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

点击翻译按钮获取中文摘要

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.