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R J Pomerantz

Publications and source records attributed to R J Pomerantz.

At least 109 records · Page 6Linked to original sources
PubMed1994

Association of alterations in NF-kappa B moieties with HIV type 1 proviral latency in certain monocytic cells.

Human immunodeficiency virus type 1 (HIV-1) replication is controlled by a complex array of virally encoded and cellular proteins. A wide spectrum of levels of HIV-1 expression have been demonstrated in various cells, both in cell culture and in vivo. Molecular mechanisms leading to restricted HIV-1 replication may differ between certain cell types. It is now demonstrated that HIV-1 proviral latency in the monocytic cell line U1, in which only extremely low levels of HIV-1 expression are detected in the baseline unstimulated state, is associated with alterations in nuclear factor-kappa B (NF-kappa B) moieties demonstrated in these cells by electrophoretic mobility shift assays (EMSAs) and in situ UV cross-linking studies. A predominance of p50 NF-kappa B moieties and possibly p50 homodimers or closely related species, rather than the p50-p56 heterodimer of NF-kappa B that is the predominant NF-kappa B species in most T lymphocytic and monocytic cells, is demonstrated in the nuclei of U1 cells. This pattern of NF-kappa B-related moieties differs from the latently infected T lymphocytic cell line ACH-2, and from the U937 monocytic line, the parental cell line of the U1 cellular clone. As such, these data suggest that different proximal mechanisms may lead to restricted HIV-1 replication in various cell types.

Base Sequence↗
PubMed1994

Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains.

A variety of genetic therapies or intracellular immunization techniques hold promise as modalities to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vivo. We have recently demonstrated that a single-chain variable fragment (SFv) construct, derived from a monoclonal antibody that binds to the HIV-1 regulatory protein Rev, can be expressed intracellularly and potently inhibits HIV-1 replication. This single-chain intracellular antibody, which avidly binds to the effector domain of Rev, is now demonstrated to dramatically inhibit various diverse laboratory and primary clinical strains of HIV-1. Potent suppression of HIV-1 replication by this modality is maintained over several months in long-term cultures. As well, the intracellular expression of anti-Rev SFv is shown to alter HIV-1 replication by specifically affecting Rev function. Importantly, no alterations in HIV-1 internalization, reverse transcription, or initial transcription of multiply spliced viral mRNAs are demonstrated in SFv-immunized cells, as compared to controls. Thus, these studies extend the understanding of the molecular mechanisms involved in the inhibition of lentivirus replication, by these intracellular antibody constructs.

Amino Acid Sequence↗
PubMed1994

Detection of HIV-1 proviral DNA in sperm from HIV-1-infected men.

OBJECTIVE: Sexual transmission is a major mode of the spread of HIV-1, although the cellular and molecular mechanisms are poorly defined. In this study, we sought to assess the cellular reservoirs of HIV-1 proviral DNA in the semen of HIV-1-infected men. DESIGN AND METHODS: An in situ polymerase chain reaction (IS-PCR), which amplifies specific genes within intact cells, was used to evaluate levels of HIV-1 provirus in seminal cells from HIV-1-infected men in various stages of clinical disease. RESULTS: Initial studies demonstrated HIV-1 provirus in relatively low numbers (1:100 to 1:6000) of both the seminal mononuclear cells and sperm from certain HIV-1-infected men. To extend these findings, 94 seminal samples from HIV-1-infected men were evaluated. HIV-1 proviral DNA was detected in seminal cells of a significant percentage of HIV-1-infected men (45%) at all stages of clinical immunodeficiency. Both seminal mononuclear cells and sperm (35 and 33% of samples studied, respectively) harbored HIV-1 proviral sequences. HIV-1-harboring sperm are shown to stain positively for HIV-1 in the mid-pieces of these cells, with rarer staining of the sperm heads. CONCLUSIONS: HIV-1 proviral DNA can be demonstrated by IS-PCR in seminal mononuclear cells and sperm from certain HIV-1-infected men. The role played by proviral DNA in these cells in the sexual transmission of this retroviral agent will require further study.

CD4 Lymphocyte Count↗
PubMed1994

Intravirion reverse transcripts in the peripheral blood plasma on human immunodeficiency virus type 1-infected individuals.

Variable levels of viral DNA have been demonstrated within human immunodeficiency virus type 1 (HIV-1) virions purified from cell cultures. In the present studies, it is demonstrated that DNase-resistant viral DNA is associated with HIV-1 virions purified from the peripheral blood plasma of both symptomatic and asymptomatic HIV-1-infected individuals. The differences in viral DNA copy numbers, detected by quantitative PCR in various regions of the HIV-1 genome, indicated that the intravirion HIV-1 DNA is frequently, but perhaps not totally, the result of partial reverse transcription. These in vivo data suggest that it may be valuable to further investigate the impact of virion-associated viral DNA upon the efficiency of intra- and interhost HIV-1 transmission modes.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.

Astrocytes↗
PubMed1994

The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication.

The p53 tumor suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act as a transcriptional activator and repressor both in vitro and in vivo. Consistent with its role in regulating transcription are recent observations that the N-terminal acidic domain of p53 binds directly to the TATA box-binding protein subunit of the general transcription factor, TF IID. It is now demonstrated that wild-type p53 (wt-p53) inhibits human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed chloramphenicol acetyltransferase activity in a cotransfection assay system. Importantly, this effect of wt-p53 on the HIV-1 LTR was also demonstrated by in vitro transcription assays. In addition, the Sp1 sites and the TATA box of the HIV-1 LTR are demonstrated to be the primary sites involved with p53-induced effects on this viral promoter. The upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) binding sites, decrease the p53-induced inhibitory effects on viral transcription. In the presence of the HIV-1 TAR sequence and Tat protein, the HIV-1 LTR also becomes less sensitive to wt-p53-induced inhibition. By using a retroviral vector delivery system, mutant forms of p53 genes were expressed in two HIV-1 latently infected cell lines, ACH-2 and U1. In the ACH-2 cell line, which is now demonstrated to contain an endogenous mutant form of p53 (amino acid 248, Arg to Gln), additional mutant p53 proteins did not alter HIV-1 replication. In U1 cells, which completely lack endogenous p53, overexpression of mutant p53 led to an increase in HIV-1 replication. Thus, these data indicate a possible functional role for wt-p53 and mutant p53 proteins in the control of HIV-1 replication patterns and proviral latency.

Base Sequence↗
PubMed1994

Cellular latency in HIV-1 infection.

There are multiple levels of transcriptional regulation that a cell can bring to bear on either native cellular genes or integrated proviral genes. One such level of regulation is inducible sequence-specific transcriptional factors, such as NF-kappa B, which have been extensively investigated with respect to the HIV-1 LTR. This level of control can be modified by organization of the integrated HIV-1 provirus into higher order chromatin structures such as nucleosomes and chromatin domains. The overall level of proviral transcriptional activity within any given cell depends on the relative contribution of each of these levels of control.

Acquired Immunodeficiency Syndrome↗
PubMed1994

In situ polymerase chain reaction and HIV-1.

The study of low-copy viral or genomic DNA sequences by in situ hybridization is often limited by sensitivity. The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology, and cell biology. One of the limitations of in situ hybridization, however, is detection and quantitation of very low levels of nucleic acid targets where the signal is insufficient to distinguish it clearly from background noise. The advent of polymerase chain reaction technology to amplify target nucleic acids provides an opportunity to develop new technologies to examine this end of the spectrum of gene expression or infection.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Polymerase chain reaction in situ: intracellular amplification and detection of HIV-1 proviral DNA and other specific genes.

The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology and cell biology. We report here a unique, highly sensitive and specific technique which can be utilized to detect a single copy of human immunodeficiency virus type I (HIV-1) provirus and other genes, at the single cell level, by in situ amplification of a portion of a gene sequence. In this method, a polymerase chain reaction (PCR) can be carried out in situ, in fixed cells, on specially designed glass slides. After amplification one can detect the amplified signals by the in situ hybridization method, utilizing either biotinylated probes or 32P-labelled probes. The early molecular events in the retroviral life-cycle of HIV-1, in specific target cells, are demonstrated utilizing in situ PCR. The techniques utilized in this procedure and various potential uses of this methodology are described.

Cell Line↗
PubMed1993

Sodium butyrate treatment of cells latently infected with HIV-1 results in the expression of unspliced viral RNA.

To investigate potential mechanisms for HIV-1 proviral latency, we generated a set of chronically HIV-1 infected and stably long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT)-transfected TE671/RD cells, and studied both their virus production and LTR-driven reporter gene expression. Established tissue culture models of retroviral latency in lymphoid and monocytoid cell lines have demonstrated that the induction of virus production is associated with a shift in HIV-1-specific mRNA from a predominance of singly and multiply spliced mRNA's to the production of full-length HIV-1 RNA. We found a similar pattern in TE671/RD cells, but in contrast to U1 and ACH2 cells, could not induce viral replication by exposure to phorbol myristate acetate (PMA) alone. We demonstrated instead that production of full-length viral RNA, viral replication, and LTR-driven CAT expression could be induced by exposure to sodium butyrate. The most proximate effect of sodium butyrate is inhibition of cellular histone deacetylase(s) which results in disruption of nucleosomes relieving one level of restriction to gene expression. Consistent with this mechanism of action, we further found that sodium butyrate's effects: (i) act synergistically with PMA and TNF-alpha; (ii) are independent of protein synthesis; (iii) do not affect the constitutively expressed creatine phosphokinase gene; (iv) do not map to a discrete sequence motif in the viral LTR; and (v) are not blocked by N-acetyl cysteine but (vi) are blocked by novobiocin, an inhibitor of cellular topoisomerase II. These data show that a similar pattern of restricted viral RNA expression exists in this nonlymphoid cellular model of HIV-1 latency. In contrast however, these results suggest that in these cells there is an additional block to viral gene expression, which is overcome with sodium butyrate. These results are discussed in the context of histone-mediated repression of HIV-1 gene expression.

Acetylcysteine↗
PubMed1993

HIV-1 latency due to the site of proviral integration.

Cell lines that are nonproductively infected with human immunodeficiency virus type 1 (HIV-1), such as the T-lymphocyte ACH2 clone, have been proposed to represent in vitro models of proviral latency. We have previously shown that such cells exhibit an aberrant pattern of viral RNA expression, with a large excess of fully spliced viral RNAs. Here, by superinfecting the ACH2 cells with a second HIV-1 virus, we demonstrate the primary mechanism of HIV-1 proviral latency in this cell line. In the dually infected cells, the exogenous virus was transcribed at high levels. However, expression from the provirus originally present in the ACH2 cells was not quantitatively increased. Still, its RNA production was shifted from a blocked early-stage to a fully productive pattern. This was interpreted to be a consequence of the high levels of Rev produced by the exogenous virus, acting in trans to promote the cytoplasmic export of all incompletely spliced viral mRNAs made in the cells. Comparable numbers of copies of each provirus were present in the dually infected cells, and both viruses grew equally well, once passed onto fresh cells. These results indicate that the low levels of transcriptional activity of the HIV-1 virus present in ACH2 cells are secondary to specific characteristics of the region of the cellular genome in which the proviral DNA is integrated.

Base Sequence↗
PubMed1993

The role of CD8-positive lymphocytes in the control of HIV-1 infection of peripheral blood mononuclear cells.

The role of CD8-positive lymphocytes from HIV-1-seropositive individuals in the inhibition of HIV-1 replication in CD4-positive lymphocytes, either from HIV-1-seropositive or -seronegative individuals, has been described. It has been suggested that CD8-positive lymphocytes isolated from HIV-1-seropositive individuals are 'primed' against certain HIV-1 antigens, hence inducing HIV-1-specific resistance. In the present studies, we show that CD8-positive lymphocytes from HIV-1-uninfected individuals also induce a significant decrease in HIV-1 replication from peripheral blood mononuclear cells (PBMC) acutely infected with HIV-1 in vitro. Unfractionated PBMC from 16 HIV-1-seronegative individuals were infected with HIV-1. In parallel experiments, PBMC from these individuals were depleted of CD8-positive lymphocytes. Viral replication was measured by a syncytia-formation assay, as well as by measuring HIV-1 p24 antigen levels in the culture supernatants on day 10 post-infection. Removal of CD8-positive lymphocytes resulted in the increased replication of HIV-1 in vitro. Reconstitution with syngeneic CD8-positive lymphocytes to CD8-positive lymphocyte-depleted wells resulted in significant decreases in HIV-1 replication. Reconstitution with allogeneic CD8-positive lymphocytes or supernatants from syngeneic or allogeneic CD8-positive lymphocyte cultures also resulted in some decrease in HIV-1 production, but it was not statistically significant. The status of HIV-1 replication in unfractionated, CD8-positive lymphocyte-depleted and CD8-positive lymphocyte-reconstituted PBMC cultures was further confirmed at the level of HIV-1 transcription. In situ hybridization of cultured cells, with a biotinylated HIV-1 gag probe, revealed increased numbers of cells actively transcribing HIV-1-specific RNA in the CD8-positive lymphocyte-depleted cell cultures, as compared with unfractionated PBMC. Thus, removal of CD8-positive lymphocytes from PBMC obtained from HIV-1-seronegative individuals resulted in a significant enhancement of HIV-1 replication in vitro.

Adult↗
PubMed1993

Human immunodeficiency virus type I provirus is demonstrated in peripheral blood monocytes in vivo: a study utilizing an in situ polymerase chain reaction.

Human immunodeficiency virus type 1 (HIV-1) infects a variety of cell types in vivo. Monocyte/macrophages may be the major reservoir for HIV-1 in the solid tissues of HIV-1-infected individuals. Conflicting data have been reported, though, regarding the presence of HIV-1 provirus in peripheral blood monocytes isolated from HIV-1-seropositive humans. We have evaluated monocytes from the peripheral blood of eleven HIV-1-infected individuals utilizing a new, highly sensitive and specific in situ polymerase chain reaction. We demonstrate HIV-1 provirus in 73% (8/11) of these samples. None of these monocyte samples was demonstrated to contain cells expressing high levels of HIV-1-specific RNA, by standard in situ hybridization. The evaluation of the HIV-1 genome in peripheral blood monocytes of certain infected individuals may assist in the understanding of HIV-1 proviral latency and pathogenesis, in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Alcohol intake increases human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells.

The effects of alcohol consumption on various T lymphocyte subset functions and on the degree of susceptibility of peripheral blood mononuclear cells (PBMC) to human immunodeficiency virus (HIV) type 1 infection and replication in vitro were investigated. PBMC from 60 HIV-1-seronegative healthy volunteers were studied before and after ingestion of alcohol beverages. After alcohol consumption, there was significantly increased HIV-1 replication (P < .001) in PBMC, as determined by HIV-1 p24 antigen levels in the culture supernatants, than in cultures obtained before alcohol ingestion. There was a decreased ability of lymphocytes, obtained after alcohol consumption, to produce interleukin-2 and soluble immune response suppressor activity after stimulation with concanavalin A. The data show that alcohol ingestion increases HIV-1 replication in human PBMC infected with HIV-1 in cell culture. This may be due to alcohol-induced functional impairment of various subsets of lymphocytes in the peripheral blood. Thus, HIV-1 replication may be augmented by alcohol in HIV-1-infected individuals, and alcohol intake may increase an individual's risk for acquiring HIV-1 infection.

Adolescent↗
PubMed1993

Human immunodeficiency virus type 1 replication in peripheral blood mononuclear cells in the presence of cocaine.

Previous studies have shown that cocaine consumption alters the normal immune functions of animals. Several epidemiologic studies have indicated an association between cocaine consumption and an increased risk for AIDS. In the present studies, unstimulated peripheral blood mononuclear cells (PBMC) isolated from 8 healthy, human immunodeficiency virus (HIV) type 1-seronegative volunteers were exposed to cocaine or one of its by-products, in vitro, at concentrations compatible with blood levels found during clinical abuse of cocaine. PBMC treated with cocaine had significantly increased levels of HIV-1 replication after infection in vitro compared with untreated PBMC. The major cocaine by-product, benzoylecgonine, did not appear to exert any significant potentiating effect on HIV-1 replication. Cocaine or its by-product did not significantly increase HIV-1 replication in persistently HIV-1-infected T lymphocytic or monocytoid cell lines. These data indicate that exposure of PBMC but not chronically HIV-1-infected cell lines to cocaine can result in increased HIV-1 replication.

Adjuvants, Immunologic↗
PubMed1993

High percentages of CD4-positive lymphocytes harbor the HIV-1 provirus in the blood of certain infected individuals.

OBJECTIVES: HIV-1 infection of humans leads to states of immunosuppression. Therefore, we sought to determine precise levels of HIV-1 infection of cells in vivo, as these data may assist in the understanding of the pathogenetic processes involved in HIV infection. DESIGN AND METHODS: We have developed an in situ polymerase chain reaction (IS-PCR), which allows amplification of various genetic elements within intact cells. Initial studies using this technique have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells (PBMC) of infected individuals than have been demonstrated in many previous studies using standard PCR techniques. This study describes a combined protocol in which an immunomagnetic bead separation technique is used with IS-PCR to specifically determine cellular reservoirs for HIV-1 and levels of infected cell types in the peripheral blood. RESULTS: CD4-positive lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected patients evaluated. The percentages of HIV-1-infected CD4-positive lymphocytes increased significantly with advancing stages of disease. These procedures also demonstrated that, with the exception of small percentages of infected peripheral blood monocytes, the CD4-positive lymphocyte is clearly the major cellular reservoir for HIV-1 in the peripheral blood. CONCLUSIONS: These data suggest that, in certain infected individuals, high levels of CD4-positive lymphocytes may harbor the HIV-1 provirus. Thus, the levels of infected lymphocytes are consistent with possible direct effects of HIV-1 on lymphocyte depletion in vivo.

CD4-Positive T-Lymphocytes↗
PubMed1993

Frequency of cells positive for HIV-1 sequences assessed by in situ polymerase chain reaction.

OBJECTIVE: HIV-1 infection of humans causes immunosuppression. The determination of precise levels of HIV-1 infection of cells in vivo may assist in the understanding of the pathogenetic processes involved in clinical infection with this virus. Studies using polymerase chain reaction have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells of infected subjects than many previous studies. METHODS: We developed a new, highly sensitive, polymerase chain reaction method that amplifies selected genetic regions within intact single cells. We used this technique before and after immunomagnetic bead separation to determine the proportion of unfractionated peripheral blood mononuclear cells and CD4+ lymphocytes carrying the HIV-1 provirus in infected subjects in different stages of disease. RESULTS: None of the peripheral blood mononuclear cells from 11 HIV-1-seronegative subjects were positive for HIV-1 provirus by the in situ polymerase chain reaction method. In 56 subjects infected with HIV-1, the percentage of peripheral blood mononuclear cells with HIV-1 ranged from 0.1 to 13.5%. CD4+ lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected subjects evaluated. The percentages of HIV-1-infected CD4+ lymphocytes increased significantly with an advancing stage of disease. The proportion of peripheral blood mononuclear cells infected with HIV-1 appeared to be at least 10 times higher than previously described. CONCLUSION: These data suggest that in certain infected patients, high levels of infected lymphocytes may harbor the HIV-1 provirus.

CD4-Positive T-Lymphocytes↗