Novel serological biomarkers for BBB dysfunction during HIV-1 infection

描述（由申请人提供）： 目前采用联合抗逆转录病毒疗法（cART）治疗艾滋病毒感染患者，可有效抑制病毒复制，预防免疫抑制并延长寿命。尽管有这些进展，即使在cART的存在下，慢性神经炎症也不能完全预防，导致HIV相关神经认知障碍（HAND）的发展。上述临床观察提示了定量生物标志物的必要性，以帮助检测和监测由HIV感染引起的中枢神经系统（CNS）并发症。血脑屏障（BBB）功能障碍是HIV-1神经发病机制中的一个众所周知的现象，可能导致神经变性。我们提出了反映血脑屏障损伤的新生物标志物，即检测含有脑内皮细胞释放的紧密连接蛋白（TJP）的细胞外微泡（eMV）。我们发现eMV从脑内皮释放是炎症和血管重塑的结果。重要的是，对脑内皮细胞（EC）的相同促炎性损伤诱导屏障通透性和紧密连接复合物的生化变化。该项目在开发生物标志物发现的创新工具和鉴定由于慢性HIV相关神经炎症而发生在BBB的新型病理生理现象方面都是独一无二的。在第一个目标中，我们将配置文件和时间顺序定义的存在TJPs在eMV分泌的结果，相关的炎症刺激和HIV病毒毒素。这些测定将与屏障完整性的测量平行进行，从而允许eMV脱落和BBB功能障碍之间的相关性。由于结构蛋白识别eMV的细胞来源，第二个目标探索eMV携带TJP作为BBB功能障碍的血清学生物标志物的基础的概念。使用最灵敏的基于ELISA的可用技术，我们已经建立了原型ELISA来测量含有TJP的血液循环eMV的水平。使用来自HIV-1感染者的患者血清，我们提供了概念验证结果，即这种新的生物标志物与HAND的诊断相关。第三个目标中概述的实验剖析了脑EC微泡形成和TJP包装的生物发生中涉及的合理分子机制。它不仅将提供对eMV形成如何触发的理解，而且还将揭示细胞内促进BBB保护的药理学干预的靶点。

DESCRIPTION (provided by applicant): Current treatment of HIV-infected patients with combined antiretroviral therapy (cART) has provided effective suppression of virus replication, prevented immunosuppression and increased lifespan. Despite these advances, even in the presence of cART, chronic neuroinflammation cannot be fully prevented, leading to development of HIV-associated neurocognitive disorders (HAND). The above clinical observation has prompted the necessity for quantitative biomarkers to help in detection and monitoring of central nervous system (CNS) complications resulting from HIV infection. Dysfunction of the blood-brain barrier (BBB) is a well-known phenomenon in HIV-1 neuropathogenesis potentially leading to neurodegeneration. We propose novel biomarkers reflecting BBB injury, namely detection of extracelluar microvesicles (eMVs) containing tight junction proteins (TJP) released from brain endothelium. We discovered that eMV release from the brain endothelium occurs as a result of inflammation and vascular remodeling. Importantly, the same pro- inflammatory insult on brain endothelial cells (EC) induces barrier permeability and biochemical changes to tight junction complexes. This project is unique in both the development of innovative tools for biomarker discovery and identification of a novel pathophysiologic phenomenon occurring at the BBB as consequence of chronic HIV-associated neuroinflammation. In the first aim, we will profile and chronologically define the presence of TJPs in eMV secreted as a consequence of relevant inflammatory stimuli and HIV virotoxins. These determinations will be made in parallel with measurement of barrier integrity, thus allowing for correlations between eMV shedding and BBB dysfunction. Because structural proteins identify the cellular origin of the eMV, the second aim explores the notion of eMV carrying TJP as the basis for a serological biomarker for BBB dysfunction. Using the most sensitive ELISA-based available technology, we have built prototype ELISAs to measure the level of blood circulating eMVs containing TJP. Using patient serum from HIV-1 infected individuals, we provide proof-of concept results that this novel biomarker correlates with diagnosis of HAND. Experiments outlined in the third aim dissect plausible molecular mechanisms involved in the biogenesis of brain EC microvesicle formation and packaging of TJP. It will offer, not only an understanding of how eMV formation is triggered, but also will reveal targets within the cell for pharmacological intervention that promote BBB protection.