RETROVIRUS INDUCED T AND NEURAL CELL DEATH

我们研究的总体目标是勾勒出 逆转录病毒诱导的致病机制(S) 免疫缺陷和神经紊乱。我们一直在 利用Moloney鼠白血病病毒(MoMuLY)及其 这些研究的突变体。其中一个突变体是ts1， 在鼠类逆转录病毒中是独一无二的，因为像艾滋病毒一样，它 破坏T细胞和神经元。尽管Ts1似乎 直接破坏T细胞，Ts1诱导的神经元死亡 似乎是间接的。这项工作的主要目标是 提案将试图描述这些机制的特征 参与Ts1诱导的这两种类型的细胞死亡。 希望这次调查将加强我们的 了解与以下相关的致病过程 逆转录病毒诱导的细胞破坏。我们的具体目标 包括： 1.确定T细胞可能参与的机制 被Ts1杀死： A.调查导致以下情况的事件顺序 通过解决以下问题对细胞死亡(PCD)进行编程 问题：1)Ts1包膜蛋白与 胸腺细胞表面(受体)触发不平衡信号 转导？如果是，这是否涉及PCD？：ii)是 未整合的病毒DNA数量增加 在Ts1感染的T细胞中？如果是这样，这种情况以前是否发生过？ 核酸内切酶(S)激活与核损伤？Iii)是否 病毒包膜蛋白在Ts1中的胞质内积累 被感染的T细胞发生在核或DNA损伤之前？如果 因此，Ts1包膜基因的激活是从潜伏期开始的吗？ 足以诱发PCD的综合形式：7；四) 在核或DNA损伤之前，细胞因子的变化： 如果是这样的话，这种细胞毒性作用能否被特定的 这些细胞因子的抗体或拮抗剂7；v)是 抑制Ts1参与的癌基因，如bcl2- 诱发性PCD？是这样的，这种情况什么时候会发生在PCD之前。 B.确定上述观察到的事件是否 体外研究可以用胸腺和 从Ts1获得的临时脾T细胞 受感染的小鼠。 2.确定可能涉及的机制 神经细胞死亡导致γTs1感染。 A.确定和评估年内感染的频率 神经系统的不同细胞类型，以及 确定信封的细胞内位置 通过使用以下方法在受感染的小鼠中表达蛋白质 方法：i)免疫组织化学；ii)电子 显微镜，三)原位杂交；和四)共聚焦 显微镜。 B.评估兴奋性氨基酸在Ts1中的作用- 使用以下方法诱导神经元变性 方法：1)氨基酸分析；2)原位定位 谷氨酸和天冬氨酸；以及iii)在体内 N-受体拮抗剂的药理实验 甲基-D-天冬氨酸(NMDA)受体。 C.评估细胞因子在Ts1诱导中的作用 神经元死亡。

The overall goal of our research is to delineate the pathogenic mechanism(s) involved in retrovirus-induced immunodeficiencies and neurologic disorders. We have been using Moloney murine leukemia virus (MoMuLY) and its mutants for these studies. One of these mutants, is ts1, is unique among murine retroviruses because like HIV, it destroys T cells and neurons. Although ts1 appears to destroy T cell directly, the neuronal death induced by ts1 appears to be indirect. The primary objectives of this proposal will attempt to characterize the mechanisms involved in these two types of cell death induced by ts1. It is hoped that this investigation will enhance our understanding of the pathogenic processes relevant to retrovirus-induced cell destruction. Our specific aims are: 1. To determine the possible mechanisms involved in T-cell killing by ts1: A. To investigate the sequence of events leading to program cell death (PCD) by addressing the following questions: 1) Does the binding of ts1 envelope protein to thymocyte surface (receptor) trigger imbalanced signal transduction? If so, does this involve in PCD?: ii) Is there an increase in the amount of unintegrated viral DNA in ts1 infected T cells? If so, does this occur before endonuclease(s) activation and nuclear damage? iii) Does intracytoplasmic accumulation of viral env protein in ts1 infected T cells occur before nuclear or DNA damage? If so, is activation of ts1 envelope gene alone from a latent integrated form sufficient to induce PCD:7; iv) Does alteration in cytokines precede the nuclear or DNA damage: Is so, can this cytotoxic effect be blocked by specific antibodies or antagonists to these cytokines 7; v) Is suppression of oncogenes such as bcl-2 involved in ts1- induced PCD? Is so, when does this occur before PCD. B. To determine whether the events observed in the above in vitro studies can be reproduced ex vivo using thymic and splenic T cells obtained on a temporal basis from ts1 infected mice. 2. To determine the possible mechanisms involved in neuronal cell death induced y ts1 infection. A. To identify and assess the frequency of infection in different cell types of the nervous system, and to determine the intracellular location of the envelope proteins in infected mice by using the following approaches: i) Immunohistochemistry; ii) Electron microscopy, iii) In situ hybridization; and iv) Confocal microscopy. B. To evaluate the role of excitatory amino acids in ts1- induced neuronal degeneration by using the following methods; i) Amino acid analysis; ii) In situ localization of glutamate and aspartate; and iii) In vivo pharmacological experiments with antagonist to the N- methyl-D-aspartate (NMDA) receptor. C. To evaluate the role of cytokines in ts1-induced neuronal death.