Novel serological biomarkers for BBB dysfunction during HIV-1 infection

描述(由申请人提供)： 目前对艾滋病毒感染患者的联合抗逆转录病毒疗法(CART)有效地抑制了病毒复制，防止了免疫抑制，并延长了寿命。尽管取得了这些进展，但即使在CART存在的情况下，慢性神经炎也不能完全预防，从而导致艾滋病毒相关神经认知障碍(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的生物标志物，即检测从脑内皮细胞释放的含有紧密连接蛋白(TJP)的细胞外微囊泡(EMVS)。我们发现，由于炎症和血管重塑，EMV从脑内皮细胞释放出来。重要的是，对脑内皮细胞(EC)同样的促炎损伤会导致屏障通透性和紧密连接复合体的生化变化。该项目在开发发现生物标记物的创新工具和识别由于慢性艾滋病毒相关神经炎而在血脑屏障发生的一种新的病理生理现象方面是独一无二的。在第一个目标中，我们将按时间顺序描述和定义由于相关炎症刺激和HIV病毒毒素而分泌的EMV中TJP的存在。这些测定将与屏障完整性的测量并行进行，从而允许EMV脱落和BBB功能障碍之间的相关性。因为结构蛋白识别EMV的细胞起源，第二个目的是探索EMV携带TJP作为BBB功能障碍的血清学生物标记物的基础的概念。利用最灵敏的基于EL ISA的技术，我们已经建立了检测含有TJP的血液循环EMVS水平的ELISA原型。使用HIV-1感染者的患者血清，我们提供了概念验证结果，即这种新的生物标记物与手部诊断相关。第三个目标中概述的实验剖析了脑内皮细胞微泡形成和TJP包装的生物发生中可能涉及的分子机制。它不仅将提供对EMV形成是如何触发的理解，而且还将揭示细胞内促进血脑屏障保护的药物干预的靶点。

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.