Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility

BACKGROUND: The aim of this study was to examine the role of apoptosis and reactive oxygen species (ROS) in inducing DNA damage in ejaculated spermatozoa. METHODS: We examined ejaculated spermatozoa from 31 patients examined for infertility and 19 healthy donors for apoptosis, production of ROS and DNA damage using annexin V binding, chemiluminescence assay and sperm chromatin structure assay. RESULTS: The percentage of spermatozoa that underwent apoptosis in the whole ejaculate and mature fraction was higher in the patients than in the donors (P<0.001 and P=0.009, respectively). Levels of ROS in the whole ejaculate and immature fraction were higher in the patients than in the donors (P=0.002 and P=0.009). Apoptosis was significantly correlated with ROS within patients in the whole ejaculate [r (95% confidence interval)=0.53 (0.19-0.86)] and in the mature [0.71 (0.39-1.00)] and immature spermatozoa [0.75 (0.45-1.00)]. Only apoptosis and the DNA fragmentation index (DFI) were significantly correlated within patients in the whole ejaculate [0.57 (0.18-0.97)]. CONCLUSIONS: DNA damage may be induced by oxidative assault. Apoptosis may not contribute significantly to the DNA damage.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Apoptosis of colorectal adenocarcinoma (COLO 201) by tumour necrosis factor-alpha (TNF-α) and/or interferon-gamma (IFN-γ), resulting from down-modulation of Bcl-2 expression

Fas antigen is constitutively expressed in the normal colon epithelium, but considerably diminished in most colorectal carcinomas. In the present study, we examine the relationship between Fas antigen expression and apoptosis using the colorectal carcinoma cell line COLO 201, on which a low grade of Fas antigen is expressed. Anti-Fas antibody had no effect on the induction of apoptosis of COLO 201. However, TNF-α and/or IFN-γ, independently and additively, up-regulated Fas antigen expression on COLO 201 and induced apoptosis in a dose-dependent manner. Both cytokines also increased the COLO 201 sensitivity to anti-Fas antibody, resulting from the down-modulation of Bcl-2 and the up-regulation of Bax. These findings indicate that cytokine(s) plus anti-Fas antibody (which mimics natural Fas ligand) are more effective in inducing apoptosis of COLO 201 than cytokine(s) alone. These findings suggest that immunotherapy in combination with cytokine(s) and lymphokine-activated killer (LAK) cells will become a more effective therapy for cancer than cytokine(s) or LAK cells alone, since the Fas ligand is expressed on activated T cells, natural killer cells and macrophages.     

肺鳞状细胞癌中死亡相关蛋白激酶的表达

目的　检测肺鳞状细胞癌中死亡相关蛋白激酶 (DAP K)mRNA表达及细胞凋亡 ,探讨DAP K与细胞凋亡的关系及其在肺鳞状细胞癌发生、发展中的作用。方法　用原位分子杂交法检测 6 0例肺鳞状细胞癌、9例癌旁肺组织DAP KmRNA表达 ;用原位末端标记TUNEL法检测相应组织中细胞凋亡 ,计算凋亡指数 (AI)。结果　肺鳞状细胞癌的DAP KmRNA阳性表达率为 4 6 7% ,癌旁肺组织为 6 7 7% ,其阳性率高于肿瘤组织 (P <0 0 1 )。在肺鳞状细胞癌中 ,高分化癌DAP KmRNA阳性率为 70 % ,低分化癌为 2 3 3% ,高分化癌的DAP KmRNA阳性率高于低分化癌 (P <0 0 1 )。肺鳞状细胞癌的细胞AI为(0 6 72 8± 0 4 2 6 1 ) % ,癌旁肺组织中支气管肺泡上皮细胞AI为 (1 0 2 89± 0 2 4 33) % ,癌旁肺组织的AI高于肿瘤组织 (P<0 0 1 )。在肺鳞状细胞癌中 ,高分化癌的AI为 (0 5 82 3± 0 1 92 2 ) % ,低分化癌为 (0 4 4 6 0± 0 1 92 5 ) % ,高分化癌的AI高于低分化癌 (P <0 0 1 )。DAP KmRNA呈阳性表达的肺癌 ,其AI为 (0 5 31 7± 0 2 0 97) % ;DAP KmRNA呈阴性者 ,其AI为 (0 4 872± 0 1 91 8) % ,两组间差异有显著性 (P <0 0 5 )。在连续切片上 ,DAP KmRNA阳性细胞的分布区域与凋亡阳性细胞的分布相似。DAP KmRNA呈阳性表达     

Chitooligosaccharides protect cultured hippocampal neurons against glutamate-induced neurotoxicity

Chitooligosaccharides (COSs), the biodegradation product of chitosan, have demonstrated a diverse array of biological activities. Here we report the protective effect of COSs (M.W. 800) against glutamate-induced neurotoxicity in cultured hippocampal neurons. The cell viability assessments, together with Hoechst 33342 staining and flow cytometry for cell apoptosis analysis, indicated that glutamate (125 μM)-induced cell apoptosis in cultured hippocampal neurons was attenuated in a concentration-dependent manner by COSs pretreatment. After measurement with Fluo 4-AM, COSs were found to depress glutamate-induced elevation in intracellular calcium concentration ([Ca²⁺]_c). The enzymatic assay indicated that COSs antagonized glutamate-evoked activation of caspase-3. These results collectively suggest that COSs prevent cultured hippocampal neurons from glutamate-induced cell damage by interfering with an increase in [Ca²⁺]_c and inhibiting caspase-3 activity.     

Acellular Bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2-deficient mice

Mice deficient for the inhibitory G protein subunit alpha2 (Galphai2(-/-)) spontaneously develop a progressive inflammatory bowel disease resembling ulcerative colitis, and have a T helper 1 (Th1)-dominated immune response prior to onset of colitis, which is further augmented after the onset of disease. The present study was performed to investigate whether the Galphai2(-/-) mice were able to down-regulate the Th1-dominated inflammatory mucosal immune response and/or induce an anti-inflammatory Th2/T regulatory response and thereby diminish the severity of colitis following treatment with acellular Bordetella pertussis vaccine. The acellular vaccine against B. pertussis, the causative agent of whooping cough, has been demonstrated to induce a Th2-mediated response in both man and mice. We therefore treated Galphai2(-/-) mice intraperitoneally with a three-component acellular B. pertussis vaccine. The treated Galphai2(-/-) mice showed significantly increased interleukin (IL)-10 production in intestinal tissue, associated with significantly reduced colitis and decreased mortality, compared to untreated Galphai2(-/-) mice. The attenuation of colitis in Galphai2(-/-) mice was due, at least partly, to the B. pertussis surface antigen filamentous haemagglutinin (FHA), which almost completely inhibited proliferation of CD4(+) T cells and stimulated apoptosis of activated CD4(+) T helper 1 cells. In conclusion, the three-component acellular B. pertussis vaccine containing filamentous haemagglutinin increases the production of IL-10 in the intestinal mucosa, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2(-/-) mice.     

Pigmentary degeneration indwed by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina

Pigmentary degeneration of the retina was induced by a single intraperitoneal Injection of 75mgkg of N-methyl-N-nitrosourea (MNU) In female Brown-Norway colored rats at 50 days of age, which were then observed at 24, 48 and 72 h and 7, 21,35 and 150 days after the treatment. MNU-treated rats showed selective destruction of the photoreceptor cells by an apoptotic mechanlsm 24 h after the treatment, and the destruction was completed by day 7. During the photoreceptor cell degeneration, proliferation of Miller cells and infiltratlon of macrophages was prominent 72h and 21 days aRttr the treatment, respectively. Müller cell proliferation and macrophage infiltratbn corresponded to degenerative photo-receptor cell phagocytosis, and prollferating Müller cell processes responded to stabilize the damaged retina. Pigment epithelial cell detachment from the Bruch's membrane was seen 72 h after the treatment, and migration within all layers of the retina was seen at day 7 when photoreceptor Cells were lost. At 21, 35 and 150 days after the treatment, lack of photoreceptor cells and deposition of pigment epithelial cells within the retina but not in contact to vascular endothe-lial cells were characteristic. MNU-induced photoreceptor apoptosis followed by Miiller cell and macrophage reaction then pigment epithellal cells deposition withln the retina partially resembles retinitis pigmentosa in humans.     

PTEN基因对人肝癌细胞系HHCC作用的研究

目的：研究PTEN基因对人肝癌细胞系HHCC细胞恶性表型及凋亡的影响。方法：将PTEN真核表达载体pBabe-PuroPTEN及空质粒pBabe-Puro，通过脂质体介导的基因转染方法，转入该基因表达缺失的HHCC细胞系中，嘌呤霉素筛选获得稳定表达PTEN基因的转染细胞。通过平板克隆形成试验、DNA凝胶电泳、相差显微镜和电镜，观察PTEN基因对人肝癌细胞系HHCC恶性表型及凋亡的影响。结果：转染后的细胞，经原位杂交、免疫组织化学检测证实，有PTEN mRNA及其蛋白的表达；平板克隆形成试验证实，转染PTEN基因后，细胞形成克隆的能力降低；转染PTEN的细胞在相差显微镜和电镜下可出现典型的凋亡形态学改变；DNA琼脂糖凝胶电泳呈现出明显的梯状条带。结论：外源性PTEN基因导入HHCC细胞后，可降低HHCC的细胞的恶性表型，并促进凋亡发生。     

Generation of peroxynitrite and apoptosis in placenta of patients with chorioamnionitis: possible implications in placental abruption

The reaction of nitric oxide (NO) and superoxide results in the formation of peroxynitrite, a potent and relatively long-lived oxidant. In infectious diseases, these molecules are not only bactericidal but also toxic to host cells. Chorioamnionitis is often complicated by premature rupture of membranes and can be associated with placental abruption. These diseases are significant causes of premature low-birth-weight deliveries and consequently the morbidity and mortality of neonates. Lipopolysaccharide, bacterial endotoxin, is known to be elevated in the amniotic fluid of patients with chorioamnionitis. Lipopolysaccharide is known to induce the formation of NO and superoxide. We report here that nitrite/nitrate, stable metabolites of NO, were increased in serum from patients with chorioamnionitis. Immunohistochemical studies demonstrated enhanced expression of inducible NO synthase and formation of nitrotyrosine, a footprint of peroxynitrite, in the placentae from patients with chorioamnionitis and also in patients with placental abruption. Furthermore, apoptotic cell death was also increased in the placentae from patients with both diseases. These results suggest that chorioamnionitis and a portion of placental abruption may share a common cascade of placental injury. Nitric oxide and its metabolities may play an important role in this cascade.     

Immune deficiency following thermal trauma is associated with apoptotic cell death

Thermal injury-associated specific immune deficiency occurs despite indicators of systemic activation of the lymphoid compartment. We investigated the possibility that postburn immune failure and T cell activation are causally related through activation-induced (apoptotic) cell death. The relationship between the cellular immune response and cell mortality was examined in cultures of peripheral blood mononuclear cells (PBMC) from 14 immunosuppressed patients with extensive burns (35–90% total body surface area). Impaired cellular immunity coincided with significantly reduced cell viability as ascertained by propidium iodide staining and dye reduction assays. Following stimulation with the mitogenic lectin, phytohemagglutinin (PHA), the majority of DNA in patient cultures was fragmented, suggesting the occurrence of apoptotic cell death. Even without stimulation a portion of patient cells was apoptotic as indicated by oligonucleosomal bands on agarose gel electrophoresis. Exogenous interleukin-2 or phorbol ester markedly reduced constitutive as well as PHAinduced DNA fragmentation.In situ demonstration of DNA strand breaks in freshly isolated patient PBMC, by a TdT-based labeling technique, confirmed that a larger fraction (up to 60%) of circulating lymphocytes was undergoing apoptosis on the periphery. These novel observations suggest that apoptosis may play a major role in thermal injury-related cellular immunodeficiency.