitney test) to the respective group in controls. the mean serum levels of tsh, ft4, ft3, lh, fsh and prolactin in the study group are depicted in table 1. significantly higher serum tsh levels were noted in the infertile cases with euthyroidism (p<0.01) and hypothyroidism (p<0.001) when their distributions were compared to their respective control groups. the rise in serum ft4 and ft3 in the infertile group with hyperthy - roidism was found to be significantly higher as compared to the control group with hyperthy - roidism (p<0.001). serum ft4 value was significantly lower (p<0.01) in the infertile group with hypothyroidism when compared to the control group with hypothyroidism. hyperprolactinemia was depicted in 41% of the infertile women while it was the case in only 15% in the control group. the mean serum prolactin concentration in the infertile cases with euthyroidism was significantly higher (p<0.001) than the control group with euthyroidism. the infertile women with hypothyroidism had significantly higher prolactin levels than the other three groups (the controls and the infertile subjects with euthyroidism and hyperthyroidism) (p<0.001). the serum lh and fsh levels in the infertile patients with hyperthyroidism were found to be significantly higher than the control group with hyperthyroidism (p<0.001 and p<0.05, respectively). menstrual disturbances observed in the control and infertile groups were 18.7% and 61.2%, respectively (table 2). the majority of the cases (82.6%) as well as the controls (66.7%) who presented with menstrual disturbances, had oligomenorrhea. fifty percent of the subjects with hypothyroidism had menstrual irregularities, presented with amenorrhea. distribution of menstrual irregularities and anovulatory cycles in the controls and the infertile group p<0.001 by chi square test among the infertile women, 54% showed non - secretory endometrium in premenstrual endo - metrial samples suggestive of the presence of an anovulatory cycle. serum tsh levels were found to be positively correlated with prolactin levels in the cases (r=0.4, p=0.01). in this study, the majority of infertile as well as fertile women were euthyroid. however, the distribution of thyroid dysfunction in the study group was somehow different hyperthyroidism being more prevalent in the controls whereas hypothyroidism was more prevalent in the infertile group. some investigators had claimed an association between mild iodine deficiency with hyperthyroidism and less frequently with hypothyroidism in the population (15, 16, 17). a relatively higher occurrence of hypothyroidism in infertile women, when compared to the control group in this study, reflects the tendency of infertile patients towards thyroid insufficiency or the vice versa. the prevalence of hypothyroidism in women of reproductive age (20 - 40 years) varies between 2% to 4% (18, 19). relatively higher crude prevalence rate of hypothyroidism (8%) in the infertile women found in our study could be due to special referral pattern of the patients who were referred to the hospital based on suspicion of thyroid abnormalities. a higher occurrence of hyperprolactinemia (41%) was seen in the infertile group as compared to the controls (15%) in this study. this higher propensity of hyperprolactinemia is in agreement with the findings of kumkum (20) who had depicted a prevalence of 46% in their study. as per the study, we observed a greater percentage of infertile patients with hypothyroidism exhibiting hyperprolactinemia (46.1%). choudhary and goswami (21) observed hyperprolactinemia in 16.6% and singh in 57% of women with hypothyroidism (22). fifty - eight percent of infertile women with raised serum prolactin levels showed nonsecretory endometrium suggestive of anovulation. kumkum (20) showed an incidence of anovulation in hyperprolactinemia patients to be 73%. prevalence of ovulatory dys - function, as one of the causes of female infertility, has been variously reported in different studies : 31.4% (22) and 51.4% (9). menstrual abnormalities were detected in about 60% of the infertile cases in this study, which is nearly similar to that observed by kumkum (20) who had reported the abnormality to be 57.6% in their study. anovulatory cycles were present in 54% of the cases, which corroborates with the finding of kumkum (50%). maximum percentage of menstrual abnormality presented by the infertile group was oligomen - orrhea (82%) whereas kumkum (20) depicted the state to be smaller (50%). in the study done by krasses (24), the prevalence of menstrual irregularities (mainly oligomenorrhea) reached 23% among 171 hypothyroid patients, while being only 8% in 214 controls (p<0.05). the authors had shown an association between the severity of menstrual abnormalities and higher serum tsh concentrations. we reported irregular menstrual cycles, mainly amenorrhea, in 31% of the cases with hypothyroidism. our study revealed an association between menstrual irregularities with raised serum prolactin levels (p<0.001) rather than tsh concentrations. a higher incidence of amenorrhea could be linked to hyperprolactinemia that was seen in the majority of patients with hypothy - roidism. joshi (25) evaluated 53 hyperthyroid patients and found 5.8% of them to be infertile. in contrast to hypothyroidism, most women with hyperthy - roidism do not have fertility problems, although 25% may have irregular menses (26). joshi found menstrual irregularities in 65% of hyperthyroid women compared to 17% in healthy controls (25). krasses indicated that menstrual disturbances in thyrotoxicosis are 2.5 times more frequent than in the general population (26). hyperprolactinemia resulting from longstanding primary hypothyroidism has been implicated in ovulatory dysfunctions ranging from inadequate corpus luteal progesterone secretion when mildly elevated to oligomenorrhea or amenorrhea when circulating prolactin levels are high (27). amenorrhea occurs in hypothyroidism due to hyper - prolactinemia resulting from a defect in the positive feedback of estrogen on lh, and because of lh and fsh suppression. our study revealed a significant association between abnormal menstrual patterns, as well as anovulatory cycles, with hyperprolactinemia in the infertile group (p<0.001). for these reasons, tsh and prolactin are commonly - ordered clinical tests in evaluating infertile women. the main drawback of the present study was the number of participants in the study. only 80 controls could be included in the study as compared to 160 cases due to the stringent inclusion criteria and non - compliance. there was a higher crude prevalence of hypothyroidism and hyperprolactinemia in the infertile women as compared to the fertile ones in the control group. hence, assessment of serum tsh and prolactin levels are mandatory in the work up of all infertile women, especially those presenting with menstrual irregularities. to avoid the slightest traces of selection bias, the data should be extrapolated to the general population with care, as the study has been a hospital - based one due to the difficulties in recruiting counterparting controls. for a better calculation of the intended prevalence	introductionthe increased prevalence of upper normal limit of tsh and raised anti - thyroperoxidase antibody titer indicate, relatively more frequent occurrence of compensated thyroid function in infertile women. this finding necessitates considering such cases for a thorough investigation of pituitary - thyroid axis. in addition, as some patients may exhibit the clinical picture of hypothyroidism despite normal tsh and free thyroxin (ft4) concentrations, this hospital - based study was undertaken to review the impact of thyroid status on the menstrual function and fertility of the subjects.materials and methodsin this study, we investigated 160 women with primary infertility who attended the biochemistry department, maulana azad medical college (mamc), new delhi for hormonal evaluations. eighty fertile women with similar age and socioeconomic status were enrolled as the controls. the association between thyroid dysfunction and levels of serum prolactin, lh and fsh as their menstrual status were reviewed.resultsthe majority of the infertile and fertile women were euthyroid. in infertile group, the crude prevalence of hypothyroidism was slightly higher in the infertile group in comparison with that of the general population. there was a positive correlation between serum tsh and prolactin levels in the infertile subjects. menstrual disorders (mainly oligomenorrhea), were reported by about 60% of the infertile women. hyperprolactinemia was depicted in 41% of the infertile women while it was only 15% in the control group. the infertile women with hypothyroidism had significantly higher prolactin levels when compared to the subjects with hyper- or euthyroidism. there was a significant association between abnormal menstrual patterns and anovulatory cycles, as observed on endometrial examination of infertile subjects with raised serum prolactin levels.conclusionthere is a greater propensity for thyroid disorder in infertile women than the fertile ones. there is also a higher prevalence of hyperprolactinemia in infertile patients.
there are only four reported cases in the medical literature describing typical bronchial carcinoids metastasizing to the brain. little is known about the pathogenesis and presentation of this disease due to the very small patient population. a 67-year - old hispanic female presented to our hospital with a three - week history of right arm numbness and poor coordination. computed tomography (ct) with intravenous contrast of the brain and subsequent magnetic resonance imaging demonstrated multiple enhancing nodular densities throughout the brain. ct with intravenous contrast of the chest, abdomen, and pelvis revealed a left hilar mass and a medial left upper lobe mass. although metastases to the central nervous system are very frequent with small cell carcinomas, their presence is very uncommon in well - differentiated neuroendocrine tumors such as the one we present here. this case raises questions about whether these tumors contain biomarkers that might predict a more aggressive behavior and if these patients might benefit from aggressive interventions similar to those taken in small cell carcinomas, such as prophylactic cranial radiation. accounting for approximately 12% of all lung malignancies in adults and roughly 2030% of all carcinoid tumors, bronchial carcinoids are neuroendocrine tumors made up of peptide and amine - producing cells originating from the embryologic neural crest [1, 2, 3, 4 ]. carcinoids originate most commonly from the gastrointestinal tract, but can also originate from the lung, thymus, and ovary. bronchial carcinoids are broken down into typical and atypical tumors based on their pathologic tumor grade. typical bronchial carcinoids are slow growing tumors that rarely metastasize, whereas atypical tumors metastasize early to the hilar or mediastinal nodes and are associated with a higher recurrence rate [5, 6 ]. the average age of adults diagnosed with typical and atypical bronchial carcinoid tumors is 45 and 55 years, respectively [7, 8, 9 ]. typical and atypical bronchial carcinoids have five - year survival rates ranging from 87100 to 3095%, respectively [7, 10, 11, 12, 13, 14, 15, 16, 17,18,19,20,21,22,2324 ]. bronchial carcinoids occur in 0.22 per 100,000 individuals per year, with a higher incidence among women and caucasians [1,2,3, 25, 26 ]. a major risk factor for tumor development is smoking, which accounts for up to half of all bronchial carcinoids and an even greater percentage of atypical carcinoids [5, 30 ]. patients with the autosomal dominant syndrome of multiple endocrine neoplasia type 1 (men1) are also at increased risk for bronchial carcinoid tumor development. we report a case of a patient who presented with a typical carcinoid that metastasized to her brain. a 67-year - old hispanic female presented to the emergency department complaining of a 3-week history of right arm numbness and poor coordination, during which eating, putting on makeup, and combing her hair had become increasingly difficult. her past medical history was significant for well - controlled essential hypertension and recently diagnosed behet 's syndrome. she smoked one pack a day for 37 years, quitting 15 years prior to the onset of symptoms. on admission, physical examination revealed right hand paresis and abnormal cerebellar function, as seen with the finger - to - nose test. all laboratory results were within normal limits. computed tomography (ct) with intravenous contrast of the brain and subsequent magnetic resonance imaging demonstrated multiple enhancing nodular densities throughout the brain, measuring up to 1.3 cm in maximum diameter (fig. 1). ct with intravenous contrast of the chest, abdomen, and pelvis revealed a 2.5 2.7 3.5-cm left hilar mass encasing the left upper lobe apical segmental artery (fig. 2) and a 2.1 2.1 2.7-cm medial left upper lobe mass abutting the anterior mediastinum. bronchoscopic evaluation confirmed the findings of imaging studies and endobronchial biopsy reported typical carcinoid histology (fig. 3), with stains positive for cd56, synaptophysin, ttf-1, and napsin, and negative for p63, ck5, and chromogranin. a ct - guided percutaneous lung biopsy was performed on the hilar site to confirm the diagnosis given the rarity of central nervous system metastasis with this histology. this report, in addition to a report from a second opinion tissue sample referral center, concurred with the diagnosis. the patient was placed on steroid taper and her metastatic brain lesions were treated with stereotactic radiosurgery (16 gy at 60% isodense) with complete improvement of neurologic symptoms. surgical evaluation deemed the patient unresectable and plans were made to start concurrent chemotherapy and radiation. written informed consent was obtained from the patient for publication of this paper and accompanying images. a copy of the written consent form is available for review by the editor - in - chief of this journal. there are only four reported cases in the medical literature describing typical bronchial carcinoids metastasizing to the brain. the patients were reported in japan, israel, and germany, and their ages ranged from 45 to 76 years. neuroendocrine tumors of the lung exist as a continuum from well - differentiated typical carcinoids to atypical carcinoids to extremely poorly differentiated small cell carcinomas. according to the 2004 who criteria for the diagnosis of neuroendocrine tumors, there are specific pathological criteria that need to be met when describing each subset of tumor (table 1). although metastasis of small cell carcinomas has been well described, little is known about the pathogenesis and presentation of typical bronchial carcinoid metastasis due to the very small patient population. in addition to exploring the tumor 's genetics and clinical tendencies, we must also understand the treatment modalities for this malignancy in order to ultimately improve patient survival. researchers have suggested several mechanisms to explain bronchial carcinoids ability to metastasize, including dendritic cell apoptosis, deletions of 11q and 4q25, dna methylation, and aneuploidy dna content. other signals or genes that have been implicated include cell cycle inhibitor rb1, drs tumor suppressor gene, phospholipase c beta 3, and apoptosis signal caspase-8. markers for malignant bronchial carcinoid tumor transformation include gastrin - releasing peptide, bcl-2, p53, pou factor brn3adel, and tyrosine kinase receptors c - kit, pdgfr alpha / beta, and egfr [46, 47 ]. additionally, cd44, nm23, and ki-67 have all been shown to have prognostic significance for this tumor [46, 47 ]. although the most common metastatic site of all carcinoid tumors is the liver, extrathoracic metastatic disease with bronchial carcinoid tumors is very rare, with one study citing a rate of 5% in a sample of 525 patients. symptomatic patients can have hemoptysis from tumor hyper - vascularity or cough with recurrent pneumonia due to proximal airway obstruction. about three - fourths of bronchial carcinoids are centrally located and have a pink to red vascular mass appearance with intact bronchial epithelium. bronchial carcinoids, in addition to merkel cell carcinomas, pheochromocytomas, medullary thyroid carcinomas, and pancreatic neuroendocrine tumors, arise from neuroendocrine cells and have the capacity to secrete biologically active neuroamines and neuropeptides. despite the fact that less than 5% of patients exhibit hormonally related symptoms, typical and atypical bronchial carcinoids have the potential to cause carcinoid syndrome and cushing 's syndrome through ectopic production of serotonin and adrenocorticotropic hormone [48,49, 50,51 ], respectively. 60% of atypical tumors express somatostatin receptors and can be imaged with radiolabeled octreotide [11, 52, 53, 54,55 ]. although this scan can image the entire body and identify metastatic disease outside the lung very easily, it has a very low specificity for disease detection [55, 56 ]. currently, there are no screening guidelines for bronchial carcinoid tumors and their presence can only be identified and confirmed through the use of radiographic studies and follow - up biopsy with immunohistological staining of the secreted products synaptophysin, neuron - specific enolase, and chromogranin. in terms of treatment modalities, surgical resection with lymph node dissection offers the only chance of a cure from these tumors. consensus guidelines from the national comprehensive cancer network suggest the use of chemotherapy and radiation for resected stage ii or iii atypical carcinoids, but not for typical carcinoids. although randomized trials have revealed only minimal activity, metastatic bronchial carcinoid patients are often treated with small cell carcinoma - related regimens including prophylactic cranial radiation with cisplatin plus etoposide with or without paclitaxel or single agent temozolomide. although metastases to the central nervous system are very frequent with small cell carcinomas, their presence is very uncommon in well - differentiated neuroendocrine tumors such as the one we present here. this case raises questions about whether these tumors contain biomarkers that might predict a more aggressive behavior and if these patients might benefit from aggressive interventions similar to those taken in small cell carcinomas, such as prophylactic cranial radiation. additionally, more data will need to be collected on metastatic typical bronchial carcinoid patients in order to better characterize the optimal radiographic and laboratory screening tests and the subsequent treatment modalities that will result in the best overall outcomes.	introductiontypical bronchial carcinoid tumors are known for their relatively indolent behavior. there are only four reported cases in the medical literature describing typical bronchial carcinoids metastasizing to the brain. little is known about the pathogenesis and presentation of this disease due to the very small patient population.case presentationa 67-year - old hispanic female presented to our hospital with a three - week history of right arm numbness and poor coordination. computed tomography (ct) with intravenous contrast of the brain and subsequent magnetic resonance imaging demonstrated multiple enhancing nodular densities throughout the brain. ct with intravenous contrast of the chest, abdomen, and pelvis revealed a left hilar mass and a medial left upper lobe mass. histopathological findings were consistent with a neuroendocrine neoplasm of bronchial origin.conclusionalthough metastases to the central nervous system are very frequent with small cell carcinomas, their presence is very uncommon in well - differentiated neuroendocrine tumors such as the one we present here. this case raises questions about whether these tumors contain biomarkers that might predict a more aggressive behavior and if these patients might benefit from aggressive interventions similar to those taken in small cell carcinomas, such as prophylactic cranial radiation.
the zebov makona strain seed stock originated from serum from a fatal case during the 2014 outbreak in gukdou, guinea (zebov isolate homo sapiens - wt / gin/2014/ makona - gueckedou - c07, accession number kj660347.2) and was passaged twice in authenticated vero e6 cells obtained from atcc (atcc, crl-1586) [11, 12 ]. six healthy adult cynomolgus macaques (macaca fascicularis) of chinese origin (weight range, 4.37.0 kg ; age range, 48 years) were used for these studies. in an initial study, 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration into the medial canthus of each eye, whereas 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration to the oropharynx. in a second study, 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration into the medial canthus of each eye, while 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration to the oropharynx. all 6 animals underwent physical examinations, had swab specimens collected from mucosal surfaces, and had blood specimens collected at the time of challenge and on days 2, 3, 4, 5, 6, 7, 8, 10, 15, 21, and 28 (for the 10 pfu study) or 3, 5, 7, 9, 14, 21, and 28 (for the 100 pfu study) after virus challenge. animal studies were completed under biosafety level 4 biocontainment at the galveston national laboratory (gnl) and were approved by the university of texas medical branch (utmb) institutional laboratory animal care and use committee, in accordance with state and federal statutes and regulations relating to experiments involving animals, and the utmb institutional biosafety committee. rna was isolated from samples using the viral rnamini kit or rneasy kit (qiagen), using 100 l of blood / swab in 600 l of buffer avl or 100 mg of tissue, respectively, per the manufacturer 's instructions. primers / probe targeting the vp30 gene of all known zebov strain sequences were used for quantitative reverse transcription polymerase chain reaction (qrt pcr), with the probe used here being 6-carboxyfluorescein (6fam)-59-ccgt caatcaaggagcgcctc39 - 6 carboxytetramethylrhodamine (tamra) (life technologies). zebov rna was detected using the cfx96 detection system (biorad) in one - step probe qrt pcr kits (qiagen), with cycle conditions and genomic rna standard determination as previously described [11, 12 ]. virus titration was performed by a plaque assay with vero e6 cells obtained from plasma samples as previously described [11, 12 ]. in brief, increasing 10-fold dilutions of the samples were adsorbed to vero e6 monolayers in duplicate wells (200 l) ; the limit of detection was 5 pfu / ml. total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing ethylenediaminetetraacetic acid, using a laser based hematologic analyzer (beckman coulter). serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glutamyl transferase, glucose, cholesterol, total protein, total bilirubin, blood urea nitrogen, creatinine, and c - reactive protein by using a piccolo point - of - care analyzer and biochemistry panel plus analyzer discs (abaxis). tissue samples from major organs were collected for histopathological and ihc examination, immersion fixed in 10% neutral buffered formalin, and processed for histopathologic analysis as previously described [11, 12 ]. for ihc analysis, specific anti - zebov immunoreactivity was detected using an anti - zebov vp40 protein rabbit primary antibody (integrated biotherapeutics) at a 1:4000 dilution. tissue sections were processed for ihc analysis, using the dako autostainer (dako). secondary antibody used was biotinylated goat anti - rabbit immunoglobulin g (igg ; vector laboratories) at 1:200 followed by dako lsab2 streptavidin horseradish peroxidase (dako). the zebov makona strain seed stock originated from serum from a fatal case during the 2014 outbreak in gukdou, guinea (zebov isolate homo sapiens - wt / gin/2014/ makona - gueckedou - c07, accession number kj660347.2) and was passaged twice in authenticated vero e6 cells obtained from atcc (atcc, crl-1586) [11, 12 ]. six healthy adult cynomolgus macaques (macaca fascicularis) of chinese origin (weight range, 4.37.0 kg ; age range, 48 years) were used for these studies. in an initial study, 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration into the medial canthus of each eye, whereas 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration to the oropharynx. in a second study, 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration into the medial canthus of each eye, while 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration to the oropharynx. all 6 animals underwent physical examinations, had swab specimens collected from mucosal surfaces, and had blood specimens collected at the time of challenge and on days 2, 3, 4, 5, 6, 7, 8, 10, 15, 21, and 28 (for the 10 pfu study) or 3, 5, 7, 9, 14, 21, and 28 (for the 100 pfu study) after virus challenge. animal studies were completed under biosafety level 4 biocontainment at the galveston national laboratory (gnl) and were approved by the university of texas medical branch (utmb) institutional laboratory animal care and use committee, in accordance with state and federal statutes and regulations relating to experiments involving animals, and the utmb institutional biosafety committee. rna was isolated from samples using the viral rnamini kit or rneasy kit (qiagen), using 100 l of blood / swab in 600 l of buffer avl or 100 mg of tissue, respectively, per the manufacturer 's instructions. primers / probe targeting the vp30 gene of all known zebov strain sequences were used for quantitative reverse transcription polymerase chain reaction (qrt pcr), with the probe used here being 6-carboxyfluorescein (6fam)-59-ccgt caatcaaggagcgcctc39 - 6 carboxytetramethylrhodamine (tamra) (life technologies). zebov rna was detected using the cfx96 detection system (biorad) in one - step probe qrt pcr kits (qiagen), with cycle conditions and genomic rna standard determination as previously described [11, 12 ]. virus titration was performed by a plaque assay with vero e6 cells obtained from plasma samples as previously described [11, 12 ]. in brief, increasing 10-fold dilutions of the samples were adsorbed to vero e6 monolayers in duplicate wells (200 l) ; the limit of detection was 5 pfu / ml. total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing ethylenediaminetetraacetic acid, using a laser based hematologic analyzer (beckman coulter). serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glutamyl transferase, glucose, cholesterol, total protein, total bilirubin, blood urea nitrogen, creatinine, and c - reactive protein by using a piccolo point - of - care analyzer and biochemistry panel plus analyzer discs (abaxis). tissue samples from major organs were collected for histopathological and ihc examination, immersion fixed in 10% neutral buffered formalin, and processed for histopathologic analysis as previously described [11, 12 ]. for ihc analysis, specific anti - zebov immunoreactivity was detected using an anti - zebov vp40 protein rabbit primary antibody (integrated biotherapeutics) at a 1:4000 dilution. tissue sections were processed for ihc analysis, using the dako autostainer (dako). secondary antibody used was biotinylated goat anti - rabbit immunoglobulin g (igg ; vector laboratories) at 1:200 followed by dako lsab2 streptavidin horseradish peroxidase (dako). no study has examined the pathogenicity of the makona strain of zebov in nhps when given at low doses and using routes of exposure more likely to be associated with a natural outbreak. in an initial study, we exposed 4 cynomolgus macaques to a target dose of 10 pfu by either the oral route (2 animals) or the conjunctival route (2 animals ; the actual dose of 10 pfu received was determined by back titration of each inoculum). surprisingly, none of the 4 animals showed any evidence of clinical illness, and there were no changes in any hematologic or serum biochemical parameters during the course of the study (table 1). no infectious virus was detected by plaque assay in plasma from any of the 4 animals, and viral rna was below the limit of detection as assessed by qrt - pcr in whole - blood specimens from these animals. with the exception of the nasal swab obtained on day 10 from subject o-2 (1.62 10 genome copies / ml), we were unable to isolate infectious virus from the day 10 nasal swab samples from this animal. analysis of sera from all 4 animals at days 21 and 28 after exposure showed only low anti - zebov gp elisa igg titers in subject o-1 (1:100 at day 21 and 1:50 at day 28). histopathologic and ihc staining for zebov antigen revealed no significant lesions and no viral antigen present in tissues of any of the animals. table 1.clinical description and outcome of zaire ebolavirus (zebov)challenged nonhuman primatessubject no.sexroutedose, pfuclinical illnessclinical pathologyviremia level, log10 pfu / mlanti - zebov igg titero-1moral10none, survivednone1:100 (21) ; 1:50 (28)o-2foral10none, survivednone0 (21, 28)c-1mconjunctival10none, survivednone0 (21, 28)c-2fconjunctival10none, survivednone0 (21, 28)o-3moral100fever (d 7), loss of appetite (d 79), depression (d 7, 8), mild rash (d 7, 8), emesis (d 7), hematemesis (d 8), animal euthanized in the morning of d 8leukocytosis (d 7, 8), thrombocytopenia (d 7, 8), hypoalbuminemia (d 8), > 2-fold increase in alt level (d 7), > 7-fold increase in alt level (d 8), > 8-fold increase in ast level (d 7), > 10-fold increase in ast level (d 8), > 4-fold increase in alp level (d 7), > 7-fold increase in alp level (d 8), > 8-fold increase in bun level (d 8), > 5-fold increase in cre level (d 8), > 3-fold increase in ggt level (d 7), > 6-fold increase in ggt level (d 8), > 4-fold increase in crp level (d 5), > 10-fold increase in crp level (d 7, 8)6.60 (d5) ; 8.75 (d7) ; 8.79 (d8)ntc-3mconjunctival100loss of appetite (d 10), survivedlymphopenia (d 7)1.70 (d14)1:50 (21)days after zebov challenge are in parentheses. fever is defined as a temperature > 1.4c higher than the baseline level or either 0.7c higher than baseline and 39.7c or 0.6c higher than baseline and 40c. mild rash is characterized by focal areas of petechiae covering 1.4c higher than the baseline level or either 0.7c higher than baseline and 39.7c or 0.6c higher than baseline and 40c. mild rash is characterized by focal areas of petechiae covering < 10% of the skin. lymphopenia and thrombocytopenia are defined as a 35% drop in numbers of lymphocytes and platelets, respectively. leukocytosis is defined by a 2-fold increase in the white blood cell count, compared with baseline. abbreviations : alp, alkaline phosphatase ; alt, alanine aminotransferase ; ast, aspartate aminotransferase ; bun, blood urea nitrogen ; cre, creatinine ; crp, c - reactive protein ; ggt, glutamyl transferase ; igg, immunoglobulin g ; nt, not tested ; pfu, plaque - forming units. as it appeared that exposure of cynomolgus macaques to a very low dose of zebov makona by either the oral or conjunctival route did not result in a productive infection causing clinical disease, we performed a second study to determine whether exposure to a slightly higher dose would cause disease in macaques. two animals were exposed to a target dose of 100 pfu by either the oral route (1 animal) or the conjunctival route (1 animal ; actual doses of 120 pfu and 150 pfu were determined by back titration of the oral and conjunctival inocula, respectively). the animal exposed by the oral route (subject o-3) initially showed signs of clinical illness on day 7, which was characterized by fever, anorexia, and the presence of a macular rash (table 1). a high circulating viremia level was noted in subject o-3 beginning at day 5 and increasing at day 8. lesions observed by histopathologic staining were consistent with zebov infection and included histiocytosis of axillary and inguinal lymph nodes, lymphocytolysis of white pulp and deposition of fibrin in red pulp of spleen, sinusoidal leukocytosis, rare multifocal necrotizing hepatitis, and numerous pleomorphic intracellular eosinophilic viral inclusion bodies in hepatocytes. these lesions had correlative strong positive ihc staining for anti - zebov vp40 antigen of mononuclear cells (macrophages, monocytes, and dendritic) in red and white pulp of the spleen, sinusoidal spaces of the liver and rare hepatocytes, and mononuclear cells within sinuses of the inguinal and axillary lymph nodes (figure 1a, 1c, 1e, and 1 g). conversely, the animal exposed by the conjunctival route (subject c-3) did not show any evidence of clinical illness, with the exception of lymphopenia on day 7 and anorexia on day 10 ; this animal survived to the study end point (table 1). low - level viremia was detected by plaque assay at day 14 in plasma from subject c-3 (table 1), while viral rna genomes were detected by qrt - pcr in nasal swabs obtained on days 7 and 9 and the oral swab sample obtained on day 9. a low anti - zebov gp enzyme - linked immunosorbent assay igg titer (1:50) was noted in subject c-3 at day 21. histopathologic examination of tissues from this animal failed to show lesions that were consistent with zebov infection. however, weak positive ihc staining for anti - zebov vp40 antigen of mononuclear cells in red pulp of the spleen and in subcapsular and medullary sinuses of the inguinal and axillary lymph nodes (figure 1b, 1f, and 1h) was noted at the study end point, although the liver was free of any antigen - positive cells (figure 1d). figure 1.comparison of zaire ebolavirus (zebov) antigen in representative tissues of cynomolgus monkeys infected via the oral or conjunctival routes. a, stained spleen tissue section showing strong, diffuse cytoplasmic immunolabeling (brown) of dendriform mononuclear cells in the red and white pulp of a zebov - orally infected animals. b, stained spleen tissue section showing weak, diffuse cytoplasmic immunolabeling of the endothelium and rare dendriform mononuclear cells in the red pulp of a zebov - conjunctivally infected animal. c, stained liver tissue section showing strong, diffuse cytoplasmic immunolabeling of kupffer cells and rare sinusoidal - lining cells and hepatocytes in a zebov - orally infected animal. d, stained liver tissue section showing no immunolabeling of zebov - conjunctivally infected animal. e, stained inguinal lymph node (ing ln) section showing strong, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal. f, stained ing ln section showing very weak, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal. g, stained axillary lymph node (ax ln) showing strong, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal. h, stained ax ln showing moderate, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal. spleen and liver representative images were taken at 40x original magnification and inguinal and axillary lymph node images at 20x original magnification from orally infected animal o-3 (a, c, e, and g) or conjunctivally infected animal c-3 (b, d, f, and h). comparison of zaire ebolavirus (zebov) antigen in representative tissues of cynomolgus monkeys infected via the oral or conjunctival routes. a, stained spleen tissue section showing strong, diffuse cytoplasmic immunolabeling (brown) of dendriform mononuclear cells in the red and white pulp of a zebov - orally infected animals. b, stained spleen tissue section showing weak, diffuse cytoplasmic immunolabeling of the endothelium and rare dendriform mononuclear cells in the red pulp of a zebov - conjunctivally infected animal. c, stained liver tissue section showing strong, diffuse cytoplasmic immunolabeling of kupffer cells and rare sinusoidal - lining cells and hepatocytes in a zebov - orally infected animal. d, stained liver tissue section showing no immunolabeling of zebov - conjunctivally infected animal. e, stained inguinal lymph node (ing ln) section showing strong, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal. f, stained ing ln section showing very weak, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal. g, stained axillary lymph node (ax ln) showing strong, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal. h, stained ax ln showing moderate, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal. spleen and liver representative images were taken at 40x original magnification and inguinal and axillary lymph node images at 20x original magnification from orally infected animal o-3 (a, c, e, and g) or conjunctivally infected animal c-3 (b, d, f, and h). previous studies have clearly demonstrated that low doses of the kikwit or mayinga strains of zebov administered to macaques by parenteral injection or small - particle aerosol result in uniformly lethal disease [79 ]. additionally, oral or conjunctival exposure of macaques to a high dose of zebov mayinga resulted in severe disease and near uniform lethality. our results show that low doses (10 pfu) of zebov makona administered by the oral or conjunctival routes resulted in minimal replication of virus and failed to cause a lethal infection. for example, the dose required to produce a lethal infection may be higher for exposure of conjunctival or pharyngeal mucosal membranes than for parenterally injected virus or small - particle aerosols delivered deep into the lungs. parenteral exposure simulates an accidental needle stick ; aerosol exposure mimics deliberate release ; and mucosal surface exposure simulates the most likely mode of contact during a natural outbreak. current knowledge suggests that low - dose exposure from a laboratory accident or intentional release represents a significant risk of lethal zebov infection to any exposed individual, whereas it appears that the threshold of the lethal dose for oral or conjunctival exposure may be at least 10-fold higher than parenteral or aerosol exposure. while this was a narrowly focused study, our findings are a first step to understanding how much infectious virus is needed for transmission to occur in a natural outbreak. alternatively, as has been suggested in other studies, there may be phenotypic differences between the makona strain and the historical mayinga and kikwit strains. as in our current study, the numbers are still too low with the makona strain to have a completely sound comparison with the mayinga and kikwit strains, but initial steps must be taken to further our understanding. future studies should focus on determining whether low doses of zebov makona are lethal by the more conventional intramuscular route and by the small - particle aerosol route and to determine whether low doses of other zebov strains are lethal if administered by the oral or conjunctival routes.	nonhuman primate (nhp) models of ebola virus (ebov) infection primarily use parenteral or aerosol routes of exposure. uniform lethality can be achieved in these models at low doses of ebov (100 plaque - forming units [pfu ]). here, we exposed nhps to low doses of ebov (makona strain) by the oral or conjunctival routes. surprisingly, animals exposed to 10 pfu by either route showed no signs of disease. exposure to 100 pfu resulted in illness and/or lethal infection. these results suggest that these more natural routes require higher doses of ebov to produce disease or that there may be differences between makona and historical strains.
the term hemophagocytosis describes the pathologic finding of activated macrophages, engulfing erythrocytes, leukocytes, platelets, and their precursor cells. hlh is an unusual syndrome characterized by fever, splenomegaly, jaundice, and the pathologic finding of hemophagocytosis (phagocytosis by macrophages of erythrocytes, leukocytes, platelets, and their precursors) in bone marrow and other tissues. we report an adult patient who presented with systemic inflammatory response syndrome and features consistent with severe sepsis and septic shock, who subsequently received a diagnosis of secondary hlh. we reviewed the relationship between hlh and septic shock from the perspective of an intensivist. a 50 years female presented to intensive care unit (icu) with fever, rash, and loss of appetite. she was admitted in an outside hospital with 24 h of epigastric abdominal pain radiating to her flanks. there was no history of night sweats, weight loss, new medications, or toxin exposure. on admission, she presented with a temperature of 38.5c, a heart rate of 120/min, and an erythematous scaling rash covering over 50% of her body surface area, with increased scaling over her scalp. her abdomen was soft and nontender, with splenomegaly palpable 3 cm below the costal margin. there was no peripheral lymphadenopathy, and the rest of her physical exam was unremarkable. her initial blood work consisted of an elevated ferritin and lactic acid dehydrogenase (ldh) level, pancytopenia, acute kidney injury, hepatitis, and hypofibrinogenemia. broad - spectrum empirical antibiotics were administered for the presumed diagnosis of sepsis, but without apparent benefit. over a period of 24 days, she developed pressor - dependent hypotension, acute respiratory distress syndrome (ards), acute kidney injury, bicytopenia (anemia and thrombocytopenia), deranged liver functions (transaminitis), and coagulopathy ; and increased inflammatory markers c - reactive protein and patent cooperation treaty. the clinical course in this patient met the criteria for severe sepsis and septic shock except in that it was unclear whether associated infections were causative. persistent fever, severe pancytopenia, hyperferritinemia, hypertriglyceridemia, increased ldh, and splenomegaly eventually prompted bone marrow aspiration (bma) leading to the diagnosis of hlh. no family history of hlh was found, and a primary familial form was considered unlikely. according to the computed tomography (ct) scan and bone marrow biopsy results, no hematological malignancy was thought to be responsible for a secondary form. despite an extensive multi - site sampling and serological studies, laboratory tests for viral infection including epstein - barr virus, cytomegalovirus, herpes simplex virus, viral hepatitis b and c, hiv were unremarkable. prolonged fever, splenomegaly, bicytopenia, hypofibrinogenemia, hyperferritinemia, and hypertriglyceridemia confirmed the diagnosis of hfs. the delay in diagnosis due to its rare incidence leads the patient to secondary sepsis in spite of aggressive and symptomatic treatment. hlh is a rare disease characterized by uncontrolled proliferation of mature histiocytes, hemophagocytosis, and up - regulation of inflammatory cytokines. the more typical findings are fever, peripheral cytopenia affecting two lineages at least, hepatosplenomegaly, hypertriglyceridemia and/or hypofibrinogenemia, and hemophagocytosis. recently, the histiocyte society updated its guidelines and proposed to add three diagnostic criteria : hyperferritinemia decreased or absent natural killer (nk) cell activity, and high soluble interleukin-2 receptor serum levels. they were present in our patient, who developed high persistent fever, severe pancytopenia, splenomegaly on abdominal ct scan, and very high plasma levels of ferritin, triglycerides, and ldh. the hlh-2004 diagnostic criteria considered the gold standard are largely based on studies of children with familial hlh. many of the criteria have poor operating characteristics when considered in the differential diagnosis of septic shock in adults. fever, anemia, thrombocytopenia, hypofibrinogenemia, and hypertriglyceridemia are common features of both syndromes. several studies show that hyperferritinemia consistent with hlh (> 500 g / l) occurs in the majority of adults with septic shock. the sensitivity of diagnostic criteria such as hypertriglyceridemia, hypofibrinogenemia, and splenomegaly may be no better than about 50%. the sensitivity of nk cell activity and soluble cd-25 levels approaches 100%, but these tests are not widely available. a strict requirement to fulfill five hlh-2004 diagnostic criteria in the adult patient in an icu could delay potentially lifesaving therapy. other available hlh-2004 criteria should be used in conjunction, with consideration of the limitations in their operating characteristics and logistics in the icu. in our opinion, hlh treatment should be considered in patients with the clinical syndrome of unremitting severe sepsis and/or septic shock with bicytopenia or pancytopenia, elevated ferritin levels, prompting for a bma, which will show histiocytic hemophagocytosis. therapy should be started in all cases with high suspicion after diagnostic tests have been initiated, but regardless of whether the results of all examinations have been obtained. among patients who are acutely ill or deteriorating, we suggest hlh - specific therapy based on the hlh-94 protocol or enrollment in a clinical trial rather than treatment based on the hlh-2004 protocol. hlh-94-based therapy includes etoposide and dexamethasone given at tapering doses over 8 weeks, with intrathecal methotrexate and hydrocortisone for those with central nervous system involvement. additional supportive measures include transfusion of blood products as indicated, induction of amenorrhea, and use of prophylactic antibiotics. the response to initial therapy is a major factor in determining the need for additional therapy including hematopoietic cell transplant. a short course of intravenous (iv) methylprednisolone (12 mg / kg / d) is often sufficient in treating hs associated with secondary infections. this treatment is given in concert with adequate anti infectious agents, and it must be administered for the shortest time necessary for correction of the most life - threatening symptoms associated with hs because of the added immunosuppression in the context of severe infection. new antiviral drugs such as cidofovir or ribavirin and rituximab (anti - cd20 antibody) may be useful. consideration should be given to the use of iv immunoglobulin in infection - associated hs, to minimize the risk of immunosuppression. importantly, however, the initiation of hlh - specific therapy for severely ill patients should not be delayed while awaiting resolution of a system infection. clinicians are encouraged to enter patients in clinical trials such as the hybrid immunotherapy for hlh trial in the united states (hit - hlh ; nct01104025) or the euro - hit - hlh trial in europe (eudract201100205214). further study of hlh, may provide important insights into the optimal treatment of sepsis, perhaps leading to the identification of a subset of patients with sepsis in which tissue damage secondary to a hyper inflammatory state might be ameliorated by immunosuppressive therapy.	hemophagocytic lymphohistiocytosis is a clinic pathologic entity characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. it is a potentially lethal disorder due to an uncontrolled immune response to a triggering agent. hps may be primary, or secondary to malignancy, infections, auto - immune diseases, and pharmacotherapy. hps is a rare, but life - threatening complication. herein, we described a female patient with hps with secondary sepsis. our objective was to raise the importance of early diagnosis of hfs by presenting a representative case.
over 160 million people worldwide are chronically infected with the hcv virus. besides the liver related complications of hcv infection such as liver cirrhosis and hepatocellular carcinoma, chronic infection is associated in several studies with extrahepatic disorders, including metabolic derangements. though not many studies exist, robust data connect hcv infection with atherosclerosis and consequently its complications as stroke and coronary heart disease. in our days when the hcv infection can be treated in more than 90% of hcv infected patients, it is most important for clinicians to deal with the extrahepatic derangements which can diminish patients ' life expectancy and alter their quality of life. the pathophysiological basis of the evidenced correlation between hcv infection and atherosclerosis is incompletely understood. hcv infection represents a chronic inflammatory state where an imbalance between th1 and th2 is observed. studies have demonstrated that patients with chronic hcv infection exhibit higher il-6 and tnf - alpha, inf, and il-2 levels and a higher ratio of proinflammatory / anti - inflammatory cytokines. hcv promoted inflammatory cytokines may contribute to the development of atherosclerosis through the enhancement of intracellular adhesion molecules, expression of anti - endothelium antibodies, and generation of oxidative stress (oxs) and insulin resistance (ir). what is more severe is that fibrosis and the associated cascade of proinflammatory and profibrogenic pathways generated in the liver might promote carotid atherosclerosis. furthermore, hcv rna sequences have been isolated within carotid plaques and this in turn may suggest the possibility of an active infection of the carotid plaque itself. moreover, another important mechanism by which chronic hcv infection can promote the development of atherosclerotic plaques is its well described correlation with proatherogenic conditions such as insulin resistance [8, 9 ] and diabetes type 2. ir induces a broad range of toxic systemic effects, including dyslipidemia, hypertension, hyperglycemia, increased production of advanced glycosylation end products, increased inflammatory tone, and a prothrombotic and prooxidative state. patients with ir are highly vulnerable to the development of accelerated atherosclerosis as well its clinical sequelae, including coronary artery disease and myocardial infarction, carotid artery disease, and ischemic stroke. multiple explanations have been proposed in order to elucidate the mechanism of the development of ir in hcv infection. primarily it was assumed that chronic inflammation and the observed in hcv infection upregulation of inflammatory markers such as tnf - alpha and il-6 and the deregulation of adipocytokines (leptin, adiponectin) were a leading step, but recent studies failed to prove this assumption. it is now believed that the hcv virus itself and moreover the hcv core protein are the main driving factor by their interactions with socs3 or socs7 expression and ppr- and ppar - a [11, 12 ]. as far as type 2 diabetes and hyperglycemia is concerned its relationship with atherosclerosis is well established and its pathophysiological basis has been extensively studied. oxidative stress, abnormal no - mediated vasodilation, and increased macrophage lipid uptake, leading to foam cell formation, are only some features of the complex pathway by which hyperglycemia promotes atheromatosis. finally, liver steatosis, observed in hcv infection, and its association with hyperhomocysteinaemia are also factors predisposing to atherosclerosis. steatosis is a common finding in hcv infected patients especially among patients infected with genotype 3 (gt 3) which seems to have a direct steatogenic effect as steatosis in infection with gt 3 is well correlated with the levels of intrahepatic viral replication. even if hcv related steatosis has not been proven to directly cause atheromatosis at least four studies have, independently of the metabolic syndrome, directly linked steatosis to atheromatosis. it is not therefore irrational to hypothesize that this effect can be attributed to hcv related steatosis also. it should be mentioned that hcv patients tend to have a more favorable lipid profile possibly due to the straightforward interaction of the hcv virion, which uses the ldl receptor to infect hepatocytes, with the host lipid metabolism (figure 1). in 2002 for the first time a study by ishizaka. proposed a link between hcv infection and carotid atherosclerosis. since then several studies by various researchers, executed in different countries, provided evidence that hcv infection is independently associated with carotid plaques with a prevalence from 38% to 64% [37, 38 ] as also an independent predictor of increased carotid intimal medial thickness (imt) (table 1). a study published in 2003 by tomiyama., where 87 anti - hcv positives and 7427 anti - hcv negative subjects were enrolled, showed that hcv infected subjects had increased arterial stiffness compared to hcv negative controls. moreover a large egyptian study by mostafa. which included 329 anti - hcv positives and 725 anti - hcv negative patients showed that patients with active disease, when adjustment for known cardiovascular risk factor was executed, had a higher risk for atherosclerosis compared to subjects with past infection. a recent study by petta. not only confirmed the higher incidence of carotids plaques in hcv infected patients but also correlated the presence of carotid plaques with the severity of liver fibrosis as it was estimated by liver biopsy. the study enrolled 174 gt 1 biopsy proven hcv patients and 174 control matched subjects. multivariate logistic regression analysis showed that older age (odds ratio [or ] 1.047, 95% confidence interval [ci ] 1.0141.082, p = 0.005) and severe hepatic fibrosis (or 2.177, 95% ci 1.0434.542, p = 0.03) were independently linked to the presence of carotid plaques. in patients 55 years the prevalence of carotid plaques was similar in those with or without severe fibrosis (25/43, 58.1% versus 22/43, 51.1% ; p = 0.51). finally, a study executed by adinolfi. in italy suggested that hcv - related steatosis is both a good marker for identifying atherosclerosis - prone individuals and an early mediator of atherosclerosis. the writers came to the conclusion that hcv - related steatosis modulates atherogenic factors such as inflammation and the dysmetabolic milieu, therefore favoring the development of atherosclerosis. once more, in this study, it was observed by the researchers that chronic hcv infection predisposes individuals to the premature development of atherosclerosis and advanced carotid changes. on the contrary a small number of studies [1921 ] failed to prove such an association, though it must be underlined that a meta - analysis executed by huang., which included 11 studies among them the studies of tien, calsikan, and masia, studies which failed to prove such an association, revealed that hcv infection is significantly associated with carotid atherosclerotic burden. taking into account the abovementioned data hcv infection must be considered as a risk factor for carotid atheromatosis. in the era of new and more efficacious treatments for chronic hcv infection the burden of the nonliver related complications of the hcv infection may become of great significance for the prior hcv infected patients ' life expectancy. as a result of this, as it was also stated by petta., it may be indicated that hcv patients aged 55 or more, those with severe fibrosis, and those with hcv related liver steatosis should undergo ultrasonography screening for carotid atherosclerotic disease. recent data have pointed out a correlation between hcv infection and increase risk for cerebrovascular disease. in a large study executed in the united states where 10,259 anti - hcv seropositive patients and 10,259 matched anti - hcv seronegatives were included, the hazard ratio (hr) of death from stroke was 2.20. in another study executed in taiwan which enrolled 23,785 subjects (1,307 anti - hcv positive subjects) the hr for cerebrovascular death was 2.18 for seropositives to anti - hcv, this positive correlation was also underlined in a recent study where 820 subjects were enrolled, where the multivariate analysis showed an or of 2.04 for stroke among hcv patients compared to anti - hcv negative patients. finally, a large meta - analysis of the latest studies conducted in this field suggested that hcv infection significantly increased the risk of stroke (or = 1.97 ; 95% ci : 1.642.30). moreover it was proposed from a single study that hcv load is linearly correlated with the risk of stroke among hcv patients. that came to the conclusion that not only did hcv infected patients have a 23% increased risk of stroke compared to age and sex - matched subjects without hcv infections but interferon - based therapy may reduce the long term risk of stroke in patients with hcv infection. this data are furthermore supported by a recent study by enger. where it was demonstrated that hcv patients are in an increased risk of events such as unstable angina and transient ischemic attacks as it was pointed out in their study published in 2014 where 22733 hcv seropositives were enrolled. to our knowledge only two studies, one including only 21 anti - hcv positive subjects, an obviously very small number of patients capable of extracting confident results, and another which was criticized because of the heterogeneity of the study population as far as age, sex, and hypertension status of the study subjects was concerned, failed to demonstrate such a positive correlation. when all data are taken into account it can be safely argued that hcv infection increases the risk of cerebrovascular events and moreover data point to the direction that hcv eradication treatment can prevent these events, a fact highly important in our days when new and more effective treatment strategies against the hcv infection exist. despite the existence of conflicting evidence, a link between hcv infection and increase cardiovascular risk can be discerned [5, 46 ]. the pioneer study in this field was published in 2004 by vassalle. where the authors suggested that seropositivity represented an independent predictor for cad with an odds ratio of 4.2 (95% ci : 1.4 to 13.0, p = 0.05). a large scale epidemiological study conducted in the united states by butt. among (82083 hcv infected and 89582 hcv uninfected subjects) veterans over a 5-year period showed a significantly higher prevalence of cardiac disease among hcv infected patients. in another study by alyan where 139 hcv seropositive and 225 hcv seronegative patients with angiographically documented cad were enrolled, hcv infection was documented to be an independent predictor for increased coronary atherosclerosis, as demonstrated by higher reardon severity score. recently a retrospective study including 78 hcv positive patients compared to 742 hcv negative subjects was executed, which observed higher ischemic heart events in the hcv positive patients than in the hcv negative patients (22% versus 13%, resp., additionally in a recent study where hcv monoinfected, genotype 1, naive, and nonobese (bmi 75%) in hcv seropositive patients compared to age-, race-, and sex - matched controls undergoing evaluation by coronary angiogram for suspected cad. the hcv infected patients were also presented, in a greater scale, with significant multivessel coronary artery disease (2 vessels). the authors notice that it is not clear whether the observed association between cad and chc infection is related to the known metabolic complications related to insulin resistance in patients with chronic hcv infection, or due to under treatment with antiplatelet and lipid - lowering agents because of concerns for gastrointestinal bleeding or hepatotoxicity. finally, a large recent study conducted in the us added more confirmatory data towards the direction of a positive association. pothineni. in their study among a total of 8,251 hcv antibody positive, 1,434 hcv rna positive, and 14,799 hcv negative patients came to the conclusion that there is an increased incidence of chd events in patients with hcv seropositivity and the incidence is much higher in patients with detectable hcv rna compared with patients with remote infection who are only antibody positive. on the other hand, few studies [33, 34 ] and a recent review by wong. failed to demonstrate a clear - cut association between hcv infection and cad. a large study executed in the uk by forde. did not show any correlation between hcv and mi, though it must be underlined that there was a short period of follow - up of the subjects and moreover chronic hcv infection was poorly proved (the authors stated that they may have included patients who spontaneously cleared the hcv virus) and additionally it was a retrospective observational study where residual confounding by unmeasured confounders is possible. as far as the study of arcari. which also failed to prove a correlation is concerned, not only was the sample size of the hcv infected patients too small but moreover there was no additional confirmatory pcr - rna executed, a fact that may have further decreased the sample. even if some studies failed to prove this correlation it is not illogical to conclude that chronic hcv infection appears to be linked with excess cardiovascular risk (table 2). based on the abovementioned data hcv infection must be considered as a pre - atherogenetic state of an increased cardiovascular risk. from a clinical point of view hcv infected patients not suitable for treatment or who have failed treatment options must be monitored for carotid atheromatosis, in order to prevent cardiovascular events. there are no formed guidelines but patients with severe liver fibrosis, hcv related steatosis, or of aged > 55, who according to research data are of high risk for carotid atheromatosis, are the most eligible candidates for assessment of the existence of carotid atheromatosis. based on the bibliography, ultrasonography of the carotid arteries with imt measurement should be offered to those hcv infected patients, in order to assess carotid atheromatosis as it is the test most commonly used in the studies conducted in this field. in order to ameliorate hcv infected patients quality of life and to prevent extrahepatic complications, patients with well proven carotid atheromatosis it must be highlighted that not all patients are amenable of receiving primary prevention with antiplatelet therapy and lipid lowering agents. the risk of bleeding as also the liver related toxicity of lipid lowering agents must be balanced against the risk of a cardiovascular event. due to the lack of studies executed in this field, more data are needed in order to further specify which patients should be screened for carotid atheromatosis. the impact of hcv genotype as also that of the virus load should be further assessed. in the era of new and more efficacious treatments for chronic hcv infection the burden of the nonliver related complications of the hcv infection may become of great significance for the prior hcv infected patient 's life expectancy. as a consequence, it is a necessity to investigate if treatment does reverse the nonliver derangements such as carotid atheromatosis observed in hcv infected patients. recent data have proven the reversion of liver fibrosis after the successful treatment of hcv infection [47, 48 ] and the diminished risk not only of hcc development but also of liver - related complications [4951 ]. moreover, the eradication of the virus inhibits the inflammatory cascade [52, 53 ]. it was already stated that patients with higher fibrosis scores showed a greater prevalence of carotid atheromatosis. it is not irrational then to hypothesize that carotid atheromatosis may also reverse the liver fibrosis and the superimposed inflammation tends to return to normal state but for the present time remains a scientific question whose answer must be provided by well - designed large randomized controlled studies.	chronic hepatitis c virus infection is associated with significant morbidity and mortality, as a result of progression towards advanced natural course stages including cirrhosis and hepatocellular carcinoma. on the other hand, the svr following successful therapy is generally associated with resolution of liver disease in patients without cirrhosis. patients with cirrhosis remain at risk of life - threatening complications despite the fact that hepatic fibrosis may regress and the risk of complications such as hepatic failure and portal hypertension is reduced. furthermore, recent data suggest that the risk of hcc and all - cause mortality is significantly reduced, but not eliminated, in cirrhotic patients who clear hcv compared to untreated patients and nonsustained virological responders. data derived from studies have demonstrated a strong link between hcv infection and the atherogenic process. subsequently hcv seems to represent a strong, independent risk factor for coronary heart disease, carotid atherosclerosis, stroke, and, ultimately, cvd related mortality. the advent of new direct acting antiviral therapy has dramatically increased the sustained virological response rates of hepatitis c infection. in this scenario, the cardiovascular risk has emerged and represents a major concern after the eradication of the virus which may influence the life expectancy and the quality of patients ' life.
crisis services are now a well - established part of the mental health systems in north america.1 these programs have been found to be successful in improving access to mental health system for those with acute emotional or mental health problems and to help persons deal with a crisis. crisis services are also a cost - effective way of reducing the costs of psychiatric hospitalization, as well as social costs, by providing professional assessment and crisis intervention in the community.2 there is evidence to suggest that the crisis services are associated with improved client satisfaction.2 interestingly, most of the published research in this area is nearly two decades old.3 a recent cochrane review reported that by supporting clients at their homes, the crisis teams can reduce repeat admissions to hospital. crisis intervention also reduced family burden, and this was preferred by both clients and their families.4 however, the same review found no differences in mortality. there is a lack of interventions in this area ; for example, another cochrane review failed to identify the impact of crisis intervention services for borderline personality disorders.5 crisis teams in different parts of the world operate differently. in canada, the crisis teams work closely with the transitional case management (tcm) teams. crisis teams in some countries work closely with home treatment teams. a number of investigations have also reported reduced length of in - client stay following introduction of crisis resolution and home treatment (crht). these studies suggest that in addition to reduced admissions, these teams reduce the duration of inpatient admissions and rates of detention. using these services can catalyze a more efficient use of in - client care.5 the crht, and tcm services offer an opportunity to provide evidence - based psychosocial interventions to improve well - being further and to prevent future relapses. evidence - based self - help, guided self - help, and low - intensity psychological help can be easily provided in these setups. there is sufficient evidence in the form of meta - analyses and literature reviews to suggest that guided self - help can be as effective as the face - to - face treatments.6,7 we, therefore, developed a formulation - driven cognitive behavioral guided self - help (fcbt - gsh) for common mental health problems that can be delivered by mental health professionals working in a crisis or tcm setting. the study used a randomized controlled design to compare the efficacy of this fcbt - gsh plus treatment as usual (tau) to tau only within crisis and tcm services. the intervention is provided by trained mental health professionals for clients under care of the crisis and tcm service. the intervention group received fcbt - gsh and tau, while the control group received tau only. the tau consisted of routine follow - up and care of the clients by crisis and tcm team. both teams offer at least once a week face - to - face contact with the clients. current clients with a diagnosis of depression, anxiety, and other common mental health problems (using dsm - iv criteria) were considered eligible for inclusion, except for those using alcohol or drugs excessively or with significant cognitive impairment and those who were experiencing acute psychotic symptoms. those who met the criteria and consented in writing were then asked to join the study and were randomly allocated to one arm of the trial. the queen s university health sciences & affiliated teaching hospitals research ethics board approved this study. out of 36 clients who completed the assessment phase and were subsequently randomized, eight dropped out of the study. of these, three participants dropped out of the intervention group, and five from the control group. outcome measure (core - om) and hospital anxiety and depression scale (hads). disability was measured using the world health organization disability assessment schedule 2.0 (whodas 2.0). the core - om8 is a self - report measure tapping four domains, including a risk to others and risk to self. the scale was found to have good reliability, validity, and sensitivity to change.8 each of the items (eg, i have felt terribly alone and isolated) is scored on a scale from 0 (not at all) to 4 (most or all of the time). it has been used in previous research to measure emotional disturbance in service settings delivering psychological interventions in primary and secondary care. hads9 is a rating scale comprised of 14 items, seven of which are designed to measure anxiety (hads - a), and seven for depression (hads - d). the hads is a widely used measure of anxiety and depression, and its psychometric properties have been described in detail.10 each of the items in hads is scored on a four - point scale from 0 to 3. the item scores are summed to provide subscale scores on the hads - d and the hads - a, which may range between 0 and 21. studies most commonly employ a cut - point of 8 (eight and above) for each of the constituent subscales, as suggested by its authors, to indicate probable caseness. whodas 2.011 was developed by the who to measure disability due to physical and psychological problems. the scale was tested across the globe and was found to be both reliable and valid.11 participants are asked to indicate how much difficulty they had in completing a certain task (eg, remembering to do important things) in the last 30 days on a scale from 1 (none) to 5 (extreme or can not do). a third - year psychiatry resident physician, two undergraduate psychology students, social workers, and other crisis team members, who had been trained in delivering cbt intervention, provided fcbt - gsh to the participants. health professionals received training in using this intervention. they were also educated on techniques described in handouts, eg, problem solving, behavioral activation, and use of thought diaries to recognize, challenge, and create alternative thoughts. they were not trained in delivering therapy as this intervention only required them to facilitate use of self - help. standard aspects of cognitive therapy, different modules of fcbt - gsh manual, as well as home work assignments were also discussed. health professionals shared the formulation, barriers to home work, and difficulties in following handouts. the study used a randomized controlled design to compare the efficacy of this fcbt - gsh plus treatment as usual (tau) to tau only within crisis and tcm services. the intervention is provided by trained mental health professionals for clients under care of the crisis and tcm service. the intervention group received fcbt - gsh and tau, while the control group received tau only. the tau consisted of routine follow - up and care of the clients by crisis and tcm team. both teams offer at least once a week face - to - face contact with the clients. current clients with a diagnosis of depression, anxiety, and other common mental health problems (using dsm - iv criteria) were considered eligible for inclusion, except for those using alcohol or drugs excessively or with significant cognitive impairment and those who were experiencing acute psychotic symptoms. those who met the criteria and consented in writing were then asked to join the study and were randomly allocated to one arm of the trial. the queen s university health sciences & affiliated teaching hospitals research ethics board approved this study. randomization was performed using www.randomization.com. out of 36 clients who completed the assessment phase and were subsequently randomized, of these, three participants dropped out of the intervention group, and five from the control group. psychopathology was measured using the clinical outcomes in routine evaluation outcome measure (core - om) and hospital anxiety and depression scale (hads). disability was measured using the world health organization disability assessment schedule 2.0 (whodas 2.0). the core - om8 is a self - report measure tapping four domains, including a risk to others and risk to self. the scale was found to have good reliability, validity, and sensitivity to change.8 each of the items (eg, i have felt terribly alone and isolated) is scored on a scale from 0 (not at all) to 4 (most or all of the time). it has been used in previous research to measure emotional disturbance in service settings delivering psychological interventions in primary and secondary care. hads9 is a rating scale comprised of 14 items, seven of which are designed to measure anxiety (hads - a), and seven for depression (hads - d). the hads is a widely used measure of anxiety and depression, and its psychometric properties have been described in detail.10 each of the items in hads is scored on a four - point scale from 0 to 3. the item scores are summed to provide subscale scores on the hads - d and the hads - a, which may range between 0 and 21. studies most commonly employ a cut - point of 8 (eight and above) for each of the constituent subscales, as suggested by its authors, to indicate probable caseness. whodas 2.011 was developed by the who to measure disability due to physical and psychological problems. the scale was tested across the globe and was found to be both reliable and valid.11 participants are asked to indicate how much difficulty they had in completing a certain task (eg, remembering to do important things) in the last 30 days on a scale from 1 (none) to 5 (extreme or can not do). a third - year psychiatry resident physician, two undergraduate psychology students, social workers, and other crisis team members, who had been trained in delivering cbt intervention, provided fcbt - gsh to the participants. health professionals received training in using this intervention. they were also educated on techniques described in handouts, eg, problem solving, behavioral activation, and use of thought diaries to recognize, challenge, and create alternative thoughts. they were not trained in delivering therapy as this intervention only required them to facilitate use of self - help. standard aspects of cognitive therapy, different modules of fcbt - gsh manual, as well as home work assignments were also discussed. health professionals shared the formulation, barriers to home work, and difficulties in following handouts. hads9 is a rating scale comprised of 14 items, seven of which are designed to measure anxiety (hads - a), and seven for depression (hads - d). the hads is a widely used measure of anxiety and depression, and its psychometric properties have been described in detail.10 each of the items in hads is scored on a four - point scale from 0 to 3. the item scores are summed to provide subscale scores on the hads - d and the hads - a, which may range between 0 and 21. studies most commonly employ a cut - point of 8 (eight and above) for each of the constituent subscales, as suggested by its authors, to indicate probable caseness. whodas 2.011 was developed by the who to measure disability due to physical and psychological problems. the scale was tested across the globe and was found to be both reliable and valid.11 participants are asked to indicate how much difficulty they had in completing a certain task (eg, remembering to do important things) in the last 30 days on a scale from 1 (none) to 5 (extreme or can not do). a third - year psychiatry resident physician, two undergraduate psychology students, social workers, and other crisis team members, who had been trained in delivering cbt intervention, provided fcbt - gsh to the participants. health professionals received training in using this intervention. they were also educated on techniques described in handouts, eg, problem solving, behavioral activation, and use of thought diaries to recognize, challenge, and create alternative thoughts. they were not trained in delivering therapy as this intervention only required them to facilitate use of self - help. standard aspects of cognitive therapy, different modules of fcbt - gsh manual, as well as home work assignments were also discussed. health professionals shared the formulation, barriers to home work, and difficulties in following handouts. there is sufficient evidence to suggest that self - help and guided self - help can be effective in helping mental and emotional health problems,6,7 especially for anxiety and eating disorders.12,13 self - help can be defined as : [] a therapeutic intervention that is based on a sound theoretical background, that uses therapeutic principles from an evidence - based intervention, and is administered through a media that does not involve direct contact with another person.13the guided self - help can be defined as self - help that involves facilitation of the self - help by a lay person (eg, a carer) or by a health professional with minimum direct contact (< 25% of the normal therapy session time). contact between the clients and the other person can take place through face - to - face contact, by telephone, by email, or any other communication method.14 [] a therapeutic intervention that is based on a sound theoretical background, that uses therapeutic principles from an evidence - based intervention, and is administered through a media that does not involve direct contact with another person.13 this fcbt - gsh was developed by fn, who has previously adapted cbt for different cultures and settings.1518 the intervention consisted of 24 handouts that could be flexibly used by a health professional. the intervention focused on psychoeducation, symptom management, changing negative thinking, behavioral activation, problem solving, improving relationships, and communication skills. the intervention consisted of essential worksheets (changing negative thinking and information sharing) plus worksheets that can be used flexibly according to a therapy plan that was driven on the basis of individuals particular needs. the intervention was delivered flexibly in that different sessions could be conducted by various team members, based on initial therapy plan. where possible, carers were engaged in this process. a typical session consisted of feedback from the last week, a discussion of barriers, and information about the next handout analyses were carried out using spssv.22 (ibm corp, armonk, ny, usa). spss frequency and descriptive commands spss explore command was used to measure normality of the data using histograms and kolmogorov smirnorov tests. for an intention - to - treat analysis, participants were included in the groups to which they were randomized, regardless of whether they received the treatment allocated to them or not. all continuous variables (eg, the baseline questionnaire scores) were compared using paired t - tests, and categorical variables (eg, gender) were compared using tests. differences in baseline and follow - up questionnaire scores between the two groups at the end of the intervention were compared using a linear regression. analyses were carried out using spssv.22 (ibm corp, armonk, ny, usa). spss frequency and descriptive commands spss explore command was used to measure normality of the data using histograms and kolmogorov smirnorov tests. for an intention - to - treat analysis, participants were included in the groups to which they were randomized, regardless of whether they received the treatment allocated to them or not. all continuous variables (eg, the baseline questionnaire scores) were compared using paired t - tests, and categorical variables (eg, gender) were compared using tests. differences in baseline and follow - up questionnaire scores between the two groups at the end of the intervention were compared using a linear regression. a total of 63 clients were referred for the intervention. out of 48 clients who fulfilled the inclusion criteria during the initial screening, 8 were excluded before baseline interviews were conducted, 4 refused immediately before baseline interview, and the remaining 36 were randomized to two groups. a total of 18 participants were randomized to the treatment group and 18 to the control group. of the 36 participants, 20 (55.6%) were female and 16 (44.4%) were male ; 24 (66.7%) were single, 6 (16.7%) married, and 6 (16.7%) were divorced / widowed or widower. in terms of psychopathology, 3 (8.3%) participants had a diagnosis of an anxiety disorder, 3 (8.3%) of depression, 5 (13.9%) of post - traumatic stress disorder, social anxiety 9 (25.0%), mixed anxiety and depression 8 (22.2%), and panic disorder with agoraphobia 8 (22.2%). table 2 provides additional differences on client variables, and table 3 provides additional differences in psychopathology of the overall sample and between the intervention and control groups at baseline. those in treatment group showed statistically significant improvement in overall psychopathology (core) (p<0.005), anxiety and depression (hads) (p<0.005), and disability (whodas 2.0) (p<0.005) at the end of the trial compared with those in tau group. this study shows that a fcbt - gsh is feasible for crisis and tcm clients and can be potentially effective in improving mental health when compared with only tau. this is the first report of an evaluation of guided self - help for clients attending crisis and tcm services. this is also the first study in which a cognitive behavioral guided self - help was delivered on the basis of the formulation. health professionals started by sharing the formulation with clients, and then developing a shared therapy plan. the case formulation is a vital element of cbt, along with assessment and intervention. a good formulation is the hypothesis about the causes of the problems and why the problem persists.19 the formulation guides the treatment. it is, however, an ongoing process and can be done at various levels, from individual symptom to disorder.20 the formulation is the only way therapy can be individualized and provides an empirical approach to hypothesis testing.21 traditionally, however, self - help and guided self - help do not use a formulation - based approach, and mainly consist of a fixed manual. we therefore decided to use a formulation that was shared and agreed between the health professional and the clients based on the client s specific needs. the health professionals working with crisis and tcm services could use the current findings in several ways. this study shows that it is feasible to deliver cbt based self help and guided self help through the frontline clinicians. a formulation can help clients understand their problems in an easy to understandable language by linking their thoughts, emotions, physical sensations, and behaviors with the events in their lives. such evidence - based interventions may motivate clients and give them hope and may also increase the clinician s feelings of being effective in providing clients with an intervention. it is also highly likely that the health professionals will feel empowered and be more effective in helping their clients by having a better understanding of their problem as a result of the positive responses from patients. although these results raise hope that such low - cost interventions can become part of the routine care, caution is warranted as no prior power calculations were conducted for this pilot study. this is a new intervention, and ideally data on patient fidelity to different components of the intervention and its relation to treatment outcomes should have been conducted. this is especially important since a formulation was the basis of this guided self - help. future research should test this intervention in well - designed studies with adequate sample sizes. this small scale study shows that cbt based self - help and guided self - help can be delivered by the front line clinicians working in crisis and tcm services. this is however, only a small scale pilot study and there is a need to conduct large scale studies with improved methodology.	backgroundcognitive behavioral therapy (cbt) is found to be effective for common mental disorders and has been delivered in self - help and guided self - help formats. crisis and transitional case management (tcm) services play a vital role in managing clients in acute mental health crises. it is, therefore, an appropriate setting to try cbt in guided self - help format.methodsthis was a preliminary evaluation of a formulation - driven cognitive behavioral guided self - help. thirty - six (36) consenting participants with a diagnosis of nonpsychotic illness, attending crisis and the tcm services in kingston, canada, were recruited in this study. they were randomly assigned to the guided self - help plus treatment as usual (tau) (treatment group) or to tau alone (control group). the intervention was delivered over 812 weeks. assessments were completed at baseline and 3 months after baseline. the primary outcome was a reduction in general psychopathology, and this was done using clinical outcomes in routine evaluation outcome measure. the secondary outcomes included a reduction in depression, measured using the hospital anxiety and depression scale, and reduction in disability, measured using the world health organization disability assessment schedule 2.0.findingsparticipants in the treatment group showed statistically significant improvement in overall psychopathology (p<0.005), anxiety and depression (p<0.005), and disability (p<0.005) at the end of the trial compared with tau group.conclusiona formulation - driven cognitive behavioral guided self - help was feasible for the crisis and tcm clients and can be effective in improving mental health, when compared with tau. this is the first report of a trial of guided self - help for clients attending crisis and tcm services.
details of the patient population, along with inclusion and exclusion criteria, have been previously described. in brief, patients 18 years of age with active up or ups extending 5 cm, but no further than 40 cm, from the anal verge were eligible for enrollment in the studies (clinicaltrials.gov : nct01008410 and nct01008423). patients had a baseline modified mayo disease activity index (mmdai) score 5 but 10, with subscale ratings 2 for rectal bleeding and endoscopic appearance. the mmdai score is the sum of 4 subscale scores : stool frequency, rectal bleeding, endoscopic appearance, and physician 's global assessment. since publication of the original mmdai, the endoscopic appearance subscale was modified to classify patients with any degree of friability with a subscale score of 2. exclusion criteria included the use of systemic, oral, topical, or rectal corticosteroids or laxatives or enemas (other than mesalamine) during the preceding 14 days. concomitant use of a stable oral 5-asa regimen was permitted at doses up to 4.8 g / d. rectal 5-asa use was discontinued during the run - in phase of the study (i.e., 47 days before randomization) and was not permitted during the studies. all authors had full access to the study data and reviewed and approved the final manuscript. details of the study design have been previously described. in brief, 2 identically designed, phase 3, randomized, double - blind, placebo - controlled, multicenter studies were conducted in the united states and russia (bucf3001 [clinicaltrials.gov identifier nct01008410 ] and bucf3002 [clinicaltrials.gov identifier nct01008423 ]). each study was comprised of a 2-week screening phase, a run - in / stabilization phase of 4 to 7 days, a 6-week treatment phase, and a 2-week follow - up phase (fig., patients received single - blind placebo rectal foam twice daily, to familiarize themselves with study drug administration. in the treatment phase of each study, patients were randomized 1:1 to receive budesonide rectal foam 2 mg/25 ml twice daily for 2 weeks, then once daily for 4 weeks, or matching placebo. a colonoscopy was required for patients with newly diagnosed uc or without a confirmed diagnosis of uc within 12 months of the screening visit. colonoscopy, if necessary, was performed no greater than 10 days and no less than 4 days before randomization. for patients not meeting the requirement for colonoscopy,, twice daily ; eos, end of study ; eot, end of trial ; q.d. subgroup analyses for the primary, key secondary, and safety endpoints were performed for patients with baseline oral 5-asa use (i.e., at the time the first study dose [budesonide foam or placebo ] was received) during the studies. the primary efficacy endpoint was the percentage of patients achieving remission (defined as mmdai endoscopy subscale score 1, mmdai rectal bleeding subscale score 0, and improvement or no change from baseline in mmdai stool frequency subscale score) at week 6. scores ranged from 0 to 3 for each mmdai subscale (endoscopy subscale score : 0 = normal or inactive disease, 1 = mild disease, 2 = moderate disease, 3 = severe disease ; rectal bleeding subscale score : 0 = no blood seen, 1 = streaks of blood with stool less than half the time, 2 = obvious blood with stool most of the time, 3 = blood alone passed ; stool frequency subscale score : 0 = normal number of stools per d for each individual patient, 1 = 1 to 2 stools more than normal, 2 = 3 to 4 stools more than normal, 3 = 5 stools more than normal ; physician 's global assessment subscale score : 0 = normal, 1 = mild disease, 2 = moderate disease, 3 = severe disease). endoscopic disease extent and activity were determined by local investigators. key secondary efficacy endpoints included the percentage of patients achieving an mmdai rectal bleeding subscale score of 0 and the percentage of patients achieving an mmdai endoscopy subscale score 1 at week 6. safety endpoints included monitoring of adverse events (aes), laboratory parameters (including morning cortisol concentrations and adrenocorticotropic hormone challenge tests), and vital signs. the safety population included all patients in the intent - to - treat population who received 1 dose of study drug. details of the patient population, along with inclusion and exclusion criteria, have been previously described. in brief, patients 18 years of age with active up or ups extending 5 cm, but no further than 40 cm, from the anal verge were eligible for enrollment in the studies (clinicaltrials.gov : nct01008410 and nct01008423). patients had a baseline modified mayo disease activity index (mmdai) score 5 but 10, with subscale ratings 2 for rectal bleeding and endoscopic appearance. the mmdai score is the sum of 4 subscale scores : stool frequency, rectal bleeding, endoscopic appearance, and physician 's global assessment. since publication of the original mmdai, the endoscopic appearance subscale was modified to classify patients with any degree of friability with a subscale score of 2. exclusion criteria included the use of systemic, oral, topical, or rectal corticosteroids or laxatives or enemas (other than mesalamine) during the preceding 14 days. concomitant use of a stable oral 5-asa regimen was permitted at doses up to 4.8 g / d. rectal 5-asa use was discontinued during the run - in phase of the study (i.e., 47 days before randomization) and was not permitted during the studies. all authors had full access to the study data and reviewed and approved the final manuscript. details of the study design have been previously described. in brief, 2 identically designed, phase 3, randomized, double - blind, placebo - controlled, multicenter studies were conducted in the united states and russia (bucf3001 [clinicaltrials.gov identifier nct01008410 ] and bucf3002 [clinicaltrials.gov identifier nct01008423 ]). each study was comprised of a 2-week screening phase, a run - in / stabilization phase of 4 to 7 days, a 6-week treatment phase, and a 2-week follow - up phase (fig., patients received single - blind placebo rectal foam twice daily, to familiarize themselves with study drug administration. in the treatment phase of each study, patients were randomized 1:1 to receive budesonide rectal foam 2 mg/25 ml twice daily for 2 weeks, then once daily for 4 weeks, or matching placebo. a colonoscopy was required for patients with newly diagnosed uc or without a confirmed diagnosis of uc within 12 months of the screening visit. colonoscopy, if necessary, was performed no greater than 10 days and no less than 4 days before randomization. for patients not meeting the requirement for colonoscopy, a sigmoidoscopy was scheduled 4 to 7 days before randomization. study design. b.i.d., twice daily ; eos, end of study ; eot, end of trial ; q.d. subgroup analyses for the primary, key secondary, and safety endpoints were performed for patients with baseline oral 5-asa use (i.e., at the time the first study dose [budesonide foam or placebo ] was received) during the studies. the primary efficacy endpoint was the percentage of patients achieving remission (defined as mmdai endoscopy subscale score 1, mmdai rectal bleeding subscale score 0, and improvement or no change from baseline in mmdai stool frequency subscale score) at week 6. scores ranged from 0 to 3 for each mmdai subscale (endoscopy subscale score : 0 = normal or inactive disease, 1 = mild disease, 2 = moderate disease, 3 = severe disease ; rectal bleeding subscale score : 0 = no blood seen, 1 = streaks of blood with stool less than half the time, 2 = obvious blood with stool most of the time, 3 = blood alone passed ; stool frequency subscale score : 0 = normal number of stools per d for each individual patient, 1 = 1 to 2 stools more than normal, 2 = 3 to 4 stools more than normal, 3 = 5 stools more than normal ; physician 's global assessment subscale score : 0 = normal, 1 = mild disease, 2 = moderate disease, 3 = severe disease). key secondary efficacy endpoints included the percentage of patients achieving an mmdai rectal bleeding subscale score of 0 and the percentage of patients achieving an mmdai endoscopy subscale score 1 at week 6. safety endpoints included monitoring of adverse events (aes), laboratory parameters (including morning cortisol concentrations and adrenocorticotropic hormone challenge tests), and vital signs. the safety population included all patients in the intent - to - treat population who received 1 dose of study drug. of the patients receiving budesonide foam (n = 267) and placebo (n = 279) in the pooled intent - to - treat population, 147 (55.1%) and 154 (55.2%) patients, respectively, reported baseline 5-asa use during the double - blind treatment phase of the studies. there were no significant differences in demographic and baseline characteristics across groups, regardless of baseline 5-asa use (table 1). demographic and baseline characteristics (intent - to - treat population) significant treatment effects favored budesonide foam versus placebo for the percentage of patients achieving remission (primary endpoint), as well as patients with an mmdai rectal bleeding subscale score of 0 at week 6 (key secondary endpoint), regardless of whether patients reported baseline 5-asa use (fig. 2). treatment with budesonide foam induced remission in a significantly greater percentage of patients with or without baseline oral 5-asa use compared with placebo at week 6 (5-asa, 42.2% versus 31.8%, respectively ; p = 0.03 : no 5-asa, 40.0% versus 14.4% ; p < 0.0001). a significantly greater percentage of patients receiving budesonide foam achieved an mmdai rectal bleeding subscale score of 0, regardless of baseline oral 5-asa use at week 6 (5-asa, 50.3% versus 35.7% ; p = 0.003 : no 5-asa, 45.8% versus 19.2% ; p < 0.0001). a significant treatment effect for achieving an mmdai endoscopy subscale score of 0 or 1 was observed in patients receiving budesonide foam versus placebo without baseline use of oral 5-asas (55.8% versus 31.2% ; p = 0.0004) ; however, while treatment with budesonide foam was favored compared with placebo, the treatment difference for the subgroup of patients with baseline oral 5-asa use did not reach statistical significance (55.8% versus 46.8% ; p = 0.08). furthermore, although the rates of response for the 2 subgroups of patients (baseline 5-asa use versus no 5-asa use) were comparable across the primary and key secondary efficacy endpoints, the treatment effect favoring budesonide foam was relatively greater in the subgroup of patients without baseline 5-asa use. patients with baseline oral 5-asa use had greater response rates with placebo compared with patients without baseline oral 5-asa use. the safety profile of budesonide foam was comparable between patients with and without baseline oral 5-asa use during the studies (table 2). the most common aes (2% in the budesonide foam group) occurred with comparable frequency in patients with and without baseline 5-asa use : blood cortisol decreased (20% versus 14%, respectively), adrenal insufficiency (3% versus 4%), headache (3% versus 2%), and nausea (1% versus 3%). of the patients receiving budesonide foam (n = 267) and placebo (n = 279) in the pooled intent - to - treat population, 147 (55.1%) and 154 (55.2%) patients, respectively, reported baseline 5-asa use during the double - blind treatment phase of the studies. there were no significant differences in demographic and baseline characteristics across groups, regardless of baseline 5-asa use (table 1). significant treatment effects favored budesonide foam versus placebo for the percentage of patients achieving remission (primary endpoint), as well as patients with an mmdai rectal bleeding subscale score of 0 at week 6 (key secondary endpoint), regardless of whether patients reported baseline 5-asa use (fig. 2). treatment with budesonide foam induced remission in a significantly greater percentage of patients with or without baseline oral 5-asa use compared with placebo at week 6 (5-asa, 42.2% versus 31.8%, respectively ; p = 0.03 : no 5-asa, 40.0% versus 14.4% ; p < 0.0001). a significantly greater percentage of patients receiving budesonide foam achieved an mmdai rectal bleeding subscale score of 0, regardless of baseline oral 5-asa use at week 6 (5-asa, 50.3% versus 35.7% ; p = 0.003 : no 5-asa, 45.8% versus 19.2% ; p < 0.0001). a significant treatment effect for achieving an mmdai endoscopy subscale score of 0 or 1 was observed in patients receiving budesonide foam versus placebo without baseline use of oral 5-asas (55.8% versus 31.2% ; p = 0.0004) ; however, while treatment with budesonide foam was favored compared with placebo, the treatment difference for the subgroup of patients with baseline oral 5-asa use did not reach statistical significance (55.8% versus 46.8% ; p = 0.08). furthermore, although the rates of response for the 2 subgroups of patients (baseline 5-asa use versus no 5-asa use) were comparable across the primary and key secondary efficacy endpoints, the treatment effect favoring budesonide foam was relatively greater in the subgroup of patients without baseline 5-asa use. patients with baseline oral 5-asa use had greater response rates with placebo compared with patients without baseline oral 5-asa use. the safety profile of budesonide foam was comparable between patients with and without baseline oral 5-asa use during the studies (table 2). the most common aes (2% in the budesonide foam group) occurred with comparable frequency in patients with and without baseline 5-asa use : blood cortisol decreased (20% versus 14%, respectively), adrenal insufficiency (3% versus 4%), headache (3% versus 2%), and nausea (1% versus 3%). the overall results of the 2 identically designed, randomized, placebo - controlled studies demonstrated that a significantly greater percentage of patients with active, mild to moderate up or ups receiving budesonide foam achieved remission at week 6 (primary efficacy endpoint) compared with placebo (41.2% versus 24.0%, respectively ; p < 0.0001). furthermore, a significantly greater percentage of patients receiving budesonide foam achieved secondary outcome measures of rectal bleeding resolution (48.3% versus 28.3% ; p < 0.0001) and endoscopic improvement (55.8% versus 39.8% ; p = 0.0002). the results of this analysis suggest that budesonide foam was efficacious and safe for induction of remission of mild to moderate up or ups in patients receiving oral 5-asa at baseline and those who were not. furthermore, baseline oral 5-asa use had no apparent effect on the safety profile of budesonide foam. baseline oral 5-asa use has been reported in a number of clinical studies of rectal therapies for the induction of remission of mild to moderate distal forms of uc ; however, these studies did not perform further efficacy or safety analyses in patients with baseline use of oral 5-asas. in a double - blind, double - dummy, randomized, comparative study of budesonide foam and budesonide enema in patients with active up or ups, response or lack of response to oral 5-asa therapy had no significant effect on the percentage of patients in clinical remission with either treatment after 4 weeks. furthermore, results of a randomized, double - blind, placebo - controlled study of patients with extensive mild to moderate uc demonstrated that concomitant treatment with oral and rectal 5-asas improved efficacy compared with oral 5-asas alone after 4 and 8 weeks. these studies suggest that baseline oral 5-asa use does not aberrantly affect the efficacy of rectal therapies in patients with up or ups. rectal 5-asas are currently the first - line treatment for patients with active, mild to moderate up or ups. pooled analysis demonstrated that treatment with rectal 5-asas was associated with significant symptomatic improvement, endoscopic improvement, and histologic improvement, as well as symptomatic remission, endoscopic remission, and histologic remission, compared with placebo in patients with distal forms of uc. however, the second - generation corticosteroid budesonide foam has demonstrated efficacy and safety in patients with mild to moderate up and ups, thus providing patients who fail treatment with rectal 5-asas a safe and efficacious therapeutic option. furthermore, patients with up or ups preferred foam to enema (83.6% versus 6.2%, respectively) in a head - to - head comparison study of these budesonide formulations. the findings of this study further support the use of budesonide foam in patients with active mild to moderate up or ups, regardless of baseline oral 5-asa use. limitations of this study include the fact that further analyses stratified by the various formulations of 5-asa and by the range of 5-asa doses were not conducted because of sample size limitations. although the combination of oral and rectal 5-asa therapies had greater efficacy than oral or rectal 5-asa monotherapy for the treatment of mild to moderate up or ups, it is possible that some patients in this study had recently discontinued rectal 5-asa monotherapy. whereas the findings of this analysis favored treatment with budesonide foam, regardless of whether patients were using 5-asas, additional prospective studies are warranted to further examine the potential benefit of oral 5-asas or other oral agents (e.g., budesonide multi - matrix) in combination with budesonide foam in patients with mild to moderate up or ups. in conclusion, budesonide foam was efficacious and well tolerated for the induction of remission in patients with active, mild to moderate up and ups receiving oral 5-asas at baseline, as well as in those who were not.	background : rectal budesonide foam is a second - generation corticosteroid efficacious for active mild to moderate ulcerative proctitis and ulcerative proctosigmoiditis. this subgroup analysis examined the impact of baseline oral 5-aminosalicylic acid (5-asa) on the efficacy and safety of budesonide foam in patients with mild to moderate ulcerative proctitis or ulcerative proctosigmoiditis.methods:patients received budesonide foam 2 mg/25 ml twice daily for 2 weeks, then once daily for 4 weeks, or placebo, with or without continued stable dosing of baseline oral 5-asas, for remission induction at week 6 (primary endpoint) in 2 identically designed, randomized, double - blind, phase 3 studies.results:of the 267 and 279 patients randomized to treatment with budesonide foam or placebo (pooled population), 55.1% and 55.2%, respectively, reported baseline 5-asa use. a significantly greater percentage of patients achieved remission with budesonide foam versus placebo, either with (42.2% versus 31.8%, respectively ; p = 0.03) or without (40.0% versus 14.4% ; p < 0.0001) baseline 5-asa use at week 6. a significantly greater percentage of patients achieved a modified mayo disease activity index rectal bleeding subscale score of 0 at week 6, regardless of baseline 5-asa use (5-asa, 50.3% versus 35.7% ; p = 0.003 : no 5-asa, 45.8% versus 19.2% ; p < 0.0001). the frequency of adverse events was comparable between groups, regardless of baseline 5-asa use.conclusions:budesonide foam was efficacious and safe for induction of remission of mild to moderate ulcerative proctitis and ulcerative proctosigmoiditis in patients receiving oral 5-asa at baseline and those who were not (clinicaltrials.gov : nct01008410 and nct01008423).
this study was conducted in accordance with a protocol approved by an institutional animal care and use committee of the national institutes of health. all laboratory work with potentially infectious materials was conducted in a biosafety level facility at the rocky mountain laboratories (division of intramural research / national institute of allergy and infectious diseases / national institutes of health). three cynomologus macaques were vaccinated with 1 dose of 10 pfu of vsvg / lasvgpc, a live - attenuated, recombinant viral vaccine in which the vsv surface glycoprotein has been replaced with those of lasv, by intramuscular injection as described (4). another age - matched control animal was vaccinated with an irrelevant vsv - based vaccine (vsvg / andvgpc) (5). at 28 days postvaccination, the 4 nhps were challenged with a lethal dose of lasv (10 50% tissue culture infectious doses [tcid50s ]) (9). the control animal showed signs of lassa fever 710 days postinoculation and was euthanized 13 days postchallenge because of severity of disease. classic indicators of lassa fever, including decreased total protein and albumin ; increased serum levels of alanine aminotransferase, aspartate aminotransferase, amylase, blood urea nitrogen, and alkaline phosphatase ; and hematologic abnormalities, including thrombocytopenia and lymphopenia, were apparent in this animal. virus isolation conducted for select tissue samples showed lasv titers of 57 log10 tcid50/g of tissue ; blood samples collected on day 10 and at the time of euthanasia (5 and 6.25 log10 tcid50/ml, respectively) showed viremia. in contrast, the 3 animals vaccinated with vsvg / lasvgpc resisted lethal lasv challenge and did not demonstrate any clinical signs of disease or any hematologic or biochemical indicators of lasv infection. at no point in the study was virus found in blood samples collected regularly from these 3 animals, even when tested by sensitive reverse transcription pcrs. an elisa with serum samples collected 45 days postchallenge demonstrated equivocal antibody titers (100) against a recombinant lasv nucleocapsid protein in 1 nhp. the other 2 animals did not show seroconversion, which suggested that vaccination caused nearly sterile immunity against lasv (table). vsv, vesicular stomatitis virus ; vhf, viral hemorrhagic fever ; lasv, lassa virus ; gpc, glycoprotein precursor ; np, nucleocapsid protein ; ebov, ebola virus ; gp, glycoprotein ; vp40 viral protein 40 ; nd, not determined. titers are indicated as reciprocal endpoint dilutions from elisas for recombinant antigens (lasv np, ebov gp, and vp40), or whole virus preparations (vsv). approximately 90 days after the original vaccination with vsvg / lasvgpc, the 3 nhps were vaccinated with a single dose of 10 pfu of vsvg / ebovgp by intramuscular injection as described (3). an additional nhp was vaccinated with a control vaccine as outlined above and served as the inoculation control. at the time of vaccination, the 3 macaques had a robust vsv - specific antibody response with titers of 25,600, as determined by a whole virus elisa (table). despite this finding, the 3 animals that received the vsv - based ebov vaccine mounted an efficient response to the ebov glycoprotein (table) and were completely protected when challenged 28 days later with a lethal dose of ebov (10 pfu) (10). hematologic and serum biochemistry values remained constant throughout the study, and virus was not found in blood samples collected regularly and tested by using real - time reverse transcription pcr. in contrast, severe ebov hemorrhagic fever developed in the control animal, which was characterized by increased serum concentrations of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase ; thrombocytopenia ; and viremia (7 log10 tcid50/ml whole blood) beginning 36 days postchallenge. this animal was euthanized 7 days postchallenge, and titration of selected tissue samples showed ebov titers of > 9 log10 tcid50/g tissue. an elisa conducted at the conclusion of the study (42 days post ebov challenge) showed increased antibody responses to vsv (titers = 102,400) and seroconversion to the ebov viral protein 40 antigen (titers 1,6006,400) in the 3 surviving nhps (table), which is consistent with published results (10). because of the remote locations where vhf agents are present, an overall shortage of health care professionals and clinics in these locations, and mobility of human populations, any vaccine against these pathogens would ideally need to elicit a protective immune response after a single vaccination. for this reason, replication - competent viral vectors are considered leading vhf vaccine candidates. as the vsvg / ebovgp vaccine heads toward clinical trials, it is necessary to clarify the potential limitations of using the vsv platform against multiple vhf agents. a major drawback for many viral vector platforms is preexisting immunity against the vector itself, which can decrease or nullify the essential protective immune response, which results in vaccine failure. design of the vsv - based vaccines, which encode and express glycoproteins from various pathogens without its own glycoprotein (11,12), suggest that preexisting immunity would not influence protective efficacy of individual vaccinations (12). results of this study demonstrate that multiple vsv vaccines can be used in a population without any deleterious effect on overall protective efficacy.	we demonstrated that previous vaccination with a vesicular stomatitis virus (vsv)based lassa virus vaccine does not alter protective efficacy of subsequent vaccination with a vsv - based ebola virus vaccine. these findings demonstrate the utility of vsv - based vaccines against divergent viral pathogens, even when preexisting immunity to the vaccine vector is present.
early and accurate assessment of the thoracolumbar spine is an important aspect of trauma reception and resuscitation. between 19% and 50% of fractures may have associated neurological damage to the spinal cord.1 missed or delayed diagnosis can lead to a multitude of problems including long - term pain, reduced quality of life, and can have devastating psychological effects. it is widely recognised that blunt trauma patients with altered mental status require imaging of the cervical and thoracolumbar spine, as clinical signs and symptoms can be unreliable.2 6 in patients with a glasgow coma scale (gcs) score of 15, much work has been done with regard to clearing the cervical spine of significant injury with established and validated gudelines.7 8 to date, there are only a few small prospective studies5 6 9 10 and a number of small retrospective analyses directed at the assessment of the thoracolumbar spine.24 1114 differences in clinical anatomy make direct translation of the results from studies on the cervical spine unreliable. the greater mass, longer distance from spinous processes to anterior body and relative immobility of the thoracolumbar vertebrae compared to the cervical vertebrae are key differences. despite these differences in a recent systematic review it has been proposed that those patients who are awake, without evidence of intoxication, with normal mental, neurological and physical examinations can be cleared clinically.15 the aim of this study was to test this hypothesis by looking at patients presenting with thoracolumbar fractures with a gcs score of 15. the alfred hospital is one of two adult tertiary trauma referral centres in melbourne, australia, and serves the statewide population of victoria of approximately five million. it has an annual emergency department (ed) census of over 45 000 patients, with more than 1200 major trauma (injury severity score (iss) > 15) admissions per annum. the diagnostic imaging evaluation of the thoracolumbar spine of patients presenting to the emergency and trauma centre includes anteroposterior and lateral views of the thoracic and lumbar spine. patients with a higher suspicion of fracture or those with pain or tenderness undergo ct scanning of the thoracic and/or lumbar spine. patients already having ct scanning of the chest or abdomen have reformatted images of the thoracic and lumbar spine developed without additional scanning. the ct scanner used was a ge lightspeed vct 64-slice scanner (general electrical company, ge healthcare, milwaukee, usa). the alfred trauma registry, funded as part of the victorian state trauma system, collects trauma data concurrent with the inpatient episode. data are collected according to a defined dataset by experienced registry staff and regularly audited. the registry collects data on all patients admitted for more than 24 h to the alfred trauma service, trauma patients with an iss of more than 15 and patients admitted for over 72 h post - injury admitted under other units. patients with iss less than 15 and isolated trauma to the vertebrae were therefore included, provided they spent over 72 h in hospital. all patients diagnosed with a thoracolumbar fracture presenting to the ed between january 2006 and december 2008 were identified from the alfred trauma database and included in the this study. data collected from the trauma registry included patient demographics, mechanism of injury, the first recorded gcs score on arrival to the ed, abbreviated injury scale codes with descriptions and iss. a subgroup of patients with an initial gcs score of 15 was identified for analysis. a retrospective explicit chart review of these patient records was conducted by dsg and fr and audited by a third operator (bm). any records with ambiguous, missing or unknown data were reviewed by all three operators and discussed at study coordination meetings held at regular intervals. thoracolumbar pain was coded as a symptom if mentioned in either the initial assessment notes of ambulance personnel, nursing or medical staff. data on prehospital analgesia given and finding of tenderness on log - roll during secondary survey were gathered from chart reviews, while blood alcohol levels were obtained from the alfred pathology service. continuous data are presented as mean with sd, whereas ordinal data are presented as medians with interquartile ranges. student 's t test was used to calculate the significance between two continuous variables, whereas the wilcoxon rank sum test was used to compare ordinal variables. the alfred hospital is one of two adult tertiary trauma referral centres in melbourne, australia, and serves the statewide population of victoria of approximately five million. it has an annual emergency department (ed) census of over 45 000 patients, with more than 1200 major trauma (injury severity score (iss) > 15) admissions per annum. the diagnostic imaging evaluation of the thoracolumbar spine of patients presenting to the emergency and trauma centre includes anteroposterior and lateral views of the thoracic and lumbar spine. patients with a higher suspicion of fracture or those with pain or tenderness undergo ct scanning of the thoracic and/or lumbar spine. patients already having ct scanning of the chest or abdomen have reformatted images of the thoracic and lumbar spine developed without additional scanning. the ct scanner used was a ge lightspeed vct 64-slice scanner (general electrical company, ge healthcare, milwaukee, usa). the alfred trauma registry, funded as part of the victorian state trauma system, collects trauma data concurrent with the inpatient episode. data are collected according to a defined dataset by experienced registry staff and regularly audited. the registry collects data on all patients admitted for more than 24 h to the alfred trauma service, trauma patients with an iss of more than 15 and patients admitted for over 72 h post - injury admitted under other units. patients with iss less than 15 and isolated trauma to the vertebrae were therefore included, provided they spent over 72 h in hospital. all patients diagnosed with a thoracolumbar fracture presenting to the ed between january 2006 and december 2008 were identified from the alfred trauma database and included in the this study. data collected from the trauma registry included patient demographics, mechanism of injury, the first recorded gcs score on arrival to the ed, abbreviated injury scale codes with descriptions and iss. a subgroup of patients with an initial gcs score of 15 was identified for analysis. a retrospective explicit chart review of these patient records was conducted by dsg and fr and audited by a third operator (bm). any records with ambiguous, missing or unknown data were reviewed by all three operators and discussed at study coordination meetings held at regular intervals. thoracolumbar pain was coded as a symptom if mentioned in either the initial assessment notes of ambulance personnel, nursing or medical staff. data on prehospital analgesia given and finding of tenderness on log - roll during secondary survey were gathered from chart reviews, while blood alcohol levels were obtained from the alfred pathology service. continuous data are presented as mean with sd, whereas ordinal data are presented as medians with interquartile ranges. student 's t test was used to calculate the significance between two continuous variables, whereas the wilcoxon rank sum test was used to compare ordinal variables. there were 1161 patients with thoracolumbar fractures over the study period, with a total of 1902 fractures. the average age was 44.318.6 years with a male to female ratio of 2.8 : 1 and a median iss of 16 (922). gcs, glasgow coma scale score. of the 536 patients presenting to ed with a gcs score of 15, 354 (66.0%) patients received prehospital opioid analgesia. the presence of thoracolumbar pain was documented in 325 (60.6%) on initial assessment, while tenderness on log - roll was elicited in 323 (60.3%) patients. a positive blood alcohol level was defined as any value greater than 0 g per 100 ml blood. the sensitivities of prehospital opioid analgesia, pain to thoracolumbar area, tenderness on log - roll and a positive blood alcohol, together with a combination of all variables are presented in table 1. sensitivity of clinical features for thoracolumbar fractures number of patients with documentation. sensitivity (95% ci) of clinical variable subgrouped by mechanism of injury mba, motorbike accident ; mva, motor vehicle accident. all patients with a lumbar vertebral fracture complained of pain and/or received prehospital analgesia and/or had tenderness on log - roll examination. there were 28 (5.2%) patients who were diagnosed with a thoracic vertebral fracture, but received no prehospital opioid analgesia, did not complain of pain on initial presentation and had no tenderness to the thoracolumbar spine elicited on log - roll. the median iss in this group of patients was 13.5 (919), which was not significantly different to the iss for the overall group (p=0.252). there were seven patients who sustained their injury after a low fall (1 m), seven motor vehicle crashes, six motorcycle crashes, three bicycle related and two pedestrians. of these 28 patients, there were 18 patients with a concurrent fracture of the cervical spine, four (14%) had rib fractures and six (22%) had other non - spinal fractures. tertiary survey of these patients revealed tenderness to the thoracolumbar spine in only two cases, both of whom had concurrent cervical spine fractures. non - significant fractures were defined as involving only one column (stable fractures) and not requiring operative fixation. of the above group, 25 (89%) patients sustained non - significant stable fractures including 10 anterior compression fractures, eight superior end plate fractures, four transverse process fractures, one spinous process fracture and two other minor abnormalities. the remaining three (11%) patients sustained clinically significant fractures defined as those involving two or more columns (unstable fractures according to the denis classification)16 or those requiring operative fixation. this included a 25-year - old motorcyclist sustaining a t5 on t6 fracture dislocation, with cord compression, requiring operative fixation. there was also a 75-year - old patient with a three column fracture from a fall from standing height and a 33-year - old pedal cyclist with a two column fracture. neither required operative fixation. this study shows that history and examination could exclude clinically significant fractures of the lumbar vertebrae but could not exclude all fractures of the thoracic vertebrae. in major trauma patients, we have shown that it is possible to exclude clinically significant thoracic fractures based on history and clinical examination in a subgroup of patients. this subgroup was defined by blunt trauma with a gcs score of 15, the absence of documented alcohol or prehospital morphine, the absence of pain on history and tenderness on log - roll, the absence of neurology and the absence of a cervical spine fracture. a very small proportion of patients (4.7%) were found to have a thoracic fracture in the presence of the above criteria, but were limited to those with a single column injury and none required no operative management. painless thoracic vertebral fractures have previously been described,17 and functional assessment using axial loaded movements have been proposed to determine clinical significance. we suggest that should these patients subsequently develop pain, they should be re - examined and have imaging to exclude stable spine injury. the most devastating clinical consequence of a missed or delayed diagnosis of a thoracolumbar vertebral fracture is the onset and progression of neurological deficits,18 19 due to movement at the fracture site, soft tissue swelling, or the development of an epidural haematoma secondary to prophylactic anticoagulation. however, the risk of this occurring has not been clearly quantified in the literature. there is also the potentially positive impact of diagnostic certainty on recovery, rehabilitation, workers compensation, and psychological wellbeing, which requires further clarification. accurate diagnosis of all injuries remains the ultimate aim during the initial assessment of injured patients. there is currently some evidence suggesting a mortality benefit when all major trauma patients are analysed.20 however, the use of extensive radiography is time consuming, expensive and results in unnecessary radiation exposure, with a potential long - term increased risk of radiation - induced carcinogenesis. the benefit of whole - body ct to trauma patients based on mechanism alone, who exhibit minimal clinical symptoms and signs, remains unknown. adopting a clinical practice of whole - body ct in this subset of patients exposes them to the long - term risks of ionising radiation and is unlikely to be associated with significant benefit. obtaining a history and a thorough clinical examination should still play a central role in the assessment of trauma patients. in our group of patients, the primary difference in excluding thoracolumbar vertebral fractures compared to those of the cervical vertebrae is in the early mobilisation of patients post - history and examination. while the cervical vertebrae may be mobilised effectively in an awake supine patient, axial loading of thoracolumbar vertebrae through mobilisation is the most effective clinical manoeuvre to detect any pain from a stable fracture. second, the presence of a cervical spine fracture has previously been shown to be associated with another spinal fracture,21 and this was confirmed in this study, necessitating imaging of the rest of the spine. the presence of any neurological deficit without pain is a further obvious variable, which necessitates imaging of the thoracolumbar vertebrae. contrary to our findings, a negative physical examination alone has previously been reported to be reliable at excluding injury. samuels and kerstein12 retrospectively reviewed 99 charts, in which 15 patients had thoracolumbar fractures. of the 55 charts in which patients had no pain or tenderness ; there were no missed fractures. however, that series did not report how severely injured patients were and disregarded other factors, which may influence the decision to image the thoracolumbar spine. the findings of our study are similar to others recommending routine imaging of the thoracolumbar spine. frankel found that 40% of 65 patients with fractures had pain or tenderness. these patients had associated injuries and high blood ethanol levels, making it hard to determine the exact reason for the absence of clinical features. cooper reported a review of 183 fractures in 110 patients who were neurologically intact and had a gcs score of 1315. about a third of these patients had no pain or tenderness, had fractures. the absence of clinical findings was significantly related to the presence of another major injury, defined as abbreviated injury scale scores of 3 or more. comparison with the findings of our study is difficult, due the small number of patients reviewed and the inclusion of patients with gcs scores of less than 15 in these studies. to date inclusion criteria to the trauma registry excluded those patients who were discharged within 24 h and those less severely injured. patients with clinically significant thoracolumbar spine fracture are unlikely to be included in the discharged group. with regard to missing data, very few patients did not have findings of pain on log - roll documented. using clinical assessment to determine imaging will miss some thoracic vertebral fractures, but the clinical significance of these fractures is likely to be minimal. the ideal technique of assessing the thoracolumbar spine on log - roll remains unclear.15 being a retrospective review in a large trauma centre, there was a high likelihood of variation in technique for examination ranging from gentle palpation to percussion. this would clearly impact on the presence or absence of tenderness on log - roll. however, it has previously been noted that the log - roll procedure is a team effort and it would be obvious to team members if pain or tenderness were present.15 furthermore, only two patients had tenderness elicited on tertiary survey following a non - tender initial examination, suggesting a measure of agreement. the spectrum of patients presenting to a large trauma centre is likely to be different to a community hospital. although laboratory alcohol levels would not be available immediately, intoxication could be assessed initially based on clinical suspicion or breath alcohol levels. blood alcohol level is a routine test performed on all patients who meet trauma call - out criteria. we can safely conclude that in patients in whom blood alcohol was not measured or was negative ; a gcs score of 15 with history and clinical examination could exclude a clinically significant thoracolumbar spine fracture. if alcohol or non - alcohol intoxicant variables were available for all patients, it would further improve the sensitivity of the rule. the effect of a distracting injury on the assessment of the thoracolumbar spine can not be determined from this retrospective review. terregino 6 looked at 183 clinically evaluable patients, of whom 17 had thoracolumbar fractures, whose only symptoms predictive of a fracture were pain and tenderness. it is possible to exclude a clinically significant lumbar vertebral fracture post - history and examination in a defined group of major trauma patients, but not a thoracic vertebral fracture. a clinically significant thoracic fracture may be excluded in patients with a gcs score of 15, not under the documented influence of alcohol or prehospital morphine, the absence of pain or tenderness on log - roll, the absence of neurology, and the absence of a concurrent cervical vertebral fracture. prospective studies are needed to develop algorithms for evaluating the thoracolumbar vertebrae in blunt trauma, based on the needs and aims of individual trauma systems.	introductionthe aim of this study was to test the hypothesis that all blunt trauma patients, presenting with a glasgow coma scale (gcs) score of 15, without intoxication or neurological deficit, and no pain or tenderness on log - roll can have any thoracolumbar fracture excluded without imaging.materials and methodsall patients diagnosed with a thoracolumbar fracture presenting to the emergency department of a major trauma centre and having an initial gcs score of 15 were included in the study. variables collected included type of fracture, mechanism of injury, the presence of pain or tenderness on log - roll, ethanol levels and prehospital opioid analgesia.resultsthere were 536 patients with thoracolumbar fractures, of which 508 (94.8%) patients had either pain, tenderness or had received prehospital opioid analgesia. a small subgroup of 28 (5.2%) patients who received no prehospital opioid analgesia, did not complain of pain and had no tenderness to the thoracolumbar spine elicited on log - roll. this subgroup was significantly older (p=0.033) and a high proportion of patients (64.3%) had a concurrent fracture of the cervical spine. within this subgroup, a clinically significant unstable thoracic fracture was present in three patients, with all three patients exhibiting symptoms and signs of neurological injury or having a concurrent cervical vertebral fracture.conclusionsin this population of blunt trauma patients with a gcs score of 15, not under the influence of alcohol or prehospital morphine administration, the absence of pain or tenderness on log - roll can exclude a clinically significant lumbar vertebral fracture, but does not exclude a thoracic fracture.
the presence of 0.05). the pesa sub - group outcomes were significantly better than those of tese, particularly in terms of pregnancy rate and embryo quality rate (p < 0.05) (figure 1). icsi is indicated in the treatment of male infertility due to severe oligospermia and cryptozoospermia (12). currently, the standard of care for sperm source selection remains unclear. in this study, we compared freshly ejaculated sperm pregnancy outcomes with tese and pesa ejaculated sperm using icsi in this population. in our study, the clinical pregnancy outcome in group 2 (pesa or tese) was higher than that of the control group. the two highest quality embryos from both groups were transferred into the uterus on day 3 although others prefer 3 days to 5 days transfer according to embryo selection criteria (13). with the advent of art and icsi in the 1990s, there were significant improvements in fertility rates (14). severe oligoasthenozoospermia, cryptozoospermia, and non - obstructive azoospermic patients can be successfully cured with icsi (15). in cryptozoospermic cases, only very meticulous methods of sperm collection with repeated semen samples allow the retrieval of a rare sperm. in addition, the absence of or insufficient number of spermatozoa in the semen of these patients on the day of the ovum retrieval can lead to the cancellation of an icsi procedure. these situations require sperm extraction from the epididymis or testis, or in some cases, ovum cryopreservation in the next time of successfully ejaculated sperm. in order to avoid the cancellation of icsi in these cases, most infertility centers provide an alternative surgically sperm extraction (pesa or tese) (10) synchronous to oocyte retrieval (16) ; however, there is a debate about outcomes of ejaculated sperm and extracted sperm in literature. therefore, we conducted a prospective cohort study to highlight outcome differences in these two methods of sperm retrieval. although a body of research implies that the freshly ejaculated sperms are better than surgically retrieved sperms for icsi, we found contrary results. reached to this conclusion that the fertilization rates are not affected by the source of the sperm but the quality of embryos gets better with ejaculated sperm when compared with sperm from tese which corroborated in a case report on men with cryptozoospermia (10, 17). nagy. (1995) and aboulghar. (1997) reported that the fertilization and pregnancy rates by testicular spermatozoa were significantly lower than those of ejaculated and epididymal spermatozoa in non - obstructive azoospermia (10, 15). the sperm extraction proportion from testicular and epididymis in our study was similar but fertility outcomes in pesa sub - group was significantly better than outcomes of tese sub - group, similar to the results of alrabeeah. in contrast to our study results, some researchers believe that the fertilization rate of testicular extracted spermatozoa from non - obstructive azoospermic men is comparable to ejaculated spermatozoa in men with unexplained fertility and also stated that the fertilization rate of severe oligospermia or cryptozoospermia is similar to that of testicular spermatozoa source (15, 18, 19). in this study, cleavage rate (74.62% and 83.43%) as well as implantation rate (21.35% and 35.17%) were significantly higher in embryo transfer, similar to the study by popal. embryo quality and hence cleavage rate between two groups were not different, but the quality of embryo in sperm - extracted group was better than that of sperm - ejaculated group (< 0.05). the sperm encounters in the genital tract with hazardous environment, contaminating cells, and biochemical agents (e.g. reactive oxygen species). also, some preliminary researches have shown the sperm may damage when passing through the male reproductive tract by decreasing the quality of sperms in terms of dna integrity (6, 20, 21). we demonstrated that the surgically extracted sperm will do better than ejaculated sperm in icsi and improve pregnancy outcomes in men with idiopathic cryptozoospermia.	backgroundcryptozoospermia (co) is a situation in which spermatozoa can not be observed in a fresh semen sample unless an extended centrifugation and microscopic search are performed. co patients are suggested to use only intracytoplasmic sperm injection (icsi) as infertility treatment. but still there is debate about the choice of sperm source in cryptozoospermic men candidate for icsi.objectivesthis study was conducted to evaluate fertility outcomes in men with idiopathic cryptozoospermia who were treated using icsi with freshly ejaculated sperm and testis sperm extraction (tese) or percutaneous epididymal sperm aspiration (pesa).methodsin this prospective cohort study carried out in an academic institution, 83 out of 92 couples with cryptozoospermia undergoing their first icsi cycle were recruited. these patients were randomly allocated to two groups : group one (n = 42) who produced freshly ejaculated sperm and, group two (n = 41) who produced a sample by tese or pesa. the groups were analyzed and compared in terms of fertilization rate, cleavage rate, embryo quality, implantation rate, and clinical pregnancy rate.resultsthere was a significant difference in fertilization rate, embryo quality, implantation rate, and pregnancy rates between the group of surgically extracted sperm and those of naturally ejaculated sperm using conventional icsi (p < 0.05).conclusionssperm quality extracted by percutaneous pesa and tese procedures increases fertility outcomes compared to naturally ejaculated sperm in men with idiopathic co. more specifically, embryo quality, which is most relevant to fertility outcome, improved when surgically extracted sperm was used for icsi.
external ventricular drainage (evd) is an important neuro - surgical procedure performing under emergent conditions. the ideal target for ventricular placement is usually within the ipsilateral frontal horn just anterior to the monro foramen2). even though the freehand technique using superficial anatomical landmarks is traditional and generally accepted method for evd, the accuracy rate of evd catheter placement has been reported about 39.9% to 84%3457). evd tips locating in nonventricular space have been reported to be 8.2% to 22.4%457). in 1985, the ghajar guide was introduced for ventricular catheter placement1), but it was unfamiliar with most neurosurgeon. currently, navigation guided evd may increase the accuracy of placement of evd, but it requires a lot of time and general anesthesia. thus, it is not suitable and reasonable in consideration of an emergent procedure and cost effectiveness of evd. this study was approved by the institutional review board for the medical instrument and its registration number is 2009 - 08. it was designed to direct a ventricular catheter along a course pointing the inner canthus and the tragus. it was composed of three portions, main body, rectangular pillar and an arm pointing the tragus. main body shaped a large letter t which was composed of horizontal and vertical portions. vertical portion has a role to direct a ventricular catheter toward the right and left inner canthi, respectively, and horizontal portion has a shallow longitudinal opening to connect the rectangular pillar and move it back and forth. rectangular pillar is 228 cm in size and has a longitudinal central hole of 4 mm in diameter to insert an evd catheter into the frontal horn of the lateral ventricle. vertical portion pointing the inner canthus was made to be placed coaxially with the central hole of the pillar. on the lateral surface of this pillar, there is a longitudinal groove running parallel with the central hole of the pillar to insert the arm pointing the tragus (fig. the rectangular pillar was connected with the horizontal portion of main body through its opening using a screw. the arm pointing the tragus was inserted into the longitudinal groove of the pillar. to position the tip of verticalportion toward the inner canthus, the main body was slightly moved from side to side centering the burr hole. the direction of the arm pointing the tragus was also controlled by back and forth movement and turn of the pillar attached to the main body. evd catheter through the central hole of the pillar was finally inserted into the ventricle in depth of 5.5 cm from the dura mater (fig. if the thick blood had been pushed out of the ventricle through the evd catheter due to the increased intracranial pressure in a case of intraventricular hemorrhage (ivh), ivh was aspirated using a syringe of 10 cc. between april 2012 and december 2014, 57 emergency evds were performed in 52 patients using this device in the operating room. admission diagnoses in 52 patients were aneurysmal subarachnoid hemorrhage in 21, intracerebral hemorrhage (ich) associated with ivh in 21 (thalamus 15, caudate nucleus 5, pons 1), pure ivh in 2, moyamoya disease in 2, cerebellar infarction in 2, meningitis in 1, ventriculitis in 1, ruptured arteriovenous malformation in 2. bilateral evds were performed in 5 patients who had thalamic hemorrhage and ruptured avm, aneurysmal subarachnoid hemorrhage, respectively. this study was approved by the institutional review board for the medical instrument and its registration number is 2009 - 08. it was designed to direct a ventricular catheter along a course pointing the inner canthus and the tragus. it was composed of three portions, main body, rectangular pillar and an arm pointing the tragus. main body shaped a large letter t which was composed of horizontal and vertical portions. vertical portion has a role to direct a ventricular catheter toward the right and left inner canthi, respectively, and horizontal portion has a shallow longitudinal opening to connect the rectangular pillar and move it back and forth. rectangular pillar is 228 cm in size and has a longitudinal central hole of 4 mm in diameter to insert an evd catheter into the frontal horn of the lateral ventricle. vertical portion pointing the inner canthus was made to be placed coaxially with the central hole of the pillar. on the lateral surface of this pillar, there is a longitudinal groove running parallel with the central hole of the pillar to insert the arm pointing the tragus (fig. the rectangular pillar was connected with the horizontal portion of main body through its opening using a screw. the arm pointing the tragus was inserted into the longitudinal groove of the pillar. to position the tip of verticalportion toward the inner canthus, the main body was slightly moved from side to side centering the burr hole. the direction of the arm pointing the tragus was also controlled by back and forth movement and turn of the pillar attached to the main body. evd catheter through the central hole of the pillar was finally inserted into the ventricle in depth of 5.5 cm from the dura mater (fig. if the thick blood had been pushed out of the ventricle through the evd catheter due to the increased intracranial pressure in a case of intraventricular hemorrhage (ivh), ivh was aspirated using a syringe of 10 cc. between april 2012 and december 2014, 57 emergency evds were performed in 52 patients using this device in the operating room. admission diagnoses in 52 patients were aneurysmal subarachnoid hemorrhage in 21, intracerebral hemorrhage (ich) associated with ivh in 21 (thalamus 15, caudate nucleus 5, pons 1), pure ivh in 2, moyamoya disease in 2, cerebellar infarction in 2, meningitis in 1, ventriculitis in 1, ruptured arteriovenous malformation in 2. bilateral evds were performed in 5 patients who had thalamic hemorrhage and ruptured avm, aneurysmal subarachnoid hemorrhage, respectively. all ventricular punctures were accomplished at one time. catheter tip located in the frontal horn in 52 evds and in the 3rd ventricle in 2, in the wall of the frontal horn of the lateral ventricle in 3. small hemorrhage along to catheter tract occurred in 1 evd. even though 3 evd catheters located in the wall of the frontal horn of lateral ventricle, csf was well drained. even though freehand insertion of an evd using superficial anatomical landmarks is the most common method practiced by young neurosurgical trainees, the catheter tip locations have been reported to be unsatisfactory. evd catheter placement has been reported to be 39.984% in the ipsilateral frontal horn4567), 2.712.4% in the contralateral frontal horn457), 18% in the lateral ventricle body7), 1.810.4% in the subarachnoid space57), approximately 10% in the brain parenchyme57), 1.822.4% in the extraventricular space45), and 8.219.5% in the third ventricle457). they recommended to use neuronavigation, ultrasonography, or other guidance techniques to position the catheter tip accurately in the frontal horn of the lateral ventricle. to increase the accuracy of ventriculostomy at kocher 's point, it was designed to direct a catheter along a course that lies at a right angle to the cranial surface. this device is rigid and consists of three equal - length standards that are applied to the patient 's scalp and a central tube at the apex of the formed pyramid for passage of the catheter. currently, navigation guided evd may increase the accuracy of placement of evd, but it requires a lot of time, room for procedure and frequent general anesthesia. in consideration of an emergent procedure and cost effectiveness of evd, navigation guided evd is not suitable. in this presentation, evd device could be used very conveniently and quickly. when performing evd using this device, the location of evd catheter tip ca n't be reach enough in the frontal horn due to the slight backward movement of the catheter in the process removing the device after ventricular puncture or due to the sight midline shift. in this study, 3 patients showed that catheter tip located in the ventricular wall of the frontal horn. however, in all patients, csf was well drained and well functioning up to the removal of evd. there was no problem for csf drain through the evd catheter because the ventricular puncture had been already accomplished. in addition, the accuracy of the direction of catheter tip toward the frontal horn was 100%. if the surgeon use this device, extraventricle location of the catheter tip will be never happened. if this device is used in patient with a slight midline shifting, evd catheter tip can be located in the contralateral frontal horn. the depth of an evd catheter inserting into the ventricle seems to be suitable to be positioned at a depth of 5.5 to 6 cm from the dura mater. an accurate placement of the ventricular catheter tip is also very important, particularly, in view point of the intraventricular thrombolytic therapy in ivh and the direct conversion of an evd to ventriculoperitoneal shunt. if the neurosurgical residency use this device more than several times during the training course, the accuracy of evd by freehand technique will be also improved because of the familiar direction of evd device for ventricular puncture. this device for evd guides to provide an accurate position of catheter tip safely and easily.	to introduce a new device for catheter placement of an external ventricular drain (evd) of cerebrospinal fluid (csf). this device was composed of three portions, t - shaped main body, rectangular pillar having a central hole to insert a catheter and an arm pointing the tragus. the main body has a role to direct a ventricular catheter toward the right or left inner canthus and has a shallow longitudinal opening to connect the rectangular pillar. the arm pointing the tragus is controlled by back and forth movement and turn of the pillar attached to the main body. between april 2012 and december 2014, 57 emergency evds were performed in 52 patients using this device in the operating room. catheter tip located in the frontal horn in 52 (91.2%), 3rd ventricle in 2 (3.5%) and in the wall of the frontal horn of the lateral ventricle in 3 evds (5.2%). small hemorrhage along to catheter tract occurred in 1 evd. csf was well drained through the all evd catheters. the accuracy of the catheter position and direction using this device were 91% and 100%, respectively. this device for evd guides to provide an accurate position of catheter tip safely and easily.
olive flounder, paralichthys olivaceus has a flattened oval body, mainly occurs at the benthos in water depths of 10200 m, and is widely distributed in korea and east asia (han., 2007). it is the most important cultured fish in korea, with approximately 40,922 tons of this species having been produced in 2012 (park., 2012 ; park., 2015) ; correspondingly large nursery and transport processes are associated with the mass culture of olive flounder (hur., 2007 ; park., 2012 ; park., 2015). however, the lack of knowledge of the physiological changes that occur in the olive flounder during transport has resulted in large mortalities. olive flounder produced at jeju island, korea, are transported predominantly by truck and ship, and the temperature and dissolved oxygen concentration are controlled during the sometimes long transport period by pumping air, and adding liquefied oxygen and ice to improve fish survival. to minimize stress and reduce the chance of fish mass mortality (ferreira., 1984) during handling and long periods of transportation, an effective high - density transport method is essential to reduce costs and minimize losses (ferreira. the development of new equipment and transport procedures is having a positive effect on the transportation of live fish. for example, the use of non - toxic salt has been shown to reduce stress and result in higher survival rates (tomasso., 1980 ; carmichael., 1984 ; carmichael & tomasso, 1988), and the use of low concentrations of calcium chloride is also a cheap and effective improvement in the handling and transportation of live fish (grizzle., 1985 ; the use of anesthetics is also an effective procedure for transporting fish (park., 2009a). anesthetic use in aquaculture reduces fish energy use, enables efficient transportation, increases ease of handling when measurements are taken, reduces pain and trauma for the fish, reduces handling stress (park., 2009b), and reduces bacterial infections, especially during surgery. water - temperatureinduced anesthesia, which is recognized as stable in fish, can be used without a mandatory period of withdrawal prior to human consumption, as is required for chemical anesthetics (summerfelt & smith, 1990). the mode of application as well as any local regulations and legislation dictate the choice of anesthetic. currently, only tricaine methanesulfonate (ms-222) and metomidate are registered for veterinary use in fish, although researchers have access to many other compounds not available for public use (park., 2011). lengthy withdrawal periods are mandatory for chemical anesthesia of food fish prior to harvest, and this has led to an interest in the search for less persistent and more natural anesthetics ; among these is clove oil (park., the active ingredient in clove oil is eugenol (4-allyl-2- methoxyphenol), which is inexpensive and a safe anesthetic that has been used as a substitute for commonly used compounds including ms-222 (park., 2011). compared with ms-222, clove oil is characterized by more rapid induction of anesthetic effects, prolonged recovery times, and a narrower range of safe doses in at least three teleost species (sladky., 2001). clove oil is also safe, inexpensive, and non - toxic for the environment, and does not require a withdrawal period compared with other anesthetic chemicals (park., 2008). clove oil has been studied as an anesthetic in a number of species (park., the aim of this study was to determine the optimum concentrations of clove oil for anesthesia in olive flounder over a range of temperature conditions. stress responses in the fish and water parameters were also analyzed during a simulated transport experiment involving various clove oil concentrations. in march 2016, specimens of olive flounder, paralichthys olivaceus (standard length : 46.6 0.45 cm ; body weight : 500.7 11.67 g), were obtained from the institute of marine living modified organisms, pukyung national university, korea. the fish were transported to the fishery genetics and breeding sciences laboratory of the korea maritime and ocean university, korea, and reared in a recirculating culture system consisting of a circulation pump, an aeration system, and a temperature control system. the temperature of the water in the tube were controlled so that it was equal to the temperature of the water in the anesthetic and recovery phases of the experiment. all fish were fasted for 24 h prior to the commencement of experiments, and were adapted to 400-l glass tubes, which were maintained at the same temperatures as the experimental water tempeatures (15, 20, and 25c). for the anesthetic experiment, 20 specimens of each experimental group were randomly removed from the breeding tube using a net, and transferred to water in an aerated 50 l plastic aquarium, to which anesthetic solution (clove oil containing 85% eugenol ; sigma, usa) had been added. when a fish had become anesthetized, it was immediately transferred to a recovery tank. the time to achieve anesthesia (anesthesia time) and the recovery time the anesthetic effects of clove oil were investigated at five concentrations (10, 20, 30, 60, 90, 120 and 150 ppm) ; these were prepared using a stock solution of clove oil dissolved in 95% methanol (sigma, usa) at a ratio of 1:10. the anesthesia time was measured from when the fish was placed in the anesthetic - containing water to when it was perfectly sedated and had minimal opercular movement (stage a6, table 1), while the recovery time was measured from the time the fish was placed in the recovery tank to when it again exhibited normal swimming and responsiveness to visual stimulation (stage r6, table 1). table 1 is decision - based framework for categorizing anesthetic effects, derived from park. (2011), and shows the stages of anesthesia and recovery that were used as endpoints in the present study, including a6 (minimal opercular movement only) and r6 (normal swimming, responsiveness to visual stimuli) (summerfelt and smith, 1990 ; park., 2011). during the experiment the fish showed several stages in the development of anesthesia, including slowed swimming and side - to - side rolling (stage a2) through to opercular movement only and no reaction when it was flipped onto its other side (stage a6). at stage a6, individual fish recovery was defined as the point at which erratic swimming began, and included the fish righting its balance and flipping itself back over when turned, (stage r5) and normal swimming, as well as responsiveness to visual stimuli (stage r6). to investigate the stress response of fish to anesthetic exposure, blood samples were extracted from five randomly selected fish prior to exposure (0 h), and at 1, 6, 12, 24, and 48 h following anesthetic treatment. the blood was transferred to capillary tubes and analyzed following centrifugation at 200 g for 10 min. the plasma was collected and stored at 80c (sw - uf-200 freezer ; samwon freezing engineering, busan, korea) until analyzed. the cortisol concentration in 50 l samples was measured using a cortisol radioimmunoassay kit (coat - a - count tkco cortisol ria kit ; dpc, usa). the sample was added to 100 ml of antiserum and incubated for 45 min at 37c, then 1,000 ml of separation reagent was added. the mixture was placed in a refrigerator at 4c for 15 min, then centrifuged at 1,200 g for 15 min. the supernatant was assayed using an automatic gamma radiation counter (cobra ; packard, usa). the plasma glucose concentration was analyzed according to the method of raabo & terkildsen (1960), using a glucose assay kit (kit 510, sigma, st louis, mo, usa) ; the production of h2o2 by glucose oxidase in the presence of o - dianisidine was measured as an absorbance increase at 450 nm. blood lactic acid concentrations were analyzed using an automatic blood analyzer (reflotron ; boehringer mannheim, germany). at 10 days following analysis of the anesthetic effect and the stress response, the respiration of fish was assessed using a respirometer chamber comprising a sealed acrylic resin box having a wall thickness of 8 mm and overall dimensions of 10 cm (width) 50 cm (length) 10 cm (height). the hose providing inflow water to the respirometer was equipped with a temperature - controlled heater to maintain the water at 20 0.3c, cartridge filters (10 m and 3 m) to remove particles from the incoming water, and a flow - through ultraviolet lamp to reduce the likelihood of oxygen consumption by microbes. the water salinity was maintained optimum anesthetic salinity by brackish water, and optimum anesthetic salinity of each group were analyzed by anesthetic experiment. the respirometer chambers were prepared and labeled according to the measurement times and anesthetic concentrations. to determine the most appropriate experimental concentrations of clove oil and lidocaine - hcl, we performed a pilot experiment for 6 h. to prevent mortality in the experimental sample, the anesthetized fish were maintained at several stages of anesthesia during the pilot study, ranging from normal swimming, opercular movement, and general movement (stage a1), to slow swimming and side - to - side rolling (stage a2) (summerfelt and smith, 1990 ; park., 2011). the anesthetic effects of clove oil were determined at four concentrations : 0 (control), and 1, 2, and 3 ppm. prior to measuring the dissolved oxygen (do) concentration, the concentrations of nh4 and co2 were measured using a spectrophotometer (dr2800, hach, loveland, colorado, usa), and the respiration frequency (gill cover movement) of the fish was measured using a counter and a digital timer. the do concentration was measured with an o2 electrode and a multi - data logger system (oxyguard, denmark). one- and two - way analysis of variance (anova) were performed for the data among treatments, and duncan s test was performed when significant differences were found (p < 0.05 ; spss statistics package spss 9.0 ; spss inc. unless otherwise stated, differences were considered to be statistically significant at p < 0.05. no samples of olive flounder, paralichthys olivaceus died from stress as a result of exposure to the anesthetic during the experiment. table 2 shows the results of a twoway anova test for the effect of clove oil dose and water temperature on anesthesia. the exposure and recovery times were affected by the dose concentration of clove oil, and by water temperature. table 3 shows the anesthetic effects of clove oil at each concentration and water temperature. the anesthesia time decreased significantly as both the concentration of clove oil and the water temperature increased. for each temperature, the anesthesia time decreased as the concentration of clove oil increased. furthermore, at each concentration of clove oil, the anesthesia time decreased as water temperature increased. the recovery time in each temperature increased significantly as the concentration of clove oil increased. in addition, the anesthesia time for each concentration of clove oil decreased as the water temperature increased. thus, the recovery time at each temperature was related to changes in the concentration of clove oil, and the recovery time increased as water temperature increased. based on an optimal anesthesia time of approximately 1 min, the optimal concentrations of clove oil for olive flounder were 120 ppm at 15c, and 90 ppm at 20c and 25c. the ratio of the recovery time to the anesthetic exposure time, as a function of the concentration of clove oil and the water temperature, is shown in fig. the recovery time / exposure time ratio gradually increased as the clove oil concentration increased in each water temerature treatment. at 90 ppm, 120 ppm, and 150 ppm, the ratio increased significantly as water temperature increased ; however, at other concentrations the ratio showed no clear relationship with water temperature. results of clove oil dose and water temperature on anesthesia among olive flounder were shown in table 3. values in the same column not sharing common superscripts are significantly different (p < 0.05). 2 shows the stress response of the fish to clove oil anesthesia over 48 h. the plasma cortisol concentration at each temperature increased significantly up to 12 h. after 1 h of exposure the plasma cortisol concentrations were 8 1.4, 10 1.0, and 14 1.3 g / dl at 15c, 20c, and 25c, respectively. after 6 h the plasma cortisol concentrations were 18 1.0, 23 1.2, and 30 1.3 g / dl at 15c, 20c, and 25c, respectively. thus, the rate of increase in the plasma cortisol concentration increased with increasing water temperature up to 12 h. after this time point, the cortisol concentration at each temperature decreased significantly to 48 h. at 12 h the plasma cortisol concentrations were 35 1.7, 36 1.8, and 35 1.4 g / dl at 15c, 20c, and 25c, respectively, while at 24 h were 20 2.4, 13 3.0, and 10 1.7 g / dl, and at 48 h were 5 0.6, 2 0.5, and 1 0.4 g / dl, respectively. thus, the rate of decrease in the plasma cortisol concentration increased as the water temperature increased. actually n=20 for each experiment because the means and se were calculated separately for each group. different letters on error bars are significantly different among elapsed time in anesthesia groups (p<0.05). the do concentrations, respiratory frequencies, ph values, and nh4 and co2 concentrations in the clove oil treatments are shown in tables 4 and 5, and figs. 3, 4, and 5, respectively. the do concentration, ph value, and the respiratory frequency decreased in all experimental groups up to 6 h (table 4 and figs. 3 and 4), whereas the nh4 and co2 concentrations increased in all experimental groups (table 5 and fig. measurement times the do concentration in all clove oil treatments was higher than in the control group (table 4). at 6 h the ph values were 6.1 0.064, 6.35 0.068, 6.57 0.061, and 6.82 0.066 in the control group and the 1, 2 and 3 ppm clove oil treatments, respectively, and ph values in all clove oil treatments were significantly higher than that of control group (fig. the co2 and nh4 concentrations and the respiratory frequencies decreased with increasing clove oil concentration (p<0.05). at 6 the respiratory frequencies were markedly less than in the control group (2 3 1.9, 23 1.5, 23 1.4, and 42 3.4, respectively ; fig. the co2 concentration in the 1, 2, and 3 ppm clove oil treatments were lower than that in the control group (table 5). with respect to nh4, at all measurement times the concentrations in the 1, 2, and 3 ppm clove oil treatments were lower than that in the control group (0.07 0.006, 0.06 0.009, 0.05 0.005, and 0.09 0.007 mg values in the same column not sharing common superscripts are significantly different (p<0.05). values in the same column not sharing common superscripts are significantly different (p<0.05). actually n=20 for each experiment because the means and se were calculated separately for each group. different letters on error bars are significantly different between no anesthesia and clove oil anesthesia groups (p<0.05). actually n=20 for each experiment because the means and se were calculated separately for each group. different letters on error bars are significantly different between no anesthesia and clove oil anesthesia groups (p<0.05). actually n=20 for each experiment because the means and se were calculated separately for each group. different letters on error bars are signifiantly different between no anesthesia and clove oil anesthesia groups (p<0.05). determining the safety and reliability of anesthetics, and the associated recovery time, is a prerequisite for choosing the most appropriate anesthetic for a given species. the anesthesia time is the time required for an animal to reach the anesthetized condition for the species, and the recovery time is the time required for the animal to completely recover (summerfelt & smith, 1990). the results of this study indicated that clove oil is an effective anesthetic for p. olivaceus. based on the criteria that anesthesia should be achieved within 3 min, that recovery should occur in < 10 min, and that no mortality occur (park., 2011), we assessed a range of concentrations of clove oil (10120 ppm) and exposure water temperatures of 20c and 25c. our results clearly indicated that shorter anesthesia times were achieved with higher concentrations of clove oil. the observed anesthesia times for olive flounder, paralichthys olivaceus exposed to clove oil were similar to previous reports of its effect on other bony fishes including sockeye salmon, oncorhynchus nerka, rock bream, oplegnthus fasciatus, and kelp grouper, epinephelus bruneus (woody., 2002 ; park., 2008 and 2009b). the dose response curves for olive flounder exposed to clove oil were negatively exponential, with higher doses resulting in reduced times to anesthesia stage a6 (see table 1). optimum anesthetic concentrations are usually expected to induce anesthesia within 3 min, with recovery occurring within 10 min (park., 2011). we set the optimum anesthesia concentration at approximately 1 min, and at this level found that the optimal clove oil concentrations for olive flounder were 120 ppm at 15c, and 90 ppm at 20c or 25c. for other species the optimal clove oil concentration for achieving an anesthesia time of approximately 1 min include 80 ppm at 9.4c for sockeye salmon, 150 ppm at 24c and 100125 ppm at 26c for rock bream, and 50 100 ppm at 22c for kelp grouper (woody., 2002 ; park., 2008 and 2009b). the relationship between water temperature and anesthesia time followed a negative exponential curve, with increasing water temperature resulting in decreased anesthesia time. a similar inverse relationship has also been reported in studies of anesthesia by clove oil in other fish species, including european sea bass, dicentrarchus labrax and gilthead sea bream, sparus aurata (constantinos., 2005). for these species, lower temperatures resulted in significantly longer induction of anesthesia and recovery times (constantinos., 2005). in addition, kelp grouper anesthetized with clove oil, and greenling, hexagrammos decagrammus anesthetized with lidocaine - hcl exhibited similar trends (park., 2003 ; constantinos., 2005 ; park., 2008). 1988) reported an anesthesia time of 93.0 7.5 s for olive flounder at 100 ppm lidocaine - hcl, and that the optimum concentration of lidocaine - hcl was 200 ppm. comparison of the anesthetic potency of clove oil and lidocaine - hcl showed that olive flounder were more sensitive to clove oil than to lidocaine - hcl ; clove oil more effectively immobilized fish at lower doses than did lidocaine - hcl in the same anesthesia time. if the ratio of the recovery time to the anesthesia time is greater than 1, the recovery time is longer than the anesthesia time. at each water temperature in our study, the ratio of the recovery time to the anesthesia time increased as the anesthesia concentration increased. that is, as the anesthetic concentration increased, the increase in the recovery time was greater than the decrease in the anesthesia time. in contrast, as the anesthesia concentration was reduced, the rate of decrease in the recovery time was greater than the rate of increase in the anesthesia time. similar results demonstrating proportional changes in the clove oil concentration and the ratio of the recovery time to the anesthesia time have been reported for rock bream (park., 2009b). cortisol is an indicator of a stress response in fish subject to various stressors, and is involved in metabolism and ecologic balance (mommsen., 1999). in addition, cortisol affects the body balance, carbohydrates, proteins, and lipids (mommsen., 1999). cortisol levels in fish suggest that anesthesia, salinity, do, ph, nh4, and acidic conditions can affect fish metabolism and hormone levels (mommsen., 1999). (1999), it means they made the annually changeable temperature, fresh water to sea water, salinity, waste water and do. plasma cortisol is recognized as a useful indicator of stress in fish (schreck, 1982 ; park., 2008). elevated plasma cortisol levels were reported in red drum (sciaenops ocellatus) simultaneously exposed to the anesthetics ms- 222 and quinaldine (massee., 1995). barton & iwama (1991) stated that usually, phenomenon that plasma cortisol concentration of fishes rises by stress is first order reaction, phenomenon that plasma glucose concentration rises is result of second - order first order reaction by hormone rise reaction by stress. the plasma cortisol concentration in anesthetized olive flounder returned to approximately the concentration in control fish 48 h post - administration of clove oil. if the metabolic rate is high, the anesthetic is absorbed more rapidly through the gills (summerfelt & smith, 1990). the metabolism of olive flounder at 25c was expected to be higher than at the other temperatures tested. the results showed that the anesthesia and recovery times at 25c were faster than at the other temperatures tested. to confirm and interpret these observations the patterns of decline in the do concentration in water in the control, clove oil, and lidocaine - hcl treatments were consistent with the trends reported in studies of the amur minnow, rhynchocypris steindachneri and winter flounder, pleuronectes americanus (park. these results suggest that high levels of stress induced by handling or netting cause high levels of oxygen consumption during the early stages of transportation. (1984), who used benzocaine - hcl as an anesthetic in the transport of java tilapia, oreochromis mossambicus. in general, both studies showed that following application of the anesthetic there was a reduction in fish metabolism, which is an indication of declining oxygen consumption. in this study there appeared to be a positive relationship between the concentration of clove oil and the do concentration, insofar as the group treated with the highest concentration of clove oil exhibited the smallest decline in do (ferreira., 1984). in a similarly designed experiment by park. (1998), involving amur minnow as the test organism, the same trends in do concentration and ventilation rates was observed over 2 h as were observed over 6 h in our study. park. (1998) used lower lidocaine - hcl concentrations (5, 10, and 20 ppm) and a lower temperature (18c, compared with 26c in this experiment), but nearly identical trends were found. this is an important comparison because it indicates the wide - ranging effects of clove oil over a broad spectrum of temperatures. (1995) performed a transportation experiment that involved the playfish, xiphophorus maculatus (gnther) being treated with three anesthetics : 2-phenoxyethanol (200 ppm), quinaldine sulfate (10 ppm), and ms-222 (30 ppm). at 16 h following anesthetic administration the nh4 concentration in the water in the 2-phenoxyethanol treatment was only 65% that in the control, 20% that in the quinaldine sulfate treatment, and relatively lower than in the ms-222 treatment. the trends in nh4 concentrations exhibited by the five experimental groups were consistent with the trends reported by park. (1998 and 2009a), insofar as the overall reduction in nh4 excretion was directly related to the anesthetic - induced decline in metabolism. this study showed that clove oil is effective for anesthetizing olive flounder, and that the anesthetic effect was influenced by water temperature. park. (2011) reported that the anesthetic effects of clove oil or lidocaine - hcl were significantly affected by water temperature and the anesthetic concentration, decreasing proportionately as these parameters increased. (2011) suggest that the anesthetic effects of clove oil are similar to those of lidocaine - hcl. the results of our experiment suggest that clove oil reduced the metabolic activity of olive flounder, thus reducing excretion of nh4 and oxygen consumption by the fish. clove oil is a cost - effective and efficient anesthetic, and is safe for use and non - toxic to the fish or users (park., 2003). in conclusion, clove oil is an effective anesthetic for use in improving the transport of olive flounder and minimizing the stress for olive flounder. the results of this study will improve the safe laboratory handling and transport of olive flounder, which are commonly required for use in research studies and experiments. the results of our study should also be useful for aquaculturalists and fish transporters wanting to minimize the stress imposed on fish during transport.	the optimum concentrations of clove oil as an anesthetic for olive flounder (paralichthys olivaceus) and the stress response of the fish to clove oil anesthesia were determined over a range of water temperatures, and investigated in a simulated transport experiment using analysis of various water and physiological parameters. while the time for induction of anesthesia decreased significantly as both the concentration of clove oil and water temperature increased, the recovery time increased significantly (p<0.05). the plasma cortisol concentration in fish at each temperature increased significantly up to 12 h following exposure (p<0.05), then decreased to 48 h (p<0.05). the do dissolved oxygen concentrations, ph values, and the fish respiratory frequencies decreased over 6 h following exposure to clove oil in all experimental groups (p<0.05), whereas the nh4 + and co2concentrations in all experimental groups increased up to 6 h (p<0.05). the ph values and do concentrations increased with increasing clove oil concentration (p<0.05) in the 6 h following exposure, and the co2 and nh4 + concentrations and the respiratory frequencies decreased with increasing clove oil concentration (p<0.05). the results of this experiment suggest that clove oil reduced the metabolic activity of olive flounder, thus reducing nh4 + excretion and o2 consumption. in conclusion, clove oil appears to be a cost - effective and efficient anesthetic that is safe for use and non - toxic to the fish and users. its use provides the potential for improved transportation of olive flounder.
the hepatitis b virus (hbv), which belongs to the hepadnavirus family, is a small circular dna virus containing a nucleocapsid and an envelope. hbv nucleocapsid contains a relatively small and incompletely double stranded 3.2 kb dna genome, viral polymerase and core protein. its envelope is composed of viral surface proteins enclosed by a lipid membrane from host cells 1, 2. in the serum of infected patients, there are both mature virion with viral dna and subviral particles without viral dna 3, 4. sub viral particles are overwhelmingly in excess to infectious particles, which is the majority of the two types 3, 4 the life cycle of hbv is believed to begin when the virus attaches to the host cell membrane via its envelope proteins. then, the viral membrane fuses with the cell membrane and the viral genome is released into the cells 5, 6 after the viral genome reaches the nucleus, the viral polymerase converts the partial double stranded dna (dsdna) genome into covalently closed circle dna (cccdna). the cccdna is believed to be the template for further propagation of pre - genomic rna, which directs the synthesis of viral dna and mrna that encode all the viral proteins 2, 7, 8 hbv core particles are assembled in the cytosol following the encapsidation of pre - genomic rna, which is then degraded during the reverse transcription of pre - genomic rna into a complementary strand of dna 9. hbv surface proteins are initially synthesized and polymerized in the rough endoplasmic reticulum (rer). these proteins are transported to the post er and pre - golgi compartments where budding of the nucleocapsid follows 10. the assembled hbv virion and sub - viral particles are transported to the golgi for further modification of its glycans in the surface proteins, and then are secreted out of the host cell to finish the life cycle. however, the early steps of hbv infection including of the penetration of virus and release of its genome into host cells is uncertain. in general, the early step of virus infection in which the virus enters the cell can be divided into three stages : attachment, fusion, and entry. enveloped viruses usually entry by an attachment to the host cells, which usually is via the interaction of viral surface protein with the specific receptor on the cell membrane 11. the fusion of a viral envelope and cell membrane and the following viral genome release finally trigger the viral infection 12, 13. the first requires a fusion of the viral envelope with the plasma membrane, leading to the release of the viral nucleocapsid. hiv is an example of a virus that uses this type of mechanism to enter 12. in the second mechanism, an endosomal compartment first takes up the attached virus. later, the viral genome is released from this endosomal compartment into the cytoplasm by a process that may or may not depend upon a lowering of the ph to activate a virus - mediated fusogenic activity. in some virus / cell systems, such as those involving influenza virus 14 or paramyxoviruses 11, 13 exogenously adding protease generates infective virions from otherwise non - infective ones the following transport of the viral genome to nucleus and the start of the virus replication finally complete the early steps of virus life cycle. a major limitation to study hbv early steps of life cycle is lack of an in vitro infection system. although hbv is considered highly efficient in establishing infection in people following parenteral exposure, tissue culture cells are curiously refractory for hbv infection 5, 15, 16, 17. although there is a growing body of data, recently most of the published information about the early stages of hepadnavirus infection is derived from duck hepatitis b virus (dhbv), since robust dhbv infectable tissue culture systems do exist. there are, however, significant differences between the duck and human hepatitis viruses. for example, and perhaps of greatest relevance, human hbv envelope polypeptides, the likely mediators of entry, thus, the degree to which information from dhbv applies to human hbv attachment and entry may be limited. the hbv surface protein antigens (hbsag) are comprised of three carboxyl - co - terminal hbs proteins termed large (lhbs), middle (mhbs) and small (shbs, also called major) protein 3, 4. lhbs and mhbs also share the highly hydrophobic, repetitive, membrane - spanning s domain. in addition, mhbs has a 55 amino acid region called pres2, lhbs has an additional 109 - 120 amino acid long region called pres1 (dependent on the viral subtype) in their n - terminus (figure 1 a) 4. although hbv surface proteins must certainly mediate early steps in the virus life cycle, the precise role for each glycoprotein in the entry and egress of the virus is controversial. the studies of hepadnavirus in cell culture, especially with explanted human or duck primary liver cells, strongly suggest that lhbs is directly involved in the viral attachment 18, 19, 20. the putative attachment site of hbv located in the pres1 was first reported by neurath and his colleagues using anti - pres1 antibody 21, 22. they found that the antibody against peptide corresponding to the hbv pres1 domain was virus - neutralizing and protective 21. in 1989, pontisso and his colleagues, using the membranes of surgically obtained human liver as a target, further confirmed the role of lhbs in the hbv attachment 23. recently, the attachment site of lhbs was functionally narrowed down to the amino acids 2147 of pres1 by employing synthetic peptides. the results suggest that this binding site was not only required but also sufficient to attach specifically hepg2 cells (figure 1 b) 24, 25. moreover, the antibody with this site has neutralized the hbv infection in chimpanzee 19, 21. it is interesting that, by mutagenesis studies and single cell attachment analysis, paran and his colleagues found that the qldpaf sequence within this pres1 region was crucial for cell attachment 25. further evidence to support this hypothesis is that this sequence is also found in the other virus and bacterial functioning as an adhesion or attachment determinant 25. this suggests that the qldpaf sequence may have a more general role in viral infection. there are differing reports about the role of mhbs in the generation of hbv in the transfected cells and in mediating hbv infection 8, 26. mhbs seem to be absent in certain hbv variants from chronically infected patients 27, 28, suggesting a non essential function in the viral infection. on the other hand, misfolded mhbs seems to be able to interfere with the assembly of hbv virions in the persistently producing virus cell line hepg2.215 29, 30. anti - pres2 antibody (that recognizes mhbs) can block hbv infection of primary human liver cell in vitro 31, 32 and chimpanzee liver cells in vivo 33 suggesting that mhbs are probably also involved in viral entry. hbv pres2 can specifically bind the poly - human albumin in vitro 34, 32, 35 and the mono human albumin in vivo 36, 37, more investigation is needed to determine whether this binding capability is involved in the viral attachment. shbs, the most abundant viral glycoprotein, certainly has a role in virus secretion 24. its role in virus uptake is supported by the studies of leenders and his colleagues 38. using recombinant hbs protein, they have performed binding studies with adult human hepatocytes, rat hepatocytes, human fibroblasts, human peripheral blood mononuclear cells and plasma membranes derived from these cell types. the results suggest that shbs was able to bind specifically to human hepatocytes, human fibroblasts and human blood mononuclear cells but not the rat hepatocytes. the binding of shbs with human hepatocytes was further supported by the observation from bruin 39. they have found that the particles only composed by shbs can bind the human hepatocyte plasma membrane and this binding resulted in the internalization of the gold labeled sub viral particles 25. however, despite the literature about hbv surface proteins, determination of which of the proteins is centrally responsible for hbv attachment and entry is elusive. recently, synthetic beads conjugated to viral proteins were used to quantitatively measure virus cell attachment, at the level of single cell resolution, by light microscopy 25. in addition to attachment, entry of the hbv protein conjugated beads into the cells can be easily detected by scanning and transmission electron microscopy. it was found that the beads conjugated with the recombinant pres1 protein containing the prominent qldpaf motif, but lacking the small hbsag regions, show efficiently attach to cells. interestingly, beads conjugated with particles containing the whole repertoire of the surface proteins were twice as active in attachment, compared to pres1 alone. thus, it appears that it is possible hbv uptake involves multiple attachment sties 17. it is also unclear as to the identity of the cellular macromolecules that are responsible for virus attachment. since hbv is highly infectious, in vivo, the relative refractoriness of liver cells, grown in culture, has been something of a puzzle. the studies duck hepatitis b virus (dhbv) infection with primary duck hepatocytes have been particularly revealing. in 1994, then in 1995, kuroki. reported that a glycoprotein in duck hepatocytes with the molecular weight 180 kilo dalton, called gp180, can be co - precipitated with dhbv 40, 41. moreover, the interaction of gp180 and dhbv can be blocked by monoclonal anti duck large protein antibodies. subsequent cdna cloning revealed the binding protein to be a carboxypeptidase h - like molecule, now classified as duck carboxypeptidase d (dcpd). li and tong have confirmed those 42, 43 and further demonstrated the carboxypeptidase d, a protease existing in the avian hepatocyte, exiting in the mammalian hepatocytes either. despite the fact that the specific pres1 domain for attachment of the lhbs to human liver cells and/or cell lines has been repeatedly verified, the cellular protein that is responsible for this binding has not been found yet. research has thus relied heavily upon human hepatoma cell lines 44. using a synthesized peptide with the sequence of hbv putative attachment site in pres1, falco. have isolated a membrane protein called hep - bp from hepg2 cells, which can specifically bind hbv pres1 peptide. furthermore, they found that after the transfection of corresponding cdna of hep - bp into hepg2 cells, the virus binding capacity increased by 2 orders of magnitude compared with untransfected cells. chinese hamster ovary cells, which normally do not bind to hbv, acquired susceptibility to hbv binding after transfection 45. the sequence analysis suggests this protein is highly homologous with the squamous cell carcinoma (scca1), a serine protease inhibitor of ovalbumin family that has been used as a tumor marker for diagnosis of scc 46. however, the lack of the tissue specificity and low level expression in the liver cells reduce the possibility of it as a relevant receptor of hbv infection. it is worthy that hep - bp is a serine protease inhibitor and is over - expressed in the cancer cells 47. the over - expression of another serine protease inhibitor kazal type (spik) in the hepg2 and huh 7 cells was also observed by us. the possible role of this protease inhibitor in the hbv infection will be discussed later. using human hepatoma cell line or the membrane of human liver cells, the protein that bind hbv mhbs or shbs has been reported by several groups. in 1989, potisso. reported that human liver plasma membranes contain receptors for mhbs via associated with polymerized human serum albumin 32. subsequently, buine identified human liver endonexin ii (eii) as a specific shbs binding protein 48. a sialoglycoprotein was also reported as a receptor for shbs binding in the uptake of hbv 49, 50. however, it is noted that no direct evidence to show that the interaction of hbv with those receptors could result in the real infection. therefore, the full understand of the hbv attachment and it receptors perhaps could only be achieved after a susceptible cell line has been established. the attachment of hbv surface protein to the host cell is essential for hbv entry. the fusion of viral protein and cell membrane allows the release of the viral genome into cytosol. in contrast, viral fusion is dependent on a specific domain within the viral surface protein, known as the " fusion motif, which is normally composed of a series of hydrophobic amino acids 51, 52, 53, 54 and is independent of specific cell receptors and thus relatively less cell specific. attachment possibly occurs at a low temperature of 4 c. however, fusion requires a temperature of at least 20 c. in 1993, we reported that hbv has a conservative region in the n - terminus of its s domain, which encloses a hydrophobic sequence with 13 amino acids 51. this sequence has all the characteristics of fusion motif of other human viruses such as paramyxoviruses and retroviruses including of hiv, rsv and influenza virus 5. more important, this region includes a core hydrophobic sequence : flg - ll - ag, which is supposed to trigger the viral cell fusion 55, 56. the fusogenesis of this sequence was confirmed by rodriguez - crespo. using the synthesized peptides containing this region of hbv and woodchuck hepatitis virus (whv), they have demonstrated that these peptides can induce the fusion of bio - membranes, liposome leakage and haemolytic activity in vitro 57, 58. we also have found that the peptides containing this sequence can trigger the syncytia of hepg2 cell from without (unpublished evidence). it is noteworthy that pre - digestion of hbv with a bacterial protease v8 allowed the hbv and whv entry into the unsusceptible cell line such as hepg2 to initiate the infection 5, 6. however, v8 protease cleaves at a proteolytic sensitive region called pest region that is just adjacent to the putative fusogenic domain of hbv suggests that the viral infection probably is the consequence of the exposure of hbv fusion domain (figure 1 b) 5,6. the proteolysis to make the viral fusion domain that does not locate at the n - terminus accessible for the infection was seen in the other virus 14, 59, 60. our unpublished data showed that the binding of v8 digested hbv to the hepg2 cells was ph dependent. at a mild acid condition, for example, ph5.5, the binding ability of those hbv to hep g2 cells was increased 60% than that at neutral condition. considering of the possible exposure of hbv fusion domain by the digestion of v8 protease, this result implies that the low ph might help hbv fusion. low ph helps viral fusion was observed in other human virus such as semliki forest virus and influenza virus in the endocytosis way 14, 13, 11, 61. however, it was demonstrated that exposing the virus to the low ph triggers translocation of the internally sequence of pres1 domain onto the viral surface 62, 63. this supports the hypothesis that the low ph in the endosome may induce the conformation change of hbv surface proteins, furthermore, to release its nucleocapsid after viral - cell fusion. the results on the role of ph in the uptake of dhbv are inconsistent 64, 65. using lysosometropic agents, for example, ammonium chloride and chloroquine, which can raise the ph in the endosome, offensperger. in contrast, rigg. reported that the treatment of ammonium chloride did not affect the uptake of dhbv 65. in spite both groups used the duck primary liver cell as an experimental infection system, however, the condition of the cell growth seems somehow different. recently, breiner and schaller have demonstrated that the uptake of dhbv by primary duck hepatocyte (pdh) requires endocytosis 66. however, the endocysis is a feature usually associated with low ph - triggered fusion mechanism. altogether, while a strict low ph dependency may not apply to dhbv to the same extent as for certain other viruses, low ph may still significantly facilitate entry of the virus 17. most data about hepadnavirus internalization and transport to the nucleus was generated from the studies of the uptake of dhbv by duck primary hepatocytes (pdh). however, the entry route for dhbv is still unclear. as we have mentioned before one is via direct fusion with the host cell membrane like hiv 67 and the other is via endocytic pathway like semliki forest virus and influenza virus 13, 14. this hypothesis was supported by the observation of the conformation change of the large surface protein of dhbv at low ph condition 62. however, the more detail investigation was from the kinetic study of the uptake of dhbv by duck primary hepatocyte. in 1999, they observed that dhbv dna appeared in the cytosol of pdh as early as one hour after post - incubation of virus with cells at 37 c 68. nevertheless, dhbv dna only can be detected in the nuclear fractions after at least 4 hours post - incubation. the cccdna and single - stranded dna were not detected until 48 hours post inoculation (p.i.) 68. comparing with the in vivo infection, cccdna can be detected in hepatocytes as fast as 6 hours after inoculation of ducks with the same dhbv strain 68, there was 40 hours delay during dhbv localization to the nucleus and formation of cccdna in vitro system 68. however, the study of hbv infection to human primary hepatocytes shows that the cccdna of hbv appeared in the nucleus after two days of the post inoculation, this is agreement with the observation from dhbv 69. the result from the kinetic study on the internalization and transportation of hbv by the hepg2 cells is somehow different with the result from the primary hepatocytes. however, this cell - line is generally refractory with the infection of serum derived hbv 5, 15 in spite the post attachment internalization of hbv in hepg2 cells was observed by several groups 15, 49. according to the observation from the qiao 's group, the internalization of hbv by hepg2 cells happened immediately after the viral attachment, and reached its peak within the cytosol two hours after post incubation at 37 c. however, the nuclear transport of hbv dna was not completed even 16 hours following viral absorption 15. the disability of transport of hbv dna to nucleus implies that the release of hbv nucleocapsid in the hepg2 cells was blocked by some unknown facts after the viral internalization. perhaps deficiencies in viral transport to the nucleus are part of the reason that hepg2 cells are refractory to hbv infection. considering the proteolysis enhances in vitro hbv infection of hepg2 cells, it is possible that the block of hbv nucleocapsid transport is a consequence of the lack of necessary protease digestion. it is still unclear whether the internalization of hbv requires a permeable domain with the sequence of plssifsrigdp at middle of pres2, which was reported by oess and his colleagues recently (figure 1b) 70. it was shown that this domain mediated the internalization of a fused protein into hela cells 70. cd spectroscopy revealed the sequence of plssifsrigdp consists of a hydrophobic region that contains internal, charged, hydrophilic residues and had the ability to fold into an amphipathic alpha - helix, which has been identified conferring the unexpected property of cell permeability in other viruses 71. examples include the antennapedia homeo domain 53, hiv - tat transcription factor 72, herpes simple virus vp22 73 and lactoferrin 74. it is interesting that this permeable domain is just adjacent with pest - fusion region of hbv (figure 1 b). if the refractoriness of hepg2 cells to hbv infection is related to the lack of requisite proteolysis, the conclusion might be that the hepg2 cells perhaps do not have these specific proteases or they do have these proteases, however, they are inactivated by an unknown agent. to find clues as to the nature of hepg2 refractoriness to hbv infection, we have compared the gene expression profile of hbv susceptible primary human liver and hepg2 cells, with an eye on expression of rna specifying proteases and their inhibitors, using a gene array system. the results show that there were no detected differences in the protease profile. however, the expression of a serine protease inhibitor kazal (spik) was more than a thousand fold higher in hepg2 cells than in human liver cells (unpublished data). at the same time, the expressions of the most other genes were found to be approximately equal amongst hepg2 cells and human liver cells (unpublished gene array data). this suggests a role for spik in the altered phenotype of hepg2 cells with respect to liver cells and possibly a role in their susceptibility differences to hbv infection. these finding that sipk was over expressed in hepg2 cells, relative to primary liver tissue, was confirmed by reverse - transcription pcr (rt - pcr) and northern blot analysis. as shown in figure 2, two specific primer sets, pi1/ pi2 and pi3/pi4, were used to amplify spik mrna by rt - pcr. pi1/pi2 directly amplifies the spik gene from the 11 bp to233 bp and pi3/pi4 directly amplifies the entire spik gene (figure 2a). the rt - pcr band generated from primers pi3/pi4 was at the predicted size and slightly larger than that one generated from primers pi1/pi2 (324 base pairs versus 224 base pairs (figure 2b). in addition, these bands were not generated from spik dna (gene sequences), because the control that lacked the enzyme reverse transcriptase in the reaction mixture did not show any band (figure 2b, lane 9). compared to human liver cells, the expression of spik gene in the hepg2 cells was overwhelming. using either of the primer sets, spik specific bands can be easily detected in the hepg2 cells, but hardly in the human liver cells (figure 2b). this difference was not a result of the variation of loading, because the expression of the thimet oligopeptidase gene in the same condition did not show any difference (figure 2c). the over - expression of the spik gene in hepg2 cells has also been confirmed by northern blot analysis. ten micrograms rna from hepg2 cells and human liver cells were resolved on a 1% non - denatured agarose gel, subsequently transferred to a nylon membrane, and then hybridized with a p labeled spik specific probe derived from the spik gene by pcr. in agreement with the results from rt - pcr, figure 3 shows that the spik mrna can only be detected in the hepg2 cells. the signal in the human liver cells was invisible (figure 3, northern blot, lane 2 & lane 3). the undetectable signal from human liver cells was not as a result of the unequal loading. the equality of rnas were applied on to the gel as evidenced by the equivalent ethidium bromide (eb) staining of ribosomal rna in both samples (figure 3, eb stain lane 2 and lane 3. similar increase of the spik expression was observed in yet another hepatoma cell line huh7, which is also refractory to hbv infection similarly to hepg2 (figure 3. taken together, these results demonstrate that spik gene is over - expressed in cells that are not susceptible to hbv infection, such as hepg2 and huh7 cells. in contrast, the expression of spik maintains at undetectable level in the hbv infectible cells such as the normal human liver cells. although, this needs to be further investigated, it is also worth noting that if over expression of sipk is common to hepatoblastoma and other cancer cells, it could be a useful biomarker for early detection of cancer (lu, in progress). as we have mentioned before, the major limitation to study hbv entry is the lack of an in vitro infection system, which can support the entire life cycle of hbv. human primary hepatocyte cultures derived from liver explants have been shown to be susceptible to hbv infection, but only for a limited amount of time following explantation 75, 76. besides of the human primary hepatocytes, there are several systems derived from hepatoma cells so far were used for the study of hbv infection in vitro 68, 77, 78, 16, 79. this includes the manipulation of the cultured hepg2 cells with dimethyl sulfoxide (dmso) or polyethyleneglycol (peg), which has been reported to enhance hbv infection in vitro 68, 77. currently, a cell line susceptible to hbv infection was reported by grippon. however, the high dose of dmso and 2 - 4% peg were used in this system to maintain susceptibility greatly limits their application for study the natural infection of hbv. therefore, although cultured cells can be shown to support hbv replication, there is yet no readily reproducible tissue culture system for hbv infectivity. the finding of over expression of spik in the hepg2 cells and huh 7 cells may suggest a strategy to establish a stable cell line that is susceptible for hbv infection by silencing those over expressed proteases inhibitor. admittedly, the cause and effect relationship between over expression of sipk and refractoriness to hbv is a long shot. never the less, it could be tested by straightforward techniques that were in use in the lab for other purposes. antisense oligonucleotides have been used previously to silence specific genes 80. in one experiment, hepg2 cells were pre - treated with 10 nm anti - sense or sense oligo corresponding to the translation initiation region of spik gene. after 2 days, cells were then infected with hbsag positive patient 's serum (10 viruses /ml). the progeny hbv both in the medium and in the cytosol were examined. by 3 days after the inoculation, the amount of hbsag in the culture medium fell below the level of detection. however, the level of hbsag in the culture medium from hepg2 cells pre - treated with anti - sense of spik significantly increased on the 4th day and 5th day post infection (p.i.), then decreased but maintained in a detectable level until harvest (7 days post infection, figure 4). on the other hand, hbsag in the culture medium from cells that were treated with sense oligo of spik were at background levels during entire period (figure 4). a similar result was found in the untreated hepg2 cells (data not shown). hepg2 cells were susceptible to hbv infection after treatment with spik specific anti - sense oligo was confirmed by the results from southern blot. at 8th day post infection, the replicative form of hbv in cytoplasm was detected feebly in infected cells that were pre - treated with anti - sense of spik (figure 5. however, no hbv dna was found in cells treated with spik sense oligo or when untreated (figure 5. the signals from infected cells were not very intense (this may be due to the less efficiently transport of anti - sense oligo to the cells), but were reproducibly present. however, the fact that hepg2 cells appear to be successfully infected by hbv following marginal down regulation of spik gene expression supports our hypothesis that the suppression of the over - expression of spik gene might reinstate the susceptibility of hepg2 cell to hbv infection by the restoration of the activity of indispensable protease. currently, a new technology called rna interference (rnai) or short interfering rna (sirna) was developed to silence target gene expression 81, 82, 83. using this method a stable cell line, in which the spik gene is silenced, could be established by using of the specific sirna, and then be selected by co - expressing a selectable gene such as the neo gene through screening with g418 84. after screening with g418, all surviving cells would be expected to generate the specific sirna silencing the targeted spik expression. the early results are, never the less, provocative, and beg further investigation. an effort, for example, to establish a susceptible cell line to hbv infection by silencing sipk is now under way. the structure of hepatitis b virus surface proteins and its interesting regions for infection. a. the structure of hbsag. b. the location of the interesting regions for hbv infection. 1g rna from hepg2 cells and human liver cells were reversely transcripted and amplified by pcr using specific primer for spik. lanes 1 to 4 were amplified with primers pi1/pi2 ; 5 to 8 were amplified with primers pi3/pi4. the samples from human liver cells were loaded in the column 1,2,5,6, (duplicated). c : rt - pcr detection of th gene of the human liver cells and hepg2 cells. 10g rna from huh7 (1), hepg2 (2) and human liver cells purchased from clonetech (3) were resolved in the 1% non - denatured agarose gel, then, transferred to nylon membrane. the image of ethidium bromide (eb) staining was taken from gel before transference. secreted hbsag in the hepg2 cells pre - treated with anti - sense of spik. hepg2 cells were pre - treated with 10nm / ml anti - sense or sense oligo of spik two days. the secreted hbsag in the culture medium was detected by auszyme kit (abbott lab. 10 hepg2 cells were pre - treated with anti - sense or sense oligo of spik or left untreated, then infected by patients ' serum. after removal of nuclei, hbv dna in cytoplasma was isolated, resolved in 1% agarose gel and detected by southern blot with hbv specific probe.	hepatitis b virus (hbv) is a human pathogen, causing the serious liver disease. despite considerable advances in the understanding of the natural history of hbv disease, most of the early steps in the virus life cycle remain unclear. virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. current knowledge on the early steps of hbv life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis b virus (dhbv) with duck primary duck hepatocytes. however, considering of the difference of the surface protein of hbv and dhbv both in the composition and sequence, the degree to which information from dhbv applies to human hbv attachment and entry may be limited. a major obstacle to the study hbv infection is the lack of a reliable and sensitive in vitro infection system. we have found that the digestion of hbv and woodchuck hepatitis virus (whbv) by protease v8 led to the infection of hepg2 cell, a cell line generally is refractory for their infection [lu. j virol. 1996. 70. 2277 - 2285. lu. virus research. 2001. 73(1) : 27 - 4 ].. further studies showed that a serine protease inhibitor kazal (spik) was over expressed in the hepg2 cells. therefore, it is possible that to silence the over expressed spik and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of hepg2 cells for hbv infection. the establishing a stable cell line for study of the early steps of hbv life cycle by silencing of spik is discussed.
acute rejection, observed after renal transplantation, has a significant impact for long - term renal allograft survival. even though its incidence was reduced by new immunosuppressive drugs, it remains a major problem after renal transplantation. acute rejection is a cell - mediated immune response that is initiated by the recognition of cd t cells by the major histocompatibility complex (mhc) class ii antigens on antigen - presenting cells (apcs) of the graft [14 ]. mhc class ii antigen activates cd t - lymphocytes, which subsequently release cytokines. released cytokines target vascular endothelial cells, the first cells to be recognized by the host 's immune system, and induce the expression of adhesion molecules and chemokines implicated in t - lymphocyte adhesion and extravasation. initial transient adhesion of t - lymphocytes mediated principally by selectins induce the rolling of the t - cells and their subsequent activation and adhesion on the endothelium via other adhesion molecules such as icam-1 and vcam-1. this adhesion stage provides the necessary signal for full t - cell activation leading to t - lymphocytes transendothelial migration. endothelial cells forming the interface between donor and recipients are the first donor cells to be recognized by the host 's immune system. endothelial cells coating the capillaries that act as a barrier between the donor organ and recipient bears the mhc class ii molecules. they are highly responsive to cytokines and express adhesion and other molecules implicated in t - lymphocyte adhesion and extravasation. such endothelial control in cell migration requires an effective intercellular adhesion, so called cell junction, between the endothelial cells. adherent junctions mediate the physical contacts between cells and anchor the actin cytoskeleton. cell - cell adhesion structures have been studied and some membrane proteins have been described such as pecam-1 or cd31 ; 51 and 21 integrins, v - cadherin, and vascular endothelium cadherin (ve - cadherin) [1012 ]. the extravasation of lymphocytes in rejecting renal allografts is thought to take place in activated peritubular capillaries. to date, the exact mechanisms involved in acute rejection after solid organ transplantation are not completely understood. in a previous work we have demonstrated that ve - cadherin is under expressed in acute rejection in a murine heterotopic heart transplantation model and in acute rejection following human heart transplantation. murine macroarrays results were validated in mice and in humans by immunohistochemistry and quantitative real - time - polymerase chain reaction (q - pcr). in this study we have performed immunohistochemical staining of ve - cadherin in human biopsies after renal transplantation. we demonstrate that ve - cadherin is also under expressed in acute rejection after renal transplantation. this study comprised transplant biopsy specimens from 15 patients (66% of men with mean age of 47 15 years). all patients received deceased donor kidney transplants. after transplantation, all patients received induction therapy with anti - lymphocyte globulin or anti il-2 receptor and triple - therapy immunossupression (cyclosporine, steroids, and mycophenolate mofetil). details relating to important clinical parameters in each group are given in table 1. needle kidney transplant biopsies were performed solely to define the diagnosis and management of patients with acute deterioration of allograft function in accordance with institutional guidelines. renal allograft tissues were obtained from transplant recipients undergoing ultrasound guided biopsies from december 2002 to august 2003. biopsies were performed between 1 day and 16 years after renal transplantation (453, 4 1118, 8 days). specimens were formalin fixed and paraffin embedded or snap frozen in liquid nitrogen. paraffin sections (4 m) were stained with periodic acid schiff (pas) and graded for acute rejection using the banff criteria. fifteen kidney transplant biopsies showing no rejection (n = 5), acute rejection grade ia (n = 5), or acute rejection grade iia (n = 5) were examined. positive control was performed using human spleen tissues that show a large number of endothelial cells. sections were incubated for 1 hour with a mouse antihuman ve - cadherin monoclonal antibody (1:50, chemicon international, temecula, ca, usa) followed by a goat antimouse immunoglobulin (dako, france), which was then revealed by the streptavidin - biotin immunoperoxidase method. sections were incubated for 1 hour with a mouse anti - human cd34 monoclonal antibody (1:50, dako, france) followed by an antimouse biotin (dako, france) which was then revealed by the streptavidin - biotin immunoperoxidase method. sections were assessed by two independent observers (ar, bm), scored according to the number of ve - cadherin stained endothelial cells in peritubular capillaries and the number of cd34 stained cells. sections were analyzed with a leica dmlb microscope (leica microsystems, germany) directly coupled to a 3ccd colour camera (jvc, argenteuil, france). quantification of ve - cadherin and cd34 staining was evaluated using image analysis software (perfect image, claravision orsay, france). positive ve - cadherin and cd34 labelled peritubular endothelial cells were counted in 10 high - power fields for each sample (x630 magnification). kruskal - wallis nonparametric test was used to compare clinical characteristics between the 3 groups. in order to take into account the variability between fields for each sample, variance analysis for repeated measures when this global test was significant, fisher lsd post - hoc test was further used when comparing between 2 groups. the mean time between transplantation and allograft biopsy was 431 1081 days (range 14298 days). there was no statistical difference between donor age, donor and recipient sex or ischemic time. serum creatinine (mean sd) was not statistically different between groups. all patients with histopathologic diagnosis of acute rejection received steroid treatment after the biopsy was obtained. all human biopsies were constituted by adequate fragments according to banff criteria showing no acute rejection (n = 5), grade ia acute rejection (n = 5), or grade iia acute rejection (n = 5) (see figure 1). in the no acute rejection group the peritubular capillary endothelial cells have a normal shape and an elongated cytoplasm. in the acute rejection grade ia the lumen size is unchanged but populated with numerous inflammatory cells adhering to swollen endothelial cells. in the acute rejection iia, the lumen is dilated, some swollen endothelial cells seemed to be detached from the basement membrane and inflammatory cells have migrated from the lumen to the interstitium. ve - cadherin staining was differentially expressed between the 3 groups (p =.02). in no acute rejection group ve - cadherin stained cells represented 1.65 0.71% of the surface examined compared to 1.62 0.34% in acute rejection grade ia and 0.75 0.31% in acute rejection grade iia (see figure 2). ve - cadherin expression was significantly reduced in acute rejection grade iia when compared to no acute rejection (p =.01) and to acute rejection grade ia (p =.02). there was no difference in ve - cadherin expression between acute rejection grade ia and no acute rejection group (p =.7). figure 3 shows ve - cadherin staining as a strong, thin, linear staining on peritubular endothelial cells in renal biopsies showing no rejection or acute rejection grade ia compared to acute rejection grade iia. cd34 staining confirmed the staining of ve - cadherin on peritubular endothelial cells (see figure 3). the working hypothesis in this study is that the endothelium lining the vessel wall of the donor kidney is first to be in contact with host cells and is subjected to immunological interactions. these molecules can facilitate the adhesion and transmigration of lymphocytes in the renal tissue leading to damage and ultimately to graft failure. we have previously demonstrated by macroarrays, immunohistochemistry, and q - pcr that ve - cadherin is under expressed in acute rejection following murine and human heart transplantation. in the current investigation, we bring evidence for the first time that ve - cadherin is implicated also in acute rejection following renal transplantation. lymphocyte adhesion to the vascular lining accompanies the first stages of the acute rejection reaction. the ability of endothelial cells to adhere lymphocytes is not a constant or a static phenomenon. several cytokines and adhesion molecules have shown to increase lymphocyte binding to and penetration through endothelial cells. in response to cytokines and to the expression of endothelial cells adhesion molecules, binding to endothelium is necessary, but not a sufficient request for the leukocyte to get into tissue. this stage is followed by a firm adhesion, and then by a rapid transmigration of leukocytes through endothelial intercellular junctions. renkonen. suggested and confirmed the hypothesis that the peritubular capillary endothelium is the site of entry of lymphocytes into rejecting kidney allografts. the authors showed that the increase in lymphocyte binding to peritubular capillaries precede the peak of leukocyte accumulation in the graft. light and electron microscopy revealed a marked activation of peritubular capillary endothelial cells in allografts, whereas these alterations were less severe or absent in syngenic controls and normal kidneys. ve - cadherin is an endothelial - specific membrane protein, present in adherent junctions of endothelial cells and responsible for the endothelial cell - cell adhesion. lampugnani. studied cultured endothelial cells monolayers by immunofluorescence microscopy. they described ve - cadherin as an endothelial - specific membrane protein with a thin, sharp continuous line highlighting the margins of each cell. it was present at the appositional surfaces of cultured cells only on reaching confluency. they observed that if the endothelial permeability was increased its distribution was punctuated and could be found only at intercellular contacts in a few areas. since then, various authors demonstrated the properties of cell - cell adhesion of the ve - cadherin and the relationship of its diminution with the increase of the endothelial permeability [1720 ]. in our study, we found the same kind of staining described as a thin, continuous line in the margins of renal peritubular human endothelial cells. cell adhesion molecules, mediating leukocyte adhesion to endothelial cells, have been shown to affect gene transcription in these cells. previous studies [1, 11, 12 ] have demonstrated that through their adhesion, leukocyte could transfer intracellular signals to endothelial cells in different ways. del maschio. suggested that these intracellular signals could induce endothelial intercellular disorganisation. they found that leukocyte adhesion to endothelial cells was related to a reduced ve - cadherin expression that could increase endothelial permeability. an alternative explanation for adherent junction 's disorganisation is the release of lytic enzymes from the pmn that can be responsible for ve - cadherin digestion and junction disassembly. such potential release of proteolytic enzymes, by activated leukocytes, may lead to cleavage of the extracellular domain of ve - cadherin, and an increase in the permeability in areas bearing deposited leukocytes. moreover, other authors [23, 24 ] have reported changes in cytosolic ca level in endothelial cells during the adhesion of polymorphonuclear leukocyte. the polymorphonuclear leukocyte adherence could induce a series of endothelial intracellular responses leading a detachment of catenins from ve - cadherin. it is conceivable that in acute rejection after solid organ transplantation activated lymphocytes adhering to vascular endothelial cells could affect the latter cells by inducing the disappearance of ve - cadherin from endothelial adherent junctions. such action could result in a significant increase in endothelial permeability due to the disassembly of endothelial adherent junctions. ve - cadherin was also investigated in other settings than acute rejection after solid organ transplantation. studied the immunohistochemical expression of ve - cadherin in an animal model of renal ischemia. the authors observed that 24 hours after ischemia, the majority of the renal microvasculature did not stain for ve - cadherin. they observed also that 72 hours after ischemia, ve - cadherin staining in the renal microvasculature was similar to that observed under physiological conditions. studied the in vitro effect of the uremic retention solute p - cresol observed in chronic renal failure. they observed an increase in endothelial permeability associated with a decreased staining of junctional ve - cadherin. in summary, in this study we have demonstrated for the first time a correlation between the expression of ve - cadherin present in peritubular endothelial cells and acute allograft rejection after human renal transplantation. the under expression of ve - cadherin in peritubular capillaries in acute rejection after kidney transplantation could be responsible for the lymphocyte transmigration into interstitial tissues leading to graft dysfunction. preventing the down regulation of ve - cadherin could conceivably prevent severe acute graft rejection.	genes involved in acute rejection (ar) after organ transplantation remain to be further elucidated. in a previous work we have demonstrated the under - expression of ve - cadherin by endothelial cells (ec) in ar following murine and human heart transplantation. serial sections from 15 human kidney banff - graded transplant biopsies were examined for the presence of ve - cadherin and cd34 staining by immunohistochemistry (no ar (n = 5), ar grade ia (n = 5), or ar grade iia (n = 5)). quantification of peritubular ec staining were evaluated and results were expressed by the percentage of stained cells per surface analysed. there was no difference in cd34 staining between the 3 groups. ve - cadherin expression was significantly reduced in ar grade iia when compared to no ar (p =.01) and to ar grade ia (p =.02). this study demonstrates a reduced ve - cadherin expression by ec in ar after renal transplantation. the down - regulation of ve - cadherin may strongly participate in human ar.
the nhs was started in 1976 when 121,700 married female registered nurses aged 3055 years received a questionnaire on health status and potential risk factors for major chronic diseases. ever since, participants were sent questionnaires biennially and the response rates have been 90% (8). the nhs was approved by the institutional review board of the brigham and women 's hospital (boston, ma). for the present analysis, we used the return of the 1984 questionnaire, which was completed by 97,510 participants, as baseline because information for family history was first collected in 1982 and the first extended food frequency questionnaire (ffq) was administered in 1984. in this 1984 questionnaire, women with a history of type 2 diabetes at baseline (n = 329) were excluded. furthermore, we excluded participants with cancer (except for nonmelanoma skin cancer) or cardiovascular diseases at baseline (n = 4,934), because the diagnosis of these diseases could have interfered with self - reports of lifestyle and family history. women with incident types of diabetes other than type 2 diabetes or unconfirmed type 2 diabetes were also excluded (n = 1,480). moreover, women with missing information for diabetes status, family history of diabetes, date of birth, weight, or height were excluded (n = 1,787). as a result there were no differences between the total study population (n = 81,757) and the population included for this analysis (n = 73,227) for baseline characteristics, except for slight differences in mean age (50.6 and 50.2 years), the proportion of participants with obesity (13.3 and 12.3%), mean weight change since age 18 years (28.3 and 27.6 kg), and the proportion of participants reporting to drink no alcohol (31.5 and 30.6%) or > 10 g of alcohol / day (23.6 and 24.0%). women were asked to report whether any of their first - degree family members (father, mother, and/or siblings) ever had diabetes in the questionnaires mailed in 1982, 1988, and 1992. no questions about the type of diabetes in family members were included. to assess obesity in the parents, a series of pictograms of body shape were included in the 1988 questionnaire (9). the pictograms estimate relative obesity, with values ranging from 1 (very lean) to 9 (very obese). women were asked to choose the pictogram that best described the body shape of their natural mother and their natural father at age 50 years. previous research has shown that these pictograms can provide a reasonably accurate estimate of measured bmi of the parents 15 years in the past (r = 0.74 for mothers and 0.63 for fathers) (9). according to the chosen image, both father and mother were categorized as nonobese (image 15) or obese (image 69). dietary information was collected using a semiquantitative ffq included in the questionnaires mailed to the participants every 24 years. the reported portions were converted to gram weights per serving and intakes of nutrients were computed by multiplying the frequency of consumption by the nutrient content in grams. physical activity was assessed every 24 years, asking about the time spent on vigorous and moderate physical activity on an average day in the last month. information on the highest educational degree of the participant and her husband, father 's and mother 's occupation, and race was assessed in the 1992 questionnaire. bmi was calculated as weight in kilograms divided by the square of height in meters. in 1980, women were asked to report their weight at age 18 years, which we used to calculate bmi at age 18 and weight change since age 18. in 1986, 1996, and 2000, waist and hip circumferences were assessed by questionnaires that included measurement instructions and a tape measure. the validity of self - reported weight and circumference measurements was assessed in 140 participants from the nhs aged 4165 years. pearson correlations between self - reported and technician - measured weight, waist circumference, and hip circumference were high (r = 0.97, 0.89, and 0.84, respectively) (12). women who reported having diabetes in the biennial questionnaires the national diabetes data group criteria (13) were used to confirm diagnosis of diabetes according to 1) an elevated glucose concentration (fasting plasma glucose of 7.8 mmol / l, random plasma glucose of 11.1 mmol / l, or plasma glucose 11.1 mmol / l after an oral glucose load) and at least one symptom related to diabetes (excessive thirst, polyuria, weight loss, or hunger) ; 2) no symptoms, but elevated glucose concentrations on two occasions ; or 3) treatment with insulin or oral hypoglycemic medication. for cases of type 2 diabetes identified after 1998, the american diabetes association criteria (14) were used, lowering the cutoff point for fasting plasma glucose concentrations to 7.0 mmol / l. in a subsample, type 2 diabetes that was confirmed by the supplementary questionnaire was consistent with medical record reviews by an endocrinologist in 98% of the women (15). person - time for each participant was calculated from the date of return of the 1984 questionnaire to the date of diagnosis of type 2 diabetes, death, or june 2004, whichever came first. cox proportional hazards analysis was used to estimate the relative risk (rr) for type 2 diabetes according to family history. to evaluate the contribution of different covariates on the association between family history and diabetes risk, we used multivariate models with the following adjustments : 1) age ; 2) age, socioeconomic status (ses) (occupation of the father, occupation of the mother, husband 's highest degree, and participant 's highest degree), and race ; 3) age, ses, race, and lifestyle (smoking, physical activity, polyunsaturated / saturated fat intake ratio and intakes of coffee, alcohol, fruit, vegetables, sugar - sweetened beverages, whole grains, red meat, trans - fat, and total energy) ; and 4) age, ses, race, lifestyle, and bmi. additional analyses were performed to evaluate whether inclusion of waist circumference or a combination of bmi at age 18 and weight change since age 18 in the statistical model would alter the results. in addition, we conducted a sensitivity analysis evaluating the effect of blood glucose testing on the results. since 1998, women were asked biennially to report whether they had been screened for fasting blood glucose in the previous 2 years. we repeated our analysis for follow - up cycles from 1996 to 2004 restricted to women who reported to have been tested for blood glucose during a 2-year follow - up period. all covariates were updated during follow - up whenever new information was obtained. for dietary variables we used cumulative updating to reduce within - person variation (16). the proportion of the association explained by including different covariates in the model was calculated based on the change in regression coefficients, using the method of lin. all p values were two - tailed, and p 40 we evaluated whether various diabetes risk factors could explain the association between family history and risk of type 2 diabetes. adjustment for socioeconomic status and race did not substantially change the association between family history and diabetes risk, whereas adjustment for lifestyle explained part of the association (table 1). to further evaluate this finding consumption of alcohol, red meat, and sugar - sweetened beverages explained 4.8% (95% ci 4.35.3), 1.1% (0.81.3), and 2.8% (2.43.2), respectively, of the association between family history of diabetes and risk of type 2 diabetes, whereas other lifestyle variables did not significantly contribute to explaining the association. adjustment for bmi also weakened the association between family history of diabetes and risk of type 2 diabetes (table 1), explaining 21.1% (19.422.9) of the association. we conducted a sensitivity analysis restricted to women who underwent blood glucose screening for the 19962004 period in which this information was available. among the screened women, the effect of additionally adjusting for ses and race, lifestyle, and adiposity was essentially the same as that for the complete study population (supplementary table a2, available in an online appendix). to further explore the role of adiposity in the association between a family history of diabetes and incident diabetes, we ran various models, adjusting for waist and hip circumference or adolescent bmi and adult weight change (table 2). neither consideration of measures of body fat distribution nor adolescent bmi explained a substantially greater proportion of the association between family history and diabetes risk compared with the model that only included bmi. rr of type 2 diabetes according to family history of diabetes adjusted for various measures of body fatness family history of diabetes denotes diabetes in 1 first - degree family members. all anthropometric variables were modeled as continuous variables : bmi (weight in kilograms divided by the square of height in meters), waist circumference (centimeters), hip circumference (centimeters), and weight change (kilograms). restricted to participants with complete data on bmi at age 18 years (4,822 diabetes events during 1,295,965 person - years of follow - up). adjusted for age, race, ses, and lifestyle as described in the footnotes to table 1. restricted to participants with complete data on waist and hip circumference with follow - up starting in 1986 when data on waist and hip circumference were first collected (3,201 diabetes events during 834,231 person - years of follow - up). in women who had at least one parent with obesity, the baseline prevalence of overweight (bmi 25.029.9 kg / m) was 30% and the prevalence of obesity (bmi 30.0 kg / m) was 17% compared with 25 and 10%, respectively, in women who did not have parents with obesity (p 40 we evaluated whether various diabetes risk factors could explain the association between family history and risk of type 2 diabetes. adjustment for socioeconomic status and race did not substantially change the association between family history and diabetes risk, whereas adjustment for lifestyle explained part of the association (table 1). to further evaluate this finding, we included all factors separately in the model. consumption of alcohol, red meat, and sugar - sweetened beverages explained 4.8% (95% ci 4.35.3), 1.1% (0.81.3), and 2.8% (2.43.2), respectively, of the association between family history of diabetes and risk of type 2 diabetes, whereas other lifestyle variables did not significantly contribute to explaining the association. adjustment for bmi also weakened the association between family history of diabetes and risk of type 2 diabetes (table 1), explaining 21.1% (19.422.9) of the association. we conducted a sensitivity analysis restricted to women who underwent blood glucose screening for the 19962004 period in which this information was available. among the screened women, the effect of additionally adjusting for ses and race, lifestyle, and adiposity was essentially the same as that for the complete study population (supplementary table a2, available in an online appendix). to further explore the role of adiposity in the association between a family history of diabetes and incident diabetes, we ran various models, adjusting for waist and hip circumference or adolescent bmi and adult weight change (table 2). neither consideration of measures of body fat distribution nor adolescent bmi explained a substantially greater proportion of the association between family history and diabetes risk compared with the model that only included bmi. rr of type 2 diabetes according to family history of diabetes adjusted for various measures of body fatness family history of diabetes denotes diabetes in 1 first - degree family members. all anthropometric variables were modeled as continuous variables : bmi (weight in kilograms divided by the square of height in meters), waist circumference (centimeters), hip circumference (centimeters), and weight change (kilograms). restricted to participants with complete data on bmi at age 18 years (4,822 diabetes events during 1,295,965 person - years of follow - up). adjusted for age, race, ses, and lifestyle as described in the footnotes to table 1. restricted to participants with complete data on waist and hip circumference with follow - up starting in 1986 when data on waist and hip circumference were first collected (3,201 diabetes events during 834,231 person - years of follow - up). in women who had at least one parent with obesity, the baseline prevalence of overweight (bmi 25.029.9 kg / m) was 30% and the prevalence of obesity (bmi 30.0 kg / m) was 17% compared with 25 and 10%, respectively, in women who did not have parents with obesity (p < 0.0001). table 3 shows the risk of type 2 diabetes according to four categories of diabetes and obesity in the parents. compared with that for women without a parental history of diabetes or obesity, the rr of type 2 diabetes was similar for women with a parental history of both obesity and diabetes and those with only a parental history of diabetes but substantially weaker for women with only a parental history of obesity. the association between having only a parental history of obesity and risk of type 2 diabetes was fully explained by the higher bmi of the participants (table 3). rr of type 2 diabetes according to a parental history of diabetes and obesity history of diabetes and/or obesity in either the father or the mother. in this prospective cohort study of 73,227 women with 20 years of follow - up, we observed a direct association between a family history of diabetes and incidence of type 2 diabetes, which was similar for having a paternal or a maternal history of diabetes. a substantial part of this association could be explained by the higher bmi in individuals with a family history of diabetes. lower alcohol consumption and higher consumption of red meat and sugar - sweetened beverages modestly contributed to explaining the association between family history of diabetes and risk of type 2 diabetes. consistent with our findings, earlier research reported rrs of type 2 diabetes in individuals with self - reported parental history of diabetes ranging from 1.87 to 2.26 (13). in the national health and nutrition examination survey, adjustment for sex, race, age, hypertension, household income, and education had no appreciable effect on the association between family history of diabetes and prevalence of type 2 diabetes, but adjustment for bmi did weaken the association (18). in our study, generalized adiposity as reflected by bmi rather than circumference measures of fat distribution contributed to the association between family history of diabetes and diabetes risk. this finding agrees with studies in which having a family history of diabetes was associated with a higher bmi but not independently with a higher waist - to - hip ratio (57). furthermore, the quebec family study revealed that total body fat, but not fat distribution, shared common familial determinants (19). consumption of red meat, sugar - sweetened beverages, and alcohol explained part of the association between family history of diabetes and risk of type 2 diabetes. higher consumption of red meat and sugar - sweetened beverages and alcohol abstinence (compared with moderate alcohol consumption) have been associated with a higher risk of type 2 diabetes (2022). consistent with the current finding, a parental history of diabetes was associated with lower alcohol consumption in a population - based study in the netherlands (5). higher consumption of red meat and sugar - sweetened beverages and alcohol abstinence (instead of moderate consumption) may thus modestly contribute to the higher risk of type 2 diabetes in individuals with a family history of diabetes. for a factor to explain a proportion of the association between a family history of diabetes and incident type 2 diabetes, it has to be both associated with risk of type 2 diabetes and shared by family members. in our study, established risk factors for diabetes such as smoking and lack of physical activity (2) did not contribute to the association between a family history and incident type 2 diabetes, suggesting that these factors did not cluster strongly within families at the age studied. this finding, however, does not imply that individuals with a family history of diabetes can not benefit from adopting these lifestyle factors to lower their risk of developing type 2 diabetes (23). furthermore, the prospective design limited the probability of differential misclassification of risk factors, and selection bias was limited because of the high response rates for the follow - up questionnaires. first, diabetes was assessed by self - report, which may have led to misclassification. however, our validation study using medical records indicated that the reporting of diabetes was accurate in this medically knowledgeable population. second, individuals with a family history of diabetes have a higher probability of being tested for diabetes compared with the general population (24), which increases their chance of having diabetes detected once it develops. however, in a sensitivity analysis restricted to women who underwent blood glucose testing, the effects of adjustment for lifestyle factors and adiposity were essentially the same as those for the complete study population. third, family history of diabetes was not specific to type 2 diabetes, which might have influenced our results. fourth, some measurement error in the assessment of adiposity, family history, and lifestyle is likely to have occurred. this has probably led to an underestimation of the proportion of the association between family history of diabetes and risk of type 2 diabetes that can be explained by adiposity and lifestyle factors. however, we can not completely exclude the possibility that correlated error in the assessment of adiposity and lifestyle and the assessment of family history of obesity and diabetes has led to an overestimation of the explained proportions of the association between family history and risk of type 2 diabetes. finally, our study only included registered nurses, which limited the variation in ses and, to a lesser extent, lifestyle behaviors. in more diverse populations, ses, lifestyle factors, and adiposity may explain a greater proportion of the association between family history of diabetes and diabetes risk. to summarize, our prospective findings confirm that having a first - degree family member with diabetes is a strong risk factor for type 2 diabetes with a similar risk associated with having a maternal and a paternal history of diabetes. a substantial part of the association between having a family history of diabetes and 20-year incidence of type 2 diabetes could be explained by excess adiposity, whereas dietary factors including consumption of alcohol, red meat, and sugar - sweetened beverages might also play a role. further studies on this topic with more detailed measures of family history of diabetes, adiposity, and lifestyle factors are warranted. however, the current findings in combination with recent studies of genetic risk variants (3,4) suggest that most of the association between family history of diabetes and diabetes risk remains unexplained. further research on novel genetic and lifestyle risk factors, lifestyle in other periods of life, epigenetic risk factors, and specific gene - environment interactions is warranted to identify additional factors that mediate the strong association between having a family history of diabetes and personal risk of type 2 diabetes.	objectiveto evaluate to what extent the association between family history of diabetes and risk of type 2 diabetes can be explained by excess adiposity and lifestyle risk factors.research design and methodswe analyzed data from 73,227 women who participated in the nurses ' health study cohort. a family history of diabetes was defined as having at least one first - degree family member with diabetes. lifestyle factors, weight, and height were assessed by using validated questionnaires, and bmi was calculated. the relative risk of type 2 diabetes was estimated using cox proportional hazards analysis.resultswe documented 5,101 cases of type 2 diabetes during 20 years of follow - up. the age - adjusted relative risk of type 2 diabetes in participants with a family history was 2.27 (95% ci 2.142.40) compared with the risk in those without a family history of diabetes. participants with a family history of diabetes had a higher bmi and were more likely to have a parental history of obesity. bmi explained 21.1% (19.422.9) of the association between family history of diabetes and risk of type 2 diabetes. intakes of red meat, alcohol, and sugar - sweetened beverages explained 1.1% (0.81.3), 4.8% (4.35.3), and 2.8% (2.43.2) of this association, respectively.conclusionsthese results suggest that excess adiposity and, to a lesser extent, specific dietary habits can explain a substantial part of the association between having a family history of diabetes and risk of type 2 diabetes.
penile squamous cell carcinoma (scc) is a rare but challenging urological tumour with a potentially devastating course of disease. only a few studies with larger scale patient cohorts have been published due to the low prevalence. the heterogeneous clinical appearance and different diagnostic and therapeutic strategies make it difficult to perform randomized trials. furthermore, the analysis is complicated by the fact that in earlier decades, different tumour classifications were used and that the now widely used tnm classification has undergone changes through regular updates. no prospective randomized clinical trials were found (jul 2013), therefore there is an overall low level of evidence. the current eau guidelines on penile cancer (2009) were used as a standard reference. penile carcinoma occurs in western countries, depending on the region, with an incidence of 0.5 to 1.6 cases per 100,000 men and represents approximately 0.5% of all malignant tumours in men. higher prevalence of the disease was detected in several asian, african and south american countries. penile carcinoma is a tumour of aging men, with a strong increase of incidence in the 6th decade of life and a peak around the 80s. scc is the most common malignant tumour of the penis, in addition to the very rare malignant lesions such as basal cell carcinoma, malignant melanoma, sarcoma, m. paget, lymphoreticular tumours and metastases. the possible risk factors are low standards of hygiene, phimosis, recurrent balanitis, high number of sexual partners (early age at first sexual intercourse), presence of hpv infection, circumcision practice, strong tobacco consumption, genital ultraviolet radiation and penile trauma. the known data suggests that the critical exposure period for certain etiologic factors is before puberty. however, international scientific societies (including the american academy of pediatrics) have not yet declared a general recommendation for neonatal circumcision because of insufficient data for its preventive effect. human papillomavirus (hpv) infection was identified to play a role in the development of penile cancer by interfering with the cell cycle regulation. dna sequences of hpv (most frequently types 16 and 18) were detected in about 4080% of tumour tissue samples in primary penile and metastatic lesions. the current data regarding the prognostic importance of hpv dna in tumour tissue is still contradictory. tobacco products seem to support the malignant cell transformation in existing hpv infection or local bacterial chronic inflammation [15 ]. the diagnosis is delayed in up to 50% of patients with scc because of shame, guilt, fear, ignorance and carelessness. the reasons for delay on the medical side are often prolonged systemic and local treatment before the biopsy of the lesion (figure 1). two thirds of the patients have an organ confined disease at the time of initial diagnosis. primary metastatic sites are the regional inguinal lymph nodes (up to 45%, depending on the histological pattern) and later, the pelvic nodes. therefore, a proper examination of the inguinal lymph node status is crucial for the evaluation of the prognosis and overall survival. an overall stage independent 5year survival rate is 50%, but only 27% of patients with lymph node metastases survive the first five years after diagnosis. detectable distant metastases (lung, liver, bone, or brain) are rare and occur late in the course of the disease. penile carcinoma is characterized by a permanent progressive course that leads to death within two years in the majority of untreated patients. 56yr patient with pt3g3 penile scc (ulcer with infiltration of urethra) and clinical enlarged inguinal lns on the left side. in addition, it has been found that the presence of vascular invasion is of significant prognostic importance. the initial classification scheme from 1921, by broders, was revised in 1991 by maiche, adding that the assessment of poorly differentiated tumour components is particularly important. recently there was an alignment of the tnm system of uicc (union internationale contre le cancer) and that of the ajcc (american joint committee on cancer). in addition to the inspection and palpation of the external genitalia, the careful examination of the bilateral inguinal region for enlarged lymph nodes is crucial. it may be difficult to detect lymph nodes in obese patients and in patients previously operated in the groin. the enlarged inguinal lymph nodes can cause skin necrosis, chronic inflammation and lead to death due to sepsis or erosion of the femoral vessels in advanced metastatic disease. the scc antigen, a glycoprotein belonging to the family of serine protease inhibitors, is a serological marker for scc (lung, cervix, esophagus, etc.), but also increases in benign diseases (e.g. psoriasis). significant increases in serum values were measured only in patients with widespread lymph node metastases or organ metastases. further prospective studies should analyse the value of scc examination for therapy control or follow up. several studies detected hypercalcemia without the presence of bone metastases, but associated with a lymph node involvement. although the inguinal lymph nodes are well accessible for palpation, the clinical examination makes a false prediction of the true pathological status in about 40% of patients. half of these cases are enlarged due to metastasis, the other half by a nonspecific inflammation. this means that in about 50% of cases an unnecessary lymphadenectomy, which is associated with high morbidity, is performed. on the other hand, 1025% of patients without clinical signs have occult lymph node metastases. ultrasound examination, although easy to use, has been shown to be imprecise due to high examiner variability. ct and mri imaging techniques are able to detect lymph nodes > 0.51 cm (sensitivity 36%). only the size of the node can be reliably assessed but inflammation and metastasis can not be differentiated. however, ct and mri are useful for lymph node evaluation in obese patients or those after inguinal operations and for detection of pelvic lymph node metastases or distant metastases. positron emission tomography (pet) is already used successfully for the detection of malignant tumours and metastases of other tumour entities. some studies reported an increased 18f fdg (18f fluorodeoxyglucose) uptake in penile carcinoma and its metastases. in combination with ct, the known general problems of fdg pet are the lack of possibility to detect micro metastases and the false positive results in inflamed lymph nodes [11, 11, 13 ]. in patients without evidence of lymph nodes involvement, but with histopathological risk factors, a clarification of the situation should be achieved with minimal morbidity. patients with detectable lymphadenopathy benefit from an exact diagnosis when inflammatory changes can be reliably found and operations avoided. there are several current approaches, such as the fine needle biopsy, the lymph node biopsy, dynamic sentinel lymph node biopsy with lymph node mapping, the sentinel lymph node resection, and the superficial or modified lymph node resection. the fine needle biopsy (fnb) with aspiration cytology is easily done and especially useful in patients with palpable lymph nodes. the inguinal lymph nodes are evaluated with high resolution ultrasound (7.5 mhz at least). subsequently, an ultrasound or ct guided biopsy and the cytopathological examination of aspirate are done. if the result is negative, fnb can be repeated after a course of antibiotic treatment. due to the large number of false negative results and an unsatisfactory sensitivity (3971%), this method is considered to be unreliable for tumour staging in patients with negative clinical findings. the principle of the sln (sentinel lymph node) is based on the theory that there are one or more lymph nodes in a primary metastatic region of tumour. the dsnb (dynamic sentinel node biopsy) can be easily done and has significantly fewer complications than the radical inguinal lymphadenectomy. lymphoscintigraphy with tc99 m labelled colloidal human albumin is performed a day before the surgery (figure 2a). intraoperative measurement of the radioactive activity is performed with a gamma camera, and the slns are localised and excised (mapping) (figure 2b). in addition, patent blue can be injected at the primary lesion in order to simplify the detection of the lymph nodes. the complication rate is 56% (radical lymphadenectomy up to 40%) and most complications can be treated conservatively. the inguinal lymph node dissection is performed only if dsnb is positive, in order to prevent overtreatment. this method is less useful in patients with palpable lymph nodes due to the high false negative rates caused by the blockage of lymph vessels by tumour cells [17, 18 ]. there are only a few studies that have analysed the reliability of the intraoperative lymph node mapping as a staging procedure in penile carcinoma (m.d. false negative rates of 0% to 29% with a significant fluctuation of the sensitivity (71 93%) were detected. the good results (overall 7% false negative, 93% sensitivity) in the largest two center study (n = 323) by leijte. are explained by the modification and technical improvement of the method over years. in addition, fnb and the histopathological work up with standardized application of immunohistochemistry were introduced. in this method, the initial false negative rate of 22% could be reduced to less than 5%. specialised clinics is limited by the small number of patients and complexity of the procedure [1421 ]. sentinel lymph nodes are marked on both sides (the illustration was kindly provided by md hautzel and md antke, nuclear medicine clinic, university of duesseldorf). penile carcinoma occurs in western countries, depending on the region, with an incidence of 0.5 to 1.6 cases per 100,000 men and represents approximately 0.5% of all malignant tumours in men. higher prevalence of the disease was detected in several asian, african and south american countries. penile carcinoma is a tumour of aging men, with a strong increase of incidence in the 6th decade of life and a peak around the 80s. scc is the most common malignant tumour of the penis, in addition to the very rare malignant lesions such as basal cell carcinoma, malignant melanoma, sarcoma, m. paget, lymphoreticular tumours and metastases. the possible risk factors are low standards of hygiene, phimosis, recurrent balanitis, high number of sexual partners (early age at first sexual intercourse), presence of hpv infection, circumcision practice, strong tobacco consumption, genital ultraviolet radiation and penile trauma. the known data suggests that the critical exposure period for certain etiologic factors is before puberty. however, international scientific societies (including the american academy of pediatrics) have not yet declared a general recommendation for neonatal circumcision because of insufficient data for its preventive effect. human papillomavirus (hpv) infection was identified to play a role in the development of penile cancer by interfering with the cell cycle regulation. dna sequences of hpv (most frequently types 16 and 18) were detected in about 4080% of tumour tissue samples in primary penile and metastatic lesions. the current data regarding the prognostic importance of hpv dna in tumour tissue is still contradictory. tobacco products seem to support the malignant cell transformation in existing hpv infection or local bacterial chronic inflammation [15 ]. the diagnosis is delayed in up to 50% of patients with scc because of shame, guilt, fear, ignorance and carelessness. the reasons for delay on the medical side are often prolonged systemic and local treatment before the biopsy of the lesion (figure 1). two thirds of the patients have an organ confined disease at the time of initial diagnosis. primary metastatic sites are the regional inguinal lymph nodes (up to 45%, depending on the histological pattern) and later, the pelvic nodes. therefore, a proper examination of the inguinal lymph node status is crucial for the evaluation of the prognosis and overall survival. an overall stage independent 5year survival rate is 50%, but only 27% of patients with lymph node metastases survive the first five years after diagnosis. detectable distant metastases (lung, liver, bone, or brain) are rare and occur late in the course of the disease. penile carcinoma is characterized by a permanent progressive course that leads to death within two years in the majority of untreated patients. 56yr patient with pt3g3 penile scc (ulcer with infiltration of urethra) and clinical enlarged inguinal lns on the left side. in addition, it has been found that the presence of vascular invasion is of significant prognostic importance. the initial classification scheme from 1921, by broders, was revised in 1991 by maiche, adding that the assessment of poorly differentiated tumour components is particularly important. recently there was an alignment of the tnm system of uicc (union internationale contre le cancer) and that of the ajcc (american joint committee on cancer). in addition to the inspection and palpation of the external genitalia, the careful examination of the bilateral inguinal region for enlarged lymph nodes is crucial. it may be difficult to detect lymph nodes in obese patients and in patients previously operated in the groin. the enlarged inguinal lymph nodes can cause skin necrosis, chronic inflammation and lead to death due to sepsis or erosion of the femoral vessels in advanced metastatic disease. there are no tumour specific serum markers. the scc antigen, a glycoprotein belonging to the family of serine protease inhibitors, is a serological marker for scc (lung, cervix, esophagus, etc.), but also increases in benign diseases (e.g. psoriasis). significant increases in serum values were measured only in patients with widespread lymph node metastases or organ metastases. further prospective studies should analyse the value of scc examination for therapy control or follow up. several studies detected hypercalcemia without the presence of bone metastases, but associated with a lymph node involvement. although the inguinal lymph nodes are well accessible for palpation, the clinical examination makes a false prediction of the true pathological status in about 40% of patients. half of these cases are enlarged due to metastasis, the other half by a nonspecific inflammation. this means that in about 50% of cases an unnecessary lymphadenectomy, which is associated with high morbidity, is performed. on the other hand, 1025% of patients without clinical signs have occult lymph node metastases. ultrasound examination, although easy to use, has been shown to be imprecise due to high examiner variability. ct and mri imaging techniques are able to detect lymph nodes > 0.51 cm (sensitivity 36%). only the size of the node can be reliably assessed but inflammation and metastasis can not be differentiated. however, ct and mri are useful for lymph node evaluation in obese patients or those after inguinal operations and for detection of pelvic lymph node metastases or distant metastases. positron emission tomography (pet) is already used successfully for the detection of malignant tumours and metastases of other tumour entities. some studies reported an increased 18f fdg (18f fluorodeoxyglucose) uptake in penile carcinoma and its metastases. in combination with ct, sensitivities of 7588% were detected but large randomized series are still missing. the known general problems of fdg pet are the lack of possibility to detect micro metastases and the false positive results in inflamed lymph nodes [11, 11, 13 ]. in patients without evidence of lymph nodes involvement, but with histopathological risk factors, a clarification of the situation should be achieved with minimal morbidity. patients with detectable lymphadenopathy benefit from an exact diagnosis when inflammatory changes can be reliably found and operations avoided. there are several current approaches, such as the fine needle biopsy, the lymph node biopsy, dynamic sentinel lymph node biopsy with lymph node mapping, the sentinel lymph node resection, and the superficial or modified lymph node resection. the fine needle biopsy (fnb) with aspiration cytology is easily done and especially useful in patients with palpable lymph nodes. the inguinal lymph nodes are evaluated with high resolution ultrasound (7.5 mhz at least). subsequently, an ultrasound or ct guided biopsy and the cytopathological examination of aspirate are done. if the result is negative, fnb can be repeated after a course of antibiotic treatment. due to the large number of false negative results and an unsatisfactory sensitivity (3971%), this method is considered to be unreliable for tumour staging in patients with negative clinical findings. the principle of the sln (sentinel lymph node) is based on the theory that there are one or more lymph nodes in a primary metastatic region of tumour. the dsnb (dynamic sentinel node biopsy) can be easily done and has significantly fewer complications than the radical inguinal lymphadenectomy. lymphoscintigraphy with tc99 m labelled colloidal human albumin is performed a day before the surgery (figure 2a). intraoperative measurement of the radioactive activity is performed with a gamma camera, and the slns are localised and excised (mapping) (figure 2b). in addition, patent blue can be injected at the primary lesion in order to simplify the detection of the lymph nodes. the complication rate is 56% (radical lymphadenectomy up to 40%) and most complications can be treated conservatively. the inguinal lymph node dissection is performed only if dsnb is positive, in order to prevent overtreatment. this method is less useful in patients with palpable lymph nodes due to the high false negative rates caused by the blockage of lymph vessels by tumour cells [17, 18 ]. there are only a few studies that have analysed the reliability of the intraoperative lymph node mapping as a staging procedure in penile carcinoma (m.d. false negative rates of 0% to 29% with a significant fluctuation of the sensitivity (71 93%) were detected. the good results (overall 7% false negative, 93% sensitivity) in the largest two center study (n = 323) by leijte. are explained by the modification and technical improvement of the method over years. in addition, fnb and the histopathological work up with standardized application of immunohistochemistry were introduced. in this method, the initial false negative rate of 22% could be reduced to less than 5%. the value of this technique for not specialised clinics is limited by the small number of patients and complexity of the procedure [1421 ]. 53yr patient with pt1g3 penile scc and clinical inapparent inguinal lns (histology : no malignancy). sentinel lymph nodes are marked on both sides (the illustration was kindly provided by md hautzel and md antke, nuclear medicine clinic, university of duesseldorf). the extent and the complete removal of the inguinal lymph node metastases is the most important prognostic factor in patients with penile carcinoma. bilateral inguinal lymphadenectomy was the standard therapy for intermediate and high risk tumours for a long time ; however, the indication has changed in recent years due to the further development of diagnostic options such as dsnb. the regional inguinal lymph nodes are divided into superficial and deep lymph nodes (divided by fascia lata). the inguinal region is sectored in five clinically relevant zones (daseler zones, figure 2c). bilateral ln dissection is indicated in the presence of inguinal lymph node metastases. however, extended ln dissection is associated with high perioperative complications (3070%), including prolonged lymphatic fistula, skin oedema and necrosis, wound infections, thrombosis and embolization. in recent series, the morbidity is significantly lower due to improved perioperative care, as well as advances in modified resection technique with careful preservation of the dermis, the scarpa 's fascia, and the great saphenous vein. pelvic lymph nodes should not be excised if inguinal lymph nodes are tumour free because there are no direct lymph vessels from the primary tumour to the pelvic lymph nodes. if the lymph node of cloquet or at least two superficial inguinal lns are affected a rate of metastatic pelvic lymph nodes of 2356% was detected. in these cases 53yr patient with pt1g3 penile scc and clinical inapparent inguinal lns (histology : no malignancy). the region is divided into five zones : central zone (v), superior (i) and inferior (iv) medial zones, serior (ii) and inferior (iii) lateral zones. in penile carcinoma, limited experience has shown that adjuvant chemotherapy may improve the long term survival of patients after radical resection of positive lymph nodes and that neoadjuvant chemotherapy permits resection after downsizing in about 50% of patients with primary inoperable inguinal metastases the response rates are low (50%). commonly occuring severe side effects which can restrict or significantly complicate the use of chemotherapy in elderly patients with impaired organ function, have found that a total of 18 different chemotherapy regimens (mean response rates 80% and lasting remissions in 50% of six patients in an small uncontrolled pilot series. the prognosis of patients with distant metastases is still very poor (median survival of 6 mo). have treated the largest group (n = 40) in a phase ii multicenter study with the cbm regimen. only one patient with retroperitoneal lymph node metastases achieved a complete remission and two patients with lung metastases achieved a partial remission. the doses required for effective tumour therapy (60 gy) lead to high rates of major complications (e.g. fistula of urethra, stricture, necrosis, fibrosis of corpora cavernosa, pain, and oedema). are performed for the treatment of the primary tumour lesions in selected cases. according to the eau guidelines, prophylactic irradiation of the palpable lymph nodes in addition, it complicates the evaluation of a possible recurrence in the post radiation fibrotic tissue. for not enlarged inguinal lymph nodes at low risk stage and in patients with negative sln biopsy results, a close follow up should be done, if an appropriate compliance of the patient is provided. most important are the clinical examination of the groins, ultrasonography, and in selected cases additional imaging techniques such as pet ct. about 90% of recurrences occur within the first five years and mostly at the site of the primary lesion [1, 14, 15, 37 ]. the extent and the complete removal of the inguinal lymph node metastases is the most important prognostic factor in patients with penile carcinoma. bilateral inguinal lymphadenectomy was the standard therapy for intermediate and high risk tumours for a long time ; however, the indication has changed in recent years due to the further development of diagnostic options such as dsnb. the regional inguinal lymph nodes are divided into superficial and deep lymph nodes (divided by fascia lata). the inguinal region is sectored in five clinically relevant zones (daseler zones, figure 2c). bilateral ln dissection is indicated in the presence of inguinal lymph node metastases. however, extended ln dissection is associated with high perioperative complications (3070%), including prolonged lymphatic fistula, skin oedema and necrosis, wound infections, thrombosis and embolization. in recent series, the morbidity is significantly lower due to improved perioperative care, as well as advances in modified resection technique with careful preservation of the dermis, the scarpa 's fascia, and the great saphenous vein. pelvic lymph nodes should not be excised if inguinal lymph nodes are tumour free because there are no direct lymph vessels from the primary tumour to the pelvic lymph nodes. if the lymph node of cloquet or at least two superficial inguinal lns are affected a rate of metastatic pelvic lymph nodes of 2356% was detected. in these cases 53yr patient with pt1g3 penile scc and clinical inapparent inguinal lns (histology : no malignancy). the region is divided into five zones : central zone (v), superior (i) and inferior (iv) medial zones, serior (ii) and inferior (iii) lateral zones. in penile carcinoma, limited experience has shown that adjuvant chemotherapy may improve the long term survival of patients after radical resection of positive lymph nodes and that neoadjuvant chemotherapy permits resection after downsizing in about 50% of patients with primary inoperable inguinal metastases. however, chemotherapy alone in metastatic disease is not curative. the response rates are low (50%). commonly occuring severe side effects which can restrict or significantly complicate the use of chemotherapy in elderly patients with impaired organ function, have found that a total of 18 different chemotherapy regimens (mean response rates 80% and lasting remissions in 50% of six patients in an small uncontrolled pilot series. the prognosis of patients with distant metastases is still very poor (median survival of 6 mo). have treated the largest group (n = 40) in a phase ii multicenter study with the cbm regimen. only one patient with retroperitoneal lymph node metastases achieved a complete remission and two patients with lung metastases achieved a partial remission. the doses required for effective tumour therapy (60 gy) lead to high rates of major complications (e.g. fistula of urethra, stricture, necrosis, fibrosis of corpora cavernosa, pain, and oedema). are performed for the treatment of the primary tumour lesions in selected cases. according to the eau guidelines, prophylactic irradiation of the palpable lymph nodes in addition, it complicates the evaluation of a possible recurrence in the post radiation fibrotic tissue. for not enlarged inguinal lymph nodes at low risk stage and in patients with negative sln biopsy results, a close follow up should be done, if an appropriate compliance of the patient is provided. most important are the clinical examination of the groins, ultrasonography, and in selected cases additional imaging techniques such as pet ct. about 90% of recurrences occur within the first five years and mostly at the site of the primary lesion [1, 14, 15, 37 ]. scc of the penis is a rare, biologically aggressive tumour. in advanced stages, the prevention and early detection are crucial, as an advanced disease with metastatic lymph node involvement worsens the prognosis considerably. the previously used radical bilateral inguinal lymphadenectomy can be avoided in most cases of early detection. in patients with unsuspicious clinical palpation of the inguinal region, intensive diagnostics (fnb, dsnb) should be done to exclude the metastases before these patients can be qualified for follow up regimen. in the case of lymph node metastases modified inguinal lymph node dissection is indicated and if necessary, followed by the consecutive pelvic lymph node dissection. a cisplatin based chemotherapy is indicated in adjuvant, neoadjuvant or advanced palliative setting but the individual benefit and risk of toxicity should be evaluated prior to therapy (figure 3). a practical approach to provide larger scale studies with good evidence algorithm for the treatment of ln metastases [according to eau guidelines 2009 (1) ].	introductionpenile carcinoma has an incidence of 4,000 cases in europe. the therapy and prognosis depend decisively on the lymph node status. lymph node metastases are detected in 2365% cases depending on the histopathological pattern. due to improved diagnostic methods an early detection of tumor stage is possible. multimodal therapeutic concepts can offer curability for a subset of patients, even those suffering from advanced disease.material and methodscurrent data on penile cancer based on a selective review of the literature by pubmed and the eau guidelines 2009.resultsinvasive diagnostic tools, such as fine needle biopsy (fnb) and dynamic sentinel node biopsy (dsnb), improved the diagnosis of lymph node status considerably and reduced the morbidity in specialized centers. the application of 18f fdg pet / ct for metastases detection needs further evaluation due to inconsistent results. inguinal lymphadenectomy is the therapeutic standard in case of metastases proof. it was possible to reduce the complications due to the new modified operation techniques. patients with extended lymph node and distant metastases have a poor prognosis. different systemic polychemotherapy regimes are applied currently and are associated with poor outcome (response rates < 50%) and high morbidity. neoadjuvant chemotherapy is recommended in patients with unresectable and relapsing lymph node metastases.conclusionscurrently, inconsistent therapy regimens are applied for metastatic penile cancer. standardization is urgently needed through the development of high quality studies and long term registers in order to lower the morbidity and increase the efficiency of diagnosis and therapy.
as surgeons gain more experience and instrumentation technology advances, more challenging procedures are attempted. acute and chronic inflammation, fistulization, and abscesses make dissection and mobilization difficult. in 1995, we published our experience with 25 patients and demonstrated that laparoscopic - assisted ileocolic resection was feasible in patients with cd. in that early report, resections in patients with a large mass or fistulae often minimally invasive techniques have become applicable in a greater number of patients with complex cd - related pathology, such as secondary surgery and the presence of fistulizing disease or abscesses. as a result, criteria for selecting which patients might be candidates for laparoscopic - assisted ileocolic resection (laicr) have become more liberal. the aim of this study was to review our experience with laicr in patients with cd during the past 13 years. specific attention was paid to assessing the adequacy and safety of this procedure, the incidence of intra- and postoperative complications, and an assessment of the adequacy of the resection as defined by disease - free margins. in an attempt to assess changes in patient selection and our performance during this 13-year experience from a prospectively maintained practice database, all patients undergoing attempted laicr for cd between 1992 and 2005 were reviewed. gross and microscopic evaluation of all specimens was performed by pathologists without specific knowledge of the conduct of the surgical procedure. surgical technique was generally uniform, and the senior authors were all familiar with laparoscopic intestinal techniques. the specimen was extracted through the incision, and the mesenteric divisions as well as the anastomosis were preformed extracorporeally. based on previously reported data, conversion to an open technique (laparotomy) was defined as a fascial incision > 6 cm in length. to evaluate any change in our experience or outcomes during this 13-year period, we divided the entire group arbitrarily into 3 time intervals (1992 to 1997, 1998 to 2001, 2002 to 2005). data were complete in 158 including 95 women (60%) and 63 men (40%). one hundred twenty - four patients (78.4%) underwent primary icr, 31 patients (18.9%) underwent secondary re - section, and 3 (1.8%) patients underwent tertiary resection. thirty - eight patients (24%) had fascial incision > 6 cm and were considered to have been converted to laparotomy (cl). however, the conversion rate to laparotomy in patients for whom this was not a primary resection was 61%. this was not statistically different from the conversion rate in patients undergoing primary icr, which was 24%. average laicr operating time was 144 minutes compared with 172 minutes in the cl group. these usually occurred during the laparoscopic portion of the case and were, in most cases, the reason for conversion. postoperative complications consisted mostly of prolonged ileus (5 patients), anastomotic leaks (2 patients), one portal and mesenteric vein thrombosis, and one bleeding episode requiring reoperation. postoperative hospital stay was 5.9 days in the laicr group versus 7.4 days in the cl group (p 6 cm as a conversion to laparotomy. numerous authors have reported that the use of minimally invasive techniques shortens postoperative length of hospital stay, and the current study confirms this. the most common reason for conversion was the presence of adhesions (47%) (figure 2). unlike our first report from 1995, the presence of a mass and fistulae accounted for only 15% of the reasons for conversion to laparotomy. it is possible that in the later time interval, experience was gained, and cases with more complex pathology, such as fistula or abscess, could be completed laparoscopically. although more intraoperative complications occurred in the cl, most of the complications occurred during the laparoscopic portion of the procedure (enterotomies, bleeding). 2 parameters were compared : the length of small and large bowel resected and the involvement of the surgical margins. the data suggest that when a laparoscopic procedure was performed, shorter lengths of bowel were resected. this difference was significant when comparing the length of the right colon resected in the laicr group and the cl group. the length of the crohns ' involvement of the ileum varies, and it is therefore impossible to evaluate it is possible that mobilization of the ascending colon near the hepatic flexure during the laparoscopic portion was suboptimal, thus limiting the extent of the colon resection. evaluation of the different time intervals reveals no statistically significant differences when comparing operating time, length of stay, conversion to laparotomy, intra- or postoperative complications, length of large and small bowel resected, or positive resection margins. of the entire group of 158 patients, the relevance of microscopically positive margins has been evaluated previously. while these reports demonstrated no increase in anastomotic complications or higher recurrence rates when resection margins are microscopically positive, the difference in margin positivity between the laicr and cl groups is striking (figure 3). laicr is a safe and feasible procedure for patients with cd. compared with our earlier reports, secondary and tertiary icr were performed more commonly. advanced minimally invasive techniques and surgical devices enable surgeons to perform resections in patients with more complex crohns ' pathology than previously.	background : this study reviews our experience with laparoscopic - assisted ileocolic resection in patients with crohn 's disease. the adequacy and safety of this procedure as measured by intraoperative and postoperative complications were evaluated. special attention was paid to the group in which laparoscopy was not feasible and conversion to laparotomy was necessary.methods:between 1992 and 2005, 168 laparoscopic - assisted ileocolic resections were performed on 167 patients with crohn 's ileal or ileocolic disease. follow - up data were complete in 158 patients.results:in 38 patients (24%), conversion to laparotomy was necessary. previous resection was not a predictor of conversion to laparotomy. average ileal and colonic length of resected specimens was 20.9 cm and 6.5 cm, respectively, in the laparoscopic group, versus 24.9 cm and 10.6 cm in the converted group. twenty of 120 specimens (16.6%) in the laparoscopic group were found to have margins microscopically positive for active crohn 's disease. none of the 38 specimens in the converted group had positive ileal margins.conclusions:laparoscopic-assisted ileocolic resection can be safely performed in patients with crohn 's disease ileitis. the finding of positive surgical margins following laparoscopic resections compared with none among conventional resections has to be thoroughly evaluated.
currently, complete surgical resection represents the only curative treatment for lung cancer1. however, the majority of lung cancer survivors who undergo lung resection experience pulmonary complications ; one - third of these patients report dyspnea, and one - fifth suffer from severely diminished pulmonary function, including respiratory muscle weakness2. pulmonary rehabilitation (pr) has been proposed as an adjunctive therapy to decrease the risk of postoperative pulmonary complications3. however, patients may not be able to consistently engage in pr, for personal or economic reasons (e.g., lacking the funds to travel to rehabilitation centers)4. therefore, there is a need for alternative methods, such as home - based pr, to maintain physical functioning in a more economical manner5. few studies have addressed pr education for caregivers, who typically facilitate home - based exercises during the outpatient period. this study evaluated the effects of caregiver education on pr (i.e., respiratory muscle strength and dyspnea) in lung cancer patients following lung resection. patients scheduled for lung resection at the department of thoracic surgery of a national university hospital, between march 2013 and november 2013, whose caregivers had not previously received education pertaining to pr, were selected for the study. a total of 50 patients agreed to participate following an explanation of the study procedure, and written informed consent was obtained from all participants. ethics approval was obtained from the inje university faculty of health science human ethics committee. the subjects were randomly assigned to experimental (n = 25) and control (n = 25) groups the day before surgery. of these, 3 and 6 subjects dropped out of the experimental and control groups, respectively, during the 4-week study period, due to violation of the exclusion criteria. therefore, the final experimental and control groups were comprised of 22 and 19 subjects, respectively. the subjects mean age was 60.2210.89 years ; their mean height was 163.01 8.77 cm, mean weight was 61.3911.35 kg, and average bmi was 23.013.34. caregiver education on pr included guidance pertaining to splinted coughing, airway clearance and breathing, and stretching and strengthening exercises. the control group received general advice from the department of thoracic surgery (once per week for 30 minutes) pertaining to pain management, postoperative care, use of an incentive spirometer and nebulizer, and mobilization of the upper limbs and trunk. in the case of subjects who were not able to visit the hospital, respiratory muscle strength was assessed using a microrpm device (micro medical ltd., maximum expiratory pressure (mep) were measured using the method described by black & hyatt6, and these served as indices of inspiratory and expiratory muscle strength. dyspnea was evaluated using the modified borg scale, ranging between 0 (no dyspnea) and 10 (most severe dyspnea). data pertaining to the general characteristics of subjects are provided as means se, with intergroup homogeneity assessed using and independent t - tests. group differences in scores before the experiment, and 2 weeks (baseline) and 6 weeks after surgery, were assessed using repeated - measures analysis of variance. mip, mep and modified borg scale values were more significantly improved in the experimental group compared to the control group, but there were no group differences (p<0.05, table 1table 1.comparison of respiratory muscle strength and dyspneamean sdcontrolexperimentalmip (cmh2o)baseline62.521.463.818.24 weeks68.021.271.519.0mep (cmh2o)baseline62.4224.361.615.64 weeks66.532.571.216.8mborgbaseline3.081.162.451.464 weeks2.631.611.771.40p<0.05). refai.7 reported an association between respiratory muscle weakness after lung resection and increased incidence of pulmonary complications. recovery of respiratory muscle function after surgery is important because lung resection leads to impairments in these muscles. nomori.8 reported a 4.3% increase in mip, and 6.4% increase in mep, 212 weeks after surgery. these data are consistent with our finding that mip and mep increased between weeks 26 in the control (8% and 6%, respectively) and experimental (12% and 15%, respectively) groups. however, these group differences were not significant. previous studies demonstrated that smaller lung resection areas are associated with greater differences in respiratory muscle strength before and after surgery8, 9. we speculate that respiratory muscle strength did not differ significantly among our groups because all subjects had undergone video - assisted thoracoscopic surgery, which requires a minimal incision in the respiratory muscles interacting with the chest wall. dyspnea caused by lung resection is an important determinant of patients quality of life10. our study commenced 2 weeks after the surgery ; the experimental group was characterized by a decrease in dyspnea over time, but this decrease was not significant compared to the control group. differences between our results and those of this previous study may be due to the use of indirect and direct interventions, respectively. in conclusion, our data suggest that caregiver education on pr can improve respiratory muscle strength and dyspnea.	[purpose ] this study evaluated the effects of caregiver education on pulmonary rehabilitation of patients who have undergone lung resection for cancer. [subjects ] patients were divided into experimental (n = 22) and control (n = 19) groups. [methods ] the caregivers of the experimental group patients received education on pulmonary rehabilitation, while the control group patients received general management advice for 4 weeks. [results ] pulmonary muscle strength (maximum inspiratory pressure and maximum expiratory pressure) was increased significantly in the experimental group compared to the control group. modified borg scale scores were decreased significantly in the experimental vs. control group. [conclusion ] providing caregivers with education pertaining to pulmonary rehabilitation was associated with improved pulmonary function in lung cancer patients following lung resection.
chronic obstructive pulmonary disease (copd) is a common disease and a major cause of death worldwide. it is the fourth main cause of death behind cardiac disease, cancer, and cerebrovascular disease in the united states, with a disease - related death incidence of 43.2 per 100,000 population. the death rate due to other diseases has declined gradually in recent years, but that due to copd has continued to rise. copd will become the third main cause of death globally in 2020 according to the world health organization (who). in taiwan, copd was the seventh main cause of death in 2007 ; the incidence of copd - related death was around 22.2 per 100,000 population. for people over 70 years old, the prevalence rate peaked at 8.8% in 1998 and reached a plateau thereafter. the final years for copd patients are marred by progressive functional decline, poor quality of life, and oxygen dependency. patients die mostly from acute exacerbation of copd / lung infection, respiratory failure, and complications secondary to other comorbidities. patients over 80 years old had the highest risk of hospitalization (14.2 times that of patients aged 40 - 49 years). to improve the quality of life of patients with copd, palliative care was added to the standard clinical management of copd in western countries at the beginning of the 21 century. palliative care is a treatment designed to relieve the symptoms caused by cancer and other diseases, and is centered on the patient and the patient 's family to optimize quality of life by anticipating and reducing suffering. palliative care often addresses the physical, intellectual, emotional, social, and spiritual needs of the patient, and is broader than hospice care, which usually focuses on the dying person and her / his family. palliative care was introduced in taiwan more than two decades ago, and most recipients were cancer patients. non - cancer patients, including patients with copd, have been covered for palliative care by health insurance since september 2009. additional information regarding end - of - life palliative care is needed to promote the practice of palliative care and educate patients, family, and medical staff on its use in copd. the purpose of this article was to investigate the difference of clinical practice pattern between copd patients with palliative care and without at the end of life at an acute care teaching hospital in northern taiwan. this was a retrospective medical record review of patients who had died in saint paul 's hospital (taoyuan, taiwan) between september 1, 2007 and december 31, 2009. saint paul 's hospital is an acute care hospital that has a multidisciplinary team that provides palliative care for hospitalized patients with end - stage disease. all patients were under the care of a primary care physician and a palliative care team consisting of physicians, specialist nurses, social workers, catholic chaplains, and volunteers. the study was approved by the institutional review board (irb) of the hospital. eligible patients were 18 years of age, had diagnosed copd (icd-9 codes 491.x, 492.x, and 493.x) by pulmonary function test or clinicians judgments, and were admitted to the hospital for acute care. copd patients were determined to be terminal during their hospital stay if they met any of the following criteria : (1) respiratory failure post - intubation ; (2) persistent hypoxia (pao2 100 beats per minute), or tachypnea (respiratory rate > 20/min) after o2 therapy ; (3) unstable hemodynamics with hypotension (sbp 100 beats per minute), or tachypnea (respiratory rate > 20/min) after o2 therapy ; (3) unstable hemodynamics with hypotension (sbp < 90 mmhg) ; or 4) cachexia with chronic malnutrition status. these enrollment criteria were comparable with global initiative for obstructive lung disease (gold) criteria stage iii or stage iv. for those patients who were later referred to palliative care, they were either referred by primary care physician 's decision or actively recruited by the palliative care team if a do not resuscitate (dnr) consent form had been signed after hospitalization. to ensure adequate time to assess patients distressing symptoms, patients who remained in the emergency department, diagnosed with cancer, or admitted for < 24 h were excluded from the study. data collected included demographics, results of pulmonary function test within last 1 year, hospital medical care, symptoms and medical costs during hospitalization. hospital medical care included days of hospitalization, days in the intensive care unit, ventilator usage, number of invasive procedures or treatments, medications, the record of signed dnr consents, and the record of hospice care referral. symptoms were recorded daily by the same specialist nurse who initially evaluated the patients and were chosen based on prior findings. total medical costs were retrospectively calculated from reimbursement received from the bureau of national health insurance during the time patient was hospitalized. patients were divided into those who did not receive palliative care (non - palliative group) and those who received palliative care (palliative group) to identify differences related to end - of - life palliative care in patients with copd. differences between subpopulations were tested using the chi - square test (= 0.05). totally, 91 patients [median age, 82.1 years (55 - 97) ; man : woman ratio, 3:1 ] were enrolled in the study. the top three comorbidities were hypertension (38%), chronic kidney disease (38%), and congestive heart failure (33%). the leading reasons for admission were acute exacerbation of copd / lung infection (60%), heart disease (20%), and non - pulmonary infection (16%). there was no between - group difference in age, gender, forced expiratory volume in 1 s (fev1), and main reason for admission. patients in the non - palliative group had a higher prevalence of cerebrovascular disease and liver cirrhosis ; there were no between - group differences in other comorbidities. the median hospital stay was 16 days (1 - 61) for all patients. the median intensive care unit (icu) stay was 8 days (1 - 42) for all 73 patients (80%) admitted to icu. sixty - one patients (67%) died in icu finally [table 2 ]. medical care utilization in copd patients over one - third of patients had received computed tomography (ct) examinations and around 10% of patients had received bronchoscopy or panendoscopy. the most common invasive procedures were feeding tube placement (93%), foley catheter insertion (86%), and invasive ventilation (52%). fifty - nine patients (65%) had signed dnr consent forms, but 40 patients (44%) had been given cardiopulmonary resuscitation (cpr) at the end of life. the most common medications prescribed were antibiotics (91%), inotropic agents (74%), and parenteral or oral steroids (66%). around 13% and 11% of patients received regular doses of nonsteroid anti - inflammatory drugs (nsaids) and opioids, respectively. analysis of end - of - life care revealed that the median hospital stay was longer in the palliative group (26 vs. 11 days for the non - palliative group) [figure 1 ]. there was a noticeably lower death rate in icu in the palliative group (41% vs. 73% for the non - palliative group), but no between - group difference in the rate of icu care, length of icu stay, and ventilator usage. cumulative survival rates of all patients (n = 91), patients in the palliative group (n = 17), and patients in the non - palliative group (n = 74) there was no between - group difference in the rate of invasive diagnostic procedures, invasive treatment, and medication usage. however, the frequency of invasive ventilation was higher in the non - palliative group (57% vs. 29%, p = 0.04). patients in the palliative group had significantly higher rate of signing dnr consent forms (100% vs. 57%, p < 0.001) and lower cpr rate (12% vs. 51%, p < 0.001) when compared to patients in the non - palliative group. seventeen out of 91 patients (18%) had received palliative end - of - life care. the median time from admission to palliative care referral and median duration of palliative care was 19 days (0 - 56 days) and 6 days (2 - 33 days), respectively. in the 15 patients (88%) where palliative care was requested by family members, the major reason for palliative care referral was unwillingness to let the patient suffer (59%) and acceptance of the possibility that palliative care might be beneficial (29%). two patients (12%) requested palliative care themselves because they were unwilling to suffer any longer. totally, 20 different symptoms in 17 patients were noted in the daily records maintained by specialist palliative care nurses, with an average of 10.6 (4 - 15) per patient. the most prevalent symptoms were breathlessness (100%), fatigue (94%), fever (88%), mental change (82%), and general edema (82%) [figure 2 ]. symptoms characteristics and their prevalent rate in patients under palliative care (n = 17) patients who were referred to palliative care spent more days in the medical care than non - referred patients. the total medical cost was similar between those given and not given palliative care ; however, the mean daily cost was significantly lower for patients referred to palliative care because of longer time in medical care (p 0.001) [table 3 ]. totally, 91 patients [median age, 82.1 years (55 - 97) ; man : woman ratio, 3:1 ] were enrolled in the study. the top three comorbidities were hypertension (38%), chronic kidney disease (38%), and congestive heart failure (33%). the leading reasons for admission were acute exacerbation of copd / lung infection (60%), heart disease (20%), and non - pulmonary infection (16%). there was no between - group difference in age, gender, forced expiratory volume in 1 s (fev1), and main reason for admission. patients in the non - palliative group had a higher prevalence of cerebrovascular disease and liver cirrhosis ; there were no between - group differences in other comorbidities. the median hospital stay was 16 days (1 - 61) for all patients. the median intensive care unit (icu) stay was 8 days (1 - 42) for all 73 patients (80%) admitted to icu. the median duration of ventilator support was 5 days (0 - 42). sixty - one patients (67%) died in icu finally [table 2 ]. medical care utilization in copd patients over one - third of patients had received computed tomography (ct) examinations and around 10% of patients had received bronchoscopy or panendoscopy. the most common invasive procedures were feeding tube placement (93%), foley catheter insertion (86%), and invasive ventilation (52%). fifty - nine patients (65%) had signed dnr consent forms, but 40 patients (44%) had been given cardiopulmonary resuscitation (cpr) at the end of life. the most common medications prescribed were antibiotics (91%), inotropic agents (74%), and parenteral or oral steroids (66%). around 13% and 11% of patients received regular doses of nonsteroid anti - inflammatory drugs (nsaids) and opioids, respectively. analysis of end - of - life care revealed that the median hospital stay was longer in the palliative group (26 vs. 11 days for the non - palliative group) [figure 1 ]. there was a noticeably lower death rate in icu in the palliative group (41% vs. 73% for the non - palliative group), but no between - group difference in the rate of icu care, length of icu stay, and ventilator usage. cumulative survival rates of all patients (n = 91), patients in the palliative group (n = 17), and patients in the non - palliative group (n = 74) there was no between - group difference in the rate of invasive diagnostic procedures, invasive treatment, and medication usage. however, the frequency of invasive ventilation was higher in the non - palliative group (57% vs. 29%, p = 0.04). patients in the palliative group had significantly higher rate of signing dnr consent forms (100% vs. 57%, p < 0.001) and lower cpr rate (12% vs. 51%, p < 0.001) when compared to patients in the non - palliative group. seventeen out of 91 patients (18%) had received palliative end - of - life care. the median time from admission to palliative care referral and median duration of palliative care was 19 days (0 - 56 days) and 6 days (2 - 33 days), respectively. in the 15 patients (88%) where palliative care was requested by family members, the major reason for palliative care referral was unwillingness to let the patient suffer (59%) and acceptance of the possibility that palliative care might be beneficial (29%). two patients (12%) requested palliative care themselves because they were unwilling to suffer any longer. totally, 20 different symptoms in 17 patients were noted in the daily records maintained by specialist palliative care nurses, with an average of 10.6 (4 - 15) per patient. the most prevalent symptoms were breathlessness (100%), fatigue (94%), fever (88%), mental change (82%), and general edema (82%) [figure 2 ]. symptoms characteristics and their prevalent rate in patients under palliative care (n = 17) patients who were referred to palliative care spent more days in the medical care than non - referred patients. the total medical cost was similar between those given and not given palliative care ; however, the mean daily cost was significantly lower for patients referred to palliative care because of longer time in medical care (p 0.001) [table 3 ]. this study describes the basic characteristics of copd patients at the end of life at an acute care teaching hospital in northern taiwan. they were predominantly men (man : woman ratio, 3:1), elderly, and suffered from multiple comorbidities, mainly hypertension, congestive heart failure, or chronic kidney disease. the main reasons for admission were acute exacerbation of copd / lung infection or complications of other chronic comorbidities. the characteristics of our patients in the present study differed from those of patients with advanced cancer in other studies on end - of - life care in taiwan. cancer patients were younger (mean age, around 65 years), had fewer comorbidities, and were admitted mainly for treatment of cancer and its related complications. these differences made it more difficult to predict the medical care needs of copd patients based on the needs of cancer patients. it is easier to predict the prognosis of cancer based on disease stage than it is for the prognosis of copd. outcomes in copd patients are more difficult to predict because comorbidities are more numerous and the reasons for disease progression vary. our data showed that more than two - thirds of patients had received feeding tubes, foley catheters, antibiotics, inotropic agents, and steroids during hospitalization. even though 65% of patients had signed these data show that the goal of care was to prolong life rather than relieve symptoms. around 80% of patients in this study had been admitted to icu, and 67% of the patients finally died in icu. the data are almost eightfold higher than those for cancer patients who received icu care at the end of life in taiwan. as many deaths occur in icu, deficiencies in end - of - life care in this setting require particular attention. medical staff trained to use end - of - life care tools there were no between - group differences in length of icu stay, duration of ventilator usage, rate of invasive diagnostic procedures, and frequency of invasive procedures and medications in this study. the median hospital stay for all patients was 16 days, while the median number of days from admission to palliative referral was 19 in the palliative group. palliative care was requested in all patients by either the patients themselves or family members, and the main reason was unwillingness to suffer. in patients referred to palliative care, invasive procedures with limited effect were frequently performed after admission. these may account for the longer median hospital stay in the palliative group in this study. the patients in the palliative group also had a higher dnr consent rate (100% vs. 57%), lower rate of death in icu (41% vs. 73%), and lower rate of cpr (12% vs. 51%) partially due to the greater knowledge of palliative care services by patients and family members and partially reflecting the possible benefit of palliative care. each patient had an average of 10.6 different symptoms in agreement with other studies on copd (which reported around 7.1 - 14 symptoms). the prevalence of these symptoms was similar in our patients and in patients with other chronic illnesses and advanced cancer. it confirmed that cancer and non - cancer patients follow a common pathway to death. because the prevalence of symptoms is similar for cancer and non - cancer patients, the need for symptom relief should be the same. breathlessness and pain are probably the most noticeable symptoms in dying copd patients and occur in around 56 - 97% and 21 - 78% of these patients, respectively. optimal symptom control requires early identification, continuous reassessment, communication, and appropriate medications. in our study, all patients in the palliative group had breathlessness and 47% had pain. assuming the same prevalence rate in all 91 patients, nsaids and opioids were prescribed regularly in only 13% and 11%, respectively, reflecting that pain in copd patients in taiwan was underrecognized, underassessed, and undertreated. copd patients at the end of life need not only to prolong life but also to have access to hospice care. encouraging medical staff to use hospice for end - of - life care in copd patients is necessary and urgent in taiwan. several studies comparing end - of - life care in copd patients and lung cancer patients have found that clinical symptoms are the same in copd patients and lung cancer patients, but copd patients receive more invasive treatment, fewer medications to relieve symptoms, and have less access to palliative care. one is life expectancy is difficult to predict and the other is the failure by physicians and patients to recognize that copd is a terminal disease. the challenge is timing the hospice intervention to meet the current needs of the patient. the body mass index, airway obstruction, dyspnea, and exercise capacity (bode) index and study to understand prognoses and preferences for outcomes and risks of treatments (support) criteria have both been used to predict survival in advanced copd patients. patients with the highest bode index had a 30 - 40% 2-year mortality, and the 6-month mortality rate was 40% for patients who support criteria. the national hospice and palliative care organization (nphco) referral guidelines have been suggested as hospice care referral criteria in copd patients in taiwan. however, parameters recommended by the guidelines were insensitive and not useful for predicting life expectancy at the time of acute exacerbation in copd patients, and therefore were of limited benefit. accurate prediction of a 6-month survival time the standard window of time for referral to hospice care in patients with nonmalignant disease is difficult, and leads patients and family to hesitate about requesting hospice care. this may be a missed opportunity for patients to receive proper evaluation of symptoms and to be referred to hospice for comprehensive, appropriate end - of - life care, which is an important reason behind having a better understanding of prevalent symptoms in end - stage noncancer patients in taiwan. copd prognosis is difficult to determine with many patients dying of exacerbations or comorbidities, making it difficult to determine when a referral to hospice care is appropriate. because predicting lifespan is difficult in copd patients, the timing of palliative care should depend on their need rather than their predicted survival. surveys have shown repeatedly that most patients with copd and other chronic illnesses anticipate their own death and welcome frank discussions and guidance from their physicians. however, physicians tend to overestimate the life expectancy and underestimate the needs of patients with advanced stage non - malignant illnesses. medical staff should discuss living will and hospice needs with copd patients at the point that the patient needs hospitalization but before any invasive procedure is performed to resolve an acute complication, such as exacerbation of copd / lung infection or stroke. to the best of our knowledge, this article is the first to evaluate the pattern of end - of - life care and prevalence of symptoms presenting in terminal copd patients in taiwan. notably, this study confirmed that copd patients at the end of life are polysymptomatic. these patients received too many medical care aims of prolong life, received too little symptom relief management, and had too little access to palliative care. hospice offers a comprehensive multidisciplinary approach to relieving the distress of any terminal disease and can support their families both before and after the patient dies. we believe that copd patients in taiwan need and have the right to receive the same kind of palliative care as cancer patients receive in taiwan. first, this study was a single - site study with a small sample size and selection bias. therefore, the study result was unable to represent the characteristics to the whole population. multi - site studies on a large scale are needed to verify if these findings can be generalized to the whole health - care system in taiwan. second, in order to ensure the accuracy of the clinical symptom records, we used only the records of patients who had received palliative care ; therefore, the symptoms we recorded are not representative of those of all copd patients at the end of life. finally, 60% patients were admitted to hospital because of acute exacerbation of copd and 67% of all the patients died in icu after admission. in addition, all of the recruited patients died after admission, and the data of the proportion of people admitted with an exacerbation of copd who die and the proportion of those admitted to icu who do not survive are not available in our study. copd patients are polysymptomatic approaching the end of life and this characteristic should be taken into account in providing appropriate end - of - life care in the same way as for cancer patients.	background : we investigated the difference of clinical practice pattern between end - stage chronic obstructive pulmonary disease (copd) patients with and without palliative care at the end of life in taiwan.materials and methods : a total of 91 copd patients who died in an acute care hospital were enrolled from one community teaching hospital in northern taiwan between september 1, 2007 and december 31, 2009. the patients were divided into palliative (n = 17) and non - palliative care (n = 74) groups. demographics and medical care data obtained through retrospective review of medical records were analyzed to determine significant between - group differences.results:there were no between - group differences in intensive care unit (icu) utilization, duration of icu stay, duration of ventilator usage, invasive diagnostic procedures, invasive treatments, medications, and total medical cost. patients in the palliative group had longer hospital stays (median 26 days vs. 11 days, p < 0.01) and higher rate of do - not - resuscitate orders (100% vs. 51%, p < 0.001), but lower rates of icu mortality (73% vs. 41%, p = 0.026), invasive ventilation (57% vs. 29%, p = 0.04), cardiopulmonary resuscitation (12% vs. 51%, p < 0.001), and daily medical cost (250 us dollars vs. 444 us dollars, p < 0.001).conclusion : palliative care was underutilized and referral was delayed for copd patients. copd patients are polysymptomatic approaching the end of life and this characteristic should be taken into account in providing appropriate end - of - life care in the same way as for cancer patients. palliative care for copd patients is urgently needed in taiwan and should be promoted.
there is an increasing concern amongst the women and girls about the appearance of their external genitalia. many women and adolescent girls present with labia hypertrophy. a young girl presenting with labia hypertrophy is quite unusual. a child with severe hypertrophy of bilateral labia minora patient a, youngest of six female siblings, was born at term in a hospital through the lower segment caesarean section. at 8 years of age, she noticed progressive increasing size of labia, which caused her pain while prolonged sitting, walking and daily activities. the increased size of labia affected her psychologically as she considered herself abnormal among her siblings. she disclosed the enlargement to her elder sister almost after 6 months of noticing it, and the patient consulted the plastic surgery clinic at the age of 8 years. on clinical examination, her labia minora was found to be thickened and enlarged more than 5.5 cm from the free edge of the labia majora with associated hypertrophy of the clitoral hood [figures 1 and 2 ] (type 6 : franco 's classification ; severe hypertrophy : ricci and pardo classificatio) [tables 1 and 2 ]. pre - operative view of labia minora hypertrophy size of labia minora measuring 5.5 cm in its width franco classification of labia minora hypertrophy ricci and pardo classification of labia minora hypertrophy for surgical correction on work - up, her hormonal assay revealed no abnormality. her thyroid profile, serum luteinizing hormone, serum follicle - stimulating hormone, serum testosterone and serum estradiol levels were within normal limits. skin and subcutaneous tissues in the labia minora were found to be hyperplastic. extended linear excision was performed which included excision of hypertrophied clitoral hood [figures 3 and 4 ]. after 24 h, the patient was advised to clean the genitalia with application of antiseptic ointment. linear excision of labia minora and clitoral hood reduction immediate post - operative result (a and b) post - operative result after two weeks with good aesthetic appearance histological examination showed acanthosis and chronic non - specific inflammation of underlying dermis [figure 6a and b ]. (a and b) histological picture illustrating mild acanthosis with mild chronic non - specific inflammation with congestion (40, h and e) hodgkinson and hait first described labia minora hypertrophy in 1984 ; though description regarding circumcision of labia minora and clitoris has been mentioned vaguely in many old scriptures. juvenile labia minora hypertrophy [jlmh ] is a less common clinical condition and becomes rarer in a developing country like india where social taboos and customs are stringently enforced in the common lives. in such milieu, jlmh cases are detected only when symptoms significantly affect a patient 's functional and psychological well - being. hence, whenever a surgeon in a developing country like india diagnoses a jlhm patient, it should be addressed properly, and relevant procedure should be carried out to relieve the patient from distressing symptoms. although no standard consensus has been established yet to classify labia minora hypertrophy ; many do exist depending on the size and severity of the hypertrophy [tables 1 and 2 ]. arbitrary landmarks taken in these measurements make it difficult to diagnose hypertrophy on a standard scale. some have measured size from midline horizontally, and some have measured from free edge.. surgical decision should largely be dependant on labia size as well as symptoms addressed by the patient. a number of studies have been published in context to labia minora hypertrophy and their management, but only a few have addressed the occurrence of hypertrophy in adolescent age and most of their patients fall in 2040 age group. although age is not a criterion for labiaplasty, most of the surgeons refrain from doing it at an early age. our patient was an 8-year - old when she noticed labial hypertrophy, which is rare. all the relevant investigations, including hormonal assays and usg were normal suggestive of an idiopathic, isolated and abnormal morphology of the labia. the size of the labia minora as well as presenting symptoms should be taken into account, while planning labiaplasty in any age group, especially in adolescents. a better classification system for labia minora hypertrophy and standardization regarding functional and cosmetic labiaplasty need to be established in this era of ever increasing vulvovaginal surgeries.	labia minora hypertrophy is a relatively uncommon surgical entity being popularised in the realm of vulvovaginal plastic surgeries. apart from the unaesthetic appearance of the hypertrophied minora, these cases are also associated with itching, hygiene problem, pain while sitting down, sports activities, difficulty in wearing tight clothing, bleeding and discomfort while or after sexual intercourse, social embarrassment, insecurity and psychological diminution of confidence and self - esteem. in a country like india, due to sociocultural reasons, patients hesitate to consult a doctor for such deformities. most of the patients suffer in silence for years. although common in the west, very few surgeons in the country perform this simple and rewarding surgery. here, we are presenting a case of premenarchal juvenile labia minora hypertrophy (jlmh) in an 8-year - old child. labial hypertrophy in this age group is uncommon. we were unable to find hypertrophy of labia minora in the eight - year - old child on english literature search.
living donor kidney transplantation is the treatment of choice for patients with end - stage renal failure for several reasons. the transplant is performed when the donor and the recipient are in optimum medical condition and at a time that is convenient for them and for their families. moreover, recipients of living donor kidney grafts enjoy greater long - term graft survival and a better quality of life than do recipients of cadaveric kidney grafts. it reduces the number of patients on the wait list for a cadaveric kidney and therefore increases the likelihood that patients with no potential living donor can undergo transplantation. this is particularly important because the gap between the number of cadaveric donors and the number of patients on the waiting list is increasing due to significant reduction in traffic accidents as well as the promotion of more healthy lifestyles with emphasis on exercise and improved dietary habits and the subsequent reduction in the incidence of stroke. an increase in the number of living donors (including living - unrelated donors) may ameliorate this trend. almost all studies that report medical outcomes of living kidney donors more than a year from donation are retrospective. although practical for evaluation of long - term outcomes, retrospective studies are vulnerable to certain methodological pitfalls and biases that may limit their interpretability. most important is the potential for selection bias, which may alter findings if there is a difference between included and nonincluded donors either as a result of non - participation or because the study investigators are unable to locate the subject. in studies that follow living kidney donors, donors in good health may be more likely to participate because of greater survival or greater ability to meet the requirements of participation. the aim of this study is to evaluate the impact and safety of kidney donation on living kidney donors in a prospective fashion. donors included in this study were male or female and donors were of a minimum of 21 years and a maximum of 65 years of age. donor has been fully informed and has given written informed consent. while criteria for exclusions were donor has hypertension or diabetes mellitus, donor is pregnancy or breastfeeding, donor is known to be hcv, hbv, and hiv positive, donor has significant liver disease, defined as having during the past 28 days continuously elevated ast and/or alt levels 3 times greater than the upper value of the normal range of the investigational site, donor with malignancy or history of malignancy, or active systemic or localized major infection, donor is participating or has participated in another clinical trial, evidence of infiltrate, cavitation or consolidation on x ray obtained during the screening / baseline evaluation, subjects with a screening / baseline hemoglobin 30 kg / m at time of donation. these preliminary and short - term data demonstrate that factors associated with t2 dm in kidney donors are similar to those in the general population and donors screened carefully at the time of donation do not appear to have an acceleration diabetic kidney disease. significant increases in the levels of serum uric acid in the first three months after donation were reported in our donors, which could be explained by the mild transient impaired renal function in the early postdonation period. in the current study, there was a significant increase in the mean levels of serum triglycerides which was stabilized afterwards. on the other hand, there were no significant differences in the mean levels of serum total cholesterol, ldl, or hdl in the first three months, but their levels showed significant increase at 3, 6, 12, and 24 months. according to tavakol., 2009, this could explain the increase of serum lipids among our donors who developed postdonation significant increase in body mass index. it is worth mentioning that 11 female donors have got successful postdonation pregnancies. in accordance, ibrahim., 2009, reported that pregnancy in kidney donors has generally been viewed to be favorable and determined fetal and maternal outcomes in a large cohort of kidney donors. in accordance to our study, ibrahim and their colleagues, 2009, stated that a total of 2102 women have donated a kidney at their institution : 1589 donors responded to pregnancy surveys, 1085 reported pregnancies, and 504 reported none. in this large survey of previous living donors in a single center, fetal and maternal outcomes and pregnancy outcomes after kidney donation were similar to those reported in the general population. obese potential live kidney donors should be advised to maintained ideal body weight in order to avoid proteinuria, hypertension, and diabetes mellitus. proteinuria increases with marginal significance but appears to be of no clinical consequence. despite the reduction in gfr in the early post donation period, although living kidney donation is a safe procedure which carries a minimal risk in comparison with the heroic act and the major benefits for the transplant recipients, the proper selection and the pretransplant assessment of the potential kidney donors together with postdonation meticulous and regular (lifelong) follow - up is strongly recommended. future controlled, prospective studies with long periods of follow - up will better delineate safety and identify donors at lowest risk for long - term morbidity. a world registry of living donors is necessary to evaluate the real magnitude of the long - term risk to living donors.	virtually, all studies reporting the outcomes of living kidney donation beyond the first year from donation were retrospective. in this prospective study, the outcome of 81 consecutive living kidney donors was thoroughly evaluated. clinical, laboratory, and radiological assessments were carried out at predonation (basal), 3, 6, 12, and 24 months after donation. the mean age at time of donation was 37.8 9.8 years and the majority was females (75.3%). the mean bmi increased significantly after donation (p < 0.04). the mean serum creatinine levels (mg / dl) were 0.75 0.14, 1.01 0.22, 0.99 0.21, 0.98 0.20, and 0.94 0.20 (p < 0.0001). likewise, the mean levels of measured creatinine clearance (ml / min) were 148.8 35.7, 94.7 26.6, 95.5 24.6, 96.7 20.2, and 101.6 26.2 (p < 0.0001). the mean 24 hours urinary protein excretion (gm / dl) were 0.09 0.03, 0.19 0.18, 0.16 0.09, 0.18 0.25, and 0.17 0.12 (p < 0.0001). there were significant increases in the means of the longitudinal and transverse diameters of the remaining kidney over time (p < 0.001). out of 42 female donors, eleven female donors have got successful postdonation pregnancies. there were no reported surgical complications, either intra- or postoperative. long - term follow - up is necessary for all living kidney donors through local institutional and world registries. this trial is registered with clinicaltrials.gov nct00813579.
mycobacterium abscessus is the most pathogenic and chemotherapy - resistant rapidly growing mycobacteria (rgm). it accounts for 80% of lung disease caused by rgm and is the second most common rgm species present in extrapulmonary disease (following m. fortuitum) (1 - 4). m. abscessus is resistant to all antituberculosis drugs including isoniazid, rifampin, ethambutol, and pyrazinamide. m. abscessus is considered to be susceptible to amikacin, cefoxitin, and clarithromycin, and moderately susceptible to imipenem (1 - 4). however, m. abscessus is not an easy organism to treat, and long - term treatment with multiple antimicrobial agents is usually required. some studies have reported that in vitro susceptibilities to several antimicrobial agents are correlated with clinical responses to treatment in rgm infections (5, 6). in addition, all clinically significant isolates should be tested against selected antimicrobial agents (1, 2). in korea, m. abscessus is the second most common etiology of lung disease caused by nontuberculous mycobacteria (ntm) (following the m. avium - intracellulare complex). it accounts for 21 to 33% of ntm lung disease in korea (7 - 9) ; however, in vitro antimicrobial susceptibility test results for this organism have never been investigated in korean population. therefore, we examined the in vitro susceptibilities of m. abscessus isolates from patient respiratory specimens that displayed m. abscessus lung disease. from july 2005 to december 2006, 74 nonduplicate isolates of m. abscessus (one isolate per patient) recovered from respiratory clinical samples were collected for use in this study. ntm species identification was performed using the polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) method based on the rpob gene (10). all patients met the diagnostic criteria recommended by the american thoracic society for m. abscessus lung disease (11). antimicrobial susceptibility testing was performed at the korean institute of tuberculosis according to the guidelines set forth by the national committee for clinical laboratory standards (nccls) (12, 13). we tested eight antimicrobial agents against m. abscessus isolates : amikacin (sigma, st. louis, mo, u.s.a.), cefoxitin (sigma), imipenem (msd, seoul, korea), tobramycin (sigma), clarithromycin (hanmi, seoul, korea), ciprofloxacin (ildong, seoul, korea), moxifloxacin (bayer, seoul, korea), and doxycycline (sigma). minimal inhibitory concentrations (mics) of all tested drugs were determined by the broth microdilution method and interpreted according to the guidelines described by the nccls document m24-a in 2003 (12). the mic of moxifloxacin for this rgm has not yet been described by the nccls. in its place, we followed the recommendation for aerobic organisms in nccls m100-s11 in 2001 (13). the colonies present on the culture medium were transferred to 15-ml tubes of 7h9 broth (becton dickinson, piscataway, nj, u.s.a.) with 0.02% tween 80 (junsei chemical, tokyo, japan). the suspension was mixed vigorously on a vortex mixer for 15 to 20 sec until the turbidity matched the density of a 0.5 mcfarland standard. the suspension was then diluted a few times following consideration of the final inoculum concentrations in the well. serial 2-fold dilutions of antimicrobial agents were prepared and added across the 96-well plates (becton dickinson). the concentrations of antimicrobial agents in the wells were determined based on the nccls recommendation (12, 13). the final inoculum suspension (150 l) was transferred to the wells of a microtiter tray containing 150 l of each antimicrobial type and dilution. the inoculated tray was covered and incubated at 30 for over 3 days in room air. endpoint mics of all the antimicrobial drugs were read as the first well in which no growth occurred. staphylococcus aureus atcc 29213 and mycobacterium peregrinum atcc 700686 were also used for quality control purposes. the susceptibility categories of all antimicrobial agents were determined according to the breakpoints recommended by the nccls and are presented in table 1 (12, 13). the results of antimicrobial susceptibility tests are presented in table 2. of the parenteral antibiotics, amikacin (99%, 73/74) and cefoxitin (99%, 73/74) were active against most m. abscessus isolates. amikacin demonstrated better in vitro activity against m. abscessus than tobramycin (36%, 27/74). imipenem (55%, 36/74) had in vitro activity against a moderate number of isolates. of the oral antibiotics, clarithromycin (91%, 67/74) was active against the majority of isolates. moxifloxacin (73%, 54/74) had better in vitro activity than ciprofloxacin (57%, 42/74). doxycycline was the least active, inhibiting only 7% (5/74) of the m. abscessus isolates. m. abscessus isolates have been determined to be uniformly resistant to standard antituberculous agents. for pulmonary diseases caused by m. abscessus, it is recommended that susceptible oral antibiotics (including clarithromycin or azithromycin) be administered in combination with parenteral medications (amikacin, cefoxitin, or imipenem) (1, 2). due to its variable in vitro susceptibilities to some drugs, antibiotic susceptibility testing of all clinically significant isolates is recommended, although the correlation between in vitro susceptibility results for m. abscessus and clinical response for specific antimicrobial agents has not been established (1, 2). our results in this study demonstrated that the resistance rates of m. abscessus isolates to various antibiotics are high. in addition, ciprofoxacin, moxifloxacin, and imipenem exhibited moderate activity (> 50% susceptibility), but the susceptibility of m. abscessus to doxycyline was poor. traditionally, amikacin was the most active agent against rgm species. according to a study by swenson., 95% of m. abscessus, 99% of m. fortuitum, and 88% of m. chelonae isolates were inhibited by this agent with a mic of 16 g / ml (5). intravenous amikacin is administered at a dose of 10 to 15 mg / kg daily to adult patients with normal renal function. this dose elicits peak serum levels in the low 20 mg / ml range. in this study, 99% of m. abscessus isolates were susceptible to amikacin and 36% were susceptible to tobramycin. this agent is administered at a dose of up to 12 g / day intravenously in divided doses. in this study, this agent has been reported to be moderately susceptible against m. abscessus (14). this study demonstrated that imipenem inhibits 55% of m. abscessus isolates at the breakpoint of < 8 g / ml and 88% of isolates at the intermediate breakpoint of < 16 g / ml. therefore, imipenem may be useful in clinical treatment regimens for m. abscessus, especially in situations when cefoxitin can not be used due to adverse effects. clarithromycin is representative of this class and has been considered to be the most effective (12). in this study, 91% of m. abscessus brown. reported that the newer macrolides generally show better activity against rgm species, and among them, clarithromycin is the strongest (15). oral fluoroquinolones have also been used in the treatment of rgm diseases. ciprofloxacin is the class representative of the older fluoroquinolones (i.e., ciprofloxacin, ofloxacin, and levofloxacin). in the current study, we tested ciprofloxacin and moxifloxacin (newer 8-methoxyfluoroquinolone), and both antimicrobials showed high activities, with 57 and 73%, respectively, of m. abscessus isolates susceptible. even though fluoroquinolone can not be used as a single - drug therapy due to the risk of developing mutational resistance (16), both ciprofloxacin and moxifloxacin could be used as alternative oral agents during combination treatment for m. abscessus lung disease. doxycycline is a member of the tetracycline antibiotics and has shown poor activity against m. abscessus and m. chelonae in previous studies. in the current study, only 21% of m. abscessus isolates were susceptible or intermediate and approximately 80% were resistant to this agent. doxycycline was the least active agent among all those tested in this study. in this study, ntm species identification was performed using the pcr - rflp method based on the rpob gene. several genes such as 16s rrna, hsp65, and rpob gene have been used to identify ntm to the species level. however, some gene target is often limited by the lack of sequence divergence among closely related mycobacterium species. for example, 16s rrna internal transcribed spacer assay can not differentiate m. abscessus from m. massiliense or m. bolletii, although this assay has proven to be valuable to distinguish m. abscessus and m. chelonae (17). accurate identification of isolated ntm species is important since distinguishing these species is clinically relevant. in conclusion, the variations in susceptibility within m. abscessus isolates to currently available antimicrobials suggest that the antimicrobial susceptibility testing of all clinically significant m. abscessus isolates be needed. in addition, our results offer clinicians choices for empirical treatment when m. abscessus lung disease is diagnosed and the in vitro susceptibilities are not available.	mycobacterium abscessus is the second most common etiology of pulmonary disease caused by nontuberculous mycobacteria in korea. although antimicrobial susceptibility tests are important for appropriate patient management in m. abscessus lung disease, the tests have never been investigated in korea. seventy - four isolates of m. abscessus recovered from patient respiratory samples were tested against eight antimicrobial agents following the guidelines set forth by the national committee for clinical laboratory standards. of the parenteral antibiotics, amikacin (99%, 73/74) and cefoxitin (99%, 73/74) were active against most isolates. imipenem (55%, 36/66) and tobramycin (36%, 27/74) had activity against moderate number of isolates. of the oral antibiotics, clarithromycin (91%, 67/74) was active against the majority of isolates. moxifloxacin (73%, 54/74) and ciprofloxacin (57%, 42/74) had activity against a moderate number of isolates. doxycycline was the least active, inhibiting only 7% (5/74) of isolates. in conclusion, the variations in susceptibility within m. abscessus isolates to currently available antimicrobials suggest that the antimicrobial susceptibilities of any clinically significant m. abscessus isolate be needed individually.
the tight link between the innate and adaptive immune systems is governed by signaling molecules such as cytokines, transcription factors, and several innate receptors. cytokines significantly modulate the inflammatory process, which plays an important protective role in innate immunity. interleukin-10 (il-10), initially known as cytokine synthesis inhibitory factor, is a pleiotropic cytokine and demonstrates dominant immunosuppressive function. il-10 is produced from variety of cells, including t cells, b cells, neutrophils, eosinophils, epithelial cells, keratinocytes, mesangial cells, monocytes / macrophages, nk cells, and tumor cells. il-10 gene has been cloned and characterized from several vertebrates such as human, mouse, chicken, bottlenose dolphin, pekin duck, turkey, seabass, grass carp, zebrafish, common carp, goldfish, and rainbow trout [314 ]. the il-10 has been reported to downregulate il-1, tnf, and il-6 expression in vivo, whereas it stimulates nk cells resulting in antibody - dependent cell mediated cytotoxicity. oxidative stress has also been reported to be modulated by il-10 as it suppresses reactive oxygen intermediate species generation [16, 17 ]. il-10 suppresses inflammatory responses elicited by activated macrophages, inhibits nitric oxide production, downregulates major histocompatibility complex (mhc) class ii expression, inhibits synthesis of a number of macrophage - derived proinflammatory factors such as il-1, tumor necrosis factor alpha (tnf), il-12, and cyclooxygenase-2, and thereby downregulates the host immune response to invading pathogens [1820 ]. in mammals, different activities of il-10 appear to be mediated through formation of oligomeric complexes of il-10 with its receptors expressed on the cell surface. homodimeric structure of il-10 has been demonstrated in other species such as gold fish and seabass [9, 13 ]. differential expression of il-10 in different tissues of a number of fish species upon infection with microorganisms or stimulation with lps indicates its role in immunomodulation in fishes as well [11, 12, 14, 2225 ]. recombinant il-10 from gold fish significantly reduced the expression of proinflammatory cytokines in monocytes activated with heat - killed aeromonas salmonicida, whereas it stimulated the expression of suppressor of cytokine signaling-3 (socs-3) in these cells. similarly, carp il-10 downregulated oxygen radical and nitrogen radical production by both macrophages and neutrophils and upregulated socs-3 expression. these investigators demonstrated that carp il-10 stimulated a subset of cd8 memory t cells and downregulated cd4 memory th1 and th2 responses. in grass carp also, the il-10 upregulated the cellular activity of peripheral blood lymphocytes and played an essential role in tgf-1 mediated immune regulation. thus, il-10 is one of the major cytokines which may affect the disease status of vertebrates including fishes. indian major carp labeo rohita belongs to the carp family cyprinidae and is commercially one of the most important and highly favored fish in the indian subcontinent. a number of gram negative bacteria have been associated with fish pathogenic conditions [2729 ]. changes in the levels of cytokines during infectious stage play a vital role in immune suppression and disease progression. it is important to understand the mechanisms involved in the protection against many of these pathogens. since fish suffer from many bacterial and viral infections, which result in major economic losses, it is imperative to understand their immune system to control infection. cytokines are widely used as adjuvants in mammals, whereas in fish their possible use as vaccine adjuvants is minimally explored. understanding of biochemical and functional characteristics of various cytokines of fish may also pave the way for their use to develop strategies for controlling infections. in the present study, we report high level expression and characterization of il-10 of l. rohita and its effect on expression profile of immune related genes in l. rohita. for recombinant expression in escherichia coli, synthetic cdna corresponding to lril-10 gene was designed according to codon preference in e. coli. ndei and xhoi restriction sites were included in the synthetic cdna at the 5 and 3 ends, respectively, for convenient cloning in the expression vector, pet22b(+) (novagen, usa). recombinant puc57-lril-10 harboring the synthetic codon biased lril-10 gene fragment of 483 nucleotides (including the restriction sites, encoding the mature l. rohita il-10 of 157 amino acid residues) was obtained from genscript, usa. the gene fragment encoding the mature rlril-10 was excised out from puc57-lril-10 plasmid using ndei and xhoi (neb, usa) and was further cloned into pet22b(+) (novagen, usa) digested with same enzymes. putative recombinants designated as pet.lril-10 were analyzed by restriction enzyme digestion and confirmed by automated dna sequencing (dna sequencing facility, department of biochemistry, university of delhi south campus, new delhi). the expressed product from this construct would be of 163 amino acid residues including a 6x histidine tag at the c - terminus of the protein. for expression and localization analysis of rlril-10 protein, plasmid pet.lril-10 was transformed into e. coli bl21 (de3) cells. briefly, the transformed cells were grown in luria - bertani broth (difco) containing 100 g / ml ampicillin under continuous shaking (220 rpm) at 37c until optical density reached 0.6. recombinant protein expression was induced by addition of 1 mm isopropyl 1-thio--d - galactopyranoside (iptg) and the cultures were then allowed to grow further for 5 h. cells were harvested by centrifugation at 3000 g for 5 min at 4c and resuspended in 10 mm tris - hcl, ph 7.0, in the absence or presence of 0.5 m arginine and lysed by sonication (5 pulses of 1 s for 40 cycles). the cell lysates were centrifuged at 12,000 g for 20 min at 4c. the soluble and insoluble fractions separated by centrifugation were analyzed in sds - page (12%) by coomassie brilliant blue (cbb - r250) staining. the soluble rlril-10 protein was purified using hispur cobalt resin (thermo scientific, usa) as per the manufacturer 's instructions. briefly, the solubilized rlril-10 was mixed with the resin and left for overnight binding with end - to - end shaking at 4c. the slurry was centrifuged at 1200 g in a swinging - bucket rotor for 2 min. the flow through was discarded and nonspecifically bound proteins were removed by washing the resin twice in wash buffer (10 mm tris - hcl, ph 8.0, and 10 mm imidazole). the rlril-10 protein was eluted with elution buffer (10 mm tris - hcl, ph 8.0, and 150 mm imidazole). eluted fractions were analyzed in sds - page (12%) and the fractions containing rlril-10 were pooled together and dialyzed in 10 mm tris - hcl, ph 7.0, using a 3.5 kda cut - off dialysis membrane (spectrum laboratories, usa). the dialyzed rlril-10 was concentrated using amicon ultracentrifugal filter device (millipore, usa) and stored at 20c in small aliquots. the integrity of the purified rlril-10 was confirmed by maldi - tof - ms analysis (sandor proteomics, hyderabad). pierce lal chromogenic endotoxin quantitation kit (thermo scientific, usa) was used as per the manufacturer 's direction to determine lps levels in the purified rlril-10. bca protein estimation kit (bio - rad, usa) was used to determine protein concentration in different fractions as per the manufacturer 's protocol. to assess the oligomeric state of the purified rlril-10, the recombinant protein was analyzed by sds - page under both nondenaturing condition (unboiled in the absence of reducing agent) and denaturing condition (boiled in the presence of reducing agent) as detailed in the legend. purified rlril-10 protein (10 g) was emulsified in freund 's complete adjuvant (1 : 1 ratio) and injected intraperitoneally (i.p.) into balb / c mice. first booster with the same amount of antigen emulsified in freund 's incomplete adjuvant (1 : 1 ratio) was given on day 15. sera were prepared from the blood samples collected on days 21 and 35 after immunization. briefly, the blood was allowed to clot for 2 h at room temperature and the serum was collected by centrifugation at 3000 g. the relative titer of antibodies in the generated polyclonal antisera against rlril-10 was determined by direct elisa using antigen - excess assay. purified rlril-10 (5 g) was coated on polypropylene elisa plate using coating buffer (0.2 m carbonate - bicarbonate buffer, ph 9.2) and incubated for 1 h at 37c. nonspecific sites were saturated by 5% fat - free skimmed milk in pbs - tween 20 solution (pbst). different dilutions of anti - rlril-10 antisera were added with 2% bsa in pbst and incubated for 1 h at 37c. hrp - conjugated goat antimouse igg was then added to each well and incubated at 37c for 1 h. wells were thoroughly washed between successive incubations with pbst. the color was developed with orthophenylenediamine (opd, sigma - aldrich chemical co., usa) in citrate phosphate buffer, ph 5.5 (0.5 mg / ml), containing h2o2 (1 l / ml, added just prior to use). the reaction was terminated by the addition of 2 n h2so4 and absorbance at 490 nm was measured in a microplate reader (biotek power wave xs, usa). western blot analysis using anti - his monoclonal antibody or anti - rlril-10 antisera was performed essentially as described earlier. protein samples resolved on the sds - page were transferred onto the nitrocellulose membrane and the blot was then blocked with 5% nonfat milk (difco) in phosphate buffered saline containing 0.1% tween 20 (pbst) overnight at 4c. this was followed by incubation with primary antibody diluted in pbst (sigma - aldrich chemical co., usa) for 1 h at room temperature, followed by three pbst washes. secondary antibody (anti - mouse hrp, sigma - aldrich chemical co., usa) was then added (1 : 5000 dilution in pbst) to react for 1 h. after washing the blot extensively with pbst, the blot was developed by addition of 3,3-diaminobenzidine (0.05% dab) after addition of hydrogen peroxide in pbs (30%, 1 l h2o2/ml of dab solution) as described earlier. the reaction was terminated by washing the membrane several times with water. alternatively, the blot was developed using pierce ecl western blotting substrate (thermo scientific, usa) and the images were acquired in a biospectrum 500 imaging system (uvp, cambridge, uk). for 2-d gel electrophoresis analysis, rlril-10 (~250 g) was precipitated using acetone and resuspended in 0.5 ml solubilization buffer (7 m urea, 2 m thiourea, 2% w / v chaps, 5 mm dithiothreitol (dtt), and 1% (v / v) ipg 310 buffer (ge healthcare, usa)). samples were loaded onto 7 cm polyacrylamide ipg strips (ph 310 linear, ge healthcare, usa) by in - gel rehydration at 50 v for 11 h. focusing was conducted at 20c in an ettan ipgphor (amersham, usa) as described earlier. second dimension electrophoresis was performed by 12% sds - page analysis followed by staining with coomassie brilliant blue r-250 (sigma - aldrich chemical co., usa). the spots were excised out and analyzed by maldi - tof - ms for protein identification (sandor proteomics, hyderabad). far - uv (200250 nm) cd spectra of the rlril-10 (0.2 mg / ml in 10 mm tris - hcl, ph 7.0) at 25c were recorded using a spectropolarimeter (jasco j-815 ; path length, 0.1 cm ; scan speed, 50 nm / min). secondary structure contents of the refolded protein from the cd measurements were calculated using the k2d2 software (http://www.ogic.ca/projects/k2d2/). a fluorescence spectrum of the rlril-10 (0.2 mg / ml in 10 mm tris - hcl, ph 7.0) was monitored using a spectrofluorimeter (cary eclipse, varian optical spectroscopy instruments, australia). sample was excited at a wavelength of 280 nm (slit width 10) and emission spectra were monitored at the wavelength range of 300 nm and 500 nm (slit width 10) at scan of 30 nm / min. the steady state of rlril-10 was graphically represented by plotting fluorescence intensity at 334 nm on y - axis and gradual increase of temperature from 20c to 90c at the interval of 5c incubated for 20 min at each temperature was plotted on x - axis. kinetics of rlril-10 unfolding were represented by plotting fluorescence intensity at 334 nm versus incubation time. to detect reactive oxygen species (superoxide anion (o), hydrogen peroxides (h2o2), and hydroxyl radicals (oh) production by phagocytic cells), oxidative burst activity was measured by reduction of nbt by the method of das. with minor modification. the optimum concentration of rlril-10 required to induce nbt reduction was determined in a preliminary experiment. blood samples (100 l) from each fish (in triplicate) were treated with equal volume of pbs or rlril-10 (1000 ng in 100 l pbs) and allowed to incubate for 1 h at 25c. equal volume of 0.2% nbt (sigma - aldrich chemical co., usa) was then added to the samples and incubated further for 1 h at 25c to allow formazan granules formation. intracellular reduction of nbt was determined by solubilizing the formazan crystals with dimethyl formamide (srl, india), centrifugation at 2000 g for 5 min, and measurement of absorbance of the supernatant at 540 nm. to study the immunoregulatory properties of rlril-10, 12 rohu juveniles (weighing ~50 g) were injected with 100 ng of rlril-10 (i.p.). spleen tissues were aseptically collected in rnalater (sigma - aldrich chemical co., usa) from rlril-10-administered fish at 3, 6, 12, and 24 h after injection (three fish for each time point). semiquantitative pcr was performed for the expression of il-1, il-6, il-8, mhci, mhcii, tnf, and nkef genes. gene specific primers were obtained from xcelris, ahmedabad, india, based on the primer sequence information used earlier [3335 ]. the constitutively expressed housekeeping gene, -actin, was used both as a positive control and for normalization. to assess the level of expression of the target genes, densitometric analysis was performed using alpha ease fc imaging software (alpha innotech corp.. the ratios of immune related genes/-actin product were subsequently calculated after subtraction of the background pixel intensity for each gene of interest and used to assess the differences in expression levels between control and the various infected samples. induction of e. coli bl21 (de3) cells, harboring plasmid pet.rlril-10, with iptg at 37c for 5 h resulted in high level of expression of 6x histidine - tagged rlril-10 as a band of the expected size of ~18 kda was seen in the induced cell lysate (figure 1(a), lane i). absence of this band at the same position in uninduced cell lysates indicated tight control of expression (figure 1(a), lane u). analysis of the soluble (s) and insoluble fractions (p) prepared by sonication of the induced cells revealed rlril-10 to have been expressed only in the insoluble fraction. induction with lower concentrations of iptg (0.1 mm to 0.5 mm) or at lower temperatures (6, 15, 18, and 25c) could not also direct the expression of rlril-10 to the soluble fraction (data not shown). presence of 0.5 m arginine in sonication buffer resulted in localization of the majority of the rlril-10 in the soluble fraction (figure 1(a), lane sa) with little remnants in pellet fraction (pa). detection of a single band at the expected position in western blot analysis of the soluble fraction (obtained by sonication in lysis buffer containing 0.5 m arginine) using anti - his antibody (figure 1(b), lane 1) confirmed the authenticity of the 6x histidine - tagged rlril-10. as presence of arginine in the sonication buffer hindered effective binding of the rlril-10 with the affinity resin, arginine was removed from the soluble fraction using pd10 column (ge healthcare, usa) prior to affinity chromatography. using hispur cobalt resin - based affinity chromatography, the recombinant rlril-10 was purified to near homogeneity (figure 1(c), lane 1). a very faint band at ~36 kda possibly indicated the presence of dimeric rlril-10. oligomeric state of the rlril-10 was confirmed by sds - page analysis under nondenaturing and denaturing conditions. as evident from figure 1(d), the unboiled rlril-10 under nonreducing conditions showed a band of ~36 kda corresponding to the dimeric form of the recombinant protein (lane 1). however, the rlril-10 dimer got fully converted into ~18 kda band corresponding to the monomeric form of the recombinant protein when the sample was analyzed under reducing conditions after boiling for 10 min (lane 2). approximately 70 mg of the purified soluble rlril-10 was obtained per liter of e. coli culture at shake flask level. lps concentration in the purified rlril-10 was determined to be 0.015 pg/g of purified protein, which is within the permissible limit of the lps for immunization studies. two - dimensional gel electrophoresis of the rlril-10 revealed the presence of 6 spots (figure 2). the 6 spots were distributed like a string forming the so - called train between pi values of 7 and 9.5, indicating the presence of several isoforms of rlril-10 differing in their pis. western blot analysis of the rlril-10, resolved on 2-d gel, with anti - rlril-10 antibody confirmed the spots to be isoforms of rlril-10 (figure 2(b)). maldi - tof - ms of three of the spots (figure 2(a), numbered 13) resulted in generation of the individual peptide with the monoisotopic mass in the range of 567.2444973.573 (spot 1), 524.0074973.538 (spot 2), and 534.1754098.731 (spot 3) for three spots, respectively (supplementary data, panel a in figures s1s3 in supplementary material available online at http://dx.doi.org/10.1155/2016/3962596). search of the sequences obtained from the peptide mass fingerprinting (pmf) of the digested protein using mascot search engine identified and confirmed these spots as il-10 protein from l. rohita (panel b in supplementary data, figures s2s4), thus confirming these spots to be isoforms of rlril-10. the secondary structure of rlril-10 was analyzed by far - uv circular dichroism spectroscopy. as evident from figure 3(a), the rlril-10 exhibited predominantly -helical structure with characteristic double minima in ellipticity at 208 nm and 222 nm. the -helical content of the rlril-10 was determined to be 91.67% whereas strand was found to be only 0.3%. an excitation wavelength of 280 nm was used to excite all 8 tyr residues. the rlril-10 gave emission maxima at 334 nm indicating intact tertiary structure (figure 3(b)). a significant decrease in the emission maxima was observed when the protein was heated at 80c indicating loss of tertiary structure. in order to study the steady state of unfolding of the rlril-10, this analysis showed consequent decrease in fluorescence intensity at 334 nm with gradual increase of temperature (figure 3(c)). when the protein was heated at a fixed temperature (60c) for different time periods, kinetics of rlril-10 unfolding showed gradual decrease in fluorescence intensity at 334 nm with increase in incubation time (figure 3(d)). in order to establish functional activity of the rlril-10 in modulating superoxide production, peripheral blood cells of l. rohita were treated with different concentrations of the recombinant protein and the optimum concentration of rlril-10 to induce nbt reduction was found to be 10 g / ml (supplementary data, figure s4). addition of rlril-10 in nbt assay clearly showed a significant decrease in the phagocytic activity in all five samples as compared to control ones (figure 4). the rlril-10 was found to effectively downregulate the expression of the proinflammatory cytokines (il-1, il-8, mhcii, and mhci) in l. rohita, whereas an increase in the expression of nkef level was noticed (figure 5). il-1 expression started to decline from three hours onwards and remained significantly lower till 12 h (p 0.005) when compared to 0 h. although there was a slight rise in il-1 expression at 24 h, when compared to its levels at 12 h, it still remained lower than that at 0 h (figure 5(a)). similarly, a decline in il-8 levels was observed from 3 h onwards which became significantly lower at 12 h (p 0.005), followed by an increase at 24 h (figure 5(b)). the rlril-10 resulted in a significant decrease in mhcii expression at 3 h (p 0.05) and 24 h (p 0.005) (figure 5(c)), whereas mhci expression levels (figure 5(d)) were significantly lower at 24 h (p 0.05) after an initial rise in its level at 3 h. no statistically significant change in tnf expression was noted at any of the time points (figure 5(e)). unlike these molecules, a gradual increase in the expression of natural killer enhancing factor (nkef) was observed from 3 h onwards (figure 5(f)). the nkef levels were significantly higher at 12 h (p 0.05) when compared to its expression levels at 0 h. a significant decline (p 0.05) in the il-6 levels was observed at 3 h after rlril-10 treatment in comparison to the control. at subsequent intervals, il-10 is a main anti - inflammatory cytokine that is produced by a variety of immune cells. cells of monocytic / macrophage lineage are primary targets of il-10 and its pleiotropic role is well documented. cloning and characterization of il-10 gene from different species revealed the existence of interspecies variation and heterogeneity at structural and functional levels. also, expression patterns of il-10 showed great variation between / within fish and other vertebrates. while il-10 has been characterized in number of bony fishes, structural and functional characterization of il-10 from l. rohita, an economically important freshwater carp, has not been investigated. the present study describes the expression, purification, and biophysical and functional characterization of recombinant il-10 of l. rohita. overexpression of rlril-10 in e. coli bl21 (de3) resulted in inclusion bodies of mis - folded and aggregated protein. as refolding and subsequent purification from inclusion bodies are difficult and produce low yields of bioactive protein, an effective strategy to obtain soluble rlril-10 was used by including l - arginine (0.5 m) in the sonication buffer. inclusion of arginine in the culture medium has been reported to result in expression of proteins in their active form. while lower concentrations of arginine in solvents are used for refolding of the proteins expressed as inclusion bodies, extraction of active soluble folded proteins from insoluble pellets obtained by lysis of e. coli cells has been achieved by using higher concentrations of arginine. addition of varying concentrations of arginine to the culture medium as reported by schffner. resulted in low cell density and thus affected the yield of soluble protein. in the present study, inclusion of arginine during sonication resulted in directing the protein in soluble fraction, thus significantly reducing the amount of arginine that would otherwise be required if added in culture medium. high yield of soluble rlril-10 was thus recovered using single step purification from heterologous expression system. authenticity of the rlril-10 was confirmed by maldi - tof - ms, where mass of the peptide generated by tryptic digestion matched with those of il-10 protein sequence in the database. cd spectroscopy of the rlril-10 showed predominantly alpha helical secondary structure of the rlril-10, which is in conjunction with crystal structure of both the dimeric forms and engineered monomeric form of human il-10 [39, 40 ]. changes in the conformation of the rlril-10 at neutral ph as a function of temperature indicated gradual loss in the helical content with an increase in temperature thus indicating unstable structure of il-10. protein sequence of mature il-10 contains six phenylalanine residues and eight tyrosine residues with intrinsic fluorescence properties. however, for tertiary structure characterization, we only measured tyr fluorescence because its quantum yield was high enough to give a good fluorescence signal. the native folded state of rlril-10 contained tyrosine residues within the core of the protein, whereas in a partially folded or unfolded state they become exposed to solvent. in a hydrophobic environment tyrosine thermal denaturation resulted in unfolding of the protein thus exposing the tyr residues to a hydrophilic environment reflected by a decreased fluorescence intensity. dimeric nature of human il-10, demonstrated by its crystal structure, has been shown to be essential for its functional activity. human il-10 consists of two 160 amino acid residue - long polypeptide chains. in humans, il-10 dimer is thought to be the result of an evolutionary mechanism of protein oligomerization often referred to as 3d domain swapping. this suggests that il-10 evolved from a monomeric protein by exchanging structural domains (-helices e and f for il-10) with another monomer to create the dimer. similar to humans, biologically active recombinant il-10 of viral and murine organisms has also been shown to exist as dimer. oligomeric state of the rlril-10 was confirmed by western blot analysis of rlril-10 using unboiled samples under nonreducing conditions together with denatured samples under reducing conditions. our results show that rlril-10 exists as a dimer as reported for its homologues from other species. the dimeric state of il-10 has been reported to be concentration dependent and an increase in dimeric forms with an increase in concentration of recombinant viral and murine il-10 has been reported. two - dimensional gel electrophoresis showed presence of several isoforms of rlril-10 which differed in their pi (pi range 7.5 to 9.5). the slight charge microheterogeneity in the rlril-10 could arise due to posttranslational modification, deamidation of the protein during purification or storage, and differential oxidation - reduction state [4446 ]. il-10 plays a major role in withstanding inflammation through regulating proliferation, differentiation, and activation of immune cells. the biologically active rlril-10 was shown by inhibition of superoxide production as a measure of phagocytosis and its pleiotropic immunoregulatory properties. phagocytosis is accompanied by an oxidative burst with an increase in the production of reactive oxygen species. our results are in agreement with earlier reports put forth by bogdan., who reported recombinant mouse il-10 to inhibit primed peritoneal macrophages function by markedly decreasing the release of reactive oxygen species. a marked reduction in production of reactive oxygen species by recombinant goldfish il-10 has also been reported in monocytes primed with aeromonas salmonicida and recombinant goldfish ifn-. in the present study, a significant decrease in superoxide production activity of leucocytes of l. rohita possibly resulting from inhibitory effect of il-10 on h2o2 production was observed even without priming. decreased phagocytic activity was indeed rlril-10 induced as no effect was observed when the treatment was given with heat - denatured rlril-10 (data not shown). however, piazzon. did not observe any effect of recombinant carp il-10 on free radicals production in unstimulated carp neutrophils and macrophages. it is to be noted that the rlril-10 concentration used in the present study was much higher (corresponding to 10 g / ml) than that reported used by piazzon. for carp il-10 (0.0050.5 u / ml) or by grayfer. for recombinant goldfish il-10 (up to 1 g / ml) using primed cells functional analysis of the rlril-10 in vivo (administration in l. rohita) showed that it downregulated most of the proinflammatory cytokines and upregulated natural killer enhancing factor in spleen. these results are consistent with anti - inflammatory function reported for il-10 homologues from other species [6, 7, 13, 20 ]. this negative regulation is mediated by the jak1/stat3 pathway, and stat3 is the primary mediator of il-10 effects. downregulation of il-1 expression as early as 3 h after rlril-10 treatment is expected as il-1 is one of the early response proinflammatory cytokines that enables organisms to respond to infections, inducing an inflammatory cascade, along with other defensive responses. in goldfish also recombinant il-10 administration resulted in decreased levels of il-1. reported opposite patterns of expression of il-10 and il-1 during different stimulations in spleen tissue of atlantic cod. in this report, a decrease in il-1 with an increase in il-10 expression and vice versa confirms the anti - inflammatory effect of il-10. although it had been described as a proinflammatory cytokine, its anti - inflammatory and immunosuppressor properties have also been reported. il-10 has generally been reported to downregulate il-6 expression together with other proinflammatory cytokines such as il-1 and tnf. lps - mediated production of il-6 has also been reported to be inhibited by il-10 and this effect of the il-10 is blocked by stat3. thus, a significant decline in il-6 transcript levels 3 h after rlril-10 exposure in the present study is in agreement with the earlier reports [15, 48, 50 ] and possibly points towards its pronounced anti - inflammatory role rather than proinflammatory one. il-10 has been known to inhibit antigen specific t cell responses by regulating expression of mhcii on the surface of monocytes. downregulation of mhcii by rlril-10 observed in the present study is in line with the reports put forth by lin., who showed that the ril-10b isoform produced by human cytomegalovirus inhibited mhcii expression on granulocyte macrophage progenitors. earlier studies have also shown that mouse cytomegalovirus infection resulted in premature and transient activation of host il-10 very early during infection resulting in a significant and selective reduction of mhcii expression on cell surface. downregulation of mhcii expression by rlril-10 in l. rohita showed that the function of il-10 on regulation of antigen presentation has remained conserved across species. although a generalized downregulation of mhcii was noticed in the present study after rlril-10 exposure in l. rohita, a significant decline at 3 and 24 h might be indicative of differential transient expression at individual level. earlier studies have established that the inhibition of production of reactive oxygen species is directly linked to inhibition of tnf synthesis. however, in our studies, though we observed inhibition of phagocytic activity of peripheral blood cells of l. rohita in vitro, tnf expression in spleen of treated fishes was not significantly altered at all the study intervals when compared to 0 h. however, a decrease of ~40% was noted at 3 h. downregulation of proinflammatory cytokines by il-10 indicates its central role in protection of cells against excessive immune responses and excessive tissue damage. the activation of natural killer cells might contribute to the clearance of the pathogen and facilitate antigen acquisition from dead cells for cross - priming by activated apcs, providing a link between the innate and the adaptive immune responses [54, 55 ]. thus, the present study provides comprehensive biophysical and functional characterization of il-10 from l. rohita, a freshwater indian major carp. soluble rlril-10 thus produced in large amounts using heterologous expression system exhibited structural (helical and oligomeric state) and biological properties (anti - inflammatory) reported for il-10 from other species and have remained conserved. the present study will help in better understanding of the il-10 modulated immune responses in l. rohita. as il-10 of l. rohita is established to generate an anti - inflammatory response, application of the rlril-10 to control excessive immune response in fish can be explored.	interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. though it is one of the major cytokines, il-10 from the indian major carp, labeo rohita, has not yet been characterized. in the present study, we report large scale production and purification of biologically active recombinant il-10 of l. rohita (rlril-10) using a heterologous expression system and its biophysical and functional characterization. high yield (~70 mg / l) of soluble rlril-10 was obtained at shake flask level. the rlril-10 was found to exist as a dimer. far - uv cd spectroscopy showed presence of predominantly alpha helices. the tertiary structure of the purified rlril-10 was verified by fluorescence spectroscopy. two - dimensional gel analysis revealed the presence of six isoforms of the rlril-10. the rlril-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1, tnf, and il-8, and augmented the nkef transcript levels in spleen of l. rohita. anti - inflammatory role of the rlril-10 was further established by inhibition of phagocytosis using nbt reduction assay in vitro. the data indicate that the dimeric alpha helical structure and function of il-10 of l. rohita as a key regulator of anti - inflammatory response have remained conserved during evolution.
adipose tissue functions as a repository for long - term energy storage and as a critical regulator of energy balance, satiety, glucose metabolism, and other physiological processes. white adipose tissue (wat) stores and releases fatty acids and glycerol in response to the energetic needs of the organism, whereas brown adipose tissue (bat) produces heat to maintain thermostasis upon cold - exposure. the most common form of lipodystrophy is congenital generalized lipodystrophy (cgl), which affects approximately one in ten million individuals and is characterized by a near complete absence of adipose tissue. cgl type 1 is caused by mutations in 1-acylglycerol-3-phosophate o - acyltransferase 2 (agpat2), and mice null for agpat2 exhibit a complete lipoatrophy. mutations in seipin cause cgl type 2, or berardinelli - seip congenital lipodystrophy type 2 (bscl2), and mice null for bscl2/seipin, or with adipose - tissue specific loss, are also lipodystrophic,,. several additional genes have been implicated in cgl and familial partial lipodystrophy, including akt2 and a subunit of phosphatidylinositol 3 kinase (pi3k),,,,,. hepatomegaly from fatty liver and splenomegaly are common in lipodystrophic patients, with hyperinsulinemia and hypertriglyceridemia often present. akt, a ser / thr kinase, is activated upon engagement of the insulin receptor and mediates hormonal regulation of many aspects of energy homeostasis in insulin - responsive tissues, including adipose tissue. akt can also be activated by insulin growth factor 1 (igf-1),,. three isoforms of akt have been identified, of which akt1 and akt2 account for the majority of akt in adipocytes,. akt1 is expressed nearly ubiquitously and is critical in the control of growth, while the akt2 isoform is expressed predominantly in the adult insulin - responsive tissues, including liver, muscle, and adipose. mice lacking akt2 are insulin - resistant but spared from severe metabolic abnormalities due to compensation by akt1,,. in this study, we deleted akt1 and akt2 selectively in adipocytes of mice, thus interrupting a critical insulin - signaling pathway. we found that loss of these akt isoforms in adipocytes caused profound lipodystrophy, associated with fatty liver and systemic insulin resistance. all mice were on a c57/b6 background and were maintained on normal chow diets from laboratory rodent diet, lab diet 5001. cre - positive male mice heterozygous for akt1 and akt2 floxed alleles were bred to females homozygous for akt1 and akt2 floxed alleles. this breeding scheme generated cre - positive experimental mice homozygous floxed for both genes and cre - negative male mice homozygous floxed for both genes that served as controls. cre - positive males heterozygous for both floxed genes were generated and exhibited a wild type phenotype (data not shown). all mouse experiments were reviewed and approved by the university of pennsylvania institutional animal care and use committee in accordance with the guidelines of the national institutes of health. organs were harvested for histopathology from 12-week - old mice, fixed in formalin for 24 h, rinsed in 70% ethanol, and stored in 70% ethanol at 4 c overnight. sectioning and staining of the liver, spleen, kidney, and pancreas were performed by the molecular pathology and imaging core at the university of pennsylvania, supported by nih grants dk050306, ca098101, and dk049210. sectioning of the epididymal fat pads was performed by the pathology core at the children 's hospital of philadelphia research institute. slides were deparaffinized in xylene (3 3 min), then rehydrated in an ethanol series of 100% (3 3 min), 95% (1 3 min), 80% (1 3 min), dh2o (1 5 min). slides were stained in hematoxylin for 30 min, rinsed in dh2o, and rinsed in tap water for 5 min. slides were dipped in acid ethanol 812 to destain them, rinsed in tap water (2 1 min), and rinsed in dh2o (1 2 min). slides were then dehydrated in an ethanol series : 95% (3 5 min), 100% (3 5 min), and then in xylene (3 15 min) before drying and mounting a coverslip. rat anti - mouse f4/80 monoclonal antibody c1:a3 - 1 from abd serotec was used for staining sections from the epididymal fat for macrophage, natural killer cells, and f4/80 positive antigen presenting cell infiltrate. blood samples were taken from 8 to 12 week old mice between 12 and 2 pm. adiponectin, leptin, and resistin levels were measured using elisa kits from linco by the radioimmunoassay and biomarkers core at the penn diabetes research center, supported by nih grant dk19525. liver triglycerides were measured by weighing 100 mg liver and lysing in 400 ul cell lysis buffer (50 mm tris, ph 7.4, 140 mm nacl, 0.1% triton - x) (250 mg / ml homogenate). samples were diluted 10 fold and incubated in 1% deoxycholate at a 1:1 ratio for 10 min, then incubated for 10 min in thermo - scientific infinity triglyceride reagent. samples were read on a tecan plate reader at 500 nm. for glucose tolerance tests, 812 week old male mice were fasted for 1618 h overnight and injected with 1 g / kg glucose intraperitoneally. blood samples were taken from the tail in sarstedt microvette lithium heparin tubes (ref 16.433.100) at time 0 for fasting insulin measurements and 15 min post injection. blood glucose levels were taken from the tail on a onetouch ultra glucometer at fasting, and post - injection at 15, 30, 60, and 120 min. for the meal challenges, 812 week old male mice were fasted for 1618 h overnight and then refed normal chow for 2 h. crystal chem ultra - sensitive mouse insulin elisa kits were used for all insulin measurements. all mice were on a c57/b6 background and were maintained on normal chow diets from laboratory rodent diet, lab diet 5001. cre - positive male mice heterozygous for akt1 and akt2 floxed alleles were bred to females homozygous for akt1 and akt2 floxed alleles. this breeding scheme generated cre - positive experimental mice homozygous floxed for both genes and cre - negative male mice homozygous floxed for both genes that served as controls. cre - positive males heterozygous for both floxed genes were generated and exhibited a wild type phenotype (data not shown). all mouse experiments were reviewed and approved by the university of pennsylvania institutional animal care and use committee in accordance with the guidelines of the national institutes of health. organs were harvested for histopathology from 12-week - old mice, fixed in formalin for 24 h, rinsed in 70% ethanol, and stored in 70% ethanol at 4 c overnight. sectioning and staining of the liver, spleen, kidney, and pancreas were performed by the molecular pathology and imaging core at the university of pennsylvania, supported by nih grants dk050306, ca098101, and dk049210. sectioning of the epididymal fat pads was performed by the pathology core at the children 's hospital of philadelphia research institute. slides were deparaffinized in xylene (3 3 min), then rehydrated in an ethanol series of 100% (3 3 min), 95% (1 3 min), 80% (1 3 min), dh2o (1 5 min). slides were stained in hematoxylin for 30 min, rinsed in dh2o, and rinsed in tap water for 5 min. slides were dipped in acid ethanol 812 to destain them, rinsed in tap water (2 1 min), and rinsed in dh2o (1 2 min). slides were then dehydrated in an ethanol series : 95% (3 5 min), 100% (3 5 min), and then in xylene (3 15 min) before drying and mounting a coverslip. rat anti - mouse f4/80 monoclonal antibody c1:a3 - 1 from abd serotec was used for staining sections from the epididymal fat for macrophage, natural killer cells, and f4/80 positive antigen presenting cell infiltrate. blood samples were taken from 8 to 12 week old mice between 12 and 2 pm. adiponectin, leptin, and resistin levels were measured using elisa kits from linco by the radioimmunoassay and biomarkers core at the penn diabetes research center, supported by nih grant dk19525. liver triglycerides were measured by weighing 100 mg liver and lysing in 400 ul cell lysis buffer (50 mm tris, ph 7.4, 140 mm nacl, 0.1% triton - x) (250 mg / ml homogenate). samples were diluted 10 fold and incubated in 1% deoxycholate at a 1:1 ratio for 10 min, then incubated for 10 min in thermo - scientific infinity triglyceride reagent. for glucose tolerance tests, 812 week old male mice were fasted for 1618 h overnight and injected with 1 g / kg glucose intraperitoneally. blood samples were taken from the tail in sarstedt microvette lithium heparin tubes (ref 16.433.100) at time 0 for fasting insulin measurements and 15 min post injection. blood glucose levels were taken from the tail on a onetouch ultra glucometer at fasting, and post - injection at 15, 30, 60, and 120 min. for the meal challenges, 812 week old male mice were fasted for 1618 h overnight and then refed normal chow for 2 h. crystal chem ultra - sensitive mouse insulin elisa kits were used for all insulin measurements. to determine the role of akt in regulating adipocyte function and survival, we generated mice lacking both akt1 and akt2 using the adipoq - cre driver. adipo - akt knockout (ko) mice were severely lipodystrophic at 812 weeks of age such that all subcutaneous and cutaneous fat depots, including interscapular brown adipose tissue, were completely absent (figure 1a). the epididymal wat was about 10 smaller (as a percentage of body weight) in adipo - akt ko mice as compared to control mice (figure 1b). hematoxylin and eosin staining of tissue sections showed that the residual epididymal wat (ewat) in ko mice contained a modest number of enlarged adipocytes (figure 1c). the ko ewat also displayed increased infiltration of f4/80 + macrophages (figure 1d). the levels of important adipokines, such as leptin, adiponectin, and resistin, were dramatically decreased in adipo - akt ko mice relative to controls (figure 1e). the body weight of adipo - akt ko mice was increased about 15% by 5 weeks of age, with controls weighing 17.3 g and adipo - akt ko mice weighing 20.5 g (figure 1f). this increase in body weight was maintained at 9 weeks of age with control mice weighing 21.8 g and adipo - akt ko mice weighing 26.0 g (figure 1f). adipo - akt ko mice had livers weighing 2.7 times that of control animals when normalized for body weight at 812 weeks of age. livers in adipo - akt mice constituted 13% of body weight, and were 3.5 g on average, as compared to 4.9% of body weight and about 1 g in controls (figure 2a, b). the greater size of ko livers was associated with a large increase in liver triglyceride levels. control mice had an average of 9.2 mg triglyceride / g of liver whereas adipo - akt ko livers had 169.1 mg triglyceride / g of liver (figure 2c). histopathological analysis of livers from adipo - akt ko mice revealed a disorganized morphology with an increase in both the number and size of lipid droplets (figure 2d). in addition to hepatomegaly, the adipo - akt ko mice displayed enlarged pancreases and kidneys (figure 3a). on the other hand, adipo - akt ko mice did not exhibit splenomegaly, which is often associated with lipodystrophy,,, (figure 3a). histopathology of both the spleen and kidney revealed no obvious abnormalities (figure 3b, c). in contrast, the pancreases of adipo - akt ko mice had enlarged islets (figure 3d). adipo - akt ko mice had glucose tolerance comparable to control mice at 812 weeks of age (figure 4a). however, while control mice had fasting insulin levels of 0.5 ng / ml, levels in the ko mice were appreciably higher at 2.5 ng / ml, indicative of significant insulin resistance (figure 4b). to further investigate the ability of adipo - akt ko mice to regulate blood glucose in the face of insulin resistance, we fasted mice overnight and challenged them with a meal of normal chow. two hours after refeeding, blood glucose levels were significantly greater in the adipo - akt ko mice than the controls (figure 4c). consistent with the results from gtt, the insulin levels were elevated in fasted adipo - akt ko and increased even further following feeding (figure 4d). plasma non - esterified fatty acids (nefa) levels were also measured during fasting and after a 2 h feeding challenge on normal chow. adipo - akt ko mice had low basal nefa levels of 0.7 mmol / l compared to control mice that had fasting nefa of 1.6 mmol / l. postprandially, nefa levels in control mice are suppressed to 0.68 mmol / l while in adipo - akt ko mice nefa levels were not changed (figure 4e). to determine the role of akt in regulating adipocyte function and survival, we generated mice lacking both akt1 and akt2 using the adipoq - cre driver. adipo - akt knockout (ko) mice were severely lipodystrophic at 812 weeks of age such that all subcutaneous and cutaneous fat depots, including interscapular brown adipose tissue, were completely absent (figure 1a). the epididymal wat was about 10 smaller (as a percentage of body weight) in adipo - akt ko mice as compared to control mice (figure 1b). hematoxylin and eosin staining of tissue sections showed that the residual epididymal wat (ewat) in ko mice contained a modest number of enlarged adipocytes (figure 1c). the ko ewat also displayed increased infiltration of f4/80 + macrophages (figure 1d). the levels of important adipokines, such as leptin, adiponectin, and resistin, were dramatically decreased in adipo - akt ko mice relative to controls (figure 1e). the body weight of adipo - akt ko mice was increased about 15% by 5 weeks of age, with controls weighing 17.3 g and adipo - akt ko mice weighing 20.5 g (figure 1f). this increase in body weight was maintained at 9 weeks of age with control mice weighing 21.8 g and adipo - akt ko mice weighing 26.0 g (figure 1f). adipo - akt ko mice had livers weighing 2.7 times that of control animals when normalized for body weight at 812 weeks of age. livers in adipo - akt mice constituted 13% of body weight, and were 3.5 g on average, as compared to 4.9% of body weight and about 1 g in controls (figure 2a, b). the greater size of ko livers was associated with a large increase in liver triglyceride levels. control mice had an average of 9.2 mg triglyceride / g of liver whereas adipo - akt ko livers had 169.1 mg triglyceride / g of liver (figure 2c). histopathological analysis of livers from adipo - akt ko mice revealed a disorganized morphology with an increase in both the number and size of lipid droplets (figure 2d). in addition to hepatomegaly, the adipo - akt ko mice displayed enlarged pancreases and kidneys (figure 3a). on the other hand, adipo - akt ko mice did not exhibit splenomegaly, which is often associated with lipodystrophy,, histopathology of both the spleen and kidney revealed no obvious abnormalities (figure 3b, c). in contrast, the pancreases of adipo - akt ko mice had enlarged islets (figure 3d). adipo - akt ko mice had glucose tolerance comparable to control mice at 812 weeks of age (figure 4a). however, while control mice had fasting insulin levels of 0.5 ng / ml, levels in the ko mice were appreciably higher at 2.5 ng / ml, indicative of significant insulin resistance (figure 4b). to further investigate the ability of adipo - akt ko mice to regulate blood glucose in the face of insulin resistance, we fasted mice overnight and challenged them with a meal of normal chow. two hours after refeeding, blood glucose levels were significantly greater in the adipo - akt ko mice than the controls (figure 4c). consistent with the results from gtt, the insulin levels were elevated in fasted adipo - akt ko and increased even further following feeding (figure 4d). plasma non - esterified fatty acids (nefa) levels were also measured during fasting and after a 2 h feeding challenge on normal chow. adipo - akt ko mice had low basal nefa levels of 0.7 mmol / l compared to control mice that had fasting nefa of 1.6 mmol / l. mmol / l while in adipo - akt ko mice nefa levels were not changed (figure 4e). our findings indicate that akt1 and/or akt2 are required for adipose maintenance in vivo. the severity of insulin resistance in the adipo - akt ko mice and their inability to regulate blood glucose postprandially follows the pattern observed in human patients with lipodystrophy, many of whom develop frank diabetes following puberty,,. mutations in agpat-2 and bscl-2/seipin account for most human lipodystrophy patients, cgl type i and cgl type ii, respectively,, and mice null for these genes have lipodystrophic and insulin resistant phenotypes,. several other genes have been manipulated in mice resulting in lipodystrophy, notably the azip / f-1 dominant negative mouse, which interferes with adipocyte development, the lamin a (lmna) dominant negative mouse that inhibits adipose renewal, and adipocyte - specific loss of peroxisome proliferator - activated receptor gamma (ppar), a transcription factor required for adipocyte differentiation and maintenance,,,. studies performed in vitro established that akt signaling is necessary for adipogenesis in cultured cells,,,,. akt1 is required for adipogenesis in mouse embryo fibroblasts (mefs) and 3t3l1 preadipocytes, while akt2 is dispensable,,. the isoform specificity in vitro are in conflict with in vivo data from akt1 null mice, that have an overall smaller body size but do not exhibit a lipodystrophic phenotype,. this suggests that either non - autonomous factors influence adipogenesis, or that akt2 can support adipogenesis in the whole animal. additionally, akt2 null mice have been suggested to develop an age - dependent lipodystrophy. our data expand on these studies and document a critical role for akt signaling in maintaining mature adipocytes. the adipoq - cre transgene is thought to be mainly expressed in mature adipocytes,,,. (2013) showed that recombination does not occur in the stromal vascular fraction of adipose tissue depots,. consistent with this, berry and rodeheffer (2013) found that highly purified adipogenic precursor cells in wat do not express adipoq - cre. interestingly, a recent study shows that adiponectin is expressed at the embryonic stage in adipocyte precursors of inguinal wat but not in adult preadipocytes. it is unclear if adipoq - cre is also expressed at sufficient levels in this population to induce efficient recombination. moreover, it is not known if adipose precursors of other depots, such as the epididymal wat similarly express adiponectin during embryogenesis. the adipoq - cre mediated deletion of akt in our model may thus reflect defects in adipocyte differentiation and maintenance, depending on the depot. there are three akt isoforms, akt1 - 3, that function in the insulin signaling pathway. each of these isoforms is expressed in adipocytes, but akt2 is the most abundant isoform,. mice null for akt1 and akt2 are born with dwarfism, are not viable, and mefs derived from these animals are unable to undergo adipogenesis. akt3 is expressed in differentiated adipocytes, but it is clearly not sufficient to preserve adipocyte insulin / igf signaling in the absence of both akt1 and akt2. the insulin and insulin - like growth factor 1 (igf-1) pathways converge on the insulin receptor substrate and phosphoinositide-3-kinase (pi3k), upstream of akt,,. loss of akt1 and akt2 in adipocytes likely ablates both insulin signaling and igf-1 signaling, potentially contributing to the severity of the lipodystrophy. concurrent deletion of the insulin receptor and insulin growth factor 1 receptor (igf1r) using ap2-cre causes lipodystrophy with a greater reduction in bat size than wat when compared to the ir knockout alone, suggesting that igf1r signaling has a more important role in bat than in wat,. interpretation of these data is confounded by the use of the ap2-cre. while the requirement for ir and igf1r signaling may differ between depots, all depots depend on akt - function. the adipo - akt ko mice display profound hepatomegaly and a small but significant renomegaly along with increased pancreas weight (figure 2, figure 3d). this organomegaly is also observed in some human lipodystrophic patients, with hepatomegaly often being most prominent,,,. hyperinsulinemia potentially contributes to the increased body size and organomegaly of adipo - akt ko mice through enhanced ir / igfr-1 signaling. another possible contributor to organomegaly is increased food intake as a result of reduced circulating levels of leptin (figure 1e), a critical regulator of energy homeostasis,,,,. increased food intake and decreased movement were observed in the agpat / lipodystrophy model and normalized with leptin supplementation. though not measured in this study, it is likely the adipo - akt ko mice would exhibit a similar phenotype. surprisingly, adipo - akt ko mice had low plasma nefa levels, a phenomenon also observed in some human lipodystrophic patients. similarly, the adipo - akt ko mice had refed nefa levels that were not significantly differently from either their fasted levels or the fed levels in control mice. in many mouse models of lipodystrophy interestingly, genetic background influences lipid profiles in lipodystrophic azip / f-1 mice. on a fvb background azip / f-1 mice have abnormally high nefa levels, whereas nefa levels are similar between control and azip / f-1 mice on a c57/b6 background. similarly, agpat2 and bscl-2/seipin null mice, both in the c57/b6 background, have normal nefa levels,. these mice, and the c57/b6 azip / f-1 mice, have significantly decreased fasting nefa levels, similar to the adipo - akt ko mice. by contrast, adipocyte - specific deletion of bscl-2/seipin results in a milder lipodystrophy and these mice do not have reduced fasting nefa levels. given the severity of lipodystrophy in the adipo - akt ko mice, it follows that they have no available adipose stores to undergo lipolysis and release fatty acids in response to a fasting state. our mouse model recapitulates many of the features observed in a familial lipodystrophy syndrome caused by loss of function mutations in akt1 and akt2, including insulin resistance and hepatomegaly. our study also demonstrates that akt signaling, likely acting downstream of the insulin and igf-1 receptors, is critically required for proper adipose expansion and/or maintenance.	objectiveadipose depot mass is tightly regulated to maintain energy homeostasis. akt is a critical kinase in the insulin - signaling cascade that is required for the process of adipogenesis in vitro. however, the role of akt in the maintenance and/or function of mature adipocytes in vivo had not been examined.methodsto study this, we deleted akt1 and akt2 in adipocytes of mice using the adipoq - cre driver.resultsstrikingly, mice lacking adipocyte akt were severely lipodystrophic, having dramatically reduced gonadal adipose and no discernible subcutaneous or brown adipose tissue. as a result, these mice developed severe insulin resistance accompanied by fatty liver, hepatomegaly and with enlarged islets of langerhans.conclusionsthese data reveal the critical role of adipocyte akt and insulin signaling for maintaining adipose tissue mass.
there is a large discrepancy between the supply and demand of surgical services in the developing world, especially for nations in sub - saharan africa. in 2008, of the estimated 235 million surgical procedures performed annually, only 3.5% was performed in developing countries. this inequality also holds true in the field of orthopaedic surgery and the delivery of musculoskeletal care. over the past decade, many countries in africa have experienced economic growth, which has been associated with a marked increase in motor vehicle traffic. this has led to an increase in injuries from road traffic accidents (rta) secondary to a lack of concomitant investment in infrastructure, implementation of traffic and safety laws, and noncompliance with the use of seat - belts and helmets. there has been longstanding and growing interest from the developed world in assisting the developing world, including providing surgical services. involvement in the early 19th century began with a religious focus through mission trips that commonly had evangelical motives. the mission trip concept has evolved beyond religious goals to include trips geared towards providing medical and surgical care in the developing world during periodic short trips by medical teams. the typical goals of such trips are to treat patients in need, decrease the local burden of disease through direct patient care and to build local capacity through education and training. in 2012, there were approximately 6000 such medical missions with approximately $ 250 million raised for funding. in recent years, a growing number of teams of well - intentioned international orthopaedic surgeons have volunteered their time and expertise on trips to the developing world. these medical missions are designed to perform a high volume of surgical procedures over a short period of time. during these trips, a large amount of material resources are transferred to the developing world in order to create a functional operating room facility in the target country. such surgical missions are assumed to be effective in achieving their goals, however, this approach to care raises several questions related to the medical, economic, and social state of the target country including : (1) what happens to patients that develop a post - operative complication after the surgical team returns to their native country ? (2) are there facilities available for patients to receive treatment for the complication ? (3) are the intellectual and material resources at the facility adequate to provide the appropriate treatment for complications ? (4) is the infrastructure suitable for patients to travel from their homes to the facility ? (5) assuming that a treatment facility and resources are available, is the treatment affordable to the patient ? (6) is the treatment culturally acceptable to the patient ? intertwined amongst the decision to perform surgical procedures in the developing world are the ethics of doing so just because one is capable of performing a procedure, should it be done ? is a procedure such as a total hip replacement, which is appropriate in a developed country, also appropriate in a country with fewer resources for postoperative care ? systems limitations locally within the target country may result in patients being left in worse positions than they were prior to treatment. we present a case performed in tanzania that resulted in the development of an untreatable post - operative complication. this testimony highlights the urgent need for a different approach to providing surgical services in the developing world, but also emphasizes the need for open forums for discussion of international morbidity and mortality cases. sm is a woman who at age 31 resided in a small rural village on the outskirts of nairobi, kenya. she lived with her parents and her two younger brothers in a 20 ft 20 ft dwelling. at the age of 5, she was afflicted with a condition that affected several of her major joints. her condition was not treated medically due to a lack of family resources as well the limited availability of local treatment options. she was fortunate to be well educated and has completed high school after which she obtained a clerical position at a printing a press and was able to work daily, although with severe pain and deformities. the pain was located in the left groin and was present at rest, but exacerbated with motion. by the age of 29, the left hip was auto - fused to her pelvis resulting in no range of motion and a significant residual deformity. by this point, most of the symptoms were consistent with low back pain and degenerative disk disease due to increased loads experienced across the lumbosacral spine secondary to the auto - fusion of the left hip. in july 2012, at 33 years of age, the patient sought treatment from a rheumatologist in nairobi and was told that she would need a total hip replacement (thr). however, a thr in nairobi would cost $ 7000, which was cost - prohibitive. although discouraged, the patient tenaciously sought alternative routes for getting the appropriate treatment. by november 2012, due to the severe soft - tissue contracture (e.g., severe adduction contracture) surrounding the left hip, she was unable to urinate comfortably and was unable to have children because vaginal delivery would not be possible. in november 2012, a rheumatology conference was held in nairobi. a rheumatology colleague from one of the authors (nps) home university in the united states attended the conference and met the patient. intrigued by her story, she contacted the author upon her return and asked him to engage in her care. in february 2013, the author was scheduled to travel to a hospital in arusha, tanzania for a short teaching trip, which is approximately 250 miles from the patient s home. it was agreed upon to meet the patient in arusha, along with new radiographs of the pelvis and left hip as well baseline labs including a sedimentation rate (esr) and c - reactive protein (crp). in february 2013 tuk (an open air carriage so named because of the sound of the motor) for 9 h to meet the surgeon and the orthopaedic team at the arusha lutheran medical center in tanzania. the patient s esr and crp were 33 (range 020) and 3.2 (range 06), respectively. the mildly elevated esr (normal crp), although not specific for any given inflammatory process, was consistent with a diagnosis of rheumatoid arthritis, but inconsistent with an infectious process. based on her normal crp the patient stated that the problems she had with her hip permeated her entire life. there was a chronic rheumatoid nodule present at the superolateral aspect of her left hip projecting through the skin. the hip was in a fixed position of 40 of flexion, 20 of adduction, and 10 of internal rotation. this case presented a challenging ethical problem the patient had a problem that could be addressed with a technically challenging thr, but what if there was a complication post - operatively ? did the benefit of changing this woman s status in her village outweigh the risk associated with the surgical procedure ? the local hospital offered a prosthesis that the surgical team had left behind from a surgical mission the previous year. since there was no surgical or implantation fee for the patient, her only financial responsibility would be the post - operative hospital costs amounting to $ 1000 us, which was feasible for her due to donations by relatives and friends. the decision was made to proceed with the surgical procedure after a detailed informed consent process. the patient was made aware of and demonstrated understanding of the alternatives, risks and benefits of a thr. a pre - operative consent form was signed by the patient and witnessed by the surgeon (nps). the morning of surgery, the patient received pre - operative antibiotics and underwent spinal anesthesia. there was no gross evidence of intraarticular infection at the time of surgery. due to the unique anatomic considerations of the patient a small pelvis with very poor bone quality secondary to disuse osteopenia a combination of components from different orthopaedic implant manufacturers were mixed and matched in order to achieve a stable final construct (fig. her host bone was extremely osteoporotic and was unable to accept the only cementless acetabular device that was available for implantation given her size ; having no other options, this cup was cemented in place. the patient tolerated the procedure well and was brought back to the recovery room in stable condition. her leg length had been restored, and soft - tissue contractures had been adequately addressed (fig. 3a and b). however, due to scant persistent serosanguinous wound drainage for several days, she was placed on intravenous antibiotic treatment. while this was associated with diminution of the drainage, it also added to her total cost burden. the patient s wound eventually healed, and she was discharged to home after a 3-week hospital stay. once back at home in nairobi, kenya, the patient completed an extensive self - directed physical therapy program outlined by the operative surgeon, since therapy services were not readily available due to high cost. the patient maintained contact with the surgeon via email with periodic updates on a monthly basis. she had been doing extremely well and was very happy with her surgical outcome. in october 2013, approximately 7 months following the index surgical procedure, the patient sent an email stating that she had new onset left hip pain. the pain began insidiously, and had become constant, both at rest as well as with activity. an x - ray of her hip that was reviewed by the operative surgeon (nps) demonstrated no interval change in component position and no peri - prosthetic fracture (fig. it was recommended that the patient obtain a new esr and crp to rule - out deep space peri - prosthetic infection. a set of inflammatory markers was obtained (esr 136 and crp 12, respectively) which was suggestive of a deep space peri - prosthetic infection. accordingly, she was advised to seek evaluation by an orthopaedic surgeon for a hip aspiration, as obtaining synovial fluid from her left hip would be the only way to definitively diagnose the presence of a deep space infection around her left thr, which is potentially very severe. effective treatment of a deep space peri - prosthetic infection typically requires using several expensive resources (e.g., 6 weeks of intravenous antibiotic therapy through a peripherally inserted central catheter), a multi - disciplinary team approach (i.e., orthopaedic surgery with expertise in revision surgery, infectious disease, and a pathologist (needed for evaluation for intra - operative specimen obtained for final microbiology), and one or more additional operations. in kenya, this treatment would cost several thousand us dollars, assuming that the full complement of intellectual and material resources are available at a given hospital facility. prior to surgical intervention, the patient was suffering from significant pain, deformity, limitations with activities of daily living, as well as the social stigma associated with her condition. however, she now is not only in pain, but also likely has a deep - space infection, with an organism with unknown virulence, which can result in a general deterioration of her health, systemic sepsis, and possibly death without appropriate timely treatment. this case demonstrates the ethical challenges that can be created from performing surgical procedures in the developing world without concomitant access to appropriate patient follow - up or resources for treating post - operative complications. while the current system is inadequate to manage the burden of disease, these inadequacies may be exacerbated at times by post - operative complications resulting from well - intentioned surgical missions. consequently, there is need for a new paradigm which provides a more sustainable solution with increased local capacity to treat complications associated with surgical procedures. arguably, this should be considered prior to medical missions and discussed with local personnel and communities as trips are being planned. in addition, until an adequate infrastructure is developed, the informed consent process for surgery in this setting should include a frank discussion about not only potential surgical complications, but also the possibility that they may not be able to be managed appropriately given constrained local resources. this care also raises the very compelling argument, that in retrospect, a total hip replacement may not have been the best choice for this patient. although her preoperative labs were not suggestive of a septic process, the possibility exists that a total hip was done in the setting of an indolent tuberculous infection. moreover, other, more simple surgical procedures, more in - line with the current local standards of care may have been more appropriate for this patient. one could argue, that in this case a simple left femoral subtrochanteric osteotomy with tenotomies and a period of plaster of paris immobilization, could have allowed for the correction of her deformity, improvement of her status, while minimizing the risks that are inherent in a total hip arthroplasty. surgeons across the globe strive to better themselves and optimize patient care. in an ideal world unfortunately, complications are very real and a legitimate concern of every surgery performed. when a complication does occur, it is paramount to critically evaluate the problem, rectify it, and to attempt to prevent future events from occurring. they have been used since the early 1900s as a means to learn from complications and have been a cornerstone in medical education. they were first implemented in surgical departments and later spread to internal medicine and other specialties. as the focus on quality assurance has grown, the core purpose of mmcs should be to call attention to the shortcomings in an organization or patient care that potentially contributed to a complication or the death of a patient and to implement actions aimed at avoiding the reoccurrence of such events. as more resources and opportunities arise to provide medical assistance in the developing world, it is critical to evaluate the impact this is having. for example, there have been calls to ensure that long - term outcomes from mission - based surgeries are measured to help ensure that there are indeed net benefits to patients and communities. nonetheless, even with the best of intentions, complications can and will happen. in the developed world, mmcs are typically held in a grand rounds type forum and allow discussions amongst people involved directly in the case and those who are third party observers. in the international forum, our ability to convene as a group is difficult, but this should not preclude our ability to conduct mmcs. in the electronic age, a written forum such as this will allow an open forum to discuss our complications and to develop solutions and potentially a network of qualified individuals to assist in managing these complex issues. moreover, a collection of these cases will likely be a rich source of data to drive policy improvement as well as education and training. in addition, consideration should be given to developing other means to promote productive conversations regarding such cases. this might include a secure wiki or similar web - based platform that does not require broad bandwidth in which surgeons and other stakeholders, both locally and internationally, could comment on cases thereby creating an opportunity to elicit important perspectives. the presented case illustrates many of the difficulties in caring for individuals in the developing world, raising several important questions : (1) how can such complications be prevented in the future ? (2) what are the possible ways of managing a complication with the resources at hand once it occurs ? while, the answers to these questions may not be evident at this point in time, ideally a forum such as a written mmc can help provide rich descriptions of the issues and problems that are encountered so as to increase awareness of them so that appropriate solutions can be identified. in this way, those engaged in well - intentioned medical missions can meet their obligations to help and minimize potential harms in a world that is characterized by marked disparities of resources. all edits / alteration are assured that not to distort scientific meaning of the information presented.	highlightsthere is a large discrepancy between the supply and demand of surgical services in the developing world, especially for nations in sub - saharan africa.there has been longstanding and growing interest from the developed world in assisting the developing world, including providing surgical services.this case demonstrates the ethical challenges that can be created from performing surgical procedures in the developing world without concomitant access to appropriate patient follow - up or resources for treating post - operative complications.consequently, there is a need for a new paradigm which provides a more sustainable solution with increased local capacity to treat complications associated with surgical procedures.until adequate infrastructure is developed, the informed consent process for surgery in this setting should include a frank discussion about not only potential surgical complications, but also the possibility that they may not be able to be managed appropriately given constrained local resources.in the interim, a forum such as a written morbidity and mortality conference can help provide rich descriptions of the issues and problems that are encountered so as to increase awareness of them so that appropriate solutions can be identified.
the porphyrias, a group of disorders characterized by a variable catalytic defect of one of the seven enzymes in the biosynthesis of heme, are classified clinically as acute or non - acute porphyrias based on their clinical manifestations with or without neurovisceral attacks [1, 2, 3 ]. the major manifestations of acute porphyrias are neurological, including neuropathic abdominal pain, peripheral neuropathy, mental disturbance and seizures. the most commonly reported types of seizures are complex partial seizures and tonic - clonic seizures [4, 5 ], even though eeg documentation of such seizures have been sparse. status epilepticus has also been rarely reported. here, we report two patients with hereditary coproporphyria (hcp) who presented with status epilepticus and an eeg documentation. a 49-year - old right - handed man with a past medical history of berger 's disease presented to the emergency room on december 13, 2009, for a 1-min - long tonic - clonic seizure and increasing confusion over the past week., he developed continuous right hand myoclonic jerks compatible with epilepsia partialis continua (epc). repeat brain mri performed 3 days after admission disclosed non - enhancing high t2 and flair signal changes over the bilateral temporal lobes (fig. 1). brain pet revealed focal hypermetabolism over the bilateral frontal and temporal lobes, predominantly over the left side. bacteriological, viral serological (including herpes simplex virus pcr) workup, immunologic parameters as well as toxicology screen revealed no abnormal findings. while the confusion progressively waned, epc persisted despite several antiepileptic drug trials (phenytoin, valproic acid, carbamazepine, clobazam, lamotrigine). a probable diagnosis of hpc was eventually made when urine coproporphyrins and fecal total porphyrins returned to twice the normal range (even when testing was made 9 days after admission while the patient was improving). these abnormal results could not be explained by other conditions which have previously been associated with secondary coproporphyrinurias such as abuse of toxic substances, liver diseases, malignancies, diverse hematological diseases, etc.. over the last 3 years, the patient has continued to present non - disabling epc (manifesting only as very low - amplitude right hand myoclonic jerks) and has had a total of four tonic - clonic seizures in the context of alcohol consumption. he remains with mild memory problems and is currently treated with levetiracetam. on the last brain mri (2 years after initial setting), t2 signal changes had decreased significantly (fig. a 30-year - old man, known only for a generalized anxiety disorder, was brought to the emergency room on november 16, 2011, in convulsive status epilepticus preceded over the last 3 days by nausea, vomiting and headaches. initial management included intravenous benzodiazepines and phenytoin followed by intubation and a perfusion of midazolam and propofol. the patient then evolved to epc as he presented continuous left hand and mouth myoclonic jerks, very rarely associated with altered consciousness. 4) showed diffuse slowing, sometime associated with right frontotemporal periodic lateralized epileptiform discharges. mri disclosed right hemispheric gyriform increased signal on t2-weighted images, restricted diffusion on dwi / adc images and mild midline shift to the left. brain pet revealed focal hypermetabolism over the right frontal and temporal lobes, and to a lesser extent in the right occipital region. as in the first patient, bacteriological, viral serological workup as well as immunologic parameters failed to disclose a plausible cause. his condition eventually worsened, with decreased level of consciousness leading to intubation, despite large spectrum antibiotics, acyclovir, several antiepileptic drug trials (phenobarbital, levetiracetam, lacosamide, topiramate, vigabatrin, gabapentin) and immunotherapy (intravenous immunoglobulins, plasma exchanges). diagnosis of porphyria was made when results from urine, fecal and serum porphyrins returned elevated at 3 weeks after admission. one month and a half after admission and despite hematin therapy and withdrawal of porphyrinogenic drugs, the patient eventually died from brain edema secondary to the status epilepticus, after he had suffered a porphyric attack from an isoflurane trial. later, genetic testing confirmed hcp with a rare homozygous missense g189s mutation in the coproporphyrinogen iii oxidase gene. a 49-year - old right - handed man with a past medical history of berger 's disease presented to the emergency room on december 13, 2009, for a 1-min - long tonic - clonic seizure and increasing confusion over the past week., he developed continuous right hand myoclonic jerks compatible with epilepsia partialis continua (epc). repeat brain mri performed 3 days after admission disclosed non - enhancing high t2 and flair signal changes over the bilateral temporal lobes (fig. 1). brain pet revealed focal hypermetabolism over the bilateral frontal and temporal lobes, predominantly over the left side. bacteriological, viral serological (including herpes simplex virus pcr) workup, immunologic parameters as well as toxicology screen revealed no abnormal findings. while the confusion progressively waned, epc persisted despite several antiepileptic drug trials (phenytoin, valproic acid, carbamazepine, clobazam, lamotrigine). a probable diagnosis of hpc was eventually made when urine coproporphyrins and fecal total porphyrins returned to twice the normal range (even when testing was made 9 days after admission while the patient was improving). these abnormal results could not be explained by other conditions which have previously been associated with secondary coproporphyrinurias such as abuse of toxic substances, liver diseases, malignancies, diverse hematological diseases, etc.. over the last 3 years, the patient has continued to present non - disabling epc (manifesting only as very low - amplitude right hand myoclonic jerks) and has had a total of four tonic - clonic seizures in the context of alcohol consumption. he remains with mild memory problems and is currently treated with levetiracetam. on the last brain mri (2 years after initial setting), t2 signal changes had decreased significantly (fig. a 30-year - old man, known only for a generalized anxiety disorder, was brought to the emergency room on november 16, 2011, in convulsive status epilepticus preceded over the last 3 days by nausea, vomiting and headaches. initial management included intravenous benzodiazepines and phenytoin followed by intubation and a perfusion of midazolam and propofol. the patient then evolved to epc as he presented continuous left hand and mouth myoclonic jerks, very rarely associated with altered consciousness. 4) showed diffuse slowing, sometime associated with right frontotemporal periodic lateralized epileptiform discharges. mri disclosed right hemispheric gyriform increased signal on t2-weighted images, restricted diffusion on dwi / adc images and mild midline shift to the left. brain pet revealed focal hypermetabolism over the right frontal and temporal lobes, and to a lesser extent in the right occipital region. as in the first patient, bacteriological, viral serological workup as well as immunologic parameters failed to disclose a plausible cause. his condition eventually worsened, with decreased level of consciousness leading to intubation, despite large spectrum antibiotics, acyclovir, several antiepileptic drug trials (phenobarbital, levetiracetam, lacosamide, topiramate, vigabatrin, gabapentin) and immunotherapy (intravenous immunoglobulins, plasma exchanges). diagnosis of porphyria was made when results from urine, fecal and serum porphyrins returned elevated at 3 weeks after admission. one month and a half after admission and despite hematin therapy and withdrawal of porphyrinogenic drugs, the patient eventually died from brain edema secondary to the status epilepticus, after he had suffered a porphyric attack from an isoflurane trial. later, genetic testing confirmed hcp with a rare homozygous missense g189s mutation in the coproporphyrinogen iii oxidase gene. porphyrias are uncommon, complex metabolic disorders caused by deficiencies in the activities of the seven out of eight enzymes of the heme biosynthetic pathway. they are generally subdivided into acute and non - acute porphyrias on the basis of their major clinical manifestations. the acute porphyrias are well - defined genetic disorders of heme biosynthesis characterized by acute life - threatening attacks of nonspecific neurologic symptoms. these acute porphyrias are comprised of acute intermittent porphyria (aip), hcp, variegate porphyria (vp) and 5-aminolevulinic acid dehydratase porphyria (adp) in which aip is the most frequent type. the combined prevalence of the acute porphyrias is approximately 5 cases per 100,000 persons, they are more common in women than in men, and often generally develop during adult life. acute porphyric attacks can be exacerbated by a variety of factors, including porphyrinogenic drugs, alcohol, endogenous hormones particularly progesterone, pregnancy, cigarette smoking, metabolic stress induced by infections or surgery and reduced caloric intake. misdiagnoses of porphyrias are common because the signs and symptoms of acute attacks are variable and may mimic many other diseases. they can present with acute neurovisceral attacks, which may be accompanied by skin lesions in hcp and vp. abdominal pain, reported approximately in 8595% of patients, is often accompanied by nausea, vomiting, constipation and diarrhea. other symptoms include acute psychiatric symptoms, seizures, tachycardia and hypertension, and peripheral neuropathy. the most common types of seizures reported in the literature consist of complex partial or tonic - clonic seizures. a summary of these previously reported cases is presented in table 1 [6, 7, 8, 9, 10 ] along with our cases. status epilepticus was part of the initial presentation for all subjects with the presence of visceral symptoms in two. the type of status varied greatly from convulsive status epilepticus to non - convulsive status epilepticus, complex partial status and epc. non - surprisingly, confirmation of diagnosis led to modification of the treatment regimen with good outcome for all patients except one (case 2). out of the previously five reported cases, 3 patients had aip, 1 patient had vp and 1 had an unknown type of acute porphyria, while both of our patients had hcp. current understanding of the pathogenesis of seizures suggests the hepatic production of a neurotoxic substance, presumably ala [a -aminobutyric acid (gaba) analogue ] and/or pbg which may interact with gaba or glutamate receptors. a combination of endothelial dysfunction, hypoperfusion, and vasoconstriction in this setting of neurotoxicity can lead to a compromise of the blood - brain barrier and brain edema. mri may help detect changes occurring in the brain at the time of the acute attack though lesions are non - specific (cortically and/or subcortically, anterior or posterior, without or with mild enhancement, generally but not always reversible) [11, 12 ]. seizures, when they occur, are a therapeutic challenge because most aeds (phenobarbital, carbamazepine, clonazepam, phenytoin, primidone, ethosuximide, valproic acid, lamotrigine, felbamate, tiagabine, topiramate) may exacerbate attack of acute porphyria. drugs reported to be relatively safe include gabapentin [6, 8, 14 ], levetiracetam, and possibly oxcarbazepine. although rare, their recognition is important as it modifies significantly patient management and probably their outcome.	purposethe porphyrias are a defect in the biosynthesis of heme which can be associated with different neurological symptoms during acute attacks such as peripheral neuropathy, mental disturbance and seizures. so far, there have only been a few case reports of status epilepticus, none of which were of epilepsia partialis continua (epc). we present here two cases of hereditary coproporphyria (hcp) manifesting epc as part of the clinical presentation.methodthe patients medical charts, eeg and imaging studies were carefully reviewed.resultscase 1 is a 49-year - old male who first presented a tonic - clonic seizure. case 2 is a 30-year - old male who came to the emergency room for a convulsive status epilepticus. both evolved to epc over the next days. epc persisted despite several antiepileptic drug trials. diagnosis of hcp was confirmed by a high level of urine, fecal and serum porphyrins in both cases and by genetic testing in one. over the last 3 years, the first patient has continued to present non - disabling epc and has had four tonic - clonic seizures associated with alcohol consumption. the second patient died from brain edema one month and half after admission.conclusionacute porphyrias should be included in the differential diagnosis of new onset status epilepticus, including epc. their recognition is important as it modifies significantly patient management, since many anticonvulsants are porphyrogenic.
both interstitial granulomatous dermatitis (igd) and palisaded neutrophilic granulomatous dermatitis (pngd) are rare disorders typically associated with systemic autoimmune conditions. they probably represent different aspects of a disease spectrum encompassing the concept of autoimmunity - related granulomatous dermatitis (argd). the present case shows that the clinicopathological correlation in argd does not always clearly fit with the classical presentations of igd or pngd. a number of autoimmune conditions are associated with cutaneous symptoms. interstitial granulomatous dermatitis (igd) the typical clinical presentation corresponds to a linear rope present on the trunk and axillae. on histopathological examination, igd is characterized by a sparse palisaded histiocytic infiltrate with little neutrophilic debris and eosinophils associated with a necrobiotic aspect of the collagen matrix. in the literature, there is some overlap between igd and palisaded neutrophilic granulomatous dermatitis (pngd). lesions of pngd typically correspond to papules occasionally admixed with small crusts on the elbows. other related disorders include rheumatoid papules, churg - strauss granuloma, superficial ulcerating rheumatoid necrobiosis, necrobiotic granuloma, palisading granuloma, cutaneous extravascular necrotizing granuloma, and rheumatoid neutrophilic dermatitis. here, a 61-year - old woman presented with skin lesions over the elbows and the left thenar region. they corresponded to erythematous and discrete hyperkeratotic papules which had been present for a few weeks (fig. immunohistochemistry revealed the presence of cd3 + t lymphocytes predominating in the perivascular area (fig. they were associated with cd68 + and mac387 + histiocytes as well as factor xiiia+ dermal dendrocytes (dd1). the pattern of dd1 distribution was similar to that of cd68 + histiocytes (fig. it was characterized by the elevation of anti - smooth muscle antibodies (1:160) and antinuclear antibodies (1:1,280). there were no detectable antibodies directed against nuclear antigens, keyhole limpet hemocyanin and mitochondria. hepatitis b and c serologies were negative. at the time of presentation, the patient 's liver tests were in the normal range. however, they recurred a few months later, and some other lesions appeared on the thighs. in some cases, there is a mismatch between the clinical presentation and the histopathological patterns of igd and pngd. indeed, these two conditions possibly represent a continuum or progression of a single disease process corresponding to autoimmunity - related granulomatous dermatitis (argd) [6, 8 ]. the clinical associations between argd, autoimmune disease and lymphoproliferative disorders have been intermixed between both igd and pngd diagnoses in the literature [6, 9, 10, 11, 12, 13, 14 ]. it is acknowledged that the inflammatory cell infiltrate is sparse to moderate in igd, while pngd has a dense neutrophilic and interstitial histiocytic infiltrate. only a single case of igd with autoimmune hepatitis association has been reported so far. some other autoimmune comorbidities including arthritis, antiphospholipid syndrome [18, 19 ] and hematologic malignancy have been reported in association with igd. in addition, interstitial granulomatous drug reactions have been linked to tnf inhibitors, ace inhibitors, diuretics [21, 22, 23, 24, 25, 26, 27, 28 ] and soy products in food.	aimboth interstitial granulomatous dermatitis (igd) and palisaded neutrophilic granulomatous dermatitis (pngd) are rare disorders typically associated with systemic autoimmune conditions. they probably represent different aspects of a disease spectrum encompassing the concept of autoimmunity - related granulomatous dermatitis (argd).case reporta 61-year - old woman presented with argd and autoimmune hepatitis. the clinical presentation suggested pngd, while histopathology was consistent with igd.discussionthe association of argd with autoimmune hepatitis is apparently a rare event. the present case shows that the clinicopathological correlation in argd does not always clearly fit with the classical presentations of igd or pngd.
treadmill walking gait analyses are frequently conducted due to their ease of collecting multiple strides of data, minimal space required and ability to finely control speed. the question of whether a treadmill (tm) walking gait replicates an overground (og) walking gait on a level surface has been extensively studied (alton., 1998 ;, 2001 ; lee and hidler, 2008 ; riley., 2007). from a spatiotemporal perspective, tm walking on a level surface generally simulates og walking (lee and hidler, 2008 ; riley., 2007). treadmills are advantageous to simulate common outdoor terrain changes in laboratory setting, such as uphill and downhill locomotion ; however, these conditions have not been compared between treadmill and overground walking. previous research into how spatiotemporal parameters change during uphill and downhill walking compared to level walking was inconclusive. half of these overground studies of inclination reported increased (mcintosh., 2006) or decreased (kawamura., these varied speeds make it difficult to attribute changes found due to inclination instead of the effect of speed. in contrast, other researchers found no change in speed with inclination concurrent with decreased step length in downhill walking (mcvay and redfern, 1994 ; redfern and dipasquale, 1997). with speed held constant across multiple inclinations, one study found no changes in stride length (lay., 2006). in contrast, a treadmill study with speed held constant found increases in step time as inclination increased from level to uphill (tulchin., 2010). studies examining the influence of inclination have exclusively examined treadmill or overground walking with no comparison across modes at different levels of inclination. in the workplace it is common for military personnel and first responders to carry loads on both flat and inclined terrain. during level walking, studies indicated that double support increased as load carried increased (ghori and luckwill, 1985 ; harman., 2000 ; kinoshita, 1985 ; martin and nelson, 1986). however, these increases were modest, less than 5% of the gait cycle. additionally, studies have reported mixed results for changes in stride length and cycle time during load carriage (harman., 2000 ; kinoshita, 1985 ; martin and nelson, 1986). these studies were conducted on level terrain, and as downhill walking resulted in shorter step lengths, it is possible that double support will increase more during declined walking when subjects carry loads compared to without any load. no study to date has comprehensively assessed spatiotemporal variables during tm and og walking at multiple inclinations and large, workplace relevant loads. therefore, the purpose of this study was to compare spatiotemporal variables for the uphill, level and downhill walking gait between treadmill and overground modes at a constant speed. we hypothesized that there would be no difference in spatiotemporal mean values and a decrease in spatiotemporal variability, as measured by standard deviation, between treadmill and overground modes at any of the inclinations or load conditions. we further hypothesized that there would be differences in spatiotemporal parameters among three levels of inclination with subjects increasing stride length and percentage of double support while decreasing the step rate with uphill walking compared to level and downhill walking. finally, we hypothesized that as load increased, these changes due to inclination would increase in magnitude. ten subjects aged 20.6 2.8 years, body height 1.70 0.05 m and mass 72.3 5.9 kg volunteered to participate in this study. ethical approval was granted by the institutional review board at the united states army research institute of environmental medicine. an a priori power analysis revealed that for a large effect size of 1.0, using =0.05 and =0.80, 10 subjects were necessary to adequately power the study. subject instrumentation included several retro - reflective markers placed on the heel, toe, sternum and bilateral acromia. for the lowest load (00 kg) condition, subjects wore standard military issue physical training uniforms (t - shirt and shorts), combat boots, plus a helmet and carried a simulated m16a1 rifle in both hands. in the 20 kg load, subjects wore clothing and equipment from the 00 kg load condition plus a standard military issue ballistic protective vest and the load carrying vest of a standard military issue backpack. the vests were loaded with soldier equipment (i.e., simulated ammunition, simulated fragmentation grenades, canteen, etc.) to achieve the 20 kg load. for the 40 kg load, subjects wore the 20 kg configuration plus a standard military issue backpack weighted with soldier equipment to reach the total load. for the data collection sessions, og data were collected on a 12 m grade - adjustable walkway. the tm walking trials were conducted on a split - belt treadmill (amti, watertown, ma, usa) with two belts (107 x 56 cm) in a fore / aft configuration. subjects completed four separate sessions : one orientation session and then three sessions of og and tm walking with one inclination (downhill, -6% grade ; level, 0% grade or uphill, + 6% grade) and three load conditions (00 kg, 20 kg and 40 kg) per session. the grades were chosen to ensure energy expenditure changes were significantly different from level walking while keeping heart rate under 80% of the maximum heart rate (duggan and haisman, 1992 ; wanta., 1993). the loads were chosen to fall within the guidelines defined in field manual 21 - 18 for a fighting load (20 kg (ghori and luckwill, 1985 ; harman., 2000) found small increases (< 5%) in percentage of double support as the load increased, which were similar to the current findings concerning double support percentage. furthermore, how these spatiotemporal changes cause alterations in injury risk and metabolic measures currently is unknown. we hypothesized that the tm data would yield lower average variability, or standard deviation, than the og data. surprisingly, the sd results did not indicate a consistent lower sd during treadmill walking. the results demonstrated that variability between modes during og was significantly lower in some instances (figure 3b) and significantly higher in others (figure 2b). these inconsistent results indicated that tm walking did not systematically influence the sd of spatiotemporal parameters compared to overground walking. the small sample size is one limitation of this study as we were not statistically powered to find less than a large effect (1.0). changes in the load of up to 40 kg, inclination of 6 percent grade away from level (i.e., uphill or downhill) and the mode (treadmill and overground) produced small, yet statistically significant changes in spatiotemporal parameters. due to the small magnitude of changes, treadmill walking appears to closely replicate the spatiotemporal parameters of overground walking. variability, as assessed by standard deviations, was not systematically lower during treadmill walking compared to overground walking.	abstractinfluences of load carriage and inclination on spatiotemporal parameters were examined during treadmill and overground walking. ten soldiers walked on a treadmill and overground with three load conditions (00 kg, 20 kg, 40 kg) during level, uphill (6% grade) and downhill (-6% grade) inclinations at self - selected speed, which was constant across conditions. mean values and standard deviations for double support percentage, stride length and a step rate were compared across conditions. double support percentage increased with load and inclination change from uphill to level walking, with a 0.4% stance greater increase at the 20 kg condition compared to 00 kg. as inclination changed from uphill to downhill, the step rate increased more overground (4.3 3.5 steps / min) than during treadmill walking (1.7 2.3 steps / min). for the 40 kg condition, the standard deviations were larger than the 00 kg condition for both the step rate and double support percentage. there was no change between modes for step rate standard deviation. for overground compared to treadmill walking, standard deviation for stride length and double support percentage increased and decreased, respectively. changes in the load of up to 40 kg, inclination of 6% grade away from the level (i.e., uphill or downhill) and mode (treadmill and overground) produced small, yet statistically significant changes in spatiotemporal parameters. variability, as assessed by standard deviation, was not systematically lower during treadmill walking compared to overground walking. due to the small magnitude of changes, treadmill walking appears to replicate the spatiotemporal parameters of overground walking.
abnormal variation of the posterior corneal curvature may occur in two forms : the generalized posterior keratoconus, characterized by an regular increase of the curvature of the entire posterior corneal surface has, and the circumscribed posterior keratoconus, in which a localized paracentral or central posterior corneal indentation is seen. in the generalized form, the corneal stroma typically remains clear. in contrast, the circumscribed posterior keratoconus shows stromal opacities overlying the localized anterior ectasia of the posterior surface, which may occupy the full stromal thickness. circumscribed posterior keratoconus is usually bilateral and sporadic, but familial cases have been also documented. despite the anterior protrusion in some cases, the 60-year - old white male of mediterranean origin presented for a cataract extraction on his left eye. visual acuity was 20/25 in the right eye and light perception in the left eye due to cataract formation. the patient denied history of injury, reporting only a bilateral ocular infection in childhood was reported. slitlamp examination revealed a bilateral paracentrally localized depression of the posterior curvature measuring 3 mm in diameter. there was scarring in the overlying corneal stroma (figures 1, 2, and 3). the posterior depression was clearly detectable using ultrasound biomicroscopy (humphrey, zeiss, oberkochen) (figure 5) and slit - scanning topography analysis (orbscan, bausch and lomb) (figure 6). corneal thickness measured 450 m within the lesion and 540 m in the adjacent healthy cornea using the orbscan system. the refractive power of both the posterior and anterior corneal curvature was 50 to 56 diopters within the paracentral area. following phacoemulsification and posterior chamber lens implantation visual acuity increased to 20/50 in the left eye. the clinical and topographic findings in this patient are consistent with the paracentral keratoconus posterior circumscriptus. this is the first report on ultrasound biomicroscopy to visualise the local anterior bulging of the posterior corneal surface with concomitant thinning of the stroma. light microscopy of this abnormality has shown focal disorganization of basal epithelium and basement membrane, a replacement of bowman 's layer by fibrous tissue, a thinned stroma with an irregular arrangement of the central collagen lamellae, and a variable appearance of descemet 's membrane with posterior excrescences indentating the vacuolated endothelium correspond to the corneal guttae seen in specular reflection. iron deposits are present in the basal and suprabasal epithelium, corresponding to the brownish epithelial line observed clinically, indicating an irregularity of the anterior corneal surface. visualisation of the posterior keratoconus using corneal topography analysis has been reported so far in a few cases [7, 8 ]. the light microscopy findings suggest an early pathogenic mechanism probably originated in the fifth or sixth month of gestation. it is classified as one of the anterior chamber cleavage anomalies (mesenchymal dysgenesis), as there are other anterior segment and systemic developmental abnormalities, as well as melanin depositions surrounding the posterior depression and iridocorneal adhesions. however, not all cases share this phenomenon. acquired cases occur and are usually associated with trauma [9, 10 ]. the mechanism in such cases involves an oblique penetrating injury with splitting of the inner corneal layers. differential diagnosis also includes congenital disorders as peter 's anomaly and congenital hereditary endothelial dystrophy but they are usually found in new borns. inflammation process as perforated corneal ulcer may also be taken into consideration, but it is usually unilateral. in most of the cases of posterior keratoconus the vision is not affected, rarely it may be associated with other ocular abnormalities as polar cataract, lenticonus, and ectopia lentis.	this paper documents a rare nonprogressive developmental disorder bilateral circumscribed posterior keratoconus in a 60-year - old man referred for a cataract surgery. for the first time ultrasound biomicroscopy was used to visualise the local anterior bulging of the posterior corneal surface with concomitant thinning of the stroma. the amount of localized posterior depression, corneal thickness and the refractive power of both the posterior and anterior corneal curvature were measured using slit - scanning topography analysis (orbscan).
endothelial and epithelial monolayers and their associated basement membranes form physical and functional barriers separating distinct tissue compartments. these barriers are frequently breached, e.g., by immune cells as part of their homeostatic or inflammatory trafficking, by tumor cells during metastatic spread, or during tissue reorganizations in development. first, often the migrating cell is guided toward the barrier by extracellular guidance cues such as chemokines. second, adhesion of the migrating cell to the barrier is mediated by sequential cell - cell interactions, as exemplified in the leukocyte extravasation cascade (reymond. third, transmigration across the barrier usually occurs via entry portals, which may either be developmentally regulated (nourshargh., 2010, seifert and lehmann, 2012) or induced by the migrating cell itself (nourshargh., 2010, vestweber, 2015). despite their apico - basal asymmetry, endothelia, and epithelia are often penetrated in both directions : e.g., neutrophils usually extravasate blood vessels from luminal to interstitial but can also transmigrate in the reversed direction (intravasation) (woodfin., likewise, tumor cells intravasate blood vessels upon leaving the primary tumor and extravasate to colonize distant organs (reymond., 2013). hence, rather than acting as pre - patterned gradients, which stereotypically guide the cells across the barrier, the directional cues governing transmigration might act locally and dynamically. lymphatic endothelial cells (lecs) of the lymphatic capillaries form a barrier for intravasating leukocytes and tumor cells. dendritic cells (dcs) enter these vessels on their way from the periphery to the lymph nodes, where dcs activate lymphocytes as part of the adaptive immune response (frster., 2008). dcs are guided from the interstitium toward the lymphatic capillaries by gradients of the chemokine ccl21 (weber., 2013), which is homeostatically secreted by the lecs (nakano and gunn, 2001). after arrival at the lymphatic capillary, dcs penetrate the basement membrane (pflicke and sixt, 2009) and the lec junctions (baluk. the molecular mechanisms directing dcs into the vascular lumen, which we investigate here, are largely unclear. dcs are attracted to the vicinity of lymphatic capillaries via gradients of ccl21 (weber., 2013). notably, ccl21 immunostainings in non - permeabilized tissues show a very faint interstitial signal compared to a strong signal localizing to lecs in permeabilized tissues (johnson and jackson, 2010, tal., 2011, weber., 2013). these depots are observed as perinuclear puncta (johnson and jackson, 2010, weber., 2013), which co - stain for the golgi marker golph4 (figure 1a) and disperse upon golgi - endoplasmic reticulum (er) disrupting brefeldin a treatment (weber., 2013). in addition to these trans - golgi depots, ccl21 locates to intracellular vesicles (figures 1a and s1a) (johnson and jackson, 2010), which might represent chemokine en route to homeostatic secretion or stores for on demand release. to test if intravasation of dcs changes ccl21 distribution, we layered in vitro - generated dcs on mouse ear explants. in this setup, dcs enter the tissue within minutes and migrate along gradients of interstitial ccl21 to ultimately intravasate into the dermal lymphatic vessels (pflicke and sixt, 2009, weber., 2013, weber and sixt, 2013) (figure 1b). in lymphatic capillaries, which had been entered by many dcs, we found that perinuclear ccl21 depots were dispersed and overall diminished (figures 1c and 1d). this change in ccl21 distribution was not observed at sites where few dcs intravasated or in ears loaded with dcs deficient for the ccl21 receptor ccr7 (figure s1b). ccr7 dcs can not read the interstitial ccl21 gradient and therefore do not interact with the lymphatic capillaries (frster., 2013).figure 1dendritic cell entry into mouse dermal lymphatic capillaries induces mobilization of ccl21(a) mouse dermal lymphatic capillary stained for lyve1 (green), golph4 (red), ccl21 (white), and nuclei (dapi, blue). zoom - in of boxed area is shown below.(b) tamra - labeled dcs (red) and lyve1-stained lymphatic capillaries (green) after 3 hr 30 invasion. yellow lines indicate the plane of orthogonal section, and yellow arrow highlights a transmigration event.(c) lyve1 (green), ccl21 (white), and nuclei (dapi, blue) of the ear dermis after 3 hr 30 in presence or absence (control) of tamra - labeled dcs (red). white arrows indicate dispersion of ccl21 (yellow arrow in control).(d) dot blot graph shows ratio of signal in high intensity ccl21 depots to ccl21 in other areas of lecs. columns represent mean values sd of control (n = 3) and + dc (n = 5) samples of 300 m long lymphatic vessel stretches.(e) transmission electron micrograph of ccl21 staining of dermis. black arrows indicate overlapping lec tips at cell - cell junctions, yellow arrow detachment of lecs from interstitial extracellular matrix, white arrow rearrangement of collagen bundles, and blue arrow a silver - amplified ccl21 immunogold label.(f) quantification of extracellular ccl21 staining at sites of dc - associated tissue alterations (see e) compared to intact area in same sample or sample devoid of dcs. bar graph shows mean sd of two (dc) and four (+ dc) independent ear samples. scale bars, 10 m (a) ; 50 m (b and c) ; 10 m in zoom - in images ; and 500 nm (e).see also figure s1. dendritic cell entry into mouse dermal lymphatic capillaries induces mobilization of ccl21 (a) mouse dermal lymphatic capillary stained for lyve1 (green), golph4 (red), ccl21 (white), and nuclei (dapi, blue). (b) tamra - labeled dcs (red) and lyve1-stained lymphatic capillaries (green) after 3 hr 30 invasion. yellow lines indicate the plane of orthogonal section, and yellow arrow highlights a transmigration event. (c) lyve1 (green), ccl21 (white), and nuclei (dapi, blue) of the ear dermis after 3 hr 30 in presence or absence (control) of tamra - labeled dcs (red). (d) dot blot graph shows ratio of signal in high intensity ccl21 depots to ccl21 in other areas of lecs. columns represent mean values sd of control (n = 3) and + dc (n = 5) samples of 300 m long lymphatic vessel stretches. black arrows indicate overlapping lec tips at cell - cell junctions, yellow arrow detachment of lecs from interstitial extracellular matrix, white arrow rearrangement of collagen bundles, and blue arrow a silver - amplified ccl21 immunogold label. (f) quantification of extracellular ccl21 staining at sites of dc - associated tissue alterations (see e) compared to intact area in same sample or sample devoid of dcs. bar graph shows mean sd of two (dc) and four (+ dc) independent ear samples. scale bars, 10 m (a) ; 50 m (b and c) ; 10 m in zoom - in images ; and 500 nm (e). to study whether mobilization of ccl21 upon dc intravasation was associated with extracellular ccl21 enrichment, we employed immunometal transmission electron microscopy. in line with the immunofluorescence analysis (figure 1a), intracellular ccl21 was found enriched in the nuclear periphery (figure s1c) and, more sparsely, in intracellular vesicles of control samples (figure s1d). extracellular ccl21 was mostly detected within 1 m distance of the basolateral side of lecs with no evidence of ccl21 gradients extending from the basolateral to the luminal side of the lecs (figure 1e). in samples exposed to dcs, local presence of dcs was associated with poorly organized interstitial collagen and partial detachment of the lecs from the interstitium (figures 1e, s1e, and s1f). importantly, these signatures of dc intravasation were associated with extracellular accumulations of basolateral, but not luminal, ccl21 (figure 1e-1f). these accumulations were most prominent at lec - lec junctions of detached endothelia (figure 1e), whereas sheer proximity of dcs seemed insufficient to discharge chemokine (figure s1 g). based on these findings, we hypothesize that contact between dcs and lecs triggers acute ccl21 secretion. triggered exocytosis of chemokine is reminiscent of exocytotic targeting of the adhesion molecule pecam to areas of lymphocyte transmigration at the blood endothelium (mamdouh., 2003). it might provide local guidance or act as a landmark for following dcs, which have been shown to intravasate with increased efficiency (martin - fontecha., 2003). to directly study ccl21 secretion, we established a traceable in vitro setup, where we lentivirally expressed variants of ccl21 in human dermal lecs, which recapitulated localization to the golgi secretory pathway (compare figures 2a and s1a) (de jong., 2005, de jong., 2008). secretion of ccl21 into the cell culture supernatant was dependent on the n - terminal signaling peptide (figures 2a and 2b). surprisingly, exclusion of the ccl21n - mcherry from the secretory pathway led to nuclear localization, possibly owing to a potential bipartite nuclear localizing signal in mature ccl21 (aa 2150). the loss of the heavily cationic c terminus (ccl21c) did not affect localization to the golgi secretory pathway (figure 2a) but instead prevented binding of ccl21 to the cell culture dish surface (figure s2a) and lead to increased ccl21 in the cell culture supernatant (figure 2b). this was in line with previous findings, showing that the c terminus immobilizes ccl21 to charged surfaces and glycosaminoglycans (hirose., 2002).figure 2ccl21-positive vesicles localize to golgi - secretory pathway and traffic along microtubules(a) giantin (white) and nuclear staining (dapi, blue) of ccl21-mcherry (red), ccl21n - mcherry, or ccl21c - mcherry expressing lecs. insets show golgi localized mcherry signal (yellow arrows) with or without staining of golgi marker giantin.(b) quantification of mcherry intensities in supernatants of ccl21-mcherry, ccl21n - mcherry, and ccl21c - mcherry expressing lecs. dot blot graph shows a mean sd of pooled data from two independent experiments, n = 4 for each construct. intensities are normalized to media background, which is set as 1.(c) basolateral non - directed tracks of tirf - imaged ccl21-mcherry - positive vesicles are indicated with white, directed tracks heading toward golgi area with green, directed tracks toward cell periphery with red, the golgi apparatus with a dashed yellow line, and the boundaries of cell contact surface with dashed white line. yellow arrow indicates a white trajectory representing random movement within a confined area.(d) tirf imaging of ccl21c - mcherry (red) and egfp - tubulin- (green) expressing primary lec. white arrow highlights one ccl21c - mcherry positive vesicle (red) throughout the image series. scale bars, 10 m (a) ; 2 m (c and d).see also figure s2. ccl21-positive vesicles localize to golgi - secretory pathway and traffic along microtubules (a) giantin (white) and nuclear staining (dapi, blue) of ccl21-mcherry (red), ccl21n - mcherry, or ccl21c - mcherry expressing lecs. insets show golgi localized mcherry signal (yellow arrows) with or without staining of golgi marker giantin. (b) quantification of mcherry intensities in supernatants of ccl21-mcherry, ccl21n - mcherry, and ccl21c - mcherry expressing lecs. dot blot graph shows a mean sd of pooled data from two independent experiments, n = 4 for each construct. (c) basolateral non - directed tracks of tirf - imaged ccl21-mcherry - positive vesicles are indicated with white, directed tracks heading toward golgi area with green, directed tracks toward cell periphery with red, the golgi apparatus with a dashed yellow line, and the boundaries of cell contact surface with dashed white line. (d) tirf imaging of ccl21c - mcherry (red) and egfp - tubulin- (green) expressing primary lec. white arrow highlights one ccl21c - mcherry positive vesicle (red) throughout the image series. scale bars, 10 m (a) ; 2 m (c and d). to trace the intracellular trafficking routes of ccl21, we performed real - time total internal reflection fluorescence (tirf) microscopy. tracking of ccl21-mcherry - positive vesicles showed two modes of behavior : some vesicles rapidly advanced along linear trajectories, whereas others moved randomly within confined areas (figure 2c). linear tracks connected golgi area and the lec periphery (figure 2c), matching the distribution pattern of microtubules (figure s2b). accordingly, ccl21 was microtubule - associated as revealed by immunostaining of untagged ccl21 and ccl21c (figure s2b) and live imaging of ccl21c - mcherry (figure 2d ; movie s1). microtubule disruption by nocodazole abolished the component of directed movement (movie s1) confirming that ccl21 is transported along microtubules, which is analogous to the transport of insulin in secretory pancreatic beta - cells (boyd. similarly to insulin, some of the linear trajectories of ccl21 vesicles were directed toward the cell periphery and some toward the golgi area, suggesting regulation of vesicle dwell time at the lec periphery (zhu., 2015). tirf imaging revealed that occasionally ccl21-mcherry vesicles were discharged into the extracellular space. regulated discharge of plasma membrane - docked vesicles is often initiated by calcium (ca) binding to synaptotagmins, which triggers the actual membrane fusion event (sdhof, 2012). accordingly, the ca ionophore ionomycin triggered robust ccl21-mcherry and ccl21c - mcherry secretion as directly observed with tirf microscopy as a sudden and transient increase in ccl21 intensity (figures 3a and 3d ; movie s2). such flashes are due to the exponential increase in tirf signal once the quantum of fluorescent probe enters the glass - water interface below the cell (axelrod, 1981), which is then followed by rapid dilution of the radially diffusing probe. bulk mcherry signal in the lec culture supernatant was increased by ionomycin (figure 3b). further, basal secretion seemed at least partially dependent on ca as chelation of intraendothelial ca with bapta - am causing a 30% reduction in ccl21c - mcherry secretion (figure 3c).figure 3regulation of ccl21 secretion(a) tirf microscopy of ccl21-mcherry vesicles (white) of a single lec after addition of 5 m ionomycin. yellow arrows indicate vesicles at time point 0, secretion events are highlighted by white arrows. see movie s2.(b) quantification of mcherry intensities of ccl21c - mcherry expressing lec culture supernatant subsequent to 6 control or 5 m ionomycin treatment. bar graph shows mean sd of pooled samples from 3 independent experiments, n = 5 (control) and 6 (ionomycin).(c) quantification of mcherry intensities of culture supernatants of ccl21c - mcherry expressing lecs 4 hr after washout of 1 hr long control or 10 m bapta - am treatment. bapta - am is a cell - permeant ca chelator, which is trapped in cells and binds intracellular calcium via carboxylic acid functional groups thus severely decreasing intracellular - free calcium. bar graph shows mean sd of pooled samples from three independent experiments, n = 7 for each condition. intensities of (b) and (c) are normalized to media background, which is set as 1.(d) quantification of pooled data from all tirf experiments on single vesicle secretion. data points show secretion frequency in control, 5 m ionomycin-, or 500 ng / ml latrunculin b - treated ccl21-mcherry or ccl21c - mcherry expressing single lecs as indicated in the figure. the dot blot graph shows mean sd secretion frequency in pooled data of single cells from 6 independent experiments, cell number (n) is indicated in the figure.(e) data points show ccl21-mcherry vesicle speed, displacement, and duration in the tirf imaging plane (basolateral membrane) in control and 13 latrunculin b (500 ng / ml)-treated single lecs, which displayed highest ccl21 secretion (see figure 3d latrunculin b column). the dot blot graph shows mean sd of vesicle tracks (51513/cell) in pooled data of single cells (n = 12 for control and 13 for latrunculin b) of three independent experiments. regulation of ccl21 secretion (a) tirf microscopy of ccl21-mcherry vesicles (white) of a single lec after addition of 5 m ionomycin. yellow arrows indicate vesicles at time point 0, secretion events are highlighted by white arrows. (b) quantification of mcherry intensities of ccl21c - mcherry expressing lec culture supernatant subsequent to 6 control or 5 m ionomycin treatment. bar graph shows mean sd of pooled samples from 3 independent experiments, n = 5 (control) and 6 (ionomycin). (c) quantification of mcherry intensities of culture supernatants of ccl21c - mcherry expressing lecs 4 hr after washout of 1 hr long control or 10 m bapta - am treatment. bapta - am is a cell - permeant ca chelator, which is trapped in cells and binds intracellular calcium via carboxylic acid functional groups thus severely decreasing intracellular - free calcium. bar graph shows mean sd of pooled samples from three independent experiments, n = 7 for each condition. intensities of (b) and (c) are normalized to media background, which is set as 1. (d) quantification of pooled data from all tirf experiments on single vesicle secretion. data points show secretion frequency in control, 5 m ionomycin-, or 500 ng / ml latrunculin b - treated ccl21-mcherry or ccl21c - mcherry expressing single lecs as indicated in the figure. the dot blot graph shows mean sd secretion frequency in pooled data of single cells from 6 independent experiments, cell number (n) is indicated in the figure. (e) data points show ccl21-mcherry vesicle speed, displacement, and duration in the tirf imaging plane (basolateral membrane) in control and 13 latrunculin b (500 ng / ml)-treated single lecs, which displayed highest ccl21 secretion (see figure 3d latrunculin b column). the dot blot graph shows mean sd of vesicle tracks (51513/cell) in pooled data of single cells (n = 12 for control and 13 for latrunculin b) of three independent experiments. see movie s3. ionomycin did not trigger secretion of all plasma membrane - associated vesicles (movie s2). some of these vesicles might not be plasma membrane - docked, but rather sequestered within the cortical actin meshwork, which has to be penetrated before the vesicles can directly contact the membrane (nakata and hirokawa, 1992, oheim and sthmer, 2000, giner., 2007). when we visualized the cortical actin meshwork of lecs with lifeact - egfp, lateral movements similar to that of ccl21-mcherry vesicles were apparent (movie s3). actin disassembly by latrunculin b substantially increased the frequency of secretion events (figure 3d ; movie s3), likely because of enhanced contact between vesicles and secretory machinery. accordingly, the lecs displaying latrunculin b - induced secretion showed increased movement of vesicles in the basolateral plane as well as in z - direction (revealed by disappearance of vesicles from the tirf field) (figure 3e ; movie s3). these results suggest that the cortical actin meshwork controls ccl21 secretion by restricting membrane - docking of vesicles. the finding that ca influx triggers ccl21 secretion in lecs (figures 3a, 3b, and 3d ; movie s2) together with our in situ finding that dcs cause release of ccl21 (figures 1e and 1f) suggested that dc - lec interactions might induce ca signals in lecs similar to ca signals in blood endothelia when being transmigrated by polymorphonuclear leukocytes (huang., while ca signaling can be induced in several ways, we were struck by our in situ observation of dcs probing lecs with a blunt foot - like processes (figures 4a and 4b), which were reminiscent of lymphocyte processes physically penetrating blood endothelium (carman., 2007, shulman., 2011). dc protrusions considerably deformed lecs and even detached them locally from the underlying extracellular matrix (figures 4a, 4b, s1e, and s1f). this suggested that dcs exert pushing forces on lecs, which might promote communication via extended cell - cell contacts. to investigate whether direct interaction was required for ca signals, lecs were grown to confluency, and dcs setting, dcs effectively transmigrated the monolayer (maddaluno., 2009) as revealed by phase contrast live cell imaging (figure 4c ; movie s4). transmigration was dependent on the secretion of ccl21, as non - infected or ccl21n - mcherry - infected lecs were only occasionally penetrated by dcs (figure s2d ; movie s4). importantly, dcs interacting with the apical side of lecs and dcs in transit through the monolayer induced an increase in ca concentration in lecs as revealed by time - lapse imaging of a ca sensor (figure 4d ; movie s5). at the population level, lecs showed a low basal frequency of ca transients, which was increased 4.5-fold upon addition of dcs (figure 4e). unlike dcs, dc - conditioned media did not induce an increase in ca concentration (figure 4e) excluding a major role for secreted factors. further, pre - treatment of dcs with mycalolide b, an irreversible actin depolymerizing agent (hori., 1993, saito., 1994), which prevents formation of actin - based cell protrusions, did not abolish ca transients (figure 4e). these results suggest that molecular engagements at the dc - lec interface are sufficient triggers. earlier, dc expressed 2 integrins (lfa-1) and its lymphatic endothelial ligand icam have been shown to be necessary for in vivo and in vitro transmigration of inflamed but not homeostatic endothelia, where icam is not expressed (johnson., 2006, johnson and jackson, 2010, vigl., 2011, accordingly, 2 integrins were dispensable for the dc entry into the dermal lymphatic capillaries as shown before (figure s2e) (lmmermann., 2008). at sites of intravasated 2 deficient (itgb2) dcs, ccl21 depots were dispersed from golgi similar to wild - type dc samples (figures s2e and s2f). thus, other molecular interactions at the dc - lec interface, such as the plexin1a - semaphorin3a axis (takamatsu., 2010), l1 (maddaluno., 2009), or yet unidentified factors might trigger the signal.figure 4endothelial ca signaling facilitates dc transmigration in vitro(a) transmission electron micrograph at a site of dc - lec interaction at ear dermis lymphatic capillary. interstitium (i), lumen (l), lec, and dc are marked.(b) immunoelectron micrograph of whole mount preparation labeled for ccl21 (silver amplified gold particles) at a site of dc - lec interaction in ear dermis. the yellow arrows in (a) and (b) indicate the endothelial detachment from interstitium.(c) phase contrast imaging of an in vitro transmigration event from the apical to basolateral side of the lec monolayer. white arrow points at leading edge, yellow arrow at cell body, and blue arrow at trailing edge of transmigrating dc. see movie s4.(d) epifluorescence imaging of oregon green bapta - am - treated lecs (green) and tamra - labeled dcs (red). blue arrow indicates dc leading edge and yellow arrow lec displaying a transient ca peak upon dc contact. below the image, the kymograph and the corresponding line graph of the oregon green bapta - am intensity in the lec (highlighted with yellow arrow) are shown. see movie s5.(e) quantification of lecs displaying ca peaks upon addition of fresh media, dcs, dc - conditioned media, or mycalolide b - treated dcs. fresh media and dc samples represent eight independent experiments (n = 18 and 19, respectively), of which two experiments included mycalolide b - treated dcs (n = 6), and three experiments included conditioned media samples (n = 4). data are normalized to average of dcs sample, which is set as 1.(f) quantification of dc transmigration on control or 10 m bapta - am (b - am)-treated ccl21-mcherry or ccl21c - mcherry expressing monolayers. dot blot graph shows mean sd of pooled samples from three independent experiments, n = 7 for bapta - am - treated ccl21c - mcherry expressing lecs and eight for all other conditions. scale bars, 500 nm (a and b) ; 50 m (c) ; and 10 m (d).see also figure s2. endothelial ca signaling facilitates dc transmigration in vitro (a) transmission electron micrograph at a site of dc - lec interaction at ear dermis lymphatic capillary. interstitium (i), lumen (l), lec, and dc are marked. (b) immunoelectron micrograph of whole mount preparation labeled for ccl21 (silver amplified gold particles) at a site of dc - lec interaction in ear dermis. the yellow arrows in (a) and (b) indicate the endothelial detachment from interstitium. (c) phase contrast imaging of an in vitro transmigration event from the apical to basolateral side of the lec monolayer. white arrow points at leading edge, yellow arrow at cell body, and blue arrow at trailing edge of transmigrating dc. (d) epifluorescence imaging of oregon green bapta - am - treated lecs (green) and tamra - labeled dcs (red). blue arrow indicates dc leading edge and yellow arrow lec displaying a transient ca peak upon dc contact. below the image, the kymograph and the corresponding line graph of the oregon green bapta - am intensity in the lec (highlighted with yellow arrow) are shown. (e) quantification of lecs displaying ca peaks upon addition of fresh media, dcs, dc - conditioned media, or mycalolide b - treated dcs. fresh media and dc samples represent eight independent experiments (n = 18 and 19, respectively), of which two experiments included mycalolide b - treated dcs (n = 6), and three experiments included conditioned media samples (n = 4). (f) quantification of dc transmigration on control or 10 m bapta - am (b - am)-treated ccl21-mcherry or ccl21c - mcherry expressing monolayers. dot blot graph shows mean sd of pooled samples from three independent experiments, n = 7 for bapta - am - treated ccl21c - mcherry expressing lecs and eight for all other conditions. scale bars, 500 nm (a and b) ; 50 m (c) ; and 10 m (d). to directly investigate whether lymphatic endothelial ca signaling was necessary for the transendothelial migration of dcs, we selectively chelated endothelial ca with bapta - am. we compared the responses to both ccl21-mcherry and ccl21c - mcherry, because full - length ccl21-mcherry accumulates on the cell culture dish surface (figure s2a), which is not seen in vivo (figure 1e). thus, we presumed that the truncated chemokine more faithfully mimics the in vivo situation. both ccl21-mcherry and ccl21c - mcherry supported transmigration but ca chelation reduced the number of transmigration events only in ccl21c - mcherry expressing monolayers (62.4% reduction, figure 4f ; movie s6), which lack the unphysiological accumulation of ccl21 (see figure s2a) and thus lack a prepatterned ccl21 gradient across the monolayer (similarly to the tissue context, figure 1e). these results suggest that dcs induce a transient ca signal in lecs, which triggers acute ccl21 secretion and possibly enables opening of the endothelial junctions, as described for blood endothelia (huang., 1993). although the decisive role of chemokines in leukocyte extravasation is well established, it has remained unclear if and how chemokines act during the actual transmigration process. our finding that ccl21 is acutely secreted upon contact between dcs and lecs extend data by shulman. (2011), which suggested that the chemokine ccl2 is released by blood endothelia in response to interactions with extravasating lymphocytes. while we show that a direct dc - lec contact is sufficient to trigger chemokine release even in the absence of cytoskeletal force generation (figure 4e), we found that applying mechanical pushing forces on lecs, and thus increasing lec plasma membrane tension (apodaca, 2002), can also trigger ca flux and ccl21 release in vitro (figures s2g s2i). this was in line with the observation that transmigrating dcs considerably deformed lecs in dermal tissue (figures 4a and 4b) and might suggest that molecular and mechanical triggers perpetuate the intravasation process in concert. earlier, long range ccl21 gradients were shown to guide dcs to the vicinity of lymphatic vessels (weber., 2013). however, lec penetration could not be explained by a simple concentration gradient across the endothelium as intra - luminal ccl21 concentrations are lower than those on the abluminal basement membrane (weber., 2013, russo., the acute chemokine release that we demonstrate exposes the leading edge of dcs to a burst of very steep ccl21 gradient, which will likely motivate the cell to locally push into the endothelium. at sites of loose button - like lec - lec junctions (baluk., 2007), physical pushing might open the junction and allow entry from the confined interstitial environment into the lymphatic capillary lumen where physical resistance is lower (figure 4 g). in addition, local chemokine release might boost transmigration by attracting more transmigrating cells toward the specific entry portal, or provide local guidance and thereby determine the actual site of penetration. taken together, our data show that the barrier cells take a more active role in their penetration than previously anticipated. wild - type c57bl/6j (charles river laboratories), ccr7 (frster., 1999) and itgb2 (wilson., 1993) male and female mice were bred and maintained according to the local rules (institutional review board approval 66018/3-ii/3b/2010). mice were sacrificed at the age of 68 weeks for the ear preparation and at the age of 812 weeks for the bone marrow extraction. ears of sacrificed mice were prepared as previously described (weber., 2013). ear sheets were fixed with 4% paraformaldehyde (pfa) in pbs at room temperature for 25 or prepared for the dc lymphatic capillary intravasation assay as follows : the ventral half of the ear was mounted between a 0.5 ml microcentrifuge tube lid and the body of the tube, of which the lower part was cut off. the 1.5 cm well formed by the cut tube was filled with 200 l of r10 with or without 50,000 (figures 1b1f) or 100,000 (figures s2e and s2f) tamra - labeled dcs. unattached dcs were washed off with r10 after 30 incubation at + 37c and 5% co2 and incubated in r10 for additional 13 hr (figures 1b1f). as itgb2 dcs are slower in invading ear explants, itgb2 dcs and wild - type controls were kept on the ear explants for 7 hr (i.e., for the whole duration of the experiment) (figures s2e and s2f). the experiments were terminated by washing the ear once with pbs followed by fixation with 4% pfa in pbs at room temperature for 25. for the quantification of extra - lec ccl21, we used two independent samples devoid of dcs (control) and four independent samples with dcs (+ dcs). all extracellular ccl21-immunometal particles within 1 m distance to the interstitial (basolateral) side of the lymphatic vessel endothelial cell were quantified. the particles were classified based on their location either in the intact area or in the area displaying dc - associated tissue alterations (i.e., disorganized collagen bundles and/or local detachment of lecs from the interstitial extracellular matrix). finally, the particle number was normalized to the corresponding endothelial length. the bulk secretion of ccl21 was measured by analyzing mcherry intensities in the culture supernatant. to measure the amounts of ccl21-mcherry, ccl21n - mcherry, and ccl21c - mcherry (figure 2b), culture supernatant was collected 48 hr after full confluency of the lec culture and the last media change. for the experiments shown in figure 3, we used ccl21c - mcherry because it has low affinity to culture substrate (figure s2a), and the changes in secretion should be immediately reflected in the mcherry intensity in the supernatant. here, culture supernatant was collected subsequent to 6 5 m ionomycin (molecular probes), 4 hr after washout of 1 hr 10 m bapta - am (life technologies, b6769) treatment or corresponding control (dmso) treatments. results were normalized to the background fluorescence of the mv2 culture media. for the information on measurement and quantification (figure 2b), ionomycin (final concentration 5 m, molecular probes) was added to the ccl21-mcherry expressing lecs and imaging was started 10 s later whereas latrunculin b (500 ng / ml, merck millipore 428020) and nocodazole (500 nm, sigma, m1404) were added on the lecs 30 prior to imaging. averages of all the tracks / cell, which lasted for longer than 2.5 s, are shown in figure 3e. only events in which the vesicles were observed prior to the flash and vesicle - like signal was severely decreased or totally absent after the flash were quantified as a secretion event (figure 3d). activated dcs were centrifuged (300rcf, 5) and resuspended in mv2 (promocell) culture media. a total of 40,000 dcs (ccl21c - mcherry monolayers ; figures 4f and s2d ; movie s4) were added on 1.5 cm well of days 45 confluent lecs. due to more efficient transmigration of dcs on ccl21-mcherry monolayers in comparison to ccl21c - mcherry monolayers, the acquired movies were quantified for the number of dcs beneath the monolayer at 1 hr for ccl21-mcherry or at 3 hr for ccl21c - mcherry expressing lec monolayers (figure 4f ; movie s6). for quantification of transmigration efficiency on non - infected or ccl21-mcherry full - length or mutant construct expressing lec monolayers (figure s2d), a percentage of transmigrated dc of all dcs was determined. for the ca - sensor assays, fresh media, dc - conditioned media, or either tamra - labeled non - treated or mycalolide b - treated dcs were carefully added on lec monolayers treated for 50 with 10 m oregon green bapta - am. the time - lapse imaging was started after 10. for the cell population analysis (figure 4e), imaging lasted for 10 and the number of lecs displaying transient ca peaks was quantified from acquired videos. all the ca sensor experiments were carried out in the absence of serum or added growth factors. prism5 and prism6 software (graphpad softwares) was used to test the normality of the data (dagostino and pearson omnibus normality test or kolmogorov - smirnov normality test) and for subsequent parametrical t test (two - tailed with welch s correction) or non - parametrical mann - whitney tests. figure legends indicate number of independent experiments and biological replicates (n) used for statistical analysis. generation and labeling of dcs, ear sheet staining, preparation of transmission electron microscopy samples, construction of lentiviral expression plasmids, virus production, lec culture and infections, lec staining, microscopy setups, mechanical pushing of the lecs, image analysis, and identification of putative nuclear localizing signal in ccl21 are outlined in the supplemental experimental procedures.	summarytrafficking cells frequently transmigrate through epithelial and endothelial monolayers. how monolayers cooperate with the penetrating cells to support their transit is poorly understood. we studied dendritic cell (dc) entry into lymphatic capillaries as a model system for transendothelial migration. we find that the chemokine ccl21, which is the decisive guidance cue for intravasation, mainly localizes in the trans - golgi network and intracellular vesicles of lymphatic endothelial cells. upon dc transmigration, these golgi deposits disperse and ccl21 becomes extracellularly enriched at the sites of endothelial cell - cell junctions. when we reconstitute the transmigration process in vitro, we find that secretion of ccl21-positive vesicles is triggered by a dc contact - induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. altogether, our data demonstrate a chemokine - mediated feedback between dcs and lymphatic endothelium, which facilitates transendothelial migration.
restoring posterior teeth with resin - based composite materials continues to gain popularity among clinicians, and the demand for such aesthetic restorations is increasing. indeed, the most common aesthetic alternative to dental amalgam is resin composite. long - term studies have shown that the bond strength of resin - bonded dentin decreased over time due to collagen degradation within the hybrid layer [2, 3 ]. meiers and shook 1996 indicated that residual bacteria might proliferate from the smear layer beneath restorations. therefore, the adjunctive use of antibacterial solutions after cavity preparation may be considered a method to reduce the incidence of postoperative sensitivity by eliminating viable bacteria and their toxins from the restoration - tooth interface. chlorhexidine (chx) is widely used as an antimicrobial agent for disinfection before placement of restorations. chx acts as matrix metalloproteinase (mmp) inhibitor, so it has beneficial effects on the preservation of dentin bond strength. chx also minimizes the convective and evaporative water fluxes from the underlying dentin, thus enhancing the bonding capacity of the self - etch adhesive. sodium hypochlorite (naocl) is widely used as chemomechanical caries removal and in dentin bonding techniques, because of its antimicrobial and tissue dissolving properties. since the smear layer composition is similar to the originating tissue (50 volume % mineral and 30 volume % collagen), the application of (naocl) over the smear layer covered dentin would eliminate its collagen phase resulting in reduction in the smear layer compactness. this property enhanced the bonding of the self - etching adhesive as it might increase the diffusively of the acidic monomers, through water - filled channels between particles of smear layer enlarging them to reach and interact with the underlying dentin surface. it has been widely used to dissolve the mineral phase of dentin without altering the structure of dentin collagen. the purpose of this study was to evaluate the effect of disinfectant agent on shear bond strength between dentin and two types of resin composite and to evaluate the failure pattern. the null hypothesis was that disinfectant agent has no effect on bond strength between resin composites and dentin. eighty molars were used in this study ; a prior patient 's consent was obtained. approval of al - azhar university, faculty of oral and dental medicine, egypt (under number 456/2013), was also obtained. the inclusion criteria included teeth that needed to be extracted due to periodontitis, pericoronitis, and unerupted or impacted teeth. the exclusion criteria included teeth that were decayed or damaged during the extraction and also those teeth that were congenitally affected such as enamel hypoplasia or amelogenesis / dentinogenesis imperfecta. once the teeth were extracted, they were stored in distilled water at 4c and used within two months following extraction. before the study, all teeth were scaled and cleaned using pumice and rubber cups. the teeth, including the roots, were embedded inside a cylindrical - shaped mold filled with self - cured acrylic resin (acrostone dental factor, uk) till the cervical line with the occlusal plane being parallel to the floor. after completing the polymerization of the acrylic resin, the tooth in the set acrylic resin was removed from the mold and the occlusal enamel of the teeth was removed perpendicular to the long axis of teeth with a low - speed diamond disk saw (isomet ; buehler, lake bluff, il, usa) and then fissure bur was used to complete the preparation until 1 mm beyond the dentinoenamel junction. the specimens were divided into four main groups : a (n = 20), according to the proposed dentin surface pretreatment:(a1)a control group without pretreatment;(a2)pretreatment with chlorhexidine gluconate 2% (consepsis, ultradent, usa);(a3)pretreatment with naocl 4% (central drug house (p), new delhi, india);(a4)pretreatment with edta 19% (file - eze, ultradent, usa). a control group without pretreatment ; pretreatment with chlorhexidine gluconate 2% (consepsis, ultradent, usa) ; pretreatment with naocl 4% (central drug house (p), new delhi, india) ; pretreatment with edta 19% (file - eze, ultradent, usa). the disinfectant in every group was applied using a disposable brush tip, left undisturbed for 20 seconds, then rinsed with water for 10 seconds, and dried with absorbent paper. each main group was divided into two subgroups (n = 10) according to type of adhesive system:(b1)etch and rinse;(b2)self - etch adhesive. for subgroup (b1) the dentin surface of each specimen was etched with 37% phosphoric acid (condicionador, dentsply, brazil) for 15 seconds, rinsed with water for 20 seconds, and dried with absorbent paper. then the self - priming adhesive (prime & bond 2.1, dentsply, brazil) was applied using a fully saturated brush tip and lightly air - dried for 5 seconds and light - cured for 20 seconds with halogen light curing unit (cromalux - e mega - physics dental rastatt, germany) with a light output of 600 mw / cm. for subgroup (b2) (adper easy one 3 m- espe, ag seefeld, germany) self - etching adhesive was applied and left undisturbed for 20 seconds, lightly air - thinned for 5 seconds, and light - cured for 20 seconds by the same curing unit. each subgroup was further divided into two subgroups (n = 5) according to the type of resin composite:(c1)microhybrid resin composite (tph, dentsply, brazil);(c2)nanohybrid resin composite (tetric evoceram, ivoclar - vivadent, schaan, liechtenstein). microhybrid resin composite (tph, dentsply, brazil) ; nanohybrid resin composite (tetric evoceram, ivoclar - vivadent, schaan, liechtenstein). either type of composite was carefully applied to the treated dentin surface by placing the material into cylindrical - shaped split teflon mold with an internal diameter of 3 mm and a height of 3 mm. composite was placed incrementally in 2 layers, 1.5 mm each ; each layer was light - cured for 20 seconds with the previous light curing unit. all samples were individually and horizontally mounted on a computer controlled materials testing machine (model lrx - plus ; lloyd instruments ltd., fare ham, uk) with a load cell of 5 kn and data were recorded using computer software (hexogen - mt ; lloyd instruments). samples were secured to the lower fixed compartment of testing machine by tightening screws (figure 1). shearing test was performed by compressive mode of load applied at resin - tooth interface using a mono - beveled chisel shaped metallic rod attached to the upper movable compartment of testing machine traveling at cross - head speed of 0.5 mm / min. both surfaces of each fractured specimen were examined using usb digital microscope (scope capture digital microscope, guangdong, china) at 30x magnification and were photographed using image analysis software (scope capture 1.1.1.1. ltd.) in order to determine the mode of failure. statistical analysis. one - way anova followed by tukey 's post - hoc test were performed to detect significance between groups. for the control group, the highest mean shear bond strength (11.3 2.2 mpa) was recorded for nanohybrid composite bonded to dentin specimen using etch - and - rinse adhesive, while the lowest mean shear bond strength (7.8 2.7 mpa) was recorded for microhybrid composite bonded to dentin specimen using self - etch adhesive. shear bond strength for specimens treated with chlorhexidine ranged from (9.2 2.2 mpa) for nanohybrid composite bonded to dentin using etch - and - rinse adhesive to (14.3 1.5 mpa) for microhybrid composite bonded to dentin using etch - and - rinse adhesive. for (naocl) group, specimens treated with naocl and bonded to nanohybrid composite resin using self - etch adhesive showed the highest mean shear bond strength (14.6 1.5 mpa) while those bonded to microhybrid composite using etch - and - rinse adhesive recorded the lowest mean shear bond strength (10.3 1.5). for edta group, microhybrid composite bonded to dentin specimens using self - etch adhesive showed the highest mean shear bond strength (16.3 1.9 mpa), while the lowest mean shear bond strength (8.3 0.9) was recorded for those specimens bonded to nanohybrid composite using etch - and - rinse adhesive. regardless of composite type or bonding agent, totally it was found that edta treated dentin recorded the highest statistically significant (p < 0.05) mean shear bond strength (12.9 1.5 mpa) followed by naocl treated dentin (12 1.5 mpa) and then chlorhexidine treated dentin (11.5 0.3 mpa), while the control group showed the lowest statistically significant shear bond strength (9.5 0.6 mpa) (figure 3). regardless of composite or disinfectant, it was found that self - etch bonding agent recorded higher shear bond strength mean value (12 2.1 mpa) than total etch bonding agent (10.9 1.8 mpa) (figure 4). regardless of disinfectant or bonding agent, it was found that microhybrid composite recorded higher shear bond strength mean value (11.8 2.6 mpa) than nanohybrid composite (11.1 1.6 mpa) (figure 5). regarding mode of failure, adhesive mode of failure represented 80% with 20% mixed failure in the control group (no pretreatment) with self - etch adhesive using microhybrid composites, chlorhexidine group with etch - and - rinse adhesive using nanohybrid composite, edta group with etch - and - rinse adhesive using both microhybrid and nanohybrid composites, and with self - etch adhesive using nanohybrid composite. adhesive mode of failure represented 60% with 40% mixed failure in chlorhexidine group with etch - and - rinse adhesive using microhybrid composite, naocl group with etch - and - rinse adhesive using both microhybrid and nanohybrid composites, and with self - etch adhesive using nanohybrid composite. however, adhesive failure represented only 20% with 80% mixed failure only in naocl group with self - etch adhesive using microhybrid composite. furthermore, cohesive failure only demonstrated 20% in chlorhexidine group with self - etch adhesive using nanohybrid composite. failure mode percentages of all groups are illustrated in figure 6 and mixed failure mode is shown in figure 7. regardless of composite type or bonding agent, it was found that edta treated dentin recorded the highest shear bond strength followed by naocl treated dentin and then chlorhexidine treated dentin while the control group showed the lowest shear bond strength. our result is in agreement with previous studies [911 ] which attributed the improvement in bond strength to the removal of the smear layer, which prevents direct contact of the self - etching adhesive with dentin ; consequently, removal of the smear layer facilitates the formation of a stronger and more homogeneous hybrid layer, while other studies found that treatment of dentin with edta produced no significant difference in bond strength compared to that produced with groups which were etched with phosphoric acid [12, 13 ]. this disagreement may be attributed to their use of edta as etching material instead of the phosphoric acid, so they used edta in high concentration for long durations while in the current study edta was used as a cavity disinfectant in lower concentration before dentin etching. sodium hypochlorite application prior to acid etching significantly increased the bond strength of both adhesive systems used. this result is in agreement with the result of previous studies [14, 15 ]. they attributed the increase bond strength to the elimination of collagen layer which was removed by application of naocl leading to a better penetration of the adhesive into intertubular dentin. this increase in bond strength may be also due to removal of smear layer by naocl. complete removal of smear layer might enhance the bonding to dentin as it facilitates the penetration of resin monomer leading to complete infiltration of the demineralized layer by numerous resin tags. on the other hand, it was reported that sodium hypochlorite significantly decreased the bond strength to dentin, which is in contrast to our results. they showed that naocl damages the organic component of dentin ; therefore, organic monomers do not sufficiently penetrate into the demineralized dentin, resulting in a lack of proper bond strength, while another study reported that sodium hypochlorite does not influence the bond strength to dentin. the disagreement of the result of those studies with the present study may be attributed to differences in sample preparation methods, application mode, and time. in this study 4% sodium hypochlorite was used for 20 sec prior to dentin etching, while the previous studies used naocl in different concentrations after dentin etching. these results are inconsistent with certain studies which showed that a chx cavity disinfectant had an adverse effect and produced significantly lower bond strengths [18, 19 ]. on the other hand, some studies reported that chx had no influence on the shear bond strength to dentin [20, 21 ]. the disagreement in the results of those studies with the present study may be attributed to differences in modes of use of chx : before etching, after etching, rinsing off, or not rinsing, also the form of material (gel or solution) and time of application. using of a chx before etching was shown to not to affect bonding to dentin, however, reduced dentin bond strengths usually when a chx was used after etching, but rinsing the chx off before bonding produced bond strengths that were similar to no - cleanser controls. rinsing away chx prior to bonding will most likely prevent undesired material interactions. among several factors that may interfere with the quality of bonding, the type of adhesive systems used is of great importance. it was found that etch - and - rinse adhesive recorded statistically nonsignificant higher shear bond strength mean value than self - etching adhesive. the obtained data is consistent with previous studies which reported that the dentin bond strength of self - etching adhesives was comparable to that of the etch - and - rinse systems [14, 23 ]. one of the advantages of self - etching adhesives is that dentin conditioning and priming occur simultaneously, resulting in the formation of a strong void - free hybrid layer, while other studies [24, 25 ] found that etch - and - rinse adhesive showed higher bond strength than self - etch adhesives. in contrast, giriyappa and chandra, 2008, showed that the self - etching primer had higher mean shear bond strength than total etch adhesive. in this study, groups treated with disinfectants recorded statistically significant higher shear bond strength for self - etch bonding agent than etch - and - rinse bonding agent. since self - etching adhesives have higher ph values than the phosphoric acid used and are not rinsed away, the smear layer or its components are incorporated into the bonded layers. for strong self - etching adhesives, the smear layer and smear plugs should be dissolved to overcome the main problems during using self - etching adhesives. so in the current study, the increased bond strength of self - etch was attributed to removal of the smear layer and smear plugs by the effect of used edta, naocl, or chx. the lowest shear bond strength was recorded for microhybrid composite bonded to dentin specimens with self - etch adhesive without any pretreatment (control), while the highest shear bond strength in the study was for microhybrid composite bonded to dentin specimen with self - etch adhesive treated with edta. this may be attributed to the self - etch which has the problem of the smear layer and smear plugs that interfere with bonding. the effect of dentin pretreatment with edta on shear bond strength of the other group may be due to the complete removal of smear layer. however, bond strength is multifactorial in nature, having many variables affecting it. therefore, further studies might be of importance in determining the effect of using edta, sodium hypochlorite, or chx prior to the application of the different adhesives in the market. all groups showed percentage of adhesive failures but we observed that the failure mode was predominantly adhesive for control group with increased percentage of mixed failure for groups of disinfectants. this result is in agreement with other studies [18, 28, 29 ]. on the other hand, our result is in disagreement with the result of another study, because the failure mode was predominantly mixed. in control groups, there was no difference between etch - and - rinse adhesive and self - etch adhesive, which is in accordance with certain studies which found that failure mode of both adhesives was mostly adhesive. the increased percentage of mixed failure on groups of disinfectants was attributed to the increased shear bond strength which clearly was reflected by the mode of failure of the bonding system. they reported that the major mode of failure in specimens with low bond strengths was adhesive failure, while cohesive fractures in dentin or composite were seen at higher bond strength. the surface treatment of dentin before bonding positively affects the shear bond strength between resin composite and dentin especially with self - etch adhesive.	objective. the aim of this study was to evaluate the effect of different disinfectant agents on bond strength of two types of resin composite materials. methods. a total of 80 sound posterior teeth were used. they were divided into four groups (n = 20) according to the dentin surface pretreatment (no treatment, chlorhexidine gluconate 2%, sodium hypochlorite 4%, and edta 19%). each group was divided into two subgroups according to the type of adhesive (prime and bond 2.1 and adper easy one). each subgroup was further divided into two subgroups according to the type of resin composite (tph spectrum and tetric evoceram). shear bond strength between dentin and resin composite was measured using universal testing machine. data collected were statistically analyzed by t - test and one - way anova followed by tukey 's post hoc test. results. it was found that dentin treated with edta recorded the highest shear bond strength values followed by sodium hypochlorite and then chlorhexidine groups while the control group showed the lowest shear bond strength. conclusions. the surface treatment of dentin before bonding application has a great effect on shear bond strength between resin composite and dentin surface.
goodpasture s syndrome or anti - glomerular basal membrane (anti - gbm) disease is a rare disorder first described by earnest goodpasture in 1919. the disorder is characterized by formation of antibodies against non - collagenous domain of the 3 chain of type iv collagen of the gbm. combination of rapidly progressive glomerulonephritis and pulmonary haemorrhage caused by this anti - gbm formation is known as goodpasture s syndrome. pulmonary haemorrhage is present in adult cases, yet children can develop isolated renal disease without pulmonary involvement. pulmonary involvement with goodpasture s syndrome this disease is especially rare in patients under the age of 5 and can lead to poor patient or renal outcome. delay in diagnosis and initiating treatment, together with technical difficulties in treatment, can result in failure to halt the progress of the disease and in permanent loss of renal function. we describe a case of goodpasture s syndrome in a 3-year - old child with successful rescue of renal function and amelioration of anti - gbm antibody over the next 3 years with continued mild proteinuria and hypertension from the initial event. according to the literature, only one previous case has been reported with successful renal rescue. to our knowledge, our patient is the youngest to survive isolated goodpasture s syndrome without permanent loss of renal function but does have adverse prognostic indicators. a previously healthy 3-year - old girl was admitted to our hospital with anaemia, gastroenteritis, fever, tiredness and anasarca for 3 weeks. her haemoglobin was 6.3 g / dl with a haematocrit of 20.3%, and her serum creatinine was 114.92 the 24-h catheterized urine collection showed 598 mg of protein and a creatinine clearance of 23 ml / min/1.73 m. her c3 was 209 mg / dl, c4 was 35.6 mg / dl and aso was 400 iu / ml (normal < 100 iu / ml). a presumptive diagnosis of glomerulonephritis was made, and as her creatinine continued to rise, a kidney biopsy was performed. six out of eight glomeruli had severe fibrinoid necrosis and cellular crescent formation (figure 1). there was no interstitial fibrosis or tubular atrophy, and the vessels were unremarkable with no features of vasculitis. the immunofluorescence examination showed a classical global and bright linear capillary wall staining for igg (figure 2). light microscopy : kidney biopsy of a 3-year - old girl with anti - gbm disease. immunofluorescence : kidney biopsy of a 3-year - old girl with anti - gbm disease. her anti - gbm antibody level was 352 au / ml (normal < 19 au / ml) (figure 3). the rest of the serology including ana, anti - dna and anca was negative on multiple occasions. based on the kidney biopsy and anti - gbm antibody level, a diagnosis of goodpasture s syndrome was made. the levels of anti - glomerular basement membrane antibody, urinary protein excretion rate and renal function (creatinine clearance) in a 3-year - old girl with goodpasture s syndrome for initial 30 weeks. she was treated with intravenous (iv) steroid pulse (three doses) and plasmapheresis. four weeks after admission, when her plasmapheresis was discontinued, her anti - gbm antibody levels rebounded, necessitating ongoing plasmapheresis treatment for a total duration of 7 weeks (figure 3). at 7 weeks, when her anti - gbm antibody level was 28 au / ml (normal < 19 au / ml), monthly intravenous infusions of cyclophosphamide treatment was initiated for the next 6 months followed by increasing intervals. oral prednisolone was continued at 30 mg / day (2 mg / kg / day) for the next 3 months before tapering. it required 4 months of aggressive immunosuppression to rescue and maintain her creatinine clearance in the normal range (figure 3). her anti - gbm antibody levels were abnormal for 7 months after the onset of disease but were detectable up to a year. as her creatinine clearance improved, she had a spike in proteinuria to 1.8 g / day (figure 3). she continued to remain hypertensive from the onset of illness for which she was treated with enalapril 3 mg / day (0.2 mg / kg / day). at 1 year, due to persistent abnormal proteinuria and hypertension the biopsy showed anti - gbm - mediated focal sclerosing glomerulonephritis with focal global glomerulosclerosis in 7 of the 64 glomeruli on biopsy. five of the remaining glomeruli had segmental sclerosis (figure 4), and the others were unremarkable. there was 10% interstitial fibrosis with tubular atrophy, but the blood vessels were unremarkable. light microscopy of repeat kidney biopsy a year after presentation of anti - gbm disease. electron microscopy of repeat kidney biopsy a year after presentation of anti - gbm disease. arrow shows immune deposits. following the second kidney biopsy, the decision was made to continue cyclophosphamide infusions at six monthly intervals with the addition of mycophenolate mofetil (mmc) suspension at the dose of 200 mg orally twice a day (20 mg / kg / day). prednisolone taper and enalapril were continued. at 3 years after initial presentation her creatinine clearance is 88.4 ml / min/1.73 m, proteinuria is 408 mg / day and anti - gbm antibody is still undetectable. she was treated with intravenous (iv) steroid pulse (three doses) and plasmapheresis. four weeks after admission, when her plasmapheresis was discontinued, her anti - gbm antibody levels rebounded, necessitating ongoing plasmapheresis treatment for a total duration of 7 weeks (figure 3). at 7 weeks, when her anti - gbm antibody level was 28 au / ml (normal < 19 au / ml), monthly intravenous infusions of cyclophosphamide treatment was initiated for the next 6 months followed by increasing intervals. oral prednisolone was continued at 30 mg / day (2 mg / kg / day) for the next 3 months before tapering. it required 4 months of aggressive immunosuppression to rescue and maintain her creatinine clearance in the normal range (figure 3). her anti - gbm antibody levels were abnormal for 7 months after the onset of disease but were detectable up to a year. as her creatinine clearance improved, she had a spike in proteinuria to 1.8 g / day (figure 3). she continued to remain hypertensive from the onset of illness for which she was treated with enalapril 3 mg / day (0.2 mg / kg / day). at 1 year, due to persistent abnormal proteinuria and hypertension, the kidney biopsy was repeated. the biopsy showed anti - gbm - mediated focal sclerosing glomerulonephritis with focal global glomerulosclerosis in 7 of the 64 glomeruli on biopsy. five of the remaining glomeruli had segmental sclerosis (figure 4), and the others were unremarkable. there was 10% interstitial fibrosis with tubular atrophy, but the blood vessels were unremarkable. light microscopy of repeat kidney biopsy a year after presentation of anti - gbm disease. electron microscopy of repeat kidney biopsy a year after presentation of anti - gbm disease. arrow shows immune deposits. following the second kidney biopsy, the decision was made to continue cyclophosphamide infusions at six monthly intervals with the addition of mycophenolate mofetil (mmc) suspension at the dose of 200 mg orally twice a day (20 mg / kg / day). at 3 years after initial presentation, the patient is asymptomatic but has controlled hypertension and proteinuria. her creatinine clearance is 88.4 ml / min/1.73 m, proteinuria is 408 mg / day and anti - gbm antibody is still undetectable. we report a unique case of goodpasture s syndrome in a young child in whom we successfully rescued renal function with aggressive immunosuppression. her ongoing mild proteinuria, hypertension and development of focal sclerosing glomerulonephritis in subsequent kidney biopsy a year later forebode a guarded prognosis. at 3 years, since the onset of the disease, she has near normal renal function. there are only a handful of cases of anti - gbm disease developing in extremely young children. one possibility may be the difficulty of diagnosing mild cases which can be mistaken for post - streptococcal glomerulonephritis (psgn) that classically occurs at a similar age. there are distinguishing characteristics such as low c3, normal c4 and absence of rapidly progressive glomerulonephritis that distinguish psgn from anti - gbm antibody disease. in our case, the patient had exposure to streptococci based on positive aso, but because of normal c3 and c4, and rising creatinine, we performed a kidney biopsy early in the illness and were able to successfully diagnose and ultimately rescue renal function. on the second kidney biopsy, the patient developed focal sclerosing glomerulonephritis presumably due to anti - gbm disease, which has not been reported in younger children. despite aggressive immunosuppression and normalization of anti - gbm antibody in the serum 6 months after the onset of the disease, the kidney biopsy showed deposition of anti - gbm antibody a year after the onset of the disease. a 6-year - old boy has been described in literature to have presented with an anti - gbm - mediated crescenteric glomerulonephritis, and following plasmapheresis and immunosuppressant therapy had a favourable outcome with normal renal function two and a half years later. most cases of anti - gbm disease in paediatric patients have acute presentations with diffuse glomerular disease and severe fibrinoid necrosis along with cellular crescent formation with subsequent glomerular scarring and severe chronic disease leading to end - stage renal disease. being that there is direct antibody formation to the non - collagen domain of type iv collagen, the involvement is typically diffuse, with all glomerular lesions being of the same age. an 11-month - old child with anti - gbm disease showed diffuse glomerular involvement with necrosis and cellular crescent formation. despite plasmapheresis and extensive immunosuppressive therapy, the development of diffuse involvement of glomeruli with necrosis and cellular crescent formation due to anti - gbm disease in older children, teenagers and young adults from ages 824 years has been described in literature [2,6,912 ]. renal outcome in these series of papers has been often unfavourable despite aggressive immunosuppression. even though our patient presented with anti - gbm glomerulonephritis with diffuse glomerular necrosis and crescent formation, on repeat kidney biopsy, she had focal glomerular sclerosis with mild chronic tubulointerstitial injury. the recovery of renal function with aggressive immunosuppression was consistent with mild chronicity on follow - up biopsy. it had been hypothesized that full basement membrane expression of type iv collagen does not occur until 3 years of age and that the full expression of the 3 antigen is at 3 years of age and beyond. some researchers have argued that between the ages of 3 months and 3 years, the 3 antigen is in a transition period that may lead to decreased anti - genicity prior to age 3 years. in alport syndrome, the lack of full expression of 3 antigen prior to age 3 years could potentially lead to milder anti - gbm disease that can be mistaken for psgn as mentioned above. others have postulated that young patients that develop anti - gbm glomerulonephritis might have precocious development of mature basement membrane formation or an autoimmune response to an unknown basement membrane antigen that is not age - dependent. the initiating stimulus (antigen) that leads to however, hypotheses include exposure to viral and bacterial infections, unknown toxins, or induced glomerular trauma such as in lithotripsy. hla type dr15 and dr4 are common in patients with anti - gbm disease with or without pulmonary involvement. in conclusion, anti - gbm disease, even in the absence of pulmonary involvement, should be considered in the differential diagnosis of rapidly progressive glomerulonephritis even at a young age. unfavourable renal outcome is not uniform, and early diagnosis with aggressive long - term immunosuppression can lead to favourable renal outcome.	goodpasture s syndrome has been documented in only a handful of children under the age of four. we describe a 3-year - old girl presenting with anaemia and renal failure whose kidney biopsy showed anti - glomerular basement membrane (gbm) disease. she was treated aggressively with pulse steroids, plasmapheresis and monthly infusions of cyclophosphamide. after months of aggressive immunosuppression, her renal function normalized, and her anti - gbm antibody disappeared. a year after the onset, she underwent a second kidney biopsy for persistent proteinuria and hypertension that surprisingly showed focal sclerosing glomerulonephritis, an unreported finding at this age. the biopsy showed deposition of antibody on the gbm despite the fact that anti - gbm antibody had normalized in the serum 5 months earlier. mycophenolate mofetil was added to the immunosuppression at that point. at her 3-year follow - up, creatinine clearance was 88.4 ml / min/1.73 m2, proteinuria was 408 mg / day and blood pressure was controlled with enalapril 0.2 mg / kg / day. she has not had a relapse or abnormal anti - gbm antibody for 30 months, but her renal prognosis remains guarded. to our knowledge, this is the youngest patient to have a successful rescue of renal function after isolated goodpasture s syndrome.
all reagents and solvents were obtained from commercial suppliers and were used as received without further purification. nmr spectra were recorded on a 400 mhz (h), 100 mhz (c) nmr spectrometer at 25 c. chemical shifts () are reported in parts per million referenced to the nmr solvent residual peak, and coupling constants (j) are reported in hertz. all of the reactions were monitored using tlc and lc ms (conducted using an ion - trap mass spectrometer system coupled with an hplc system). melting points were acquired in triplicate using an automatic melting point instrument and are reported as ranges. ir spectra were recorded on an ft - ir spectrometer as a neat oil or solid. a 500 ml three - neck flask with a stir bar was oven - dried and charged with isopropyl methyl ketone (80 g, 79.8 mmol) dissolved in meoh (150 ml). br2 (140 g, 87.8 mmol) was added dropwise to the reaction mixture over a period of 30 min, after which time the mixture was allowed to stir for 3 h. water (200 ml) was added, and the reaction mixture was allowed to stir overnight. the contents were transferred into a separatory funnel containing water (600 ml), and this solution was extracted with ether (3 250 ml). the collected organic layers were washed with water (3 250 ml), nahco3 (1 250 ml), and brine (1 250 ml), dried over na2so4, filtered, and evaporated to give 10 as a pale - yellow oil (139 g, 97%). h nmr (cdcl3, 400 mhz) 3.86 (s, 2h), 2.51 (d, j = 7.0 hz, 2h), 2.202.11 (m, 1h), 0.92 (d, j = 6.7 hz, 6h). although there was a small amount (5%) of 2 bromide 11 present, the crude material was used directly in the next step. a mechanical stirrer was attached to a 1000 ml three - neck round - bottom flask. the flask was then charged with pph3 (250 g, 954 mmol), to which was added ethyl acetate (300 ml). bromomethyl acetate (145 g, 947 mmol) was dissolved in 200 ml of ethyl acetate, and this solution was slowly added to the reaction mixture. after the addition was complete, the reaction mixture was allowed to stir overnight. the fluffy white solid was collected by filtration, washed with ether, and air - dried overnight to give 380 g of compound. the white solid thus obtained was transferred to a 1000 ml beaker, to which were added naoh solution (1 n, 300 ml) and water (300 ml). the resulting slurry was stirred for 20 min, and then the contents were transferred to a separatory funnel. after extraction with ch2cl2, the combined organic layers were washed with water and brine, dried over na2so4, filtered, and evaporated to give white crystalline solid 13 (301 g, 96% yield). h nmr (cdcl3, 400 mhz) 7.487.65 (m, 15h), 3.60 (br s, 3h), 2.98 (br s, 1h). to an oven - dried 2 l three - neck flask were added 13 (224 g, 670 mmol) and anhydrous toluene (750 ml). to the flask ketone 10 (60 g, 335 mmol) dissolved in anhydrous toluene (100 ml) was charged into the addition funnel and then added to the reaction mixture dropwise. the reaction mixture was heated at 90 c for 3 h to give a thick yellow suspension, which was then cooled and filtered. to the filtrate was added methyl bromoacetate (51 g, 335 mmol), and the mixture was stirred at 90 c for 2 h. once again the reaction mixture was cooled to give a precipitate, which was filtered, washed with cold toluene, and then evaporated to give a crude oil that upon purification by flash chromatography using hexane / ethyl acetate (2:1) as the mobile phase gave olefin 5 (25 g, 38% yield). h nmr (cdcl3, 400 mhz) 7.06 (d, j = 16.0 hz, 1h), 6.66 (d, j = 16.0 hz, 1h), 3.81 (s, 3h), 2.50 (d, j = 7.0 hz, 2h), 2.242.14 (m, 1h), 0.94 (d, j = 7.0 hz, 6h) ; c nmr (cdcl3, 100 mhz) 199.5, 166.1, 139.8, 130.2, 52.3, 50.5, 24.7, 22.5 ; ft - ir (neat) 2957.7, 2873.8, 1729.5, 1698.5, 1466.8, 1436.7, 1368.7, 1298.5, 1272.5, 1196.6, 1171.5, 1113.8, 1062.7, 1027.7, 979.6, 857.8, 702.8 cm. to an oven - dried 500 ml three - neck flask with a stir bar were attached a reflux condenser and an addition funnel. the assembly was flushed with argon for 15 min and charged with vinylmagnesium chloride (199 ml, 1.6 m in thf, 320 mmol). to this was added anhydrous et2o (100 ml), and the reaction mixture was cooled in an ice bath for 30 min. isovaleraldehyde (25 ml, 290 mmol) dissolved in et2o (50 ml) was added dropwise to the reaction mixture under a positive flow of argon with constant stirring over a period of 40 min. the reaction mixture was allowed to stir overnight with gradual warming to room temperature. after confirmation of reaction completion by tlc analysis, the reaction mixture was quenched by adding saturated aqueous nh4cl (100 ml) and then transferred to a separatory funnel. extraction with et2o, washing of the organic layers with water and brine, drying over na2so4, filtration, and evaporation of solvents gave alcohol 16 (25 g, 76% crude) as a pale - yellow oil. h nmr (cdcl3, 400 mhz) 5.905.81 (m, 1h), 5.22 (td, j = 17.2, 1.4 hz, 1h), 5.08 (td, j = 10.4, 1.3 hz, 1h), 4.194.14 (br m, 1h), 1.791.69 (m, 1h), 1.64 (br s, 1h), 1.491.43 (m, 1h), 1.351.28 (m, 1h), 0.92 (dd, j = 6.6, 2.8 hz, 6h) ; c nmr (cdcl3, 100 mhz) 141.6, 114.3, 71.5, 46.2, 24.5, 23.0, 22.3 ; ft - ir (neat) 3338.9, 2955.7, 2925.8, 2870.8, 1644.9, 1468.8, 1423.8, 1384.8, 1367.8, 1308.9, 1152.8, 1091.8, 1055.8, 1016.8, 988.6, 918.6, 841.8, 661.8 cm. note : alcohol 16 is appreciably volatile, so care must be taken while evaporating the solvents after workup. a round - bottom flask with a stir bar was oven - dried and then flushed with argon for 10 min. methyl acrylate (5 equiv), grubbs second - generation catalyst (0.005 equiv), and cui (0.006 equiv) were charged into the flask under a positive flow of argon, and to this mixture was added anhydrous et2o (1 m). argon was bubbled through the reaction mixture for 15 min while the assembly was set up for reflux using an oil bath. after 10 min, alcohol 16 (1 equiv) was added, and the mixture was allowed to reflux for 5 h. the reaction was monitored by tlc (2:1 hexanes / etoac ; kmno4 staining). after completion of the reaction, the solvents were evaporated, and the crude mixture was purified by flash chromatography using a hexane / etoac (2:1) solvent system. the pure fractions were collected together, evaporated, and dried under vacuum to give alcohol 18 (75% yield) as a dark oil. h nmr (cdcl3, 400 mhz) 6.96 (dd, j = 15.7, 5.0 hz, 1h), 6.04 (dd, j = 15.6, 1.6 hz, 1h), 4.394.34 (br m, 1h), 3.75 (s, 3h), 1.79 (obscured d, 2h), 1.77 (d, j = 5.2 hz, 1h), 1.541.46 (m, 1h), 1.411.34 (m, 1h), 0.95 (d, j = 7.0 hz, 6h) ; c nmr (cdcl3, 100 mhz) 167.0, 150.9, 119.4, 69.4, 51.7, 45.7, 24.5, 23.1, 22.1 ; ft - ir (neat) 3441.9, 2955.7, 2870.8, 1724.6, 1704.5, 1658.7, 1467.8, 1436.7, 1385.8, 1367.8, 1304.6, 1276.6, 1194.7, 1168.5, 1078.7, 1035.7, 983.6, 926.8, 863.8, 840.8, 714.8 cm. mno2 (106 g, 1.22 mol) was heated in an oven at 100 c overnight and then added to a flask containing 11 (30 g, 175 mmol) in ch2cl2 (200 ml). the reaction mixture was allowed to stir for 10 h, after which conversion was complete by tlc analysis. the solvents were evaporated to give pure product 5 (23 g, 79% yield) as a pale - yellow oil. the h nmr, c nmr, and ft - ir data matched those listed above for the material prepared by the previous method. an oven - dried 100 ml flask with a stir bar was flushed with argon for 15 min and charged with vinylmagnesium chloride (18 ml, 1.6 m in thf, 28 mmol). to this solution was added anhydrous et2o (30 ml), and the reaction mixture was cooled in an ice bath for 30 min. aldehyde (25 mmol) dissolved in et2o (10 ml) was added dropwise to the reaction mixture under a positive flow of argon with constant stirring over a period of 5 min. the reaction mixture was allowed to stir overnight with eventual warming to room temperature. after confirmation of reaction completion by tlc analysis, the reaction mixture was quenched by adding saturated aqueous nh4cl (100 ml) and then transferred to a separatory funnel. extraction with et2o, washing of the organic layers with water and brine, drying over na2so4, filtration, and evaporation of solvents gave the desired alcohol, which was purified by flash chromatography using an appropriate hexane / etoac gradient. colorless oil (2.1 g, 58% yield) ; h nmr (cdcl3, 400 mhz) 7.367.33 (m, 2h), 7.287.25 (m, 3h), 6.005.92 (m, 1h), 5.27 (td, j = 17.2, 1.4 hz, 1h), 5.16 (td, j = 10.5, 1.3 hz, 1h), 4.404.35 (br m, 1h), 2.932.88 (m, 1h), 2.842.79 (m, 1h) ; c nmr (cdcl3, 100 mhz) 140.1, 137.7, 129.6, 128.5, 126.6, 115.0, 73.7, 43.8 ; ft - ir (neat) 3376.8, 3063.9, 3027.9, 2919.9, 1644.9, 1603.9, 1495.8, 1454.8, 1424.8, 1117.8, 1076.8, 1029.6, 990.6, 921.6, 854.8, 744.5, 697.3, 668.6 cm. brown oil (1.95 g, 68% yield) ; h nmr (cdcl3, 400 mhz) 7.42 (d, j = 8.4 hz, 2h), 7.19 (d, j = 8.2 hz, 2h), 5.985.90 (m, 1h), 5.29 (td, j = 17.1, 1.2 hz, 1h), 5.16 (td, j = 10.3, 1.2 hz, 1h), 5.10 (br, 1h), 2.04 (br s, 1h) ; c nmr (cdcl3, 100 mhz) 141.5, 139.8, 131.6, 128.0, 1221.6, 115.7, 74.7 ; ft - ir (neat) 3342.8, 2981.9, 2871.9, 1640.9, 1591.8, 1485.6, 1401.7, 1241.8, 1191.8, 1100.8, 1070.6, 1033.7, 1009.4, 986.5, 924.4, 815.5, 794.5, 719.6 cm. for other metathesis reactions, the same procedure as used for the synthesis of alcohol 18 was followed using the appropriate acrylate and olefin reactants. pale - yellow oil (0.300 g, 87% yield) ; h nmr (cdcl3, 400 mhz) 7.377.33 (m, 2h), 7.307.23 (m, 3h), 7.03 (dd, j = 15.7, 4.1 hz, 1h), 6.09 (dd, j = 15.7, 1.8 hz, 1h), 4.574.54 (br m, 1h), 3.76 (s, 1h), 2.992.95 (m, 1h), 2.842.78 (m, 1h) ; c nmr (cdcl3, 100 mhz) 166.9, 149.1, 136.7, 129.5, 128.7, 127.0, 120.2, 71.7, 51.7, 43.2 ; ft - ir (neat) 3446.9, 3028.9, 2950.9, 1704.5, 1658.7, 1602.9, 1495.8, 1454.8, 1435.7, 1309.6, 1273.5, 1195.6, 1166.5, 1100.6, 1076.7, 1030.6, 983.6, 926.7, 855.7, 823.8, 746.6, 698.3 cm. data for the e isomer : h nmr (cdcl3, 400 mhz) 7.287.21 (m, 6h), 7.187.11 (m, 4h), 6.95 (dd, j = 15.6, 4.5 hz, 1h), 6.02 (dd, j = 15.6, 1.7 hz, 1h), 5.09 (s, 2h), 4.464.40 (br m, 1h), 2.872.83 (m, 1h), 2.712.65 (m, 1h) ; c nmr (cdcl3, 100 mhz) 166.2, 149.5, 136.7, 135.9, 129.5, 128.7, 128.5, 128.2, 128.1, 127.0, 120.3, 71.8, 66.3, 43.2 ; ft - ir (neat) 3421.9, 3030.9, 2942.9, 1715.5, 1655.7, 1603.9, 1454.7, 1377.8, 1302.6, 1266.5, 1159.4, 1100.6, 1077.7, 1027.6, 982.6, 908.8, 855.8, 735.5, 695.2 cm ; hrms (es - tof) m / z calcd for c18h18o3na [m + na ] 305.1154, found 305.1154. off - white solid (0.326 g, 78% yield) ; mp 65.071.9 c ; h nmr (cdcl3, 400 mhz) 7.377.33 (m, 2h), 7.297.24 (m, 3h), 6.92 (dd, j = 15.6, 4.7 hz, 1h), 6.00 (dd, j = 15.6, 1.7 hz, 1h), 4.544.49 (br m, 1h), 2.992.94 (m, 1h), 2.822.76 (m, 1h), 1.51 (s, 9h) ; c nmr (cdcl3, 100 mhz) 165.7, 147.6, 136.9, 129.5, 128.7, 126.9, 122.5, 80.5, 71.8, 43.3, 28.1 ; ft - ir (neat) 3486.8, 2977.8, 1693.5, 1656.6, 1602.8, 1493.9, 1454.8, 1391.8, 1367.7, 1326.7, 1297.6, 1252.7, 1213.7, 1151.6, 1111.6, 1071.8, 1011.7, 995.7, 981.7, 941.7, 863.7, 848.8, 746.7, 710.7, 698.4 cm ; hrms (es - tof) m / z calcd for c15h20o3na [m + na ] 271.1310, found 271.1310. brown oil (0.351 g, 91% yield) ; h nmr (cdcl3, 400 mhz) 7.50 (d, j = 8.5 hz, 2h), 7.21 (d, j = 8.4 hz, 2h), 7.00 (dd, j = 15.6, 4.9 hz, 1h), 6.15 (dd, j = 15.6, 1.7 hz, 1h), 5.30 (br d, j = 4.88 hz, 1h), 3.73 (s, 3h) ; c nmr (cdcl3, 100 mhz) 166.8, 148.3, 139.8, 131.9, 131.9, 128.2, 122.3, 120.1, 72.8, 51.8 ; ft - ir (neat) 3419.8, 2951.8, 1703.5, 1658.6, 1590.8, 1486.7, 1436.6, 1401.7, 1274.5, 1274.5, 1166.5, 1091.5, 1070.5, 1039.6, 1009.4, 980.5, 924.7, 879.8, 825.5, 761.7, 719.7 cm. pale - yellow solid (0.350 g, 86% yield) ; mp 63.565.7 c ; h nmr (cdcl3, 400 mhz) 7.50 (d, j = 8.6 hz, 2h), 7.387.33 (m, 5h), 7.23 (d, j = 8.1 hz, 2h), 7.04 (dd, j = 15.6, 5.0 hz, 1h), 6.20 (dd, j = 15.6, 1.7 hz, 1h), 5.35 (br t, j = 3.6 hz, 1h), 5.19 (s, 2h), 2.10 (d, j = 4.0 hz, 1h) ; c nmr (cdcl3, 100 mhz) 166.0, 148.3, 139.7, 135.7, 132.0, 128.5, 128.3, 128.2, 122.4, 120.4, 72.9, 66.4 ; ft - ir (neat) 3461.8, 2987.8, 1685.6, 1646.7, 1589.8, 1482.1, 1454.8, 1395.7, 1382.7, 1326.7, 1280.6, 1218.7, 1181.6, 1106.7, 1088.6, 1069.6, 1010.6, 998.6, 949.7, 825.7, 806.7, 767.7, 748.5, 698.5 cm ; hrms (es - tof) m / z calcd for c17h15bro3na [m + na ] 369.0102, found 369.0102. white solid (0.315 g, 85% yield) ; mp 51.053.4 c. data for the e isomer : h nmr (cdcl3, 400 mhz) 7.51 (d, j = 8.3 hz, 2h), 7.24 (d, j = 8.3 hz, 2h), 6.89 (dd, j = 15.6, 4.8 hz, 1h), 6.05 (dd, j = 15.8, 2.0 hz, 1h), 5.32 (br t, j = 4.6 hz, 1h), 2.10 (d, j = 3.4 hz, 1h), 1.48 (s, 9h) ; c nmr (cdcl3, 100 mhz) 165.5, 146.5, 140.0, 131.9, 128.2, 122.7, 122.2, 80.8, 72.9, 28.1 ; ft - ir (neat) 3440.8, 2982.8, 1683.7, 1652.8, 1588.9, 1482.8, 1454.8, 1393.8, 1365.7, 1340.7, 1315.8, 1299.8, 1257.7, 1234.7, 1149.5, 1092.7, 1066.7, 1045.7, 1007.6, 979.6, 822.5, 762.7 cm ; hrms (es - tof) m / z calcd for c14h17bro3na [m + na ] 335.0259, found 335.0259. to a dry 100 ml flask with a stir bar were added the dess martin periodinane reagent (1.2 equiv) and dichloromethane (1 m). to the mixture was added a solution of the allylic alcohol (1.0 equiv) in minimal dichloromethane. the reaction mixture was allowed to stir for 30 min and then monitored by tlc to confirm complete transformation of the alcohol to the ketone. following this, an equal volume of ether was added, and the mixture was transferred to a separatory funnel containing a saturated nahco3 solution. the organic layer was washed with na2s2o3 solution, saturated bicarbonate solution, water, and then brine. evaporation of the solvents gave the desired ketoester in nearly quantitative yield, free of detectable side products. pale - yellow oil (0.111 g, 94% yield) ; h nmr (cdcl3, 400 mhz) 7.377.29 (m, 2h), 7.30 (obscured d, 1h), 7.21 (obscured d, 2h), 7.12 (d, j = 16.1 hz, 1h), 6.74 (d, j = 16.1 hz, 1h), 3.92 (s, 2h), 3.79 (s, 3h) ; c nmr (cdcl3, 100 mhz) 196.5, 165.8, 138.6, 132.8, 131.1, 129.5, 128.9, 127.4, 52.4, 48.8 ; ft - ir (neat) 2952.8, 1723.5, 1687.5, 1603.8, 1496.7, 1454.7, 1438.6, 1414.8, 1348.7, 1310.5, 1256.4, 1213.6, 1199.5, 1170.4, 1077.6, 1029.7, 1011.6, 981.4, 940.7, 915.6, 858.6, 739.5, 700.4 cm. yellow oil (0.256 g, 99% yield) ; h nmr (cdcl3, 400 mhz) 7.437.37 (m, 5h), 7.357.29 (m, 3h), 7.217.20 (br, 2h), 7.15 (d, j = 15.9 hz, 1h), 6.68 (d, j = 15.9 hz, 1h), 5.32 (s, 2h), 3.92 (s, 2h) ; c nmr (cdcl3, 100 mhz) 196.5, 165.2, 138.9, 135.2, 132.8, 131.2, 129.5, 128.9, 128.7, 128.5, 128.4, 127.4, 67.2, 48.7 ; ft - ir (neat) 3031.9, 2961.9, 1722.5, 1696.5, 1636.8, 1496.8, 1454.7, 1376.8, 1274, 1212.6, 1165.4, 1073.7, 975.5, 732.5, 695.2 cm ; hrms (es - tof) m / z calcd for c18h16o3na [m + na ] 303.0997, found 303.0997. yellow oil (0.152 g, 86% yield) ; h nmr (cdcl3, 400 mhz) 7.367.33 (m, 2h), 7.307.28 (br d, 1h), 7.227.20 (br d, 2h), 7.02 (d, j = 15.7 hz, 1h), 6.67 (d, j = 15.5 hz, 1h), 3.91 (s, 2h), 1.50 (s, 9h) ; c nmr (cdcl3, 100 mhz) 196.9, 164.6, 137.7, 133.6, 133.0, 129.5, 128.9, 127.3, 82.1, 48.5, 27.9 ; ft - ir (neat) 2979.8, 1695.5, 1636.8, 1496.8, 1476.8, 1455.8, 1393.8, 1368.6, 1305.5, 1257.6, 1148.2, 1071.7, 1031.8, 976.5, 841.7, 730.6, 697.4 cm ; hrms (es - tof) m / z calcd for c15h19o3 [m + h ] 247.1333, found 247.1334. activated mno2 (10 equiv, obtained from sigma - aldrich and heated at 100 c overnight before use) was added to a flask containing the alcohol (1 equiv) in ch2cl2 (1 m). the reaction mixture was allowed to stir for 12 h as the conversion was monitored by tlc analysis. the solvents were evaporated to give the pure product, typically in high yield and free of detectable side products. amber solid (0.311 g, 83% yield) ; mp 70.374.6 c ; h nmr (cdcl3, 400 mhz) 7.62 (d, j = 15.4 hz, 1h), 7.28 (d, j = 8.8 hz, 2h), 7.08 (d, j = 8.7 hz, 2h), 6.91 (d, j = 15.4 hz, 1h), 3.85 (s, 3h) ; c nmr (cdcl3, 100 mhz) 188.3, 165.8, 135.9, 135.3, 132.6, 132.3, 130.3, 129.3, 52.4 ; ft - ir (neat) 2952.8, 1721.6, 1666.6, 1626.7, 1581.6, 1485.8, 1438.7, 1396.7, 1324.7, 1301.5, 1220.7, 1194.7, 1170.5, 1067.5, 1007.7, 993.6, 943.6, 833.6, 821.6, 755.4, 661.7 cm. yellow solid (0.250 g, 77% yield) ; mp 97.2101.3 c ; h nmr (cdcl3, 400 mhz) 7.88 (d, j = 5.8 hz, 1h), 7.86 (d, j = 8.6 hz, 2h), 7.66 (d, j = 8.6 hz, 2h), 7.417.40 (br m, 5h), 6.94 (d, j = 15.5 hz, 1h), 5.29 (s, 2h) ; c nmr (cdcl3, 100 mhz) 188.4, 165.3, 136.2, 135.3, 135.2, 132.7, 132.3, 130.3, 129.3, 128.7, 128.6, 128.5, 128.4, 67.3 ; ft - ir (neat) 3064.9, 2956.8,1717.4, 1667.5, 1628.7, 1582.6, 1499.9, 1485.8, 1455.7, 1397.6, 1371.7, 1324.7, 1292.3, 1217.8, 1164.3, 1111.7, 1071.5, 1006.6, 978.6, 969.4, 949.6, 912.7, 862.7, 838.6, 811.8, 753.2, 731.7, 700.4, 663.6 cm. yellow solid (0.300 g, 82% yield) ; mp 86.690.0 c ; h nmr (cdcl3, 400 mhz) 7.86 (d, j = 7.0 hz, 2h), 7.74 (d, j = 15.5 hz, 1h), 7.64 (d, j = 7.0 hz, 2h), 6.80 (d, j = 15.5 hz, 1h), 1.53 (s, 9h) ; c nmr (cdcl3, 100 mhz) 188.8, 164.6, 135.4, 135.0, 134.9, 132.2, 130.4, 129.1, 82.1, 28.0 ; ft - ir (neat) 2971.8, 2930.8, 1709.5, 1663.6, 1622.7, 1582.6, 1566.7, 1480.8, 1453.8, 1397.7, 1369.7, 1299.5, 1285.6, 1251.7, 1149.4, 1069.6, 1004.5, 969.5, 864.6, 837.6, 756.4, 697.8, 666.6 cm ; hrms (es - tof) m / z calcd for c14h16bro3 [m + h ] 311.0205, found 311.0283.	a direct and scalable route to -keto-,-unsaturated esters, useful intermediates in medicinal chemistry and natural products synthesis, is reported. the key step involves the use of grubbs second - generation olefin metathesis catalyst for cross - metathesis of alkyl acrylates and 2 allylic alcohols. the metathesis step is followed by oxidation to give the desired products in high yield on scales of up to 25 g.
burn injury is a major global problem with special importance in the east mediterranean region, including iran. in iran, burn injuries have high incidence and mortality rate, in that at least eight people die from burn every day. it is ranked as the third cause of mortality after traffic accidents and trauma. despite the survival of many of such patients, due to the recent advances in medical treatment, psychological problems and deformities are still among the main consequences of burn injuries. in that, devastating and traumatic effects of burn injuries on all physical, emotional, psychological, social, and economic dimensions have challenged the iranian health care system in treating, rehabilitating, and providing palliative care to such patients. heavy burdens of burn injuries such as loss of function, altered appearance, and psychological distress are a threat to the patient 's successful return to the life he / she had before burn and to society. patients may experience severe feelings of anger, guilt, and disappointment affecting their mental health and readiness for involvement in their treatment programs. in addition, the pain caused by a devastating experience, functional and aesthetic impairment, and altered body image and social roles negatively impact the patient 's essence, particularly his / her self - concept. hopelessness, loss of meaning, and existential distress among such survivors can be differentiated from depression and is recognizable in palliative care settings. it is associated with chronic medical illness, disability, body disfigurement, fear of loss of dignity, social isolation, and a subjective sense of incompetence feeling of greater dependency on others or the perception of being a burden. because of the sense of impotence or helplessness, some of the survivors can predictably progress to a desire to die or to commit suicide. these patients need complex mental efforts to redefine their identity in struggling against difficult condition after burn injury. consequently, investigating how to deal with changes in self - concept in burn patients, who are experiencing complex treatment interventions, has been considered to be very important. in a limited number of relevant studies, there are reports of different positive / negative experiences of identity, roles, lifestyle, relationships, and physical function as the factors affecting self - concept in burn patients. in this respect, a number of psychological theories have shown that patients with deformities have lower self - esteem level ; however, other experimental research has not supported this claim. some researchers have suggested that the observed changes in self - concept, measured by the questionnaire, lack adequate reliability. on the other hand, despite a number of cross - sectional and qualitative studies on the potential impact of changes in the appearance and functions on self - esteem, a few of them have addressed compatibility with these disorders. therefore, researchers have suggested more in - depth investigations on the achievement and preservation of self - concept in this group of people. in this regard, conducting qualitative studies seems to be useful, due to their ability in describing slow recovery experience and giving more in - depth understanding of how to achieve a new self - concept, which makes post - burn life bearable. with respect to the effect of social, cultural, and religious dimensions of this experience, the limited number of qualitative studies, and also different cultural and religious contexts in iran, investigating burn patients experiences of how the new self - concept is formed in life after burn injury seems to be essential. since focusing attention on self - concept in palliative care is vital, this study was conducted to investigate the burn patients experiences of preserving self - concept in life after burn injury in iran. due to lack of a comprehensive plan for palliative care for burn survivors in our country, this study can be used as a conceptual framework for palliative care program in iran. the present work is a qualitative study, which seeks to determine, using qualitative content analysis, the true meaning and message of the deep interview conducted with the patients having survived burns. a purposeful sampling was used to achieve the purpose of the research. to ensure primary access to the participants, the burn patients above 20, from whose burn tragedy a minimum of six months had elapsed and who had been discharged from burn wards of sina hospital, tabriz, iran were included in the present study conducted in 2010 - 2013. in a qualitative research, sampling is often started purposefully aiming to select people who have experienced the phenomenon in question, and are able to offer their experiences ; i.e. those rich in information to participate actively and help the researcher to understand their lives and their social interactions in a better way. this process continues by theoretical sampling, in which the selection of each new participant depends on previous samples or participants and the data obtained from them. selection of the next subjects depends on who were selectedfirst and what information has been obtained from them. in the course of the study, purposive sampling is gradually replaced with theoretical sampling. at the start of the sampling, the researcher was looking for people who, as the key knower, were rich sources of information about burn experience ; thus, could help to have a better understanding about the phenomenon of preserving self - concept. sampling was performed atfirst through an objective - based method and after formation of initial concepts and their characteristics and spectrum, the next objectives were selected via theoretical sampling to further complete the concepts and the obtained classes, and further discover the relation between classes. individuals selected for the study included people who had been contacted over the phone, and having expressed their willingness to participate in the study ; they were assured their information would be kept confidential. subsequently, considering the factors affecting the burn patients experience, 17 people were selected for the study trying to ensure maximum variety of the subjects in terms of gender, age, profession, education, the burn type, percent and severity, and of course their willingness to participate in the study. sample size was determined by data saturation when the researchers found that no new information is emerging. general characteristics of the participants (p 17) the main method used for data collection was in - depth unstructured interviewing with open - ended questions. this type of interview is appropriate for qualitative research because of its flexibility and depth. interview with open - ended questions allowed the participants to fully explain their experiences about the phenomenon under study. all interviews were conducted by the third researcher and started off with a general question : the following questions were asked based on the information provided by the participant and were focused on clarifying the main question that was the process of preserving self - concept. the length of personal interviews varied between 45 and 120 minutes and was 82.5 minutes, on average. all interviews were recorded, and were immediately analyzed word - by - word using maxqd10 software. given that in a qualitative research the researcher has to be immersed in the information, he listened to the interviews and reviewed the typed texts several times. in the first step, all the interviews were recorded and then immediately transcribed verbatim and used as the main data of research. in the second step, after listening carefully to the tape - recorded interviews several times and reading the transcribed material, the text was divided into meaningful units. in the third step, the meaningful units were abstracted and labeled with codes. according to the experiences of the participants, the visible and hidden meanings were identified as sentences or paragraphs, and then they were summarized and labeled with codes. in the fourth step, based on the similarities, differences and proportions, the codes implying the same theme were classified in the same category. eventually, the main theme, which is considered aspects of experience structure was written down. using the group members comments and suggestions, the vague and obscure points requiring more attention were discovered and revised in the next interview. concepts were specified based on the inner content of the text, which was reviewed and revised according to all data. during the study long - term relationship with burn survivors increases trust and makes them more willing to share the truth, which in turn increases generation of real and authentic data. member checking or respondent validation was used to verify data accuracy and to ensure data validity. sharing with participants some parts of the interview and his interpretations of their words, the researcher discussed his own interpretation and the meaning of participants words with them to achieve identical ideas and concepts. member checking, reading the transcribed interviews, revision of initial codes, categories and concepts as well as receiving the participants feedback were all the techniques used for data validation. data credibility was confirmed by peer review and the interview transcripts, codes, and categories were extracted. additionally, the results were examined by three faculty members and there was compatibility between the extracted data. the researcher shared the findings with some burn survivors who were not participating in the study and data compatibility was confirmed by them as well. interviewing with different participants, using direct quoting, and providing examples and rich data representation made the data transferability and fittingness possible. data dependability was provided by immediate transcription of interview and re - examination of all data using the external checking. the accuracy of the data collection and analysis process according to the methodology principles was approved by professors and advisors, while it was reviewed as well in the meetings held every nine months, with experts and skilled people within the field of qualitative research ; the ambiguities or drawbacks were discussed and analyzed, and consensus was finally achieved. this study is a part of a phd dissertation in nursing from tabriz university of medical sciences, which was approved by the ethics committee of that university. a letter providing information about the study and the rights of participants was distributed to the participants. participants were told that they were free to accept or reject to participation in the research. the present work is a qualitative study, which seeks to determine, using qualitative content analysis, the true meaning and message of the deep interview conducted with the patients having survived burns. a purposeful sampling was used to achieve the purpose of the research. to ensure primary access to the participants, the burn patients above 20, from whose burn tragedy a minimum of six months had elapsed and who had been discharged from burn wards of sina hospital, tabriz, iran were included in the present study conducted in 2010 - 2013. in a qualitative research, sampling is often started purposefully aiming to select people who have experienced the phenomenon in question, and are able to offer their experiences ; i.e. those rich in information to participate actively and help the researcher to understand their lives and their social interactions in a better way. this process continues by theoretical sampling, in which the selection of each new participant depends on previous samples or participants and the data obtained from them. selection of the next subjects depends on who were selectedfirst and what information has been obtained from them. in the course of the study, purposive sampling is gradually replaced with theoretical sampling. at the start of the sampling, the researcher was looking for people who, as the key knower, were rich sources of information about burn experience ; thus, could help to have a better understanding about the phenomenon of preserving self - concept. sampling was performed atfirst through an objective - based method and after formation of initial concepts and their characteristics and spectrum, the next objectives were selected via theoretical sampling to further complete the concepts and the obtained classes, and further discover the relation between classes. individuals selected for the study included people who had been contacted over the phone, and having expressed their willingness to participate in the study ; they were assured their information would be kept confidential. subsequently, considering the factors affecting the burn patients experience, 17 people were selected for the study trying to ensure maximum variety of the subjects in terms of gender, age, profession, education, the burn type, percent and severity, and of course their willingness to participate in the study. sample size was determined by data saturation when the researchers found that no new information is emerging. the main method used for data collection was in - depth unstructured interviewing with open - ended questions. this type of interview is appropriate for qualitative research because of its flexibility and depth. interview with open - ended questions allowed the participants to fully explain their experiences about the phenomenon under study. all interviews were conducted by the third researcher and started off with a general question : the following questions were asked based on the information provided by the participant and were focused on clarifying the main question that was the process of preserving self - concept. the length of personal interviews varied between 45 and 120 minutes and was 82.5 minutes, on average. all interviews were recorded, and were immediately analyzed word - by - word using maxqd10 software. given that in a qualitative research the researcher has to be immersed in the information, he listened to the interviews and reviewed the typed texts several times. in the first step, all the interviews were recorded and then immediately transcribed verbatim and used as the main data of research. in the second step, after listening carefully to the tape - recorded interviews several times and reading the transcribed material, the text was divided into meaningful units. in the third step, the meaningful units were abstracted and labeled with codes. according to the experiences of the participants, the visible and hidden meanings were identified as sentences or paragraphs, and then they were summarized and labeled with codes. in the fourth step, based on the similarities, differences and proportions, the codes implying the same theme were classified in the same category. then, the categories and sub - categories were formed. eventually, the main theme, which is considered aspects of experience structure was written down. using the group members comments and suggestions, the vague and obscure points requiring more attention were discovered and revised in the next interview. concepts were specified based on the inner content of the text, which was reviewed and revised according to all data. during the study, some methods were used to ensure data accuracy and stability. long - term relationship with burn survivors increases trust and makes them more willing to share the truth, which in turn increases generation of real and authentic data. member checking or respondent validation was used to verify data accuracy and to ensure data validity. sharing with participants some parts of the interview and his interpretations of their words, the researcher discussed his own interpretation and the meaning of participants words with them to achieve identical ideas and concepts. member checking, reading the transcribed interviews, revision of initial codes, categories and concepts as well as receiving the participants feedback were all the techniques used for data validation. data credibility was confirmed by peer review and the interview transcripts, codes, and categories were extracted. additionally, the results were examined by three faculty members and there was compatibility between the extracted data. the researcher shared the findings with some burn survivors who were not participating in the study and data compatibility was confirmed by them as well. interviewing with different participants, using direct quoting, and providing examples and rich data representation made the data transferability and fittingness possible. data dependability was provided by immediate transcription of interview and re - examination of all data using the external checking. the accuracy of the data collection and analysis process according to the methodology principles was approved by professors and advisors, while it was reviewed as well in the meetings held every nine months, with experts and skilled people within the field of qualitative research ; the ambiguities or drawbacks were discussed and analyzed, and consensus was finally achieved. this study is a part of a phd dissertation in nursing from tabriz university of medical sciences, which was approved by the ethics committee of that university. a letter providing information about the study and the rights of participants was distributed to the participants. participants were told that they were free to accept or reject to participation in the research. the text obtained from interviewing 17 participants with burn experience was considered as the analysis unit [table 1 ]. the concept of self - locating as the essence of participants experience in transition from different stages (i.e. occurrence of accident, transferring to hospital and hospitalization, discharging from hospital and returning to home, and finally to society) was extracted from following four categories : (a) self - exploration (exploring the changes in one 's life), (b) others exploration (exploring the changes in the life of family members and the relationship between self and others), (c) position evaluation (self - position analysis), and (d) self - concept preservation. findings showed that participants were involved with mental processes in transition from the accident phase to returning to society, and in an effort to preserve self - concept against the threat in different physical, emotional - psychological, spiritual, behavioral, and value dimensions. this mental process included self - exploration, others exploration, and position evaluation, which helped the understanding of the current position and choosing facilitating activities for that transition. at the first stages after an accident where burn injury posed only physical threat, self - exploration mainly included focusing on severe and unbearable feeling of pain, dealing with wounds, and wandering in oppressive atmosphere of the burn ward. a participant described the experience of pain as follows : when they took us for dressing, when they poured water on the wounds, it was like knocking nails into the body, one by one. the wounds were terrible and the ward had a very bad atmosphere (p. 6). self - exploration continued after discharge, returning to home, and gradual reduction of physical pain by exploring bodily changes. according to a participant, i could see unhealed skin grafts, wounds on my face, i needed my family 's help in doing even a small thing (p. 1). the inevitable continuation of life and the need for interacting with others were the common experience of the majority of participants from the third stage, i.e. return to society. at this stage, over this course, the goal of self - exploration was to investigate the results from such efforts as covering of scars and cosmetic surgeries to correct deformities and physical limitation. a young female participant said : it made me sad when i saw people looking at me strangely in the street. i underwent a number of surgeries but my hands were not still in good shape and i had a burn scar on my face. because of that, i always had gloves and tried to blur the burn site by wearing make - up (p. 7). for the participants, the fourth stage was like a period that they had time to think about all dimensions and events. for some participants, burn memories continued to affect how they control and value their lives, even after 10 or 20 years. at this stage, helpful and hindering thoughts ran through the participants mind on a set of frightening and traumatic memories of the previous stages. that is why self - exploration took place through self - struggling, engaging in inner dialog, fantasizing, and dreaming. according to a participant, i always feel pain and agony, they never leave my heart, when i think by myself. i was dreaming the past, [when ] i was not burnt and went out with my friend. focusing on the burn consequences in family, exploring changes in interactions, and paying attention to social issues by the participants are among the characteristics of this category. in the initial stages, with participants wandering in the gloomy atmosphere of the burn ward and observation of other patients and families in agony, the others exploration mechanism gets activated in parallel with self - exploration. another participant talked about her experience : there were two rooms at the end of the corridor in the hospital for seriously injured patients. from there, dead bodies were taken out one after another (p. 3). the same participant added about the effect of burn on his / her family : you can imagine what happened to my mother when she heard about it. her eyes filled with tears) (p. 1). after being placed in a new position at home, exploration of the others continued by investigating the effect of burn on personal life and relationships. a number of participants called the cost of treatment - induced economic burden on the family and/or the time spent for caring them as imposing strict conditions on family life. since i was burnt, my sister devoted her life to look after me. i know how much he suffers from the heavy burden of all these costs and agonies (p. 11). by joining them, participants began to investigate the changes in the way others interact with them, through the same mechanism. a young man said : some of my friends were ashamed of walking with me on the street. because everybody looked at me with curiosity or pity (p. 8). another participant said, when you go to a public place, it seems that something is going on, all look carefully. findings from the participants experiences showed that they arrive at a new position based on : (a) analyzing and comparing their conditions after burn with before that, and (b) evaluating and judging their individual and social positions, and their distance away from their dreams, goals, and desires in life. for some participants, this new position was interpreted as an unusual and unnatural position. in this regard, the participants expressed such experiences as changing to someone else, duality. for example : after a while when i recognized that i was not the same person, that i had changed, i felt very desperate. at home i was a person, outside someone else (p. 6). while some participants imagined themselves in a position caused by bodily and capability changes, where they could be the same person again with the same spirits, beauty, and close family ties by healing of their wounds ; they, considered burn as something that had only affected their appearance and capabilities : eventually, one comes to terms with it, but you are no longer the same person, i used to work hard ! i am the same person with the same characteristics though, i only have problem with my leg (p. 4). burn patients employed such strategies as putting efforts and struggling, substituting the roles, dealing with deformities, paying attention to health, coping with pain, getting themselves busy, escape, violence, getting along, secrecy, and absorbing energy resource in transition from the accident to society. the result of their efforts was self - concept preservation, ranging from maintaining the previous body image, being aware of the new body image, accepting the new body image, and rewriting the self - concept. during the first stage, which was mainly filled with the feeling of pain and agony, participants sought to preserve their previous image by denying the severity of injuries and hiding their pain and agony from their families, as well as misrepresenting the conditions. these efforts contributed to intensified pain caused by strict treatments and cares, as well as the agony of being dependent on others. in addition, participants felt that they could prevent imposing more pain and agony to their families by means of such behaviors. after that immense pain, when my parents came, i sat comfortably and upright to prevent them from becoming sadder (p. 1) by arriving at home, participants mainly tried to preserve themselves from the understood changes, to improve their awareness, and to obtain new information. for example, a participant said : when i was discharged from the hospital, i still had problem in my leg, but no one instructed me. when i found my leg severely infected, i visited the specialized hospital on my own (p. 4). the other part of this awareness was acquired through interactions, examining others reactions to the wounds, and seeking information from them. a participant said : when the family came and saw my conditions, they said these wounds would gradually crumple, you should do something. then, they suggested that i visit a good doctor in tehran1 (p. 7). at this stage, the participants attention was focused on health and efforts to obtain information about and awareness of their new body image. with participants entering into society and due to the consequent need for more interaction with others, they have to accept their new body image to preserve themselves in these interactions. indeed, getting used to the new conditions and shifting from disturbing experiences to new ones such as the feeling of gratitude, calmness, feeling of pleasure, and raising spirit on the one hand, and performing such activities as self - care, role substitution, engaging in something, and improving personal relationships on the other hand, contributed to this acceptance. accepting the new body image paves the way for normalizing the new life by acquiring new skills and behaving and interacting with others more easily. in this way and by getting involved with the normal flow of life, the participants not only followed their normal roles, but also to some of them the injury was like an opportunity for spiritual growth. the participants said : after the burn accident, my attitudes towards plenty of things changed, i grew up, now i know what to do when someone gets sick. how a burn patient suffers, how to behave toward him / her (p. 7). i was jealous and thankless, but now i prostrate a thousand times a day (p. 9). although self - preservation moved toward the acceptance of the new body image with acquiring awareness of it, this study could not distinguish a clear time border between the two stages. findings showed that living with burn injuries was a live experience of an internal journey for the participants ; a mental journey from awareness to acceptance, where one moves from home to society, and life activities move from falling into the habit to getting normal. however, they were not sure about the duration of this experienced journey, most of them mentioned a period between four and eight years, during which they got used to living with burn injuries. regardless of returning to a normal life, memories of the burn accident still remained in the mind of participants who had passed a long time since the accident. preserving self at this stage, which was occurring in parallel to other life stages, was in a mental context of the past memories, successes and current losses, and dream of future achievements. when i look at my pictures, i do nt want to see that my condition has changed, my story has completely altered. now, i am obsessive, depressed, i can not stand anything, i am so exhausted (p. 6). the participants activities for preserving self - concept at this stage were in the form of a reciprocal effort to obtain or maintain independence, change life priorities, focus on life 's achievements and successes, and/or display such behaviors as threatening others, escaping, and getting along passively. finally, depending on the factors, affecting individual and social life, the action / interaction process makes them rewrite themselves appropriately or inappropriately. according to a participant : you can never forget those memories, but when time passes and you see yourself ahead of others in several things, these are actually helpful. it is no longer important, looks no longer matter, you have other priorities, you just do your own business, i think financial independence is the most important factor, meaning you are not dependent on others, and then entertainment and other enjoyments (p. 1). findings showed that participants were involved with mental processes in transition from the accident phase to returning to society, and in an effort to preserve self - concept against the threat in different physical, emotional - psychological, spiritual, behavioral, and value dimensions. this mental process included self - exploration, others exploration, and position evaluation, which helped the understanding of the current position and choosing facilitating activities for that transition. at the first stages after an accident where burn injury posed only physical threat, self - exploration mainly included focusing on severe and unbearable feeling of pain, dealing with wounds, and wandering in oppressive atmosphere of the burn ward. a participant described the experience of pain as follows : when they took us for dressing, when they poured water on the wounds, it was like knocking nails into the body, one by one. the wounds were terrible and the ward had a very bad atmosphere (p. 6). self - exploration continued after discharge, returning to home, and gradual reduction of physical pain by exploring bodily changes. according to a participant, i could see unhealed skin grafts, wounds on my face, i needed my family 's help in doing even a small thing (p. 1). the inevitable continuation of life and the need for interacting with others were the common experience of the majority of participants from the third stage, i.e. return to society. at this stage, over this course, the goal of self - exploration was to investigate the results from such efforts as covering of scars and cosmetic surgeries to correct deformities and physical limitation. a young female participant said : it made me sad when i saw people looking at me strangely in the street. i underwent a number of surgeries but my hands were not still in good shape and i had a burn scar on my face. because of that, i always had gloves and tried to blur the burn site by wearing make - up (p. 7). for the participants, the fourth stage was like a period that they had time to think about all dimensions and events. for some participants, burn memories continued to affect how they control and value their lives, even after 10 or 20 years. at this stage, helpful and hindering thoughts ran through the participants mind on a set of frightening and traumatic memories of the previous stages. that is why self - exploration took place through self - struggling, engaging in inner dialog, fantasizing, and dreaming. i always feel pain and agony, they never leave my heart, when i think by myself. i was dreaming the past, [when ] i was not burnt and went out with my friend. focusing on the burn consequences in family, exploring changes in interactions, and paying attention to social issues by the participants are among the characteristics of this category. in the initial stages, with participants wandering in the gloomy atmosphere of the burn ward and observation of other patients and families in agony, the others exploration mechanism gets activated in parallel with self - exploration. another participant talked about her experience : there were two rooms at the end of the corridor in the hospital for seriously injured patients. from there, dead bodies were taken out one after another (p. 3). the same participant added about the effect of burn on his / her family : you can imagine what happened to my mother when she heard about it. after being placed in a new position at home, exploration of the others continued by investigating the effect of burn on personal life and relationships. a number of participants called the cost of treatment - induced economic burden on the family and/or the time spent for caring them as imposing strict conditions on family life. since i was burnt, my sister devoted her life to look after me. i know how much he suffers from the heavy burden of all these costs and agonies (p. 11). by joining them, participants began to investigate the changes in the way others interact with them, through the same mechanism. a young man said : some of my friends were ashamed of walking with me on the street. because everybody looked at me with curiosity or pity (p. 8). another participant said, when you go to a public place, it seems that something is going on, all look carefully. findings from the participants experiences showed that they arrive at a new position based on : (a) analyzing and comparing their conditions after burn with before that, and (b) evaluating and judging their individual and social positions, and their distance away from their dreams, goals, and desires in life. for some participants, this new position was interpreted as an unusual and unnatural position. in this regard, the participants expressed such experiences as changing to someone else, duality. for example : after a while when i recognized that i was not the same person, that i had changed, i felt very desperate. at home i was a person, outside someone else (p. 6). while some participants imagined themselves in a position caused by bodily and capability changes, where they could be the same person again with the same spirits, beauty, and close family ties by healing of their wounds ; they, considered burn as something that had only affected their appearance and capabilities : eventually, one comes to terms with it, but you are no longer the same person, i used to work hard ! i am the same person with the same characteristics though, i only have problem with my leg (p. 4). at the first stages after an accident where burn injury posed only physical threat, self - exploration mainly included focusing on severe and unbearable feeling of pain, dealing with wounds, and wandering in oppressive atmosphere of the burn ward. a participant described the experience of pain as follows : when they took us for dressing, when they poured water on the wounds, it was like knocking nails into the body, one by one. the wounds were terrible and the ward had a very bad atmosphere (p. 6). self - exploration continued after discharge, returning to home, and gradual reduction of physical pain by exploring bodily changes. according to a participant, i could see unhealed skin grafts, wounds on my face, i needed my family 's help in doing even a small thing (p. 1). the inevitable continuation of life and the need for interacting with others were the common experience of the majority of participants from the third stage, i.e. return to society. at this stage, over this course, the goal of self - exploration was to investigate the results from such efforts as covering of scars and cosmetic surgeries to correct deformities and physical limitation. a young female participant said : it made me sad when i saw people looking at me strangely in the street. i underwent a number of surgeries but my hands were not still in good shape and i had a burn scar on my face. because of that, i always had gloves and tried to blur the burn site by wearing make - up (p. 7). for the participants, the fourth stage was like a period that they had time to think about all dimensions and events. for some participants, burn memories continued to affect how they control and value their lives, even after 10 or 20 years. at this stage, helpful and hindering thoughts ran through the participants mind on a set of frightening and traumatic memories of the previous stages. that is why self - exploration took place through self - struggling, engaging in inner dialog, fantasizing, and dreaming. i always feel pain and agony, they never leave my heart, when i think by myself. i was dreaming the past, [when ] i was not burnt and went out with my friend. focusing on the burn consequences in family, exploring changes in interactions, and paying attention to social issues by the participants are among the characteristics of this category. in the initial stages, with participants wandering in the gloomy atmosphere of the burn ward and observation of other patients and families in agony, the others exploration mechanism gets activated in parallel with self - exploration. another participant talked about her experience : there were two rooms at the end of the corridor in the hospital for seriously injured patients. from there, dead bodies were taken out one after another (p. 3). the same participant added about the effect of burn on his / her family : you can imagine what happened to my mother when she heard about it. after being placed in a new position at home, exploration of the others continued by investigating the effect of burn on personal life and relationships. a number of participants called the cost of treatment - induced economic burden on the family and/or the time spent for caring them as imposing strict conditions on family life. since i was burnt, my sister devoted her life to look after me. i know how much he suffers from the heavy burden of all these costs and agonies (p. 11). by joining them, participants began to investigate the changes in the way others interact with them, through the same mechanism. a young man said : some of my friends were ashamed of walking with me on the street. because everybody looked at me with curiosity or pity (p. 8). another participant said, when you go to a public place, it seems that something is going on, all look carefully. findings from the participants experiences showed that they arrive at a new position based on : (a) analyzing and comparing their conditions after burn with before that, and (b) evaluating and judging their individual and social positions, and their distance away from their dreams, goals, and desires in life. for some participants, this new position was interpreted as an unusual and unnatural position. in this regard, the participants expressed such experiences as changing to someone else, duality. for example : after a while when i recognized that i was not the same person, that i had changed, i felt very desperate. at home i was a person, outside someone else (p. 6). while some participants imagined themselves in a position caused by bodily and capability changes, where they could be the same person again with the same spirits, beauty, and close family ties by healing of their wounds ; they, considered burn as something that had only affected their appearance and capabilities : eventually, one comes to terms with it, but you are no longer the same person, i used to work hard ! i am the same person with the same characteristics though, i only have problem with my leg (p. 4). burn patients employed such strategies as putting efforts and struggling, substituting the roles, dealing with deformities, paying attention to health, coping with pain, getting themselves busy, escape, violence, getting along, secrecy, and absorbing energy resource in transition from the accident to society. the result of their efforts was self - concept preservation, ranging from maintaining the previous body image, being aware of the new body image, accepting the new body image, and rewriting the self - concept. during the first stage, which was mainly filled with the feeling of pain and agony, participants sought to preserve their previous image by denying the severity of injuries and hiding their pain and agony from their families, as well as misrepresenting the conditions. these efforts contributed to intensified pain caused by strict treatments and cares, as well as the agony of being dependent on others. in addition, participants felt that they could prevent imposing more pain and agony to their families by means of such behaviors. after that immense pain, when my parents came, i sat comfortably and upright to prevent them from becoming sadder (p. 1) by arriving at home, participants mainly tried to preserve themselves from the understood changes, to improve their awareness, and to obtain new information. for example, a participant said : when i was discharged from the hospital, i still had problem in my leg, but no one instructed me. when i found my leg severely infected, i visited the specialized hospital on my own (p. 4). the other part of this awareness was acquired through interactions, examining others reactions to the wounds, and seeking information from them. a participant said : when the family came and saw my conditions, they said these wounds would gradually crumple, you should do something. then, they suggested that i visit a good doctor in tehran1 (p. 7). at this stage, the participants attention was focused on health and efforts to obtain information about and awareness of their new body image. with participants entering into society and due to the consequent need for more interaction with others, they have to accept their new body image to preserve themselves in these interactions. indeed, getting used to the new conditions and shifting from disturbing experiences to new ones such as the feeling of gratitude, calmness, feeling of pleasure, and raising spirit on the one hand, and performing such activities as self - care, role substitution, engaging in something, and improving personal relationships on the other hand, contributed to this acceptance. accepting the new body image paves the way for normalizing the new life by acquiring new skills and behaving and interacting with others more easily. in this way and by getting involved with the normal flow of life, the participants not only followed their normal roles, but also to some of them the injury was like an opportunity for spiritual growth. after the burn accident, my attitudes towards plenty of things changed, i grew up, now i know what to do when someone gets sick. how a burn patient suffers, how to behave toward him / her (p. 7). i was jealous and thankless, but now i prostrate a thousand times a day (p. 9). although self - preservation moved toward the acceptance of the new body image with acquiring awareness of it, this study could not distinguish a clear time border between the two stages. findings showed that living with burn injuries was a live experience of an internal journey for the participants ; a mental journey from awareness to acceptance, where one moves from home to society, and life activities move from falling into the habit to getting normal. however, they were not sure about the duration of this experienced journey, most of them mentioned a period between four and eight years, during which they got used to living with burn injuries. regardless of returning to a normal life, memories of the burn accident still remained in the mind of participants who had passed a long time since the accident. preserving self at this stage, which was occurring in parallel to other life stages, was in a mental context of the past memories, successes and current losses, and dream of future achievements. when i look at my pictures, i do nt want to see that my condition has changed, my story has completely altered. now, i am obsessive, depressed, i can not stand anything, i am so exhausted (p. 6). the participants activities for preserving self - concept at this stage were in the form of a reciprocal effort to obtain or maintain independence, change life priorities, focus on life 's achievements and successes, and/or display such behaviors as threatening others, escaping, and getting along passively. finally, depending on the factors, affecting individual and social life, the action / interaction process makes them rewrite themselves appropriately or inappropriately. according to a participant : you can never forget those memories, but when time passes and you see yourself ahead of others in several things, these are actually helpful. it is no longer important, looks no longer matter, you have other priorities, you just do your own business, i think financial independence is the most important factor, meaning you are not dependent on others, and then entertainment and other enjoyments (p. 1). findings showed that the participants achieve the self - locating issue through such mechanisms as self - exploration, others exploration, and position evaluation. this concept refers to a new understanding of one 's position in the process of surviving and returning to society. morse (1995) illustrated this process in the form of vigilance, enduring, and suffering. concept, which is subjective in nature, manifests as preserving the previous body image, awareness of the new body image, acceptance of the new body image, and finally rewriting the self - concept, at each stage of transition toward returning to society. concept prepares the subjective context for choosing the strategies that fit one 's current position at each transition stage. however, based on the effective external, internal, and interpersonal factors, entering into and transition from this path differ from person to person. based on a qualitative meta - analysis of the studies, investigating human responses to threats from diseases, morse (1997) combined two theories of illness constellation model and preserving self, a five - stage model, in which self - comforting strategies were embedded for preserving the self. however, the described stages in this theory have been properly given as a comprehensive model based on other theories ; the presented concepts as well as the border of each stage is not clearly developed. in addition, in terms of the new concept, a clear description of people 's mental processes has not been provided. this study provides further description of the burn patients mental process by explaining such concepts as self - exploration, others exploration, and position evaluation. in addition, it presents a number of assumptions about self - concept over the post - burn course. during the course from occurrence of the accident to physical stability, self - exploration is usually done by focusing on severe and intolerable feeling of pain, facing with the wounds of the selves and of the other patients, and wandering in the oppressive atmosphere of the burn ward. in addition, observing the suffering endured by the family members is the result of the exploration of the others within this period. by means of position evaluation, one searches for the conditions, where he / she is capable of tolerating the current pain and suffering. consequently, he / she looks for the strategies to be employed for preserving his / her previous integrity by downplaying the damages and preserving his / her previous image. rejecting the severity of damage at the time of the accident and during the first days of hospitalization, denying the pain, and pretending to be looked calm and elegant when visiting the family are all proofs of repression of emotions by the participants of this study moreover, other experiences such as refusing to see the wounds in some studies are examples of trying to deny the severity of the damage and preserving the previous body image. at this stage, when the pain is severe, its onset puts major stress on the patient, in a way that it attracts all of his / her energy and attention to self - preservation. the patient feels that he / she is threatened and humiliated. at this stage, maintaining emotional control to endure all types of fear or phobia of physical threat is essential. morse (1997) showed that suppressed emotions, during the enduring to survive, places the person in a state of shut down. the present study showed that at this stage, self - locating helps the patient in enduring the suffering from severe pain, aggression toward bodily realm, and dependence on family in doing daily activities. however, other studies show that when there is a severe pain, people employ pain preventing strategies like distraction, shutting it out, focusing on something like staring at the ceiling, and/or disembodying. in a grounded theory - based study, which has investigated the processes from the rehabilitation aspect, suspecting, reading the body, observing, overwhelming, maintaining emotional control, and accepting help are introduced as the first human responses to the threat against his entirety at the vigilance stage. after rescue and survival, the results from this study indicate that at this stage, the main mental activity of the participants is having awareness of their new selves. this awareness is acquired through focusing on the altered appearance or others reaction to self. based on the present study, this awareness in a group of patients is limited to bodily changes and in the other group is represented in the form of a global change. it sometimes is understood based on the expressed experiences, i.e. changing to someone else. at this stage, self - locating will help self - image awareness, i.e. understanding one 's present position. when people admit their fears along this path, they will begin controlling and investigating their body. acknowledging the seriousness of the situation indicates people 's awareness of selves, environment, and anything making them involved internally or externally. in a phenomenological study, moi. (2008) extracted the concept of a new bodily awareness as the essence of burn patients experience of life. a new and difficult awareness of the limitations, acquired by seeing a strange and vulnerable body, sadness, disability, and insecurity. in this regard, a number of researchers have mentioned the experience of inner dialogue, i.e. communications that patients have in their heads, which help and guide them toward patience. awareness of the altered appearance and the new self - image, as a part of burn injured patients experience, is one of the most important psychological variables affecting their life quality it plays an important role in short - term and long - term adaptability. nonetheless, despite acquiring extensive knowledge from psychometric evaluations, further information is still required to understand the differences observed in personal experiences of such patients. the importance of the present findings, compared to the majority of quantitative studies that have investigated the short - term effects of burn injuries, is their focus on long - term experiences of burn patients. in addition, the social reception level, adaptability and emotions, and such characteristics as introspection are all affecting adaptability with new situations. in this respect, findings from the present study showed that after 1 - 8 years and acquisition of awareness of the new position, the patient gradually enters another stage by observing wound healing and normalization of others behaviors, listening to the experiences of the preceding burn patients, and specially finding out that the appearance of burn scars is downplayed by close friends and relatives. at this stage, self - locating encourages people to endeavor to seek ways of acquiring further capabilities. then, by going through this path, acceptance of the new self - image is cultivated. despite what was said, the risk of dissatisfaction with the new body image always affects adaptability and life quality of such people. by accepting the new body image and returning to normal life, exploration of the self and the others focuses on the values, beliefs, and life expectations achievement level. burn patients value and judge the quality of life by evaluating the achievements, as well as comparing their life before burn with their post - burn life, and/or with the life of others. deformities can lead to lack of social skills, with increased level of shyness, social anxiety, rejection, and behaviors associated with fear and shame. they can also impair one 's satisfaction with life and his / her functions by entering the patient into a faulty social cycle. in this studies show that psychosocial problems, as important causal factors, precede burn injuries in causing post - injury adaptive immune reactions ; in that, in some moderate burn - injured patients, returning to a normal life has been challenging even after full recovery. in addition, those who receive further social support from friends and relatives, during the position evaluation stage, enjoy a more positive body image and self - esteem than others. the present study identified the concept of self - locating as the essence of patients experiences at different stages after burn. this phenomenon, described in form of self - exploration, others exploration, position evaluation, and self - concept preservation subcategories, will help clearing and understanding relevant comprehensive theories. however, further investigations are recommended to understand the consequence of this phenomenon in the palliative care process of burn survivors. burn survivors are engaged with a complex mental process at all stages from post - accident, transfer, and hospitalization to returning to home and society. in this way, they achieve self - locating through exploration of the self and the others and position evaluation. self - locating refers to understanding the present position and choosing such activities that preserve them from changes. the obtained results can be used as a conceptual framework for understanding the burn survivors response to self - concept changes in life after burn. in addition, the concepts presented in this study can be employed to design rehabilitation and palliative care models in compliance with the existing cultural context in iran.	background : burn injury is a devastating experience affecting all aspects of a person 's essence, including his / her identity and perception. these patients require complex cognitive efforts to redefine their identity to deal with difficult condition after burn injury and preserve self - concept. the experience of life after burn injury is generally a solitary one, closely related to the patients cultural and religious context. therefore, this study was conducted aiming at investigating burn patients experiences regarding how to preserve self - concept in life after burn injury in iran.materials and methods : this qualitative study was carried out using qualitative content analysis and in - depth unstructured interviews with 17 surviving burn subjects.results:during the qualitative content analysis process, the concept of locating as the essence of the participants experience was extracted as follows : (a) self - exploration (exploring the changes in one 's life), (b) others exploration (exploring the changes in the life of family members and the relationship between self and others), (c) position evaluation (self - position analysis), and (d) self - concept preservation.conclusion:the present study has developed new understandings of mental experiences of burn patients self - concept by describing the concept of self - locating. it helps us in classifying and understanding the concepts described in comprehensive theories developed in this area. they do this by focusing on what burn patients experience for choosing self - preservation strategies and having a meaningful life. the finding can be used as a conceptual framework for palliative care program in iran.
sexual dysfunction in women is characterized by persistent and recurrent problems in sexual response or desire, which cause affliction and affect their relationship with their partner.1,2 sexual dysfunctions may occur at any age and affect roughly 40% of women at some point in their lifetime, with 12% of women reporting afflictive sexual problems.3 there are different types of sexual dysfunctions, characterized as (1) sexual interest / arousal disorder ; (2) orgasmic disorder ; and (3) genitopelvic pain / penetration disorder.4 it is important to explain to the patient that transient sexual problems are common ; however, if the problem persists over months or causes affliction to the patient or their partner, then the cause of the dysfunction should be investigated.5,6 there are several risk factors that may contribute to the emergence of female sexual dysfunction. these include the patient s well - being ; important events in romantic relationships ; partner s sexual problems ; gynecological factors including childbirth, hysterectomy, vaginal pain, or pelvic pain ; urinary tract alterations ; and drug side effects (use of beta blockers, anti - depressant, or anti - psychotic drugs).711 several therapeutic alternatives are used in the treatment of female sexual dysfunction ; however, it is important to identify any possible contributing physical and emotional factors before beginning treatment.9,11,12 therapeutic approaches include stress management and management of any relationship problems, treatment of vaginal dryness, dyspareunia, and investigation of possible drug side effects.10,11 the use of testosterone in the form of gels, creams, ointments, or in oral form is not recommended for pre - menopausal women, and carry the risk of side effects in addition to containing very high doses of testosterone.1316 the use of medications for erectile dysfunction has proven to be largely ineffective.9,15,17 dehydroepiandrosterone (dhea) has been shown to be effective in improving sexual interest and satisfaction among women with adrenal insufficiency ; however, the benefit of supplementation in women with normal adrenal function has not been confirmed.1820 in this study, we evaluated the use of tribulus terrestris extract to treat female sexual dysfunction. t. terrestris is a plant belonging to the zygophyllacea family, with many common names including caltrop, cat s head, and bindii. the leaves and roots are used for medicinal purposes in traditional medicine in india and china and were also in use in ancient greece.21 the primary study objective was to evaluate the efficacy of t. terrestris extract in female sexual dysfunction. this was a qualitative quantitative study performed at hospital das clnicas de terespolis (terespolis rj, brazil). subjects were included based on inclusion and exclusion criteria, followed by hospital records verification. inclusion criteria for female patients are that they should be of reproductive age (over 18 years old), with a clinical presentation of sexual dysfunction, who were treated with tablets containing 250 mg t. terrestris extract (brand name androsten) at the dose of 1 tablet three times per day for a period of 90 days. the study protocol was evaluated and approved by the fundao educacional serra dos rgos ethical committee (approval no. exclusion criteria were pregnant or breastfeeding women, patients presenting hypersensitivity to any component of the study medication, and any other diseases or conditions that, in the opinion of the investigator, exclude the patient from the study. all study data were recorded in the clinical research form, in which subjects were identified using 3-digit sequential numbers. in addition to physical examination results (vital signs, weight, body mass index [bmi ]), safety analysis included the following laboratory test results : complete blood count, amylase, glucose and fasting glucose, serum prolactin, follicle - stimulating hormone, leutenizing hormone, thyroid - stimulating hormone, serum potassium, blood urea nitrogen, serum creatinine, total and fractionated billirrubins, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase. the primary safety and tolerability measures included any changes in vital signs and physical examination in relation to pretreatment, any changes in clinical laboratory examinations in relation to pretreatment, and the occurrence of adverse events after the first dose of study medication. the secondary safety measure was the evaluation of overall tolerability of the study medication performed post - treatment by the study physician, using the same classifications of very good, good, fair, or the primary efficacy analysis included results of the female sexual function index (fsfi), a validated self - report measure of female sexual function. this is a 19-item questionnaire that is subdivided into six domains : desire, arousal, lubrication, orgasm, satisfaction, and pain. the scoring system followed the instructions given by the authors who developed and validated the questionnaire. the maximum possible score is 36 points and the minimum possible score is 2 points.22 secondary efficacy measures included the patient and physician assessments, in which both the subject and the physician rated the patient s overall condition on a scale of 110 points, with 1 corresponding to the worst assessment and 10 the best. the study physician also evaluated the overall efficacy and safety of the study medication as very good dhea levels together with total and free testosterone were also included in the efficacy analysis (reference ranges : dhea : 15170 ng / ml ; serum testosterone : 363 ng / dl ; free testosterone : 245 pmol / ml). at the end of the treatment period, subjects who completed the treatment cycle were asked to rate their willingness to continue treatment on a scale of 1 (very unwilling) to 10 (very willing). the clinical research form was filled, coded, and the data were analyzed using graphpad prism version 5.1 software. frequency tables were generated and central tendency measures were calculated (mean, median, mode). as appropriate, we used the student s t - test or the repeated - measures analysis of variance (anova) for continuous variables and fisher s test or the test for categorical variables (= 0.05). sixty subjects (41.67%) were married, while 55 (38.19%) were divorced, 22 (15.28%) were single, and 7 (4.86%) were widows. ethnicity (self - reported) was as follows : asian (n = 5), caucasian (n = 60), black (n = 26), and mulatto (n = 53). the relevant medical history including use of contraceptives, reproductive, and menstrual information together with data on depression is summarized in table 1. mean dhea level at pretreatment was 57.83 ng / ml (30.11), while there was a statistically significant (p 6 points in the patient assessment and 25 patients had scores > 6 points in the physician assessment. table 4 summarizes the results of the physical examination performed pre- and post - treatment. the results show that the treatment was effective in terms of improvement in the fsfi questionnaire, with the majority of patients showing improved post - treatment scores together with statistically significant improvements in the total score of the questionnaire as well as in most of the individual domain scores. although side effects were reported, there were no reports of serious adverse effects during the treatment period. the safety evaluations including laboratory tests and physical examination results did not undergo significant alterations during the treatment period. overall, t. terrestris has a good safety profile ; previously reported side effects include irritation of the gastric mucosa and gastric reflux. the side effects reported during the treatment period do reflect this information, with an overwhelming majority of side effects related to the gastrointestinal tract.23,24 however, these effects were transient and, as mentioned previously, no serious adverse effects were reported. the assessment of the female patient presenting with sexual dysfunction should take into account a wide variety of factors, including an important emotional component. interestingly, during pretreatment assessment, we observed a relatively high number of patients self - reporting depression during the assessment of medical history, a finding that reflects the data present in the literature.25,26 t. terrestris is often used in the treatment of infertility, decreased libido, and erectile dysfunction. it is also used among athletes to increase muscle resistance and improve performance in sports, although scientific evidence to support this effect is lacking.2731 the active components of t. terrestris include the steroidal glycoside saponins furostanol and spirostanol, which are found in the leaves of the plant.27 protodioscin is a chemical substance derived from the t. terrestris plant that has been confirmed to increase sexual desire and improve erections by means of conversion of protodioscin to dhea.29,32 in animal studies, increased testosterone levels along with increased dehydrotestosterone and dhea are suggestive of aphrodisiac activity.2731 the aphrodisiac pro - erectile properties were co - related with a release of nitric oxide from nerve endings enervating the corpus cavernosum of the penis.24 however, the literature is lacking additional studies to confirm these effects.30,33 indeed, our results showing a statistically significant increase in dhea among the treated patients are interesting as this effect can also be observed among treated female subjects. however, the lack of correlation between increasing dhea levels with both free testosterone and serum testosterone levels is confounding. contrary to expectations,34,35 mean testosterone levels decreased among the total treated subjects, despite the presence of a clinical response. it may be because the dhea levels in women have a greater impact on fsfi domains in relation to testosterone, explaining our positive results. a larger, prospective, randomized, comparative study would likely provide further insight into the effect of t. terrestris extract on testosterone levels in women. it may also be of interest to examine the effects of different doses of t. terrestris extract in future study. the co - occurrence of enhanced female sexual function and increased dhea levels is suggestive of physiological alterations underlying clinical improvement following treatment. our results strongly support the safety and effectiveness of t. terrestris extract in the treatment of female sexual dysfunction.	this is a qualitative quantitative study based on hospital records of female patients of reproductive age, presenting sexual dysfunction, and treated with 250 mg tribulus terrestris extract (1 tablet thrice daily for 90 days). safety monitoring included vital signs, physical examination, laboratory tests, and occurrence of adverse events. efficacy analysis included results of the female sexual function index (fsfi), dehydroepiandrosterone (dhea) levels together with total and free testosterone, and the patient and physician assessments. there was a statistically significant improvement in total fsfi scores (p < 0.0001) post - treatment, with improvement among 106 (88.33%) subjects. there was a statistically significant (p < 0.0001) increase in the level of dhea, while the levels of both serum testosterone (p = 0.284) and free testosterone decreased (p < 0.0001). most adverse events recorded were related to the gastrointestinal tract. physical examination showed no significant changes post - treatment. based on the results, it is concluded that the t. terrestris extract is safe and effective in the treatment of female sexual dysfunction.
the glutathione - s - transferase (gst) proteins represent an extended family with high diversity depending on the organism [13 ]. these enzymes are usually able to conjugate glutathione (gsh) to hydrophobic molecules and thus are involved in biotransformation pathways. the addition of gsh onto molecules, previously oxidized by detoxification phase i enzymes, results in the formation of compounds which are usually less toxic and more soluble. additionally, these proteins can exhibit other activities such as thiol transferase or peroxidase activities [46 ], suggesting that they could directly participate in the cellular response to oxidative stress. in a recent paper, we have studied the diversity of fungal gsts, considering zygo-, asco- and basidiomycetes. the major result of this study is that it suggests a link between the number of gst encoding sequences in the analyzed genomes and the fungal way of life. indeed, 46 and 27 gst - encoding genes have been found in the genomes of postia placenta and phanerochaete chrysosporium, respectively, compared to only 10 for the pathogen ustilago maydis or 11 for the yeast saccharomyces cerevisiae. p. placenta and p. chrysosporium are ligninolytic basidiomycetes, which use unspecific oxidative reactions to degrade lignin and, by extension, recalcitrant compounds. in p. in contrast, p. placenta is thought to degrade lignin by secreting various small iron - binding molecules initiating fenton reactions. beside these extracellular systems, oxidation of recalcitrant molecules could also occur from the action of various cell - wall linked oxidases such as cytochrome p450 monooxygenases. in the genome of both these fungi, a huge number of cytochrome p450 monooxygenase - related genes have been detected in comparison with other fungi [11, 12 ]. such a diversity of cytochrome p450 monooxygenases can be related with the high occurrence of gst encoding genes in their genome and thus to their ability to metabolize recalcitrant compounds found in their natural ecosystem. another interesting point is the overrepresentation of a particular class of gsts called ure2p both in the genome of p. placenta and p. chrysosporium. for these species, the ure2p class represents one - third of the total identified gsts (9/27 for p. chrysosporium and 17/46 for p. placenta). chrysosporium ure2p sequences cluster into two different subclasses. the first one, enclosing 8 of the 9 sequences, is related to the single ure2p isoform of s. cerevisiae. in yeast this protein is known to act as a negative regulator of the nitrogen catabolite regulation (ncr) in response to primary nitrogen source by disabling gln3p to activate transcription. moreover, the inability of a ure2 deleted mutant strain (ure2) to grow in presence of hydrogen peroxide (h2o2) has been demonstrated, and a relationship between diminishing levels of gsh and peroxide sensitivity was established. it was suggested that the susceptibility of the ure2 strain to the exogenous h2o2 can result from increased gsh degradation due to the deregulated localization of the gamma - glutamyl transpeptidase activating factors gln3p / gat1p. the last p. chrysosporium sequence, named pcure2p1, clusters in another group containing gsta (an4905) from aspergillus nidulans. gsta contributes to metal detoxification and contrary to the yeast isoform, it is not involved in the ncr. in this study, we performed an exhaustive analysis of ure2p sequences from fungal genomes, focusing on p. chrysosporium to correlate the diversity of sequences with the diversity of functions. the liquid culture medium consists of 5 mm sodium acetate ph 4.5, 1% glucose, 1 mm ammonium tartrate, 1% (v / v) base medium (20 g / l kh2po4, 5 g / l mgso4, 1 g / l cacl2), 7% (v / v) trace medium (1.5 g / l nitrilotriacetate, 3 g / l mgso4, 1 g / l nacl, 0.1 g / l feso47h2o, 0.1 g / l cocl2, 0.1 g / l znso47h2o, 0.1 g / l cuso4, 10 mg / l alk(so4)12h2o, 10 mg / l h3bo3, 10 mg / l namoo42h2o) and 225 m mncl2. the fungal inoculation was made by adding 2.510 spores (od650 = 0.5) per flask containing 100 ml of liquid medium. for the cultures on wood, the fungus was first grown for 3 days on malt agar plates, and autoclaved wood chips were then placed on the fungal mat. the fungus was also grown in liquid medium containing polycyclic aromatic hydrocarbons (pah). for this condition, the fungus was then transferred into new flasks containing pah. to prepare these flasks, a stock solution of pah was solubilised in hexane and added to the flasks to reach final quantities of 2.25 mg phenanthrene, 0.225 mg fluorene, 0.225 mg fluoranthene, and 0.225 mg anthracene per flask. 100 ml of liquid culture medium, the composition of which is described above but without glucose, was added in each flask after complete evaporation of hexane. for the global phylogenetic analysis, 189 sequences from both ascomycetes and basidiomycetes were found in ncbi genome database (http://www.ncbi.nlm.nih.gov/genome/) using blastp with all p. chrysosporium ure2p sequences as template. the deeper analysis focusing on basidiomycetes was performed using data from the fungal genomics program of the joint genome institute (jgi) (http://genome.jgi-psf.org/phchr1/phchr1.home.html) using blastp with p. chrysosporium ure2p sequences as template. alignments were done with muscle and the phylogenetic tree was constructed with the neighbour - joining method. predictions of subcellular localization were done using wolfpsort (http://wolfpsort.org/) and mitoprot (http://ihg2.helmholtz-muenchen.de/ihg/mitoprot.html) softwares. synteny was determined through the fungal genomics program of the joint genome institute (jgi). all references of the sequences used in this study are given in additional data. gene expression was checked by semiquantitative reverse transcription polymerase chain reaction (rt - pcr). total rna isolation was performed using the rneasy plant mini kit (qiagen, hilden, germany). rnase - free dnase treatment (qiagen) was applied according to the manufacturer 's protocol to avoid genomic dna contamination. reverse transcription (rt) reactions were performed with 500 ng of total rna using the masterscript kit (prime) according to the manufacturer 's protocol. rt products were amplified by pcr in the following conditions : dna denaturation for 1 min at 95c and 33 cycles at 95c for 5 s, 52c for 45 s, and 72c for 1 min using go taq dna polymerase (promega). the suitability of the extracted rna for rt - pcr amplification was checked by performing rt - pcr control experiments in the same amplification conditions with the ubiquitin encoding gene. pahs from total culture media were successively extracted three times with dichloromethane (dcm) (v / v) in separation funnels. dcm extracts were evaporated in a speedvac concentrator (thermo scientific rc1010), and dried samples were dissolved in acetonitrile for hplc analyses using a dionex ultimate 3000 system with a 5 m agilent eclipse pah of 4.6 150 mm c18 reverse phase column maintained at 30c. the compounds were detected and identified through dionex uv photodiode array detector at 254 nm. the elution solution of 70% acetonitrile and 30% h2o was used with a flow rate of 2 ml / min. p. chrysosporium pellets grown in liquid cultures with or without pah as indicated below were crushed, and observations were made with an epifluorescence microscope (nikon e600) with an hq - fitc - bp filter cube (chroma) for excitation at 345 nm and emission at 485 nm (dapi). amplifications of gst cdnas were performed from rt products obtained as described above, using the high proof herculase dna polymerase (stratagene). the pcr products were cloned into the ncoi and bamhi sites of the pet-3d vector (novagen) resulting in a construction devoid of a his - tag. the recombinant plasmid was then used to transform escherichia coli strain bl21 (de3) cotransformed by the helper plasmid psbet in order to provide the rare t - rnas for agg and aga codons. after induction with isopropyl -d-1-thiogalactopyranoside (iptg), proteins were purified using a combination of gel filtration and anion exchange chromatography as described in rouhier.. the activities of the recombinant proteins were assayed spectrophotometrically in 1 ml reaction medium using 8.5 m pcure2p4, 1.3 m pcure2p6, and 0.55 m pcure2p1. gst activity was assayed in 50 mm phosphate buffer ph 6.5, 2 mm chlorodinitrobenzene (cdnb) and 0.2 to 12.5 mm gsh. the ph dependency of pcure2p4 and pcure2p6 activities was assessed in 50 mm phosphate buffer ph 5.8 to ph 8.0 using 4 mm cdnb and 6 mm gsh. the other activities were assayed in 30 mm tris - hcl ph 8, 1 mm edta buffer, 1 mm -hydroxyethyl disulphide (he d), dehydroascorbate (dha) or peroxides as substrates, 0.2 to 12.5 mm gsh, 180 m nadph, and 0.5 iu of purified glutathione reductase [2123 ]. ure2p sequences have been identified within available genome database from ncbi using blastp and pcure2p amino acid sequences as template. concerning bacterial genomes, no similar sequence was found for archaea while some were identified for spirochaetales and proteobacteria except the epsilon subdivision. among eukaryotic organisms, only stramenopiles, dictyosteliida, and ure2p amino acid sequences from ascomycete and basidiomycete genomes have thus been collected and analyzed through a phylogenetic approach (figure 1). two main subclasses, previously called ure2p and cluster 2 [2, 7 ], are distinguishable and have been renamed ure2pa and ure2pb, respectively. these sequences were grouped according to the fungal taxonomy, suggesting a recent diversification of ure2p sequences among each phylum. while ure2pb sequences from saccharomycotina and pezizomycotina are close to each other, saccharomycotina sequences are rather related to basidiomycotina. we then focused our analysis on basidiomycotina by performing an exhaustive analysis of ure2p sequences in all available basidiomycete genomes from the jgi fungal genomics program. ure2p sequences have been found in all the species tested. a phylogenetic analysis of the sequences allowed classifying them in the ure2pa or ure2pb subclasses (data not shown). two species (malassezia globosa and schizophyllum commune) possess only one sequence from the ure2pa subclass, some other (agaricus bisporus, coniophora puteana, stereum hirsutum, fomitiporia mediterranea, heterobasidion annosum, serpula lacrymans, pleurotus ostreatus, coprinopsis cinerea, cryptococcus neoformans, puccinia graminis, laccaria bicolor, and tremella mesenterica) possess only sequences from ure2pb, while the other fungi exhibit both isoforms. globally, the fungi exhibiting the highest number of sequences are wood decaying fungi (figure 2, grey shadowing), however some species also involved in wood degradation show a relatively small number of ure2p sequences (auricularia delicata, schizophyllum commune, heterobasidion annosum, serpula lacrymans, pleurotus ostreatus, and tremella mesenterica). thus, the diversification of ure2p sequences does not seem to be related with fungal evolution, nor with the wood degrading properties of the fungi, suggesting the occurrence of other environmental pressures. globally the ure2p synteny is not conserved among agaricomycotina, except for ure2pb sequences in few species which possess only these isoforms (s. lacrymans, l. bicolor, and h. annosum) (figure 3). ure2pb genes are surrounded with putative mitochondrial aminomethyltransferase, cytochrome c oxidase assembly protein, dna methylase and rna binding protein. however the putative link between ure2pb and either mitochondrial metabolism or nucleic acid modification pathways has not been documented. the expression of all p. chrysosporium ure2p coding genes was monitored in two conditions using semiquantitative rt - pcr (figure 4). the first one corresponds to ligninolytic conditions (figure 4(a)), and the second is a condition where the fungus is exposed to genotoxic pahs. in this condition, p. chrysosporium was able to dissipate between 15 to 30% of the initially added pah in the culture medium after 10 days of incubation, the highest removal occurring with fluorene and fluoranthene. taking advantage of the fluorescent properties of pah, we were able to detect their intracellular accumulation in p. chrysosporium in our culture conditions after 10 days of treatment (figure 4(b)). this accumulation occurred both in hyphae and spores, not in vacuoles but rather in lipid vesicles as shown before in fusarium solani. no difference was highlighted concerning gene expression in ligninolytic (wood) compared to nonligninolytic (malt) conditions (figure 4(a)) : pcure2p4 was not expressed, pcure2p3, pcure2p5, and pcure2p6 were slightly expressed, while the others were constitutively expressed in both conditions. pcure2p1, pcure2p7, and pcure2p8 were constitutively expressed with and without pah treatment, while the pcure2p4 and pcure2p6 genes were specifically induced after pah treatment (figure 4(b)). pcure2p4, pcure2p6, and pcure2p8 genes are repeated in tandem but are not regulated in the same manner suggesting a different regulatory system between pcure2p8 and the two others. pcure2p2, pcure2p3, pcure2p5, and pcure2p9 are not or very slightly expressed in both conditions. the amino acid sequences of pcure2p4 and pcure2p6 are very similar to one another showing 83.2% identity. the proteins have the classical organization of gst, that is, a gsh - binding domain (thioredoxin domain or g - site) and a -helical domain (gst c - domain) (figure 5). the trx - domain of these proteins is highly conserved, exhibiting amino acids known to be involved in the interaction with gsh in the s. cerevisiae isoform (scure2p) [26, 27 ]. asn124 and arg164 (scure2p numbering) are shared by scure2p, pcure2p1, pcure2p4, and pcure2p6. glu180 and ser181 residues are only conserved in scure2p, pcure2p4, pcure2p6 but not in pcure2p1. another interesting point is the presence of a putative mitochondrial targeting peptide in pcure2p1, suggesting a potential role for this protein in the organelle. the major difference between the yeast isoform and the other fungal sequences is the presence of the prion domain in scure2p. this gln / asn rich sequence at the n - terminal end is required for aggregation properties of the protein. however, deletion of this n - terminal region has no effect on the stability or folding of the protein in vitro. among all the fungi analysed, only sequences from saccharomycotina exhibit the n - terminal prion domain, suggesting that the other proteins have evolved separately in the other fungi, the most likely hypothesis being the acquisition of this property in saccharomycotina. the differences in the amino acid sequences are likely to influence the catalytic properties of the corresponding proteins. to test this hypothesis, the recombinant pcure2p1, pcure2p4, and pcure2p6 proteins have been produced in escherichia coli and purified. for both enzymes, the highest specific activity was measured around ph 6.5 (figure 6). the affinity of pcure2p4 and pcure2p6 are, respectively, 2.62 mm 0.26 and 3.12 mm 0.20 for gsh, and 2.46 mm 0.62 and 2.49 mm 0.71 for cdnb. contrary to scure2p, pcure2p4 and pcure2p6 had no gsh peroxidase activity whatever the peroxide used (hydrogen peroxide, t - butyl peroxide, or cumene peroxide). the purified recombinant pcure2p1 was not active with both cdnb and peroxides but did reduce he d and dha, thus exhibiting thioltransferase activity. while the single ure2p isoform of s. cerevisiae has been well studied for its prion properties, its involvement in the ncr, and its role in the oxidative stress response [13, 14, 28, 29 ], this study is the first description of ure2p in other fungi. this class is extended in basidiomycetes and especially in saprophytic fungi, suggesting a putative link between these gsts and the degradative properties of the fungi. in many species, ure2p genes are duplicated in tandem, revealing a monophyletic phylogenetic relationship between genes as described recently for insect gsts. moreover, a tyrosine hydroxyl group thought to act as a hydrogen bond donor to the sulphur of gsh thus lowering its pka to stabilize a nucleophilic thiolate is conserved in the first 15 amino acid residues of fungal sequences. the presence of this residue at the end of the first -sheet defines a subgroup of gsts called y - gst type. based on the taxonomic distribution, it has been suggested that this type has evolved more recently compared to the so - called s / c - gst type found in other gst classes such as omega. we have tried here to decipher the significance of this diversification and the likely physiological roles of these enzymes. pcure2p4 and pcure2p6 from the ure2pa subclass exhibit glutathionylation activity with cdnb, while pcure2p1 from the ure2pb subclass possesses only deglutathionylation (also called thioltransferase) activity. incidentally, scure2p, which clusters into the ure2pa subclass possesses deglutathionylation and peroxidase activity. however, none of the tested pcure2p exhibits the peroxidase activity described in yeast, suggesting other functions in cell. to test this hypothesis, complementation tests using a s. cerevisiae ure2p mutant the results showed that pcure2p1, pcure2p4, and pcure2p6 are not able to restore the phenotype of the mutant concerning both the sensitivity to h2o2 and its function in the ncr. however, a homologue of the yeast gln3p is present in the genome of p. chrysosporium (phchr 43861), suggesting that ncr could exist in this fungus. the main conclusion is that pcure2p1, 4, and 6 do not have the same activity as scure2p in protecting cells against oxidative, heavy metal, or aromatic compounds stress, even if pcure2p4 and pcure2p6 belong to the same subclass as the yeast isoform. we can hypothesize that pcure2p proteins are not directly involved in rescuing oxidative stress such as gsts exhibiting peroxidase activities, but rather acts to detoxify specific substrates. the specific expression of pcure2p4 and pcure2p6 genes after pah treatment is in accordance with the enzymatic data. pahs are aromatic molecules that are degraded by at least three mechanisms in fungi : one uses the cytochrome p-450 system which is composed of a superfamily of monooxygenases, one uses the fenton reaction, and the other uses the soluble extracellular enzymes of lignin catabolism, including lignin peroxidase (lip), manganese peroxidase (mnp), and laccases which are nonspecific and oxidize a wide variety of organic compounds [37, 38 ]. however, in our conditions, no lip or mnp activity was detected in the culture medium ; we rather observed an intracellular storage of pah in lipid vesicles as previously shown in fusarium solani. we can hypothesize a role of pcure2p4 and 6 in intracellular pah glutathionylation, transport, or oxidative stress rescue., the homologue angsta contributes to metal and xenobiotic detoxification, as evidenced by the sensitivity to selenium, silver, nickel, sulphanilamide, and pyrrolnitrin of strains lacking a functional copy of gsta. the analysis of the soluble proteome of p. chrysosporium has revealed the presence of pcure2p1 in a standard culture condition with a relative low abundance. moreover, the authors have shown a 3-fold upregulation of the protein after copper treatment. another study revealed an induction of pcure2p1 gene in response to nonylphenol. because of its putative mitochondrial localization a bacterial homolog of pcure2p1 called yfcg with 43% amino acid identity has been characterized in escherichia coli. since this ure2pb isoform is largely represented among fungi (figure 2), the question of a putative bacterial origin is open. in conclusion, we showed in this study that ure2p gsts in p. chrysosporium do not have the same role as scure2p. the repartition into 2 distinct subclasses in many fungi suggests that these proteins have evolved separately inside the ure2p class, adapting their functional specificities to environmental constraints. it is indeed assumed that the genes that are under external pressure may evolve to carry out myriad functions with diverse substrate specificities through local duplications followed by diversification. the putative link between the saprophytic properties of the fungi and the function of the ure2p proteins remains to be elucidated to consider the use of these gsts with high substrate specificities as environmental biomarkers.	the glutathione - s - transferase (gst) proteins represent an extended family involved in detoxification processes. they are divided into various classes with high diversity in various organisms. the ure2p class is especially expanded in saprophytic fungi compared to other fungi. this class is subdivided into two subclasses named ure2pa and ure2pb, which have rapidly diversified among fungal phyla. we have focused our analysis on basidiomycetes and used phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. the results show that among the nine isoforms found in p. chrysosporium, two belonging to ure2pa subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. moreover, we have highlighted differential catalytic activities and substrate specificities between ure2pa and ure2pb isoforms. this diversity of sequence and function suggests that fungal ure2p sequences have evolved rapidly in response to environmental constraints.
the worldwide population is aging and consequently the incidence of osteoporosis is increasing. this systemic, metabolic disease is most distressing to patients and healthcare providers in the occurrence of osteoporotic fractures of the hip and spine and occasionally, various other sites throughout the skeleton. osteoporotic fractures throughout the skeleton can cause physical deformity, loss of height, respiratory distress, appetite suppression, constipation, weight loss, emotional anguish, and eventually mortality. it has been well documented that those with osteoporotic fractures are at greater risk of subsequent fractures, particularly in the first post fracture year (lindsay 2001). thus, those who have experienced one osteoporotic fracture must cope with the constant fear of successive fractures. in the past several decades, osteoporosis detection and the pharmacological improvement in bone mineral density (bmd) and reduction of osteoporotic fractures incidence has improved markedly. several different classifications of drugs (bisphosphonates, selective estrogen receptor modulators, calcitonin, parathyroid hormone) have shown in a variety of clinical trials to increase bmd and reduce fracture rates with varying efficacy. despite these successes, there is a need for nonpharmacologic therapies used separately or adjunctively to prevent osteoporotic fractures. this review will explore the current and future options for the nonpharmacological prevention of osteoporotic fractures. preservation of muscular and skeletal mass is an important component in the care of patients with increased risk of osteoporotic fractures. a recent cochrane review concluded that aerobic, weight bearing, and resistance exercises were effective in increasing bmd of the spine in postmenopausal women and that regular walking was effective in building bmd at the hip (bonaiuti 2002). surprisingly, controlled studies have not shown that exercise activities are effective in the reduction of osteoporotic fractures. few studies have examined the ability of women diagnosed with osteoporosis to improve bone mineral density and reduce fractures via an exercise training program (klentrou 2005). a recent comprehensive review of musculoskeletal rehabilitation literature confirmed that there is still no conclusive evidence that coordinated, multidisciplinary care is more effective than conventional hospital care in the rehabilitation of patients with osteoporotic fractures (pfeifer 2004). it has been hypothesized that the lack of conclusive evidence for exercise in the prevention of osteoporotic fractures is largely due to the difficulty in conducting large clinical trials of exercise with elders diagnosed with disease states. the recommendation of regular, weight bearing and strength training exercise for patients at risk for falls and osteoporotic fractures is generally believed to be safe and effective with supervision. additionally, clinicians should consider the recommendation of physical therapy when appropriate. according to the national osteoporosis foundation, exercise recommendations for patients at risk for osteoporotic fractures should include the referral to a specifically trained physical therapist. after a thorough physical assessment, exercise activities should focus on : body mechanics and posture, balance, gait and transfer training, resistance weights and progressive aerobic activities (nof 2003). while the optimal beneficial ratio between exercise and bone mass has yet to be proven, referral to a physical therapist, who is trained in osteoporosis care and cognizant of the issues regarding risk for osteoporotic fractures may be indeed be beneficial. in addition to low bone density, patients at risk for osteoporotic fractures experience other risk factors for falls. sensory deficits, living alone, advanced age, musculoskeletal weakness, improper footwear, diminished reflexes and coordination, medications, and comordid conditions may contribute to a propensity for falls in this population. elders who have fallen previously are at risk for future falls due to lack of confidence and potentially uncorrected environmental hazards. several studies have been conducted to examine interventions that might reduce risk factors and decrease the incidence of falls. an educational intervention comprised of informational brochures and post - discharge telephone counseling recently completed in a canadian emergency department was no more effective than usual care in reducing fear of falls or recurrent falls in community - dwelling patients. researchers concluded that future strategies must be more comprehensive than simple education to prevent falls (rucker 2006). a year long study of 620 elders completed recently in australia demonstrated that an individualized fall prevention program consisting of exercise and sensory improvements reduced some risk factors but did not prevent falls (lord 2005). despite the dearth of research evidence, modifications to reduce fall risk should be explored with each patient in an individualized and thoughtful manner. generally, advice should include : visual and auditory improvements with vision correction or hearing aids if appropriate, improvement of household lighting, elimination of obstacles such as electrical cords and throw rugs, installation of assistive devices in the bathroom and kitchen, and use of canes and walkers for improved ambulation. treatment of comorbid conditions to reduce the risk of dizziness, electrolyte imbalances, blood pressure fluctuations, blood sugar irregularities, or side effects of prescribed medications should also be explored. it has been well documented that osteoporotic hip fractures can be extremely costly in both economic and quality of life indicators. when an elder with osteoporosis of the hip falls and sustains the typical fracture of the femoral neck, it often signals a downward spiral of pain and loss of independence. commonly, an osteoporotic hip fracture results in the end of living unassisted in the community for previously independent elders. while pharmacologic therapies have shown to reduce hip fracture incidence, many elders are not identified as osteoporotic, or adherent to therapy if prescribed. external hip protectors, which consist of hard plastic inserts in specially designed elastic briefs, have been developed and researched in the last decade as a means of protection against hip fractures caused by falls. in studies of nursing home patients, the use of hip protectors has reduced the incidence of osteoporotic hip fractures (harada 2001 ; lauritzen 2003 ; schoor 2003). conversely, there is little evidence to support the use of hip protectors outside the nursing home setting (birks 2003 ; sawka 2005). the economic benefits of hip protectors have been explored in several studies (waldegger 2003 ; honkanen 2005 ; meyer 2005 ; oliver 2005). the major problem regarding the use of hip protectors is compliance and continued use of the devices in both nursing home and community dwelling women with osteoporosis (burl 2003 ; patel 2003). unfortunately, current designs of the hip protectors are costly and somewhat difficult to apply, particularly for frail elders who are at most risk for hip fracture. future modifications to style, reduction in cost, and ease of application may increase the use of hip protectors and thus strengthen their effectiveness in hip fracture prevention. in addition to the modalities discussed above, several alternative therapies for the prevention of osteoporotic fractures and fracture pain have been explored. a device that delivers dynamic motion therapy has been explored as a drug - free way to halt bone loss and grow new bone, which would ultimately reduce fracture incidence. this device is a platform that transmits high - frequency, low intensity mechanical forces through the patient s feet and up through the skeleton. patients are instructed to stand on the platform at least five days per week for 20 minutes each day. currently it is approved and marketed in canada, austria, germany, ireland, the united kingdom, malaysia, israel, australia, and new zealand. in the united states, evidence of the efficacy of this technology on animal models for the improvement in quantity and quality of bone has been documented (rubin 2002). the preclinical work led to human studies in children with low bone mass due to disabling conditions (ward 2004) and postmenopausal women (rubin 2004). both of these studies resulted in increases in bmd in patients subjected to the dynamic motion therapy treatment. the underlying theory of dynamic motion therapy is that bone is very sensitive to mechanical stimulus and that it can adapt its structure to become denser and stronger when functional demands are placed on it. in a recent study, investigators postulated that increasing bone mass in young women may ultimately reduce the risk of osteoporosis in the elderly. in this year long study of 48 young women (1520 years) with low bone density and a history of at least on skeletal fracture, daily use of dynamic motion therapy for a period of at least 10 minutes resulted in increases in bone and muscle mass in the treatment group. if these increases could be maintained in larger groups and preserved through adulthood, this intervention may truly be a truly preventive fracture therapy (gilsanz 2006). researchers are currently examining this technology to determine if it would be additionally useful to combat bone loss that occurs in astronauts during long - term space flights. other modalities have been explored to assess the effect on bone density enhancement and ultimately fracture prevention. recently a randomized, prospective study of the effects of tai chi on bone mineral density concluded that tai chi as an exercise intervention is beneficial for retarding bone loss which ultimately may help to reduce fracture risk (chan 2004). it has also been postulated that chiropractic care, acupuncture, and chinese herbal medicine may assist in the improvement of bone density and ultimately the reduction of painful osteoporotic fractures (ernst 2003 ; yingxia 2002 ; xu and lawson 2004). however, fractures should not represent the end of medical options or assistance as several helpful modalities utilized by various health care professionals exist. pharmacological therapies can do much to improve bone density and reduce the incidence and severity of fractures. in addition to the appropriate prescribed drugs, clinicians need to be informed and encouraging about the use of nonpharmacological measures to assist patient at risk for osteoporotic fractures.	osteoporosis is a systemic, metabolic disease that can result in debilitating fractures. the lasting effects of vertebral and hip fractures can cause acute and chronic pain, deformity, and emotional distress. research evidence and clinical experience have determined that weight bearing and strength training exercise, fall prevention efforts, hip protectors, and some alternative therapies may assist patients in avoiding the pervasive and lasting effects of osteoporotic fractures. clinicians should consider the recommendations of nonpharmacological measures to assist patients at risk for experiencing the culminating event of this destructive disease.
orthopaedic surgeons may find it difficult to get rotationally anatomical reduction of a shaft fracture of the humerus. if rotational control is not achieved, nonunion is more likely to occur.2 in addition, malrotation of the humeral component of total elbow replacement influences laxity and causes maltracking.3 the rotational deformity of the distal humerus may limit the motion of the ipsilateral shoulder joint.4 few technical tip or study have introduced to prevent malrotation of the humerus. the authors tried to find a simple landmark for prediction of rotational status during the operation.. however, the position and shape are similar when the left and the right bicipital groove structures are compared in each individual.57 the objective of this study was to evaluate the reliability of the bicipital groove as a point of reference for the prediction of the rotational state of the humerus, by comparing the contralateral bicipital groove images on standard radiographs. the mean age of the 62 men was 34 7.5 years (range, 24 - 53), and the mean age of the 38 women was 33 6.8 years (range, 22 - 48). subject who had a history of shoulder disease or fracture of the humerus were excluded. first, the subject was placed in the supine position with the shoulder abducted to 90 ; the elbow flexion was to the same at 90 when the forearm was pronated fully. the images were obtained in the cephalic view at a 45 angle, a point with a clear outline of the lesser and greater tuberosity [figure 1 ]. images of the bicipital groove, at 45 of external rotation, were used in this study [figure 2 ]. the angular orientation of the bicipital groove has been referenced to the transepicondylar axis at about 55 in prior studies.89 then, standard line were drawn on the medial border of ulna from the tip of olecranon to the styloid process of the forearm [figure 3a ]. at the neutral rotation point, the shape of the proximal humerus was recorded and transferred to the right monitor of a c - arm image intensifier (oec series 9800 ; oec medical systems, salt lake city, utah, usa) as a reference point for the rotational state. the subject arm was in a random position, rotationally, with the shoulder abduction at 90. the observer group was composed of one expert surgeon (a), one orthopaedic resident (b), and one medical student (c). all three observers stood in front of the monitor, where they could not see the individual. they compared the images of the bilateral bicipital groove on the monitors with regard to the contour and proximal and distal width of the bicipital groove, distance of the interval from the lateral cortex of the proximal humerus to the lesser tuberosity, with the naked eyes [figure 2c ]. an assistant rotated the arm being examined inward or outward, and the point where the subjective image of the arm showed a similar and symmetrical shape to the image of the bicipital groove, previously recorded on the right side of the monitor [figure 3b ], was noted. the distribution and mean angular discrepancy were calculated using the values obtained from the three observers. the interobserver reliability was evaluated by calculating the intraclass correlation coefficient (icc) using pasw 17.0 statistical software (spss inc, chicago, il, usa) for windows (microsoft, redmond, wa, usa). preparing the image of the contralateral shoulder. (the individual is in the supine position with the shoulder abducted at 90 (a), with a fully pronated forearm (b), and the image intensifier is at 45 in the cephalad view (c)) fluoroscopic images of the proximal humerus. (internal rotation at 45 (a), neutral (b), external rotation at 45 which the line of the greater and lesser tuberosity make clear contour of the bicipital groove on radiograph in 45 degree rotated externally (c)) comparing the subjective shoulder with the contralateral shoulder. (lines are drawn on the medial border of the ulna from the tip of the olecranon to the styloid process on the subjective arm (a). an assistant rotate the subjective arm with random rotation being examined inward or outward until an observer select the similar image with the contralateral one (b) and measure the angular difference from the neutral point (c)) all observations which were expressed as an absolute value were placed within 15 of the neutral point. the discrepancy for the mean angular measurements were from -4 to + 4 in 72% of the assessments for each observer and 99% of the assessments were within 10 [table 1 ]. those of each observer was 3.6 (3.0) in observer a, 3.2 (2.3) in observer b and 3.4 (2.7) in observer c. these differences were not statistically significant based on analysis of variance (anova) (p = 0.495) [table 2 ]. in addition, the intraclass correlation coefficient (icc) was calculated to evaluate the interobserver reliability. the icc value was calculated to be 0.847, which indicates a high interobserver reliability (> 0.70). the bicipital groove of the proximal humerus has been extensively studied and its anatomy well documented.81011 some investigators have used the bicipital groove as a reference point with computed tomography.912 the relationship of the bicipital groove with humeral retroversion in cadavers was studied.1314 several investigators have suggested that the position of the bicipital groove relative to the humeral head is comparable.1315 however, most investigations of the bicipital groove focused on retroversion of the humerus for the prevention of malrotation, which was suggested as a cause of hemiarthroplasty failure.15 the purpose was to evaluate the reliability of the bicipital groove as a point of reference for the prediction of the rotational state of the humerus on two - dimensional radiograph of c - arm image intensifier during operation especially for humeral shaft fractures. this study assumed that the anatomical structures of the bicipital grooves were the same on both the right and left sides. delude.6 and hernigou.7 reported no meaningful difference in comparisons of the left side and right side. boileau.5 suggested that assessment of the rotational state of the humerus may be most accurately achieved based on the contralateral bicipital groove. cassagnaud.,17 by contrast, reported a considerable difference between right and left side measurements. at a rotation of 45 externally, the humeral head was rotated until the base of the head was perpendicular to the axis of the proximal humerus.15 in addition, the greater tuberosity was brought into relative clear prominence.18 the 45 externally rotated view showed relatively clear outlines of the contours of the bicipital groove that could be easily compared to the contralateral side with the naked eye. there is a relatively wide range of variation in the bicipital groove angle from 5 to 97, with a mean value of 55.5.9 in cadavers, the measured angle of the bicipital groove was about 55.8. hempfing.15 suggested that the centre of the bicipital groove differs from the epicondylar axis by about -10. this means that the epicondyles of the distal humerus are approximately 45 in relation to the internal rotation. thus, this position could show a relatively neutral rotational state of the humerus, hypothetically. in this study, the c - arm image intensifier was at a 45 in the cephalad view, instead of rotating the arm. it was difficult for the assistant to rotate the shoulder of an individual more externally. the purpose of this study was to determine an accurate and simple method that could be applied in the operating room setting. similar to evans and wales19 evaluation of the rotational state, using the radial tubercle as a land mark or as kim.20 used the lesser trochanter of the femur as a landmark for the correction of the rotational status during surgery for a fracture of the shaft of the femur. to the best of our knowledge, this is the first study to consider the bicipital groove as a useful landmark for estimating the rotational state of the humeral shaft fracture in the operating room setting. this methodology also could benefit for operating the corrective osteotomy and malunion of humeral shaft fractures. li.1 found that the degree of malrotation correlated with a decreased range of motion in patents who underwent intramedullary nailing. rotational deformities were took place according to patient 's position during surgery.21 in the operating room, the procedures were performed by comparison of the injured side with the asymptomatic, contralateral side that was evaluated before surgery using the c - arm image intensifier. first, a 90 degree abduction and 45 external rotation antero posterior view of standard radiographs of the shoulder was performed. the shape of the bicipital groove, of the contralateral side, was then stored preoperatively in the c - arm image intensifier 's second monitor. before inserting the device, the proximal portion was rotated while the elbow was extended fully and the forearm pronated exactly until the shape of the greater tuberosity and interval of the bicipital groove was identical on both sides. this might be an effective method for successful surgical treatment of humeral shaft fractures with plate osteosynthesis or intramedullary nailing. this can be performed before the insertion of the locking screws, the surgeon controls the rotation of the proximal fragment. the two dimensional radiograph of c - arm image intensifier obtained was less precise than a three dimensional image and the comparison of the contour of the bilateral bicipital groove with the naked eyes of the observers was not objective. however, our methodology is cost effective, give less radiation exposure than the preoperative ct evaluation. further observation of clinical outcome and confirmation of these findings will be required in a larger study. a simple radiological contour of the bicipital groove is a useful landmark. comparing the shape of the bicipital groove in the 45 degrees externally rotated standard view bilaterally, was an effective method for estimating the rotational state of the humerus, intraoperatively.	background : accurate reduction of rotational displacement for transverse or comminute fracture of humeral shaft fracture is difficult during operation. the purpose of this study was to evaluate the reliability of the bicipital groove as a point of reference for the prediction of the rotational state of the humerus on two dimensional images of c - arm image intensifier during operation for humeral shaft fractures.materials and methods : one hundred subjects, 62 male, 38 female, aged 22 - 53 years were recruited contralateral bicipital groove on the 45 degrees externally rotational standard anterior - posterior view recorded before surgery. three observers, watched only contour of bicipital groove in monitor of c - arm image intensification with naked eye without looking at the subject and predicted rotational state of the humerus by comparing the contour of the opposite side of bicipital groove. the angle of discrepancy from real rotational position was then assessed.results:the mean (sd), angular discrepancy between the neutral point and the predicted angle was 3.4(2.7). a value within 5 was present in 72% of cases. all observations were within 15. there was no interobserver variation (p = 0.47). the intraclass correlation coefficient (icc) was 0.847.conclusion:contour of the bicipital groove on simple radiograph was a useful landmark. comparing the contour of the bicipital groove in the 45 degrees externally rotational standard view bilaterally, was an effective method for reduction of rotational displacement of the humerus.
the vein of marshall (vom) is an embryonic remnant of the left superior vena cava.1) the ligament of marshall (lom), which contains nerves, fibrous tissues, and muscle bundles (marshall bundle), is adjacent to the vom.2) the lom is a trigger of ectopic beats initiating paroxysmal atrial fibrillation (af)3) and is innervated by the arrhythmogenic autonomic nerve.4) moreover, the lom is connected to the neighboring left atrium (la) including the perimitral area and lateral ridge, and may be a part of the re - entrant circuit. therefore, the lom may play a role in the initiation and maintenance of af or atrial tachycardia (at). recently, the feasibility of injection of pure ethanol into the vom was demonstrated ; the adjunctive effect to pulmonary vein isolation (pvi) was indicated.5) in addition, vom ethanol infusion assists in consisting achieving bidirectional perimitral isthmus (pmi) block.6) however, the effectiveness of the infusion in persistent atrial fibrillation (peaf), which is an extensive substrate in the la, is unclear. in the present case, af was terminated by ethanol infusion adjunctive to complex fractionated atrial electrogram (cfae) guided ablation in a patient with peaf. a 64-year - old male presented with a 6-month history of symptomatic drug refractory peaf. the patient was admitted for catheter ablation of af. transthoracic echocardiogram revealed normal ejection fraction of 55 - 60% with mildly enlarged la of 43.8 mm in the anterior - posterior diameter. cardiac magnetic resonance (cmr) imaging revealed late gadolinium enhancement (lge) in 20% of the la surface including the anterior and posterior wall. cfaes in the la were mapped for 6 seconds using ensite navx system three - dimensional automated software (endocardial solutions, st. the cfaes were predominantly located at perimitral, septal area and left atrial appendage base (fig. activation pattern at the high clustering of cfae was mapped using high density 20-pole catheters (afocus ii ; st. the activation patterns obtained from three af beats of unipolar recordings at each cfae site were classified into four categories : 1)complete reentry, 2) incomplete reentry, 3) wave collision, and 4) wave breakup with conduction block. we preferentially targeted cfae areas with complete or incomplete reentry pattern because these sites might play an important role as rotor. in this case, only the perimitral area had re - entrant, rotor - like electrical activity (fig., we assumed that the perimitral area could be the critical site for af perpetuation. therefore, we decided to infuse ethanol into the vom as an add - on to radiofrequency ablation. at first, cannulation into coronary sinus was performed by ablation catheter (therapy coolflex ; st. paul, mn, usa) and 8 fr sheath (sl1 sheath ; st. paul, mn, usa). to confirm the existence and feasibility of vom, (7 fr ; cordis, miami, fl, usa) was subselectively engaged into the vom and then a hi - torque floppy ii angioplastic guidewire 0.014 (abbott, santa clara, ca, usa) was advanced into the vom as far as possible. a ryujin plus otw 2.020 mm angioplasty balloon catheter (terumo, tokyo, japan) was advanced into the vom over the angioplastic wire and inflated at the ostium of vom. engagement of the balloon catheter into the vom was confirmed by injecting contrast medium into the vom (fig. 2b), and 1 cc of 100% ethanol was injected into the vom three times (fig. each injection was delivered over 1 minute through a balloon lumen and flushed with normal saline. within 30 seconds after the first ethanol injection la local activation time mapping demonstrated macro - reentrant at, the circuit of which involved reentry around the mitral annulus in a counterclockwise fashion (fig. bidirectional block of pmi line was achieved after ablation within cs adjunct to pmi ablation (fig. this case report demonstrates that ethanol infusion into the vom can effectively eliminate cfaes at pmi. in addition, we suggest that activation mapping at cfaes area during af can identify the critical cfaes that maintain the peaf. even though the underlying etiology of cfae has not been fully elucidated, cfaes are considered as slow conduction or pivot sites, which represent continuous reentry of the fibrillatory waves.7) based on these theories, cfae guided ablation is widely used for modifying the extensive substrate of the la and/or right atrium in patients with peaf. however, whether cfae reflects slow conduction or pivot site, or only wave collision that overlaps different wavelets at the same area is contentious. moreover, 90% of continuous cfae sites do not overlap over lge on cmr and occur at non - lge and patchy lge sites.8) therefore, identification of the critical pivot site perpetuating af among the continuous fractionated electrogram is necessary. in the present case, we performed unipolar activation mapping at the cfae areas using a high density circular catheter to identify critical pivots and preferentially targeted the sites with complete or incomplete reentry pattern because these areas might be crucial for the perpetuation of af. among three cfaes sites, only the pmi area had complete reentry pattern and ablation at this site easily lead to termination of af. therefore, this case suggests the usefulness of unipolar mapping to identify the true cfae sites which is related to the persistence of af. however, there is limitation of unipolar mapping, which includes difficulty in positioning the lasso catheter at the la septum, la lateral ridge and below the la appendage. the present case highlights that peaf can be terminated and organized to at by ethanol infusion into the vom. valderrbano.5) demonstrated the feasibility of ethanol infusion into vom adjunct to pvi ; ethanol infusion decreased voltage around pmi and the la lateral ridge. in addition, ethanol infusion in the vom has an adjunctive effect in perimitral flutter ablation.6) the proximal portion of lom has complex muscle fiber connections to cs adjacent to pmi.1) the mid - portion of lom is directly connected to the la lateral ridge and pulmonary veins.1) these muscle fiber connections can act as an anchor of re - entry. therefore, ethanol infusion into the vom can lead to substrate modification at pmi and la lateral ridge. moreover, the pmi area and la lateral ridge are too thick to achieve transmural lesion only by endocardial ablation. therefore, ethanol infusion in the vom can be an effective intervention for achieving transmural scar and facilitating durable conduction block at this area. however, in this case, bidirectional block of pmi was not achieved by only ethanol infusion, but required endocardial pmi and intra - cs ablation adjunct to ethanol infusion. the transmurality and bidirectional block could be achieved by pmi ablation in only 15% of patients.9) this finding suggests that ablation of pmi concurrent with endocardial or epicardial lateral ridge may be needed to achieve transmural lesion in most cases. therefore, ethanol infusion into vom can be applied to the patients with peaf, especially when cfaes clusters at pmi area and the la lateral ridge.	we report the case of a 64-year - old male with persistent atrial fibrillation (af) terminated by ethanol infusion into vein of marshall as add - on therapy. three - dimensional automated complex fractionated atrial electrogram (cfae) during af revealed clustering of cfae at perimitral isthmus (pmi) and its unipolar mapping showed rotor - like activation, which was suggested to be critical in the perpetuation of af. af was organized to atrial tachycardia (at) by 100% ethanol infusion in the vein of marshall. adjunctive radiofrequency ablation at pmi successfully terminated at and led to bidirectional block of pmi.
strong evidence shows that pilonidal disease originates in the epidermis, in a midline stretched hair follicle. early pilonidal changes are amplified due to deep tissue disruption from moisture, anaerobic conditions, hairs, and bacteria. on the basis of these observations, a new paradigm of origin emerges that pilonidal disease is exclusively an epidermal problem, rather than a deep tissue problem, which is the theory behind current recommendations for wide excision [2, 3 ]. therefore, the focus of treatment should be actions to change the conditions that attack epidermis, rather than wide excision which attacks deep and healable tissue. the most difficult complication that may follow surgery for pilonidal disease is a persistent unhealed midline wound, most commonly seen after laying open or excision of primary disease. although the authors are familiar with all procedures, karydakis and z - plasty procedures are among the common procedures done currently, so they were chosen in the study. karydakis procedure is basically a gluteal advancement flap. unlike other flap procedures (i.e., rhomboid flap, z - plasty, and gluteal rotation flap) which are fasciocutaneous, karydakis flap is adipocutaneous flap ; it is, therefore, technically easier, less bloody, and less time consuming ; it obviously has a better cosmetic outcome as it leaves a single, lateral, longitudinal scar ; and it requires significantly shorter hospital stay. the present study was done to study karydakis flap reconstruction as a treatment of symptomatic pilonidal sinus disease and to compare karydakis flap reconstruction and double z - plasty as a treatment of symptomatic pilonidal sinus disease. with proper ethical permission, a prospective randomized trial was conducted on 50 cases of symptomatic or recurrent pilonidal sinuses reported in outpatient department of j.l.n. the sample size is decided according to the past experience of cases of pilonidal sinuses and local guidelines. the cases are randomized on the basis of hospital registration number by the initial screening personnel who did not get involved in operative part. after proper preoperative workup and antibiotic prophylaxis, first group (gp 1) undergoes wide excision with karydakis flap reconstruction and the second group undergoes wide excision with double z - plasty. karydakis technique consisted of a vertical eccentric elliptical incision extended down to the postsacral fascia ; complete removal of unhealthy tissue, including a rim of normal tissue around the cyst and sinus tract ; mobilization of the medial wound edge by undercutting the adipose tissue at a depth of 1 cm ; advancement of flap across the midline to the postsacral fascia ; and suturing of its edges to the lateral wound edges. close attention was paid to ensure adequate mobilization and tension free fixation of flap, which was lateralized 1 cm from midline (figures 1(a)1(d)). in group 2 (gp 2), z - plasty was performed by a vertical elliptical incision extended down to the postsacral fascia ; complete removal of unhealthy tissue with a rim of normal healthy tissue ; two oblique incisions given by making an angle of 60 degrees with the vertical line (figures 2(a) and 2(b)). the flaps are suitably interdigitated and sutured in three layers according to depth of wound so as to avoid negative spacing. all patients were asked to pay attention to hygiene rules, to not sit or use a semisitting position for 15 days, and to depilate hairs around their gluteal region as necessary for every 3 months beginning after hospital discharge. data obtained during hospital admission and at 10, 21, and 30 days after discharge included complications, duration of hospitalization healing, work off period, and degree of satisfaction. the surgical procedure was considered unsuccessful (early failure) if a given patient experienced purulent discharge, abscess, or complete wound dehiscence for which further treatment was required within 30 days of the operation. all similar events that occurred following an uneventful recovery period were considered recurrent disease. the data was analysed using unpaired student 's t - test and the proportions were analysed using fisher exact test. the most common age of presentation in both groups was 2130 years (mean age : 28 years). in both groups, the incidence in male patients was 9 times more than that in females. in both groups, the incidence was more common in patients with coarse body hair density (8084%) and among the patients with a deep natal cleft (88%). grossly, primary disease was present in 82% of the patients among which 87.8% were males. recurrent disease was present in 20% of patients and all of them are males and all these patients have deep natal cleft. in both groups, the common clinical presentations were small pus discharging sinus (56%), multiple discharging sinus (28%), and swelling with pus discharging sinus (16%). the duration of hospitalization for karydakis procedure was significantly lesser than that for double z - plasty (mean : 4 versus 11 days, resp. similar observations are obtained for duration of wound healing (mean : 13 versus 30 days, resp., p < 0.001), work off period (mean : 11 versus 36 days, resp., p < 0.001), and the duration of presence of significant pain postoperatively (mean : 11 versus 38 days, resp., the complications observed in double z - plasty were infection of wound (60%), seroma (56%), hematoma (52%), necrosis (48%), wound dehiscence (48%), and loss of sensation (40%). the complications in karydakis procedure were infection (16%), seroma (12%), and loss of sensation (4%). there was absence of hematoma, necrosis, and wound dehiscence in karydakis procedure. recurrence developed in 32% of the cases in double z - plasty group comparable to no recurrence seen in karydakis procedure, but the patients were followed up until 6 months. recurrence information was based on the voluntary information by the patients after that in the outpatient department. all these complications were more common among patients with primary disease, deep natal cleft, and coarse body hair density (table 1). pilonidal sinus disease is an acquired condition usually seen in young adults and carries high postoperative morbidity and patient 's discomfort. through this study, we compare the two modern methods of treatment, namely, karydakis procedure and double z - plasty.(1)epidemiology : in present study, pilonidal sinus disease is an acquired condition usually seen in young adults of 21 to 30 years of age. keighley (peak age incidence is 15 to 24 years) [5, 6 ]. this higher occurrence in the 3rd decade of life is because pilonidal disease starts at the onset of puberty, when sex hormones start acting on pilosebaceous glands in the natal cleft. a hair follicle becomes distended with keratin and subsequently infected, leading to a folliculitis and an abscess which extends into the subcutaneous fat. awad and saad reported that 56.3% of pilonidal disease patients had coarse hair density and deep natal cleft. we fully agree with this, as we found that patients with recurrent disease were all males having a coarse body hair density and deeper natal cleft.(2)postoperative comparison : hospitalization, wound healing, and work off periods are relative measures of outcome. they are strongly related to personal, sociocultural, and socioeconomic levels, type of job, social assurance, and behavioral patterns. we observed shorter hospitalization period with karydakis flap technique (4.08 + 0.91 days) in comparison to double z - plasty (10.92 + 2.29 days). in the present study, the mean duration of wound healing was 12.84 + 1.70 days, the number of days in which pain lasted postoperatively was 11.52 + 1.50 days, and work off periods were 10.56 + 1.29 days in karydakis flap procedure, while in double z - plasty technique the mean days of wound healing were 30.16 8.97 days, the number of days in which pain lasted postoperatively was 37.92 8.31 days, and work off periods were 35.72 8.59 days. fazeli. (2006) in a clinical trial of 216 cases divided into three arms of 72 cases each have concluded that wounds healed much faster in the z - plasty group, 15.4 days, and the work off periods were 17.5 days. taubanakis in 1986 produced a study in which he found that mean time of healing in z - plasty was < 14 days.(3)complications : in present study, the overall complication rates were maximum for the double z - plasty group (45.07%) and the commonest complications were loss of local sensation (68%), tip necrosis (44%), infection (8%), seroma formation (4%), and wound dehiscence (4%). similarly, sayed, in his experience of 30 cases with z - plasty, reported among early complications loss of local sensation in 58%, tip necrosis in 20%, wound infection in 6.7%, and wound dehiscence in 3.3%. loss of local sensation is a known complication following z - plasty because in this technique flaps are transposed in such a way to avoid suture line in midline. to achieve this mansoory and dickson, in their experience of 127 cases with z - plasty, suggested that more than 50% of patients develop loss of local sensation and over 30% of patients develop tip necrosis. tip necrosis was also more with our technique of double z - plasty (44%) ; it was because of sharp triangles. however, it was only superficial necrosis, thereby increasing morbidity, but it does not affect overall results. in our experience, hematoma occurred in cases having deep natal cleft and coarse body hair density. this may be attributed to more dissection in deep natal cleft cases and minimal use of cautery in order not to compromise the blood supply of the flaps, resulting in inadequate hemostasis and hematoma formation. this is mainly because of occurrence of compromised blood supply in making flaps. in double z - plasty, to minimize the tip necrosis, we have observed and advise to do the following:(i)the incision to achieve transposition of flaps should be in full depth having deeper tissues along with the skin;(ii)approximation of soft tissue (subcutaneous and deeper) should be meticulous by vicryl suture;(iii)the cautery should be minimally used. epidemiology : in present study, pilonidal sinus disease is an acquired condition usually seen in young adults of 21 to 30 years of age. keighley (peak age incidence is 15 to 24 years) [5, 6 ]. this higher occurrence in the 3rd decade of life is because pilonidal disease starts at the onset of puberty, when sex hormones start acting on pilosebaceous glands in the natal cleft. a hair follicle becomes distended with keratin and subsequently infected, leading to a folliculitis and an abscess which extends into the subcutaneous fat. awad and saad reported that 56.3% of pilonidal disease patients had coarse hair density and deep natal cleft. we fully agree with this, as we found that patients with recurrent disease were all males having a coarse body hair density and deeper natal cleft. postoperative comparison : hospitalization, wound healing, and work off periods are relative measures of outcome. they are strongly related to personal, sociocultural, and socioeconomic levels, type of job, social assurance, and behavioral patterns. we observed shorter hospitalization period with karydakis flap technique (4.08 + 0.91 days) in comparison to double z - plasty (10.92 + 2.29 days). in the present study, the mean duration of wound healing was 12.84 + 1.70 days, the number of days in which pain lasted postoperatively was 11.52 + 1.50 days, and work off periods were 10.56 + 1.29 days in karydakis flap procedure, while in double z - plasty technique the mean days of wound healing were 30.16 8.97 days, the number of days in which pain lasted postoperatively was 37.92 8.31 days, and work off periods were 35.72 8.59 days. fazeli. (2006) in a clinical trial of 216 cases divided into three arms of 72 cases each have concluded that wounds healed much faster in the z - plasty group, 15.4 days, and the work off periods were 17.5 days. taubanakis in 1986 produced a study in which he found that mean time of healing in z - plasty was < 14 days. complications : in present study, the overall complication rates were maximum for the double z - plasty group (45.07%) and the commonest complications were loss of local sensation (68%), tip necrosis (44%), infection (8%), seroma formation (4%), and wound dehiscence (4%). similarly, sayed, in his experience of 30 cases with z - plasty, reported among early complications loss of local sensation in 58%, tip necrosis in 20%, wound infection in 6.7%, and wound dehiscence in 3.3%. loss of local sensation is a known complication following z - plasty because in this technique flaps are transposed in such a way to avoid suture line in midline. to achieve this mansoory and dickson, in their experience of 127 cases with z - plasty, suggested that more than 50% of patients develop loss of local sensation and over 30% of patients develop tip necrosis. tip necrosis was also more with our technique of double z - plasty (44%) ; it was because of sharp triangles. however, it was only superficial necrosis, thereby increasing morbidity, but it does not affect overall results. in our experience, hematoma occurred in cases having deep natal cleft and coarse body hair density. this may be attributed to more dissection in deep natal cleft cases and minimal use of cautery in order not to compromise the blood supply of the flaps, resulting in inadequate hemostasis and hematoma formation. this is mainly because of occurrence of compromised blood supply in making flaps. in double z - plasty, to minimize the tip necrosis, we have observed and advise to do the following : (i)the incision to achieve transposition of flaps should be in full depth having deeper tissues along with the skin;(ii)approximation of soft tissue (subcutaneous and deeper) should be meticulous by vicryl suture;(iii)the cautery should be minimally used. the incision to achieve transposition of flaps should be in full depth having deeper tissues along with the skin ; approximation of soft tissue (subcutaneous and deeper) should be meticulous by vicryl suture ; the cautery should be minimally used. however, in karydakis flap technique, infection, seroma, hematoma, loss of sensation, flap necrosis, and wound dehiscence were very less common. in both groups, wound dehiscence and infection were more common in cases having deep natal cleft and coarse body hair density. this is because, as we already know the perineum to be the most contaminated area of our body with the mid natal cleft being the bacterial harbor where maximum bacterial colonization occurs, dense body hair adversely affects maintenance of a proper hygiene, thus increasing the infection rate. so, with our experience, it is obvious that the deep natal cleft and coarse body hair density not only are risk factors for the causation of the disease but also complicate the procedure done, increasing the infection, necrosis, and wound dehiscence rate.(4)recurrences : in present study, no recurrence proves the superiority of the karydakis flap procedure over double z - plasty technique for treatment of pilonidal disease. recurrences : in present study, no recurrence proves the superiority of the karydakis flap procedure over double z - plasty technique for treatment of pilonidal disease. on comparison, karydakis flap was found superior to double z - plasty having less seroma formation, no local hematoma, and no flap necrosis. statistically, this comparison was highly significant (p < 0.001). karydakis flap has some added advantages over double z - plasty technique like keeping scar away from the midline and flattening of the natal cleft, thus reducing local recurrence rates.	the most difficult problems that follow surgery for pilonidal disease are persistent unhealed midline wound and recurrence. various innovations were proposed to deal with these problems. the adipocutaneous flap of karydakis was devised to shift the natal cleft, while z - plasty involves fasciocutaneous flap. the present prospective randomized trial was conducted on 50 cases of symptomatic or recurrent pilonidal sinuses divided randomly into two equal groups undergoing karydakis procedure and z - plasty. the duration of hospitalization for karydakis procedure was found significantly lesser than that for double z - plasty (p < 0.001). similar observations are obtained for duration of wound healing (p < 0.001), work off period (p < 0.001), and the duration of presence of significant pain postoperatively (p < 0.001). the overall complications were more in double z - plasty. recurrence developed in 32% of the cases in double z - plasty group comparable to no recurrence seen in karydakis procedure. thus, karydakis flap was found superior to double z - plasty having less seroma formation, no local hematoma, and no flap necrosis. statistically, this comparison was highly significant (p < 0.001). karydakis flap has some added advantages over double z - plasty technique like keeping scar away from the midline and flattening of the natal cleft, thus reducing local recurrence rates.
coronary artery disease (cad), one of the most common cardiovascular diseases, ranks first among fatal diseases in adults around the world [13 ]. several risk factors have been confirmed, such as smoking, hypertension, diabetes, high blood cholesterol, excessive alcohol drinking, depression, and lack of exercise. as for the underlying mechanism of cad, it is reported that cardiac atherosclerosis may have a significant influence on the occurrence and progression of the disease. however, genetic and environmental risk factors have been widely researched in the etiology of cad, and their remarkable effects on susceptibility to cad have also been identified. in recent years, accumulating evidence indicates that genetic polymorphisms may be implicated in individual susceptibility to cad, including polymorphisms within genes of aplnr, interleukin-6, cyp7a1, and pai-1. in addition, the apolipoprotein m gene (apom), located on human chromosome 6 p21.31, has been reported to be significantly related to the occurrence of cad. the apom gene codes a 22-kda protein that belongs to the apolipoprotein superfamily in structure. apom protein was first identified and determined in a study on lipoprotein by xu. in 1993. human apom cdna, with 734 base pairs, encodes a residue - long protein with 188 amino acids. apom is reportedly related to lower high - density lipoprotein (hdl) cholesterol, triglyceride - rich lipoproteins, lipoproteins containing apob, and very low - density lipoprotein (vldl). only expressed in the kidneys and liver, previous studies have suggested that one of the polymorphisms in apom gene, rs805296, was related to the susceptibility to cad [1720 ] ; however, the number of studies is relatively limited, and the results are divergent rather than conclusive due to various reasons. therefore, we comprehensively summarized all the findings on the association of apom rs805296 polymorphism with risk of cad so as to reach a more authentic conclusion by performing the present meta - analysis. the electronic databases searched for all the usable studies were : pubmed, embase, and chinese national knowledge infrastructure (cnki). apom, polymorphism or variant or mutation, and coronary artery disease or cad or atherosclerosis. in case of missing any adequate studies, we screened the articles in the reference lists of relevant studies by manual searching. all the studies were restricted to those in english or chinese. all the publications meeting the following requirements were included into our meta - analysis : (1) possessing the case and control subjects at the same time ; (2) studying the correlation between apom rs805296 polymorphism and cad risk ; (3) with sufficient data describing genotype and allele frequencies of the polymorphism in case and control groups ; (4) human studies ; and (5) the genotype distribution in control group conforming to hardy - weinberg equilibrium. those studies with case - only design, inadequate information, or duplicating other articles were excluded. for publications with similar datasets, an identical form for data extraction was designed in advance, and the whole process was completed by 2 authors separately. information to be extracted from each included article incorporated the following aspects : year of publication, name of first author, country of origin, ethnicity, genotyping method, sample sizes of cases and controls, genotypic and allelic distribution in case and control groups, and p value for hardy - weinberg equilibrium in control group. since all the publications would have control groups with genotypic and allelic frequencies consistent with hardy - weinberg equilibrium according to the selection criteria, we checked the compliance degree of hardy - weinberg equilibrium using the chi - square test for those not stating relevant data. the strength of relationship between apom rs805296 polymorphism and cad risk was evaluated with odds ratio (or) and its corresponding 95% confidence interval (95% ci) under 5 genetic models (cc versus tt, cc + tc versus tt, cc versus tt+tc, c versus t and tc versus tt). the absence or presence of statistically significant inter - study heterogeneity, tested by the -test - based q statistic, determined the use of fixed - effects model (mantel - haenszel method) or random - effects model (dersimonian and laird method). in sensitivity analysis, each single study was deleted in turn to observe the alterations of the overall results. through begg s funnel plots and egger s test the electronic databases searched for all the usable studies were : pubmed, embase, and chinese national knowledge infrastructure (cnki). apom, polymorphism or variant or mutation, and coronary artery disease or cad or atherosclerosis. in case of missing any adequate studies, we screened the articles in the reference lists of relevant studies by manual searching. all the publications meeting the following requirements were included into our meta - analysis : (1) possessing the case and control subjects at the same time ; (2) studying the correlation between apom rs805296 polymorphism and cad risk ; (3) with sufficient data describing genotype and allele frequencies of the polymorphism in case and control groups ; (4) human studies ; and (5) the genotype distribution in control group conforming to hardy - weinberg equilibrium. those studies with case - only design, inadequate information, or duplicating other articles were excluded. for publications with similar datasets, an identical form for data extraction was designed in advance, and the whole process was completed by 2 authors separately. information to be extracted from each included article incorporated the following aspects : year of publication, name of first author, country of origin, ethnicity, genotyping method, sample sizes of cases and controls, genotypic and allelic distribution in case and control groups, and p value for hardy - weinberg equilibrium in control group. all the publications would have control groups with genotypic and allelic frequencies consistent with hardy - weinberg equilibrium according to the selection criteria, we checked the compliance degree of hardy - weinberg equilibrium using the chi - square test for those not stating relevant data. the strength of relationship between apom rs805296 polymorphism and cad risk was evaluated with odds ratio (or) and its corresponding 95% confidence interval (95% ci) under 5 genetic models (cc versus tt, cc + tc versus tt, cc versus tt+tc, c versus t and tc versus tt). the absence or presence of statistically significant inter - study heterogeneity, tested by the -test - based q statistic, determined the use of fixed - effects model (mantel - haenszel method) or random - effects model (dersimonian and laird method). in sensitivity analysis, each single study was deleted in turn to observe the alterations of the overall results. through begg s funnel plots and egger s test, we detected if there existed significant publication bias across eligible studies. initially, 81 records were identified through the computer search, and 36 remained after excluding 8 studies not on humans and 37 apparently irrelevant ones. through the subsequent exclusion for reviews and letters (7), without full texts (3), duplicates (5), not about apom rs805296 polymorphism (8) and without original data (6), we eventually included 7 studies in the quantitative synthesis [18,2126 ]. table 2 shows the ors with 95% cis and p values for heterogeneity test under all the genetic models. overall, the apom rs805296 polymorphism elevated the cad risk in all the genetic contrasts (cc versus tt : or=2.13, 95% ci=1.163.91 ; cc + tc versus tt : or=1.80, 95% ci=1.502.17 ; cc versus tt+tc : or=1.91, 95% ci=1.043.51 ; c versus t : or=1.72, 95% ci=1.452.04 ; tc versus tt : or=1.78, 95% ci=1.472.15). figure 2 describes the forest plot for the association of apom rs805296 polymorphism with cad risk under the cc versus tt model. as shown in table 2, p values for heterogeneity under all the genetic models were larger than 0.05 (p=0.491 for cc versus tt model ; p=0.204 for cc + tc versus tt model ; p=0.557 for cc versus tt+tc model ; p=0.126 for c versus t model ; p=0.361 for tc versus tt model). therefore, there was no marked heterogeneity and the fixed - effects model was used for pooling the results. begg s funnel plots and egger s test were used to detected possible publication bias among the included studies from the visual and statistical perspective, respectively, and neither the shapes of funnel plots (figure 3) nor the statistical data of egger s test (p=0.260) provided evidence for the presence of obvious publication bias. in the process of sensitivity analysis, every individual study was omitted in sequence, and the changed results were observed correspondingly. no radical alteration occurred in the pooled results, suggesting that no single study substantially affected the results, and our meta - analysis outcomes were statistically robust. initially, 81 records were identified through the computer search, and 36 remained after excluding 8 studies not on humans and 37 apparently irrelevant ones. through the subsequent exclusion for reviews and letters (7), without full texts (3), duplicates (5), not about apom rs805296 polymorphism (8) and without original data (6), we eventually included 7 studies in the quantitative synthesis [18,2126 ]. table 2 shows the ors with 95% cis and p values for heterogeneity test under all the genetic models. overall, the apom rs805296 polymorphism elevated the cad risk in all the genetic contrasts (cc versus tt : or=2.13, 95% ci=1.163.91 ; cc + tc versus tt : or=1.80, 95% ci=1.502.17 ; cc versus tt+tc : or=1.91, 95% ci=1.043.51 ; c versus t : or=1.72, 95% ci=1.452.04 ; tc versus tt : or=1.78, 95% ci=1.472.15). figure 2 describes the forest plot for the association of apom rs805296 polymorphism with cad risk under the cc versus tt model. as shown in table 2, p values for heterogeneity under all the genetic models were larger than 0.05 (p=0.491 for cc versus tt model ; p=0.204 for cc + tc versus tt model ; p=0.557 for cc versus tt+tc model ; p=0.126 for c versus t model ; p=0.361 for tc versus tt model). therefore, there was no marked heterogeneity and the fixed - effects model was used for pooling the results. begg s funnel plots and egger s test were used to detected possible publication bias among the included studies from the visual and statistical perspective, respectively, and neither the shapes of funnel plots (figure 3) nor the statistical data of egger s test (p=0.260) provided evidence for the presence of obvious publication bias. in the process of sensitivity analysis, every individual study was omitted in sequence, and the changed results were observed correspondingly. no radical alteration occurred in the pooled results, suggesting that no single study substantially affected the results, and our meta - analysis outcomes were statistically robust. cad is a complex multi - genetic disease caused by synergistic effects of genetic and environmental risk factors. hereditary epidemiological studies have suggested that genetic mutations may elevate individual risk of developing cad [2931 ]. initially separated and cloned from chylomicrons, apom in plasma mainly exists in hdl particles, and very little is in triglyceride - rich lipoprotein (tgrlp) and low - density lipoprotein (ldl), suggesting apom may be associated with lipid transportation and metabolism. richter. found an important role of apom in the formation of hdl, and confirmed its protective effects against atherosclerosis. in the study by xu., the correlation between apom and indexes of lipid indicated that apom levels in plasma had a positive relation with factors against the progression of atherosclerosis, such as apoa i and hdlc, and was negatively related with factors promoting atherosclerosis development, such as triglyceride, total cholesterol, and lipoprotein (a), and that elevated levels of apom could prevent and slow the progression of atherosclerosis. the human apom gene is located in a region adjacent to that of major histocompatibility complex (mhc) in which multiple genes are related to immune response ; therefore, the apom gene is likely to participate in the regulation of immune defense. among a number of polymorphisms within the apom gene, the rs805296 variant in the proximal promoter region has been verified to have a link with plasma cholesterol, and may increase individual susceptibility to cad. in this present study, we referred to previous studies and analyzed the association between apom rs805296 polymorphism and cad risk. our results indicate that apom rs805296 polymorphism under all the comparisons could elevate the risk of cad, suggesting this polymorphism might act as a promoter for cad onset. several case - control studies have investigated the significance of apom rs805296 in cad risk in chinese populations, and obtained useful findings. using the method of polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp), huang. carried out a screening for apom rs805296 in 220 cad cases and 195 normal controls, and observed the frequency of c allele in case and control groups was 19.1% and 12.6% respectively, and this difference was statistically significant (p=0.011), which proved the polymorphism rs805296 might be a susceptible factor for cad. zhang. performed a large study recruiting 675 patients with acute coronary syndrome (acs) and 636 healthy control subjects, and found that the frequencies of both c allele and cc genotype of apom rs805296 polymorphism in the case group were significantly higher than those in the control group (p<0.01). subsequently, after the adjustment of susceptibility factors for cad, the c allele was found to be an independent risk factor for the occurrence of acs. in contrast, zheng. found no statistically significant difference in distribution of 3 genotypes of apom rs805296 polymorphism, including tt, tc, and cc, between case and control groups, and concluded that rs805296 might not be correlated with the development of cad. conducted in a chinese population, the study of zheng. obtained results that are in contrast with our present study and the other case - control studies listed above, which might be attributed to differences in number of samples, methods of genotyping, correction factors, and other risk elements. absence of heterogeneity and publications bias is the biggest strength of this meta - analysis. however, as in previous studies and meta - analyses, our meta - analysis also has some weaknesses that should be clearly presented. because all the prior studies on the association of apom rs805296 polymorphism with cad risk only focused on chinese populations, our meta - analysis solely discussed this association among chinese people, which might not be representative in other ethnic groups. in addition, the limited number of included studies and the relatively small sample size might lessen the statistical power of our results. another important aspect that should be stated is that some potential risk factors such as family history, smoking status, body mass index (bmi) and other environmental influences were not incorporated into the discussion of the present study due to limited original information of included studies. in conclusion, our meta - analysis results revealed a significant correlation of apom rs805296 polymorphism with cad risk, and showed rs805296 polymorphism might confer increased risk of cad in the chinese population. the association between apom rs805296 and onset risk of cad needs to be further verified by studies containing combined effects of genetic and environmental factors and larger sample size in multiple ethnicities.	backgroundthe present meta - analysis aimed to summarize the inconsistent findings on the association of apolipoprotein m gene (apom) rs805296 polymorphism with the risk of coronary artery disease (cad), and to obtain a more authentic result about this topic.material/methodsa total of 7 available articles were identified through electronic databases pubmed, embase, and chinese national knowledge infrastructure (cnki) and their useful data were carefully extracted. the relationship between apom rs805296 polymorphism and cad risk was assessed by odds ratios (ors) and corresponding 95% confidence intervals (95% cis), which were calculated using the fixed- or random - effects model, according to the degree of heterogeneity. hardy - weinberg equilibrium test, sensitivity test, and publication bias examination were also performed in this meta-analysis.resultsaccording to the pooled results, apom rs805296 polymorphism conferred an increased risk of cad under all the genetic contrasts : cc versus tt, cc + tc versus tt, cc versus tt+tc, c versus t, and tc versus tt (or=2.13, 95% ci=1.163.91 ; or=1.80, 95% ci=1.502.17 ; or=1.91, 95% ci=1.043.51 ; or=1.72, 95% ci=1.452.04 ; or=1.78, 95% ci=1.472.15).conclusionsapom rs805296 polymorphism may be a risk factor for developing cad.
data sources were the truven health marketscan commercial claims and encounters and the marketscan medicare supplemental databases from january 1, 2009, to september 30, 2012. records of patients with a copd diagnosis at any diagnosis position within the intake period from july 1, 2009, through september 30, 2011, were included. claims for laboratory, pathology, or radiology services were not used to identify individuals with a specific condition, because their use could incorrectly identify individuals as having that condition based on the reason for testing (eg, screening) rather than the test results ; therefore, those claims were ignored during patient selection, and diagnoses were termed non - rule - out copd. the first occurrence of non - rule - out copd diagnosis (international classification of diseases, ninth revision, clinical modification codes 490.xx, 491.xx, 492.xx, 494.xx, or 496.xx) (e - table 1) was defined as the index event, and the date of the index event was defined as the index date. the study proposal was presented to and accepted by the novartis outcomes research review forum. eligible patients were aged 40 to 90 years (inclusive), had copd, and used at least one long - acting muscarinic antagonist (lama), long - acting 2-adrenergic agonist (laba), inhaled corticosteroid (ics)/laba, or lama + ics + laba from 180 days before the index date through 180 days after the index date. patients were enrolled continuously in the medical, pharmacy benefit, and fee - for - service plan from 180 days before the index date to 360 days after the index date. comorbidities were defined as any occurrence of a specific diagnosis code from 180 days before the index date through 180 days after the index date. this time period, which does not fall completely within the baseline period or the 360-day follow - up period, was chosen to establish and solidify the baseline patient comorbidities being evaluated. comorbidities of interest were identified prospectively and consisted of chronic kidney disease (ckd) ; cardiovascular disease (cvd), including heart failure, stroke, acute myocardial infarction (mi), and peripheral vascular disease ; asthma ; depression ; diabetes ; osteoporosis ; and anemia. resource consumption was measured from 180 days prior to the index date to the index date (for baseline assessments) and from the index date through 360 days after the index date (for outcomes assessments). resource use assessments included all - cause and disease - specific ed visits, hospitalizations, office visits (defined as any office visit to any doctor), outpatient visits, and total length of hospital stay ; in this instance, disease - specific means copd- or asthma - related. the health - care costs assessed included all - cause and disease - specific costs for ed visits, hospitalizations, office visits, and other outpatient visits, as well as medical, prescription drug, and total health - care costs. other covariate variables included age, sex, region, employment status, and index medication (the first drug class used during the period). patient characteristics, comorbidities of interest, health - care use, and costs were summarized descriptively. a generalized linear model (glm) was used to evaluate which comorbidities drive total costs after accounting for patient characteristics in the total population. after examining the data, we selected glms with a log - link and distribution to evaluate the incremental all - cause costs, adjusting for baseline demographics, resource use, and comorbidities. to better understand total costs that are potentially attributable to comorbidities, the average treatment effect (change in the response by a change in a covariate) of each comorbidity was calculated by using the recycled prediction method. the predicted costs for patients with cvd were calculated based on the estimated glm (with costs as the dependent variable) by assuming all patients had cvd (regardless of whether they had cvd) while keeping other covariates as they were. the predicted costs, for which every observation is treated as if it represents patients without cvd, were obtained in the same manner. the average treatment effect was the mean difference in the predicted costs for the two groups. thus, we compared two hypothetical populations (one with cvd and one without cvd) that had the exact same values for the other independent variables in the model. cis were generated with the percentile method (the 95% lower bound ci is the 2.5th percentile of the bootstrap distribution, and the 95% upper bound ci is the 97.5th percentile of the bootstrap distribution). data sources were the truven health marketscan commercial claims and encounters and the marketscan medicare supplemental databases from january 1, 2009, to september 30, 2012. records of patients with a copd diagnosis at any diagnosis position within the intake period from july 1, 2009, through september 30, 2011, were included. claims for laboratory, pathology, or radiology services were not used to identify individuals with a specific condition, because their use could incorrectly identify individuals as having that condition based on the reason for testing (eg, screening) rather than the test results ; therefore, those claims were ignored during patient selection, and diagnoses were termed non - rule - out copd. the first occurrence of non - rule - out copd diagnosis (international classification of diseases, ninth revision, clinical modification codes 490.xx, 491.xx, 492.xx, 494.xx, or 496.xx) (e - table 1) was defined as the index event, and the date of the index event was defined as the index date. the study proposal was presented to and accepted by the novartis outcomes research review forum. eligible patients were aged 40 to 90 years (inclusive), had copd, and used at least one long - acting muscarinic antagonist (lama), long - acting 2-adrenergic agonist (laba), inhaled corticosteroid (ics)/laba, or lama + ics + laba from 180 days before the index date through 180 days after the index date. patients were enrolled continuously in the medical, pharmacy benefit, and fee - for - service plan from 180 days before the index date to 360 days after the index date. comorbidities were defined as any occurrence of a specific diagnosis code from 180 days before the index date through 180 days after the index date. this time period, which does not fall completely within the baseline period or the 360-day follow - up period, was chosen to establish and solidify the baseline patient comorbidities being evaluated. comorbidities of interest were identified prospectively and consisted of chronic kidney disease (ckd) ; cardiovascular disease (cvd), including heart failure, stroke, acute myocardial infarction (mi), and peripheral vascular disease ; asthma ; depression ; diabetes ; osteoporosis ; and anemia. resource consumption was measured from 180 days prior to the index date to the index date (for baseline assessments) and from the index date through 360 days after the index date (for outcomes assessments). resource use assessments included all - cause and disease - specific ed visits, hospitalizations, office visits (defined as any office visit to any doctor), outpatient visits, and total length of hospital stay ; in this instance, disease - specific means copd- or asthma - related. the health - care costs assessed included all - cause and disease - specific costs for ed visits, hospitalizations, office visits, and other outpatient visits, as well as medical, prescription drug, and total health - care costs. other covariate variables included age, sex, region, employment status, and index medication (the first drug class used during the period). patient characteristics, comorbidities of interest, health - care use, and costs were summarized descriptively. a generalized linear model (glm) was used to evaluate which comorbidities drive total costs after accounting for patient characteristics in the total population. after examining the data, we selected glms with a log - link and distribution to evaluate the incremental all - cause costs, adjusting for baseline demographics, resource use, and comorbidities. to better understand total costs that are potentially attributable to comorbidities, the average treatment effect (change in the response by a change in a covariate) of each comorbidity was calculated by using the recycled prediction method. the predicted costs for patients with cvd were calculated based on the estimated glm (with costs as the dependent variable) by assuming all patients had cvd (regardless of whether they had cvd) while keeping other covariates as they were. the predicted costs, for which every observation is treated as if it represents patients without cvd, were obtained in the same manner. the average treatment effect was the mean difference in the predicted costs for the two groups. thus, we compared two hypothetical populations (one with cvd and one without cvd) that had the exact same values for the other independent variables in the model. cis were generated with the percentile method (the 95% lower bound ci is the 2.5th percentile of the bootstrap distribution, and the 95% upper bound ci is the 97.5th percentile of the bootstrap distribution). patient characteristics on the index date are summarized in table 1, and health - care use and costs from the 180 days preceding the index date through the index date are summarized in table 2. the most common comorbidity was cvd (34.8%), followed by diabetes (22.8%), asthma (14.7%), anemia (14.2%), ckd (9.9%), depression (9.9%), and osteoporosis (6.9%). most patients (52.8%) had one or two comorbidities of interest. patients with ckd and anemia experienced the highest incidence of all - cause ed visits leading to hospitalizations (23.2% and 20.4%, respectively) and all - cause hospitalizations (38.0% and 33.8%, respectively). the percentages of all - cause office visits and of all - cause other outpatient visits were generally similar across the various comorbidity groups (all - cause office visits, 93.8%-95.4% ; all - cause other outpatient visits, 95.1%-97.7%) and were higher than those for patients with no baseline comorbidities of interest (83.9% and 84.6%, respectively). ics = inhaled corticosteroid ; laba = long - acting 2-adrenergic agonist ; lama = long - acting muscarinic antagonist. ckd = chronic kidney disease ; cvd = cardiovascular disease ; ics = inhaled corticosteroid ; laba = long - acting 2-adrenergic agonist ; lama = long - acting muscarinic agonist ; mi = myocardial infarction. spouse / child / dependent relation. within the period from 180 d before the index date through 180 d after the index date (inclusive). includes heart failure, stroke, acute mi, and peripheral vascular disease. baseline resource use and resource use from the index date (exclusive) through 180 d before the index date (inclusive). the prevalence of copd- or asthma - related hospitalizations was highest among patients with asthma at baseline (8.3%) ; for the other baseline comorbidities, the frequencies of copd- or asthma - related hospitalizations were similar, ranging from 3.4% (osteoporosis) to 3.9% (depression). mean all - cause total health - care costs from the 180 days before the index date through the index date were highest among patients with ckd ($ 19,405) and anemia ($ 18,011) and lowest among those with asthma ($ 10,583) and osteoporosis ($ 11,438). mean copd- or asthma - related total health - care costs were highest among patients with asthma ($ 1,845) and osteoporosis ($ 1,566). in addition to those shown in table 2, analyses were conducted based on the number of comorbidities present. patients with four or more comorbidities experienced the highest incidence of ed visits leading to hospitalizations (33.1%), compared with 22.2% in those with three comorbidities, 13.8% in those with two comorbidities, 7.4% in those with one comorbidity, and 2.5% in those with no comorbidities. similarly, the rate of all - cause hospitalizations was highest in patients with four or more comorbidities (50.4%) and lowest in those with no comorbidities (5.0%). all - cause total health - care costs increased as the number of comorbidities increased (zero comorbidities of interest, $ 4,790 ; four or more comorbidities of interest, $ 27,895), as did copd- or asthma - related total health - care costs (zero comorbidity of interest, $ 871 ; four or more comorbidities of interest, $ 2,216). during the time period from the index date through 360 days after the index date, 38.6% of patients with copd and ckd and 34.3% of patients with copd and anemia had all - cause ed visits leading to hospitalizations (fig 2a). the percentage of patients experiencing all - cause hospitalizations was highest among those with ckd (57.0%) and anemia (52.6%) (fig 2a). the percentage of patients with copd- or asthma - related hospitalizations was highest among those with asthma (17.4%) and cvd (12.0%) (fig 2a). all - cause total health - care costs during the time period from the index date through 360 days following the index date were highest among patients with copd and ckd ($ 41,288) and patients with copd and anemia ($ 38,870) (table 3). copd- or asthma - related total health - care costs were highest among patients with copd and asthma ($ 5,389) and those with copd and ckd ($ 5,117). a, b, resource use from index date through 360 d after the index date for each outcome. ckd = chronic kidney disease ; cvd = cardiovascular disease ; er = emergency room. total medical costs by comorbidity and number of comorbidities data are presented as mean (sd). costs are from index date through 360 d after the index date and are in 2012 us dollars. see table 1 legend for expansion of abbreviations. patients with four or more comorbidities of interest experienced the highest incidence of ed visits leading to hospitalizations (50.1% vs 9.5% with no comorbidities) and all - cause hospitalizations (68.7% vs 16.7% with no comorbidities) (fig 2b). all - cause total health - care costs and copd- or asthma - related total health - care costs both increased as the number of comorbidities increased (table 3). the reference group selected for the glm was female patients with copd aged 40 to 64 years, living in the south, employed, having an index medication of ics / laba fixed or loose - dose combination with no ed visits or hospitalizations regardless of relationship to asthma or copd, and who did not have ckd, cvd, asthma, depression, diabetes, osteoporosis, or anemia. in this group, a ratio, based on the impact on total health - care costs, was estimated for each variable in the model (table 4) and represents multiplicative effects. ratios for variables included in the generalized linear model arithmetic mean cost for the reference group was $ 12,408. characteristics with the greatest impact on costs included depression (ratio, 1.35), ckd (ratio, 1.43), anemia (ratio, 1.54), and cvd (ratio, 1.55) comorbidities (table 4). the average treatment effect for each comorbidity after adjusting for age, sex, geographic location, baseline health - care use, employment status, and index copd medication is shown in figure 3. for the time period from the index date through 360 days following the index date, a patient with copd and anemia had, on average, $ 10,762 more in total health - care costs than a patient with copd but without anemia. cvd and ckd increased total health - care costs by $ 9,882 and $ 8,912, respectively. difference in average total health - care cost by comorbidity from index date through 360 d after the index date in 2012 us dollars. patient characteristics on the index date are summarized in table 1, and health - care use and costs from the 180 days preceding the index date through the index date are summarized in table 2. the most common comorbidity was cvd (34.8%), followed by diabetes (22.8%), asthma (14.7%), anemia (14.2%), ckd (9.9%), depression (9.9%), and osteoporosis (6.9%). most patients (52.8%) had one or two comorbidities of interest. patients with ckd and anemia experienced the highest incidence of all - cause ed visits leading to hospitalizations (23.2% and 20.4%, respectively) and all - cause hospitalizations (38.0% and 33.8%, respectively). the percentages of all - cause office visits and of all - cause other outpatient visits were generally similar across the various comorbidity groups (all - cause office visits, 93.8%-95.4% ; all - cause other outpatient visits, 95.1%-97.7%) and were higher than those for patients with no baseline comorbidities of interest (83.9% and 84.6%, respectively). ics = inhaled corticosteroid ; laba = long - acting 2-adrenergic agonist ; lama = long - acting muscarinic antagonist. ckd = chronic kidney disease ; cvd = cardiovascular disease ; ics = inhaled corticosteroid ; laba = long - acting 2-adrenergic agonist ; lama = long - acting muscarinic agonist ; mi = myocardial infarction. spouse / child / dependent relation. within the period from 180 d before the index date through 180 d after the index date (inclusive). includes heart failure, stroke, acute mi, and peripheral vascular disease. baseline resource use and resource use from the index date (exclusive) through 180 d before the index date (inclusive). the prevalence of copd- or asthma - related hospitalizations was highest among patients with asthma at baseline (8.3%) ; for the other baseline comorbidities, the frequencies of copd- or asthma - related hospitalizations were similar, ranging from 3.4% (osteoporosis) to 3.9% (depression). mean all - cause total health - care costs from the 180 days before the index date through the index date were highest among patients with ckd ($ 19,405) and anemia ($ 18,011) and lowest among those with asthma ($ 10,583) and osteoporosis ($ 11,438). mean copd- or asthma - related total health - care costs were highest among patients with asthma ($ 1,845) and osteoporosis ($ 1,566). in addition to those shown in table 2, analyses were conducted based on the number of comorbidities present. patients with four or more comorbidities experienced the highest incidence of ed visits leading to hospitalizations (33.1%), compared with 22.2% in those with three comorbidities, 13.8% in those with two comorbidities, 7.4% in those with one comorbidity, and 2.5% in those with no comorbidities. similarly, the rate of all - cause hospitalizations was highest in patients with four or more comorbidities (50.4%) and lowest in those with no comorbidities (5.0%). all - cause total health - care costs increased as the number of comorbidities increased (zero comorbidities of interest, $ 4,790 ; four or more comorbidities of interest, $ 27,895), as did copd- or asthma - related total health - care costs (zero comorbidity of interest, $ 871 ; four or more comorbidities of interest, $ 2,216). during the time period from the index date through 360 days after the index date, 38.6% of patients with copd and ckd and 34.3% of patients with copd and anemia had all - cause ed visits leading to hospitalizations (fig 2a). the percentage of patients experiencing all - cause hospitalizations was highest among those with ckd (57.0%) and anemia (52.6%) (fig 2a). the percentage of patients with copd- or asthma - related hospitalizations was highest among those with asthma (17.4%) and cvd (12.0%) (fig 2a). all - cause total health - care costs during the time period from the index date through 360 days following the index date were highest among patients with copd and ckd ($ 41,288) and patients with copd and anemia ($ 38,870) (table 3). copd- or asthma - related total health - care costs were highest among patients with copd and asthma ($ 5,389) and those with copd and ckd ($ 5,117). a, b, resource use from index date through 360 d after the index date for each outcome. ckd = chronic kidney disease ; cvd = cardiovascular disease ; er = emergency room. total medical costs by comorbidity and number of comorbidities data are presented as mean (sd). costs are from index date through 360 d after the index date and are in 2012 us dollars. see table 1 legend for expansion of abbreviations. patients with four or more comorbidities of interest experienced the highest incidence of ed visits leading to hospitalizations (50.1% vs 9.5% with no comorbidities) and all - cause hospitalizations (68.7% vs 16.7% with no comorbidities) (fig 2b). all - cause total health - care costs and copd- or asthma - related total health - care costs both increased as the number of comorbidities increased (table 3). the reference group selected for the glm was female patients with copd aged 40 to 64 years, living in the south, employed, having an index medication of ics / laba fixed or loose - dose combination with no ed visits or hospitalizations regardless of relationship to asthma or copd, and who did not have ckd, cvd, asthma, depression, diabetes, osteoporosis, or anemia. in this group, a ratio, based on the impact on total health - care costs, was estimated for each variable in the model (table 4) and represents multiplicative effects. ratios for variables included in the generalized linear model arithmetic mean cost for the reference group was $ 12,408. characteristics with the greatest impact on costs included depression (ratio, 1.35), ckd (ratio, 1.43), anemia (ratio, 1.54), and cvd (ratio, 1.55) comorbidities (table 4). the average treatment effect for each comorbidity after adjusting for age, sex, geographic location, baseline health - care use, employment status, and index copd medication is shown in figure 3. for the time period from the index date through 360 days following the index date, a patient with copd and anemia had, on average, $ 10,762 more in total health - care costs than a patient with copd but without anemia. cvd and ckd increased total health - care costs by $ 9,882 and $ 8,912, respectively. difference in average total health - care cost by comorbidity from index date through 360 d after the index date in 2012 us dollars. our results were consistent with those of previously published work, in that a significant burden of comorbidity was associated with copd, and comorbid conditions were associated with incremental increases in resource use and health - care costs. total health - care costs during the period from the index date through 360 days following the index date were greatest among patients with copd and ckd or anemia ; copd- or asthma - related total health - care costs were greatest among patients with copd and asthma and ckd. these results were driven, in part, by high incidences of all - cause ed visits leading to hospitalizations among patients with copd and ckd or anemia, and a high incidence of copd- or asthma - related hospitalization among patients with copd and asthma. multivariable analyses adjusted for age, sex, geographic location, baseline health - care use, employment status, and index copd medication showed that the effect of comorbidities on total health - care costs was greatest for anemia. this finding is consistent with the high incidences of all - cause ed visits leading to hospitalizations and all - cause hospitalizations among patients with copd and anemia. substantial treatment effects of cvd and ckd (about $ 9,000) are likely attributable, in part, to the high incidence (about 50%) of all - cause hospitalization in both groups. cavailles reviewed the pathophysiologic and epidemiologic links between copd and the comorbidities studied here, and concluded that shared risk factors and the influence of chronic systemic inflammation are likely contributors to these relationships. smoking is a major risk factor for both copd and cvd, and cvd was the most common comorbidity in the copd population ; thus, the frequent coexistence of these two conditions is unsurprising. however, the literature also suggests that the systemic inflammation associated with copd produces a procoagulant state and endothelial dysfunction that may contribute to thromboembolic events. in fact, at least one study has shown that the link between cardiovascular events (including death) and copd is independent of smoking status and other confounding coronary risk factors. there is no evidence of a direct role for copd - related inflammation in anemia, which was identified as the most costly of the comorbidities in this study. however, older age, malnutrition, and cvd frequently accompany copd and are believed to play a role in the development of anemia in patients with copd. consistent with our findings, ornek determined that anemia significantly increased the cost of copd treatment in patients hospitalized for acute exacerbation of copd. furthermore, anemia was independently prognostic for premature mortality, hospital admissions, and cumulative duration of hospitalization in patients with severe copd receiving long - term oxygen therapy. our results support ckd as a key driver of total health - care costs as well as copd- and asthma - related health - care costs in patients with copd, although it was not as prevalent as other comorbidities studied. epidemiologic studies have confirmed copd as a risk factor for ckd, and the literature suggests that renal function is sensitive to hypoxemia and hypercarbia. additionally, arterial stiffness associated with copd may damage glomeruli, and some copd medications may have nephrotoxic effects. the presence of chronic renal failure significantly increased the cost of care in patients hospitalized for acute exacerbation of copd. finally, copd- or asthma - related total health - care costs were higher among patients with copd and asthma than among those with any other comorbidity. overlap syndrome refers to patients who have components of both conditions, and it is often used to describe elderly individuals in whom the distinction between asthma and copd is difficult to make. in a study of nearly 25,000 insured adults with copd, those with asthma had 1.6 times greater odds of having respiratory - related ed visits, hospitalizations, or both than did those with copd alone and demonstrated an approximately 50% increase in respiratory - related health - care costs. hospital cost use for copd or bronchiectasis was also evaluated in a recent analysis of the nationwide inpatient sample and the nationwide emergency department sample database of the healthcare cost and utilization project. although significant trends were not found in age - adjusted rates of hospital discharges from 2001 to 2012, ed visits from 2006 to 2011, or 30-day readmissions from 2009 to 2012, the mean charges and costs of all discharges increased considerably from 2001 to 2012, with aggregate charges for inpatient stays increasing from $ 8,023,983,422 in 2001 to $ 18,112,392,566 in 2012. this study was subject to several limitations. because it was restricted to patients who were on long - acting therapies, patients with the mildest form of copd and patients with more advanced disease who were not appropriately prescribed long - acting therapies inherent to claims data research, the clinical accuracy of the coding could not be assessed. in addition, patients with health maintenance organizations or full or partial capitated point - of - service insurance coverage were excluded from this study because the financial information for this population was incomplete. further, this analysis did not include patients on medicaid and it contained < 8% of patients with managed medicare. no information was available regarding the severity of disease and the level of treatment adherence. an analysis in the younger, working - age copd population (45 - 64 years of age) showed that these costs (which include the costs of impaired productivity at work, lost productivity because of early retirement, disability pensions paid, and tax revenue lost) are considerably higher than the direct medical cost of copd. in conclusion, these results show that a high prevalence of patients with copd and multiple comorbidities have associated high resource use and costs, especially within the all - cause use category. further research on comorbid conditions affecting the treatment adherence of patients with copd, copd pathogenic pathways, and worsening overall prognosis is necessary to elucidate the role of comorbidities in copd.	background : the morbidity and mortality associated with copd exacts a considerable economic burden. comorbidities in copd are associated with poor health outcomes and increased costs. our objective was to assess the impact of comorbidities on copd - associated costs in a large administrative claims dataset.methods:this was a retrospective observational study of data from the truven health marketscan commercial claims and encounters and the marketscan medicare supplemental databases from january 1, 2009, to september 30, 2012. resource consumption was measured from the index date (date of first occurrence of non - rule - out copd diagnosis) to 360 days after the index date. resource use (all - cause and disease - specific [ie, copd- or asthma - related ] ed visits, hospitalizations, office visits, other outpatient visits, and total length of hospital stay) and health - care costs (all - cause and disease - specific costs for ed visits, hospitalizations, office visits, and other outpatient visits and medical, prescription, and total health - care costs) were assessed. generalized linear models were used to evaluate the impact of comorbidities on total health - care costs, adjusting for age, sex, geographic location, baseline health - care use, employment status, and index copd medication.results:among 183,681 patients with copd, the most common comorbidities were cardiovascular disease (34.8%), diabetes (22.8%), asthma (14.7%), and anemia (14.2%). most patients (52.8%) had one or two comorbidities of interest. the average all - cause total health - care costs from the index date to 360 days after the index date were highest for patients with chronic kidney disease ($ 41,288) and anemia ($ 38,870). the impact on total health - care costs was greatest for anemia ($ 10,762 more, on average, than a patient with copd without anemia).conclusions : our analysis demonstrated that high resource use and costs were associated with copd and multiple comorbidities.
tooth preparation is considered to be complete clinically when the consistency of underlying dentin is hard to a sharp probe and free of caries. the carious process usually progresses as a series of exacerbations and remissions that are characterized by periods of high production of acid that are responsible for the dissolution of the hard tissues of the tooth. if allowed to proceed untreated, it results in the progressive destruction of the tooth and eventual infection of the dental pulp. carious dentin has been identified by two layers of soft dentin, the outer carious layer is infected unremineralizable with irreversible deteriorated collagen fibers, with no odontoblastic processes, insensitive and therefore, should be removed. the inner carious layer is uninfected, remineralizable with reversibly denatured collagen fibers, alive with living odontoblastic processes, sensitive, and so should be preserved. caries detector dyes have been developed to further help the diagnosis and removal of dental caries, by differentiating between infected and affected dentin. the dye stains only the infected outer carious dentin.[24 ] dye usage allows dentists to perform an ideal cavity preparation for adhesive restorations. to ensure that all carious dentin has been removed, use of dye is indicated as the last step in tooth preparation. the bonding mechanism for current adhesive agents is based on the acid removal of the smear layer and demineralization of the underlying dentin, which leaves an exposed collagen network. the application of hydrophilic primers followed by the adhesive, which encapsulate this collagen network and form a resin impregnated layer or hybrid layer. few reports are available in literatures regarding the effect of caries detection dye on the bond strength of sound and carious affected dentin. the present study has been designed to evaluate the influence of caries detection dye on the in - vitro tensile bond strength of adhesive materials to sound and carious affected dentin. materials along with their composition used in the present study are summarized in table 1. materials and their composition used in the study forty freshly extracted (both carious and non - carious) human mandibular molar teeth were selected for this study. the samples were stored in normal saline at room temperature until they were subjected to the experimental procedure. for carious affected dentin, twenty samples with coronal caries extending approximately halfway through the dentin were used in this study. the buccal carious surface was ground parallel to the long axis of the tooth to expose a flat surface of normal dentin surrounding the carious lesion. the buccal enamel was grinded with the help of carborundum disc, made smoothened by the sand paper (silicon carbide, 220 - 600 grit) and washed copiously with distilled water. to obtain carious affected dentin, grinding was performed using combined criteria of visual examination and staining with caries detector dye (kurary, japan) as described that is the dentin was hard to an explorer and no longer stained bright red with caries detector dye [figure 1 ]. flow chart showing distribution of samples into groups the samples in group - a (n=20, control group) were without application of caries detection dye on both sound and carious affected dentin surfaces. the samples in group - b (n=20, experimental group) were with application of caries detection dye on sound and carious affected dentin surfaces. the control and experimental group were further divided into two subgroups a1 and a2 and b1 and b2 with 10 samples in each as shown in the following flowchart. buccal surface of each sample was exposed from acrylic resin block. in control subgroups, dentin surfaces were etched with 37% phosphoric acid gel for 15 sec while in experimental subgroups all samples were etched after application of caries detection dye. after etching, it was rinsed with spray water and dried leaving a moist dentin surface for application of the bonding agent. the adhesive resin (single bond) was applied in a single layer on the sound and carious affected dentin surface as per the manufacturer 's instruction and photo cured for 10 sec using qth light source (3 m curing light 2500). a plastic cylindrical mould with a internal diameter of 3 mm and length 4 mm was placed atop the bonded surface. a flexible orthodontic wire to be used during testing procedure the composite resin was build up in increments and each layer was cured for 20 sec. after complete curing, the plastic mold was easily removed with the help of tweezers. debonding procedure was performed in tension on instron universal testing machine at a crosshead speed 0.5 mm / min. after testing, the fracture mode of each specimen was determined visually under 5x magnification. forty freshly extracted (both carious and non - carious) human mandibular molar teeth were selected for this study. the samples were stored in normal saline at room temperature until they were subjected to the experimental procedure. for carious affected dentin, twenty samples with coronal caries extending approximately halfway through the dentin were used in this study. the buccal carious surface was ground parallel to the long axis of the tooth to expose a flat surface of normal dentin surrounding the carious lesion. the buccal enamel was grinded with the help of carborundum disc, made smoothened by the sand paper (silicon carbide, 220 - 600 grit) and washed copiously with distilled water. to obtain carious affected dentin, grinding was performed using combined criteria of visual examination and staining with caries detector dye (kurary, japan) as described that is the dentin was hard to an explorer and no longer stained bright red with caries detector dye [figure 1 ]. flow chart showing distribution of samples into groups the samples in group - a (n=20, control group) were without application of caries detection dye on both sound and carious affected dentin surfaces. the samples in group - b (n=20, experimental group) were with application of caries detection dye on sound and carious affected dentin surfaces. the control and experimental group were further divided into two subgroups a1 and a2 and b1 and b2 with 10 samples in each as shown in the following flowchart. buccal surface of each sample was exposed from acrylic resin block. in control subgroups, dentin surfaces were etched with 37% phosphoric acid gel for 15 sec while in experimental subgroups all samples were etched after application of caries detection dye. after etching, it was rinsed with spray water and dried leaving a moist dentin surface for application of the bonding agent. the adhesive resin (single bond) was applied in a single layer on the sound and carious affected dentin surface as per the manufacturer 's instruction and photo cured for 10 sec using qth light source (3 m curing light 2500). a plastic cylindrical mould with a internal diameter of 3 mm and length 4 mm was placed atop the bonded surface. a flexible orthodontic wire to be used during testing procedure the composite resin was build up in increments and each layer was cured for 20 sec. after complete curing, the plastic mold was easily removed with the help of tweezers. debonding procedure was performed in tension on instron universal testing machine at a crosshead speed 0.5 mm / min. tensile bond strength of each group was calculated in mpa. after testing, the fracture mode of each specimen tensile strength is significantly higher in sound dentine from carious affected dentine in control subgroups as well as experimental subgroups [table 3 ]. the mean value of tensile strength within control group and experimental group anova table for tensile strength in subgroups since f is highly significant hence there are significant difference in tensile strength of with and without application of dye in experimental and control group. there are significant differences between the control subgroups (a1 vs a2) and experimental subgroups (b1 vs b2) in tensile strength values. (p<.001) [table 4 ]. comparison of tensile strength between control (without application of caries detection dye) and experimental subgroup (with application caries detection dye) caries- detector dyes have proven to be useful in the identification and removal of carious dentin. these agents made from basic fuschin in a propylene glycol base reliably stain only the dentin that is infected with bacteria and irreversibly demineralized without staining the affected dentin. therefore, the presence of stain reliably determines the part of dentin to be removed. early formulations of the caries detector included a 5% basic fuchsin solution in propylene glycol as a solvent ; however, it was replaced with 1% acid red 52 solution in the same solvent as a substitute dye because fuchsin is believed to be carcinogenic. studies demonstrated that dentin containing less than 10000 cfu / mg was normally not disclosed by the fuschin dye, whereas counts of greater than 550,000 cfu /mg dentin were readily stained. this suggests that the dye can be used to approximate the bacterial load of the dentinal surface because a certain bacterial mass apparently needs to be present before the dye is absorbed by the dentin. the mechanism by which caries disclosing agents selectively stain only carious, irreversibly demineralized dentin has been determined. both basic fuchsin and acid red stain the collagen fibers exposed by the bacteria caused dentin demineralization process. the results of this study show that the mean value of tensile bond strength of single bond was higher in the control subgroup than experimental subgroups which can be explained as that caries - affected dentin contain some substances that interfere with free radical generation or propagation, leading to improper polymerization of resins in such dentin. the peritubular dentin matrix of caries - affected dentin, which take up much more toluidine blue stain and exhibit more intense metachromasia than normal peritubular dentin, suggests the presence of mucopolysaccharides or glycoprotiens. these molecules may interfere with resin wetting of fine porosities within both intertubular and peritubular dentin and/or fusayama, observed affected dentin to have turbid, transparent and subtransparent zones. there is limited information on the structure of these zones as well as conflicting evidences about their properties. general perception is that transparent dentin is sclerotic and hypermineralized due to tubular occlusion which might act as a barrier to penetration of primers and bonding agents. although the adhesive resin may have followed the primer, it may not have copolymerized well with the primer. thus the adhesion of resins to caries - affected dentin may be inferior to that of normal dentin, due to weaker collagen and/or weaker resin even though most of the tubules in such dentin are filled with mineral deposits. these intratubular crystals are not well - packed and are softer than well - packed apatite even through they are more acid - resistant.[1015 ] in relation to the influence of dyes on the adhesion of filtekz250, the decrease in the bond strength may be due to dye solution remaining in sound and affected dentin as mentioned in other studies. single bond is an adhesive that needs to have close contact with the dentin substrate to produce the desired bond strength. dye remaining trapped in dentin may adversely affect the wetting of dentin by materials, thereby decreasing micromechanical retention of these materials. it was observed that despite the application on sound dentin and carious affected dentin, caries - detecting dyes, even after being rinsed and acid etched, were not completely removed, as evidenced by some samples with sound tissue remaining lightly colored which might have influenced the results. within the limitation of the present study, it is concluded that the tensile bond strength was higher in sound and carious affected dentin in both control and experimental group without application of caries detection dyes than that with the application of caries detection dyes.	objectives : the objective of this study was to evaluate the influence of caries detection dye on the in - vitro tensile bond strength of adhesive materials to sound and carious affected dentin.materials and methods : forty healthy and carious human molars were ground to expose superficial sound dentin and carious affected dentin. caries detector dye was applied to sound and carious affected dentin and rinsed. subsequently the dentin was etched with 37% phosphoric acid and rinsed leaving a moist dentin surface. the adhesive (single bond) was applied in single layers and light cured. a posterior composite (filtek z 250) were used to prepare the bond strength specimens with a 3 mm in diameter bonding area. control and experimental groups were made with and without application of dye respectively. each group includes both sound and carious affected dentin. after 24 hour immersion in distilled water, tensile bond strength (mpa) was measured using an instron testing machine.results:analysis of variance (anova) was used to evaluate the data. the tensile bond strength were significantly less in experimental subgroup than control subgroups.conclusion:the tensile bond strengths were higher in sound and carious affected dentin without application of caries detection dyes.
	transition metal dichacogenides represent a unique class of two - dimensional layered materials that can be exfoliated into single or few atomic layers. tungsten diselenide (wse2) is one typical example with p - type semiconductor characteristics. bulk wse2 has an indirect band gap (1.2 ev), which transits into a direct band gap (1.65 ev) in monolayers. monolayer wse2, therefore, is of considerable interest as a new electronic material for functional electronics and optoelectronics. however, the controllable synthesis of large - area wse2 atomic layers remains a challenge. the studies on wse2 are largely limited by relatively small lateral size of exfoliated flakes and poor yield, which has significantly restricted the large - scale applications of the wse2 atomic layers. here, we report a systematic study of chemical vapor deposition approach for large area growth of atomically thin wse2 film with the lateral dimensions up to 1 cm2. microphotoluminescence mapping indicates distinct layer dependent efficiency. the monolayer area exhibits much stronger light emission than bilayer or multilayers, consistent with the expected transition to direct band gap in the monolayer limit. the transmission electron microscopy studies demonstrate excellent crystalline quality of the atomically thin wse2. electrical transport studies further show that the p - type wse2 field - effect transistors exhibit excellent electronic characteristics with effective hole carrier mobility up to 100 cm2 v1 s1 for monolayer and up to 350 cm2 v1 s1 for few - layer materials at room temperature, comparable or well above that of previously reported mobility values for the synthetic wse2 and comparable to the best exfoliated materials.
dental trauma is one of the most painful and stressful injuries in young patients and requires both expedient and expert management by a dental practitioner. children are always keen to explore their surroundings without realizing the risks they are exposed to. the world health organization has defined exarticulation as the complete displacement of a tooth from its alveolar socket due to traumatic injury. in permanent dentition, it may occur as the result of fights or sport injuries, while falls against hard objects are among the frequent causes in the primary dentition. research has shown that an exarticulated tooth can be re - planted without complications if it is reinserted into the socket within 20 minutes when stored dry, and within one to three hours if placed in a suitable storage medium. when the tooth is kept dry for more than 20 minutes, its pdl cells begin to necrotize, and upon replantation, inflammation and resorption develop in proportion to the extra - oral dry time. many storage media such as milk, saliva, hank s balanced salt solution (hbss),, hbss has been proposed as the storage medium of choice for treatment of avulsed teeth by the american associations of endodontists. however, the major disadvantage of hbss and many of the other aforementioned media is that they are not easily available at places where these traumatic injuries occur. hence, there is a need to identify a storage medium that will be readily available, yet effective. aloe vera has recently gained popularity in the medicinal field because of its antidiabetic, anticancer, and antibiotic properties. its anti - inflammatory properties enhance wound healing whether it is used topically or orally. because of its increased use, the plant can be found commonly in most households, nurseries, parks and recreation areas. also, several reputable suppliers have produced a stabilized aloe vera gel for use alone or in combined formulations [68 ]. the purpose of this study was to assess the efficacy of three easily available storage media, milk, aloe vera, and egg white, in preserving the viability of pdl cells of avulsed teeth in vitro. forty - five non - carious human premolar teeth with normal periodontium and closed apices, undergoing extraction for orthodontic therapeutic purposes, were included in this study (fig. they were divided into three equal groups using simple random allocation (using a chit pull system). group i : stored in milk (control group)group ii : stored in aloe vera (experimental group)group iii : stored in egg white (experimental group). group i : stored in milk (control group) group ii : stored in aloe vera (experimental group) group iii : stored in egg white (experimental group). non - carious mature premolar teeth were extracted pasteurized, homogenized milk with fat content of 3gm/100ml (saras dairy) at room temperature was used as the control group. to prepare aloe vera gel, a kitchen knife was used to cut off the outer spiked edges of the leaves. after removing the outer skin of the leaves, the contents were blended thoroughly and filtered through a piece of muslin cloth and were placed into a glass jar with a tight fitting lid (fig. egg white was separated from the yolk and collected in a bowl using the half shelves. following extractions, the coronal third of pdl was scraped with a curette to remove cells likely to be damaged due to instrumentation. to simulate avulsion injury, teeth were kept in mud for 15 minutes immediately after removal of the damaged cells from the coronal one - third (fig. after 30 minutes, the root surface was irrigated twice with sterile isotonic saline to remove the residual storage media. the apical two - third of the root surfaces (as measured from epithelial attachment) were scraped in a petri dish to obtain the pdl cells. preparation of aloe vera gel mud for 15 minutes to simulate avulsion injury the obtained scrapings were subsequently added to a falcon tube containing 2.5 ml of phosphate buffer (fig. 4). to the above mixture, 0.5 mg of type i collagenase was added, and this mixture was incubated for 30 minutes. following incubation, pdl cells (from apical two - third of the root surface) were scraped in a petri dish the supernatant was discarded and the centrifuged residue was collected. to this, an equal volume of 0.4% trypan blue stain was added, and it was mixed well. trypan blue stains non - viable cells blue and viable cells appear colorless or pink. following staining, the number of cells (total and non - viable) was determined by counting the cells overlying a 41 mm area of the counting chamber (figs. 5 and 6). microscopic view of viable and non - viable cells in milk microscopic view of viable and non - viable cells in aloe vera the viable cell percentage was calculated as : [(total cells - stained cells)/total cells ] 100 the mean and standard deviation of the number of viable cells from different storage media were calculated and statistical analysis was performed. inter - group comparison was done using one - way analysis of variance (anova) complemented by the tukey s test. the number of cells (total and non - viable) was determined by counting the cells overlying a 41 mm area of the counting chamber (figs. 5 and 6). microscopic view of viable and non - viable cells in milk microscopic view of viable and non - viable cells in aloe vera the viable cell percentage was calculated as : [(total cells - stained cells)/total cells ] 100 the mean and standard deviation of the number of viable cells from different storage media were calculated and statistical analysis was performed. inter - group comparison was done using one - way analysis of variance (anova) complemented by the tukey s test. the teeth stored in aloe vera demonstrated significantly higher percentage of viable pdl cells (87.3%) compared to those stored in egg white (75.7%) or milk (74.0%) (table 1, graph 1). aloe vera showed higher number of viable cells when compared to milk and egg white (p=0.0001 between groups i ii and ii iii, p=0.4757 between groups i iii, table 2). the mean values of viable and nonviable and percentage of viable pdl cell count in the study groups and control group percentage of viable cells inter - group comparison using one - way anova complemented by the tukey s test. post hoc power analysis was done using g power software 3.0.10. by fixing the alpha at 0.05, the calculated effect size from the given values was 0.55 and the power of the study was 0.9 or 90%. recent clinical studies have shown that avulsed teeth replanted within five minutes have the best prognosis, resulting in much higher reattachment success. to avoid root resorption and maintain pdl cell viability, various transport media have been proposed to store the tooth in, if immediate re - plantation is not possible. the basic principle behind the use of these media is to transport the tooth to an environment similar to that of the oral cavity. various studies have found hbss, viaspan, and eagle s medium to be suitable for transportation of avulsed teeth as they preserved pdl cell viability. however, the main disadvantages of these media are high cost and lack of availability. therefore, there should be an effort to find a storage medium that is easily available and cheap. hence, the present study was carried out to compare the effectiveness of two of the most commonly available and cost effective storage media, aloe vera and egg white. therefore, noncarious mature human premolar teeth undergoing extraction for orthodontic therapeutic purposes were selected. for the same reason, teeth from young healthy individuals without periodontal disease were included in the study. on average it takes about 10 to 15 minutes for the victim and the person caring for him / her to recover from the traumatic event and act appropriately. according to pohl, pdl cells remain in a non - compromised state with up to 15 minutes of dry time. other investigators have shown that at two hours of dry time, no vital pdl cells remain. in the present study, it was decided to keep the extracted tooth in mud for at least 15 minutes to simulate avulsion injury, and to ensure that sufficient viable pdl cells are available for assessment. following a 15-minute dry time, the teeth were placed in different storage media for 30 minutes. this period was very important because the pdl cells were most susceptible to damage during this time. it is a physiological medium, which contains fewer bacteria compared to saliva ; therefore it is one of the most frequently used transport media. due to these advantages, in the present study, milk was chosen as the positive control. in the present study, aloe vera gel was chosen as a transport medium because it contains 99% water and over 75 nutrients, which include 20 minerals, 19 amino acids and 12 vitamins. the human body requires 22 amino acids to maintain good health, eight of which are essential, as the body can not synthesize them. all of these eight essential amino acids and 11 of 14 secondary amino acids are found in aloe vera. it is thought to promote healing ; therefore it can be used in surgical wounds, in root canal treatment as an analgesic dressing, and around dental implants to control inflammation. in a recent study, human kidney cell death rate egg white from a single egg contains 4.7 grams of 40 different proteins, 0.3 grams of carbohydrate, 62 milligrams of sodium, and the remainder being water. a method suggested by ragnarsson. has been quoted in the literature to evaluate the efficacy of different storage media in preserving the viability of dental fibroblasts. ragnarsson. removed fibroblasts from the root surfaces and cultured them after adding them to a storage medium. however, doyle. placed the extracted teeth directly in a storage medium. at a pre - determined time, the teeth were taken out of the medium and pdl cells were isolated to evaluate cell viability. since the method by doyle. was found to closely replicate the clinical scenario, it was followed in this study. to quantitate the number of viable pdl cells in the current study and to preserve maximum cell viability, the root surfaces were treated with type i collagenase for rapid cell retrieval and cellular integrity. trypan blue exclusion assay quickly and easily differentiates non - viable cells from viable ones. as chromophore present on cell membranes is negatively charged, it does not take up trypan blue stain unless the membrane is damaged ; consequently all the viable cells are excluded as they do not pick up the dye. trypan blue stain used in this study assessed only viability of the cells and not the actual physiologic health and metabolic capabilities of them. according to our results, maximum percentage of viable cells was found in aloe vera (87.3%), followed by egg white (75.7%) and milk (74.07%). aloe vera maintained the highest number of viable cells, which could be attributed to its composition. aloe vera also contains allantoin, which has been found to stimulate fibroblast activity and collagen proliferation. in a recent study, green tea extract also showed higher number of viable cells compared to milk, which was used as a positive control group. egg white maintained less viable cells than aloe vera, which may be attributed to its high ph (9.38), and also to the large amount of proteins in egg white, which might act as foreign bodies. in our study, milk showed the least number of viable cells (74.0%), which could be attributed to the presence of various enzymes in the milk, which could be harmful to the fibroblasts of the pdl. found that milk was unable to regenerate depleted cell metabolites and restore the viability of pdl cells. also, because of the lack of a cell energy source and ions in milk, repopulation of pdl was not permitted. milk was found to be a compatible storage medium only when it was cold and fresh. immediate re - implantation is the best treatment for exarticulated teeth, provided that the teeth have viable pdl cells at the time of re - implantation. if that is not possible, storing the tooth in a storage medium would be the next best option to preserve its viability until re - implantation is possible. within the limitations of this study, it appears that aloe vera maintains pdl cell viability better than egg white or milk. because of its superiority as a storage medium, and other medicinal properties, it is recommended that it should be made easily available in the form of gel or other formulations over the counter, and be added to first aid kits at schools and sport clubs, so that it can be easily accessed at the time of trauma / injury to teeth. further studies, including in vivo studies, are needed to investigate whether aloe vera can maintain pdl cell viability if maintained at lower temperatures or at extended extra oral dry time.	objectives : this study was undertaken to evaluate the viability of periodontal ligament (pdl) cells of avulsed teeth in three different storage media.materials and methods : forty - five premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups based on storage media used [group i : milk (control) ; group ii : aloe vera (experimental) ; group iii : egg white (experimental) ]. following extractions, the teeth were placed in one of the three different storage media for 30 minutes, following which the scrapings of the pdl from these teeth were collected in falcon tubes containing collagenase enzyme in 2.5 ml of phosphate buffered saline. the tubes were subsequently incubated for 30 minutes and centrifuged for five minutes at 800 rpm. the obtained pdl cells were stained with trypan blue and were observed under optical microscope. the percentage of viable cells was calculated.results:aloe vera showed the highest percentage of viable cells (114.38.0), followed by egg white (100.96.3) and milk (101.17.3).conclusion : within the limitations of this study, it appears that aloe vera maintains pdl cell viability better than egg white or milk.
seven consecutive patients [three men and four women aged 15 - 77 (mean, 42.7) years ] admitted to our hospital with hypoglycemia between april 1, 2001 and september 31, 2002 were retrospectively studied. the time from the onset of neuroglycopenic symptoms to diagnosis ranged from 4 to 42 months. the age, sex, and history of the patients are summarized in table 1. the inclusion criteria were coexisting hypoglycemic symptoms with low fasting serum glucose levels (< 50 mg / dl) and biochemical evidence of organic hyperinsulinemia, confirmed by a fasting test (serum insulin : glucose ratio (0.3, with high c - peptide levels). symptomatic hypoglycemia was observed and documented [plasma glucose range : 27 - 46 (mean, 35) mg / dl ] after 24 - 72 hours of fasting. concomitant insulin [range : 10.9 - 33.6 (mean, 18.4) iu / ml ] and c - peptide levels [range : 1.95 - 5.67 (mean, 3.95) ng / ml ] were not suppressed. all these patients had pathologic glucose - insulin ratios (range, 0.40 - 0.73 ; mean, 0.51). thus, hyperinsulinemic hypoglycemia due to insulin - secreting tumors was proven in all cases. radiological imaging modalities employed for the localization of these pancreatic adenomas are summarized in table 2. ultrasonography (us) was performed in three cases, endoscopic ultrasonography (eus) in two, computed tomography (ct) in seven, and intraoperative ultrasonography (ious) in four. in all cases, the detection rate of each study was evaluated. after obtaining informed consent from each patient none of our patients was receiving cardiac glycosides, and none was suffering from sarcoidosis or renal failure, conditions considered relative contraindications to intra - arterial calcium injection. all tests were carried out under local anesthesia and with continuous electrocardiographic and blood pressure monitoring. to avoid iatrogenic hypoglycemia, blood sugar levels were checked before the procedure. in patient 2, this was 37 mg / dl, and 25 g of intravenous glucose were administered, using a 50% solution. prior to angiography, a 5-french (f) cobra catheter (terumo, tokyo, japan) was placed in the right hepatic vein close to its junction with the inferior vena cava through a puncture site in the right internal jugular vein (fig. the right femoral artery was then punctured, and a 5-f yashiro catheter (terumo) was advanced to the celiac trunk and the superior mesenteric artery (sma). occasionally, a 3-f progreat microcatheter (terumo) was used for coaxial superselective catheterization of the gastroduodenal (gda) and splenic artery (spa). selective presampling angiography was performed after selectively injecting nonionic contrast agent (iomeron 300 ; ilsung, korea) into the gda, spa, and sma. for angiographic studies, digital subtraction angiography (dsa), a 5-ml bolus of calcium gluconate (0.025 meq ca / kg) diluted in saline was rapidly injected through the proximally positioned catheter in each selectively catheterized artery. the period between each injection was at least 5 minutes. before and 30, 60, and 120 seconds after calcium injection, 5 ml of hepatic venous blood was obtained. each sample was labeled according to the time interval and artery of origin, and centrifuged, and the resulting serum was assayed for insulin concentration in all patients and c - peptide concentration in six. all asvs results were analyzed by an individual blinded to all other preoperative imaging studies and operative findings. according to the findings of doppman., a positive finding is indicated by a twofold elevation of insulin levels in the 30- or 60-second samples (or both) obtained from the hepatic vein (1). a twofold rise in insulin levels following calcium gluconate injection of the gda or sma indicates that the tumor is located in the pancreatic head or uncinate process ; a twofold rise after injection into the spa suggests a corporeal or caudal location. for diagnostic accuracy, c - peptide gradients in all patients except patient 1 the diagnostic criteria for c - peptide have not been reported, but we supposed that such analysis would be useful if insulin gradients were equivocal. the results of asvs in which insulin gradients only were determined, and in which additional c - peptide gradients were established, were compared. comparisons were also made between the sensitivity of these modalities and that of other localizing modalities in terms of the operative findings and outcome of surgery. the median follow - up time was 4 (range, 1 - 10) months. at these intervals, patient histories were obtained and plasma glucose levels were determined after 24 - 72 hours of fasting. seven consecutive patients [three men and four women aged 15 - 77 (mean, 42.7) years ] admitted to our hospital with hypoglycemia between april 1, 2001 and september 31, 2002 were retrospectively studied. the time from the onset of neuroglycopenic symptoms to diagnosis ranged from 4 to 42 months. the age, sex, and history of the patients are summarized in table 1. the inclusion criteria were coexisting hypoglycemic symptoms with low fasting serum glucose levels (< 50 mg / dl) and biochemical evidence of organic hyperinsulinemia, confirmed by a fasting test (serum insulin : glucose ratio (0.3, with high c - peptide levels). symptomatic hypoglycemia was observed and documented [plasma glucose range : 27 - 46 (mean, 35) mg / dl ] after 24 - 72 hours of fasting. concomitant insulin [range : 10.9 - 33.6 (mean, 18.4) iu / ml ] and c - peptide levels [range : 1.95 - 5.67 (mean, 3.95) ng / ml ] were not suppressed. all these patients had pathologic glucose - insulin ratios (range, 0.40 - 0.73 ; mean, 0.51). thus, hyperinsulinemic hypoglycemia due to insulin - secreting tumors was proven in all cases. radiological imaging modalities employed for the localization of these pancreatic adenomas are summarized in table 2. ultrasonography (us) was performed in three cases, endoscopic ultrasonography (eus) in two, computed tomography (ct) in seven, and intraoperative ultrasonography (ious) in four. in all cases, after obtaining informed consent from each patient, asvs was performed according to the protocol proposed by doppmann. none of our patients was receiving cardiac glycosides, and none was suffering from sarcoidosis or renal failure, conditions considered relative contraindications to intra - arterial calcium injection. all tests were carried out under local anesthesia and with continuous electrocardiographic and blood pressure monitoring. to avoid iatrogenic hypoglycemia, blood sugar levels were checked before the procedure. in patient 2, this was 37 mg / dl, and 25 g of intravenous glucose were administered, using a 50% solution. prior to angiography, a 5-french (f) cobra catheter (terumo, tokyo, japan) was placed in the right hepatic vein close to its junction with the inferior vena cava through a puncture site in the right internal jugular vein (fig. the right femoral artery was then punctured, and a 5-f yashiro catheter (terumo) was advanced to the celiac trunk and the superior mesenteric artery (sma). occasionally, a 3-f progreat microcatheter (terumo) was used for coaxial superselective catheterization of the gastroduodenal (gda) and splenic artery (spa). selective presampling angiography was performed after selectively injecting nonionic contrast agent (iomeron 300 ; ilsung, korea) into the gda, spa, and sma. for angiographic studies, digital subtraction angiography (dsa), a 5-ml bolus of calcium gluconate (0.025 meq ca / kg) diluted in saline was rapidly injected through the proximally positioned catheter in each selectively catheterized artery. the period between each injection was at least 5 minutes. before and 30, 60, and 120 seconds after calcium injection each sample was labeled according to the time interval and artery of origin, and centrifuged, and the resulting serum was assayed for insulin concentration in all patients and c - peptide concentration in six. all asvs results were analyzed by an individual blinded to all other preoperative imaging studies and operative findings. according to the findings of doppman., a positive finding is indicated by a twofold elevation of insulin levels in the 30- or 60-second samples (or both) obtained from the hepatic vein (1). a twofold rise in insulin levels following calcium gluconate injection of the gda or sma indicates that the tumor is located in the pancreatic head or uncinate process ; a twofold rise after injection into the spa suggests a corporeal or caudal location. for diagnostic accuracy, c - peptide gradients in all patients except patient 1 were also analyzed. the diagnostic criteria for c - peptide have not been reported, but we supposed that such analysis would be useful if insulin gradients were equivocal. the results of asvs in which insulin gradients only were determined, and in which additional c - peptide gradients were established, were compared. comparisons were also made between the sensitivity of these modalities and that of other localizing modalities in terms of the operative findings and outcome of surgery. the median follow - up time was 4 (range, 1 - 10) months. at these intervals, patient histories were obtained and plasma glucose levels were determined after 24 - 72 hours of fasting. the results of all localization studies and surgery are presented in table 2. in no case did asvs lead to complications, and procedural - related symptomatic hypoglycemia and hypercalcemia were not encountered. insulin or c - peptide gradients were calculated as the ratios of hepatic venous insulin or c - peptide concentrations, respectively, at 30, 60, and 120 seconds after asvs to baseline. in patients 1, 2, 5 and 7, significant insulin gradients were observed after calcium injection into the gda and in patient 6 after stimulation of the gda and sma, indicating the presence of insulinomas in the pancreatic head or uncinate process (fig. 2b), and in patient 3, both insulin and c - peptide gradients increased significantly after stimulation of the spa, suggesting that the pathological source of insulin might be the pancreatic body or tail (fig. peak insulin gradients in the gda 30 seconds after stimulation and in the spa at 60 seconds were not significantly different (fig. after 30 seconds, insulin gradients decreased gradually in the gda, but in the spa showed a gradual increase. the analyst, blinded to all other preoperative imaging studies and operative findings, thus could not confidently determine the location of the insulinoma. in this case, additional c - peptide gradients were, however, helpful (fig. the c - peptide gradient peaked 60 seconds after stimulation of the spa, and in the gda was not more than 1.0. eight insulinomas, 5 - 25 (mean, 12.5) mm in size, were found and confirmed histopathologically after enucleation (table 2). all tumors except in patient 3 were less than 2 cm in size. three were in the head, two in the uncinate process and three in the tail. in patient 4, the tumors were correctly localized by us in two of three patients (sensitivity, 67%), by eus in one of two (sensitivity, 50%), by ct in four of seven (sensitivity, 57%), by angiography in three of seven (sensitivity, 43%), and by ious in four of four (sensitivity, 100%). for insulin gradients only, the sensitivity of asvs was 86% (6 of 7 patients), but with additional c - peptide gradients, sensitivity was 100% (6 of 6 patients). all patients were cured by enucleation of the insulinoma and remained free of hypoglycemia during the follow - up period. the availability of methods to accurately determine circulating levels of insulin, c - peptide, and proinsulin has led to dramatic improvements in the preoperative diagnosis of insulinomas, and to exclusion of the surreptitious administration of insulin as the cause of hypoglycemia (3). preoperative localization of a tumor has, for this reason, moved to center stage as the main focus of interest. since daggett. (6) published their findings in 1981, the question of whether imaging is necessary prior to initial insulinoma - related surgery has been a matter of debate (13 - 15). many surgical teams, meanwhile, do not rely exclusively on bimanual palpation of the pancreas combined with ious, but insist on meticulous preoperative localization, and it is to this that they attribute their high operative success rates (16 - 18). an average of 10% of insulinomas defy surgical exploration (4, 16) and correct preoperative localization minimizes the extent to which this is necessary, thus reducing morbidity (13). recent developments in the laparoscopic resection of pancreatic insulinomas, a procedure increasingly described for tumors located in the body or tail of the pancreas, emphasize, furthermore, the need for an accurate preoperative localization procedure (19, 20). hence, even though they are controversial in terms of cost - effectiveness (3, 22), preoperative localization studies are recommended (13, 15 - 17, 21). if such studies do not facilitate rational and safe surgical enucleation or resection, they are not, however, time- and cost - effective (23). the mean diameter of insulinomas is generally not more than 15 mm (2, 11, 15, 24), and non - invasive imaging modalities thus often fail to localize them. non - invasive modalities such as us, ct, and mri have shown low sensitivities (8, 9, 13, 14, 16, 21, 25), while ct and us will detect up to 60% of biochemically proven insulinomas. relatively little has so far been reported about mri in the diagnosis of islet cell tumors, and its exact role is uncertain (4). in our series the mean size of insulinomas was 12.5 mm, a fact which could explain the low detection rate of adenomas at us and ct. however, recent reports describing the findings of dynamic ct and mri have been promising (26 - 28). the sensitivities of other, invasive, localization techniques for detecting insulinomas were higher : 57 - 82% with eus (11, 12, 18, 29, 30), 35% to 65% with angiography (9, 10, 31, 32), and 77 - 100% with transhepatic portal venous sampling (tpvs) (7 - 10, 13, 14, 17, 21, 31, 32). however, these techniques have certain disadvantages, including low sensitivity in the case of angiography and eus (the sensitivity of eus depends on the location of the tumor) and higher morbidity for tpvs (4, 8, 9, 33). the sensitivity of ious combined with palpation was more than 80% in most reported series (2 - 4, 14, 16, 31, 34). however, insulinomas located in the pancreatic head or uncinate process, and thus deep within the parenchyma, were difficult or impossible to palpate, even where an experienced surgeon was involved (5), and to detect at surgery, using ious, particularly at hospitals with limited experience of this modality (9, 11, 31). (3, 7), the detection of insulinomas is based on their hormone production, not their appearance. the sensitivity of the technique is independent of lesion size. at the same time, however, since the lesion is identified by its hormone output, it is not visulalized directly and can not be located precisely. its position can be confirmed only in terms of a region of the pancreas (head / uncinate process versus body / tail). adenomas to the left of the sma can be treated by enucleation and distal pancreatectomy, whereas those to the right require enucleation. in our series, moreover, asvs is far less invasive and complicated than tpvs and can be safely carried out in combination with a normal dsa procedure (5, 8, 9). complications of asvs are rare (33), and major complications did not occur in our patients. although hypoglycemia is a severe, even life - threatening potential complication of asvs, it has not previously been reported (4). insulin levels in the hepatic vein usually peaked in samples obtained 30 or 60 seconds after calcium stimulation, and were often returning toward baseline within 120 seconds of stimulation, suggesting that insulin release is confined to the short interval during which beta cells are exposed to high serum calcium levels (9). calcium should be administered cautiously to patients with sarcoidosis, or renal or cardiac disease, particularly to those receiving cardiac glycosides (7). the inotropic effects of cardiac glycosides and calcium are synergistic ; if both are administered simultaneously, dysrhythmia may thus occur (33). insulin gradients in the gda 30 seconds after stimulation and in the spa at 60 seconds were not significantly different, and while this gradient peaked 30 seconds after the start of calcium gluconate infusion into the gda, in the spa it increased gradually. additional analysis of the c - peptide gradient showed that in the spa it peaked 60 seconds after stimulation, and in the gda it was not more than 1.0. the analyst localized the tumor to the pancreatic body or tail, and two small adenomas found in the tail were to the left of the sma. elevated insulin levels in the hepatic veins after asvs did not always equate with correct localization (8). we assumed that additional analysis of c - peptide gradients would help assess the location of a tumor when insulin gradients are equivocal, basing our assumption on two factors : first, calcium gluconate administration led not only to hyperinsulinemia, but also to increased serum proinsulin and c - peptide levels (35). second, although the secretory ratio of c - peptide to insulin is 1:1, the ratio in serum is about 5:1 to 15:1 (36). the molar concentration of c - peptide in blood is higher than that of insulin because of the hepatic clearance of the latter (36). approximately 50% of insulin is rapidly removed by its initial passage through the liver, but hepatic extraction of c - peptide is negligible (36). a relatively high concentration of c - peptide may thus reflect a subtle increase in its level, compared to that of insulin, after stimulation with calcium gluconate. in six of our patients, to our knowledge, this is the first report to describe the use of these gradients in asvs for the localization of insulinomas. however, to confirm the efficacy of c - peptide gradients, in spite of the significant additional cost of multiple c - peptide assays, further investigation is needed. a further point is that because catheterization of the left hepatic vein was technically more difficult, and also unnecessary, the right hepatic vein only was chosen (4, 8, 9, 19, 24). selective distribution of splenic and mesenteric venous blood to the left and right portal vein, respectively, may mask an adenoma in the body - tail region, but such portal flow dynamics have never been proven (24). (19) reported that where asvs was used, there was no difference in the degree of step - up between right and left hepatic vein sampling., furthermore, observed that in patients with tumors in the body and tail of the pancreas, insulin levels in samples obtained from the right hepatic vein were always elevated (9). in conclusion, asvs is a highly accurate and safe method for the preoperative localization of insulinomas. additional c - peptide gradients seem to be helpful in assessing the location of a tumor, but additional investigation is needed.	objectiveto determine the value of selective intra - arterial calcium stimulation with hepatic venous sampling using serum insulin and c - peptide gradients for the preoperative localization of insulinomas.materials and methodsseven consecutive patients [three men and four women aged 15 - 77 (mean, 42.7) years ] with hypoglycemia underwent selective intra - arterial calcium stimulation in conjunction with hepatic venous sampling. insulin gradients were calculated by an individual blinded to all other preoperative imaging studies and operative findings. in all patients except one, c - peptide gradients were also analyzed. the results were compared with the preoperative findings of ultrasonography, computed tomography, arteriography and endoscopic ultrasonography, as well as with the intraoperative findings of ultrasonography and palpation at surgery.resultseight insulinomas (mean diameter, 12.5 mm) were diagnosed after surgery. in six patients, the calcium stimulation test with insulin gradients allowed accurate localization of the pathologic source of insulin secretion. both c - peptide and insulin gradients substantially increased diagnostic accuracy. in one patient, c - peptide gradients were more helpful than insulin gradients for tumor localization.conclusionselective intra - arterial calcium stimulation with hepatic venous sampling is a highly accurate and safe method for the preoperative localization of insulinomas. additional c - peptide gradients seem to be helpful in assessing tumor location, but further study is needed.
an effective and adequate relationship between patient and physician during medical care is necessary for the provision of good care. although communication skills training is being developed in medical schools, little attention is paid and less than 5% of the curriculum is allocated to communication skills education. besides, a vast majority of continuing medical education (cme) curriculum focuses on technological and biomedical aspects of medical care. therefore, continuous training of communication skills through their practical years is necessary to refresh the previously acquired skills and to develop new techniques. medicine in the context of health system, offering services by family physicians that provide primary health care services is an area of health care in which importance for good communication has received particular attention. improving the quality of primary care is a key effort of health policy. to improve clinical performance and the quality of primary health care services, an adequate doctor many studies have confirmed that there is a strong association between the physician communication skills and the compliance and satisfaction of the patients. patient satisfaction is an important indicator for care quality in terms of acceptability and as a predictor of health outcome. the strategy of communication skills training programs curriculums can range from passive, didactic, large group presentations to highly interactive learning methods, such as workshops, small groups, and individualized training sessions. some of them are workshops that take from 16 h (2- day) to 105 h (5 days a week for 3 weeks). another format of training programs are video conference and small group session (e.g., three 2-h sessions in three successive weeks or eight 3-h sessions weekly). generally, weekly sessions take less time, in comparison with workshops. selecting a strategy for communication skills training program depends on the implementation situation. in the context of health system, because family physicians often have limited time to follow extensive courses and because constraints such as time restrictions or a limited budget, an effective, feasible, affordable, and continuous training approach is important. to finding an opportunity for training program, based on their opinion, one of the routine programs that keep a minimum disruption of the daily activities of the health networks can be selected. training program that is concurrent with selected routine program and learner participation in training program procedures can be overcome by extra time and labor. the aim of this study is design and implementing the communication skills training program, for the physicians involved in health care centers as a family physician, simultaneously with the routine program and assess its feasibility, acceptability, educational content and method appropriateness, and ability to integrating into the health system programs based on stakeholder feedback and to examine the program 's effect on physicians communication skills, knowledge, and attitude and their patient satisfaction. this study was a randomized field trial in the context of health care systems, which evaluated the effectiveness of a communication skills training program for family physicians. the effects of the program on physicians communication skills, knowledge and attitude, and their patient satisfaction were examined using data collected at three time points, baseline, immediately after the program (intervention takes 3 months), and 4 months after for long - term follow up, in two health networks, who were randomly assigned to the intervention or control during 2013. feasibility, acceptability, educational content and method appropriateness, and ability to integrating it into the health system of the program was assessed based on program evaluation data obtained from stakeholders in the intervention group. the medical ethics committee of isfahan university of medical science has approved the study design, protocols, and informed consent process (code of thesis was 3921173). to find the opportunity for communication skills training, a meeting was arranged with health system administrators and the family physician who were stakeholders, to analyze the situation with brain storming method. based on their opinion, one of the routine programs in health system is organization meetings that gather physicians together in order to coordinate and discuss about their responsibilities. concurrent holding of organization meeting and communication skills training, is a fundamental approach for time saving. another issue that was brain storming in our meeting with stakeholders, was educational topics. we picked out some topics on communication problems, which family physicians challenge in their practice, based on their point of view and the calgary cambridge guideline. thus, our intervention curriculum includes theses 12 following topics : basic communication skills : initiating the session advanced communication skills : breaking bad news taking a sexual history communicating with patients from different cultures communicating with children and adolescents communicating with patient 's family challenging consultation and communication with colleagues. this trial was carried out in two health networks in isfahan (iran 's third largest city, located in the center of iran), iran, which were selected randomly. two selected health networks were randomly assigned to the intervention or to the control group. to make sure that the changes were made by the intervention, a control group was chosen. and to prevent contamination, control and intervention groups were selected from separate health networks. the sample size of the trial was based on the main outcome, namely, patient satisfaction. the sample size was estimated to be 128 participants (64/group), with = 0.05, d = 9.3 and power = 80%. we considered 10% attrition rate, and the final sample size was estimated to be 140 patients (70/group). one physician per five patients (20% of the sample size) considered to estimate the number of physicians (14/group). from the available list (sampling frame) of the physicians who were employed in the intervention selected health network, 14 doctors were selected through a simple random sampling then sampling frame, in control group, 14 doctors were matched as demographic variables (age and gender) with intervention group. five patients of any physician were selected through convenience sampling in their workplace (a total of 70 patients for each group). the inclusion criteria were physicians who were employed in the health network as a family physician. the stakeholders that participated to evaluate appropriateness of the method of intervention included physicians and health system administrators (directorate of the district health network, health deputy, mental health program coordinators, promotion and expansion of system coordinators). program outcome measures in physicians and patients : the physicians communication skills, knowledge and attitude, and their patient satisfaction were considered as outputs of our study. physicians knowledge was assessed by using an existing physicians knowledge toward communication skills questionnaire that consisted of 12 items, which participants responded on a yes / no scale. physicians attitude toward communication skills questionnaire consisting of 10 likert - type items (5-point scale) measured physician 's attitude. in other studies, validity and reliability of this questionnaire was demonstrated., the cronbach 's alpha was 0.85 in the current sample. patient satisfaction of medical interview questioner was constructed to measure their patient satisfaction that consisted of 24 items and each item was measured on a 5-point likert scale. total score were divided on 125 (up score = 24 5 = 125) to calculate percent(%) of patient satisfaction. in all the questionnaires, higher total scores indicated better levels of understanding of each concept. patient communication skills training, a program evaluation checklist (13 items) was administered to all stakeholders after intervention to assess the stakeholders perception about the program. the checklist was based on the cdc program evaluation framework that included feasibility items (4 items), acceptability items (3 items), ability to continuing and integrating into health system items (2 items), and content and implementation method appropriateness items (4 items) ; each item was measured on a 2-point scale (1 = agree to 0 = disagree). stakeholders who were participating physicians and health system administrators completed this checklist at the end of the training program. the intervention program consists of six sessions (two topics / sessions) with 2-week intervals at the time of organization meetings. each session takes 2 h. three separate guidebooks for focal points, leaders, and learners were developed. the contents of the books were based on guidebooks on communication skills and expert opinion. the training method was case discussion. the focal points (mental health program coordinator employed in health system) were trained about method of program by the researcher. each physician chose one topic according to his interest, and then he designed cases that express communication problem of topic through his experience. for each session, focal point asked two physicians to participate in the program as a session leader. the leader raised the case of the considered topic in 10 min, then other participants discussed about solving the problem in 30 min and finally leader presented the guideline of the considered topic from leader guidebook in 20 min. in this way, each participant directly learned one topic and indirectly learned other topics through peer education. for statistical analysis, chi - square test, t - test, and repeated measure analysis of variance were used. this study was a randomized field trial in the context of health care systems, which evaluated the effectiveness of a communication skills training program for family physicians. the effects of the program on physicians communication skills, knowledge and attitude, and their patient satisfaction were examined using data collected at three time points, baseline, immediately after the program (intervention takes 3 months), and 4 months after for long - term follow up, in two health networks, who were randomly assigned to the intervention or control during 2013. feasibility, acceptability, educational content and method appropriateness, and ability to integrating it into the health system of the program was assessed based on program evaluation data obtained from stakeholders in the intervention group. the medical ethics committee of isfahan university of medical science has approved the study design, protocols, and informed consent process (code of thesis was 3921173). to find the opportunity for communication skills training, a meeting was arranged with health system administrators and the family physician who were stakeholders, to analyze the situation with brain storming method. based on their opinion, one of the routine programs in health system is organization meetings that gather physicians together in order to coordinate and discuss about their responsibilities. concurrent holding of organization meeting and communication skills training, is a fundamental approach for time saving. another issue that was brain storming in our meeting with stakeholders, was educational topics. we picked out some topics on communication problems, which family physicians challenge in their practice, based on their point of view and the calgary cambridge guideline. thus, our intervention curriculum includes theses 12 following topics : basic communication skills : initiating the session gathering information physical examination explanation and planning advanced communication skills : breaking bad news taking a sexual history communicating with patients from different cultures communicating with children and adolescents communicating with patient 's family challenging consultation and communication with colleagues. this trial was carried out in two health networks in isfahan (iran 's third largest city, located in the center of iran), iran, which were selected randomly. two selected health networks were randomly assigned to the intervention or to the control group. to make sure that the changes were made by the intervention, a control group was chosen. and to prevent contamination, control and intervention groups were selected from separate health networks. the sample size of the trial was based on the main outcome, namely, patient satisfaction. the sample size was estimated to be 128 participants (64/group), with = 0.05, d = 9.3 and power = 80%. we considered 10% attrition rate, and the final sample size was estimated to be 140 patients (70/group). one physician per five patients (20% of the sample size) considered to estimate the number of physicians (14/group). from the available list (sampling frame) of the physicians who were employed in the intervention selected health network, 14 doctors were selected through a simple random sampling then sampling frame, in control group, 14 doctors were matched as demographic variables (age and gender) with intervention group. five patients of any physician were selected through convenience sampling in their workplace (a total of 70 patients for each group). the inclusion criteria were physicians who were employed in the health network as a family physician. the stakeholders that participated to evaluate appropriateness of the method of intervention included physicians and health system administrators (directorate of the district health network, health deputy, mental health program coordinators, promotion and expansion of system coordinators). program outcome measures in physicians and patients : the physicians communication skills, knowledge and attitude, and their patient satisfaction were considered as outputs of our study. physicians knowledge was assessed by using an existing physicians knowledge toward communication skills questionnaire that consisted of 12 items, which participants responded on a yes / no scale. physicians attitude toward communication skills questionnaire consisting of 10 likert - type items (5-point scale) measured physician 's attitude. in other studies, validity and reliability of this questionnaire was demonstrated., the cronbach 's alpha was 0.85 in the current sample. patient satisfaction of medical interview questioner was constructed to measure their patient satisfaction that consisted of 24 items and each item was measured on a 5-point likert scale. total score were divided on 125 (up score = 24 5 = 125) to calculate percent(%) of patient satisfaction. this is a valid and reliable instrument base on zamani., study. in all the questionnaires, higher total scores indicated better levels of understanding of each concept. patient communication skills training, a program evaluation checklist (13 items) was administered to all stakeholders after intervention to assess the stakeholders perception about the program. the checklist was based on the cdc program evaluation framework that included feasibility items (4 items), acceptability items (3 items), ability to continuing and integrating into health system items (2 items), and content and implementation method appropriateness items (4 items) ; each item was measured on a 2-point scale (1 = agree to 0 = disagree). stakeholders who were participating physicians and health system administrators completed this checklist at the end of the training program. the intervention program consists of six sessions (two topics / sessions) with 2-week intervals at the time of organization meetings. each session takes 2 h. three separate guidebooks for focal points, leaders, and learners were developed. the contents of the books were based on guidebooks on communication skills and expert opinion. the focal points (mental health program coordinator employed in health system) were trained about method of program by the researcher. each physician chose one topic according to his interest, and then he designed cases that express communication problem of topic through his experience. for each session, focal point asked two physicians to participate in the program as a session leader. the leader raised the case of the considered topic in 10 min, then other participants discussed about solving the problem in 30 min and finally leader presented the guideline of the considered topic from leader guidebook in 20 min. in this way, each participant directly learned one topic and indirectly learned other topics through peer education. findings are shown as relative frequencies, mean, and standard deviation. for statistical analysis, chi - square test, t - test, and from the list of two health network physicians, 28 doctors (14/group) were selected. five patients of each physician participated in the study before and after intervention (70/group). physicians knowledge and attitude scale, before and after intervention were summarized in table 2. knowledge and attitudes of physicians, in the intervention group, were significantly increased after intervention (p 0. these scales were not significantly changed during 4-month follow up (knowledge p = 0. physician 's knowledge and attitude scale in the control group did not significantly change [table 2 ]. in the intervention group, before program implementation, patients were 71.48% satisfied with doctors communication skills and increased right after intervention (p = 0.004). 4 months after intervention patients satisfaction was significantly decreased from right after the intervention (p = 0.027) but still significantly higher than base (before intervention) (p = 0.005). in the control group, baseline patients satisfaction on doctors communication skills was 73.48% and it was not significantly different from two other followed - up measurements [figure 1 ]. physicians characteristics by intervention arm mean scores of physicians knowledge and attitude and mean percentage of patient satisfaction by intervention arm mean percent of patient satisfaction by intervention arm over time from 26 stakeholders, 14 were physicians who participated in training program, 12 were health system administrators. table 3 shows details of rates of agreement on program evaluation items based on stakeholder feedback. physicians rates of agreement versus personnel rates of agreement on program evaluation items were not significantly different (p > 0.05). in general, 80.5% of them agreed, that use of routine program as a strategy for training was evaluation of content and procedures appropriateness showed that on average, 80.75% of stakeholders agreed that the educational content and procedures were appropriate, 61% of stakeholders agreed that this program can be continued and sustained in the health network program, and 57.7% agreed that integrating this program to health system was feasible. from the list of two health network physicians, 28 doctors (14/group) were selected. five patients of each physician participated in the study before and after intervention (70/group). physicians knowledge and attitude scale, before and after intervention were summarized in table 2. knowledge and attitudes of physicians, in the intervention group, were significantly increased after intervention (p 0. these scales were not significantly changed during 4-month follow up (knowledge p = 0. physician 's knowledge and attitude scale in the control group did not significantly change [table 2 ]. in the intervention group, before program implementation, patients were 71.48% satisfied with doctors communication skills and increased right after intervention (p = 0.004). 4 months after intervention patients satisfaction was significantly decreased from right after the intervention (p = 0.027) but still significantly higher than base (before intervention) (p = 0.005). in the control group, baseline patients satisfaction on doctors communication skills was 73.48% and it was not significantly different from two other followed - up measurements [figure 1 ]. physicians characteristics by intervention arm mean scores of physicians knowledge and attitude and mean percentage of patient satisfaction by intervention arm mean percent of patient satisfaction by intervention arm over time from 26 stakeholders, 14 were physicians who participated in training program, 12 were health system administrators. table 3 shows details of rates of agreement on program evaluation items based on stakeholder feedback. physicians rates of agreement versus personnel rates of agreement on program evaluation items were not significantly different (p > 0.05). in general, 80.5% of them agreed, that use of routine program as a strategy for training was evaluation of content and procedures appropriateness showed that on average, 80.75% of stakeholders agreed that the educational content and procedures were appropriate, 61% of stakeholders agreed that this program can be continued and sustained in the health network program, and 57.7% agreed that integrating this program to health system was feasible. this study provides an experimental evidence that this communication skills training curriculum is well received by physicians and health system administrators and is beneficial for physicians and patients, which gives an acceptable evidence as a competency of a program to continue and integrate into a health system. implementation of training curriculum based on peer education and through the weekly routine coordination meetings can improve knowledge, attitude, and patients satisfaction with doctors communication skills in the setting of health care center physicians, as a family physician. an essential characteristic of this intervention is the strategy of implementation that is based on the number of meetings of physicians in the health network. this method is to hold a few 2-h educational meetings in which physicians discuss about communication problems. time limitation and human resources restriction, which we overcome, are two major restrictions in the health systems. the advantage of this method is that it can be proceeded without an extra time and excess manpower, and it is an important aspect of feasibility. in support of this fact, the result of this study shows 92.9% of physicians and 83.3% of administrators perceive the costs of technical resources and time spent are reasonable. also, an approximate two thirds of stakeholders agree that the program would suffer a minimum disruption of the daily activities of the health networks. other systematic reviews have also shown that weekly sessions take less time in comparison with workshops. approximately, 70% of physicians and 80% of personnel expressed that their opinions and needs in planning the program have been considered and no political conflict is claimed. most of the physicians who participated in the study reported satisfaction, enjoyment, and usefulness of the program. after some business activities, this program is a good opportunity to have a good time with colleagues. on the other hand, because physicians engage in the job as facilitators and select the educational topics based on their interest, they feel they belong to the program. the similarities between leaders and learners have been considered the core of a peer education program and pivotal in increasing the learners receptiveness of the message being delivered. preparing for leadership and assuming leadership roles may also lead to positive outcomes in peer leaders, and they develop more knowledge and positive attitudes due to their contribution. the benefits of peer education are in concurrence with other studies. among the assessing items of the appropriateness of the methodology and content of education, the maximum agreement is on educational content appropriateness maybe because of the needs and interest of the contributors in choosing the subjects. in addition, most of the stakeholders are satisfied with scheduling and educational tools that include guidebooks for focal points, facilitators, and learners. an approximate 85% of the participants were satisfied with case discussion as an educational method because they can discuss about their challenges and they are not only passive listeners. the effectiveness of group discussion training method in doctors communication skills is supported by several studies. another advantage of this study is the use of peer education that reduces the costs for health system and is pleasurable for learners because each learner experiences leadership and improves interpersonal and self - presentation skills and group work contribution. agreement rate for program continuation and integration items are approximately 60%, which are least receptive compared with other items. sustenance is one of the important part of such training program and stakeholders may underestimate its importance. perhaps due to the comprehensiveness of the training program, they assume it is sufficient. physicians knowledge and attitude have significantly increased after the intervention and kept fixed during follow up period. patients satisfaction with doctors communication skills increased approximately by 10% after the intervention that is statistically significant, but 4 months after intervention, patients satisfaction decreased slowly, although it is still more than patients satisfaction before program implementation. a study in the united kingdom, which reports the impact of communication skills training during 12-month follow up reveals that clinicians had integrated key communication skills into clinical practice. it is noteworthy that offering physicians short - term training without integrating it into the system might not be sufficient to meet a constant satisfaction improvement of the patients through the physicians communication skills and needs some refreshing meetings to create long - term sustainability. the limitations of this study are that the benefits of the program to patients must be interpreted cautiously. there is an important question to be answered : whether the modest changes (10%) in patients satisfaction are clinically meaningful. future research is needed to answer this question and determine the clinical significance of the magnitude of observed changes by examining its associations with relevant clinical indices such as patients compliance. furthermore, researchers suggest future research to study other opportunities for continuous training and cost - effectiveness analyses of different methods. to save time and human resources, policy makers can use routine program as an opportunity toward continuous education to health professionals, such as doctor	background : to have high - quality primary health care services, an adequate doctor patient communication is necessary. because of time restrictions and limited budget in health system, an effective, feasible, and continuous training approach is important. the aim of this study is to assess the appropriateness of a communication skills training program simultaneously with routine programs of health care system.materials and methods : it was a randomized field trial in two health network settings during 2013. twenty - eight family physicians through simple random sampling and 140 patients through convenience sampling participated as intervention and control group. the physicians in the intervention group (n = 14) attended six educational sessions, simultaneous organization meeting, with case discussion and peer education method. in both the groups, physicians completed communication skills knowledge and attitude questionnaires, and patients completed patient satisfaction of medical interview questionnaire at baseline, immediately after intervention, and four months postintervention. physicians and health network administrators (stakeholders), completed a set of program evaluation forms. descriptive statistics and chi - square test, t - test, and repeated measure analysis of variance were used to analyze the data.results:use of routine program as a strategy of training was rated by stakeholders highly on feasibility (80.5%), acceptability (93.5%), educational content and method appropriateness (80.75%), and ability to integrating in the health system programs (approximate 60%). significant improvements were found in physicians knowledge (p < 0.001), attitude (p < 0.001), and patients satisfaction (p = 0.002) in intervention group.conclusions:communication skills training program, simultaneous organization meeting was successfully implemented and well received by stakeholders, without considering extra time and manpower. therefore it can be a valuable opportunity toward communication skills training.

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