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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：
9532834
负责人：
GANJAM V KALPANA
金额：
$ 37.27万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
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 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9532834
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 Count Cell Culture Techniques Cell Line Cell model Cells Cocaine 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：这项研究的长期目标是研究HIV-1病毒的储备库，并坚持对中枢神经系统的研究和理解。非法药物的主要作用是建立和重新激活潜在的病毒储备库。他们的持续作用是潜伏感染的病毒细胞。此外，CNS病毒对根除HIV-1病毒构成了一个主要的全球障碍。目前的全球战略旨在进一步消除潜在的病毒宿主，包括。一种针对仅占全球总储存库仅1%的外周血液量的治疗方法和方法，是一种针对外周血的治疗方法。目前还不确定是否有可能制定类似的战略，以实现对感染艾滋病毒的CNS细胞的有效根除。未知。除了缺乏对中枢神经系统和HIV+T细胞潜伏期的初步了解外，还缺乏潜伏期的潜在影响。与药物相反，非法毒品在全球艾滋病毒感染人群中很常见，这构成了一个进一步的复杂性。许多人。众所周知，非法药物可以刺激HIV-1病毒的复制。自那以来，目前的检测方法只能测量患者的外周血细胞。 HIV病毒储存库不适用于将固体组织移植到细胞中，而不是复制能力较差的细胞，如巨噬细胞和小胶质细胞。在大脑中发现了多种星形胶质细胞。我们已经开发出一种新的、单细胞-单分子的星形胶质细胞，即Multiplex。免疫荧光技术(IF)和基于鱼类的DNA检测技术(Smira)能够检测出HIV-1阳性的细胞。复制。由于使用了自动化，大量的细胞无法被扫描。在这项新的提议中，我们将不会使用这种技术。一种新的方法是首先对细胞系模型中的潜伏期进行量化和表征，然后再研究大脑中潜在的潜伏期。衍生自小胶质细胞和星形胶质细胞的研究描绘了三种主要药物(甲基苯丙胺、可卡因)的治疗效果。和大麻)依赖于潜伏细胞形成的最高效率，以及潜伏的脱毒细胞的复活率。建立跨越血脑屏障(BBB)的潜伏期。提出了三个新的目标。在第一个目标中，我们将继续研究。这些非法药物的主要作用依赖于通过建立体外培养和体外培养来实现对潜伏感染的CNS来源的干细胞的重新激活。体内有潜伏期的模型。首先，我们将不会使用永生化的人类单核细胞系和小胶质细胞系来进行研究。再激活的动力学过程以及冰毒、可卡因和大麻对使用Smira后的再激活的影响。在这之后，我们还将进行类似的研究，使用一种新的原代中枢神经系统细胞。在目标2中，我们将继续研究非法药物的主要作用。在整个BBB的中枢神经系统细胞中建立了两个HIV储存库。在使用Smira技术检测到HIV-1+细胞之后，我们将继续努力。确定药物滥用、抗逆转录病毒药物和LRAs的相对有效范围，以确定其潜伏期。在整个BBB。在第三个目标中，我们将研究非法药物对感染艾滋病毒的人的中枢神经系统和艾滋病毒宿主的影响。采用脑部尸检样本，来自名HIV+携带者或无非法吸毒史或无非法吸毒史的人(摘自报告)。曼哈顿脑银行(NNTC)，我们将不会表征小胶质细胞和星形胶质细胞与潜伏的神经细胞的相对频率。在使用HIV-1病毒的水库中，HIV-1病毒正在积极复制病毒，而不是使用Smira病毒和最先进的病毒。高速/分辨率的整个幻灯片扫描仪，如PerkinElmer和Pannorama的250和Flash II。我们将继续研究其效果。药物滥用的发生率取决于药物再激活的比率。这项拟议的药物研究计划将为进一步研究提供有价值的研究工具。建立和重新激活中枢神经系统的潜伏期，以应对非法药物，并提供了一个更好的药物筛选和分析方法。为了更好地根除这些潜在的癌症细胞，我们需要在不久的将来。