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Biomedical subjects

M Stevenson

Publications and source records attributed to M Stevenson.

At least 19 recordsLinked to original sources

Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.

After cell infection by the human immunodeficiency virus type 1 (HIV-1), nascent viral DNA in the form of a high molecular weight nucleoprotein preintegration complex must be transported to the nucleus of the host cell prior to integration of viral DNA with the host genome. The mechanism used by retroviruses for nuclear targeting of preintegration complexes and dependence on cell division has not been established. Our studies show that, after infection, the preintegration complex of HIV-1 was rapidly transported to the nucleus of the host cell by a process that required ATP but was independent of cell division. Functional HIV-1 integrase, an essential component of the preintegration complex, was not required for nuclear import of these complexes. The ability of HIV-1 to use host cell active transport processes for nuclear import of the viral preintegration complex obviates the requirement for host cell division in establishment of the integrated provirus. These findings are pertinent to our understanding of early events in the life cycle of HIV-1 and to the mode of HIV-1 replication in terminally differentiated nondividing cells such as macrophages (monocytes, tissue macrophages, follicular dendritic cells, and microglial cells).

Adenosine Triphosphate

Metabolic and biochemical status of articular cartilage following cryopreservation and transplantation: a rabbit model.

To determine the fate of transplanted cryopreserved articular cartilage, an animal model employing the proximal humerus in the rabbit has been developed. Previous studies have been hindered by problems of postoperative joint instability, secondary injury due to immobilization, and paucity of cartilage for analysis. This experiment demonstrates the survival and function of transplanted cartilage by quantitative assessment of metabolic and biochemical parameters. Forty-five New Zealand white rabbits underwent transplantation of the right proximal humerus. In 29 animals, the proximal half of the humerus was resected and replaced by a cryopreserved osteoarticular allograft. Autograft procedures were carried out in the remaining animals. Following sacrifice at 3, 6, 9, and 12 months postoperatively, articular cartilage was analyzed for gross appearance, collagen synthesis, proteoglycan synthesis, and water, hydroxyproline, hexosamine, and hexuronic acid contents. The results indicate that the cryopreserved osteoarticular allografts retained their metabolic and biochemical integrity and behaved as viable and biologically functional units 1 year postoperatively.

Animals

HIV-1 replication and potential targets for intervention.

Intense research into fundamental processes of human immunodeficiency syndrome type 1 (HIV-1) replication has yielded knowledge that in many aspects equals or exceeds that of the oncogenic retroviruses. The availability of sensitive virus detection methods has allowed a more thorough characterization of the biology of virus persistence and latency in vivo and removed the dependence on in vitro models. As a clearer picture of the pattern of HIV-1 replication in vivo evolves, it becomes apparent that HIV-1 biology is distinct from that of the prototypic oncogenic retroviruses in several key aspects, particularly with regard to host cell range and determinants of viral permissiveness. In this respect it may be appropriate to examine the lentivirus, rather than the oncovirus model system to better understand the biology and pathogenesis of HIV-1 infection. This synopsis of recent and ongoing research developments in HIV-1 replication and pathogenesis emphasizes the determinants of host cell permissiveness, early events in virus replication, and underlying features in HIV-1 cytopathogenesis. In addition, basic viral replication processes which can be exploited for therapeutic intervention are discussed.

Animals

Effect of a diet high in monounsaturated fat from almonds on plasma cholesterol and lipoproteins.

The effect of almonds as part of a low saturated fat, low cholesterol, high-fiber diet was studied in 26 adults (13 men, 13 women). The baseline diet was modified in a similar way for all subjects by limiting meat, fatty fish, high-fat milk products, eggs, and saturated fat. Grains, beans, vegetables, fruit, and low-fat milk products were the foundation of the diet. During the almond diet period, raw almonds (100 mg/day) supplied 34 g/day of monounsaturated fatty acid (MUFA), 12 g/day of polyunsaturated fatty acid, and 6 g/day of saturated fatty acid. Almond oil was the only oil allowed for food preparation. There was a rapid and sustained reduction in low-density lipoprotein cholesterol without changes in high-density lipoprotein cholesterol. This was reflected in a total plasma cholesterol decrease from (means +/- SEM) 235 +/- 5.0 at baseline to 215 +/- 5.0 at 3 weeks, and to 214 +/- 5.0 mg/dl at 9 weeks (p less than 0.001). When the consumption of nuts high in MUFA increases the fat content of the diet, reduction rather than elevation of plasma cholesterol has to be expected, possibly due to the MUFA content of these nuts.

Adult

Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection.

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.

DNA, Viral

Posttranslational modifications within the HIV-1 envelope glycoprotein which restrict virus assembly and CD4-dependent infection.

Alterations in two highly conserved N-linked glycosylation sites within the gp120 envelope glycoprotein of human immunodeficiency virus type I (HIV-1) implicated in the phenotype of a noncytopathic HIV-1 variant were introduced independently and in combination into a cytopathic, infectious HIV-1 clone by site-specific mutagenesis. Neither mutation affected the synthesis of HIV-1 envelope glycoproteins. However, one of the mutations restricted the ability of HIV-1 envelope to localize on the cell membrane and thus markedly impaired virus assembly. The HIV-1 assembly defect could be overcome in trans if site-specific mutants were packaged in HeLa cells constitutively producing wild-type HIV-1 envelope glycoprotein. In addition to inefficient virus assembly, this mutation impaired the ability of the virus to infect CD4+ T cells, but did not affect CD4-independent infection of muscle cells. These results suggest additional functions of posttranslational modification in virus replication (i.e., envelope glycoprotein transport). Given that such modifications can restrict CD4-mediated uptake without affecting CD4-independent uptake, variations in posttranslational env processing between different HIV-1 genotypes may affect virus tropism in vivo.

Amino Acid Sequence

Predominance of distinct viral genotypes in brain and lymph node compartments of HIV-1-infected individuals.

A modified polymerase chain reaction protocol was used to amplify the entire envelope-coding region of HIV-1 directly from brain and lymph node tissue obtained at autopsy from three HIV-1-infected individuals. Molecular analysis of amplified DNA by digestion with 18 restriction endonucleases, singly and in combination, revealed different HIV-1 genotypes in the brain and lymph node compartments in each of the three individuals. This anatomic compartmentalization of HIV-1 populations may reflect different viral genomic sequences that determine tropism or differences in host immune selection pressures in the brain and lymphoid compartments that drive the emergence of distinct viral populations.

Acquired Immunodeficiency Syndrome

Lymphotropic viruses as etiologic agents of lymphoma.

DNA and RNA virus with immortalized lymphocytes are described including Epstein-Barr virus and HTLV-I. The roles of immune deficiency and genetic alterations occurring in a proliferating cell are also considered.

Animals

Transformation of rat bladder epithelial cells by introduction of a single oncogene.

Malignant transformation of cells in vitro requires the action of cooperating oncogenes. However, the action of a single activated oncogene, if placed under a strong constitutive promoter, is sufficient to transform in vitro established cells. We have investigated the role of cooperating oncogenes in the transformation of normal rat bladder epithelial cells. Effects of polyoma middle T, v-Ha-ras or activated rat c-Ha-ras, independently or in combination with v-myc on in vitro and in vivo behavior of rat bladder epithelial cells were studied. Introduction of polyoma middle T alone, c-Ha-ras or in some cases v-myc into "normal" rat bladder epithelial cells was sufficient for full malignant transformation of these cells. In addition, introduction of polyoma middle T, activated c-Ha-ras or v-myc transformed cells into nude mice, resulted in development of highly invasive adenocarcinomas. These results indicate that full malignant transformation of normal rat bladder epithelial cells did not require the action of introduced cooperating oncogenes.

Adenocarcinoma

HIV-1 replication is controlled at the level of T cell activation and proviral integration.

During progression of the Acquired Immune Deficiency Syndrome (AIDS), the human immunodeficiency virus type 1 (HIV-1) is harbored in CD4+ T cells, which act as the primary reservoir for the virus. In vitro, HIV-1 requires activated T cells for a productive infection; however, in vivo, the number of circulating T cells in the activated state that are potential targets for HIV-1 infection is low. We have investigated the ability of HIV-1 to infect resting T cells, and the consequences of such an infection. T cell activation was not required for HIV-1 infection; however, viral DNA was unable to integrate in resting T cells and was maintained extrachromosomally. Subsequent T cell activation allowed integration of extrachromosomal forms and led to a productive viral life cycle. Extrachromosomal forms of viral DNA were found to persist for several weeks after infection of resting T cells and, following T cell activation, these forms maintained their ability to integrate and act as a template for infectious virus. Several lines of evidence, including temporal analysis of HIV-1 replication and analysis of an HIV-1 integrase deletion mutant, indicated that extra-chromosomal HIV-1 DNA genomes were transcriptionally active. These results are compatible with a model whereby HIV-1 can persist in a non-productive extra-chromosomal state in resting T cells until subsequent antigen-induced or mitogen-induced T cell activation, virus integration and release. Thus agents that induce T cell activation may control the rate of HIV-1 replication and spread during AIDS progression.

Acquired Immunodeficiency Syndrome

Autonomous growth of lymphoid cells following IL-2 expression from retrovirus vectors containing HIV-1 trans-acting elements.

The human immunodeficiency virus type I (HIV-1) possesses powerful regulatory elements that control the rate of replication of HIV-1 and subsequent processing of HIV-1 genes. We have used this regulatory mechanism to drive expression of foreign genes inserted in retrovirus vectors. This approach was used to express the human IL-2 gene in IL-2-dependent mouse CTLL-2 cells to determine the role of autonomous growth in maintaining proliferation of virus-infected T lymphocytes during HTLV-1-induced adult T-cell leukemia (ATL). Expression of IL-2 sequences in IL-2-dependent mouse CTLL-2 cells resulted in autonomous growth of IL-2-independent CTLL-2 clones. Endogenous expression of IL-2 appeared to interrupt normal constraints of growth in that these IL-2-independent clones showed reduced cell-density-dependent inhibition but not a tumorigenic phenotype. IL-2-independent CTLL-2 clones did not secrete detectable quantities of IL-2 into culture supernatant and exhibited reduced sensitivity to the inhibitory effects of both IL-2 and IL-2 receptor antibody. These results suggest that the IL-2 autocrine loop within these cells involves intracellular IL-2/IL-2 receptor binding. The apparent lack of IL-2 production and poor responsiveness to IL-2 or IL-2 antibodies displayed by cell lines from ATL patients may be explained by an intracellular IL-2/IL-2 receptor autocrine loop.

Animals

Rearrangement and expression of T-cell receptor genes in marmoset lymphoid cell lines with natural killer activity.

To study the role of the T-3/T-cell receptor complex in mediating natural killer activity, we have examined T-cell receptor gene configuration and expression in five transformed marmoset cell lines with natural killer activity. All lines showed rearrangement of the beta chain of the T-cell receptor and expressed full size beta chain transcripts as well as full size delta chain transcripts. Only three lines contained full size T-cell receptor alpha chain transcripts. Of the three lines which had potential for encoding functional T-3/T-cell receptor complex, those which possessed natural killer activity also expressed T-gamma transcripts. In addition, one clone with high natural killer activity which did express high levels of T-gamma mRNA was unable to produce functional T-3/T-cell receptor complex due to truncated T-cell receptor alpha chain message. The results in this paper provide strong evidence that natural killer activity is dependent upon expression of the T-cell receptor gamma chain.

Animals

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

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

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

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

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