Microbiology / Infectious Disease

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31259


Recent Submissions

Now showing 1 - 7 of 7
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    (2013-04-12) Park, In-Woo
    Purpose: 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.
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    (2013-04-12) Liu, Ziqing
    Purpose: 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.
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    (2013-04-12) Luo, Xiaoyu
    Purpose: HIV-1 Nef is an essential pathogenic factor for acquired immunodeficiency syndrome disease progression. It binds to over 30 putative cellular components and initiates a great variety of functions. Recent studies have demonstrated that Nef can be transferred from infected cells to uninfected cells and leads to significant changes on the bystander cells. This intercellular Nef transfer is possibly linked to the massive depletion of CD4 T cells in disease progression. However, the underlying mechanism for the intercellular Nef transfer is still not fully understood. In this study, by using advanced tools, we tested two potential mechanisms for intercellular Nef transfer among CD4 T cells including direct Nef transfer through cell-cell contact and Nef secretion in the form of exosomes. Methods: Nef-GFP Jurkat cells were co-cultured with red dye-labeled uninfected Jurkat cells for 8 hours. Cell mixture was analyzed under florescence microscope. To determine the percentage of Nef transfer during HIV infection, HIV and HIV Nef-deleted virus-infected jurkat cells were collected when cells reached 50% infected. The percentage of Nef+ cells in uninfected population and how it affected by cell density was analyzed by Immunostaining and flow-cytometry. In order to test if exosomes are involved in intercellular Nef transfer among CD4 T cells, we utilized optiprep gradient centrifugation to separate exosomes from viral particles and then detect Nef expression in exosome fractions. Results: Nef was detected in the bystander Jurkat cells after co-cultured with Nef-expressing /infected Jurkat cells. Nef was transferred through nanotubes and filopodia bridges by forming connections between infected cells and uninfected cells. There was about 3-10% of uninfected cells that were found to be Nef+ in infected Jurkat cells (50% infection). The intercellular Nef transfer was enhanced by increasing the cell density. Using optiprep gradient centrifugation, virus was successfully separated with exosomes and Nef was only detected in the virus fractions but not exosome fractions. Conclusions: Taken together, our studies showed that Nef can be transferred to uninfected bystander CD4+ T cells either from Nef-expressing cells or from HIV-infected cells. This intercellular Nef transfer is mediated by direct cell-cell contact possibly through synapse or nanotube like structures. Exosome is not involved in the intercellular Nef transfer.
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    (2013-04-12) Zhang, Ziugen
    Purpose: 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.
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    (2013-04-12) Rahimian, Pejman
    Purpose: Our purpose is to determine the significance of astrocyte-released exosomes in regulation of HIV-1 Tat neurotoxicity. We hypothesize that the exosomes produced by astrocytes contribute to Tat neurotoxicity. Methods: Primary astrocyte cultures were prepared from the embryos of brain-specific and doxycycline-inducible Tat-transgenic and wild type mice and treated with 5 ug/ml doxycycline for 3 days to induce Tat expression. The Tat transgenic mice express HIV Tat gene under the dual control of a doxycycline-inducible promoter and the glial fibrillary acidic protein (GFAP) gene promoter. Addition of doxycycline induces the expression of HIV-1 Tat protein in mouse astrocytes. Supernatants from doxycycline-treated cultures were collected and incubated with SHSY5Y neuroblastoma cells for 3 days. MTT assay was performed to determine the survival of the neurons. Briefly, tetrazolium reagent was added to cultures and incubated for 5 hours in 37oC. The reduction of this reagent by NAPDH-Oxidase in the mitochondria of healthy cells will form a blue color which can be measured spectrophotometrically after dissolution with acid-isopropanol. The intensity of the blue color indicates the number of healthy cells. To determine the roles of exosomes in Tat-induced neurotoxicity, astrocyte cultures were first induced with doxycycline, the culture media were replaced with fresh complete media, the cell continued to culture for 48 hours in the presence of 5 uM or 10 uM GW4869, an inhibitor of neutral sphingomyelinase which is critical for the production of exosomes in astrocytes. The supernatants from GW4869-treated cultures were collected and determined for its neurotoxicity using the MTT assay described above. Results: Supernatants of doxycycline-induced Tat-transgenic astrocyte cultures decreased the survival of neuroblastoma cells compared to those of wild type astrocytes. Inhibition of exosome production in doxycycline-treated astrocytes with GW4869 improved the survival of neuroblastoma cells compared to those that were not treated. Conclusions: These findings confirmed that Tat expression in astrocytes in the absence of HIV infection caused neurotoxicity and showed for the first time that exosomes produced from Tat-expressing astrocytes were involved in induction of Tat neurotoxicity.
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    (2013-04-12) Fan, Yan
    Purpose: HIV-1 Tat is a major pathogenic factor for HIV-associated neurodegenerative diseases. One of the consistent hallmarks of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by increased cytoplasmic accumulation of intermediate filament glial fibrillary acidic protein (GFAP). Our previous studies show that Tat induces GFAP expression in astrocytes and GFAP is a critical regulator of Tat neurotoxicity. However, the molecular mechanisms responsible for GFAP activation-mediated Tat neurotoxicity is not known. Thus, we attempted in this study to determine the underlying unknown molecular mechanisms. Methods: Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes were either transfected with pTat.cMyc or treated with doxycycline to induce Tat expression. Cells were harvested to detect GFAP, eIF2a, ATF6, Oasis and Bip expression by Western blotting to , or to detect Tat, GFAP and XBP-1 by RT-PCR. Culture supernatants were collected and analyzed for neurotoxicity toward primary mouse or human neurons using MTT assay. GFAP, MAP-2 and Bip expression in astrocytes, Tat-expressing mouse brain and the brains of HIV-infected individuals were also determined by IF and IHC staining. Flow cytometry was performed to determine proteasomal activity by monitor expression intensity of proteasomal activity reporter pZsProSensor-1. Results: We showed that HIV-1 Tat-induced GFAP up-regulation and aggregation in astrocytes activated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways, such as PERK, IRE1, ATF6 and OASIS, We further showed that supernatants from Tat-expressing astrocytes were neurotoxic. Importantly, we showed that the neurotoxicity in these culture supernatants were significantly diminished when GFAP was null or when the cultures were treated with 4-sodium phenyl butyrate (4-PBA), a chemical chaperon capable of blocking the ER stress, suggesting that GFAP activation and aggregation-induced ER stress in the presence of Tat expression is at least in part responsible for Tat neurotoxicity. Lastly, we showed that GFAP activation and aggregation resulted in lower proteasome activity and induction of autophagy in astrocytes. Conclusions: Taking together, this study demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis.
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    (2013-04-12) Schutt, Christina
    Purpose: The purpose of our case is to help other physicians learn from this unique case of Coccidiomycosis. Methods: This is a case Report, the only materials that had been used were the patients chart information and history taken from the patient. Results: We report a case of a pulmonary cavitary lesion in a 74-year-old African American man who presented to the emergency department with right upper quadrant abdominal pain and cough of one week duration. Chest CT revealed a cavitary lesion in the right upper lobe. His diagnosis was made two weeks after his admission by bronchial lavage, at which time appropriate treatment was initiated. Conclusions: This case emphasizes the importance of a thorough history-taking when presented with a problem that has a wide differential diagnosis.