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High-grade osteosarcoma, a primary malignant bone tumour, is experiencing a global increase in reported incidence with varied prevalence. Despite advances in management, which include surgery and neoadjuvant chemotherapy often an unsatisfactory outcome is found due to poor or heterogeneous response to chemotherapy. Our study delved into chemotherapy responses in osteosarcoma patients and associated molecular expressions, focusing on CD95 receptor (CD95R), interferon (IFN)-γ, catalase, heat-shock protein (Hsp)70, and vascular endothelial growth factor (VEGF). Employing immunohistochemistry and Huvos grading of post-chemo specimens, we analysed formalin-fixed paraffin-embedded (FFPE) osteosarcoma tissue of resected post-chemotherapy specimens from Dr. Soetomo General Academic Hospital in Surabaya, Indonesia (DSGAH), spanning from 2016 to 2020. Results revealed varied responses (poor 40.38%, moderate 48.08%, good 11.54%) and distinct patterns in CD95R, IFN-γ, catalase, Hsp70, and VEGF expression. Significant differences among response groups were observed in CD95R and IFN-γ expression in tumour-infiltrating lymphocytes. The trend of diminishing CD95R expression from poor to good responses, accompanied by an increase in IFN-γ, implied a reduction in the count of viable osteosarcoma cells with the progression of Huvos grading. Catalase expression in osteosarcoma cells was consistently elevated in the poor response group, while Hsp70 expression was highest. VEGF expression in macrophages was significantly higher in the good response group. In conclusion, this study enhances our understanding of immune-chemotherapy interactions in osteosarcoma and identifies potential biomarkers for targeted interventions., (© 2024. The Author(s).)

Background & Aims: Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice., Methods: To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed., Results: We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p = 0.041)., Conclusion: We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data., Lay Summary: Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021. Published by Elsevier B.V.)

Studying the features of changes in markers of oxidative stress (OS) and inflammation indicators in COPD patients depending on the degree of bronchial obstruction is one of the priority directions for improving the prognosis and monitoring of the course of this pathology. We conducted a comparative investigation of changes in markers of OS and apoptosis at the systemic and local levels in patients with moderate to severe COPD during exacerbation and stable phase. 105 patients with COPD aged 46-67 and 21 healthy nonsmoking volunteers comparable in age were examined. COPD patients were divided into four groups: moderate COPD (GOLDII) during the exacerbation (GOLDIIex, n = 25) and in the stable phase (GOLDIIst, n = 27), severe COPD (GOLDIII) during the exacerbation (GOLDIIIex, n = 29), and in the stable phase (GOLDIIIst, n = 24). We studied the levels of such lipid peroxidation (LPO) products as diene conjugates (DC) and Schiff bases (SB) and parameters of induced chemiluminescence (Imax, total light sum-S, Imax/S) in blood serum, as well as sCD95 concentration in blood and exhaled breath condensate (EBC). The relationship between the values of the OS system indicators with sCD95, as well as with the parameters of lung function, was investigated. Multidirectional changes in OS indicator levels in COPD patients depending on the severity of obstructive airway disorders have been established. The maximum values of DC (0.26 ± 0.046 RU), Imax (0.265 ± 0.19 RLU), and Imax/S (0.13 ± 0.05) were typical for patients with moderate COPD, while the highest SB level (5.7 ± 2.3 RU) was observed in severe COPD during an exacerbation. The exacerbation of the disease was characterized by an increase in DC concentration in both GOLDIIex (0.26 ± 0.046 RU) and GOLDIIIex (0.209 ± 0.02 RU) compared to the stable moderate and severe COPD (0.202 ± 0.028 RU and 0.19 ± 0.03 RU, respectively, p < 0.05). The established decrease in high values of DC, Imax, Imax/S, and sCD95 and an increase in SB concentration in GOLD III can serve as quantitative indicators of the prognosis of the severity of the disease. The serum concentration of sCD95 in GOLDIIex (366.4 ± 70.5 U/ml) and GOLDIIst (361.4 ± 72.8 U/ml) did not differ from the control group (393.7 ± 80.9 U/ml, p > 0.05). In patients with FEV1 < 49% during the exacerbation and stable phase, the serum levels of Imax/S (0.058 ± 0.01 and 0.062 ± 0.01) and sCD95 (318.2 ± 66.3 U/ml and 321.4 ± 42.5 U/ml) were lower than the values of healthy volunteers (0.08 ± 0.01 and 393.7 ± 80.9 U/ml, respectively, p < 0.05). A positive correlation between sCD95 concentration and airway obstruction degree in all examined COPD patients was established. The revealed numerous associations between sCD95 and OS marker levels in GOLDIII indicate a relationship between systemic radical stress and apoptosis processes both in the respiratory tract and the whole body under conditions of severe inflammation. The established correlations between the values of DC, Imax, and sCD95 in the blood serum and the lung function parameters in all studied patients allow us to consider these indicators as additional prognostic indicators of disease intensification. Our work results help clarify the participation and detail of FRO and apoptosis processes in developing pathophysiological features in moderate to severe COPD in different periods and, accordingly, improve the efficiency of diagnosis and treatment of the disease., Competing Interests: The authors declare that there is no potential conflict of interest associated with this manuscript., (Copyright © 2021 Svetlana Soodaeva et al.)

Elucidation of the pharmacodynamic mechanisms of drugs capable of potentiating the effects of non-steroidal anti-inflammatory drugs is an important task. In this in vitro study, the ability of Traumeel S to influence the innate and acquired immunity was evaluated. Traumeel S was found to reduce activities of NADPH oxidase and neutrophil extracellular traps, as well as to evoke anti-inflammatory activity of lymphocyte subpopulations., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Metabolically healthy obesity (MHO) accounts for roughly 35% of all obese patients. There is no clear consensus that has been reached on whether MHO is a stable condition or merely a transitory period between metabolically healthy lean and metabolically unhealthy obesity (MUO). Additionally, the mechanisms underlying MHO and any transition to MUO are not clear. Macrophages are the most common immune cells in adipose tissues and have a significant presence in atherosclerosis. Fas (or CD95), which is highly expressed on macrophages, is classically recognized as a pro-apoptotic cell surface receptor. However, Fas also plays a significant role as a pro-inflammatory molecule. Previously, we established a mouse model (ApoE -/- /miR155 -/- ; DKO mouse) of MHO, based on the criteria of not having metabolic syndrome (MetS) and insulin resistance (IR). In our current study, we hypothesized that MHO is a transition phase toward MUO, and that inflammation driven by our newly classified CD95 + CD86 - macrophages is a novel mechanism for this transition. We found that, with extended (24 weeks) high-fat diet feeding (HFD), MHO mice became MUO, shown by increased atherosclerosis. Mechanistically, we found the following: 1) at the MHO stage, DKO mice exhibited increased pro-inflammatory markers in adipose tissue, including CD95, and serum; 2) total adipose tissue macrophages (ATMs) increased; 3) CD95 + CD86 - subset of ATMs also increased; and 4) human aortic endothelial cells (HAECs) were activated (as determined by upregulated ICAM1 expression) when incubated with conditioned media from CD95 + -containing DKO ATMs and human peripheral blood mononuclear cells-derived macrophages in comparison to respective controls. These results suggest that extended HFD in MHO mice promotes vascular inflammation and atherosclerosis via increasing CD95 + pro-inflammatory ATMs. In conclusion, we have identified a novel molecular mechanism underlying MHO transition to MUO with HFD. We have also found a previously unappreciated role of CD95 + macrophages as a potentially novel subset that may be utilized to assess pro-inflammatory characteristics of macrophages, specifically in adipose tissue in the absence of pro-inflammatory miR-155. These findings have provided novel insights on MHO transition to MUO and new therapeutic targets for the future treatment of MUO, MetS, other obese diseases, and type II diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Johnson, Drummer IV, Shan, Shao, Sun, Lu, Saaoud, Xu, Nanayakkara, Fang, Bagi, Jiang, Choi, Wang and Yang.)

Traumatic brain injury (TBI), due to its high mortality and morbidity, is an important research topic. Apoptosis plays a pathogenic role in a series of neurological disorders, from neurodegenerative diseases to acute neurological lesions.In this study, we analyzed the association between apoptosis and the Glasgow Outcome Scale (GOS), to examine the potential of apoptosis as a biomarker for a TBI outcome. Patients with severe TBI were recruited at the Department of Neurosurgery, Wujin Hospital Affiliated with Jiangsu University, between January 2018 and December 2019. As a control group, healthy subjects were recruited. The concentrations of caspase-3, cytochrome c, sFas, and caspase-9 in the cerebrospinal fluid (CSF) were analyzed by enzyme-linked immunosorbent assay (ELISA). The association between the GOS and the clinical variables age, sex, initial Glasgow Coma Scale (GCS) score, intracranial pressure (ICP), cerebral perfusion pressure (CPP), initial computed tomography (CT) findings, and apoptotic factors was determined using logistic regression. The area under the receiver operator characteristic (ROC) curve (AUC), and thus the sensitivity and specificity of each risk factor, were obtained.The levels of caspase-3, cytochrome c, sFas, and caspase-9 in the TBI group were significantly higher than those in the control group (P < .05). The logistic regression results showed that ICP and caspase-3 were significant predictors of outcome at 6 months post-TBI (P < .05). The AUC was 0.925 and 0.888 for ICP and caspase-3, respectively. However, the AUC for their combined prediction was 0.978, with a specificity and sensitivity of 96.0% and 95.2%, respectively, showing that the combined prediction was more reliable than that of the 2 separate factors.We demonstrated that caspase-3, cytochrome C, sFas, and caspase-9 were significantly increased in the CSF of patients following severe TBI. Furthermore, we found that ICP and caspase-3 were more reliable for outcome prediction in combination, rather than separately.

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.

Background and Objectives : Studies suggest that FAS/FASL polymorphisms are associated with male infertility; however, their results are still inconclusive. Therefore, this systematic review and meta-analysis aimed to summarize and clarify the overall association of FAS/FASL polymorphisms and risk of male infertility. Materials and Methods : Our search was conducted on the databases of Science Direct, PubMed and Google Scholar. For performing the meta-analysis, pooled odds ratio (OR) values with 95% confidence interval (CI) was applied in order to analyze the strength of association between the FAS/FASL polymorphisms and risk of male infertility. A total of seven relevant studies published up to September 2018 were considered. Results : FASL-844C/T genotype results of 559 patients and 623 healthy individuals were included in our study. For FAS-670A/G genotype effect, 751 patients and 821 healthy individuals were explored. Results showed that all analysis models including dominant, recessive and allelic models of FASL-844C/T and FAS-670A/G polymorphism had no significant effect on infertility in men ( p > 0.05 and p > 0.05, respectively). According to sensitivity analysis, our results were stable. Conclusion : We demonstrated that FAS/FASL polymorphisms might not be an effective factor on male reproductive health. For precise determination of FAS/FASL polymorphisms effects on male infertility, large-scale case-control studies should be performed.

Objective: Endometriosis is characterized by the growth of endometrial tissue outside the uterine cavity. The prevalence of endometriosis among women experiencing pain, infertility, or both is as high as 35% to 50%. The most common symptoms of endometriosis are dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility. Evidence has suggested that endometriosis symptoms result from a local inflammatory peritoneal reaction caused by ectopic endometrial implants that undergo cyclic bleeding. On the other hand, regular physical exercise seems to have protective effects against diseases that involve inflammatory processes such as type 2 diabetes and colon and breast cancer. On this basis, it is possible that the practice of physical exercise may have beneficial effects on endometriosis. Therefore, the objective of this study was to evaluate the possible anti-inflammatory effect of physical exercise on endometriosis experimentally induced in rats., Study Design: Seventy female Wistar rats were divided into 7groups of 10 animals each. Animals performed light exercise (swimming once a week), moderate exercise (swimming 3 times a week), and intense exercise (swimming 5 times a week) before or after endometriosis induction., Results: At the end of the experimental protocol, a reduction in the size of endometriotic lesions was observed after physical exercise regardless of its frequency, with a greater reduction in the groups practicing moderate and intense activity; an increase in FAS levels and a decrease in matrix metalloproteinases 9 and proliferating cell nuclear antigen (PCNA)levels was also observed. The immunohistochemistry results did not lead to conclusive results. As expected, oxidative stress was reduced in all groups. These results show that the practice of physical exercise could be beneficial, at least in part, for the treatment of endometriosis.

The expression of five members of the TNF receptor superfamily and two of their ligands in human pancreatic ductal adenocarcinoma were investigated in parallel by immunohistochemistry. 41 patients with histologically confirmed ductal carcinoma of the pancreas were enrolled in this study in order (i) to compare the individual TNFR-SF expression and their ligands in PDAC-cells and (ii) to investigate their correlation with survival data. All patients had undergone pancreaticoduodenectomy and were staged as pT3N1M0. Immunostaining was done on FFPE tissue sections of the tumor tissue, using antibodies directed against TRAIL-Receptor-1, -2 and -4, TRAIL, CD95, TNF-Receptor-1 and TNF-α. The intensity and quantity of immunostaining were evaluated separately for tumor cell cytoplasm and tumor cell nucleus. Immunostaining results were correlated with each other and with patient survival. All proteins were found to be expressed in the majority of the tumor cells. The expression (i) of the following members of TNFR-SF and their ligands correlated with each other: TNF-Receptor-1 and TNFα (cytoplasmatic scores, p=0.001), TNF-Receptor 1 and TRAIL (nuclear antigen expression p=0.005 and the main score p=0.001, which contains the overall intracellular antigen expression), TNF-Receptor 1 and CD95 (main score, p=0.001), TRAIL-Receptor-1 and TRAIL-Receptor-2 (nuclear parameters, p=0.023), TRAIL-Receptor-4 and TRAIL (main score p=0.041). In addition (ii), high cytoplasmatic expression of TNF-Receptor-1 and a strong cytoplasmatic and nuclear expression of CD95 correlated significantly with a better prognosis of the PDAC patients.

The profound deficiency of Th17 cells contributes to HIV disease progression. The mechanisms of their perturbation remain unclear. Recently, CCR6 + CD95 + CD4 + naïve T cells (CCR6 + CD95 + CD4 + T NA ), identified as pre-committed Th17 precursors, were recognized as a subpopulation of CD4 + T cells with stem cell properties. Following phenotypical identification, we evaluated their level in patients during chronic HIV infection and following antiretroviral therapy (ART) using flow cytometry. The levels of CCR6 + CD95 + CD4 + T NA were decreased during chronic HIV infection and correlated with CD4 + T cell counts. Immunological responders harbored higher frequency of CCR6 + CD95 + CD4 + T NA , which was associated with CD4/CD8 T cell ratio. Immunological non-responders with lower frequency of CCR6 + CD95 + CD4 + T NA failed to exhibit a correlation between CCR6 + CD95 + CD4 + T NA and CCR6 + CD95 + CD4 + T CM , and displayed elevated ratio of CCR6 + CD95 + CD4 + T CM /T NA . The number of CCR6 + CD95 + CD4 + T NA was increased following early ART. These findings shed light on the importance of targeting pre-committed Th17 precursors that enhance immune reconstitution., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Deviations in the apoptotic process have been demonstrated in prostate carcinogenesis. We aimed to evaluate especially the process of extrinsic apoptosis in the spectrum of neoplastic lesions of the prostate epithelium so as to reveal the variations in the apoptotic process., Material and Method: The study included 20 benign prostatic hyperplasia, 8 high-grade prostatic intraepithelial neoplasia and 82 prostatic carcinoma patients. Immunohistochemistry was performed on sections obtained from materials of suprapubic prostatectomy, tru-cut biopsy, transurethral resection and radical prostatectomy. While Fas and FasL were evaluated in glandular and stromal areas, DcR1 and FLIP were evaluated in only glandular areas. Intensity and extent of immunostaining for Fas and FasL antibodies were separately scored and both scores were summarized. The total score of ≥ 4 both for Fas and FasL, expressions of FLIP and DcR1determined in more than 5% of glandular areas were accepted as positive., Results: Glandular FasL positivity was observed in 63.8 and 20% of the cases with prostatic carcinoma and benign prostatic hyperplasia, respectively (p=0.001). The loss of stromal Fas expression in PCa was obvious (p < 0.001). FLIP positivity was more frequently seen in high-grade prostatic intraepithelial neoplasia and PCa., Conclusion: In prostatic carcinoma, decreased stromal Fas expression, contrary to higher glandular FasL positivity, supports the assertion that sensitivity of epithelial and stromal cells to apoptosis and their protective pathways against apoptosis undergo alterations. Increased FLIP expressions in high-grade prostatic intraepithelial neoplasia and prostatic carcinoma can also be interpreted accordingly.

Background: Critically ill patients with acute kidney injury (AKI) can be divided into two subphenotypes, resolving or nonresolving, on the basis of the trajectory of serum creatinine. It is unknown if the biology underlying these two AKI recovery patterns is different., Methods: We measured eight circulating biomarkers in plasma obtained from a cohort of patients admitted to an intensive care unit (ICU) (n = 1241) with systemic inflammatory response syndrome. The biomarkers were representative of several biologic processes: apoptosis (soluble Fas), inflammation (soluble tumor necrosis factor receptor 1, interleukin 6, interleukin 8) and endothelial dysfunction, (angiopoietin 1, angiopoietin 2, and soluble vascular cell adhesion molecule 1). We tested for associations between biomarker levels and AKI subphenotypes using relative risk regression accounting for multiple hypotheses with the Bonferroni correction., Results: During the first 3 days of ICU admission, 868 (70%) subjects developed AKI; 502 (40%) had a resolving subphenotype, and 366 (29%) had a nonresolving subphenotype. Hospital mortality was 12% in the resolving subphenotype and 21% in the nonresolving subphenotype. Soluble Fas was the only biomarker associated with a nonresolving subphenotype after adjustment for age, body mass index, diabetes, and Acute Physiology and Chronic Health Evaluation III score (p = 0.005)., Conclusions: Identifying modifiable targets in the Fas-mediated pathway may lead to strategies for prevention and treatment of a clinically important form of AKI.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Previous investigations have demonstrated the adverse impacts of fluoride on Sertoli cells (SCs), such as oxidative stress and apoptosis. SCs are the crucial cellular components that can create the immune privileged environment in testis. However, the effect of fluoride on SCs immune privilege is unknown. In this study, mouse SCs were exposed to sodium fluoride with varying concentrations of 10 -5 , 10 -4 , and 10 -3  mol/L to establish the model of fluoride-treated SCs (F-SCs) in vitro. After 48 h of incubation, F-SCs were transplanted underneath the kidney capsule of mice for 21 days, or cocultured with spleen lymphocytes for another 48 h. Immunohistochemical analysis of GATA4 in SCs grafts underneath kidney capsule presented less SCs distribution and obvious immune cell infiltration in F-SCs groups. In addition, the levels of FasL protein and mRNA in non-cocultured F-SCs decreased with the increase of fluoride concentration. When cocultured with F-SCs, lymphocytes presented significantly high cell viability and low apoptosis in F-SCs groups. Protein and mRNA expressions of FasL in cocultured F-SCs and Fas in lymphocytes were reduced, and the caspase 8 and caspase 3 mRNA levels were also decreased in fluoride groups in a dose-dependent manner. These findings indicated that fluoride influenced the testicular immune privilege through disturbing the Fas/FasL system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

T cell immunoglobulin and mucin domain 3(TIM3) is a negative regulator of cellular immunity and it is highly expressed on CD8+T cells in persistent viral infection and cancer setting as report. However, how TIM3 expressed on CD8+T cells in myelodysplastic syndrome (MDS), that is a malignant disorder, has not been clarified. Here, decreased CD8+T cells, less IFN-γ secretion in CD8+T cells and increased TIM3 on CD8+T cells had been seen. Increased TIM3+CD8+T cells with lower perforin and granzyme B expression and higher CD95 expression in MDS patients had been observed. These findings suggested that TIM3 might be related to CD8+T cells defect. Therefore, further explorations about mechanism of TIM3+CD8+T cells defect are needed, which might be helpful for adoptive T-cell therapy in MDS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Peripheral CD27 + memory B-cells become quantitatively reduced and dysfunctional in patients with cirrhosis through poorly characterized mechanisms. We hypothesized that the disappearance of CD27 + memory B-cells results from enhanced sensitivity to apoptosis caused by exposure to gut microbial translocation products. Using isolated naïve and memory B-cells from patients with cirrhosis and age-matched controls, ex vivo and activation-induced sensitivity to Fas-mediated apoptosis was assessed under relevant experimental conditions. We observed differential expression of CD95(Fas) in CD27 + B-cells from cirrhotic patients that was inversely correlated with peripheral CD27 + B-cell frequency. While memory B-cells from cirrhotic patients were resistant to Fas-mediated apoptosis ex vivo, Toll-like receptor 4(TLR4)-ligation restored Fas-sensitivity. Sensitivity to Fas-mediated apoptosis could be transferred to healthy donor memory B-cells by co-culturing these cells with plasma from cirrhotic patients, a sensitivity partially mediated by Fas and TLR4 signaling, and partially rescued via B-cell receptor crosslinking. We conclude that peripheral CD27 + memory B-cells in cirrhosis exhibit increased sensitivity to Fas-induced apoptosis in an activation-dependent manner to which endotoxin contributes, associated with reduced frequency of circulating memory B-cells. Destruction of this critical cell subset may contribute to the cirrhotic immunodeficiency state and heightened risk of systemic infections in advanced liver disease.

A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = -0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.

Purpose: Doxorubicin and trabectedin are considered active drugs in soft tissue sarcoma (STS). The combination of both drugs was hypothesized to be advantageous and safe on the basis of preclinical evidence and a previous phase I trial, respectively. The aim of this study was to compare the clinical outcome of trabectedin plus doxorubicin with doxorubicin as first-line treatment of advanced STS patients., Patients and Methods: In this open-label randomized phase II trial, the main end point was progression-free survival (PFS). Trabectedin 1.1 mg/m(2) in a 3-hour infusion plus doxorubicin 60 mg/m(2) as the experimental arm and doxorubicin 75 mg/m(2) as the control arm were administered for up to six cycles. Translational research was planned to correlate the expression of apoptotic and DNA repair genes with clinical outcome., Results: In 115 randomly assigned patients, the median PFS was 5.5 months in the control arm and 5.7 months in the experimental arm (hazard ratio, 1.16; 95% CI, 0.79 to 1.71; P = .45) in the intent-to-treat analysis. The trial was stopped for futility after the interim analysis, because the results in the experimental arm showed the risk reduction for the main end point to be < 9.64%. The proportion of patients with grade 3 or 4 thrombocytopenia, asthenia, and liver toxicity was significantly higher in the experimental arm. FAS and p53 were shown to be prognostic factors for PFS (7.0 months if FAS+ and p53-; 3.4 months if FAS+/p53+ or FAS-/p53-; and 0.7 months if FAS- and p53+; P < .001) and for overall survival., Conclusion: Trabectedin plus doxorubicin did not show superiority over doxorubicin alone as first-line treatment of advanced STS. The prognostic role of apoptotic key genes, FAS and p53, was shown to be robust enough to continue this research line., (© 2016 by American Society of Clinical Oncology.)

Microarrays allow the miniaturization and multiplexing of biological assays while only requiring minute amounts of samples. As a consequence of the small volumes used for spotting and the assays, evaporation often deteriorates the quality, reproducibility of spots, and the overall assay performance. Glycerol is commonly added to antibody microarray printing buffers to decrease evaporation; however, it often decreases the binding of antibodies to the surface, thereby negatively affecting assay sensitivity. Here, combinations of 14 hygroscopic chemicals were used as additives to printing buffers for contact-printed antibody microarrays on four different surface chemistries. The ability of the additives to suppress evaporation was quantified by measuring the residual buffer volume in open quill pins over time. The seven best additives were then printed either individually or as a 1:1 mixture of two additives, and the homogeneity, intensity, and reproducibility of both the spotted protein and of a fluorescently labeled analyte in an assay were quantified. Among the 28 combinations on the four slides, many were found to outperform glycerol, and the best additive mixtures were further evaluated by changing the ratio of the two additives. We observed that the optimal additive mixture was dependent on the slide chemistry, and that it was possible to increase the binding of antibodies to the surface threefold compared to 50 % glycerol, while decreasing whole-slide coefficient of variation to 5.9 %. For the two best slides, improvements were made for both the limit of detection (1.6× and 5.9×, respectively) and the quantification range (1.2× and 2.1×, respectively). The additive mixtures identified here thus help improve assay reproducibility and performance, and might be beneficial to all types of microarrays that suffer from evaporation of the printing buffers.

Objective: Lack of surface Fas expression is a main route for apoptotic resistance which is considered an important mechanism of tumorigenesis and tumor progression. Fas and FasL expression in 110 non-small cell lung carcinomas (NSCLCs) were investigated to evaluate their roles in pulmonary carcinogenesis and to examine the clinicopathologic significance of Fas expression with its relationship with p53 and bcl-2 over- expression., Methods: Immunohistochemical analysis using tissue microarray demonstrated that a large proportion of NSCLC patients (60%) showed lack of membranous Fas expression. The Fas-negative cases revealed the significantly lower survival rate than Fas-positive ones. Also, the loss of Fas receptor expression was found more frequently in advanced stage and higher nodal status. FasL protein was increased in most NSCLCs (89%) compared to normal lungs., Results: p53 and bcl-2 overexpression showed no association with Fas expression. Conclusively, reduced membranous Fas expression as a mechanism of apoptotic resistance is considered to play an important part of the pulmonary carcinogenesis, which may predict poor survival and have a negative prognostic influence., Conclusion: Increased FasL expression is thought to be a basis for the immune evasion in NSCLCs. The rare bcl-2 overexpression suggests that this anti-apoptotic protein is unlikely to play a role in the apoptotic resistance of NSCLCs.

Objective: To investigate the relation of Fas and Fas ligand (FasL) protein expression with carcinogenesis and metastasis of cardiac carcinoma., Methods: Immunohistochemistry was used to detect Fas and FasL protein expression in 64 cardiac carcinoma tissue samples and 20 normal gastric tissue samples. Relation between FasL and Fas expression, age and gender of gastric cancer patients, and pathological subtype and lymph node metastasis of gastric cancer was analyzed., Results: The Fas expression level was significantly higher in normal gastric tissue samples than in cardiac carcinoma tissue samples (85.0% vs. 25.0%, P<0.001), while the FasL expression level was significantly lower in normal gastric tissue samples than in cardiac carcinoma tissue samples (30.0% vs. 81.3%, P<0.001). The Fas expression level was significantly higher in invasive lymph nodes than in non-invasive lymph nodes (82.9% vs. 56.5%, P<0.003) and in well-differentiated gastric carcinoma tissue samples than in poorly-differentiated cardiac carcinoma tissue samples (50.0% vs. 18.0%, P=0.015). The FasL expression level was significantly lower in well-differentiated cardiac carcinoma tissue samples than in poorly- differentiated cardiac carcinoma tissue samples (42.9% vs. 84.0%, P=0.021). The Fas and FasL expression levels (25.0% and 81.3%) were significantly different in cardiac carcinoma tissue samples (P<0.001), but had a non-linear correlation (P=0.575)., Conclusion: Abnormal Fas and FasL expressions in cardiac carcinoma and lymph node tissues are involved in carcinogenesis and metastasis of gastric cancer.

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

Introduction: Lupus erythematosus is a multisystemic disease that is characterized by autoantibody production and immune complex deposition in such tissues as the mucosa, joints, the central nervous system, and skin. Cutaneous lupus erythematosus is categorized as acute, subacute, and chronic. Chronic cutaneous lupus erythematosus comprises discoid lupus erythematosus (DLE) and lupus profundus (LP)., Aim: To analyze the expression of proapoptotic molecules in patients with lupus erythematosus discoid and lupus profundus., Material and Methods: Descriptive study, the study groups comprised 10 cases of LP and 10 cases of DLE, and a control. Skin samples of cases and controls were processed for immunohistochemistry and by TUNEL technique. The database and statistical analysis was performed (statistical test X(2)) SPSS (Chicago, IL, USA)., Results: Apoptotic features were broadly distributed along the skin biopsies in epidermal keratinocytes as well as at dermis. By immunohistochemistry the expression of Fas receptor and Fas-L was higher in the skin of lupus patients compared with controls. We also noted differences in Fas-L, -Fas, and -Bax proteins expression intensity in discoid lupus erythematosus patients in the epidermis, and hair follicles., Conclusions: Fas and Fas-L are expressed similarly in LP and DLE.

Study Design: Immunohistochemical assessment of apoptotic markers in human cases of compressive myelopathy due to neoplastic compression., Objective: To characterize the role of apoptosis in neoplastic compressive myelopathy in human postmortem tissue with extramedullary tumor involvement., Summary of Background Data: Neoplasms, whether primary or metastatic, may lead to compression of the spinal cord and development of a compressive myelopathy syndrome. Apoptotic processes of cell death are thought to contribute to cell death in chronic compressive myelopathy because of degenerative spondylosis, but this has not previously been described in neoplastic compression., Methods: Six postmortem cases of human neoplastic compressive myelopathy were assessed for apoptosis using a panel of immunohistochemical markers including Fas, B-cell lymphoma 2 (Bcl-2), caspase-3 and 9, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF), and terminal deoxynucleotide transferase dUTP Nick End Labeling (TUNEL)., Results: Apoptosis was maximal at the site of tumor compression. Glial cells, predominantly oligodendrocytes, were immunopositive for DNA-PKcs, PARP, AIF, and TUNEL. Axons were immunopositive for caspase 3, DNA-PKcs, and AIF. Neurons were immunopositive for DNA-PKcs, PARP, AIF, and TUNEL., Conclusion: The current study demonstrates that apoptosis plays a role in human neoplastic compressive myelopathy. Necrosis dominates the severe end of the spectrum of compression. The prominent oligodendroglial involvement is suggestive that apoptosis may be important in the ongoing remodeling of white matter due to sustained compression., Level of Evidence: 4.

Purpose: The Fas-Fas Ligand interaction is one of the essential events for the induction of apoptosis whereas the exact role of their soluble forms in the reproductive system is still not fully understood. Also oxidative stress in the pathogenesis of infertility causing diseases in women and has been suggested as one of the important factors that negatively affect IVF outcome. In this study, our aim was to evaluate serum and follicular fluid levels of soluble Fas soluble Fas Ligand, malondialdehyde, superoxide dismutase and total antioxidant capacity in patients undergoing IVF and compared with controls., Methods: This study included 109 patients. Patients were classified as unexplained infertility (N = 31), PCOS (N = 19), tubal factor (N = 9) and endometriosis (N = 10) and compared with male factor infertility (N = 40) that was the control group. sFas and sFasL levels were measured by immunoassay method. MDA, SOD and TAC levels were measured by colorimetric method., Results: Patients with unexplained infertility, PCOS and tubal factor had significantly lower sFas levels compared with their controls (respectively, p < 0.01, p < 0.05, p < 0.05). However, SOD activity in unexplained infertility, PCOS and endometriosisgroupswere significantly higher than control group (p < 0.01).Decreased follicular fluid TAC levels were found in all patient groups compared with controls (respectively, p < 0.01, p < 0.05, p < 0.01, p < 0.01).Patients with tubal factor had significantly higher serum sFasL (p < 0.05), but lower follicular fluid sFasL levels (p < 0.05) compared with unexplained infertility. Tubal factor and endometriosis groups had lowerfollicular fluid TAC levels compared to unexplained infertility and PCOSgroups (p < 0.01)., Conclusion(s): In this study, serum and follicular fluid sFas levels were decreased and antioxidant activity was impaired in infertility, possibly implying increased apoptosis. Especially in unexplained infertility group changes in this parametres more remarkable.

At the present stage of study of chronic obstructive pulmonary disease (COPD) one of the problem is the definition of new criteria for the topical and systemic chronic inflammation of this disease. The aim of the research was to study the concentration of nitric oxide metabolites, the level of soluble human leukocyte antigens class I (sHLA-I) and of soluble CD95 molecules (sCD95) in the serum of blood and exhaled breath condensate (EBC) in patients with exacerbation of COPD. We investigated 49 moderate-to-severe COPD patients with exacerbation, and 21 healthy nonsmokers. The concentration of sHLA-I and sCD95 molecules was studied in serum and in EBC using the ELISA method. The nitrosative stress was evaluated by the measurement of NO2(-) levels in the serum and the concentration of ΣNO2(2)/NO3(2) in the EBC. Exacerbation of COPD is associated with increasing concentrations of NO2(2) in the serum and of the levels of ΣNO2(2)/NO3(2) in the EBC, together with the changing concentration of sHLA-I and sCD95 molecules in the both biological liquid. An association was discovered between the exacerbation of COPD and the indicators of nitrosative stress, the parameters of lung function and the concentration of sHLA-I, sCD95 molecules. The findings suggest a pathogenetic role of nitrosative stress and of soluble molecules of HLA-I and CD95 in the progression of COPD. The studied markers can be used as predictors of unfavourable prognoses of COPD and as quantitative criteria in the diagnosis of exacerbation of moderate-to-severe COPD.

Objective: To investigate the expression level of Fas-FasL in conjunctiva tissue of chronic Stevens-Johnson syndrome (SJS) patients and to analyze the role of Fas-FasL pathway in the mechanism of ocular damage related to SJS., Methods: Conjunctiva tissue was obtained during symblepharon release operation and cryostat sectioned at 5 µm. The tissue morphology was observed by electron microscopy. The expression of Fas-FasL (CD95/CD178) in conjunctiva was detected by immunohistochemistry (direct immunofluorescence). The conjunctiva tissue of normal people was also observed and tested as contrast group., Results: The pathogenic changes included incrassation of epithelium and cavity of mucous glands in the sub-epithelium. The expression of Fas in SJS patients focusing in the basal layer of epithelium appeared a lower level compared to that in normal human conjunctiva, which distributed equally in all layers of epithelium. The expressions of FasL in SJS patients and normal people were both weak., Conclusions: The low level expression of Fas-FasL in conjunctiva of SJS patients has certain effect on the chronic ocular damage.

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/high CD127 Ø/low FoxP3+, and effector T cells were defined as CD25+CD127+FoxP3 Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Background: Little is known about the release of apoptotic proteins during periodontal breakdown. This pilot study investigates the presence of factors associated with apoptosis in serum, saliva, and gingival crevicular fluid (GCF) and their association with periodontal disease severity and activity., Methods: GCF, whole saliva, and serum were obtained from 47 adult patients with chronic periodontitis (CP) and 10 healthy controls. Clinical measurements, including probing depth (PD), clinical attachment level (CAL), and radiographs, were used to classify patients into healthy, mild, and moderate/severe CP groups. Enzyme-linked immunosorbent assays were used to measure apoptosis or DNA fragmentation in GCF and active caspase-3, soluble Fas (sFas), and sFas ligand (sFasL) in saliva and serum. Western immunoblotting was used to detect Fas, FasL, sFasL, and caspase-3 expression in GCF., Results: DNA fragmentation was positively correlated with PD and CAL regardless of patient disease status (P <0.001). sFas and sFasL were present in saliva and serum, but there were no differences between groups. In GCF, the greater odds of detecting Fas, sFasL, and caspase-3 increased with increasing PD and CAL (P <0.05). In addition, sites with inflammation and PD ≥5 mm had significantly greater odds of exhibiting Fas, sFasL, and caspase-3 expression compared with sites without inflammation and PD <5 mm (P <0.05). Caspase-3 was not detected in saliva or serum. At the patient level, only FasL and disease status were significantly correlated (P <0.05)., Conclusion: Factors associated with apoptosis were detected in GCF in patients with CP.

Background: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues., Results: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis., Conclusions: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Introduction: Despite routine use of umbilical cord blood (CB) grafts as stem cell source for allogeneic stem cell transplantations, much remains unknown regarding their cell composition and correlation with clinical outcome., Methods: We analyzed material from 30 CB units used for allogeneic hematopoietic stem cell transplantation by multicolor flow cytometry. Phenotypic data were correlated with various clinical outcomes such as survival, graft-versus-host disease (GVHD), relapse, rejection, viral reactivation, and bacteremia., Results: We found that above-median frequencies of CD69+ T cells, naïve CD8+ T cells, and CD127+ B cells in the CB graft were each associated with significantly improved patient survival. Moreover, a statistically significant correlation was seen between higher levels of CD94+ T cells and herpes simplex virus and varicella zoster virus reactivation post transplantation. A similar correlation was seen for the frequency of CD95+ cells in total CD3+, as well as CD4+ and CD8+ T-cell subsets, and viral reactivation. Finally, a higher frequency of naïve CD8+ T cells was associated with the incidence of acute GVHD., Conclusion: Our study highlights the importance of further exploration of graft composition before CB transplantation as a tool for risk prediction., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Objectives: Deregulation of apoptosis related genes may be associated with poor outcome in cancer. Aim of the present study was to investigate the prognostic role of expression levels of apoptosis related proteins in stage II and III colon cancer., Methods: From tumor samples of 386 stage II and III colon cancer patients, DNA was isolated and tissue microarrays were constructed. Expression of Bcl-2, Bcl-X, BAX, XIAP, Fas, FasL and c-FLIP was evaluated and PCR-based microsatellite instability analysis was performed., Results: High FasL expressing tumors were associated with high disease recurrence rates in stage II colon cancer patients overall, as was low Bcl-X expression in microsatellite stable stage II patients. In stage II patients, a multivariable model based on FasL and Bcl-XL expression revealed a significant association with disease free survival (DFS). In stage III colon cancer patients, low Bcl-2, low BAX and low Fas expression levels were associated with worse outcome. In these patients a multivariable model based on angioinvasion and Bcl-2, Fas and FasL expression was significantly associated with DFS., Conclusions: Stage II patients with low Bcl-X and high FasL protein expression levels and stage III patients with low Fas, high FasL and low Bcl-2 expression could be considered as high risk for disease recurrence., (© 2013 Wiley Periodicals, Inc.)

Background: Mutations in the gene for the signal transducer and activator of transcription 1, STAT1, have been shown to be associated with death at an early age due to overwhelming viral infection (complete STAT1 deficiency) or, more commonly, selective deficiencies to mycobacterial or fungal infection (typically heterozygous STAT1 mutations)., Objectives: To define the molecular basis of progressive combined immunodeficiency in a group of patients with fatal infections., Methods: We studied a group of unrelated patients who displayed an unusual progressive form of combined immunodeficiency. Whole exome sequencing assisted in confirming a common genetic defect in this group, which consisted of a heterozygous mutation of the STAT1 gene. STAT1 protein level as well as function was assessed, and a detailed evaluation of the immune system, including analysis of thymus tissue, was performed., Results: Patients were found to carry de novo heterozygous mutations in STAT1 encoding T385A, I294T, or C284R amino acid substitutions. STAT1 expression appeared significantly decreased as a result of these changes but not completely absent, with diminished signaling responses. This group display progressive loss in lymphocyte number and function accompanied by increasing autoimmune features as well as severe, fatal infections., Conclusions: These findings show that some heterozygous aberrations of STAT1 can be associated with progressive combined immunodeficiency, quite distinct from the limited susceptibilities to infection previously reported for heterozygous STAT1 mutations. These mutations were not inherited, rather, arose de novo in each case. Accompanied by significant patient mortality, this finding suggests that this class of STAT1 mutation is ultimately fatal due to overwhelming infection., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

The identification of molecular prognostic markers for pancreatic cancer patients could provide insightful information for their management in the clinic. The aim of the study is to investigate whether or not the expressions of c-Myc and Fas (CD95/APO1) have prognostic relevance for overall survival (OS) in patients with pancreatic cancer. we used immunohistochemistry on tissue microarrays containing 162 pancreatic cancer specimens to assess the protein expression levels of c-Myc and Fas. Kaplan-Meier survival analysis demonstrated that high level of c-Myc cytoplasmic expression was significantly correlated with worse survival in patients with pancreatic cancer (P = 0.012), while high level of Fas cytoplasmic expression was significantly associated with better outcome of pancreatic cancer (P = 0.046). However, multivariate Cox model analysis showed that tumor differentiation, lymph node status and c-Myc cytoplasmic expression were significant independent prognostic factors for OS (P < 0.001, P = 0.023, P = 0.001, respectively). On the contrary, Fas cytoplasmic expression did not independently influence patient's prognosis (P = 0.249). Our data suggested that high level of c-Myc cytoplasmic expression may be considered as a valuable marker for prognosis of pancreatic cancer.

Background: Locoweeds cause significant livestock poisoning and economic loss all over the world. Animals can develop locoism, a chronic neurological disease, after grazing on locoweeds. Oxytropis kansuensis is a variety of locoweed that contains swainsonine as its main toxic ingredient. The purpose of this study was to investigate the apoptotic pathway induced in the cerebrum by swainsonine., Results: Twenty-four Sprague-Dawley rats were randomly divided into four groups (experimental groups I, II, III and a control group) and 6 SD rats of each group were feed in 3 cages separately. Rats were penned as groups and fed with feeds containing 15% (SW content 0.03‰), 30% (SW content 0.06‰), or 45% (SW content 0.09‰) O. kansuensis for experimental groups I, II, and III, respectively, or complete feed in the case of the control group. One hundred and nineteen days after poisoning, and all rats showed neurological disorders at different degrees, which were considered to be successful established a chronic poisoning model of O. kansuensis. rats were sacrificed and the expression of Fas, FasL, Bcl-2, Bax as well as cleaved caspase-3, -8 and -9 proteins in brain tissues were detected by Western blot. The results showed that SW treatment up-regulated Fas and Fas ligand (FasL) (P < 0.05), and that there was an increase in Bax and a decrease in Bcl-2 protein (P < 0.01). Moreover, SW treatment significantly increases the activation of caspase-3, 8 and -9, the key effectors in apoptosis pathway (P < 0.01)., Conclusion: Our data suggest that SW induces apoptosis in cells of the brain through death receptor and mitochondria-mediated, caspase-dependent apoptotic pathways in the brain tissue of SD rats.

Objective: To assess seminal plasma soluble Fas (sFas) relationship with oxidative stress and varicocele (Vx) grade in infertile men., Methods: In all, 230 men were prospectively investigated: fertile men without Vx, fertile men with Vx, infertile men without Vx, and infertile men with Vx. In their semen, seminal oxidant (malondialdehyde [MDA]), antioxidants (ascorbic acid, glutathione peroxidase [GPx], catalase [CAT], and superoxide dismutase [SOD]), and seminal sFas were assessed., Results: Either fertile or infertile men with Vx demonstrated significantly higher seminal oxidants (MDA) and significantly lower seminal antioxidants (SOD, GPx, CAT, and ascorbic acid), sFas compared with fertile or infertile men without Vx. Infertile men with or without Vx had significantly higher seminal MDA and significantly lower seminal antioxidants, sFas compared with fertile men with or without Vx. Men with Vx grade III had significantly higher seminal MDA and significantly lower antioxidants, sFas compared with Vx grade II and I, respectively. Seminal sFas demonstrated significant positive correlation with sperm count, sperm motility, sperm normal forms, seminal ascorbic acid, SOD, GPx, and CAT and significant negative correlation with seminal MDA., Conclusion: Down regulation of seminal sFas in Vx associated men is related to increased oxidative stress and is correlated with Vx grade., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Aims: The aim of this study was to assess the influence of anti-TNF agents on the expression of apoptosis-related proteins in Crohn's disease (CD) patients., Methods: The clinical, biochemical and endoscopic activity of CD was assessed with the use of tissue sampling before the initiation of therapy and after induction doses of infliximab and adalimumab. Additionally, the immunohistochemical expression of active caspase 3, TNFR1, Fas, Bcl-2, Bax, CD4 and CD8 proteins was estimated. Patients achieving deep remission were considered as responders., Results: Of the 35 patients qualified for the study, 60% achieved deep remission. In those patients, a significant decrease in the number of CD4 and CD8 positive cells was noted. Also observed was a significant increase in the expression of active caspase 3 in lamina propria mononuclear cells, which correlated with an increase of the pro-apoptotic Bax/Bcl-2 ratio. No change in Fas and TNFR1 expression was observed in those cells. Moreover, there was a significant decrease in active caspase 3 expression in enterocytes, observed independently of the Bax/Bcl-2 ratio. This correlated with a change in TNFR1 expression. No significant changes in the expression of the investigated proteins were noted in non-responders group., Conclusions: The efficacy of anti-TNF antibodies is, at least partly, dependent on apoptosis modulation. In lamina propria mononuclear cells, the increase of apoptosis is probably the result of the induction of the intrinsic pathway mediated by Bcl-2 family proteins. In enterocytes - the decrease of apoptosis is mediated by the extrinsic pathway, probably via TNFR1., (Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Background: Some inflammatory factors play an important role in recurrent oral ulceration (ROU). The genetics mechanism of expression level of inflammatory factors is not clear in ROU, but from genetics the expression level of inflammatory factors at least partly depend on the gene polymorphisms. Therfore, we decided to investigate inflammatory factors gene polymorphism and its association with the susceptibility of recurrent oral ulceration in Chinese., Methods: Genomic DNA was obtained from 42 subjects with recurrent oral ulceration, 86 subjects of healthy control individuals.Genotypes and alleles of 10 genes and 17 polymorphisms sites were analyzed by Mass-ARRAY Analyzer method. Then, the differences in distribution of each genotype and allele were compared., Results: The statistical differences in distribution of TNF-α (rs1800629 and rs1800630) genotype and allele were observed among the groups with recurrent oral ulceration and healthy control individuals (P < 0.01), while VEGFA (rs1570360, rs833061, and rs2010963), EGF (rs4444903), TNF (rs361525), IL10 (rs1800896, rs1800872), IL2 (rs2069762), IL4 (rs2243250), Fas (rs1800682, rs2234767), IL12A (rs2243115, rs568408), IL12B (rs3212227), and IFNG (rs2430561) showed no statistical differences of genotype and allele in controls as compared to those in patients., Conclusions: This study suggests that the TNF-α (rs1800629 and rs1800630) genotype is an indicator for the susceptibility of recurrent oral ulceration., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

The study aimed for addressing the expression of soluble Fas (sFas) and soluble Fas Ligand (sFasL) in human nucleus pulposus (NP) and its attendant relationship with disc degeneration. Human NP samples were collected from patients with disc degeneration and cadavers as degenerate and normal groups, respectively. Subsequently, NP cells were cultured in monolayer. ELISA was performed to identify the expression levels of sFas and sFasL in the supernatant of NP cell cultures in vitro. Quantitative real-time PCR was used to detect the expression of sFas and sFasL in human NP cells in mRNA solution. The study comprised 12 degenerate and 8 normal cadaveric NP samples. The concentration value of sFas in the supernatant was significantly higher from degenerate NP than that from normal NP at each time point. In contrast, sFasL was significantly lower at each time point. Moreover, the expression of sFas and sFasL reached the peak at various early stages of cell cultures and decreased thereafter. Furthermore, the mRNA level of Fas in degenerate NP cells was significantly higher than that in normal cells; whereas FasL showed an opposite pattern. The study is the first addressing the expression of sFas and sFasL in human NP cell cultures. Moreover, the expression of sFas and sFasL varies with culture time in vitro with different levels in degenerate and normal settings. These findings indicate that sFas and sFasL might play a role in intervertebral disc degeneration.

Recently the dogma that there is an inverse linear association between androgen receptor (AR) CAG and GGN polymorphisms and receptor activity has been challenged. We analysed the pattern of association between 21 male reproductive phenotypes and AR CAG/GGN repeat lengths in 557 proven-fertile men. A linear association was only found between sperm DNA fragmentation index (DFI) and CAG length, and between inhibin B and GGN length. Men with longer CAG then the reference (22-24), had higher oestradiol levels, whereas men with shorter CAG stretches had a higher DFI and a higher proportion of Fas-positive germ cells. Subjects with either short or long CAG had increased seminal levels of prostate-specific antigen and neutral α-glucosidase activity. Compared to men with the median GGN length of 23, those with shorter GGN repeats had higher levels of inhibin B, higher proportions of normal and progressive sperm, and a higher fraction of Fas-positive sperm, while men with longer GGN had higher oestradiol levels. These data indicate that at least for some markers of male reproductive function the association with CAG or GGN repeat length is curvilinear., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background/aim: Binding of FAS ligand (FASL) to its physiological receptor FAS, induces the activation of caspase-8, which triggers cell death. The FAS-FASL system regulates germ cell death. In this study, the role of the FAS-FASL system in male infertility was examined., Patients and Methods: 72 samples were used (age=38.76 ± 9.06 years). Basic semen analysis was performed according to the WHO Laboratory Manual. Soluble (s) forms of FAS and FASL were measured in seminal plasma using commercially available immunoassay kits., Results: Among the examined samples, 24 were normal and 48 abnormal, as evaluated by basic semen analysis. sFAS and sFASL levels in abnormal samples were slightly higher than in the normal ones. In all samples, sFAS correlated negatively with pH. In normal samples, sFAS was positively correlated with sperm concentration. In abnormal samples, sFAS strongly correlated with sFASL., Conclusion: Both factors of the FAS system were detected in seminal plasma. Further studies are necessary to shed light into the possible role of FAS-FASL system in male infertility.

Introduction: The aim of this study was to examine a potential link between apoptotic biomarkers in gingival crevicular fluid (GCF) and periodontal destruction in four cases of localized aggressive periodontitis (LAP), diagnostically enhanced by cone beam computed tomography., Case Series: This study examined the GCF in four patients diagnosed with LAP (formerly localized juvenile periodontitis) at a routine periodontal examination. The LAP diseased sites had attachment loss ranging from 5-12 mm. Atotal of 62 samples of GCF were collected from diseased sites and from contralateral, matched healthy sites with minimal or no attachment loss. All samples were assayed for apoptotic markers, including Fas/FasL, DNAfragmentation, and nitric oxide. The GCF samples were analyzed utilizing enzyme-linked immunosorbent assays for DNA fragments and nitric oxide levels, whereas Western blotting was used for Fas/FasL analyses. Our results showed a significant increase in the apoptotic markers Fas/FasL and DNA fragmentation when comparing GCF from diseased versus non-diseased sites in patients with LAP., Conclusion: To our knowledge, this is the first report of apoptotic biomarkers associated with patients diagnosed with LAP. Finding significantly increased levels of these markers in localized areas may help us understand the pathophysiology associated with this specific form of periodontitis, and, furthermore, may provide a basis for a quantifiably prognostic test when attempting to treat this disease.

Objective: To evaluate the expression of apoptosis-related genes and their correlation with prognosis in cervical cancer patients., Materials and Methods: The expressions of Survivin, Fas and FasL in tissues of cervical cancer, cervical intraepithelial neoplasia (CIN), chronic cervicitis and normal cervix wer detected by immunohistochemical staining, and the relationship between the expression of Survivin, Fas and FasL and clinical pathologic characteristics of cervical cancer was correlation analysis., Results: The positive expression rates of Survivin and FasL in cervical cancer tissues were significantly higher than those in tissues of normal cervix, chronic cervicitis and CIN (p < 0.05), but lower positive expression rate of Fas was observed in cervical cancer tissues when compared with that in normal cervix, chronic cervicitis and CIN tissues (p < 0.05). The expression of Survivin was significantly correlated with clinical staging and lymph node metastases of cervical cancer (p < 0.05). The expression of FasL was correlated with lymph node metastases, clinical staging and pathological grading of cervical cancer (p < 0.05). The expression of Survivin was negatively correlated with that of Fas (r = -0.517, p < 0.01), but positively correlated with that of FasL (r = 0.381, p < 0.01) in tissues of cervical cancer., Conclusions: The up-regulated expression of Survivin and FasL and down-regulated expression of Fas may be involved in the carcinogenesis and development of cervical cancer. The expression of FasL may be one of the prediction indexes for disease progression and prognosis in cervical cancer.

Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ cell population to numbers that can be supported by the Sertoli cells, and, possibly, selective depletion of meiotic and postmeiotic abnormal germ cells. We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men. Six hundred and four men from Greenland, Poland and Ukraine were consecutively enrolled during their pregnant wife's antenatal visits. Semen analysis was performed as recommended by the World Health Organization. Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell. DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The percentage of Fas-positive sperm cells was higher in men with high total sperm count (P<0.01), more motile sperms (P=0.04) and fewer sperm head defects (P=0.05). These associations were consistent within and across study regions. Furthermore, testosterone, follicle-stimulating hormone (FSH) and sexual hormone-binding globulin (SHBG) were significantly negatively correlated with Fas within and across regions as well. The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics. The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarify the biological significance of sperm apoptotic markers.

Aims and Background: Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary., Methods: We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay., Results: Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations., Conclusions: Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of somatic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, Fas gene without the stop codon was amplified in the present study using RT-PCR and directly cloned into the eukaryotic expression vector pAcGFP-N1. The resultant recombinant plasmid pAcGFP-bFas was then transfected into bovine follicular granulosa cells. Expression of AcGFP was observed under fluorescent microscopy, and the transcription and translation of Fas were detected by RT-PCR and western blot analysis. The methyl-tetrazolium (MTT) assay, Hoechst33342 staining and DNA ladder method were performed to determine the growth inhibition and apoptosis of the cells. The results showed that GFP expression was detected as early as 24 h after transfection. The Fas fusion gene was successfully expressed in granulosa cells as evidenced by the detection of a 994-bp fragment corresponding to the Fas mRNA by RT-PCR and a 64.5-kD band corresponding to the Fas fusion protein by western blot. Granulosa cell viability decreased significantly at 72 h after transfection, and the apoptosis rate of the cells transfected with pAcGFP-Fas was significantly higher than that of the control group. Cells in the Fas transfection group showed ladder patterns characteristic of apoptosis, and the nuclei were shrunken and densely hyperchromatic or fragmented, suggesting that Fas is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis when over-expressed. This study will aid in further understanding the mechanism of regulation of Fas on bovine oocyte formation and development., (© 2011 Blackwell Verlag GmbH.)

Background and Aim: Our purpose is to study the clinical significance of Fas/FasL expression in colon cancer and liver metastases (LM)., Material and Methods: The expression of Fas/FasL in 68 patients with colon cancer was examined immunohistochemically and correlated to the clinicopathological features of the tumors., Results: High expression of FasL, was observed in stage D and in LM (p=0.024). Fas expression was reduced in stage D tumors and in LM, when compared to earlier stages of disease (p=0.024). LM had also shown a decreased expression of Fas (p=0.016). Tumors with low FasL expression upregulate more often their Fas expression (p = 0.028). No correlation could be established regarding the patients survival., Conclusions: Low expression of Fas and high expression of FasL are more often in colon tumor stage D and in liver metastasis; these imply tumor aggression, resistance against apoptosis, and could be held as negative prognostic factors.