Genetic analysis of an HIV type 2 subtype B virus from a Spanish individual with AIDS.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
J S Epstein
Publications and source records attributed to J S Epstein.
Evidence of HIV-2 infection in Equatorial Guinea (central Africa): partial genetic analysis of a B subtype virus.
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Adult↗
Detection of acquired B antigen by monoclonal anti-B blood grouping reagents.
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ABO Blood-Group System↗
Food and Drug Administration conference on the feasibility of genetic technology to close the HIV window in donor screening.
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Antigens, Viral↗
Rapid and sensitive detection of cell-associated HIV-1 in latently infected cell lines and in patient cells using sodium-n-butyrate induction and RT-PCR.
To develop a rapid and sensitive means of detecting cell-associated human immunodeficiency virus (HIV), donor cells from HIV seropositive patients were treated with the potent viral activator sodium-n-butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of RT-PCR was estimated to be equivalent to 1 x 10(-16) grams (0.1 fg) or approximately 64 copies of the input standard viral RNA per reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH-2 and U1 cell lines were used as representatives of T-lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT-PCR assays. When peripheral blood mononuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV-positive PBMCs by RT-PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell-associated viral load in vivo and without possible mutations brought about by long-term co-cultivation assays with cells from seronegative donors.
Butyrates↗
Impaired antigen presentation to CD4+ T-cells by HIV-infected monocytes is related to down-modulation of CD4 expression on helper T-cells: possible involvement of HIV-induced cellular factors.
Defective antigen presentation by HIV-infected monocytes is related to severe immune dysfunction in patients with AIDS, although the mechanism by which this process occurs is not well defined. Here we report that reduced capacity by HIV-infected monocytes to stimulate or present antigen to CD4+ T-cells was mediated by cellular factors associated with the plasma membranes of HIV-infected monocytes. In contrast, soluble factors secreted by HIV-infected monocytes had little or no effect on T-cell stimulation. Reduced T-cell stimulation by HIV-infected monocytes was related to down-modulation of CD4 expression on helper T-cells and was not affected by the inclusion of anti-HIV-gpl20 Ab, indicating the involvement of soluble or cell-associated viral envelope protein to be less likely. Exposure of CD4+ T-cells, that had been in co-culture with HIV-infected monocytes, to uninfected monocytes partially restored impaired T-cell stimulation. Thus, for the first time we report that altered capacity of HIV-infected monocytes to stimulate and present antigen to CD4+ T-cells is related to down-modulation of CD4 expression on T-cells, and appears to occur via membrane-associated cellular factors on HIV-infected monocytes.
CD4 Antigens↗
HIV-1-Tat protein promotes chemotaxis and invasive behavior by monocytes.
Monocytes are susceptible to HIV infection and to activation by a regulatory gene product of the HIV genome, HIV-Tat. Recently, we have demonstrated that treatment with HIV-Tat up-regulates monocyte adhesion to the endothelium and increases metalloproteinase production. in the present study, we have examined the ability of the HIV-Tat protein to alter the migratory and invasive behavior of monocytes. Monocytes pretreated for 24 h with 10 ng/ml HIV-Tat exhibited enhanced migratory behavior compared with untreated monocytes in chemotaxis assays, both in the absence of a chemoattractant as well as in response to FMLP. in addition, HIV-Tat itself induced the migration of both untreated and HIV-Tat pretreated monocytes. Checkerboard analysis showed that monocytes migrated in response to an HIV-Tat concentration gradient, thus confirming the chemotactic characteristics of the HIV-Tat protein. Pretreatment of monocytes with 10 ng/ml HIV-Tat for 24 h also increased their ability to invade reconstituted extracellular membrane (Matrigel)-coated filters by 5-fold in the absence of chemoattractant. The presence of FMLP or HIV-Tat further enhanced invasion by both untreated and HIV-Tat-pretreated monocytes by more than 10-fold. Monocyte invasion was partially inhibited by the inclusion of anti-beta integrin Ab or tissue inhibitor of metalloproteinase (TIMP). Thus, for the first time, we present evidence that HIV-Tat can enhance the chemotactic and invasive behaviors of monocytes and propose an active role for HIV-Tat in the recruitment of monocytes into extravascular tissues, a process which may contribute to the destruction of tissues and cellular architecture often seen in patients with acquired immunodeficiency syndrome.
Chemotaxis, Leukocyte↗
HIV-1-Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. A mechanism of HIV pathogenesis.
Monocytes are major targets of HIV infection in patients with AIDS. In vitro infection of monocytes with HIV is associated with increased expression of beta 2 integrins, which increases both monocyte aggregation and monocyte/endothelial adhesion as well as monocyte metalloproteinase (MMP-9) expression. Treatment of primary monocytes with soluble HIV-Tat protein mimicked many of the properties of HIV infection of monocytes. Tat treatment up-regulated the expression of the beta 2 integrins, which was associated with the formation of large aggregates of monocytes and increased adhesion to endothelial monolayers. Treatment of monocytes with Tat increased their adhesion to both untreated and TNF-alpha-treated endothelial monolayers, and adhesion was inhibited by inclusion of anti-beta 2 and anti-ICAM-1 Abs. The increased adhesion of activated monocytes was accompanied by substantial disruption of the endothelial monolayers, with retraction or detachment of individual endothelial cells. Tat treatment of monocytes up-regulated the synthesis and release of the protease MMP-9, providing a potential mechanism to explain endothelial cell/basement membrane detachment. Thus, extracellular Tat is capable of activating monocytes even in the absence of HIV infection. Our studies demonstrate that many of the effects of HIV infection on monocyte homotypic and heterotypic adhesion, protease secretion, and disruption of the endothelium can be mimicked by treatment with HIV-Tat protein alone. These results suggest a mechanism where monocytes could be inappropriately activated by HIV-Tat, secreted by HIV-infected cells, causing them to extravasate into underlying tissues and ultimately contribute to tissue damage as seen during the progression of AIDS.
Cell Adhesion↗
Inhibition of HIV replication by immunoliposomal antisense oligonucleotide.
The sequence-specific suppression of HIV-1 replication using CD4 monoclonal-antibody-targeted liposomes, containing Rev antisense phosphorothioate oligonucleotides is described. Liposomes were prepared by encapsulating the 20-mer antisense DNA sequence of the rev HIV-1 regulatory gene, in the form of a phosphorothioate oligonucleotide. Specific targeting was accomplished by conjugating anti-CD4 mouse monoclonal antibody to the surface of the liposomes. HIV-1-infected H9 cells as well as peripheral blood T-lymphocytes were incubated with the immunoliposomes of antisense found to have potential antiviral effect. HIV-1 replication was reduced by 85% in antisense immunoliposome-treated H9 cells and peripheral blood lymphocytes, whereas the inhibition of HIV-1 replication was not observed using either empty immunoliposomes or immunoliposomes containing scrambled Rev phosphorothioate oligonucleotide sequences. The antiviral activity of both the free and the encapsulated oligonucleotides were assessed by p24, reverse transcriptase (RT) assays and polymerase chain reaction (PCR) analysis. Liposome preparations demonstrated minimal toxicity in H9 as well as in peripheral blood lymphocyte cell culture experiments. These in vitro culture results demonstrate the potential efficacy of immunoliposomes to inhibit HIV replication.
Animals↗
Development of a multiplex PCR assay for the simultaneous detection and discrimination of HIV-1, HIV-2, HTLV-I and HTLV-II.
BACKGROUND: Multiplex polymerase chain reaction (PCR) has been established as a general technique for the simultaneous amplification of different target sequences. Uses of multiplex include pathogens identification, linkage analysis and genetic disease diagnosis. The high sensitivity of PCR may produce false-positive results due to contamination with previously amplified material. OBJECTIVES: To develop a multiplex PCR technique that can simultaneously detect and discriminate human immunodeficiency virus types 1 and 2 (HIV-1/2) and human T-lymphotropic virus types 1 and 2 (HTLV-I/II) proviral sequences. Such a method should incorporate a system that prevents the occurrence of false-positive results. STUDY DESIGN: Combinations of four primer pairs, one for each retrovirus, were assayed in order to determine the combination of oligonucleotides as well as the PCR conditions that yield the most specific and sensitive coamplification of proviral sequences. To prevent contamination with DNA from previous PCR amplifications, the uracil N-glycosylase (UNG) system was incorporated into the coamplification format. RESULTS: A combination of primer pairs from the gag region of HIV-1, env of HIV-2, pol of HTLV-I and tax of HTLV-II yielded specific and sensitive coamplification of proviral sequences. The UNG system was incorporated and shown to be efficient in the degradation of contaminating DNA. In the evaluation of a serologically well established panel of singly and dually infected individuals, the assay detected 20/22 HIV-1, 8/10 HIV-2, 8/8 HTLV-I and 8/8 HTLV-II infections.
Cell Line↗
Scalp flaps in the treatment of baldness. Long-term results.
BACKGROUND: Scalp flaps have been used as a treatment for male pattern baldness for many years. OBJECTIVE: To assess the long-term results of these flaps. METHODS: Twenty patients who underwent a total of 31 temporo-parieto-occipital and temporo-parietal flaps 10-20 years ago were reviewed. RESULTS: Consistent maintaining of the initially obtained coverage and a low incidence of complications was demonstrated. Universally, the male pattern baldness progressed, requiring in some patients subsequent procedures. CONCLUSIONS: Scalp flaps, when performed properly with anticipation of further hair loss, are an effective treatment of male pattern baldness in the appropriately selected patient.
Adult↗
Interferon-gamma inhibits HIV-induced invasiveness of monocytes.
HIV-infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92-kd metalloproteinase (MMP-9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)-infected monocytes with interferon-gamma at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP-9 produced by HIV-infected monocytes treated with interferon-gamma 1 day prior to infection with HIV as compared with that of untreated HIV-infected monocytes. Monocytes treated with interferon-gamma 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP-9 were substantially lower than those produced by monocytes inoculated with live HIV, heat-inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat-inactivated HIV did not result in increased invasiveness or high MMP-9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4-gp120 interactions and required active virus infection. Furthermore, addition of interferon-gamma to monocytes on day 10 after infection inhibited MMP-9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon-gamma-induced down-regulation of MMP-9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN-gamma in monocytes. Down-regulation of MMP-9 in HIV-infected monocytes by interferon-gamma may play an important role in the control of HIV pathogenesis.
Antiviral Agents↗
Interferon-gamma-induced downregulation of CD4 inhibits the entry of human immunodeficiency virus type-1 in primary monocytes.
We have previously shown that the treatment of monocytes with interferon-gamma (IFN-gamma) prior to exposure with human immunodeficiency virus type-1 (HIV) results in complete inhibition of HIV infection of monocytes. In the present report, we have extended this study to obtain information on the mechanism(s) underlying IFN-gamma-induced inhibition of HIV infection of monocytes. To examine the effect of IFN-gamma on HIV entry, the first event in the infectious cycle of the virus, we amplified HIV-gag sequences in the genomic DNA and RNA of IFN-gamma treated monocytes, and found no evidence for the presence of either proviral DNA or HIV RNA sequences. These results were consistent with the absence of intracellular HIV particles either in the latent or actively replicating state as determined by flow-cytometric analysis of these cells. Furthermore, no HIV-induced cytopathic effects, such as multinucleated giant cell formation or cell death, were observed in IFN-gamma-treated monocytes after their exposure to HIV. Stimulation of IFN-gamma-treated monocytes 6 days postinfection with tumor necrosis factor-alpha (TNF-alpha), which is known to augment HIV replication in the infected cells, did not result in the induction of the HIV indicating the absence of latent HIV infection in IFN-gamma-treated monocytes. Treatment of monocytes with IFN-gamma, TNF-alpha, or with a combination of the two agents which is known to induce antimicrobial free radical nitric oxide (NO2- in the murine system did not induce NO2- production human monocytes suggesting the antiviral activity of IFN-gamma to be independent of NO2(-)-mediated killing of HIV or HIV-infected monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
CD4 Antigens↗
A trial of RSV immune globulin in infants and young children: the FDA's view.
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Bias↗
Interferon-gamma induces resistance in primary monocytes against human immunodeficiency virus type-1 infection.
Treatment of monocytes with interferon-gamma 1 day before, or at the time of infection with human immunodeficiency virus type-1 (HIV-1) induced complete resistance in monocytes against HIV-1 infection. There was no evidence of viral RNA, proviral DNA, p24 antigen, or reverse transcriptase activity through 2 weeks after inoculation. Ultrastructural examination of these cells showed no detectable virus particles. When interferon-gamma was added to monocytes 1 to 3 days post-infection, virus integration occurred, but the viral expression was either ablated (1 day post-infection) or significantly inhibited (3 days post-infection). Treatment of monocytes with interferon-gamma before or after infection with HIV-1 produced significantly higher levels of tumor necrosis factor-alpha and interleukin-8 than untreated or uninfected monocytes. These results suggest that altered regulation of cytokines may mediate antiviral activity of interferon-gamma in monocytes.
Cells, Cultured↗
Subspecialization.
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Education, Medical, Graduate↗
Enhanced diagnostic efficiency of the polymerase chain reaction by co-amplification of multiple regions of HIV-1 and HIV-2.
A method for co-amplification of multiple viral sequences of HIV-1 and HIV-2 by polymerase chain reaction was designed. The technique resulted in the specific detection of each type of virus and allowed the amplification of as few as two copies of target DNA. The amplification of multiple regions of the viral genome offers the advantage of detecting multiple target sequences, which may be essential for some viruses, such as HIV, that exhibit a high degree of variability in their gene sequences. In addition, the method permitted the detection of both virus types in the same reaction, allowing for greater utility in geographic areas where coinfections with both viruses occur and cross-reactivity in Western blots is observed. This method was applied successfully to the detection of viral DNA in clinical specimens.
Base Sequence↗
Idiopathic CD4+ T lymphocytopenia: a review and current perspective.
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Acquired Immunodeficiency Syndrome↗