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

项目摘要 药物滥用与艾滋病毒疾病的更严重的过程有关，如穷人所示。 在cART治疗的受试者中的免疫重建和共病率增加。的 与这些不良临床结局相关的机制尚未得到很好的理解， 研究队列中控制不佳的混杂因素的结果，缺乏对 药物水平、免疫功能测量不完整以及对 导致艾滋病毒持续存在。我们已经使用系统生物学，并表明，正反之间的平衡， 炎症途径是HIV持续存在的主要驱动因素。这种平衡的破坏导致穷人 在HIV感染的cART治疗的受试者中的免疫重建和潜伏性免疫缺陷的频率增加 被感染的细胞我们的初步结果表明，CO2的使用解释了 促炎和抗炎效应分子和细胞之间的平衡，导致较低水平的 这些受试者中的免疫重建和T细胞稳态的扰动， 其他队列中的艾滋病毒持续存在。我们召集了一个多学科小组， 这些障碍，并将使用公正的OMIC方法来解决我们的主要目标， 是描述可卡因引发的导致HIV持续存在的分子机制。具体目标1 我们将使用三种独立的最先进的检测方法来定量HIV的大小和细胞定位， 我们将使用转录组学对特定的细胞亚群和其他高密度读数，包括流动 细胞计数和系统血清学，以确定与HIV增加相关的效应细胞和分子 水库规模在具体的目标2中，我们将应用一种全球性和有针对性的蛋白质组学方法来验证 可卡因引发的分子途径导致艾滋病病毒持久性增加。代谢组学将用于 确定代谢物的作用，特别是短链脂肪酸和胆固醇对艾滋病毒的持久性。在 具体目标3：我们将验证我们已经通过基因表达和蛋白质组学鉴定的机制。 目标1和目标2中显示的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潜伏期的原代细胞测定，首次允许测量多个T细胞中的潜伏期再激活， 细胞亚群。LARA将使我们能够在体外证实可卡因及其代谢物可以动员上述 识别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 子集和对延迟再激活剂的响应。

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.