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Approximately 15–40% of people living with HIV develop HIV-associated neurocognitive disorders, HAND, despite successful antiretroviral therapy. There are no therapies to treat these disorders. HIV enters the CNS early after infection, in... more
Approximately 15–40% of people living with HIV develop HIV-associated neurocognitive disorders, HAND, despite successful antiretroviral therapy. There are no therapies to treat these disorders. HIV enters the CNS early after infection, in part by transmigration of infected monocytes. Currently, there is a major opioid epidemic in the United States. Opioid use disorder in the context of HIV infection is important because studies show that opioids exacerbate HIV-mediated neuroinflammation that may contribute to more severe cognitive deficits. Buprenorphine is an opioid derivate commonly prescribed for opiate agonist treatment. We used the EcoHIV mouse model to study the effects of buprenorphine on cognitive impairment and to correlate these with monocyte migration into the CNS. We show that buprenorphine treatment prior to mouse EcoHIV infection prevents the development of cognitive impairment, in part, by decreased accumulation of monocytes in the brain. We propose that buprenorphine...
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Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been... more
Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contras...
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Lymphotropic strains of human immunodeficiency virus type 1 (HIV-1), including HTLV-IIIB, replicate poorly in macrophages. We have shown previously that lymphotropic HIV-1 fuses equally well with T lymphocytes and macrophages (M. J.... more
Lymphotropic strains of human immunodeficiency virus type 1 (HIV-1), including HTLV-IIIB, replicate poorly in macrophages. We have shown previously that lymphotropic HIV-1 fuses equally well with T lymphocytes and macrophages (M. J. Potash, M. Zeira, Z.-B. Huang, T. Pearce, E. Eden, H. Gendelman, and D. J. Volsky, Virology 188:864-868, 1992), suggesting that events in the virus life cycle following virus-cell fusion limit virus replication. We report here that HIV-1 DNA is synthesized efficiently in either ADA or HTLV-IIIB infected alveolar macrophages or monocyte-derived macrophages within 24 h of virus infection, as observed by polymerase chain reaction for amplification of viral DNA sequences from the gag gene. Infection by a cloned lymphotropic HIV-1 strain, N1T-A, also leads to viral DNA synthesis. However, circular viral DNA was detected during strain ADA infection but not during HTLV-IIIB or N1T-A infection of monocyte-derived macrophages. These findings indicate that during ...
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Membranes of murine lymphoma cells expressing H-2a antigens were isolated, purified and co-reconstituted with isolated Sendai virus envelopes according to a previously published procedure (Volsky, D.J. et al., Proc. Natl. Acad. Sci. USA... more
Membranes of murine lymphoma cells expressing H-2a antigens were isolated, purified and co-reconstituted with isolated Sendai virus envelopes according to a previously published procedure (Volsky, D.J. et al., Proc. Natl. Acad. Sci. USA 1979. 76: 5440.). The resulting hybrid H-2a/Sendai virus envelope vesicles (SH-2a vesicles) were capable of binding to and fusing with mouse lymphoma cells. The fusion resulted in the implantation of H-2a antigens into membranes of target cells, as demonstrated by the presence of serologically active antigens on cultured cells 8 and 16 h after implantation.
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Membranes of murine lymphoma cells expressing H-2a antigens were isolated, purified and co-reconstituted with isolated Sendai virus envelopes according to a previously published procedure (Volsky, D.J. et al., Proc. Natl. Acad. Sci. USA... more
Membranes of murine lymphoma cells expressing H-2a antigens were isolated, purified and co-reconstituted with isolated Sendai virus envelopes according to a previously published procedure (Volsky, D.J. et al., Proc. Natl. Acad. Sci. USA 1979. 76: 5440.). The resulting hybrid H-2a/Sendai virus envelope vesicles (SH-2a vesicles) were capable of binding to and fusing with mouse lymphoma cells. The fusion resulted in the implantation of H-2a antigens into membranes of target cells, as demonstrated by the presence of serologically active antigens on cultured cells 8 and 16 h after implantation.
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The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived... more
The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzymelinked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif -negativ...
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It is thought that interference during human immunodeficiency virus type 1 (HIV-1) infection is established by downmodulation of the principal virus receptor, CD4. Here we present evidence to the contrary. At various times after primary... more
It is thought that interference during human immunodeficiency virus type 1 (HIV-1) infection is established by downmodulation of the principal virus receptor, CD4. Here we present evidence to the contrary. At various times after primary infection, we superinfected T cells in vitro by exposure to a genetically distinct viral clone or to a virus carrying the chloramphenicol acetyltransferase gene. Replication of each virus strain was determined by restriction enzyme analysis of total cellular DNA, by PCR amplification of viral DNA, or by assay of cell extracts for chloramphenicol acetyltransferase activity. We found that efficient viral interference is established within 24 h of infection at a multiplicity of infection of 1. At that time, expression of viral structural proteins was low and infected cells displayed undiminished levels of surface CD4 and were fully susceptible to virus binding and fusion. Superinfection by either cell-free HIV-1 or cocultivation was blocked. Cells resis...
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We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992).... more
We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but n...
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The nature of the interaction between human immunodeficiency virus (HIV) and human cells of astrocytic origin was studied in vitro with cultured glial cells and intact HIV or infectious molecular clones of the virus. Infection of glial... more
The nature of the interaction between human immunodeficiency virus (HIV) and human cells of astrocytic origin was studied in vitro with cultured glial cells and intact HIV or infectious molecular clones of the virus. Infection of glial cells with intact HIV was characterized by low-level expression of viral transcripts as detected by Northern blotting and in situ hybridization (less than 10 copies of HIV RNA per cell), transient virus replication, absence of viral antigens detectable by immunofluorescence, and complete lack of cytopathic effects. However, the HIV-infected glial cells persistently expressed HIV tatIII gene activity as detected by a chloramphenicol acetyltransferase assay, and HIV transcripts could be detected by in situ hybridization in 20 to 30% of cells up to 4 months after infection, suggesting that the lack of cytopathicity in HIV-exposed cells was not due to transient viral infection. To evaluate whether increased expression and replication of HIV in glial cells...
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HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by... more
HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system's microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. These observations provide unique insights into glial crosstalk during disease by supporting astrocyte-mediated...
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The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain,... more
The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about astrocytes' consequences on MP. Here, we addressed this question using co-culture systems of HIV-infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent the absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes...
Research Interests: Immunology, Intercellular Communication, Biology, Medicine, HIV, and 15 moreHumans, Mice, Female, Animals, Microglia, Cell Death, Astrocyte, Central Nervous System, Hiv Infection, Astrocytes, Human immunodeficiency virus, Cell communication, AIDS Dementia Complex, Neuroimmune Pharmacology, and coculture techniques
Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuate monocyte-macrophage transmigration across the blood-brain barrier (BBB). A key factor in mediating these events is... more
Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuate monocyte-macrophage transmigration across the blood-brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1 (MCP-1). Upregulated glial-derived MCP-1 in HIV-1-infected brain tissues generates a gradient for monocyte recruitment into the nervous system. We posit that the inter-relationships between MCP-1, voltage-gated ion channels, cell shape and volume, and cell mobility underlie monocyte transmigration across the BBB. In this regard, MCP-1 serves both as a chemoattractant and an inducer of monocyte-macrophage ion flux affecting cell shape and mobility. To address this hypothesis, MCP-1-treated bone marrow-derived macrophages (BMM) were analyzed for gene and protein expression, electrophysiology, and capacity to migrate across a laboratory constructed BBB. MCP-1 enhanced K+ channel gene (KCNA3) and channel protein expression. Elect...
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Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy,... more
Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capab...
Research Interests: Biology, Medicine, Gene expression, Multidisciplinary, Amyotrophic Lateral Sclerosis, and 13 moreHumans, HIV-1 Associated Dementia (HAD), Temporal Lobe Epilepsy, Astrocyte, Astrocytes, Molecular cloning, Neurodegenerative Disease, Malignant Glioma, Base Sequence, Glutamic Acid, Glutamate Transporter, Glutamate Receptor, and Molecular Sequence Data
Epstein-Barr virus (EBV) receptors were implanted into the membranes of receptor-negative cells, using Sendai virus envelopes as vehicles. The presence of the receptors in the target cell membrane was demonstrated by monitoring the fate... more
Epstein-Barr virus (EBV) receptors were implanted into the membranes of receptor-negative cells, using Sendai virus envelopes as vehicles. The presence of the receptors in the target cell membrane was demonstrated by monitoring the fate of radioiodinated donor membranes. Receptors could be detected for at least 36 hr after implantation. [3H]Thymidine-labeled EBV bound efficiently to receptor-implanted cells but not to control cells. Binding was inhibited by an excess of nonlabeled virus. Of the [3H]thymidine-labeled EBV DNA, 50-75% was found inside the receptor-implanted, EBV-exposed cells 24 hr after the infection. The viral genome was functionally active in B lymphocyte-derived cell lines of human, murine, and baboon origin; in T lymphocyte-derived lines of human and murine origin; in mouse fibroblasts; and in freshly explanted mouse lymphocytes, as shown by the expression of EBV-determined nuclear, early, and viral capsid antigens.
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Research Interests: Data Analysis, Flow Cytometry, Biology, Cell Cycle, Inflammation, and 15 moreDNA replication, DNA repair, Medicine, Gene expression, Molecular Mechanics, Humans, Astrocyte, Genome Analysis, Astrocytes, Epidermal Growth Factor, Nervous System, Manganese Compounds, Gene expression profiling, Interferon gamma, and Chlorides
NF-κB is a transcriptional activator that often regulates inflammatory responses. We demonstrate that human immunodeficiency virus type 1 activates nuclear localization of NF-κB in macrophages in a manner dependent upon virus strain but... more
NF-κB is a transcriptional activator that often regulates inflammatory responses. We demonstrate that human immunodeficiency virus type 1 activates nuclear localization of NF-κB in macrophages in a manner dependent upon virus strain but independent of virus replication. Through the use of an inhibitor, NF-κB activation was found to be responsible for the cytokine and chemokine induction that we recently reported.
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Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and... more
Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the ...
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Research Interests: Pathogenesis, Biology, Medicine, HIV, Brain, and 10 moreVirus, Mice, Animals, Microglia, Interferon, Clinical Sciences, Chemokine, Neurosciences, Astrocytosis, and HIV infections
Copolymer-1 (COP-1) elicits neuroprotective activities in a wide range of neurodegenerative disorders. This occurs, in part, by adaptive immune-mediated suppression of microglial inflammatory responses. Because HIV infection and immune... more
Copolymer-1 (COP-1) elicits neuroprotective activities in a wide range of neurodegenerative disorders. This occurs, in part, by adaptive immune-mediated suppression of microglial inflammatory responses. Because HIV infection and immune activation of perivascular macrophages and microglia drive a metabolic encephalopathy, we reasoned that COP-1 could be developed as an adjunctive therapy for disease. To test this, we developed a novel animal model system that reflects HIV-1 encephalitis in rodents with both innate and adaptive arms of the immune system. Bone marrow-derived macrophages were infected with HIV-1/vesicular stomatitis-pseudotyped virus and stereotactically injected into the basal ganglia of syngeneic mice. HIV-1 pseudotyped with vesicular stomatitis virus envelope-infected bone marrow-derived macrophages induced significant neuroinflammation, including astrogliosis and microglial activation with subsequent neuronal damage. Importantly, COP-1 immunization reduced astro- an...
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Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions... more
Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotr...
Research Interests: Biology, Cytokines, Cell Division, HIV, Dexamethasone, and 15 moreArticles, Cerebral Cortex, Animals, Eicosanoids, Hiv Infection, Astrocytes, Cytokine, Human immunodeficiency virus, Experimental Medicine, Fetus, Base Sequence, Cell communication, Brain Neoplasms, Arachidonic Acid, and HIV infections
Research Interests: Molecular Biology, Flow Cytometry, Biology, Virology, Medicine, and 15 moreCell Culture, HIV, Cell line, Brain, Humans, Virus, P-glycoprotein, Monoclonal Antibodies, Astrocytes, Human Brain, Glial Fibrillary Acidic Protein, Receptor, Monoclonal Antibody, Biochemistry and cell biology, and ribonuclease
Background HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte... more
Background HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection. Results Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37°C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of ...
Research Interests: Cognitive Science, Epidemiology, Biology, Cytokines, Inflammation, and 15 moreCell Adhesion, HIV, Brain, Humans, Cell fusion, Astrocyte, Astrocytes, Green Fluorescent Protein, Human immunodeficiency virus, HeLa cells, Fusion Protein, Feline immunodeficiency virus, Biochemistry and cell biology, binding sites, and HIV infections
Research Interests: Immunology, Medicine, HIV, Humans, Child, and 7 moreFemale, Male, AIDS, Immunoglobulin E, Clinical Sciences, T lymphocytes, and HIV infections
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14)... more
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold...
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The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein... more
The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.
Research Interests: Chemistry, Kinetics, HIV, Homology Modeling, Humans, and 15 moreMice, Animals, Biochemical, Mouse Model, HeLa cells, Integrase, Fluorescence anisotropy, Mechanism of action, Hiv Integrase Inhibitors, Amino Acid Sequence, HIV Integrase, Catalytic Activity, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein... more
The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.
Research Interests: Chemistry, Kinetics, HIV, Homology Modeling, Humans, and 15 moreMice, Animals, Biochemical, Mouse Model, HeLa cells, Integrase, Fluorescence anisotropy, Mechanism of action, Hiv Integrase Inhibitors, Amino Acid Sequence, HIV Integrase, Catalytic Activity, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
OBJECTIVE Almost half of HIV-positive people on antiretroviral therapy (ART) have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal (IN) insulin therapy improves cognition in... more
OBJECTIVE Almost half of HIV-positive people on antiretroviral therapy (ART) have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal (IN) insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested IN insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. DESIGN AND METHODS Insulin pharmacokinetics following IN administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with IN insulin (2.4 IU/mouse) and examined for NCI in behavioral tests and HIV burdens by QPCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neural function related genes by RT-QPCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. RESULTS IN insulin administration to mice resulted in μIU/ml levels of insulin in CSF with a half-life of about 2 h, resembling pharmacokinetic parameters of patients receiving 40 IU. IN insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; IN insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24 h of treatment cessation. IN insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. CONCLUSIONS Our preclinical studies support the use of IN insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.Thisis an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.
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Gene set enrichment analysis (GSEA) is a microarray data analysis method that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. GSEA is especially useful when gene expression... more
Gene set enrichment analysis (GSEA) is a microarray data analysis method that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. GSEA is especially useful when gene expression changes in a given microarray data set is minimal or moderate. We developed a modified gene set enrichment analysis method based on a parametric statistical analysis model. Compared with GSEA, the parametric analysis of gene set enrichment (PAGE) detected a larger number of significantly altered gene sets and their p-values were lower than the corresponding p-values calculated by GSEA. Because PAGE uses normal distribution for statistical inference, it requires less computation than GSEA, which needs repeated computation of the permutated data set. PAGE was able to detect significantly changed gene sets from microarray data irrespective of different Affymetrix probe level analysis methods or different microarray platforms. Comparison of two aged mus...
Research Interests: Computer Science, Computational Biology, Statistical Analysis, Biology, Microarray Data Analysis, and 15 moreMedicine, Gene expression, Energy Metabolism, Biological Sciences, Humans, Diabetes mellitus, Mathematical Sciences, BMC Bioinformatics, Microarray Analysis, Gene Set Enrichment Analysis, Sensitivity and Specificity, Normal Distribution, Microarray Data, Parametric analysis, and Gene expression profiling
The rate-limiting steps in infection by human immunodeficiency virus type 1 (HIV-1) deficient in the viral infectivity factor, Vif, are unknown. As a measurement of completion of the early stages of the HIV-1 life cycle, the levels of... more
The rate-limiting steps in infection by human immunodeficiency virus type 1 (HIV-1) deficient in the viral infectivity factor, Vif, are unknown. As a measurement of completion of the early stages of the HIV-1 life cycle, the levels of viral DNA were examined by polymerase chain reaction amplification during infection by vif-positive and vif-negative viruses of MT-2 and H9 cells, in which vif is required for HIV-1 replication. Viral DNA was detected within hours of infection by both viruses, but the accumulation of vif-negative virus DNA was impeded in terms of both extent and kinetics. Inefficient viral DNA synthesis correlated with restricted replication of the vif-negative virus. Increasing the input dose of vif-negative virus increased viral DNA levels within 24 h of infection but failed to overcome the block to subsequent DNA synthesis and productive infection. Infection of C8166 cells, in which vif function is dispensable, resulted in efficient DNA synthesis by vif-positive and...
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Lymphocytes from eight preleukemia patients were exposed to Epstein-Barr virus (EBV) in vitro in an attempt to establish lymphoblastoid cell lines. No signs of viral infection were detected, and no cell lines were obtained. Studies using... more
Lymphocytes from eight preleukemia patients were exposed to Epstein-Barr virus (EBV) in vitro in an attempt to establish lymphoblastoid cell lines. No signs of viral infection were detected, and no cell lines were obtained. Studies using fluorescein-labeled EBV and flow cytometry revealed an unusual and consistent deficiency in EBV receptors in all patients examined. In control studies, about 15% of the unseparated lymphocytes from healthy donors bound fluorescein-labeled EBV. In spite of the lack of EBV receptors, B-lymphocytes amounted to 10 to 20% of the preleukemia lymphocyte populations, a proportion similar to that in healthy donors. When lymphocytes from preleukemic patients were first implanted with functional EBV receptors and then exposed to EBV, synthesis of EBV-determined nuclear, early, and viral capsid antigens was induced. Subsequently, several cell lines originating from preleukemic patients' lymphocytes were established. These lines are of a B-lymphocyte origin ...
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Thirty-nine patients from the Nebraska Regional Hemophilia Center were studied for the prevalence and titers of antibodies to HTLV-III/LAV, and for clinical symptoms of possible progression toward the acquired immunodeficiency syndrome... more
Thirty-nine patients from the Nebraska Regional Hemophilia Center were studied for the prevalence and titers of antibodies to HTLV-III/LAV, and for clinical symptoms of possible progression toward the acquired immunodeficiency syndrome (AIDS). Fourteen of 26 (54%) patients with hemophilia A were positive for HTLV-III/LAV antibodies as determined by immunofluorescence and Western blotting. Retrovirus was detected in cultured lymphocytes of two out of three seropositive individuals by reverse transcriptase assay and molecular hybridization with cloned HTLV-III/LAV probe. Of the twelve factor VIII-deficient patients who were seronegative, one had received only heat-treated factor VIII concentrates, six only cryoprecipitate or fresh frozen plasma, two prothrombin complex concentrates, one had not been transfused since 1983, and two had never been transfused. None of the patients treated only with factor IX concentrates, volunteer donor plasma, or cryoprecipitate had HTLV-III/LAV antibod...
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... By analysing the differential expression of cy-tochrome P450 in rats, it was found that the bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magni-tude (0.1 to 100... more
... By analysing the differential expression of cy-tochrome P450 in rats, it was found that the bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magni-tude (0.1 to 100 micrograms of total RNA). ...
Male homosexuals at risk for developing AIDS frequently exhibit chronic lymphadenomegaly (LAD). They are at high risk for developing malignant B cell lymphomas. A study of Epstein-Barr virus (EBV) revealed marked abnormalities in these... more
Male homosexuals at risk for developing AIDS frequently exhibit chronic lymphadenomegaly (LAD). They are at high risk for developing malignant B cell lymphomas. A study of Epstein-Barr virus (EBV) revealed marked abnormalities in these patients. One hundred percent of the patients were seropositive. The patients with most severe acquired immune deficiency disorders manifested a decreased number of circulating B cells with EBV receptors and decreased lymphocyte transformation. Patients often showed defective memory T cell cytotoxic responses to autologous EBV infection in vitro. Three of five lymph node specimens contain significant EBV genome copies to suggest a significant etiologic role. In addition, a Burkitt-like lymphoma carried EBV genome. Although all of the men were seropositive for EBV, reactivation patterns were not as common as anticipated. Given the presence of EBV genome in the lymph nodes of the patients who lack anti-early antigen (EA) antibodies indicative of reactivation, we suggest that reliance on serology to indicate EBV involvement is insufficient for assessing the patient. The detection of a t(8;14) transposition in the monoclonal mu kappa Burkitt-like lymphoma containing EBV genome supports the view that cytogenetic transposition is a mechanism in lymphomagenesis.