IU Indianapolis ScholarWorks :: Browsing by Author "He, Johnny J."

Browsing by Author "He, Johnny J."

Now showing 1 - 10 of 20

Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity
(Springer Nature, 2011-03) Fan, Yan; Zou, Wei; Green, Linden A.; oh Kim, Byung; He, Johnny J.; Microbiology and Immunology, School of Medicine
Human immunodeficiency virus type 1 (HIV-1) Tat plays an important role in HIV-associated neuropathogenesis; the underlying mechanisms are still evolving. We have recently shown that HIV-1 Tat induces expression of glial fibrillary acidic protein (GFAP), a characteristic of HIV-1 infection of the central nervous system (CNS). We have also shown that the Tat-induced GFAP expression in astrocytes is regulated by p300, and that deletion of the early growth response 1 (Egr-1) cis-transacting element within the p300 promoter abolishes Tat-induced GFAP expression. In this study, we further examined the relationship between Tat and Egr-1 in astrocytes. We found increased Egr-1 protein expression in Tat-expressing human astrocytoma cells and mouse primary astrocytes. Using the Egr-1 promoter-driven firefly luciferase reporter gene assay and the site-directed mutagenesis, we demonstrated that Tat increased Egr-1 expression by transactivating the Egr-1 promoter and involving specific serum response elements (SRE) within the promoter. Consistent with these data, we showed that Tat transactivation of the Egr-1 promoter was abrogated when astrocytes were cultured in serum-reduced media. Taken together, these results reveal that Tat directly transactivates Egr-1 expression and suggest that Tat interaction with Egr-1 is probably one of the very upstream molecular events that initiate Tat-induced astrocyte dysfunction and subsequent Tat neurotoxicity.
CD4+ T cell mediated tumor immunity following transplantation of TRP-1 TCR gene modified hematopoietic stem cells
(2013-12-10) Ha, Sung Pil; Touloukian, Christopher E.; Broxmeyer, Hal E.; Gardner, Thomas A.; Harrington, Maureen A.; He, Johnny J.
Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.
Characterization of Hepatitis C Virus Infection of Hepatocytes and Astrocytes
(2014) Liu, Ziqing; Yu, Andy; He, Johnny J.; Brutkiewicz, Randy R.; Kao, Cheng C.; Sullivan, William J., Jr.
Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.
E7 PROTEINS OF HIGH-RISK (TYPE 16) AND LOW-RISK (TYPE 6) HUMAN PAPILLOMAVIRUSES REGULATE p130 DIFFERENTLY
(2010-10-15) Barrow, Lisa C.; Roman, Ann; Brown, Darron; He, Johnny J.; Nakshatri, Harikrishna
Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.
Effects of traditional Chinese medicinal herbal extracts on HIV-1 replication
(2011-03-16) Wang, Ting; He, Johnny J.; Yu, Andy; Schloemer, Robert H.
Background: The current treatment for HIV/AIDS is called highly active antiretroviral therapy (HAART) and is a combination of anti-HIV reverse transcriptase inhibitors and protease inhibitors. HAART is capable of suppressing HIV replication and subsequently improving the patients’ survival. However, the issues associated with use of HARRT such as the high cost, severe side-effects, and drug resistance have called for development of alternative anti-HIV therapeutic strategies. In this study, we screened several traditional Chinese medicinal herbal extracts for their anti-HIV activities and determined their anti-HIV mechanisms. Methods: Nine traditional Chinese medicinal (TCM) herbal plants and their respective parts derived from Hainan Island, China were extracted using a series of organic solvents, vacuum dried, and dissolved in dimethyl sulfoxide. Initial anti-HIV activity and cytotoxicity of these extracts were evaluated in HIV-infected human CD4+ T lymphocytes Jurkat. Extracts of higher anti-HIV activities and lower cytotoxicity were selected from the initial screening, and further examined for their effects on HIV-1 entry, post-entry, reverse transcriptase, gene transcription and expression using combined virology, cell biology and biochemistry techniques. Results: Four extracts derived from two different herbal plants completely blocked HIV-1 replication and showed little cytotoxicity at a concentration of 10 g/ml. None of these four extracts had any inhibitory effects on HIV-1 long terminal repeat promoter. Two of them exhibited direct inhibitory activity against HIV-1 reverse transcriptase (RT). All four extracts showed significant blocking of HIV-1 entry into target cells. Conclusions: These results demonstrated that four TCM extracts were capable of preventing HIV-1 infection and replication by blocking viral entry and/or directly inhibiting the RT activity. These results suggest the possibility of developing these extracts as potential anti-HIV therapeutic agents.
HIV Tat Impairs Neurogenesis through Functioning As a Notch Ligand and Activation of Notch Signaling Pathway
(Society for Neuroscience., 2016-11-02) Fan, Yan; Gao, Xiang; Chen, Jinhui; Liu, Ying; He, Johnny J.; Neurological Surgery, School of Medicine
Alterations in adult neurogenesis have been noted in the brain of HIV-infected individuals and are likely linked to HIV-associated neurocognitive deficits, including those in learning and memory. But the underlying molecular mechanisms are not fully understood. In the study, we took advantage of doxycycline-inducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with fewer neuron progenitor cells (NPCs), fewer immature neurons, and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes or recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat or Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Last, Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo Together, these results show that Tat adversely affects NPCs and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HIV/neuroAIDS therapeutics. SIGNIFICANCE STATEMENT: HIV infection of the CNS causes cognitive and memory deficits, which have become more prevalent in the era of combination antiretroviral therapy (cART). Neurogenesis is impaired in HIV-infected individuals. But the underlying molecular mechanisms remain largely unknown. In this study, we have discovered that HIV Tat impairs neurogenesis through the Notch signaling pathway. These findings are particularly important because Tat protein has recently been detected in the brain of HIV-infected individuals with HIV replication in the periphery being effectively controlled by cART. The current study not only further highlights the importance of HIV Tat protein in HIV/neuroAIDS, but also presents a new strategy to develop novel HIV/neuroAIDS therapeutics, particularly in the era of cART.
HIV-1 NEF: THERAPEUTIC STRATEGIES AND VIROLOGICAL SYNAPSE-MEDIATED INFECTION
(2011-03-16) Green, Linden Ann; He, Johnny J.; Blum, Janice Sherry, 1957-; Georgiadis, Millie M.; Roman, Ann
HIV-1 infection is one of the greatest public health concerns today. The current HIV/AIDS therapy is effective in halting virus multiplication and has improved the outlook of AIDS; however, high cost, side effects, and the rise of drug-resistant viral strains have posed challenges for long-term treatment and management and mandate development of alternative anti-HIV therapies. Despite the fact that a great deal of progress has been made in our understanding of the infection over the last twenty-seven years, there are many unanswered basic scientific questions and no vaccines. In this study, we focused on two aspects related to the HIV-1 protein Nef: one is development of a Nef-based anti-HIV therapeutic strategy; the other is discovery of a novel mechanism that accounts for Nef-enhanced viral infectivity. We first devised an anti-HIV therapeutic strategy that took advantage of the high virion incorporation of the Nef mutant Nef7 to deliver anti-HIV factors to the virion. We performed a series of proof-of-concept experiments, using the host anti-HIV cellular factor APOBEC3G (A3G). The Nef7.A3G fusion protein retains important properties of Nef7: higher virion incorporation efficiency, lack of PAK2 activation, and reduced CD4 and MHC I downregulation, as well the anti-HIV infectivity function of A3G. Moreover, virus-like particle (VLP)-mediated delivery of Nef7.A3G into infected CD4+ T lymphocytes leads to inhibition of HIV-1 replication in these cells. These results support the use of Nef7 as an anti-HIV therapeutic strategy for the delivery of therapeutic proteins into HIV-1 virions. HIV-1 Nef protein has long been known to enhance viral infectivity. However, the underlying molecular mechanism remained elusive. Here we show that Nef is important for VS formation and VS-mediated virus transmission from cell to cell, especially in primary cells. Nef accomplishes this by inducing the clustering of VS components CD81 and ZAP70 and by inducing formation of actin protrusions, and these functions involve specific and distinct Nef domains. These findings not only yield new insights into the regulatory function of Nef in viral infectivity, but could also lead to development of more effective anti-HIV therapies that work equally well at blocking both VS-mediated and cell-free virus infection.
IMPAIRED FUNCTION OF FANCONI ANEMIA TYPE C DEFICIENT MACROPHAGES
(2012-03-16) Liu, Ying; Haneline, Laura S.; Dent, Alexander L.; He, Johnny J.; Srour, Edward F.; Yoder, Mervin C.
Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response.
THE INHIBITOR-OF-APOPTOSIS PROTEIN SURVIVIN INCREASES P34CDC2 PHOSPHORYLATION AND ENHANCES CELL SURVIVAL AND PROLIFERATION BY PROTECTING THE WEE1 KINASE FROM DEGRADATION BY CASPASE-3
(2009-09-30T20:10:46Z) Guzman, Javier Rivera; He, Johnny J.; Broxmeyer, Hal E.; Pelus, Louis M.; Nakshatri, Harikrishna
The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 are involved in cell cycle progression and apoptosis. Activation of Cdc2 is required for its pro-apoptotic activity, which can be inhibited by phosphorylation at Tyrosine-15 (Tyr15). In transduced IL-3-dependent murine BaF3 hematopoietic cells, over-expression of wild-type-(wt)-Survivin increased Cdc2-Tyr15 phosphorylation, while over-expression of a dominant-negative-(dn)-T34A-Survivin construct decreased its phosphorylation. The increased phospho-Tyr15 levels associated with ectopic Survivin directly correlated with enhanced BaF3 cell survival in the absence of growth factors, and low phospho-Tyr15 levels observed in cells expressing ectopic dn-Survivin correlated with decreased survival. BaF3 cells transduced with Internal Tandem Duplication (ITD) mutations of the Flt3 receptor that results in increased Survivin levels, also contained increased levels of Tyr15 phosphorylated Cdc2. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were observed compared to cells expressing control vector alone. Treatment of control BaF3 cells with the caspase-3 inhibitor Ac-DEVD-CHO increased both Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion over-expression of wt-Survivin in these cells maintained high levels of Tyr15 phosphorylated Cdc2 and Wee1 protein. In MCF7 human breast cancer cells that lack caspase-3, increase of Tyr15 phosphorylated Cdc2 and Wee1 kinase protein by caspase-3, -7 or a pan-caspase inhibitor was absent, linking Survivin and caspase-3 to the increase of Wee1 and Tyr15 phosphorylation of Cdc2. To further link Survivin and Cdc2, we treated cells with AICAR and 17-AAG that inhibit Hsp90, which is known to be required for Survivin stability. Treatment of BaF3 cells expressing wt-Survivin with AICAR and 17-AAG decreased Cdc2-Tyr15 phosphorylation compared to vehicle-treated control cells. Taken together, these results indicate that Survivin protects the Cdc2-Tyr15-targeting kinase Wee1 from degradation by caspase-3 which leads to increased inhibitory Cdc2-Tyr15 phosphorylation resulting in reduced apoptosis and enhanced survival.
Involvement of p300 in constitutive and HIV-1 Tat-activated expression of glial fibrillary acidic protein in astrocytes
(Wiley, 2010-10) Zou, Wei; Wang, Zhenyuan; Liu, Ying; Fan, Yan; Zhou, Betty Y.; Yang, X. Frank; He, Johnny J.; Microbiology and Immunology, School of Medicine
HIV-1 Tat protein is an important pathogenic factor in HIV-1-associated neurological diseases. One hallmark of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by elevated GFAP expression in astrocytes. We have shown that Tat activates GFAP expression in astrocytes (Zhou, et al., Mol. Cell. Neurosci. 27:296, 2004) and that GFAP is an important regulator of Tat neurotoxicity (Zou, et. al., Am. J. Pathol. 171:1293, 2007). However, the underlying mechanisms for Tat-mediated GFAP up-regulation are not understood. In the current study, we reported concurrent up-regulation of adenovirus E1a-associated 300 kDa protein p300 and GFAP in Tat-expressing human astroytoma cells and primary astrocytes. We showed that p300 was indeed induced by Tat expression and HIV-1 infection and that the induction occurred at the transcriptional level through the cis-acting elements of early growth response 1 (Egr-1) within its promoter. Using siRNA, we further showed that p300 regulated both constitutive and Tat-mediated GFAP expression. Moreover, we showed that ectopic expression of p300 potentiated Tat transactivation activity and increased proliferation of HIV-1-infected astrocytes, but had little effect on HIV-1 replication in these cells. Taken together, these results demonstrate for the first time that Tat is a positive regulator of p300 expression, which in turn regulates GFAP expression, and suggest that the Tat-Egr-1-p300-GFAP axis likely contributes to Tat neurotoxicity and predisposes astrocytes to be an HIV-1 sanctuary in the CNS.

Browsing by Author "He, Johnny J."

Results Per Page

Sort Options