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Biomedical subjects

A L Cunningham

Publications and source records attributed to A L Cunningham.

At least 73 records · Page 4Linked to original sources
PubMed1996

Inhibition of human immunodeficiency virus replication in differentiating monocytes by interleukin 10 occurs in parallel with inhibition of cellular RNA expression.

The mechanism of inhibition of HIVBa-L replication by interleukin 10 (IL-10) in primary monocytes and macrophages at various stages of maturation was investigated using semiquantitative PCR for reverse-transcribed HIV DNA, and Northern hybridization for HIV mRNA expression in comparison with extracellular p24 antigen. Pretreatment of monocytes with IL-10 markedly inhibited expression of both unspliced and spliced HIV RNA, reaching a nadir at 7 days and recovering to normal levels by 10 days after a single application. The optimum inhibitory concentration was 25 ng/ml. Less inhibition of HIV RNA expression was observed when IL-10 was added after HIV infection of monocytes and the inhibitory effect progressively declined to negligible levels as monocytes matured into macrophages over 10 days. IL-10 also downregulated the expression of cellular genes, including the transferrin receptor, 28S rRNA, and GAPDH. The kinetics of the inhibition of cellular mRNAs correlated with the inhibition of HIV RNA and also declined as monocytes matured into macrophages. In contrast, IL-10 did not inhibit cellular mRNA expression in the macrophage cell line THP-1. Neutralizing polyclonal antibody to IL-10 reversed all its inhibitory effects. Interaction of IL-10 and TNF-alpha in combination were generally antagonistic in their effects on HIV transcription. IL-10 prevented stimulation of HIV RNA expression by TNF-alpha after preincubation with monocytes for 48 hr. IL-10 had no effect on the levels of HIV cDNA or the process of initiation and completion of reverse transcription. The inhibitory effect of IL-10 on HIV replication in maturing monocytes was probably mediated mainly by inhibition of cellular gene expression and inhibition of maturation of monocytes into macrophages and their activation, with consequent downregulation of HIV mRNA.

Cell Differentiation↗
PubMed1996

Managing HIV. Part 3: Mechanisms of disease. 3.1 Structure and function of HIV.

The discovery of HIV in 1983 is a landmark of medical science in the 20th century. HIV is a fragile but stealthy virus that thrives within the cells of the immune system itself, subverting the body's defences against disease. Knowing the structure and life cycle of the virus is the key to understanding how it is transmitted, how it causes disease and how best to prevent or control infection.

AIDS Vaccines↗
PubMed1996

Analysis of recombinant and native CD4 by one- and two-dimensional gel electrophoresis.

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.

CD4 Antigens↗
PubMed1996

Herpes simplex virus protein targets for CD4 and CD8 lymphocyte cytotoxicity in cultured epidermal keratinocytes treated with interferon-gamma.

In early recurrent herpetic lesions, CD4 T lymphocytes are the predominant infiltrating cells, and keratinocytes expressing major histocompatibility complex (MHC) class II antigens, induced by interferon-gamma (IFN-gamma), are the major site of herpes simplex virus (HSV) replication. IFN-gamma pretreatment of human keratinocytes in vitro reduced MHC class I antigen down-regulation by HSV-1 infection and induced expression of HLA-DR that was unaltered by subsequent HSV-1 infection. Incubation of these infected keratinocytes with phosphonoacetic acid (PAA) almost completely inhibited expression of four major HSV glycoproteins, although expression of early proteins was not affected. Weak CD8 T lymphocyte cytotoxicity against IFN-gamma-stimulated, HLA-DR-expressing HSV-1-infected keratinocytes was consistently directed to the immediate early/early proteins (all 9 patients tested) but against late proteins to a lesser degree (4/9 patients). However, CD4 T lymphocyte cytotoxicity was much greater and directed predominantly against late HSV-1 glycoproteins (all 9 subjects tested) in these cells.

Antiviral Agents↗
PubMed1996

Twin studies demonstrate a host cell genetic effect on productive human immunodeficiency virus infection of human monocytes and macrophages in vitro.

Biological and genetic variability is a prominent feature of human immunodeficiency virus (HIV) strains, especially in tropism, syncytium formation, and replicative capacity. To determine whether there were variable host cell effects on HIV replication in monocytes, three different strains of low-passage-number monocytotropic blood isolates of HIV and the laboratory-adapted strain Ba-L were inoculated into panels of adherent monocytes drawn from 44 different donors, and peak extracellular HIV p24 antigen titers were compared. The clinical HIV strains showed patterns of either moderate or low-level replication in most donor monocytes (20 to 4,000 pg/ml). However, within this range there was marked variation in peak titers in most donors. HIV type 1 Ba-L replicated in all donor monocytes to much higher levels with less variability (30 to 40 ng/ml). Furthermore, replication of 21 clinical blood-derived strains of HIV in blood monocytes and monocyte-derived macrophages (MDM) from pairs of identical twins and age-matched unrelated donors (URD) of the same sex were compared. In all of the seven pairs of identical twins, the kinetics of replication (measured by extracellular HIV p24 antigen) of panels of four clinical HIV type 1 isolates in monocytes were similar within pairs. However, marked and significant differences in kinetics of HIV production occurred within 10 of the 12 unrelated donor pairs (P = 0.0007). The remaining two URD pairs showed similar kinetic patterns, but only one pair had the same HLA-DR genotype. Similar results were observed with monocytes/MDMs obtained from a second bleed of the same donor. Hence, discordant patterns of HIV replication kinetics between URD monocyte pairs contrasted with concordant patterns in identical twin monocytes. These data strongly suggest a host cell genetic effect on productive viral replication in monocytes and MDMs. So far, no consistent genetic linkage of HIV replication pattern with HLA-DR genotype has been observed.

Adult↗
PubMed1996

Subtype B isolates of human immunodeficiency virus type 1 detected in Australia.

The human immunodeficiency virus type 1 (HIV-1) can be subtyped on the basis of nucleotide sequence variability. Knowledge of circulating HIV-1 genotypes or subtypes allows understanding of the origin and spread of HIV-1 in different geographical regions, and is required for rational vaccine development. A study was undertaken to determine the predominant HIV-1 subtype in Australia. Part of the HIV-1 envelope gene (including the variable domain, V3) was sequenced directly from DNA extracted from peripheral blood mononuclear cells of 17 HIV-1 seropositive people in Sydney, Australia. Phylogenetic analysis based on nucleotide sequence suggested that all patients (including individual cases acquired in New Zealand, Papua New Guinea and Thailand) were infected with HIV-1 subtype B. Octapeptides from the HIV-1 envelope V3 loop tip indicated variation but included a predominance of the most common subtype B octapeptides HIGPGRAF (4 cases), NIGPGRAF (3 cases) and PIGPGRAF (1 case). These data suggest that subtype B is the major HIV-1 strain in Australia (and probably in New Zealand and Papua New Guinea), although the importation of HIV-1 acquired overseas is likely to lead to the detection and dissemination of other subtypes in Australia.

Australia↗
PubMed1996

An HIV-1 infected long-term non-progressor (LTNP): molecular analysis of HIV-1 strains in the vpr and nef genes.

We describe a long-term non-progressive injecting drug user (IDU) who was infected with human immunodeficiency virus type-1 (HIV-1) in 1984, and has survived with stable CD4+ T-cell counts (> 800/microliters blood) without any acquired immune deficiency syndrome (AIDS) related illness. With a goal to investigate the molecular nature of HIV-1 strains infecting this patient, we amplified the nef and vpr genes directly from the fresh uncultured peripheral blood mononuclear cells (PBMCs), and carried out co-culture studies. Sequence analysis of the nef gene (from 1994 samples) showed no deletions (as has been previously reported) expected for a 7 base pair duplication at the C-terminus which prematurely terminated the nef reading frame, whereas even after repeated attempts the nef gene could not be amplified from the 1992 PBMC samples. In contrast, the vpr gene (from 1992 and 1994 samples) revealed two distinct quasispecies with no apparent defects. We observed five amino acid substitutions, between residues 83-90, at the C-terminus which has been recently implicated in G2 cell cycle arrest as an early step to HIV-1 infection. In the light of recent evidence on the role of nef gene defects/attenuations in long-term survival of HIV-1 infected patients, it may be that the nef gene defect created by gene duplication, which eliminated the cysteine-206 crucial in disulfide bond formation, may play a role in chronic HIV-1 infection in this patient. These data further suggest that deletions in the nef gene may not be the only reason for long-term non-progression of HIV-1 infection in some individuals, but the gene defects like duplication and subtle mutations in the functional motifs of both nef and vpr genes may confer similar protection in HIV-1 infected patients surviving for longer periods of time with stable CD4 counts.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus.

Antibodies, Monoclonal↗
PubMed1994

Unlinked anonymous screening of antenatal patients for antibody to human immunodeficiency virus type 1 (HIV-1).

OBJECTIVES: To determine the prevalence of HIV-1 infection in the general community by using a target population (antenatal patients) as an indicator of infection, to monitor any change in the prevalence of HIV infection in this population and provide baseline information on heterosexual spread of HIV infection into this low prevalence population. PATIENTS AND DESIGN: Between January 1989 and January 1992, 4537 unlinked anonymous antenatal sera were tested in two study groups at Westmead Hospital. Repeatedly reactive sera were confirmed by western blot and other supplementary assays as appropriate. RESULTS: No significant change in the seroprevalence of HIV-1 infection was detected between the two study periods. Of the 2208 sera tested in 1989-1990, one (0.05%) was confirmed as positive and one (0.05%) gave a non-specific reaction by enzyme immunoassay (EIA). Of the 2329 sera tested in 1991-1992, there was one (0.04%) HIV-1 antibody positive serum, and two gave non-specific EIA reactions. These results were compared with three other sample populations tested at Westmead Hospital during the same period: linked antenatal patients, antenatal methadone clinic attendees and all women tested. CONCLUSIONS: Periodic anonymous testing of a sample antenatal population (combined with screening of high risk patients) is useful for monitoring the prevalence of HIV infection in these populations and estimating any future need for generalised screening.

Adult↗
PubMed1994

Herpes simplex virus type 2 infection of heterosexual men attending a sexual health centre.

OBJECTIVE: To identify risk factors, particularly circumcision status, associated with serological evidence of herpes simplex virus type 2 (HSV-2) infection of heterosexual men. DESIGN: A cross-sectional case-control study employing an anonymous delinked interviewer-administered questionnaire, clinical examination, and a type-specific serological test for HSV-2. PARTICIPANTS AND SETTING: Three hundred consecutive heterosexual male patients at a public sexually transmissible diseases (STD) clinic in Sydney, Australia. MAIN OUTCOME MEASURES: Associations between serological evidence of HSV-2 infection and history of genital herpes or contact with genital herpes, history of other common STDs, and demographic and behavioural factors such as age, education level, number of sexual partners and lack of circumcision. RESULTS: One hundred and ninety-four patients (64.7%) had antibodies to HSV-2 but only 24% of these gave a history of genital herpes. A history of genital herpes or sexual contact with genital herpes, reported total lifetime number of sexual partners, failure to complete high school and a history of non-gonococcal urethritis or genital warts were associated with serological evidence of HSV-2 infection at the univariate level. Neither increasing age nor lack of circumcision was associated with HSV-2 infection. Following multivariate analysis only the lifetime number of partners and failure to finish high school were significantly strong predictors of HSV-2 infection. CONCLUSION: This is the highest prevalence of HSV-2 infection ever detected in an Australian population and one of the highest recorded globally. As younger men were as commonly infected as older men, and an earlier (1985) study involving the same clinic yielded a lower prevalence, it appears that a high level of ongoing HSV-2 transmission is occurring among Sydney heterosexuals. Increased awareness of this fact could enhance safer sex campaigns.

Adolescent↗
PubMed1994

The magnitude of HIV replication in monocytes and macrophages is influenced by environmental conditions, viral strain, and host cells.

It has been difficult to standardize the effects of various stimuli on HIV replication in monocytes and macrophages in different laboratories. In this study we have shown that it is possible to standardize HIV inocula, culture conditions, and HIV antigen assay to achieve reproducibility with the same viral isolate and donor monocyte and macrophage. However, changes in serum concentration, in the strain of blood-derived isolate (even at low passage), in the donor source of the monocyte/macrophage, and in the state of cellular maturation all influenced HIV production by these cells. The donor source of monocytes contributed as much to variability in HIV production as the HIV strain and results were reproducible when the same source was used repeatedly. These in vitro data suggest that genetic differences may contribute to differences in HIV replication in macrophages and consequently to tissue virus load in vivo.

Antigens, Viral↗