Alcohol's Effects on HIV-1 and the Blood: Brain Barrier

描述（由申请人提供）： HIV-1感染某些个体的CNS可能导致显著的认知和运动功能障碍，称为HIV-1诱导的脑病。此外，包括乙醇（Etoh）摄入在内的许多辅因子可能会改变HIV-1通过血脑屏障（EBB）进入CNS的渗透。了解这些与HIV-1进入CNS储库的渗透有关的辅因子在HAART时代至关重要。我们的实验室最近开发了一种完全由人CNS细胞类型组成的体外BBB系统。我们已经证明，各种病毒蛋白，特别是HIV-1 Vpr，对CNS细胞有深远的影响，导致细胞凋亡。利用互补方法，Etoh对BBB的HIV-1感染和CNS的病毒渗透的影响将是全部或特定解剖的病毒 . 据推测，Etoh可能对HIV HIV-1蛋白具有显著影响，可能通过细胞凋亡诱导BBB破坏，以及增加BBB构成细胞（包括微血管内皮细胞（MVECS）和星形胶质细胞）的感染。在具体目标1中，将探讨Etoh对BBB中关键CNS细胞的HIV-1感染（有或无抗逆转录病毒药物）的细胞和分子效应。将使用无细胞和细胞相关（例如，感染的巨噬细胞）形式的特定病毒株。在Specific Aim U中，将从机理上分析Etoh与选定的重组HIV-1蛋白对破坏该屏障的影响。将使用补充方法（包括跨内皮电阻（TEER）和ZOI紧密连接蛋白改变）评估Etoh对MV-1蛋白的作用对BBB的潜在破坏（与S.A.1相同）。Etoh与选择的重组病毒蛋白对特定BBB细胞凋亡的影响将通过TUNEL、膜联蛋白V测定和离子通道测量进行分析。还将使用缺乏特异性病毒调节和结构基因（包括Vpr、Nef、gpl 2 O和达特）的突变病毒来探测这些效应。细胞内表达这些特异性蛋白质的HIV-1载体系统将与Etoh一起使用。最后，将在Etoh的设置下分析BBB细胞上特异性趋化因子受体的改变，特别是CNS特异性趋化因子受体APJ。将分析结合相关同源趋化因子和gpl 20后对趋化因子受体表达、下调和细胞内钙通量的影响。有了这些相互关联的具体目标，提出了一系列新技术将被组合以确定Etoh对这些细胞的HW-1 I感染诱导BBB改变的精确作用。

DESCRIPTION (provided by applicant): Infection of the CNS of certain individuals by HIV-1 may lead to significant cognitive and motor dysfunction, entitled HIV-1-induced encephalopathy. In addition, a number of cofactors including ethanol (Etoh) ingestion may alter penetration of HIV-1 into the CNS through the blood:brain barrier (EBB). Under-standing these cofactors involved with penetration of HIV-1 into the CNS reservoir is of critical importance now in the era of HAART. Our laboratories have recently developed an in vitro BBB system comprised totally of human CNS cell-types. We have demonstrated that various viral proteins, especially HIV-1 Vpr, has profound effects on CNS-based cells leading to apoptotic death. Utilizing complementary approaches, the effects of Etoh on HIV-1 infection of the BBB and viral penetration of the CNS will be -1 virus in toto or specific dissected . It is hypothesized that Etoh may have significant effects with the HIV HIV-1 proteins in inducing the disruption of the BBB, possibly through apoptosis, as well as increasing infection of BBB-constituting cells, including microvascular endothelial cells (MVECS) and astrocytes. In Specific Aim 1, the cellular and molecular effects of Etoh on HIV-1 infection, with and without anti-retroviral agents, of critical CNS cells in the BBB will be approached. Specific viral strains in cell-free and cell- associated (e.g, infected macrophages) forms will be utilized. In Specific Aim U, effects of Etoh with selected recombinant HIV-1 proteins on disruption of this barrier will be mechanistically analyzed. Potential disruption of the BBB by Etoh's effects with MV-1 proteins will be assessed (as for S.A.1) using complementary methods, including trans-endothelial electrical resistance (TEER) and ZOI tight junction protein alterations. Etoh's effects with select recombinant Viral proteins on apoptosis of specific BBB cells, will be analyzed via TUNEL, Annexin V assays and ion channel measurements. These effects will also be probed using mutant viruses lacking specific viral regulatory and structural genes, including Vpr, Nef, gpl2O and Tat. HIV- I vector systems to express these specific proteins intracellularly will be utilized with Etoh. Finally, alterations of specific chemokine receptors on BBB cells, especially the CNS-specific chemokine receptor APJ, will be analyzed in the setting of Etoh. Effects on chemokine receptor expression, down- regulation, and intracellular calcium fluxes after binding to relevant cognate chemokines and gpl2O will be analyzed. With these inter-related specific aims, it is proposed that a series of new technologies will be combined towards determining the precise effects of Etoh on inducing alterations in the BBB with HW-l I infection of these cells.