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.

项目摘要该应用程序的长期目标是研究CNS中持续存在并了解的HIV-1储层非法药物在建立和重新激活潜在储层中的作用。持续的潜在感染细胞中枢神经系统构成了消除HIV-1的主要障碍。消除包括潜在水库的当前策略一种“冲击和杀伤”疗法，目的是外围血，仅占总储层的1％。目前，是否有可能设想消除HIV感染的CNS细胞的类似策略未知。除了缺乏有关中枢神经系统HIV+细胞潜伏期的知识以及潜伏期的影响逆转药物，在感染HIV的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。由于当前测量外周血的方法 HIV储层不适用于固体组织或复制较差的细胞，例如巨噬细胞，小胶质细胞和大脑中发现的星形胶质细胞。我们已经开发了一种新颖的单细胞单个分子，多重分子，免疫荧光（IF）和RNA基于RNA的基于FISH的测定（SMIRA），以检测HIV-1的细胞。复制。使用自动化，可以扫描大量单元。在此提案中，我们将采用此事首先定量和表征细胞系模型的潜伏期的新方法，然后是大脑中的潜在储层衍生的小胶质细胞和星形胶质细胞，并描绘了三种滥用药物的作用（甲基苯丙胺，可卡因和大麻）关于潜在细胞形成的效率，潜在细胞的重新激活速率以及建立跨脑障碍（BBB）的潜伏期。提出了三个目标。在AIM 1中，我们将学习非法药物通过在体外和EX中建立潜在感染的CNS衍生细胞重新激活的作用延迟的体内模型。首先，我们将使用永生的人类单核细胞和小胶质细胞系进行研究使用Smira的重新激活动力学以及甲基，可卡因和大麻对重新激活的影响。随后将使用原代CNS细胞进行类似的研究。在AIM 2中，我们将研究非法药物的作用关于在BBB跨CNS细胞中建立HIV储存剂。使用Smira检测HIV-1+细胞，我们将确定滥用药物，抗逆转录病毒药物的相对效果以及增加LRA以建立潜伏期跨BBB。在AIM 3中，我们将研究非法药物对艾滋病毒感染者中枢神经系统HIV水库的影响。采用来自艾滋病毒+患者有或没有非法吸毒史的艾滋病毒+个体的脑尸检样本（来自曼哈顿脑库（NNTC），我们将表征小胶质细胞和星形胶质细胞潜在细胞的相对频率 HIV-1正在积极复制与潜在的水库，使用Smira和最先进的水库高速/分辨率全幻灯片扫描仪，Perkinelmer Pannoramic 250 Flash II。我们将研究效果重新激活率的药物滥用。拟议的研究将为研究提供有价值的工具响应非法药物的CNS潜伏期建立和重新激活并提供药物筛查测定法将来消除这些潜在细胞。