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

描述（改编自申请人的摘要）：HIV-1可以导致一系列的 中枢神经系统（CNS）的破坏性临床状况 以认知和运动功能障碍为特征的疾病。的 这种HIV-1诱导的痴呆综合症的分子机制仍然存在， 神秘的此外，在高效抗逆转录病毒治疗的时代， 中枢神经系统也可以作为HIV-1的储存库或避难所 在联合治疗期间。我们的实验室最近开发了一种 完全由人CNS组成的体外血：脑屏障（BBB）系统 cell-tvpes，它成功地模拟了血脑屏障的细胞相互作用， vivo.此外，我们最近已经证明， Vpr蛋白在诱导人神经细胞凋亡中具有重要作用 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补充技术， 将分析BBB上选定的慢病毒蛋白。据推测 Vpr和其他可能的特异性病毒蛋白，包括达特、Nef和 包膜蛋白gp I 2 O已被证明可诱导细胞凋亡， 其他人类细胞，可能对诱导细胞程序性死亡有影响， BBB构成细胞，包括微血管内皮细胞（MVEC）和 星形胶质细胞在具体的目标一，这一假设将评估使用在 具有细胞凋亡和BBB通透性变化读数的体外BBB系统 包括TUNEL、膜联蛋白V和Caspase-8测定，此外， 跨内皮电阻（TEER）和ZO] ti，-，ht-连接蛋白 改变。两种游离重组]抗病毒蛋白，除了突变体 将在该系统中分析缺乏感兴趣基因的病毒。在 此外，利用一系列基于HIV-I的慢病毒载体系统， 选择的病毒蛋白将在相关的BBB细胞类型中表达。在 互补的具体目标二，HIV-1感染的细胞也将进行分析 因为它们诱导细胞的通透性变化和细胞凋亡的产生， 体外血脑屏障系统特别是，缺乏每种特定病毒的突变病毒， 调节和结构基因也将用于互补方法， 感染CD 4 + T细胞和原代巨噬细胞后， BBB.最后，联合抗逆转录病毒化疗对艾滋病毒的影响- 1-将分析感染的细胞与BBB的相互作用。这将是一 不同化疗方案的模型系统，在改变 感染细胞引起的通透性改变和程序性细胞死亡 在人的血脑屏障中。在HAART时代，这些将是关键的研究， 了解HAART方案组合的变化如何影响HIV-I：BBB 相互作用，这也将在系统中建模与耐药病毒 感染的外周血细胞中的菌株。在这些相互关联的研究中， 提出了一系列新技术，包括HIV-1载体， 系统和体外血脑屏障系统，被用来确定 HIV-I蛋白的精确作用，以及相关的分子机制， 诱导血脑屏障的改变。

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.