OMICs to define impact of cocaine on immunity and HIV persistence in treated HIV infection

物质滥用与更严重的艾滋病毒病程有关，表现为免疫力较差 接受CART治疗的受试者的重建和增加的共病发生率。这些机制是 与这些糟糕的临床结果有关的人没有被很好地理解，很可能是糟糕的后果- 研究队列中的受控混杂因素，缺乏对药物水平的定量评估，以及不完整 测量这些受试者的免疫功能。我们已经使用系统生物学来识别 免疫介导的保护。我们已经提供了强有力的证据表明，支持和反对 即使在接受CART治疗的受试者中，细胞内的炎症途径也受到干扰。这种平衡的破坏会导致 接受艾滋病毒感染的CART治疗的受试者免疫重建差，潜伏期增加的频率增加 被感染的细胞。我们已经通过我们在FIU的合作者接触到了一批特征良好的CART治疗患者 受试者也是可卡因吸毒者，并受到良好的可卡因使用监测。可卡因使用情况如图所示 根据我们的初步结果解释了促炎和抗炎之间的平衡进一步被扰乱 效应分子和细胞；这导致这些受试者的免疫重建水平较低 潜在地导致了更高的艾滋病毒持久性。我们将使用最先进的方法来实现我们的主要目标： 我们将描述导致免疫功能障碍和HIV持续存在的分子机制 在吸食可卡因的受试者中观察到。在特定目标1中，我们将使用多色流式细胞术来 描述促炎和抗炎细胞的分布和功能，包括Treg亚群和 确定它们与CART后T细胞动态平衡丧失和免疫重建不良的关系 不同记忆T细胞亚群的转录图谱将导致分子通路的识别 在可卡因使用者中，这与破坏CD4T细胞稳态有关；这些机械性的发现将是 通过流式细胞仪和Fluidigm BioMark TM在单细胞水平上进行验证。我们的初步结果 表明在可卡因使用者中，CD8 T细胞表达与动态平衡失调相关的标记。我们会 确定可卡因对HIV特异性CD8 T细胞反应的影响，并评估这些影响是否与 炎症环境的失衡或者是由于缺乏CD4T细胞的帮助。《特定目标2》 我们将评估和界定与可卡因对艾滋病毒持久性的影响有关的机制。我们有 开发了两种新的分析方法，将为艾滋病毒宿主的规模和 可卡因对艾滋病毒持久性的影响。Tilda将允许我们监测可诱导的HIV病毒的频率 并量化评估可卡因对建立和维持 HIV在体外感染人群中的蓄积物。劳拉将允许我们在体外确定可卡因及其 代谢产物可以影响HIV在不同记忆T细胞亚群中潜伏期的建立。

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, and incomplete measurement of immune function in these subjects. We have used systems biology to identify mechanisms of immune mediated protection. We have provided strong evidence that the balance between pro and anti- inflammatory pathways in cells is disrupted even in cART treated subjects. Disruption of this balance leads to poor immune reconstitution in HIV infected cART treated subjects and to increased frequencies of latently infected cells. We have access through our collaborators at FIU to a well-characterized cohort of cART treated subjects who are also cocaine users and who are well monitored for their cocaine use. Cocaine use as shown by our preliminary results account for further perturbation of the balance between pro and anti inflammatory effector molecules and cells; this results in lower levels of immune reconstitution in these subjects and potentially to higher HIV persistence. We will use state of the art approaches to address our major objective: we will delineate the molecular mechanisms that result in the immune dysfunction and HIV persistence observed in cocaine users cART treated subjects. In specific aim 1 we will use polychromatic flow cytometry to characterize the distribution and function of pro and anti-inflammatory cells including Treg subsets and determine their association to loss of T cell homeostasis and poor immune reconstitution post cART Transcriptional profiling of different memory T cell subsets will lead to the identification of molecular pathways in cocaine users which are associated to disrupted CD4 T cell homeostasis; these mechanistic findings will be validated at the single cell level by flow cytometry and by Fluidigm Biomark TM . Our preliminary results indicate that in cocaine users CD8 T cells express markers associated to deregulated homeostasis. We will determine the impact of cocaine on HIV specific CD8 T cell responses and assess if these are associated to the imbalances in the inflammatory environment or they are due to the lack of CD4 T cell help. In specific aim 2 we will assess and define mechanisms associated to the impact of cocaine on HIV persistence. We have developed two new assays that will provide critical insights to the magnitude of the HIV reservoir and to the impact of cocaine on HIV persistence. TILDA will allow us to monitor the frequencies of inducible HIV viruses and to provide a quantitative assessment of the impact of cocaine on establishment and maintenance of the HIV reservoir ex vivo in infected subjects. LARA will allow us to determine in vitro that cocaine and its metabolites can impact the establishment of HIV latency in different memory T cell subsets.