This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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