Mitochondrial DNA content in blood extracellular vesicles as a biomarker of neuronal mitochondrial DNA damage induced by cigarette smoking in virally suppressed, HIV-positive African Americans

项目摘要/摘要 抗逆转录病毒疗法(ART)极大地降低了机会性感染的发生率，改善了生活 艾滋病毒感染者的预期。HIV相关性神经认知障碍(HAND)与 抗逆转录病毒治疗的使用率仍然很高(15%-50%)。与此同时，手部的诊断往往是具有挑战性的。 此外，目前还没有有效的替代标记和明确的手部辅助治疗方法。自.以来 艾滋病病毒感染者的吸烟率比一般人高三到四倍。 感染艾滋病毒的非裔美国人(AA)占美国艾滋病毒携带者的42% 在美国，迫切需要解决吸烟对携带艾滋病毒的AA患者神经元功能障碍的影响。 线粒体功能障碍是各种神经疾病的标志。线粒体损伤和 线粒体DNA(MtDNA)含量降低通常发生在神经元变性之前。我们的长期目标 是开发一种临床适用的非侵入性测试来监测吸烟对神经细胞的影响 HIV阳性个体的线粒体DNA含量。这项测试将允许临床医生监测神经元mtdna的变化。 对吸烟者艾滋病病毒感染后的ART含量进行分析，以利于早期医疗干预。我们的整体 目的探讨吸烟对神经元胞外线粒体DNA含量的影响。 根据吸烟和艾滋病毒状况，从AA的外周血液中分离出水泡(NEV)。我们的中央 假说是吸烟加剧了病毒抑制的HIV阳性患者的神经元线粒体DNA损伤 AAS，导致外周血新能源汽车mtDNA释放增加。我们的理论基础是线粒体DNA含量 NEVS是一种新的非侵入性生物标志物，可用于早期检测神经元中的mtDNA损伤。我们的特定 目的是：1)量化从AA非吸烟者的外周血液中分离出的新能源汽车中的线粒体DNA含量， 包括病毒抑制的HIV阳性和HIV阴性的受试者；以及2)比较 从携带mtDNA的HIV阳性AA吸烟者外周血中分离出新城疫病毒 从病毒抑制的艾滋病毒阳性非吸烟者以及艾滋病毒阴性吸烟者中分离出的新能源汽车的内容 和不吸烟的人。在目标1中，我们将测量从日本人外周血中分离出来的新能源汽车的线粒体DNA含量 HIV阴性和HIV阳性非吸烟者囊泡内DNA实时定量聚合酶链式反应(QPCR)研究 从新能源汽车中提取。在目标2中，我们将分析新能源汽车mtDNA含量的定量变化，这些变化可能 反映与吸烟和ART相关的线粒体DNA损伤。我们的建议是创新的，因为它 提出用外周血中线粒体DNA含量作为监测新能源汽车的新生物标志物 神经元线粒体DNA损伤。我们从这项拟议的研究中得到的发现将具有重要意义，他们将建立线粒体DNA 新能源汽车中的含量作为一种新的、非侵入性的生物标志物用于实时监测神经元功能障碍 与抗逆转录病毒治疗和吸烟者的艾滋病毒阳性个体的线粒体DNA损伤有关。

Project Summary/Abstract Antiretroviral therapy (ART) drastically reduces the incidence of opportunistic infections and improves life expectancy among HIV-infected individuals. HIV-associated neurocognitive disorders (HAND) related to the use of ART remain highly prevalent (15–50%). Meanwhile, the diagnosis of HAND is often challenging. Moreover, there are no validated surrogate markers and definitive adjunctive treatment for HAND. Since the prevalence of smoking is three- to four-fold higher among HIV-infected individuals than in the general population, and that HIV-infected African Americans (AAs) represent 42% of individuals living with HIV in the U.S., there is an urgent need to address the effect of smoking on neuronal dysfunction in AAs living with HIV. Mitochondrial dysfunction is a hallmark of various neurological disorders. Mitochondrial damage and mitochondrial DNA (mtDNA) content reduction often occur prior to neuronal degeneration. Our long-term goal is to develop a clinically applicable, non-invasive test to monitor the effects of cigarette smoking on neuronal mtDNA content in HIV-positive individuals. This test will allow clinicians to monitor changes in neuronal mtDNA content in HIV-infected cigarette smokers on ART in order to facilitate early medical intervention. Our overall objective is to determine the effect of cigarette smoking on mtDNA content in neuron-derived extracellular vesicles (NEVs) isolated from the peripheral blood of AAs, according to smoking and HIV status. Our central hypothesis is that cigarette smoking exacerbates neuronal mtDNA damage in virally suppressed HIV-positive AAs, leading to increased release of mtDNA in peripheral blood NEVs. Our rationale is that mtDNA content in NEVs is a novel and non-invasive biomarker for early detection of mtDNA damage in neurons. Our specific aims are: 1) To quantify mtDNA content in NEVs isolated from the peripheral blood of AA non-smokers, including virally suppressed HIV-positive and HIV-negative subjects; and 2) To compare the mtDNA content in NEVs isolated from the peripheral blood of virally suppressed HIV-positive AA smokers with the mtDNA content in NEVs isolated from virally suppressed HIV-positive non-smokers, as well as HIV-negative smokers and non-smokers. In Aim #1, we will measure mtDNA content in NEVs isolated from the peripheral blood of HIV-negative and HIV-positive non-smokers by real-time quantitative PCR (qPCR) using intravesicular DNA extracted from NEVs. In Aim #2, we will analyze quantitative changes in mtDNA content in NEVs that may reflect mtDNA damage associated with cigarette smoking and ART. Our proposal is innovative because it proposes using mtDNA content in peripheral blood NEVs as a novel surrogate biomarker for monitoring neuronal mtDNA damage. Our findings from this proposed study will be significant they will establish mtDNA content in NEVs as a novel, non-invasive biomarker for real-time monitoring of neuronal dysfunction associated with mtDNA damage in HIV-positive individuals on ART and cigarette smokers.