Mechanisms of Transmigration of HIV Infected Cells Across the Blood Brain Barrier

描述（由申请人提供）：随着HAART的成功，HIV感染者的寿命延长，HIV介导的中枢神经系统损伤，包括HIV相关痴呆（HAD）和其他形式的神经认知障碍，越来越多地呈现为HIV感染的破坏性后果。尽管这是一个较晚的临床发现，但HIV感染的单核细胞对血脑屏障（BBB）的破坏和对中枢神经系统实质的浸润已被证明发生在感染的早期。事实上，认知障碍与神经炎症的关系比与中枢神经系统内病毒的存在更为直接。CCL2，主要的单核细胞化学引诱剂，已被证明在HAD和HIV脑炎患者中显著升高。因此，了解HIV和CCL2介导的单核细胞向中枢神经系统的迁移以及伴随的血脑屏障破坏的机制将有助于确定潜在的治疗靶点，最终目标是预防HIV相关的神经认知能力下降。我们假设HIV感染单核细胞改变了它们CCR2、细胞粘附分子、粘附物和紧密连接蛋白的表达，这些受感染单核细胞响应CCL2在中枢神经系统中的迁移导致单核细胞-血脑屏障内皮细胞的异常相互作用，促进迁移增强和血脑屏障破坏。本研究的目的是确定受HIV感染的单核细胞对CCL2趋化因子和随后的血脑屏障分解的反应增强向中枢神经系统渗透的分子相互作用。本研究的具体目标是：(1)利用微阵列、RT-PCR、Western blot和共聚焦成像技术，鉴定由于HIV感染和CCL2趋化刺激导致的单核细胞中差异表达的基因或蛋白质定位的变化，这些基因或蛋白质定位可能有助于单核细胞增强浸润；(2)利用微阵列、RT-PCR、Western blot、共聚焦成像和共免疫沉淀（ColP）研究，表征CCL2对脑微血管内皮细胞连接蛋白的影响，这些蛋白可能会增强单核细胞浸润并破坏血脑屏障的完整性；(3)利用放射标记、ColP、Western blot、放射自显像、共聚焦成像和组织染色，表征HIV感染单核细胞和人脑微血管内皮细胞中连接蛋白、连接蛋白和细胞骨架之间的动态相互作用，这些相互作用有助于单核细胞对CCL2的迁移特性增强和血脑屏障完整性的破坏；(4)利用放射标记、免疫沉淀、放射自显影以及激酶和磷酸酶抑制剂，确定受CCL2感染的HIV单核细胞在血脑屏障转移过程中细胞连接蛋白的磷酸化变化。

DESCRIPTION (provided by applicant): With the success of HAART, HIV infected individuals are living longer and HIV mediated damage to the CNS, including HIV Associated Dementia (HAD) and other forms of neurocognitive impairment, are increasingly presenting as a devastating consequence of HIV infection. Although a late clinical finding, compromise of the blood brain barrier (BBB) and infiltration into the CNS parenchyma by HIV infected monocytes have been shown to occur early in infection. In fact, cognitive impairment correlates more directly with neuroinflammation than with the presence of virus within the CNS. CCL2, the predominate monocyte chemoattractant, has been shown to be substantially elevated in patients with HAD and HIV Encephalitis. Thus, understanding the mechanisms of HIV and CCL2 mediated monocyte transmigration into the CNS and concomitant disruption of the BBB will enable the identification of potential targets of therapy, with the ultimate goal of preventing HIV related neurocognitive decline. We hypothesize that HIV infection of monocytes alters their expression of CCR2, cell adhesion molecules, and adherens and tight junction proteins, and that transmigration of these infected monocytes in response to CCL2 in the CNS results in aberrant monocyte-BBB endothelial cell interactions, promoting enhanced migration and BBB disruption. The goal of this research is to define the molecular interactions responsible for the enhanced diapedesis by HIV infected monocytes into the CNS in response to the CCL2 chemokine and the subsequent BBB breakdown. The specific goals of this research are: (1) to identify differentially expressed genes or changes to protein localization in monocytes as a result of HIV infection and CCL2 chemotactic stimulation that may cofitribute to their enhanced diapedesis using microarray, RT-PCR, Western blot, and confocal imaging; (2) to characterize the effects of CCL2 on brain microvascular endothelial cell junction proteins that may potentiate monocyte diapedesis and disrupt BBB integrity using microarray, RT-PCR, Western blot, confocal imaging, and coimmunoprecipitation (ColP) studies; (3) to characterize the dynamic interactions between junctional proteins, adaptor proteins, and the cytoskeleton in HIV infected monocytes and in human brain microvascular endothelial cells that contribute to the enhanced transmigration properties of monocytes and disruption of BBB integrity in response to CCL2 using radiolabeling, ColP, Western blot, autoradiography, confocal imaging, and tissue staining; and (4) to determine the phosphorylation changes in cell juction proteins during transmigration of HIV infected monocytes across the BBB in response to CCL2 using radiolabeling, immunoprecipitation, autoradiography, and kinase and phosphatase inhibitors.