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，艾滋病毒感染者的寿命延长，艾滋病毒对中枢神经系统的损害，包括艾滋病毒相关性痴呆(HAD)和其他形式的神经认知障碍，越来越成为艾滋病毒感染的破坏性后果。虽然临床发现较晚，但HIV感染的单核细胞破坏血脑屏障(BBB)和渗透到中枢神经系统实质已被证明发生在感染的早期。事实上，认知障碍更直接地与神经炎症有关，而不是与中枢神经系统内病毒的存在有关。CCL2是主要的单核细胞趋化物质，已被证明在HAD和HIV脑炎患者中显著升高。因此，了解HIV和CCL2介导的单核细胞迁移到中枢神经系统的机制以及伴随的血脑屏障的破坏将有助于识别潜在的治疗靶点，最终目的是预防HIV相关的神经认知功能下降。我们假设，HIV感染单核细胞改变了其CCR2、细胞黏附分子、黏附和紧密连接蛋白的表达，这些感染的单核细胞在中枢神经系统对CCL2的反应导致单核细胞与BBB内皮细胞的异常相互作用，促进了迁移和BBB破坏。本研究的目的是明确HIV感染的单核细胞对CCL2趋化因子和随后的血脑屏障破坏的反应促进了单核细胞进入中枢神经系统的分子相互作用。本研究的具体目标是：(1)利用微阵列、RT-PCR、Western印迹和共聚焦成像技术，鉴定HIV感染和CCL2趋化刺激引起的单核细胞差异表达基因或蛋白定位的改变，这些差异表达基因或蛋白定位变化可能与其促进单核细胞转运有关；(2)通过微阵列、RT-PCR、Western印迹、共聚焦成像和免疫共沉淀(COLP)研究，鉴定CCL2对脑微血管内皮细胞连接蛋白的影响，这些蛋白可能增强单核细胞的转运并破坏BBB的完整性；(3)利用放射标记、COLP、Western印迹、放射自显影、共聚焦成像和组织染色等方法，研究HIV感染单核细胞和人脑微血管内皮细胞中结合蛋白、接合蛋白和细胞骨架之间的动态相互作用，这些作用有助于增强单核细胞的迁移特性，破坏CCL2诱导的BBB完整性；以及(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HIV感染的单核细胞在跨血脑屏障迁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.