An unbiased OMICs approach to identify mechanisms of Cocaine regulation of the HIV reservoir

项目摘要 物质滥用与更严重的艾滋病毒病程有关，如穷人所示 接受CART治疗的受试者的免疫重建和合并症发生率增加。这个 导致这些不良临床结果的机制还不是很清楚，很可能是 研究队列中混杂因素控制不佳的后果，缺乏对 药物水平，对免疫功能的测量不完整，对 导致艾滋病毒持久化。我们使用了系统生物学，并证明了支持和反对之间的平衡 炎症途径是S艾滋病病毒持续存在的主要驱动因素。破坏这种平衡会导致贫穷 HIV感染的CART治疗对象的免疫重建和潜伏期频率的增加 被感染的细胞。我们的初步结果显示，可卡因的使用解释了进一步的干扰 促炎和抗炎效应分子和细胞之间的平衡，导致较低水平的 这些受试者的免疫重建和T细胞稳态的紊乱导致了 艾滋病毒在其他队列中的持久性。我们已经组建了一个多学科小组，将使我们能够克服 这些障碍并将使用公正的组学方法来解决我们的主要目标，即 是描绘可卡因触发导致艾滋病毒持续存在的分子机制。在特定目标中1 我们将使用三种独立的最先进的分析方法来量化艾滋病毒的大小和细胞定位。 储存库；我们将在特定细胞亚群和包括Flow在内的其他高密度读数上使用转录转录 细胞学和系统血清学以确定与HIV增加相关的效应细胞和分子 水库大小。在特定的目标2中，我们将应用一种全局的、有针对性的蛋白质组学方法来验证 可卡因引发的分子通路增加了艾滋病毒的持久性。代谢组学将被用于 确定代谢物，特别是短链脂肪酸和胆固醇对艾滋病毒持久性的作用。在……里面 具体目标3我们将验证我们已有的通过基因表达和蛋白质组学确定的机制 在目标1和目标2中显示与可卡因对艾滋病毒持久性的影响有关；我们将使用一种新的 HIV潜伏期的原代细胞分析首次允许测量多个T细胞的潜伏期重新激活 单元格子集。LARA将允许我们在体外证实可卡因及其代谢物可以动员上述 确定途径并监测它们对不同记忆T细胞中HIV潜伏期建立的影响 子集和对潜伏期重新激活剂的响应。

PROJECT ABSTRACT Substance abuse is associated with a more severe course of HIV disease as shown by the poor immune reconstitution in cART treated subjects and an increased rate of co-morbidities. The mechanisms that are involved in these poor clinical outcomes are not well understood most likely a consequence of poorly- controlled confounding factors in study cohorts, lack of quantitative assessment of drug levels,, incomplete measurement of immune function and poor understanding of the mechanisms that lead to HIV persistence. We have used systems biology a n d shown that t h e balance between pro and anti- inflammatory pathways a s major drivers of HIV persistence. Disruption of this balance leads to poor immune reconstitution in HIV infected cART treated subjects and to increased frequencies of latently infected cells. Cocaine use as shown by our preliminary results accounts for further perturbations of the balance between pro and anti inflammatory effector molecules and cells which results in lower levels of immune reconstitution in these subjects and perturbations in T cell homeostasis which have led to heightened HIV persistence in other cohorts. We have assembled a multidisciplinary group that will allow us to overcome these aforementioned hurdles and will use unbiased OMICs approaches to address our major objective which is to delineate the molecular mechanisms triggered by cocaine that lead to HIV persistence. In specific aim 1 we will use three independent state of the art assays to quantity the size and cellular localization of the HIV reservoir; we will use transcriptomics on specific cell subsets and other high density readouts including flow cytometry and system serology to identify the effector cells and molecules that are associated to increased HIV reservoir size. In specific aim 2 we will apply a global and targeted proteomics approach to validate molecular pathways triggered by cocaine that lead increased HIV persistence. Metabolomics will be used to identify the role of metabolites and particularly short chain fatty acids and cholesterol on HIV persistence. In Specific Aim 3 we will validate the mechanisms identified by gene expression and proteomics which we have shown in aims 1 and 2 to be associated to the impact of cocaine on HIV persistence; we will use a novel primary cell assay of HIV latency which allows for the first time to measure latency reactivation in multiple T cells subsets. LARA will allow us to confirm in vitro that cocaine and its metabolites can mobilize the above identified pathways and monitor their impact on the establishment of HIV latency in different memory T cell subsets and on the response to Latency Reactivating Agents.