ACTIVATION-INDUCED APOPTOSIS--ROLE IN AIDS PATHOGENESIS

在HIV感染过程中，CD4+细胞有一个深刻的和选择性的下降， 与进行性免疫缺陷相关的T细胞。 的 HIV感染导致CD4 T细胞耗竭的机制是 辩论。 尽管最近的数据表明， 循环和淋巴器官中， 即使在疾病的早期阶段， 用人类淋巴细胞重建SCID小鼠也认为， 直接的病毒感染和细胞病变不能解释所有的 CD4 T细胞死亡。 此外，黑猩猩可以维持一个 体内HIV慢性感染，体外c细胞病变感染， 但不会发展为获得性免疫缺陷综合征（AIDS）， 这表明，其他机制，直接病毒破坏有助于 导致CD4 T细胞减少 我们已经证明，CD4在 人CD4 T细胞，然后通过T细胞受体进行信号传导， 抗原通过凋亡导致活化诱导的细胞死亡。 这些 结果提示了AIDS中大量CD4 T细胞耗竭的机制， 特别是在面临并发感染和抗原挑战时 与其他有机体。 HIV中T细胞死亡机制的证据 感染来自几个团体，包括我们自己的团体， 来自HIV感染者的CD4（有时还有CD8）T细胞 在培养物中活化后发生细胞凋亡。 这个实验 建议的目的是调查的机制和作用， 艾滋病中的细胞凋亡 在目标1中，我们建议确认我们的体外 体内观察，并确定激活诱导的细胞凋亡 在艾滋病的发展过程中起着重要作用。 我们的体内研究将利用 从人类和非人类灵长类动物的HIV或SIV感染组织中， 原位鉴定凋亡细胞并确定是否有更多的细胞 凋亡的细胞比感染病毒的细胞多。 进展将 通过患者细胞的纵向和横截面研究来研究 和血清，以便将细胞凋亡与疾病阶段相关联， 分析灵长类动物模型中的细胞凋亡， 类似艾滋病的疾病。 在目标2中，我们将研究CD4 引发艾滋病中的细胞凋亡。 我们将确定 凋亡引发信号在HIV感染中被传递到CD4 T细胞， 对这种信号敏感的细胞，动力学和 这种信号的传播，以及特定激酶和 磷酸酶在引发事件中的作用。 这些实验将采用来自HIV感染者和未感染者的PBL 成人和儿童，感染和未感染的淋巴结组织 人和非人灵长类动物、T细胞系和人CD4转基因小鼠 结合一些实验方法，包括流动 DNA片段和蛋白质的细胞计数和生化分析 激酶和磷酸酶活化。 拟议的研究将调查细胞凋亡的一系列方面， 艾滋病，从其临床相关性到分子机制。 我们的最终 目标是为艾滋病毒感染者制定治疗干预措施， 个人，以防止发展为艾滋病。 理想情况下，这 将允许抗病毒疗法消除病毒感染，而 疾病被控制住了

During HIV infection there is a profound and selective decrease in CD4 T cells which is associated with progressive immunodeficiency. The mechanism(s) by which HIV infection lead to CD4 T cell depletion are debated. Although recent data gives evidence for a higher viral load in the circulation and in lymphoid organs that had been previously appreciated, even at early stages of disease, recent data from a model reconstituting the SCID mouse with human lymphoid cells also argue that direct viral infection and cytopathicity cannot account for all of the CD4 T cell death. In addition, the fact that chimpanzee can sustain a chronic infection with HIV in vivo, and c cytopathic infection in vitro, but does not progress to acquired immune deficiency syndrome (AIDS), suggests that mechanisms other that direct viral destruction contribute to CD4 T cell loss. We have demonstrated that crosslinking of CD4 on human CD4 T cells followed by signaling through the T cell receptor for antigen results in activation-induced cell death by apoptosis. These results suggest a mechanism for the massive CD4 T cell depletion in AIDS, particularly in the face of concurrent infection and antigenic challenge with other organisms. Evidence for this mechanism of T cell death in HIV infection has come from several groups, including our own, who have shown that CD4 (and in some cases CD8) T cells from HIV-infected individuals undergo apoptosis upon activation in culture. The experiments in this proposal are designed to investigate both the mechanism and role of apoptosis in AIDS. In Aim 1, we propose to confirm our in vitro observations in vivo, and to determine if activation-induced apoptosis plays a role in progression to AIDS. Our in vivo studies will make use of HIV- or SIV-infected tissue from humans and from nonhuman primates to identify apoptotic cells in situ and to determine whether more cells are apoptotic than are productively infected with virus. Progression will be studied by longitudinal and cross-sectional studies of patient cells and serum in order to correlate apoptosis with disease stage and by analysis of apoptosis in primate models which do or do not progress to AIDS-like disease. In Aim 2, we will investigate the mechanism of CD4 priming for apoptosis in AIDS. We will identify the means by which the apoptotic priming signal is delivered to the CD4 T cell in HIV infection, the cell which is susceptible to this signal, the kinetics and propagation of this signal, and the role of specific kinases and phosphatases in the priming event. These experiments will employ PBL's from HIV-infected and uninfected adults and children, lymph node tissue from infected and uninfected humans and nonhuman primates, T cell lines, and human CD4 transgenic mice in conjunction with a number of experimental methods including flow cytometry and biochemical analysis of DNA fragmentation, and protein kinase and phosphatase activation. The proposed studies will investigate a range of aspects of apoptosis in AIDS, from its clinical relevance to molecular mechanisms. Our ultimate goal is the development of therapeutic interventions for HIV-infected individuals, in order to prevent progression to AIDS. Ideally, this would allow anti-viral therapies to eliminate viral infection, while the disease is held at bay.