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Biomedical subjects

O Bagasra

Publications and source records attributed to O Bagasra.

At least 55 records · Page 3Linked to original sources
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

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

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↗
PubMed1993

Experimental model of congenital syphilis.

Female LSH hamsters infected with Treponema pallidum subsp, endemicum before pregnancy or during early pregnancy transmit a form of syphilis to the fetus that is similar to human congenital syphilis. The offspring develops rhinitis, skin rash, failure to thrive, and hepatosplenomegaly. T. pallidum is detectable in their livers, spleens, and nasal secretions. Immunoglobulin M antibodies are detected in the serum.

Animals↗
PubMed1992

A quantitative reverse transcriptase-polymerase chain reaction for HIV-1-specific RNA species.

The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.

Base Sequence↗
PubMed1992

Blocked early-stage latency in the peripheral blood cells of certain individuals infected with human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infections of humans have a natural history characterized by a variable but usually slow progression to an immunodeficient state. We have described a molecular model of HIV-1 proviral latency in certain cell lines, characterized by extremely low or undetectable levels of unspliced genomic HIV-1-specific RNA but significant levels of multiply spliced HIV-1-specific RNA. We have utilized a quantitative reverse transcriptase-initiated polymerase chain reaction to measure the levels of various HIV-1 RNA species in peripheral blood mononuclear cells. The median level of multiply spliced HIV-1 RNA was dramatically higher than the median level of unspliced viral RNA in asymptomatic individuals. In addition, HIV-1 RNA patterns characterized by at least a 10-fold excess of multiply spliced to unspliced viral RNA were significantly more common in asymptomatic individuals than in patients with the acquired immunodeficiency syndrome. We suggest that asymptomatic clinical HIV-1 infection is characterized by a preponderance of HIV-1-infected peripheral blood cells blocked at an early stage of HIV-1 infection. This viral expression pattern, which we have called blocked early-stage latency, may constitute a reservoir of latently infected cells in certain HIV-1-infected persons.

HIV Seropositivity↗
PubMed1992

CD14 is involved in control of human immunodeficiency virus type 1 expression in latently infected cells by lipopolysaccharide.

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in transfected monocyte-macrophage cell lines and dramatically increases HIV-1 production in the latently infected monocyte-macrophage-like cell line U1. This response to LPS, however, can only be observed after pretreatment of the U1 cells with granulocyte-macrophage colony-stimulating factor (GM-CSF). CD14, the differentiation antigen that acts as a receptor for complexes of LPS and LPS-binding protein, is now demonstrated to be involved in LPS-induced stimulation of HIV-1 replication. CD14 is shown to be expressed on a subpopulation of U1 cells only after treatment with GM-CSF and correlates with HIV-1 production stimulated by LPS. Importantly, only those U1 cells that express CD14 can be induced by LPS to upregulate HIV-1 production. In addition, a monoclonal antibody directed against CD14 can block LPS-induced stimulation of HIV-1 production from these latently infected cells.

Acute-Phase Proteins↗
PubMed1992

Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction.

BACKGROUND: Studies of human immunodeficiency virus type 1 (HIV-1) infection have attempted to quantitate the viral load correlate it with the degree of immune deficiency. In one study, only about 1 in 10,000 peripheral-blood mononuclear cells (PBMC) expressed HIV-1, but in other studies, at least 1 in 100 CD4-positive cells was infected and harbored the HIV-1 provirus. METHODS: We developed a new, highly sensitive in situ polymerase-chain-reaction (PCR) method that amplifies selected genetic regions within intact single cells. We used this technique to determine the proportion of PBMC carrying HIV-1 provirus in infected patients in different stages of disease. RESULTS: None of the PBMC from 11 HIV-1--seronegative patients were found to be positive for HIV-1 provirus by the in situ PCR method. In 56 patients infected with HIV-1, the percentage of PBMC with HIV-1 ranged from 0.1 percent to 13.5 percent. The mean percentage of infected mononuclear cells was greater in 13 patients with persistent generalized adenopathy (mean, 6.6 percent) and 19 with the acquired immunodeficiency syndrome (Stages IV-A to IV-C) (4.6 percent) than in 19 patients with asymptomatic HIV-1 infection (0.9 percent) (P less than 0.001). However, in five patients with Kaposi's sarcoma (Stage IV-D), an average of only 1.6 percent of mononuclear cells were infected. CONCLUSIONS: In HIV-1 infection, the proportion of PBMC that are infected appears to be at least 10 times higher than previously described. It is likely that most infected cells contain HIV-1 provirus in a latent or defective form that was not detected in some earlier studies.

Acquired Immunodeficiency Syndrome↗
PubMed1992

TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗