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R J Pomerantz

Publications and source records attributed to R J Pomerantz.

At least 73 records · Page 4Linked to original sources
PubMed1998

Chemokine receptors and the molecular basis for human immunodeficiency virus type 1 entry into peripheral hematopoietic stem cells and their progeny.

There exist at least two major coreceptors for human immunodeficiency virus (HIV)-1 entry into target cells, the CXCR-4 and CCR-5 chemokine receptors for T lymphocyte-tropic and macrophage-tropic strains of HIV-1, respectively. Highly purified human CD34 cells derived from umbilical cord blood were shown not to express CD4, CXCR-4, and CCR-5 on their cell membranes, as analyzed by immunofluorescent staining and flow cytometric analyses. However, expression of these molecules was inducible when highly purified CD34 cells underwent proliferation and differentiation along myeloid cell lineages, in the presence of suitable cocktails of hematopoietic growth factors. HIV-1 infectivity studies showed that macrophage-tropic strains of HIV-1 could efficiently infect differentiated CD34 cells. T lymphocyte-tropic strains could not infect CD34 cells before or after induction of receptors and coreceptors. These data suggest that HIV-1 infection of CD34 cells and their progeny depends on membrane expression of the critical CD4 receptor, as well as certain chemokine coreceptors.

Antigens, CD↗
PubMed1998

Isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (Fab RSVF2-5) that exhibits therapeutic efficacy in vivo.

A second human respiratory syncytial virus (RSV)-neutralizing monoclonal antibody was isolated and its binding site was identified. Fab F2-5 is a broadly reactive fusion (F) protein-specific recombinant Fab generated by antigen selection from a random combinatorial library displayed on the surface of filamentous phage. In an in vitro plaque-reduction test, the Fab RSVF2-5 neutralized the infectivity of a variety of field isolates representing viruses of both RSV subgroups A and B. The Fab recognized an antigenic determinant that differed from the only other human anti-F monoclonal antibody (RSV Fab 19) described thus far. A single dose of 4.0 mg of Fab RSVF2-5/kg of body weight administered by inhalation was sufficient to achieve a 2000-fold reduction in pulmonary virus titer in RSV-infected mice. The antigen-binding domain of Fab RSVF2-5 offers promise as part of a prophylactic regimen for RSV infection in humans.

Animals↗
PubMed1998

Novel inhibitory effects of gamma-glutamylcysteine ethyl ester against human immunodeficiency virus type 1 production and propagation.

The anti-human immunodeficiency virus type I (anti-HIV-1) effects of gamma-glutamylcysteine ethyl ester (gamma-GCE; TEI-2306) were examined in vitro. In initial studies using a vigorously HIV-1-producing human T-lymphocytic cell line, gamma-GCE displayed a novel biphasic repressive effect on chronic HIV-1 infection that was unlike that of other glutathione prodrugs or other reported antioxidants. In high doses, up to a concentration of 2.5 mM, at which neither glutathione (GSH) nor another GSH precursor has shown inhibitory effects, gamma-GCE potently inhibited the production of HIV-1 by a selective cytopathic effect against infected cells, while the viability and growth of uninfected cells were unaffected at the same gamma-GCE concentrations. At lower concentrations (200 to 400 microM), gamma-GCE significantly repressed the virus production from chronically HIV-1-expressing cells without affecting their viability. The discrepancy of the thresholds of the toxic doses between infected and uninfected cells was found to be more than 10-fold. Relatively high doses of gamma-GCE, utilized in acute HIV-1 infection of T-lymphocytic cells, entirely blocked the propagation of HIV-1 and rescued the cells from HIV-1-induced cell death. Furthermore, gamma-GCE at such concentrations was found to directly inhibit the infectivity of HIV-1 within 4 h. Repressive effects of gamma-GCE on acute HIV-1 infection in human primary human peripheral blood mononuclear cells were also demonstrated. Here, the anti-HIV-1 strategy utilizing gamma-GCE is removal of both HIV-1-producing cells and free infectious HIV-1 in vitro, in place of specific immunoclearance in vivo, which might lead to an arrest or slowing of viral propagation in HIV-1-infected individuals.

Anti-HIV Agents↗
PubMed1998

Natural endogenous reverse transcription of HIV type 1.

Molecular mechanisms by which human lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are sexually transmitted have yet to be fully elucidated. It is now demonstrated that endogenous reverse transcription of lentiviruses can occur within the intact virion, before infection of target cells. This is a biochemically active process and is altered by the microenvironment to which HIV-1 virions are subjected. Stimulation of endogenous reverse transcription within virion particles, without nonphysiological permeabilization, has been called natural endogenous reverse transcription (NERT). This molecular mechanism has been shown to augment HIV-1 infection in initially quiescent peripheral blood mononuclear cells (PBMCs) as well as nonproliferating macrophages. As such, this process may be important in augmenting the sexual transmission of HIV-1, as genital secretions have been shown to stimulate NERT within HIV-1 virion particles. Further studies are planned to elucidate fully this initial molecular mechanism, which may be critical in understanding the sexual transmission of HIV-1 and therefore the spread of the AIDS pandemic.

HIV Infections↗
PubMed1997

Natural endogenous reverse transcription of simian immunodeficiency virus.

It has been demonstrated that human immunodeficiency virus type 1 (HIV-1) virions are biochemically active particles, within which reverse transcription can take place even in physiological microenvironments. This process has been termed "natural endogenous reverse transcription" (NERT). In this report, we demonstrate that purified virions of simian immunodeficiency virus (SIV) also contain virus-specific DNA, which resulted from partial reverse transcription. Further, viral DNA synthesis could be initiated in SIV virions in the presence of polyamines and deoxyribonucleoside triphosphates (dNTPs), at physiological concentrations. The viral infectivity upon initially quiescent cells was significantly increased, when the levels of intravirion reverse transcripts were modulated. These data suggest that NERT of SIV may play an important role for SIV pathogenesis and transmission. In contrast to HIV-1, these hypotheses may be further directly investigated by in vivo model systems.

Blood↗
PubMed1997

Intracellular inhibition of HIV-1 replication using a dual protein- and RNA-based strategy.

Exporting unspliced human immunodeficiency virus type 1 (HIV-1) RNA from the nucleus to the cytoplasm, through an interaction between the viral regulatory Rev protein and Rev response element (RRE) RNA, is a critical step in the HIV-1 life-cycle. Disruption of either Rev or the RRE will completely inhibit HIV-1 replication. As such, a strategy for somatic gene therapy to treat HIV-1 infection by intracellular expression of an anti-HIV-1 Rev single chain variable fragment (SFv) and a ribozyme which specifically targets the RRE was developed. The anti-Rev D8SFv, which specifically targets the Rev activation domain, may be a key component of combination intracellular immunization, as it has been previously shown to potently inhibit Rev function, thereby inhibiting viral replication. In the present studies, different HIV-1 RRE region-specific hammerhead ribozymes were constructed and their anti-HIV-1 replication effects were assayed in diverse RNA polymerase (pol) II and III promoters and vector systems in cell culture. Utilizing this combination of an SFv and a ribozyme as a dual strategy to block HIV-1 replication, both at the protein and RNA level, data from these studies demonstrated that potent inhibition of HIV-1 replication can be achieved via this approach. Combination gene therapies hold promise, analogous to combination chemotherapeutic regimens, for the in vivo treatment of HIV-1 infections.

Base Sequence↗
PubMed1997

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Absence of the inducible form of nitric oxide synthase in the brains of patients with the acquired immunodeficiency syndrome.

A majority of human immunodeficiency virus type I (HIV-1)-infected-individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including acquired immune deficiency syndrome (AIDS)-dementia complex (ADC), encephalitis, and various other disorders of the CNS. A series of devastating clinical conditions in the CNS of certain HIV-1-infected-individuals may be caused by infection of cells in the brain parenchyma. ADC is characterized by cognitive dysfunction, motor difficulties, coordination abnormalities and other neurological signs and symptoms, which develop in many HIV-1-infected-individuals. The precise molecular mechanisms leading to AIDS dementia remain incompletely explained. Various mechanisms including cytokine dysregulation, toxic effects of viral proteins and release of certain toxic substances from macrophages, especially nitric oxide, have been implicated as pathogenic mediators in the development of ADC. We have examined post mortem CNS tissues collected from 22 patients, previously diagnosed with AIDS, to explore if nitric oxide is responsible for the observed pathology in ADC. As controls, we utilized tissues collected from the brains of patients who expired without AIDS or other CNS pathologies. In addition, we also utilized post-mortem brain tissues from eight patients who were diagnosed with multiple sclerosis (MS) and were found to express inducible nitric oxide synthase (iNOS) in our previous studies, as positive controls. Highly sensitive in situ reverse transcriptase-initiated polymerase chain reaction (RT-IS-PCR) studies demonstrated that iNOS mRNA was present in the CNS tissues from all the positive MS controls, but were absent in all 22 specimens from AIDS patients, as well as in the brain tissues from normal controls. We have also analyzed the tissues for the presence of the NO reaction product, nitrotyrosine, to evaluate the presence of a protein nitrosalation adduct. Nitrotyrosine was not demonstrable in any of the AIDS brains. These findings indicate that iNOS may not play a significant role in the neuropathogenesis of most cases of ADC.

AIDS Dementia Complex↗
PubMed1996

Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription.

Reverse transcription of HIV-1, without detergent or amphipathic peptide-induced permeability of the viral envelope, has been demonstrated to occur in the intact HIV-1 virion. In this report, we demonstrate that the amphipathic domains in the C terminus of the transmembrane glycoprotein (gp41) account for the natural permeability of the HIV-1 envelope to deoxyribonucleoside triphosphates, the substrates for DNA polymerization. In addition, nonphysiological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorporate into, and irreversibly terminate reverse transcripts. As a result, viral infectivity is potently inhibited. Since the lentiviral envelope with these newly demonstrated characteristics can serve as a delivery pathway for anti-reverse transcription agents, we propose a unique strategy to prevent HIV-1 interand, possibly, intrahost transmission.

Animals↗
PubMed1996

TAR- and Tat-independent replication of human immunodeficiency virus type 1 in human hepatoma cells.

The molecular mechanisms involved in the regulation of human immunodeficiency virus type 1 (HIV-1) replication may differ in various cell types and with various exogenous stimuli. TAR/Tat interactions play important roles in HIV-1-long terminal repeat (LTR)-directed transcription, and have become specific targets in molecular therapies for blocking HIV-1 replication. As we previously reported, astrocytic glial cells, which can support HIV-1 replication in cell culture and may be infected in vivo, provide an intracellular milieu in which TAR mutant HIV-1 viruses may replicate. In further studies of this molecular model, several divergent human cell types were analyzed for both TAR- and Tat-independent HIV-1 replication. Human hepatoma cell lines, which can be productively infected by HIV-1 after the hepatoma cells are transduced with the human CD4 receptor gene, were found to support high levels of HIV-1 replication. In these studies, utilizing a transient transfection system with wild-type and various TAR, Tat, or combined TAR/Tat mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated human hepatoma cells. Remarkably, in human hepatoma cells, HIV-1 replication is not only independent of TAR but also can be independent of Tat expression. It is further demonstrated, using electrophoretic mobility shift assays (EMSAs) and an in situ UV cross-linking system, that human hepatoma cells contain novel endogenous cellular proteins that bind to the proviral HIV-1 5' LTR in the downstream region, between nucleotides +38 to +125 on proviral DNA. This alternative regulatory pathway of TAR- and Tat-independent viral production may provide a new system to dissect further the interactions of Tat/TAR and determine the role of the TAR element, in its DNA form, in HIV-1 replication.

Base Sequence↗
PubMed1996

Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell.

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.

Astrocytes↗
PubMed1996

Nucleo-cytoplasmic redistribution of the HTLV-I Rex protein: alterations by coexpression of the HTLV-I p21x protein.

The function of the Rex protein of human T-cell leukemia virus type I (HTLV-I) has been demonstrated to be very similar to the Rev protein of human immunodeficiency virus type 1 (HIV-1). Both of these retroviral regulatory proteins rescue unspliced viral RNAs from the nuclei of infected cells. The Rev protein of HIV-1 has been reported to shuttle between the nucleus/nucleolus and the cytoplasm. Here, we have found that Rex also relocated out of the nucleus in the presence of actinomycin D. This effect was demonstrated in dose- and time-course-dependent manners. In comparison with previous reports on HIV-1 Rev, these effects with Rex seemed to be similar, but less distinct, which may reflect precise differences in the subcellular localization and/or shuttling pathways of Rev and Rex. Interestingly, the endogenous truncated form of the Rex protein, p21x, significantly interfered with the intracellular translocation of Rex, when coexpressed in trans. As expression of p21x occurs in various HTLV-I-infected cells, p21x may play a role in the life-cycle of HTLV-I, through regulating the dynamic subcellular distribution of the viral trans-activator, Rex.

Animals↗
PubMed1996

Cis/trans-activation of the interleukin-9 receptor gene in an HTLV-I-transformed human lymphocytic cell.

The MT-2 cell-line, which had been established through in vitro cell to cell transmission of human T-cell leukemia virus type I (HTLV-I) among human primary lymphocytes, was shown to possess multiple copies of integrated proviruses, including defective proviral genomes. By analysing a genomic clone, we identified the integration site of a single HTLV-I long terminal repeat (LTR) in the interleukin-9 (IL-9) receptor (IL-9R) gene. The integrated HTLV-I-LTR was shown to be functional as a promoter and the integration site was located in an intron upstream of the first coding exon of the IL-9R gene. Upon analysis of total cellular RNA, specific expression of HTLV-I-LTR Il-9R chimeric mRNAs in MT-2 cells was demonstrated. Cloning and characterization of these cDNAs have identified HTLV-I-IL-9R chimeric splicing, using either intact or alternative splice sites within the IL-9R gene. The potential roles of multiple interactions between IL-9, IL-9R and HTLV-I in the monoclonal expansion and transformation of MT-2 cells are explored.

Base Sequence↗
PubMed1996

Increased human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms.

Previous studies have shown that alcohol ingestion significantly increases human immunodeficiency virus type 1 (HIV-1) replication in peripheral blood mononuclear cells (PBMC) isolated and infected with HIV-1 in vitro. Whether the increased replication of HIV-1 observed after alcohol ingestion was due to unknown factors released from the gastrointestinal tract during alcohol ingestion or to certain metabolites produced by intestinal flora that degraded alcohol was investigated. In addition, cellular mechanisms involved in the increased replication of HIV-1 after alcohol exposure were evaluated. Twelve healthy HIV-1-seronegative subjects abstained from alcoholic beverages for >10 days. Nine were infused with 500 mg/kg ethanol (7.5% at 20 ml/kg/h) in saline, whereas 3 were infused with saline alone. Compared with saline-infused subjects, ethanol-infused subjects' PBMC exhibited significantly increased replication of HIV-1 when infected in vitro, which was associated with increased inhibition of CD8+ T lymphocytes' function by alcohol.

Adolescent↗
PubMed1996

Human herpesvirus type 8 DNA sequences in cell-free plasma and mononuclear cells of Kaposi's sarcoma patients.

Human herpesvirus (HHV) type 8 has been detected in both classical and AIDS-related Kaposi's sarcoma, body-cavity lymphomas, and other types of tumors. HHV-8 has also been detected in DNA from peripheral blood mononuclear cells (PBMC) of some Kaposi's sarcoma patients and more readily in B cell fractions derived from panned cell subpopulations. Two patients were followed using several methods; in situ hybridization, solution-based polymerase chain reaction (PCR), and in situ PCR. HHV-8 was intermittently detected in plasma, and detection correlated with detection in PBMC. In situ PCR demonstrated HHV-8 sequences in both peripheral blood B lymphocytes and, to a lesser extent, T lymphocytes. HHV-8 may undergo periods of viremia while at other times it is undetectable and infects circulating B cells and some T cells.

Adult↗
PubMed1996

Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry.

OBJECTIVES: The majority of HIV-1-infected individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including AIDS dementia complex, encephalitis, and various other disorders of the CNS. The present study sought to evaluate the cellular reservoirs and expression patterns of HIV-1 in brain tissue to gain further understanding of HIV-1 neuropathogenesis. DESIGN: CNS tissue, obtained post-mortem from 22 patients with AIDS and four HIV-1-seronegative controls, was analyzed. METHODS: CNS samples were evaluated using a combination of in situ DNA polymerase chain reaction (PCR), reverse transcriptase (RT)-initiated in situ PCR, and immunohistochemistry. By utilizing this triple-staining methodology, HIV-1 proviral DNA and HIV-1-specific mRNA can be identified at the single cell level. RESULTS: HIV-1 was detected in all 22 AIDS brain specimens and in none of the four brains from HIV-1-seronegative individuals. The most commonly infected cells in AIDS brains were microglia cells and macrophages, but variable levels of HIV-1 infections were demonstrated in many of the major histological cell types within the CNS, including neurons, microvascular endothelial cells (MVEC) and astrocytes. The presence of HIV-1-infected cells was not uniform with infected cells unevenly distributed throughout the brain parenchyma. The degree of HIV-1 mRNA expression varied from 39-65% of the cells in the CNS harboring HIV-1 provirus. Choroid plexus and MVEC exhibited relatively high levels of productive infection. CONCLUSION: These findings demonstrate that several cell types in the CNS, in addition to microglia or macrophages, may become infected with HIV-1 in vivo with variable levels of HIV-1 mRNA expression. The diverse cellular reservoirs for HIV-1 in the CNS may be critically linked to the molecular mechanisms involved in HIV-1 neuropathogenesis. In addition, in vivo infection of MVEC, and possibly cells in the choroid plexus, may directly contribute to penetration of the blood-brain barrier by HIV-1.

Adult↗