Immunohistochemical detection of Fas and Fas ligand in sarcomatoid renal cell carcinoma

Sarcomatoid renal cell carcinomas (SRC) are rare neoplasms associated with a very poor prognosis. The aim of this study was to evaluate biomarker expression and clinical significance in this uncommon renal cancer. Cytokeratin, epithelial membrane antigen, vimentin, desmin, smooth muscle actin, CD34, S-100 protein, MIB 1, p53, Fas and Fas ligand immunohistochemical expression was investigated in seven renal cell carcinomas with sarcomatoid changes. No significant difference between sarcomatoid and nonsarcomatoid areas was observed with the different biomarkers, excepted for Fas ligand. Fas expression was diffuse in sarcomatoid and nonsarcomatoid areas. However, Fas ligand had a higher expression in sarcomatoid in comparison to nonsarcomatoid areas. Our results showed that Fas and Fas ligand are both expressed in renal cancer. We suggest that the aggressive behavior of sarcomatoid carcinoma may be related to a higher expression of Fas ligand by tumor sarcomatoid cells. These findings may indicate that Fas ligand is a possible therapeutic molecular target for treatment of SRC.     

Fas and Fas-ligand are expressed in the uteroplacental unit of first-trimester pregnancy

PROBLEM: Fas and Fas-ligand (FasL) are thought to provide a strategy for reducing graft rejection in immunologically 'privileged' tissues by controlling injurious lymphocyte reactions. As the uteroplacental unit is often defined as an immune-privileged site, we investigated the expression of Fas and FasL in this tissue in the first trimester of pregnancy. METHOD OF STUDY: Western blotting, immunohistochemistry, and double immunofluorescence were used for this examination. RESULTS: Western blotting with purified first-trimester trophoblast cells revealed one specific band for FasL. The presence of FasL on different trophoblast populations could be confirmed by immunohistochemistry and double immunofluorescence. In the villous part of the placenta, FasL is mostly located on cytotrophoblast cells with no access to maternal blood flow, whereas in trophoblast-invaded uterine tissue, interstitial trophoblast cells, which are in close contact with maternal leukocytes, revealed a strong signal for FasL, but no staining for Fas on these cells. However, Fas was found on CD45+ maternal leukocytes. CONCLUSION: Based on our experimental findings, we speculate that the abundant presence of FasL on trophoblast cells within the maternal decidua may play an important role in the maintenance of immune privilege in the pregnant uterus by endowing fetal trophoblast cells with a defense mechanism against activated maternal leukocytes, whereas in the villous part of the placenta, the Fas FasL system seems to be involved in the regulation of placental growth.     

淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

利用噬菌体肽库技术获得与人Fas结合的多肽基序

目的　利用噬菌体随机肽库技术获得与人Fas胞外区结合的多肽及其多肽基序 ,观察多肽基序的生物学功能。方法　以人Fas胞外区与IgGFc段的融合蛋白Fas .Fc为筛选配基 ,筛选噬菌体随机九肽库 ;微量淘洗与ELISA相结合鉴定阳性克隆 ,DNA测序和分析。化学合成多肽进行竞争性ELISA以及细胞增殖抑制实验。结果　经过 4轮亲和筛选 ,微量淘洗鉴定 ,获得 4 2个阳性克隆 ;固定ELISA实验显示筛选到的噬菌体短肽能与Fas .Fc特异性结合 ,并呈剂量依赖关系 ;随机选取 13个阳性克隆进行DNA测序 ,其序列及出现几率分别为 :PRKARVDTS(2 / 13)、YKKKSLQVQ (2 / 13)、YKKKSMLQA(2 / 13)、SRKKYDQYA(4/ 13)、YARKIKPTA(2 / 13)和ARKKTEGAG(1/ 13)。经多重序列分析 ,获得多肽基序 : R/KKK A。在ELISA和竞争性ELISA实验中 ,化学合成多肽EGEFYKKKSM LQADPAK (P3)可抑制Fas与抗人Fas单抗Apo 1的结合 ,且呈剂量效应关系 ;P3不能抑制Fas与FasL的结合。细胞增殖实验表明 ,多肽可抑制Jurkat细胞增殖 ,且随多肽剂量的增加而加强。多肽与单抗Apo 1联合作用对Jurkat细胞增殖的影响与单独使用P3没有明显差别。结论　通过噬菌体随机肽库技术获得与Fas结合的多肽及其多肽基序 ,它们可能模拟了抗Fas抗体Apo 1对Fas的结合位点 ,为基于Fas凋     

Failure of TGF β1 and IL-12 to regulate human FasL and mTNF alloreactive cytotoxic T-cell pathways

Abstract: The effect of TGFβ1 and IL-12 on calcium-independent cytotoxic pathways was investigated. We have previously demonstrated that the regulatory effect of TGFβ1 and IL-12 on human alloreative CTL activity was associated with regulation of perform and granzyme B gene expression. To determine the effect of both cytokines on the alternative cytotoxic pathway involving FasL and mTNF, we first investigated the expression of both molecules on human primary alloactivated T cells. Our results show that human allostimulated T lymphocytes express FasL. Cell lysis experiments demonstrate that the FasL cytotoxic pathway is involved in the killing of specific target cells mediated by human alloreactive CTL. In addition, allogeneic stimulation induced significant mTNF expression on both CD4+ and CD8+ responder T cells. Using TNF-sensitive target cells, we also demonstrate that the mTNF-mediated cytotoxic pathway is involved in the cytotoxic activity of human primary allostimulated T lymphocytes. Neither TGFβ1 nor IL-12 had an effect on FasL or mTNF expression. Furthermore, addition of TGFβ1 or IL-12 at the initiation of the MLR had no significant effect on Fas- and mTNF-mediated cytotoxicity.
Taken together, our results provide a novel insight into the differences between regulation by cytokines of perforin-dependent and -independent cytotoxic mechanisms. Unlike their role in the perforin/granzyme B pathway, TGFβ1 and IL-12 do not appear to mediate any regulatory effect on FasL and mTNF cytotoxic pathways used by human alloreactive primary CTL.     

Correlation between the number of apoptotic cells and expression of the apoptosis-related antigens Fas, Ley and bcl-2 protein in non-Hodgkin's lymphomas

The relationship between the number of apoptotic cells and the expression of apoptosis-related antigens was examined In 56 cases of non-Hodgkin's lymphomas and in 10 cases of reactive hyperplastic lymph nodes (RHL). Apoptosis was visually quantified by the in situ end-labeling (ISEL) method, and the expression of Fas, Le^y antigens and bcl-2 protein was examined by Immunohistochemistry. The expression of Le^y antigen was observed in germinal centers of RHL and 45% of non-Hodgkin's lymphomas. The apoptotic cell count (AC) in follicular lymphomas was significantly less than that in diffuse lymphomas. The distribution pattern of apoptotic cells In follicular lymphomas was inverse to that in RHL. In follicular lymphomas, AC was lower in follicles than in inter-follicular areas. In contrast, AC was higher in follicles than in Interfollicular areas in RHL. Le^y antigen-positive lymphomas showed a significantly higher AC than the negative cases. The Fas antigen-positive lymphomas showed a higher AC than the negative cases. However, AC in bcl-2 protein-positive and negative cases was not significantly different. These results suggest that Le^y and Fas antigens appear to be involved in the apoptotic tendency of tumor cells in non-Hodgkin's lymphomas, whereas bcl-2 does not necessarily.     

Concentrations of Soluble CD95 and CD8 Antigens in the Plasma and Levels of CD8+CD95+, CD8+CD38+, and CD4+CD95+ T Cells Are Markers for HIV-1 Infection and Clinical Status

Apoptosis mediated via the CD95 (FAS/APO-1) receptor is thought to play a role in the depletion of CD4+ T cells in HIV infection. In the present study expression of the CD95 antigen on lymphocyte subsets and the plasma level of soluble CD95 (sCD95) were determined in HIV-1-infected adults. The expression of CD95 was increased on CD8 cells in all groups of HIV+ individuals, while increased expression of CD95+ cells on CD4 cells was limited to individuals with CD4 counts of <200 mm³. The proportion of CD4+ that expressed CD95 was inversely correlated with the percentage of CD4+ PBL. The concentration of sCD95 was significantly higher in the plasma of HIV-infected individuals than in normal controls. The level of sCD95 in HIV-infected subjects showed no correlation with the percentage of PBL expressing CD95, indicating that the increased level of sCD95 did not reflect release from CD95+ PBL. The plasma sCD95 concentration was significantly correlated with the percentage of CD8+ cells and, particularly, with CD8+CD38– cells. A striking inverse correlation was found between the sCD95 plasma concentration and the proportion of CD4+CD95+ cells out of the total CD4+ population. There was no correlation between the serum level of sCD95 and that of soluble CD8 (sCD8), both of which were increased in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals. Unlike the level of sCD95, the level of sCD8 in the plasma of HIV+ individuals was correlated with the percentage of CD95+ and CD8+CD38+ cells. The present study indicates that plasma sCD95 may be one of the factors that regulate apoptotic death of lymphocytes in HIV infection.