Effect of Drugs of Abuse on CNS HIV-1 Reservoirs and Neuropathogenesis

项目摘要 本申请的长期目标是研究持续存在于CNS中的HIV-1储库，并了解 非法药物在建立和重新激活潜在储存库方面的作用。潜伏感染细胞的持续存在 中枢神经系统中的HIV-1感染是HIV-1根除的主要障碍。目前消除潜在水库的战略包括 这是一种“休克和杀死”疗法，针对的是外周血，而外周血仅占总储存量的1%。 目前，是否有可能设想类似的根除HIV感染的CNS细胞的策略， 未知除了缺乏关于CNS HIV+细胞中潜伏期的知识以及潜伏期的影响外， 逆转剂、艾滋病毒感染人群中常见的非法药物构成了进一步的复杂性。许多 已知非法药物会刺激HIV-1复制。由于目前测量外周血的方法 HIV储库不适用于实体组织或复制不良的细胞，如巨噬细胞、小胶质细胞 和大脑中发现的星形胶质细胞。我们开发了一种新的，单细胞单分子，多重， 免疫荧光（IF）和基于RNA FISH的检测（SMIRA）检测HIV-1活跃的细胞 复制使用自动化，可以扫描大量细胞。在这个提案中，我们将采用这本小说 方法首先定量和表征细胞系模型中的潜伏期，然后导出大脑中的潜伏库 小胶质细胞和星形胶质细胞，并描绘了三种药物滥用（甲基苯丙胺，可卡因和 大麻）对潜伏细胞的形成效率、潜伏细胞的再活化速率和建立 血脑屏障（BBB）的潜伏期。为了夏季本科课程的目的，我们将 研究非法药物对潜在感染的CNS衍生细胞系的重新激活的作用， 潜伏期的体外模型。我们将使用永生化的人单核细胞和小胶质细胞系来研究 使用SMIRA的再活化动力学和METH的影响。

PROJECT SUMMARY The long term goal of this application is to study HIV-1 reservoirs that persist in CNS and to understand the role of illicit drugs in establishing and reactivating the latent reservoirs. Persistence of latently infected cells in CNS poses a major barrier for HIV-1 eradication. Current strategies to eliminate the latent reservoirs include a “shock and kill” therapy and is aimed at peripheral blood, which constitutes only 1% of the total reservoirs. Whether it is possible to envision similar strategies for the eradication of HIV-infected CNS cells is currently unknown. In addition to the lack of knowledge about latency in CNS HIV+ cells and the effects of latency reversing agents, illicit drugs common within the HIV-infected populations constitute a further complexity. Many illicit drugs are known to stimulate HIV-1 replication. Since the current method to measure the peripheral blood HIV reservoir is not applicable to solid tissues or to cells that replicate poorly such as macrophages, microglia and astrocytes found in the brain. We have developed a novel, Single cell-single molecule, Multiplex, Immunofluorescence (IF) and RNA FISH-based Assay (SMIRA) to detect cells in which HIV-1 is actively replicating. Using automation, a large number of cells can be scanned. In this proposal, we will employ this novel method to first quantitate and characterize latency in cell line models, then the latent reservoirs in brain derived microglia and astrocytes and delineate the effect of three drugs of abuse (methamphetamine, cocaine and cannabis) on the efficiency of formation of latent cells, the rate of reactivation of latent cells, and establishment of latency across blood-brain-barrier (BBB). For the purpose of the summer undergraduate program, we will be studying the role of illicit drugs on the reactivation of latently infected CNS-derived cell lines by establishing in vitro models of latency. We will employ immortalized human monocyte and microglial cell lines to study the kinetics of reactivation and the effect of METH using SMIRA.