Protection against retroviruses are owing to a different form of immunity. An RNA-based molecular immunity hypothesis.
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Animals↗
Biomedical subjects
O Bagasra
Publications and source records attributed to O Bagasra.
Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen.
Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.
DNA, Viral↗
Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.
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Animals↗
Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene.
Platelet factor XI is associated with the platelet plasma membrane and has an apparent Mr (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of approximately 1.9 kilobases was detected in megakaryocytic cells, and one of approximately 2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala91-Arg144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.
Alternative Splicing↗
Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia.
The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV-1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV-1 virus, will the course of the infection be altered, resulting in a prolonged symptom-free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV-1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV-1-related disease. All SS patients with a diagnosis of HIV-1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow-up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV-1 viral burden. The studies included the branched-DNA signal amplification assay, quantitative DNA-polymerase chain reaction (PCR), quantitative reverse transcription (RT)-initiated-PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow-up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV-1-infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T-lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long-term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long-term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV-1-seropositive SS patients (44%) may be asymptomatic long-term nonprogressors. In these patients, the CD4+ T-lymphocyte counts remained high and their viral burdens were remarkably lower than in non-SS HIV-1-seropositive individuals. Whereas this study does not prove an "autosplenectomy" hypothesis, it suggests that in patients with both SS and HIV-1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV-1 infection may be one.
Adolescent↗
Localization of human herpesvirus type 8 (HHV-8) in the Kaposi's sarcoma tissues and the semen specimens of HIV-1 infected and uninfected individuals by utilizing in situ polymerase chain reaction.
Kaposi's sarcoma (KS) is a form of skin cancer, most commonly found in individuals suffering from acquired immunodeficiency syndrome, or AIDS. However, before the worldwide infection of human immunodeficiency virus (HIV), the rare occurrence of KS was confined to two distinct groups of individuals. In the Western world, the classical form of KS was often found in older men (60-70 years of age) from the Mediterranean area. Another form called endemic KS, was found in Equatorial Africa. Currently, the most common cases of KS are found in individuals suffering from AIDS. This is called AIDS-associated KS. Between 30 and 40% of male, homosexual AIDS patients suffer from AIDS-associated KS. KS is also occasionally diagnosed in transplant patients receiving immunosuppressive drugs (to keep their body from rejecting the foreign organ). As opposed to cases of classic and endemic KS, the KS in AIDS patients progresses very quickly, often with a fatal outcome. Human herpesvirus type 8 (HHV-8) has been implicated as the cause of Kaposi's sarcoma (KS), but the exact connection of the virus to the neoplasm is not known. The virus has been detected within the sarcoma skin lesions, but has additionally been seen in peripheral blood cells, semen samples, prostate tissue, and other types of soft tissue tumors. In this study, we evaluated HHV-8 within the skin lesion of KS as well as in semen specimens obtained from HIV-1 infected and uninfected specimens from HIV-1-seronegative individuals. Twenty-eight tissue samples representing AIDS-associated, endemic KS, and six non-KS patients were collected for observation from different centers throughout the world. The tissues were examined utilizing in situ polymerase chain reaction (ISPCR) and hybridization to identify and localize the herpesvirus within the KS lesions. With the use of the sensitive ISPCR technique, HHV-8 DNA was detected in the spindle cells within the nodular skin lesions, as well as in the microvascular endothelial cells which line small vessels within the lesions in all forms of KS. In addition, we analysed semen specimens from HIV-1 infected and uninfected men, our analyses revealed that HHV-8 was present in the significant proportions of the HIV-1-infected-individuals' sperm, as well as in the mononuclear cells of the semen specimens. HHV-8 DNA was demonstrated, by ISPCR, in KS lesions as well as in seminal mononuclear cells and sperm of significantly high proportion of HIV-1-infected men. What role the presence of HHV-8 in the sperm cells plays in the sexual transmission of this herpesvirus will require further study. However, the reports which demonstrate that KS lesions can develop in infants of only a few weeks of age, increases the possibility that this agent may be vertically transmitted. It can be suggested that HHV-8 is relatively ubiquitous and its frequency increases with the increasing immunosuppression.
HIV Infections↗
Natural immunity against human immunodeficiency viruses: prospects for AIDS vaccines.
We have hypothesized and have presented evidence that there may be another form of immunity, other than humoral and cellular immunities, which operates against retroviruses. In order to distinguish it from the traditional immune responses, we have named this form of immunity "molecular immunity". The major goal of this hypothesis is to better define the "messenger molecules" that are critical in forming the molecular immunity against retroviruses, and to further determine the activation pathways of this relatively unexplored form of immunity. We have provided evidence that this natural immunity against retroviruses and specifically against HIV-1, can be activated and optimized, and have made some interesting observations. We believe that resistance to HIV-1 and to other retroviruses can be induced by various means, including low dose exposure, infection with replication defective viruses and exposure to non-pathogenic but genetically related viruses.
AIDS Vaccines↗
Prevention of experimental allergic encephalomyelitis by targeting nitric oxide and peroxynitrite: implications for the treatment of multiple sclerosis.
In this study we provide further evidence associating activated cells of the monocyte lineage with the lesions of multiple sclerosis (MS). Using a combination of immunohistochemistry and reverse transcriptase-dependent in situ polymerase chain reaction analysis, we have identified monocytes expressing inducible nitric oxide synthase (iNOS) to be prevalent in the plaque areas of post mortem brain tissue from patients with MS. In addition, we have obtained evidence of the nitration of tyrosine residues in brain areas local to accumulations of iNOS-positive cells. In parallel studies we have assessed the effects of inhibitors of iNOS induction, as well as scavengers of nitric oxide and peroxynitrite in the experimental allergic encephalomyelitis model. Significant therapeutic effects were seen with the inhibitor of iNOS induction, tricyclodecan-9-xyl-xanthogenate, a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and a peroxynitrite scavenger, uric acid. In particular, treatment with high doses of uric acid virtually prevented clinical symptoms of the disease. Together with our demonstration of the presence of activated macrophages expressing high levels of iNOS and evidence of peroxynitrite formation in brain tissue from patients with MS, these findings are of importance in the development of approaches to treat this disease.
Animals↗
DNA and amino acid sequence of anti-HIV-1 Rev sFv.
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Amino Acid Sequence↗
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↗
The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily.
Interferon-gamma inducible protein 10 kD (IP-10) is a highly inducible, primary response gene that belongs to the C-X-C chemokine superfamily. Despite the original cloning of IP-10 in 1985, its biological functions are still unclear although accumulating reports indicate that it is a pleiotropic molecule capable of eliciting potent biological effects, including stimulation of monocytes, natural killer and T-cell migration, regulation of T-cell and bone marrow progenitor maturation, modulation of adhesion molecule expression as well as inhibition of angiogenesis. More interest is now likely to be focused on IP-10 due to the recent cloning of an IP-10 receptor. This paper aims to highlight our current knowledge of IP-10 and its homologues as well as defining its likely involvement in regulating fibroproliferation following inflammatory lung injury.
Amino Acid Sequence↗
Detection of HIV in oral mucosal cells.
OBJECTIVE: To determine the prevalence of HIV DNA and RNA and the morphologic localization of HIV in the oral cavity of HIV-seropositive subjects. DESIGN: A cross-sectional analysis of saliva, buccal scrapings and buccal biopsies from HIV-seropositive injecting drug users (IDUs). SUBJECTS AND METHODS: Whole saliva, buccal mucosal scrapings and buccal biopsies were obtained from HIV-seropositive and seronegative IDUs. Presence of HIV DNA and RNA was assessed by polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR). RT in situ PCR was used to detect HIV tat/rev RNA in buccal mucosal scrapings. Host-cell integrated HIV-proviral DNA in buccal biopsies was detected by in situ PCR. Presence of intact HIV viral particles in buccal scrapings was assessed by electron microscopy. RESULTS: HIV DNA was detected in 40% (18/45) and HIV RNA in 69.2% (25/36) of saliva samples from HIV-seropositive IDUs. Viral particles consistent with HIV were localized in inter-epithelial spaces by electron microscopy. RT in situ PCR revealed the presence of HIV tat/rev RNA in 36% (8/22) of the seropositive samples tested. CONCLUSIONS: Our results suggest that epithelial cells can be productively infected by HIV. Epithelial cells in buccal mucosa may acquire HIV in the basal layers through contact with submucosal HIV-positive lymphocytes and/or Langerhans' cells. HIV infection may also spread by inter-epithelial cell contact. As HIV infected cells mature they travel to more superficial layers and are shed into the oral cavity.
CD4-Positive T-Lymphocytes↗
In situ PCR. Current protocol.
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Immunohistochemistry↗
Human gene therapy. Dreams to realization.
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Genetic Therapy↗
Titering replication-defective virus for use in gene transfer.
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Animals↗
Self-seal reagent: evaporation control for molecular histology procedures without chambers, clips or fingernail polish.
Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directly to the in situ reaction mixture, effectively controls evaporation during in situ procedures by creating an evaporation-limiting barrier around the periphery of a standard cover glass as the reaction proceeds. At the end of the procedure, the cover glass is easily removed by soaking in an aqueous solution. A model is presented for how Self-Seal Reagent controls evaporation while maintaining reagent concentrations. Self-Seal Reagent is shown to be effective in the detection of HIV sequences in cells by in situ PCR.
Cosmetics↗
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↗
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↗