Effects of HIV -1 Proteins on the Blood/ Brain Barrier

HIV -1 蛋白对血/脑屏障的影响

• 批准号: 6394448
• 负责人: ROGER J POMERANTZ
• 金额: $ 31.8万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-21 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6394448
• 关键词: Lentivirus; apoptosis; astrocytes; blood brain barrier; cell type; drug resistance; genetic transduction; helper T lymphocyte; human immunodeficiency virus 1; human subject; open reading frames; recombinant proteins; terminal nick end labeling; transfection /expression vector; vascular endothelium permeability; virus protein

DESCRIPTION (Adapted from Applicant's Abstract): HIV- I can lead to a series of devastating clinical conditions in the central nervous system (CNS) of certain infectedindividuals characterized by cognitive and motor dysfunction. The molecular mechanisms underlying this HIV-1-induced dementia complex remain enigniatic. In addition, in the era of highly active anti-retroviral therapy (HAART), the CNS may also act as a reservoir or sanctuary site for HIV- I during combination therapy. Our laboratories have developed recently an in vitro blood:brain barrier (BBB) system comprised totally of human CNS cell-tvpes, which successfully models the cellular interactions of the BBB in vivo. In addition, we have demonstrated recently that the HIV- I regulatory protein, Vpr, has significant effects on inducing apoptosis in human neuronal cells, as well as activating bone marrow-derived macrophages. Utilizing complementary techniques based on these preliminary studies, the effects of selected lentiviral proteins on the BBB will be analyzed. It is hypothesized that Vpr, and possibly other specific viral proteins including Tat, Nef and the envelope protein gp I 2O which have been demonstrated to induce apoptosis in other human cells, may have effects on inducing programmed cell-death in BBB-constitudng cells, including microvascular endothelial cells (MVEC) and astrocytes. In specific aim one, this hypothesis will be evaluated using the in vitro BBB system with read-outs for apoptosis and BBB permeability changes including the TUNEL, Annexin V and Caspase-8 assays, in addition to trans-endothelial electrical resistance (TEER) and ZO] ti,-,ht-junction protein alterations. Both free recombinant ]antiviral proteins, in addition to mutant viruses lacking the genes of interest, will be analyzed in this system. In addition, utilizing a series of HIV- I -based lentiviral vector systems, these selected viral proteins will be expressed in the relevant BBB cell-types. In the complementary specific aim two, HIV-1-infected cells will also be analyzed for their inductions of permeability changes and apoptosis generation in the in vitro BBB system. In particular, mutant viruses lacking each specific viral regulatory and structural gene will also be used in a complementary approach, after infection of a CD4+ T-cells and primary macrophages for effects on the BBB. Finally, the effects of combination anti-retroviral chemotherapy on HIV- 1-infected cell interactions with the BBB will be analyzed. This will be a model svstem for different regimens of chemotherapeutics, in altering infected-cells' induced alterations on permeability and programmed cell-death within the human BBB. In the era of HAART, these will be critical studies in understanding how changes in combinations of HAART regimens affect HIV-I:BBB interactions, which will also be modeled in the system with resistant viral strains in infected peripheral blood cells. Within these inter-related studies, it is proposed that a series of new technologies, including HIV- I vector systems and an in vitro BBB system, be brought to bear towards determining the precise effects of HIV- I proteins, and thus the molecular mechanisms involved, in inducing alterations in the BBB.

描述（根据申请人的摘要改编）：HIV-我可以导致一系列 某些中枢神经系统（CNS）的破坏性临床状况 具有认知和运动功能障碍为特征的不感染的不症​​状。这 这种HIV-1诱导的痴呆症复合物的分子机制仍然存在 象征性。此外，在高度活跃的抗逆转录病毒疗法的时代 （HAART），中枢神经系统也可以充当HIV-I的水库或庇护所 在组合疗法期间。我们的实验室最近开发了 体外血液：脑屏障（BBB）系统完全由人类中枢神经系统组成 细胞-TVP，成功地模拟了BBB的细胞相互作用 体内。此外，我们最近证明了HIV I调节 蛋白质VPR对诱导人神经元凋亡具有重大影响 细胞以及激活骨髓来源的巨噬细胞。利用 基于这些初步研究的互补技术， 将分析BBB上选定的慢病毒蛋白。它是假设的 该VPR，以及可能其他特定的病毒蛋白，包括TAT，NEF和 包膜蛋白GP I 2O已被证明可以诱导凋亡 其他人类细胞可能会对诱导程序性细胞死亡产生影响 BBB-Constitudng细胞，包括微血管内皮细胞（MVEC）和 星形胶质细胞。在特定目标中，将使用IN评估该假设 体外BBB系统具有用于凋亡和BBB渗透率的读出的变化 包括TUNEL，ANNEXIN V和CASPASE-8测定法 跨内皮电阻（TEER）和ZO] Ti， - ，HT结构蛋白 改变。两种游离重组]抗病毒蛋白，除了突变体 缺乏感兴趣基因的病毒将在该系统中进行分析。在 此外，利用一系列基于HIV -I的慢病毒矢量系统，这些 选定的病毒蛋白将在相关的BBB细胞类型中表达。在 还将分析互补的特定目的两个，HIV-1感染的细胞 因为它们诱导了渗透性变化和凋亡的产生 体外BBB系统。特别是，缺乏每个特定病毒的突变病毒 调节和结构基因也将以互补的方式使用 感染CD4+ T细胞和原发性巨噬细胞后，以对 BBB。最后，组合抗返回病毒化学疗法对HIV的影响 将分析与BBB的1感染细胞相互作用。这将是一个 改变化学治疗方案的模型SVSTEM，改变 感染细胞引起的渗透性和编程细胞死亡的改变 在人类BBB中。在哈特时代，这些将是 了解HAART方案组合的变化如何影响HIV-I：BBB 相互作用也将在系统中以耐药性病毒进行建模 感染的外周血细胞中的菌株。在这些相互关联的研究中， 有人提出，包括HIV-I向量在内的一系列新技术 系统和体外BBB系统，以确定确定 HIV-I蛋白的精确作用，因此涉及的分子机制， 在诱导BBB的改变时。