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CNS损伤，包括HIV相关性痴呆（HAD）和其他形式的神经认知障碍，越来越多地表现为HIV感染的破坏性后果。虽然晚期的临床发现，血脑屏障（BBB）的损害和渗透到CNS实质的HIV感染的单核细胞已被证明发生在感染早期。事实上，认知障碍与神经炎症的关系比与CNS内病毒的存在的关系更直接。CCL 2是一种主要的单核细胞趋化因子，已被证明在HAD和HIV Encephalitis患者中显著升高。因此，了解HIV和CCL 2介导的单核细胞迁移到CNS中以及伴随的BBB破坏的机制将能够鉴定潜在的治疗靶点，最终目标是预防HIV相关的神经认知下降。我们假设，HIV感染的单核细胞改变其表达的CCR 2，细胞粘附分子，和adherens和紧密连接蛋白，和这些感染的单核细胞在中枢神经系统中的CCL 2的结果在异常的单核细胞-血脑屏障内皮细胞的相互作用，促进增强迁移和血脑屏障破坏。本研究的目的是确定负责增强渗出的HIV感染的单核细胞进入中枢神经系统响应于CCL 2趋化因子和随后的血脑屏障破坏的分子相互作用。本研究的具体目的是：（1）利用微阵列、RT-PCR、Western blot和共聚焦成像技术，鉴定HIV感染和CCL 2趋化刺激后单核细胞中可能与其增强渗出有关的差异表达基因或蛋白定位的变化;（2）使用微阵列表征CCL 2对脑微血管内皮细胞连接蛋白的作用，所述连接蛋白可增强单核细胞渗出并破坏BBB完整性，RT-PCR、Western印迹、共聚焦成像和免疫共沉淀（ColP）研究;（3）使用放射性标记、ColP、蛋白质印迹、放射自显影来表征HIV感染的单核细胞和人脑微血管内皮细胞中的连接蛋白、衔接蛋白和细胞骨架之间的动态相互作用，所述动态相互作用有助于单核细胞的增强的迁移特性和响应于CCL 2的BBB完整性的破坏，共聚焦成像和组织染色;和（4）使用放射性标记、免疫沉淀、放射自显影和激酶和磷酸酶抑制剂，测定HIV感染的单核细胞响应于CCL 2而穿过BBB的迁移过程中，细胞粘附蛋白的磷酸化变化。

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.