JCV STUDY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 由人类多瘤病毒JC(JCV)引起的进行性多灶性白质脑病(PML)仍然是免疫功能低下个体死亡和发病的重要原因，即使在HAART之后，仍有大约6%的艾滋病患者受到影响。尽管有一些证据表明B细胞可能是JCV传播的媒介，但JCV感染的B细胞进入中枢神经系统(CNS)的潜在机制(S)尚不清楚。B细胞跨血脑屏障(BBB)的迁移是由黏附分子、B细胞表面的VLA-4和淋巴细胞功能相关抗原-1(LFA-1)以及趋化因子、单核细胞趋化蛋白-1(MCP-1)和白介素8(IL-8)介导的。然而，目前尚不清楚JCV感染的B细胞是否通过改变这些黏附分子和细胞因子受体CCR2和CXCR1/CXCR2的表达模式而优先跨越血脑屏障。 我们的初步研究结果表明：(1)JCV可有效感染原代培养的人脑微血管内皮细胞(HBMVE)，(2)无细胞JCV可跨越体外血脑屏障模型，(3)体外培养的HBMVE细胞不表达5-HT2AR，JCV感染HBMVE细胞和JCV跨血脑屏障的迁移不依赖5-HT2AR，(4)JCV可非生产性感染EB病毒转化的B细胞。因此，我们的中心假设是，在病毒血症期间，JCV通过感染HBMVE细胞或通过感染的B细胞或两者兼而有之，穿过血脑屏障，导致潜伏的中枢神经系统感染。 拟议的研究将通过解决两个具体目标，具体解决我们关于无细胞和B细胞相关的JCV跨血脑屏障迁移机制的知识空白： (1)研究JCV与HBMVE细胞和体外BBB模型相互作用的细胞和分子机制，包括：i)JCV在HBMVE细胞中的感染和复制的变化，以及唾液酸受体阻滞剂处理后JCV跨血脑屏障的迁移；以及ii)通过监测JCV在迁移之前、期间和之后的连接蛋白、claudin-1和-5以及occludin、TEER、细胞旁标志物(如菊糖)的表达以及转录本(如葡萄糖转运体1和大中性氨基酸转运体1)的表达来描述JCV的变化。 (2)研究JCV感染B细胞与体外血脑屏障之间的细胞和分子相互作用：1)用JCV感染B细胞，比较感染和未感染的B细胞表面黏附分子VLA-4、LFA-1和趋化因子受体CCR2、CXCR1/CXCR2的表达；II)比较感染和未感染的B细胞在体外血脑屏障中的迁移情况，观察迁移前后血脑屏障的变化。 新发现的关于无细胞和B细胞相关的JCV跨血脑屏障迁移的知识可能会导致未来对PML和其他脱髓鞘疾病的治疗干预措施的改进。 临床研究中心的参与将包括研究的整体协调，包括招募、同意和进行研究访问。招聘工作将由夏威夷大学马诺阿分校和JABSOM Kakaako校区的CRC工作人员以及密歇根大学学生健康诊所和普通公众进行。儿童权利中心将只在儿童权利委员会批准的位于JABSOM Kakaako校区的地点安排和实施考察访问。 在JABSOM Kakaako园区建立和维护设施所需的认证也将由儿童权利委员会负责。CRC将与UH ESHSO官员合作，以遵守任何安全法规和要求。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progressive multifocal leukoencephalopathy (PML), caused by human polyomavirus JC (JCV), remains an important cause of mortality and morbidity among immunocompromised individuals and affects approximately 6% of AIDS patients even in the post-HAART era. Despite some evidence of B cells being a possible vehicle for JCV dissemination, the underlying mechanism(s) of JCV-infected B cells entry into the central nervous system (CNS) is unclear. B-cell migration across the blood-brain barrier (BBB) is mediated by adhesion molecules, very late antigen-4 (VLA-4) and lymphocyte function associated-antigen-1 (LFA-1) on B cells, and chemokines, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). However, it remains unclear whether JCV-infected B cells preferentially cross the BBB by altering the expression pattern of these adhesion molecules and cytokine receptors, CCR2 and CXCR1/CXCR2. Our preliminary data demonstrate that: (1) JCV productively infects primary human brain microvascular endothelial (HBMVE) cells, (2) cell-free JCV can cross the in-vitro BBB model, (3) HBMVE cells in-vitro do not express serotonin receptor 2A (5- HT2AR), and JCV infection of HBMVE cells and JCV transmigration across the BBB is independent of 5- HT2AR, and (4) JCV can non-productively infect Epstein Barr virus-transformed B cells. Accordingly, our central hypothesis is that during viremia, JCV crosses the BBB either by infecting HBMVE cells or via infected B cells or both, resulting in latent CNS infection. The proposed research will specifically address gaps in our knowledge about the mechanism of transmigration of cell-free and B cell-associated JCV across the BBB by addressing two specific aims: (1) To characterize the cellular and molecular mechanisms of interaction of JCV with the HBMVE cells and the in-vitro BBB model, by documenting i) changes in infection and replication of JCV in HBMVE cells, and JCV transmigration across the BBB when treated with sialic acid receptor blockers, and ii) changes in BBB by monitoring the expression of junctional proteins, claudin-1 and -5 and occludin, TEER, paracellular markers, such as inulin, and expression of transcripts such as glucose transporter type 1 and large neutral amino acid transporter 1 before, during and after transmigration of JCV. (2) To characterize the cellular and molecular interactions between JCV-infected B cells and the in-vitro BBB by i) infecting B cells with JCV and comparing the expression of adhesion molecules VLA-4 and LFA-1 and chemokine receptors CCR2 and CXCR1/CXCR2 on JCV-infected and JCV-uninfected B cells and ii) comparing the transmigration of JCV-infected and uninfected B cells across the in-vitro BBB and documenting changes in BBB before, during and after transmigration. Newfound knowledge about the transmigration of cell-free and B cell-associated JCV across the BBB may lead to future improved therapeutic interventions for PML, and possibly other demyelinating disorders. Clinical Research Center's involvement will consist of the overall coordination of the study, including recruitment, consenting, and conducting study visits. Recruitment will be done by CRC staff at the University of Hawaii at Manoa and JABSOM Kakaako campuses, as well as the UH student health clinic and the general public. CRC will accommodate the PI to set up and implement study visits solely at the CRC approved sites located at the JABSOM Kakaako campus. Certification needed in setting up and maintaining the facility at the JABSOM Kakaako campus will also be the responsibility of the CRC. The CRC will work with UH ESHSO officer to comply with any safety regulations and requirements.