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Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis

滥用药物对 CNS HIV-1 储存库和神经发病机制的影响

基本信息

批准号：
9977143
负责人：
GANJAM V KALPANA
金额：
$ 62.57万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9977143
关键词：
AIDS/HIV problem Anatomy Anti-Retroviral Agents Astrocytes Automation Autopsy Biological Assay Biological Models Blood - brain barrier anatomy Brain CD4 Positive T Lymphocytes Cannabis Cell Culture Techniques Cell Line Cell model Cells Cocaine Consumption Development Drug Screening Drug abuse Frequencies Future Genetic Transcription Goals HIV HIV-1 Human Illicit Drugs Immunofluorescence Immunologic In Vitro Individual Infection Kinetics Knowledge Measures Methamphetamine Methods Microglia Modeling National NeuroAids Tissue Consortium Nature Neuraxis Neuropathogenesis Patients Population Proteins Proviruses RNA Reaction Recording of previous events Resolution Rest Role Sampling Scanning Shock Slide Solid Speed Testing Time Tissues Viral Viral Load result Virus base cell type drug of abuse illicit drug use macrophage memory CD4 T lymphocyte methamphetamine effect monocyte novel patient population peripheral blood prevent reactivation from latency response single molecule specific biomarkers tool

项目摘要

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). Three Aims are proposed. In Aim 1, we will study the role of illicit drugs on the reactivation of latently infected CNS-derived cells by establishing in vitro and ex vivo models of latency. First, we will employ immortalized human monocyte and microglial cell lines to study the kinetics of reactivation and the effect of METH, Cocaine and cannabis on the reactivation using SMIRA. This will be followed by similar studies using a primary CNS cells. In Aim 2, we will study the role of illicit drugs on the establishment of HIV reservoirs in CNS cells across BBB. Using SMIRA to detect HIV-1+ cells, we will determine the relative effect of drugs of abuse, antiretrovirals and the addition of LRAs to establish latency across BBB. In Aim 3, we will study the effect of illicit drugs on CNS HIV reservoirs in HIV-infected individuals. Employing brain autopsy samples from HIV+ individuals with or without illicit drug use history (from the Manhattan Brain Bank (NNTC), we will characterize the relative frequency of microglial and astrocyte latent cell reservoirs in which HIV-1 is actively replicating vs. those that are latent, using SMIRA and state-of-the-art, high-speed/resolution whole slide scanner, the PerkinElmer Pannoramic 250 Flash II. We will study the effect of drug abuse on the rates of reactivation. The proposed studies will provide valuable tools to study establishment and reactivation of CNS latency in response to illicit drugs and provides a drug screening assay to eradicate these latent cells in the future.

项目总结