Search PubMed⌕ Search

Biomedical subjects

M Stevenson

Publications and source records attributed to M Stevenson.

At least 163 records · Page 9Linked to original sources
PubMed1990

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.

CD4 Antigens↗
PubMed1990

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.

Amino Acid Sequence↗
PubMed1990

The reproduction of physician autonomy in Ontario medical group practice.

A belief exists about the advantages of group medical practice over solo practitioners. The paper through a survey of 105 group practices examines a number of organisational and operational factors. The results suggest that practices are not always organised in a way to maximise the benefits of group operation but are still characterised by an individualistic approach.

Decision Making, Organizational↗
PubMed1989

Inhibition of human immunodeficiency virus type 1-mediated cytopathic effects by poly(L-lysine)-conjugated synthetic antisense oligodeoxyribonucleotides.

The ability of poly(L-lysine)-conjugated and methylphosphonate-modified synthetic human immunodeficiency virus type 1 (HIV-1) antisense oligodeoxyribonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection was studied. The abundance of viral antigens in oligomer-treated cultures indicated that the oligomers did not significantly affect viral infectivity. Similarly, no significant effects on relative viral RNA accumulation were apparent. The presence of poly(L-lysine)-modified oligomer complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus titre in infected cultures. In addition, these cells were protected from HIV-1-mediated cytopathic effects while the other cultures rapidly succumbed to the cytotoxic effects of HIV-1 infection. The presence of poly(L-lysine)-conjugated oligomer resulted in the establishment of a persistent HIV-1 infection characterized by a highly productive virus infection in the absence of cell death while treatment of persistently infected cells with phorbol ester resulted in renewed cytopathicity. These results demonstrate the ability of synthetic antisense oligonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection.

Blotting, Northern↗
PubMed1988

Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for persistence in AIDS.

Masking of host cell receptors following retroviral infection is the basis for the phenomenon of virus interference. Amphotropic retrovirus vectors were used to express the HIV envelope glycoprotein in a human CD4+ cell line. Envelope expression is accompanied by a reduction in the level of surface CD4 receptor molecules and correlates with the presence of intracellular envelope-CD4 receptor complexes. Cells expressing the HIV envelope acquire a cytolysis-resistant phenotype such that infection with HIV leads to a non-cytopathic persistent virus infection. Furthermore, phorbol ester-mediated stimulation of viral replication in persistently infected cells results in renewed cytolytic effects which, due to the absence of CD4 in the cell population, are absolutely independent of syncytium formation. This study elucidates the mechanism by which viral persistence is initiated and maintained in the course of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Kinetics of osmotic water movement in chondrocytes isolated from articular cartilage and applications to cryopreservation.

The ability of chondrocytes to survive conditions encountered during cryopreservation depends on the responses of the cells to the physiochemical changes that result when water is removed from the environment of the cells in the form of ice. Cellular responses are therefore closely related to the osmotic permeability properties of the plasma membrane. In order to optimize the conditions for cryopreservation of chondrocytes, osmotic properties of the chondrocyte membrane were determined from the kinetics of volume change in hypertonic solutions at different temperatures. The hydraulic conductivity of the plasma membrane was 0.305 +/- 0.025 micron3/micron2/min/atmosphere at 24 degrees C, with an Arrhenius activation energy of 8.06 kcal/mol. These values are similar to those reported for other cell types, but the osmotically inactive volume of the chondrocytes (0.41 +/- 0.04) was significantly higher than for other cells, implying that chondrocytes have a higher dry weight ratio or that they contain a higher proportion of osmotically inactive or bound water. These results were used to calculate the osmotic responses of chondrocytes at low temperatures and to predict that the least damaging cooling rate for isolated chondrocytes in the absence of cryoprotective compounds is 10 degrees C/min. The ultimate goal of this study is the development of an analytical model applicable to the optimization of techniques for cryopreservation of intact cartilage and other tissues.

Animals↗
PubMed1988

Preferential in-vitro growth and expansion of leukemic T lymphoblasts.

An in-vitro culture system was used to selectively grow malignant cells from the bone marrow of a patient with acute T-lymphoblastic leukemia. Molecular analysis of DNA extracted from the bone marrow cells before culture showed the presence of both rearranged and germ line patterns for the T-cell beta receptor (CTB) gene, and chromosomal analysis revealed the presence of a major and a minor abnormal clone. The cells were cultured in RPMI medium supplemented with 20% fetal calf serum, 2% lymphocyte conditioned medium, L-glutamine and antibiotics. The presence of malignant cells in the cultured population was confirmed by morphologic, molecular probing and cytogenetic analysis. After four weeks in culture, DNA extracted from the cultured cells showed only the rearranged pattern for the CTB gene. Chromosomal analysis of the same cultured sample revealed only the presence of the initially predominant abnormal clone. Shortly thereafter, analysis of fresh uncultured bone marrow cells from the patient in relapse revealed that the same chromosomally abnormal clone also predominated in vivo. Thus, our results demonstrate the selective nature of this culture system and its ability to amplify leukemic T-lymphoblasts. This culture system is also useful for detecting occult malignant cells in histologically normal bone marrow.

Adult↗
PubMed1987

Expression of the T4 molecule (AIDS virus receptor) by human brain-derived cells.

Three human cell lines of astrocytic origin were evaluated for expression of a human T-lymphocyte surface glycoprotein, T4, which also serves as a cellular receptor for the human immunodeficiency virus (AIDS virus, HIV). T4 antigen was detected on the cell surface of 2 of these cell lines using monoclonal OKT-4 antibody and flow cytometry. Gene transcripts encoding the T4 molecule were detected by a ribonuclease protection assay in surface T4-positive and -negative cells. Our results suggest that astrocytes may serve as targets for HIV infection in the brain.

Antibodies, Monoclonal↗
PubMed1987

Susceptibility of human glial cells to infection with human immunodeficiency virus (HIV).

Three human brain-derived cell lines (including two of astrocytic origin) were exposed in vitro to the human immunodeficiency virus (HIV), the etiologic agent of immunodeficiency in AIDS. In all three lines, HIV transcripts were detected by in situ hybridisation in 20-30% of cells 48 h after infection. Synthesis of virus gag gene products p24 and p55 was demonstrated by immunoblotting. No cytopathic effects typical of HIV-infected human T lymphocytes were observed. Our data indicate that HIV is neurotropic, and support the hypothesis that this virus may infect astrocytes in the brain.

Antibodies, Monoclonal↗
PubMed1987

Expression of glial fibrillary acidic protein in human glioma cell lines as detected by molecular hybridization.

The expression of glial fibrillary acidic protein (GFAP) in continuous glioma and non-glioma cell lines was evaluated by molecular hybridization and immunocytochemistry. RNA transcripts from the GFAP gene were detected in two of six cell lines by Northern blot analysis of cellular RNAs using a cloned mouse GFAP cDNA probe. The probe recognized a single GFAP RNA species of 2.7 kilobases under high-stringency washing conditions. The presence of GFAP transcripts as determined by molecular hybridization corresponded to the immunocytochemical detection of GFAP using two different monoclonal antibodies to this protein. These data indicate that GFAP expression in human cells can be detected by molecular hybridization using a murine GFAP cDNA probe, illustrating the high degree of inter-species conservation of GFAP. Molecular hybridization represents a reliable and sensitive method for the detection of GFAP expression in vitro, which can be used in conjunction with or as an alternative to immunocytochemistry.

Cell Line↗
PubMed1987

A human T-cell line resistant to cytopathic effects of the human immunodeficiency virus (HIV).

Infection of human helper T lymphocytes with the human immunodeficiency virus (HIV) results in a rapid induction of cytopathic effects and cell lysis. We isolated a variant of the human T-lymphoblastoid cell line, CEM, that is fully susceptible to HIV infection but resistant to virally induced cytopathic effects. Exposure of the cells, designated CR-10, to HIV resulted in the expression of viral antigens in 100% of cells within 6-9 days. Virus-infected cells remained fully viable and could be cultivated under standard culture conditions for a desired period of time. Parental CEM cells died within 9-12 days after HIV infection. Proviral DNA could be detected in the HIV-infected CR-10 cells by Southern blot and molecular hybridization 4-5 days after infection; the relative amount of proviral DNA reached maximum at Days 6-10 and remained stable during an 8-month follow-up period. Virus production by HIV-infected CR-10 cells was documented by electron microscopy and detection of reverse transcriptase activity in cell culture supernatants. HIV-infected CR-10 cells exhibited a down modulation of the OKT-3, OKT-4, OKT-4A, OKT-8, and OKT-11 T-cell surface markers, but not of the OKT-9 (transferrin receptor). One of the HIV persistently infected CR-10 cell clones has been kept in continuous culture for over 8 months. During this period, the cells remained fully viable, 100% positive for HIV antigens, and negative for most of the T-cell surface markers tested and continued to produce biologically active HIV. The CR-10 and HIV-infected CR-10 cell lines will be useful in studies on the biology of HIV and in the isolation and large-scale propagation of this virus.

Antibodies, Monoclonal↗
PubMed1987

Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors.

Antigens, Surface↗
PubMed1987

Persistent productive infection of human glial cells by human immunodeficiency virus (HIV) and by infectious molecular clones of HIV.

The nature of the interaction between human immunodeficiency virus (HIV) and human cells of astrocytic origin was studied in vitro with cultured glial cells and intact HIV or infectious molecular clones of the virus. Infection of glial cells with intact HIV was characterized by low-level expression of viral transcripts as detected by Northern blotting and in situ hybridization (less than 10 copies of HIV RNA per cell), transient virus replication, absence of viral antigens detectable by immunofluorescence, and complete lack of cytopathic effects. However, the HIV-infected glial cells persistently expressed HIV tatIII gene activity as detected by a chloramphenicol acetyltransferase assay, and HIV transcripts could be detected by in situ hybridization in 20 to 30% of cells up to 4 months after infection, suggesting that the lack of cytopathicity in HIV-exposed cells was not due to transient viral infection. To evaluate whether increased expression and replication of HIV in glial cells would have any effect on cell growth and viability, we established HIV-positive glial cell lines by cotransfection of cells with infectious molecular clones of HIV DNA and a selectable marker gene. Three clones were isolated which produced high levels of viral particles, were strongly positive for HIV antigens by immunofluorescence, and contained greater than 1,000 copies of HIV RNA per cell. These cell lines showed no cytopathic changes (lysis, fusion), and their growth kinetics were similar to HIV- controls, but significant morphological changes were detected (cytoplasmic swelling; increased numbers of rounded, presumably detaching cells). Our results show that astrocytic cells can support a persistent, replicative HIV infection with limited pathogenic effects.

Antigens, Viral↗
PubMed1987

Detection of malignant cells in histologically normal bone marrow using culture techniques.

Cellular material retained on screens used to filter aspirated bone marrow for future autologous marrow transplantation was studied using long-term culture techniques in 20 consecutive patients. All patients had marrow aspirate and biopsy specimens that were normal histologically. Cultures from five patients grew malignant cells similar to those of the known underlying malignancy. Two types of culture methods were used in these studies. Method I consisted of a long-term bone marrow culture system which predominantly favors adherent cells, and Method II consisted of a suspension type culture system favoring expansion of mononuclear cell types. These findings suggest that tumor cells are reinfused more often at the time of autologous bone marrow transplantation than has been previously suspected. Although the clinical significance of these findings is not known, it is clear that culture techniques combined with special stains and molecular probing will allow the improved detection of occult tumor cells in bone marrow from patients undergoing autologous marrow transplantation. These observations emphasize the need for a comprehensive study of histologically normal autologous marrow using culture techniques to determine the frequency of occult involvement by viable malignant cells and the clinical implications of these findings.

Adult↗
PubMed1986

Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, has the ability to transform human B lymphocytes. No other cell type has been experimentally transformed by EBV, either by intact virions or naked viral DNA and subgenomic fragments. Two immortalized human T-lymphoblastoid cell lines have now been established by transfecting cord blood lymphocytes with purified B95-8 viral DNA enclosed in fusogenic Sendai virus envelopes (RSVE) and then exposing the cells to EBV from a P3HR-1 cell subclone. One of these lines, which has been fully characterized, is termed HBD-1. This line is positive for EBV DNA and expresses surface OKT11, OKT4, and Tac receptors, but not M-1, mu immunoglobulin chains, EBV receptors, or B-1 surface markers. The cells contain fully rearranged T-cell receptor genes and germline immunoglobulin genes. The karyotype of the cells is normal, they do not require interleukin-2 for growth, and do not contain human T-lymphotropic virus type I. However, the HBD-1 cells contain incomplete EBV genomes and express several EBV-determined antigens, including the early antigen type D, membrane antigens, but not EBV-determined nuclear antigen (EBNA). This association of the EBV genome with permanently growing hematopoietic cells of non B-cell lineage should prove useful in studies on the mechanism of EBV-mediated cell transformation.

Cell Line↗