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S M Wolinsky

Publications and source records attributed to S M Wolinsky.

At least 37 records · Page 2Linked to original sources
PubMed1997

Human immunodeficiency virus type 1 genetic evolution in children with different rates of development of disease.

The rate of development of disease varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected children. The reasons for these observed differences are not clearly understood but most probably depend on the dynamic interplay between the HIV-1 quasispecies virus population and the immune constraints imposed by the host. To study the relationship between disease progression and genetic diversity, we analyzed the evolution of viral sequences within six perinatally infected children by examining proviral sequences spanning the C2 through V5 regions of the viral envelope gene by PCR of blood samples obtained at sequential visits. PCR product DNAs from four sample time points per child were cloned, and 10 to 13 clones from each sample were sequenced. Greater genetic distances relative to the time of infection were found for children with low virion-associated RNA burdens and slow progression to disease relative to those found for children with high virion-associated RNA burdens and rapid progression to disease. The greater branch lengths observed in the phylogenetic reconstructions correlated with a higher accumulation rate of nonsynonymous base substitutions per potential nonsynonymous site, consistent with positive selection for change rather than a difference in replication kinetics. Viral sequences from children with slow progression to disease also showed a tendency to form clusters that associated with different sampling times. These progressive shifts in the viral population were not found in viral sequences from children with rapid progression to disease. Therefore, despite the HIV-1 quasispecies being a diverse, rapidly evolving, and competing population of genetic variants, different rates of genetic evolution could be found under different selective constraints. These data suggest that the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations are compatible with a Darwinian system evolving under the constraints of natural selection.

Amino Acid Sequence↗
PubMed1996

Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection.

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques.

We used the simian immunodeficiency virus (SIV)/rhesus macaque model to study events that underlie sexual transmission of human immunodeficiency virus type 1 (HIV-1). Four female rhesus macaques were inoculated intravaginally with SIVmac251, and then killed 2, 5, 7, and 9 d later. A technique that detected polymerase chain reaction-amplified SIV in situ showed that the first cellular targets for SIV were in the lamina propria of the cervicovaginal mucosa, immediately subjacent to the epithelium. Phenotypic and localization studies demonstrated that many of the infected cells were likely to be dendritic cells. Within 2 d of inoculation, infected cells were identified in the paracortex and subcapsular sinus of the draining internal iliac lymph nodes. Subsequently, systemic dissemination of SIV was rapid, since culturable virus was detectable in the blood by day 5. From these results, we present a model for mucosal transmission of SIV and HIV-1.

Animals↗
PubMed1996

Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure.

Some individuals remain uninfected with human immunodeficiency virus type-1 (HIV-1) despite multiple high-risk sexual exposures. We studied a cohort of 25 subjects with histories of multiple high-risk sexual exposures to HIV-1 and found that their CD8+ lymphocytes had greater anti-HIV-1 activity than did CD8+ lymphocytes from nonexposed controls. Further studies indicated that their purified CD4+ lymphocytes were less susceptible to infection with multiple primary isolates of HIV-1 than were CD4+ lymphocytes from the nonexposed controls. This relative resistance to HIV-1 infection did not extend to T-cell line-adapted strains, was restricted by the envelope glycoprotein, was not explained by the cell surface density of CD4 molecules, but was associated with the activity of the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. This relative resistance of CD4+ lymphocytes may contribute to protection from HIV-1 in multiply exposed persons.

CD4-Positive T-Lymphocytes↗
PubMed1996

The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.

A 32-nucleotide deletion (delta 32) within the beta-chemokine receptor 5 (CCR5) gene has been described in subjects who remain uninfected despite extensive exposure to HIV-1. This allele was found to be common in the Caucasian population with a frequency of 0.0808, but was not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCRS genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta 32 allele, whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta 32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

Alleles↗
PubMed1996

Maintaining the integrity of human immunodeficiency virus sequence databases.

Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.

Base Sequence↗
PubMed1995

Infection of Macaca nemestrina brain with human immunodeficiency virus type 1.

We previously reported that pigtailed macaques (Macaca nemestrina) became infected after intravenous inoculation with the LAI strain of human immunodeficiency virus type 1 (HIV-1), an isolate that replicates in both M. nemestrina lymphocytes and blood-derived monocyte/macrophages. In the current study we investigated the presence of HIV-1 and pathology in the postmortem brains of four of these macaques. Histopathological findings revealed focal lesions in white matter in the frontal and occipital lobes of one macaque, with myelin loss, nerve fibre loss, and gliosis within these lesions. Semi-quantitative, solution-based PCR revealed HIV-1 DNA in the brains of two of the other macaques. Using slide-based PCR-driven in situ hybridization, we studied these two macaques further and detected intranuclear, circular HIV-1 DNA in vascular endothelia and other non-neuronal brain cells. These findings indicate that M. nemestrina brain can be infected with HIV-1 in vivo and may provide a useful animal model for understanding early HIV-1 brain infection in humans.

AIDS Dementia Complex↗
PubMed1995

Changes in the viral mRNA expression pattern correlate with a rapid rate of CD4+ T-cell number decline in human immunodeficiency virus type 1-infected individuals.

The rate of disease progression varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected individuals. Several cross-sectional studies have shown an association between the stage of HIV-1 disease and the viral burden or the relative levels of viral gene expression. To study the extent of HIV-1 transcription and replication and its correlations with disease progression, we quantified serial, longitudinal samples of blood cells from 10 HIV-1-infected individuals with markedly different rates of CD4+ T-cell number decline following seroconversion. After normalization for the input nucleic acid content, multiply spliced viral mRNA and unspliced viral RNA were quantified by competitive reverse transcription-PCR using oligonucleotide primers which flank the major tat/rev/nef splice junction and span an internal region of the gag open reading frame, respectively. Coamplification of internal cRNA template controls was used to normalize for variation in the efficiency of reverse transcription and in vitro enzymatic amplification. Similarly, proviral DNA was also quantified by competitive PCR performed within the linear range of amplification. Viral RNA was detected in the blood cells of each individual from all time points regardless of the rate of CD4+ T-cell decline. Unspliced genomic viral RNA rapidly increased in the blood cells from HIV-1-infected individuals who had a precipitously declining CD4+ T-cell number. In contrast, both unspliced and multiply spliced viral mRNAs remained relatively stable in the blood cells from HIV-1-infected individuals who have had a relatively benign clinical course. These data demonstrate that the extent of viral transcription and replication correlates with the rate of CD4+ T-cell number decline and that quantifying intracellular viral RNA is of potential prognostic value.

Base Sequence↗
PubMed1995

Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo.

Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV-1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection.

CD4 Antigens↗
PubMed1994

Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Search for highly conserved viral and bacterial nucleic acid sequences corresponding to an etiologic agent of Kawasaki disease.

The use of conventional methods to detect a possible infectious cause of Kawasaki disease (KD) has been unsuccessful. Using the polymerase chain reaction and DNA hybridization techniques, we have sought evidence that a known or new herpesvirus, parvovirus, or bacterial pathogen is related etiologically to KD. Peripheral blood DNA from acute KD patients was subjected to amplification and dot-blot hybridization to detect the presence of herpesvirus DNA, and acute KD peripheral blood and serum DNA were subjected to dot-blot hybridization for the presence of parvoviral DNA. All samples were negative for both herpesvirus and parvovirus DNA. In addition, we analyzed buffy-coat white blood cell DNA, synovial fluid DNA, and frozen autopsy and formalin-fixed, paraffin-embedded myocardial tissue DNA from KD patients for the presence of highly conserved bacterial 16S ribosomal RNA gene sequences with the polymerase chain reaction, and all were negative. These results argue against a direct pathogenic role for herpesviruses, parvoviruses, and bacteria in KD. This approach to the detection of highly conserved genomic sequences among broad groups of microorganisms can be adapted for the detection of other groups of microorganisms and may yet prove useful in the search for an etiologic agent of KD.

Animals↗
PubMed1993

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Base Sequence↗
PubMed1993

Quantification of human immunodeficiency virus type 1 tat mRNA as a marker for assessing the efficacy of antiretroviral therapy.

To evaluate the use of human immunodeficiency virus type 1 (HIV-1) tat mRNA quantification as a marker for antiretroviral therapy, 10 zidovudine-naive, p24 antibody-positive subjects (Centers for Disease Control classes III and IV) were studied at the start of zidovudine treatment. HIV-1 proviral DNA content and tat mRNA levels were monitored for 20 weeks from the initiation of therapy. Levels of tat mRNA were quantified using an engineered tat cRNA internal control under conditions of linear amplification. Proviral DNA levels increased in 2 patients and decreased in 8 during 20 weeks of therapy. In each case, tat mRNA levels exhibited similar but much more pronounced changes. Results indicate that quantitative measurement of tat mRNA levels is extremely useful for monitoring antiretroviral therapy.

DNA, Viral↗
PubMed1993

Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories.

An independent quality assurance program has been established by the Virology Committee of the AIDS Clinical Trials Group in the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring polymerase chain reaction (PCR) assays for human immunodeficiency virus type 1 (HIV-1) DNA that are performed by 11 laboratories participating in multicenter clinical trials in the United States. To perform HIV-1 DNA PCR for patients in AIDS Clinical Trials Group protocols, each laboratory was initially certified by correctly testing a coded certification panel consisting of eight well-defined clinical whole-blood specimens and 30 cell pellets containing 0, 2, 5, 10, 20, or 50 8E5/LAV cells per 125,000 uninfected peripheral blood mononuclear cells. PCR was performed by one of two standardized commercial assays for amplification and nonisotopic detection of HIV-1 proviral DNA. For continuing certification, each laboratory must correctly test eight coded whole-blood samples per quarter and run three or four coded cell pellets and HIV-1 DNA copy standards with every PCR assay in real time. The PCR results for the coded pellets on each run are entered into an encrypted computer file, which immediately assesses the validity of the run. To date, 10 of 11 laboratories have correctly tested all HIV-1-positive and -negative samples in the initial certification panel on their first or second attempt. Subsequently, 9 of these 11 laboratories have continued to maintain their certified status. The use of commercial HIV-1 DNA PCR assays and an external quality assurance program have ensured that results from different laboratories are comparable and that problems with sensitivity and specificity are quickly identified.

Certification↗
PubMed1992

Infection of Macaca nemestrina by human immunodeficiency virus type-1.

After observations that Macaca nemestrina were exceptionally susceptible to simian immunodeficiency virus and human immunodeficiency virus type-2 (HIV-2), studies of HIV-1 replication were initiated. Several strains of HIV-1, including a recent patient isolate, replicated in vitro in peripheral blood mononuclear cells (PBMCs) and in CD4-positive M. nemestrina lymphocytes in a CD4-dependent fashion. Eight animals were subsequently inoculated with either cell-associated or cell-free suspensions of HIV-1. All animals had HIV-1 isolated by cocultivation, had HIV-1 DNA in their PBMCs as shown by polymerase chain reaction, and experienced sustained seroconversion to a broad spectrum of HIV-1 proteins. Macaca nemestrina is an animal model of HIV-1 infections that provides opportunities for evaluating the pathogenesis of acute HIV-1 replication and candidate vaccines and therapies.

Animals↗
PubMed1992

Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.

Multiple human immunodeficiency virus type-1 sequences from the V3 and V4-V5 regions of the envelope gene were analyzed from three mother-infant pairs. The infants' viral sequences were less diverse than those of their mothers. In two pairs, a proviral form infrequently found in the mother predominated in her infant. A conserved N-linked glycosylation site within the V3 region, present in each mother's sequence set, was absent in all of the infants' sequence sets. These findings demonstrate that a minor subset of maternal virus is transmitted to the infant.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Human papillomavirus type 16 and 18 gene expression in cervical neoplasias.

Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3' end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6-E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis.

Adenocarcinoma↗