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

描述(由申请人提供)： HIV-1感染某些人的中枢神经系统可导致严重的认知和运动功能障碍，称为HIV-1诱发脑病。此外，包括乙醇(Etoh)摄入在内的许多辅助因素可能会改变HIV-1通过血液：脑屏障(EBB)进入中枢神经系统的渗透。在HAART时代，了解这些与HIV-1渗透到中枢神经系统储存库有关的辅助因素至关重要。我们的实验室最近开发了一种完全由人中枢神经系统细胞类型组成的体外血脑屏障系统。我们已经证明，各种病毒蛋白，特别是HIV-1 vpr，对中枢神经系统细胞具有深刻的影响，导致细胞凋亡。利用互补的方法，乙醇对HIV-1感染血脑屏障和病毒对中枢神经系统的渗透的影响将被整体或特定解剖-1病毒。推测乙醇可能与HIV-1蛋白一起诱导血脑屏障的破坏，可能是通过细胞凋亡，以及增加构成血脑屏障的细胞，包括微血管内皮细胞和星形胶质细胞的感染。在具体目标1中，将探讨乙醇对血脑屏障中关键的中枢神经系统细胞在使用和不使用抗逆转录病毒药物的情况下感染HIV-1的细胞和分子的影响。将利用无细胞和细胞相关(例如，受感染的巨噬细胞)形式的特定病毒株。在特定的目标U，乙醇与选定的重组HIV-1蛋白对破坏这一屏障的影响将被机械地分析。将使用互补方法(包括跨内皮细胞电阻(TEER)和Zoi紧密连接蛋白改变)来评估乙醇对MV-1蛋白的影响对血脑屏障的潜在破坏作用(对于S.A.1)。乙醇和精选的重组病毒蛋白对特定BBB细胞凋亡的影响将通过TUNEL、Annexin V检测和离子通道测量来分析。这些影响也将使用缺乏特定病毒调控和结构基因的突变病毒来探索，包括vpr、Nef、gpl2O和Tat。在细胞内表达这些特定蛋白的HIV-I载体系统将与乙醇一起使用。最后，将在无水乙醇治疗的背景下分析血脑屏障细胞上特异性趋化因子受体的变化，特别是中枢神经系统特异性趋化因子受体APJ。将分析与相关同源趋化因子和gpl2O结合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，有人建议将一系列新技术结合起来，以确定乙醇在诱导HW-L I感染这些细胞的血脑屏障改变中的确切作用。

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.