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Biomedical subjects

R J Pomerantz

Publications and source records attributed to R J Pomerantz.

137 records · Page 8Linked to original sources
PubMed1987

Therapy of human immunodeficiency virus infections.

Although therapy is currently available for many of the infections and neoplastic complications of AIDS, there is no effective therapy for the underlying immunodeficiency state. The authors review various attempts at treatment to date, and conclude that a major decrease in HIV-associated morbidity will require a combined approach, making use of our knowledge of the epidemiology, as well as the molecular and cellular biology, of the virus.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: direct selection from a combinatorial phage display library.

A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-1 (HIV-1). The library was panned against the HIV-1 Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10(-6)M to 10(-8)M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG1 heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22-33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52-64) and to the Rev activation domain (residues 75-88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-1 regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-1 replication in human cells.

Amino Acid Sequence↗
PubMedDate not supplied

Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells.

To determine the mechanism of glucocorticoid (GC)-mediated inhibition of T cell functions, the effect of dexamethasone (DM) on T cell proliferation and interleukin-2 receptor (IL-2R) generation were studied. Dexamethasone inhibited IL-2-induced T cell proliferation by 30%-88%, relative to its concentration within the cultures. The effect of DM on expression of IL-2R alpha (Tac, p55, CD25) and beta (p75) genes in activated T cells was examined next. In T cells stimulated with purified phytohemagglutinin (PHA-p) and 4 beta-phorbol 12-myristate 13-acetate (PMA) addition of DM to the cultures resulted in a 60% reduction in IL-2R alpha and a 30% reduction in IL-2R beta membrane expression compared to T cells cultured in the absence of DM (p < 0.01). Inhibition of membrane IL-2R alpha and IL-2R beta expression by 10(-6) M DM was partially reversible by recombinant human IL-2 (rhIL-2). By Northern blot analysis, DM caused a comparable decrease in IL-2R alpha and in IL-2R beta mRNA levels to membrane receptor expression in mitogen-stimulated T cells. By in vitro transcription assays, DM regulated IL-2R alpha gene expression at a transcriptional level while transcription of IL-2R beta gene was unaffected by DM. The mechanism of action of DM on IL-2R alpha transcription was examined by determining the mRNA levels of the p50 subunit of nuclear factor kappa B (NF-kappa B), a transcription factor that stimulates IL-2R alpha gene expression. The data indicate that 10(-6) M DM increased T cell p50 NF-kappa B mRNA levels by four-fold compared to the levels obtained in the absence of DM. Further, the level of nuclear proteins capable of binding to the NF-kappa B sites in activated T cells increased in response to DM. In sum, DM regulates T cell membrane expression of IL-2R by more than one molecular mechanism.

Base Sequence↗
PubMedDate not supplied

Human studies on alcohol and susceptibility to HIV infection.

Here we review existing evidence that alcohol intake may influence the susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and the effect that alcohol may have on accelerating the onset of AIDS after the initial infection. Possible immunological and psychosocial mechanisms to explain the increased incidence of HIV-1 infection in alcoholism are discussed.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

HIV-1 regulatory protein tat induces RNA binding proteins in central nervous system cells that associate with the viral trans-acting-response regulatory motif.

OBJECTIVES: To investigate some of the cellular consequences of HIV-1 Tat expression in human astrocytic cells. This study is based on evidence that cellular factors play a critical role in facilitating transcriptional activation by Tat through its interaction with the trans-acting-response (TAR) RNA element and upstream HIV-1 long terminal repeat (LTR) promoter binding site. STUDY DESIGN-METHODS: Using the previously established astrocytic cell line of human origin stably transfected with Tat cDNA, we analyzed the formation of a nucleoprotein complex consisting of three cellular proteins associated with TAR RNA using ultraviolet (UV) crosslinking and glutathione-S-transferase (GST) pull-down assays. RESULTS: UV crosslinking experiments reveal that the molecular masses of the proteins range from 50 to 62 kd. Transient transfection studies demonstrate that the presence of these proteins correlates with the ability of Tat to transactivate the HIV-1 LTR in the absence of the trinucleotide bulge, a region within TAR that has been shown to be important for Tat-TAR interaction. A combination of GST pull-down assays and RNA binding studies demonstrates that the 50-kd protein interacts with both Tat and TAR and is likely to be NF-kappa B p50. CONCLUSIONS: Taken together, these data suggest that in the absence of a functional Tat binding site such as TAR (which tethers the viral protein to the RNA), cellular protein NF-kappa B p50 may be able to bring Tat into the RNA binding complex Tat has been shown to activate expression of a variety of cellular genes that may not contain a binding site for Tat but do contain binding sites for NF-kappa B family members. The results presented in this study may be relevant for Tat-mediated transactivation of cellular as well as viral genes, both of which might contribute to the central nervous system damage associated with HIV-1 infection.

Astrocytes↗
PubMedDate not supplied

Analysis of HIV-1 in the cervicovaginal secretions and blood of pregnant and nonpregnant women.

OBJECTIVES: To detect HIV-1 in cellular and acellular fractions of cervicovaginal secretions obtained by cervicovaginal lavage (CVL) and evaluate viral genotypes in the HIV-1-positive CVL samples. STUDY DESIGN/METHODS: This study consists of 37 HIV-1-seropositive pregnant and nonpregnant women from the United States. A total of 63 paired CVL and blood samples were collected. HIV-1 DNA from cervical cells (CC) and virion RNA from cervical supernatant (CS) was detected by gag polymerase chain reaction (PCR) assays. The HIV-1 genotypes were determined by analyzing the nested PCR-amplified V3 region sequences of the HIV-1 gp120 envelope gene. RESULTS: Within this cohort, 95% of the women were on single or combination antiretroviral therapy. Of the pregnant women, 63% of samples had HIV-1 viral DNA in the CC, and 29% of samples were positive for viral RNA in the CS. Among nonpregnant women, 71% of samples were positive for HIV-1 DNA in CC, and 46% of samples tested positive for virion RNA in CS. Plasma viral load ranged between 10,000 and 100,000 copies/mL and showed significant correlation with the detection of HIV-1 RNA in the CVL; this relation was less apparent with viral DNA in CC. The viral blood and CVL specimens were further analyzed by evaluating the genotypes of HIV-1 variants. In most patients, a high degree of similarity was observed between the viral sequences derived from blood and CVL samples. Two patients demonstrated closely related but somewhat distinct genotypic variants in CVL and blood. One subject showed clear compartmentalization in which distinct viral genotypes were observed in CVL and blood. Based on V3 loop analyses of gp120, with one exception, the cervicovaginal secretions harbored viral populations with a macrophage (CCR5)-tropic phenotype. CONCLUSIONS: This study demonstrates the unique characteris tics of HIV-1 strains in the genital secretions of a relatively large cohort of HIV-1-infected women in the United States. These results are important for further analysis of HIV-1 transmission and pathogenesis in vivo and for rational vaccine design.

Adult↗
PubMedDate not supplied

Inhibition of HIV-1 replication and infectivity by expression of a fusion protein, VPR-anti-integrase single-chain variable fragment (SFv): intravirion molecular therapies.

OBJECTIVES: To deliver antiretroviral agents or other foreign proteins into progeny virions and evaluate their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication. STUDY DESIGN/METHODS: HIV-1 encodes proteins in addition to gag, pol, and env, some of which are packaged into virus particles. One essential retroviral enzyme is integrase (IN), which has been used as a target for developing agents that inhibit virus replication. In previous studies, we demonstrated that intracellular expression of single-chain variable antibody fragments (SFvs), which bind to IN, results in resistance to productive HIV-1 infection in T-lymphocytic cells. Because the highly conserved accessory HIV-1 Vpr protein can be packaged within virions in quantities similar to those of the major structural proteins, this primate lentiviral protein may be used as a fusion partner to deliver antiviral agents or other foreign proteins into progeny virions. In these studies, the fusion proteins Vpr-chloramphenicol acetyl transferase (CAT) and Vpr-SFv-IN have been developed. Stable transfectants expressing these fusion proteins were generated from PA317 cells and SupT1 T-lymphocytic cells and analyzed using immunofluorescence microscopy. After challenge of SupT1 cells with HIV-1, p24 antigen expression was evaluated. The incorporation of these fusion proteins were evaluated by immunoprecipitation of virions using a Vpr antibody. RESULTS: Expression of the fusion proteins was confirmed by immunofluorescent staining in PA317 cells transfected with the plasmids expressing Vpr-CAT and Vpr-SFv-IN proteins. Stable transfectants expressing these fusion proteins were generated from SupT1 T-lymphocytic cells. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression, was inhibited in cells expressing Vpr-SFv-IN. It was demonstrated that Vpr-chloramphenicol acetyl transferase (Vpr-CAT and Vpr-SFv-IN proteins can be efficiently packaged into the virions and that Vpr-SFv-IN also decreases the infectivity of virions into which it is encapsidated. CONCLUSIONS: An anti-integrase single-chain variable fragment moiety can be delivered into HIV-1 virions by fusing it to Vpr. Vpr-SFv-IN decreases HIV-1 production in human T-lymphocytic cells. The benefits of "intravirion" gene therapy include immunization of target cells as well as decreasing infectivity of HIV-1 virions harboring the fusion construct. Thus, this approach to anti-HIV-1 molecular therapies has the potential to increase inhibitory effects against HIV-1 replication and virion spread.

Anti-HIV Agents↗
PubMedDate not supplied

Morphologic changes in human immunodeficiency virus type 1 virions secondary to intravirion reverse transcription: evidence indicating that reverse transcription may not take place within the intact viral core.

INTRODUCTION: In the past, retroviral endogenous reverse transcription (ERT) was considered an artificial process, secondary to permeabilization of the viral envelope by detergents or amphipathic peptides. However, recently we have demonstrated that ERT may occur in a variety of lentiviruses without detergent treatment and may lead to increased infectivity of lentivirions in initially quiescent T lymphocytes and nonproliferating cells, such as macrophages. As full-length reverse transcripts could be synthesized within lentiviral particles, it is worth evaluating the potential alterations in lentiviral morphology due to the stimulation of intravirion reverse transcription. METHODS: Using quantitative DNA-polymerase chain reaction (PCR) and transmission electron microscopy (TEM), we characterized critical alterations in human immunodeficiency virus type 1 (HIV-1) virions after stimulation of intravirion reverse transcription. RESULTS: Intravirion reverse transcription in HIV-1 virions was stimulated using deoxyribonucleoside triphosphates (dNTPs) and physiologic polyamines. Our studies indicated that HIV-1 virions, in which intravirion reverse transcription was stimulated, showed dissolution of the p24-shelled viral core and absence of the core-envelope linkage (CEL) region by TEM. These changes in the structure of the core correlate with the in vitro alterations in virion infectivity on primary cells. CONCLUSIONS: Stimulation of intravirion HIV-1 reverse transcription leads to morphologic changes in the viral particles that suggest changes in the compact viral core, which is consistent with active reverse transcription before infection of target cells. Further, via this unique approach, we suggest that intravirion or intracellular reverse transcription of HIV-1 is unlikely to take place within intact viral cores made up of p24-containing outer shells. As such, these results suggest a new approach to further dissect the intravirion or intracellular reverse transcription machinery of lentiviruses.

DNA, Viral↗
PubMedDate not supplied

The nuclear function of the nuclear diffusion inhibitory signal of human immunodeficiency virus type 1: critical roles in dominant nuclear localization and intracellular stability.

OBJECTIVES: Human immunodeficiency virus type 1 (HIV-1) Rev is a nucleocytoplasmic shuttling protein with dominant localization in the cell nucleus/nucleolus. To clarify the mechanism(s) that enables such a biased subcellular localization under the co-presence of nuclear/nucleolar targeting (NOS) and nuclear export signals (NES), the properties of another functional domain, a nuclear diffusion inhibitory signal (NIS), was investigated. STUDY DESIGN: The NIS was previously shown to interfere with passive nuclear entry of small proteins. Intracellular distribution and degradation profiles of Rev mutants that harbor mutations in the NIS were analyzed biochemically and cellbiologically. RESULTS: A NIS-deficient Rev mutant, which was no longer functional as Rev, displayed a significantly more rapid degradation profile as a consequence of its dramatic leakage into the cytoplasm. Additionally, disabling the NOS/nuclear localization signal (NLS), as well as the NIS, resulted in further rapid degradation, which also supports the hypothetical role of the nucleolus as a Rev storage site. CONCLUSIONS: It was uncovered that the NIS is playing a key role in HIV-1 Rev function by maintaining the nuclear-dominant localization and the intracellular stability of Rev.

Amino Acid Sequence↗
PubMedDate not supplied

Development of an in vitro blood-brain barrier model to study molecular neuropathogenesis and neurovirologic disorders induced by human immunodeficiency virus type 1 infection.

OBJECTIVE: An in vitro blood-brain barrier (BBB) system was developed using primary cultures of human brain-derived microvascular endothelial cells (MVECs), macrophages, neuronal cells, and human fetal astrocytes. This BBB system simulates important morphologic and permeability characteristics of the BBB in vivo. This system could be used to study human neurologic/neurovirologic disorders. STUDY DESIGN AND METHODS: Microvascular endothelial cells were cultured to 100% confluency on the upper side of 0.45-microm polyethylene tetraphthalte (PET) membrane inserts coated with MVEC attachment factor. Expression of ZO-1 (Zona Occludens 1), a protein specifically associated with tight junctions and the intercellular sealing of adjacent MVECs, was analyzed by immunocytochemical methods. The integrity of the BBB formed on the insert membrane was also assessed by the measurement of electrical current passage through the membrane; and, after the formation of complete BBB, a two-compartment system was developed using the cell culture insert upper surface, essentially a confluent monolayer of brain-derived MVECs housed on a six-well chamber surface. In the six-well chamber surface, there are different central nervous system (CNS)-based cells, ie, human astrocytes, immature neurons, mature neurons, and MVEC. The cell culture inserts were in close juxtaposition to the surface of the chamber, making an intimate contact with the cells, separated by an insert membrane. The MVEC surface of the insert was exposed to human immunodeficiency virus type 1 (HIV-1) strains 89.6, NL4-3, and IIIB. After 72 hours, the cells were fixed and used for in situ polymerase chain reaction (IS-PCR), whereas the supernatant was subjected to HIV-1 p24 antigen determination. RESULTS: Primary human brain MVECs are capable of forming tight junctions, revealed by the expression of ZO-1, as well as elevated transendothelial electrical resistance. Based on these characteristics, these cells formed an in vitro BBB, which then was used to study the transfer of HIV-1 through this barrier. It was observed that HIV-1 can infect MVEC and can cross it in vitro and infect the cells growing on the opposite side of the membrane. Infection of various CNS-based cells was confirmed by IS-PCR, as well as by HIV-1 p24-antigen determination. It was observed that the dual-tropic strain, 89.6, had a greater potential to create a breach in the in vitro BBB, followed by NL4-3 and IIIB. CONCLUSION: This model system is relevant for evaluating HIV-1 neuropathogenesis and therapeutics designed to alter HIV-1 expression in human CNS-based cells. As such, the effects of highly active antiretroviral therapy on HIV-1 infection of the human CNS, a possible drug sanctuary site, can be evaluated using this technology.

AIDS Dementia Complex↗