全反式维甲酸对兔颈动脉粥样硬化性狭窄中平滑肌细胞凋亡及其机制的研究

目的观察全反式维甲酸（ATRA）对兔颈动脉粥样硬化性狭窄后平滑肌细胞凋亡的影响。方法新西兰兔40只，随机分为3组：对照组（CON）、手术组（SUR）、治疗组（TRE），均给予高脂饮食，两周后手术组和治疗组用空气干燥法建立颈动脉粥样硬化模型，治疗组于术前给予全反式维甲酸灌胃。分别于术后两周、四周处死动物取病变处血管，HE染色后观察观察内膜增生情况，并用TUNEL法检测细胞凋亡、免疫组化检测凋亡抗原基因Fas。结果形态学观察显示内膜增生程度由强到弱为：SUR〉TRE〉CON。在治疗组凋亡细胞TUNEL阳性细胞数最多，Fas表达最明显。结论ATRA具有诱导平滑肌细胞凋亡的作用，在一定程度上抑制血管内膜增生，抑制血管损伤后再狭窄，其机制可能是通过FAS／FASL途径。     

Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity

BACKGROUND & AIMS: Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T-cell receptors (NKT cells) in APAP-induced liver injury. METHODS: C57BL/6 mice were administered a toxic dose of APAP intraperitoneally to cause liver injury with or without depletion of NK and NKT cells by anti-NK1.1 monoclonal antibody (MAb). Serum alanine transaminase (ALT) levels, liver histology, hepatic leukocyte accumulation, and cytokine/chemokine expression were assessed. RESULTS: Compared with APAP-treated control mice, depletion of both NK and NKT cells by anti-NK1.1 significantly protected mice from APAP-induced liver injury, as evidenced by decreased serum ALT level, improved survival of mice, decreased hepatic necrosis, inhibition of messenger RNA (mRNA) expression for interferon-gamma (IFN-gamma), Fas ligand (FasL), and chemokines including KC (Keratinocyte-derived chemokine); MIP-1 alpha (macrophage inflammatory protein-1 alpha); MCP-1 (monocyte chemoattractant protein-1); IP-10 (interferon-inducible protein); Mig (monokine induced by IFN-gamma) and decreased neutrophil accumulation in the liver. Hepatic NK and NKT cells were identified as the major source of IFN-gamma by intracellular cytokine staining. APAP induced much less liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice compared with that in wild-type mice. CONCLUSIONS: NK and NKT cells play a critical role in the progression of APAP-induced liver injury by secreting IFN-gamma, modulating chemokine production and accumulation of neutrophils, and up-regulating FasL expression in the liver, all of which may promote the inflammatory response of liver innate immune system, thus contributing to the severity and progression of liver injury downstream of the metabolism of APAP and depletion of reduced glutathione (GSH) in hepatocytes.     

Genetic variants in interleukin-18 gene and risk for cervical squamous cell carcinoma

Cervical cancer is strongly associated with infection of oncogenic types of human papillomavirus (HPV). However, HPV infection alone is not sufficient for progression to cervical cancer. It is now recognized that host immunogenetic background participates in the control of HPV infection and development of cervical cancer. Interleukin-18 (IL-18) is a multifunctional cytokine that induces interferon-gamma secretion and plays a central role in antitumor immunity. The aim of this study is to determine if potentially functional polymorphisms in IL-18 gene are associated with risk of HPV-induced cervical cancer in Taiwanese women. Pre-Developed TaqMan Allelic Discrimination Assay was used to genotype IL-18 −1297 T/C, −607 C/A, −380 C/G, −137 G/C, and +105 A/C polymorphisms in a hospital-based study of 470 women with cervical squamous cell carcinoma (CSCC) and 722 age-matched healthy control women. The presence and genotypes of HPV in CSCC was determined by PCR. None of the polymorphisms or any haplotype was found to have significant differences in distribution among all subjects with CSCC, those with HPV-16 positive CSCC, and controls. Our results suggest that the IL-18 −1297 T/C, −607 C/A, −380 C/G, −137 G/C, and +105 A/C polymorphisms are not associated with susceptibility to CSCC in Taiwanese women.     

Inhibition of Fas-mediated Apoptosis by Antigen: Implications for Lymphomagenesis

Apoptotic deletion of expanded B cell populations is essential in avoidance of autoimmune disease and immune regulation of some B cell malignancies. The role of CD4+ T cells in B cell apoptosis is evident from the high incidence of B cell tumors and autoimmunity in patients with T cell diseases such as the acquired immune deficiency syndrome (AIDS). We have previously demonstrated that in Epstein-Barr Virus (EBV) negative Burkitt's lymphoma (BL), a tumor derived from proliferating centroblasts of the germinal center, the malignant lymphocytes can be induced to express Fas (CD95) by ligation of CD40 at the B cell surface. Upon CD40 engagement, BL cells are sensitized to T-cell derived death signals provided by Fas ligand (FasL, CD95L). HBL-3 is a cell line derived from an AIDS-related BL in which the tumor IgM binds the human erythrocyte "i" antigen. To determine whether Fas-mediated apoptosis of BL cells is reduced in the context of antigen to which the tumor IgM binds, we stimulated HBL-3 cells with CD40 ligand (CD40L, CD154) in the presence and absence of human erythrocytes expressing the "i" antigen, and measured Fas-mediated apoptosis upon exposure to an agonistic anti-Fas antibody. We observed that HBL-3 cells were sensitized to Fas-mediated death by exposure to CD40L. When i+ RBCs were present, Fas-mediated apoptosis in HBL-3 cells was reduced by greater than 30%. In contrast, there was no reduction in Fas-mediated apoptosis in the presence of i &#109 (I+) RBCs. These findings demonstrate that Fas-mediated deletion of BL cells is inhibited upon surface IgM engagement by antigen for which the malignant clone has affinity.     

Clinical Significance of Serum Soluble Fas,Fas Ligand and Fas in Intrahepatic Lymphocytes in Chronic Hepatitis C

Hepatitis C Virus (HCV) is a major etiological agent of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. The present work was performed to study the fas system (Fas-FasL and soluble Fas) in chronic hepatitis C infection. Also, to correlate the degree of liver cell damage with the Fas system. The study was carried out on 45 patients positive for HCV RNA by nested RT-PCR in addition to 13 HCV negative control subjects. Wedge liver biopsies samples were obtained from patients and controls during abdominal operations for determination of cellular expression of Fas and Fas-L on hepatocytes and infiltrating lymphocytes respectively by flow cytometry. Histological activity index (HAI) was determined in chronic HCV patients. Also blood samples were taken from patients and controls for determination of sFas. There was statistically insignificant difference in Fas expression in hepatocytes of patients (P?=?0.34) in comparison to control. Meanwhile, there was a statistically significant decrease in FasL expression in patients compared to control (P< 0.001) and statistically significant increase in soluble Fas in patients compared to control (P < 0.001). The HAI of liver fibrosis for all patients were within mild score with mean ± SD 4 ± 0.5. From this study, we could conclude that Fas system is one of the important pathways regulating the response to HCV infection. Increased serum sFas in HCV patients is accompanied by down-regulation of Fas/Fas-L expression resulting in inhibition of apoptosis in liver cells as a process for elimination of virus infected cells and this may ultimately leads to chronicity of the disease.     

雌激素缺乏导致的骨质疏松发病过程中骨髓间充质干细胞FasL表达降低对CD4+T淋巴细胞凋亡的影响

 目的：研究雌激素在骨质疏松发病过程中导致骨髓间充质干细胞（BMMSCs）Fas配体（FasL）表达降低对CD4+T淋巴细胞凋亡的作用。方法：选用8周龄健康雌性小鼠行双侧卵巢切除术(OVX),建立绝经后骨质疏松模型。选用同一批次周龄相同、体重相近的健康小鼠行双侧卵巢附近脂肪组织部分切除,建立假手术组（sham）,Micro-CT确定模型成功建立。流式细胞术对BMMSCs行表型鉴定,将OVX组、sham组和sham+雌激素刺激组BMMSCs与激活的T淋巴细胞共培养,以Annexin V/7-AAD/CD4流式细胞仪计数3组共培养体系中CD4+T淋巴细胞的凋亡差异,用实时荧光定量PCR和Western blotting检测OVX组、sham组及不同浓度雌激素刺激BMMSCs后FasL的表达情况。结果：流式检测显示sham+雌激素刺激组CD4+T淋巴细胞凋亡程度大于sham组(P<0.05),sham组CD4+T淋巴细胞凋亡程度大于OVX组(P<0.05),实时荧光定量PCR及Western blotting显示OVX组FasL mRNA及蛋白水平表达都较sham组低。在一定范围内不同浓度雌激素刺激下的sham组BMMSCs中FasL mRNA及蛋白表达与雌激素浓度呈梯度上升。结论：雌激素能够调控BMMSCs中FasL的表达,雌激素调控BMMSCs表达FasL能够影响CD4+T淋巴细胞凋亡的改变,这可能与绝经后骨质疏松发病相关。     

Intracellular Fas ligand is elevated in T lymphocytes in severe aplastic anaemia

Increased expression of Fas receptor by haemopoietic progenitors in aplastic anaemia (AA) suggests that excessive apoptosis contributes to multilineage bone marrow (BM) failure. To investigate the role of Fas ligand (FasL) in triggering progenitor cell death, we examined FasL levels in T lymphocytes of patients with severe untreated AA (n = 8). Expression of FasL on the surface of CD3+ cells was not detectable. However, flow cytometric analysis of saponin-permeabilized cells demonstrated higher levels of intracellular FasL in AA than in normal T cells (P < 0.005), both prior to and following activation with phytohaemagglutinin. Confocal microscopy revealed that FasL-specific signals overlapped with cathepsin D staining, indicating that intracellular FasL is stored in lysosomal granules. Levels of intracellular FasL in patients examined 1 month after immunosuppression with antilymphocyte globulin and cyclosporin A were lower than prior to treatment. The caspase inhibitors, DEVD and zVAD, enhanced colony formation and prolonged survival of AA BM cells in liquid cultures by about 10-fold (P < 0.05). Taken together, these data provide further evidence that apoptosis by the Fas/FasL system plays a role in the depletion of stem cells in AA.     

In situ expression of Granzyme B and Fas‐ligand in the liver of viral hepatitis

Abstract: Background/Aims: The molecular mechanism involved in hepatocellular injury in viral hepatitis remains to be clarified. Methods: We investigated the in situ expression of effector molecules of cytotoxic T lymphocytes such as Fas‐ligand (Fas‐L), perforin and Granzyme B (Gr‐B) immunohistochemically in liver tissues from 20 patients with chronic hepatitis B (CHB) and C (CHC). The degree of cell infiltration was analysed semi‐quantitatively and compared with the histological activity index (HAI). Fas‐L was expressed in both CD4 and CD8 T‐cells in the portal tract as well as in the parenchyma. Results: Immunostaining of serial sections demonstrated that mononuclear cells at interface hepatitis and focal necrosis were mainly Fas‐L positive CD8 T‐cells. On the other hand, the expression of perforin or Gr‐B was limited to a few mononuclear cells in the portal tract and parenchyma. Semi‐quantitative analysis showed a positive correlation between HAI and the grade of infiltration of CD8 T‐cells or Fas‐L‐positive cells, while the correlation was not apparent between HAI and the number of Gr‐B positive cells. The expression of these molecules was not different between types of viruses. Conclusions: These results suggest that Fas‐L‐positive CD8 T‐cells play a major role in the pathogenesis of liver cell injury in chronic hepatitis.     

Bile salt-induced apoptosis involves NADPH oxidase isoform activation

BACKGROUND & AIMS: Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis. METHODS: The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7). RESULTS: The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Czeta (PKCzeta). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase-dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCzeta, or NADPH oxidases. CONCLUSIONS: The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.