Browsing by Subject "HCV"
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Item An Analysis of Subject Recruitment Issues for an HCV Investigational Drug Clinical Trial(2009-05-01) Lander, Aitemad A.; Oglesby, MichaelChronic hepatitis C infection is debilitating, and investigational drugs currently in phase III clinical trials are promising. It is important that efficient antiviral medication reach market if it has the potential for inhibiting viral replication. Because of subject recruitment issues, many studies fail to meet enrollment goals and therefore cannot complete the clinical trial process and bring drugs to market. Therefore, a recruitment plan must be implemented that will ensure that enrollment goals are met. The goal of this practicum was to test the hypothesis that educating the medical and non-medical community about the HCV clinical trials would increase subject enrollment. However, due to certain limitations, not enough data was gathered to provide clear and concise results.Item CELL-CELL CONTACT-MEDIATED HEPATITIS C VIRUS INFECTION AND ITS REQUIREMENT FOR HCV RECEPTORS(2013-04-12) Liu, ZiqingPurpose: Hepatitis C virus (HCV) leads to chronic infection in 80% of infected people. In these patients, HCV evades the immune system with the presence of neutralizing antibodies (nAbs), yet the underlying mechanisms have not been completely understood. Recently discovered cell-cell contact-mediated (CCCM) HCV transmission, in contrast to cell-free infection, was found to be nAbs-resistant, suggesting its important role in immune evasion. The objective of the current study was to characterize CCCM HCV infection and to determine the roles of the four known HCV receptors (CD81, scavenger receptor B1, claudin-1 and occludin) in this process. Methods: Infected donor hepatocyte Huh7.5.1 were either co-cultured with uninfected and fluorescently labeled target Huh7.5.1, or seeded into the upper chamber of a trans-well, where uninfected Huh7.5.1 were seeded in the lower chamber. After different time of culturing, the mixed donor and target cells in the co-culture assay or the target cells in the trans-well assay was immunostained for HCV core and analyzed by flow cytometry for percentage of newly infected target cells. To determine the roles of HCV receptors and cytoskeleton in CCCM HCV transfer, co-culture assay was also performed with target cells where one or all HCV receptors were knocked-down by siRNA, or with the presence of cytoskeleton inhibitors. Results: In this study, we have demonstrated that HCV spreading among hepatocytes leads to the formation of infection foci dependent upon cell density and that HCV is directly transferred from infected hepatocytes to uninfected hepatocytes. Compared to cell-free infection in the trans-well assay (undetectable within 20 hr), CCCM HCV transfer in the co-culture assay occurs very rapidly (3 hr). Knock-down of HCV receptors in target cells and cytochalasin D treatment of the co-culture greatly inhibited CCCM HCV transfer, suggesting the important roles of all four HCV receptors and intact actin cytoskeleton in this process. With a fluorescently labeled replication-competent HCV, the CCCM transfer process is further dissected by 3D live cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and post transfer. Conclusions: The study shows for the first time that CCCM HCV transfer constitutes a rapid and vital route for HCV infection and dissemination. These findings will aid in the development of new and effective strategies for preventing and treating HCV infection.Item HIV AND HCV INFECTION OF THE BRAIN IN HUMANIZED MICE(2013-04-12) Zhang, ZiugenPurpose: In this study, we wished to determine the possibility of HIV and HCV infections in the brains of these mice in preparation of using the mice to study HIV and HCV co-infection of the brain and the roles of the co-infection in HIV/HCV-associated neurological diseases . Methods: Human CD34+ human hematopoietic stem cells (HSC) and hepatocyte progenitors were co-transplanted into the Balb/CRag2-/-𝛄C-null mice, which led to efficient engraftment of human leukocytes and hepatocytes. These humanized mice were infected with HCV alone or HCV and HIV. Two months after infection, livers and brains of these mice were collected. Half of the brain was fixed and paraffin-embedded for immunostaining for HIV p24 and HCV core; the other half of the brain was used for DNA and RNA extraction. Polymerase Chain Reaction (PCR) was performed to detect HIV DNA; Strand-specific Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed to detect positive-strand and negative strand HCV RNA. Results: Three of four mice in HCV mono-infected group, one of five mouse in the HCV/HIV co-infected group showed severe fibrosis in liver. The HCV core protein and HIV P24 protein were detected in the brain, although faint. All 5 mouse infected with HIV were detected for HIV DNA in the brain. All 4 HCV mono-infected mice and all 5 HCV/HIV co-infected mice were detected for positive-strand HCV RNA in their brains, while only 3 of those brains were detected for negative-strand HCV RNA. Conclusions: These results showed that HCV and HIV are both capable of gaining access into the brain and replicating in the brain of these mice and suggest that humanized mice could be used to study the effects of HCV infection and HCV/HIV co-infection on the brain dysfunction.Item ROLE OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 NEF IN ACCELERATION OF HEPATITIS C VIRUS-MEDIATED LIVER DISEASE.(2013-04-12) Park, In-WooPurpose: HIV-1 infection has profound, adverse consequences for every stage of the natural history of HCV infection via significantly elevating HCV viral load and expediting HCV-mediated liver disease progression in the co-infected host. However, molecular details for how HIV-1 accelerates this pathogenesis are largely unknown. According to recent publications, HIV-1 Nef can be transferred from HIV-1 susceptible cells to other uninfected susceptible cells and even to non-susceptible target cells by formation of conduits or by exosomes, suggesting that Nef is a leading candidate molecule for explaining the occurrence of HIV-1-mediated opportunistic diseases in non-susceptible target tissues. Accordingly, we have investigated the role of HIV-1 and viral protein Nef in HCV-infected hepatocytes to better understand the pathobiology of HIV-1 and HCV co-infection. Methods: Infectivity of HIV-1 in human hepatocytes was monitored at the indicated time point by measuring reverse transcriptase (RT) activity in the clarified culture supernatants, and effect of Nef on the expression of HCV replicon was examined by measuring reporter gene, Luciferase (Luc), expression in the replicon cells. Transfer of Nef to target hepatocytes and subcellular distribution of lipid droplets (LD) were studied by immunofluoscence and confocal microscopic analyses as well as by flow cytometric analyses. Results: Infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the hepatocytes transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was found to be transferred from expressing T cells to hepatocytes through conduits, wherein up to 16% (average 10%) of hepatocytes harbored the transferred Nef, when the hepatocytes were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of LD, and consistently up-regulated HCV replication by 1.5~2.5 fold in the target hepatocytes, which is remarkable in relation to the initially indolent viral replication. Conclusions: HIV-1 Nef is a critical element in accelerating HCV-mediated liver pathogenesis through modulation of lipid molecules and changes in HCV replication.Item Ubiquitin-protein ligase E3A (UBE3A) mediation of viral infection and human diseases(Elsevier B.V., 2023-08-05) Chaudhary, Pankaj; Proulx, Jessica; Park, In-WooThe Ubiquitin-protein ligase E3A, UBE3A, also known as E6-associated protein (E6-AP), is known to play an essential role in regulating the degradation of various proteins by transferring Ub from E2 Ub conjugating enzymes to the substrate proteins. Several studies indicate that UBE3A regulates the stabilities of key viral proteins in the virus-infected cells and, thereby, the infected virus-mediated diseases, even if it were reported that UBE3A participates in non-viral-related human diseases. Furthermore, mutations such as deletions and duplications in the maternally inherited gene in the brain cause human neurodevelopmental disorders such as Angelman syndrome (AS) and autism. It is also known that UBE3A functions as a transcriptional coactivator for the expression of steroid hormone receptors. These reports establish that UBE3A is distinguished by its multitudinous functions that are paramount to viral pathology and human diseases. This review is focused on molecular mechanisms for such intensive participation of UBE3A in disease formation and virus regulation.