Masters Degrees (Medical Virology)
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Browsing Masters Degrees (Medical Virology) by browse.metadata.advisor "Glashoff, Richard"
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- ItemThe apoptotic potential of different HIV-1 subtype C Tat mutations in cell culture(Stellenbosch : Stellenbosch University, 2013-03) Isaacs, Shahieda; Engelbrecht, Susan; Glashoff, Richard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.
- ItemMonocytes in chronic HIV-1 infection : changes in phenotypic marker expression and their relationship with immune activation(Stellenbosch : Stellenbosch University, 2014-12) Poovan, Karmistha; Glashoff, Richard; Ipp, Hayley; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Medical Virology.ENGLISH ABSTRACT: HIV-infection is characterized by depletion of CD4+ T-cells from the gut-associated lymphoid tissue (GALT) which causes irreparable gastrointestinal tract damage and subsequent microbial translocation of bacterial products such as lipopolysaccharide (LPS), a component of Gram-negative bacteria, into systemic circulation. HIV infection also affects the functions and relative population sizes of various immune cells, such as monocytes. Monocytes are important innate immune cells as they are often the first cells recruited to sites of infection and inflammation. They then either promote inflammatory processes; elicit adaptive immune responses, through their antigen presenting ability; aid in pathogen and debris clearance or aid in damage repair. This cross-sectional study investigated functional changes to monocytes and monocyte subsets (CD14+CD16- and CD14+CD16+) in HIV+, treatment naïve individuals and healthy uninfected controls, using whole blood assays and isolated monocytes. A number of chemokine receptors associated with function and homing, and specific gut-homing receptors, were investigated. Monocyte activation, apoptotic potential and intracellular monocyte cytokine production were also investigated. All markers were evaluated using multi-parameter flow cytometry. Monocyte responsiveness to in vitro LPS stimulation and expression of the afore-mentioned chemokine receptors to viral load, CD4+ count and CD38/8 T-cell expression was also assessed. During HIV-infection monocytes appeared primed to exit systemic circulation and migrate towards the gut, as seen through elevated CD62-L (p < 0.005) and CCR7 (p < 0.005), whereas the CD14+CD16+ subset was increased (p = 0.0461) and exhibited a higher activation status through increased CD69 expression (p < 0.005) compared to the CD14+CD16- subset. An interesting observation was the significantly increased IL-10 production by the CD14+CD16+ subset (p < 0.005). An elevated CCR5 expression in total monocytes (p < 0.005) was also seen. After LPS stimulation, the HIV+ group displayed unique and significant percentage increases in the total monocyte population. The findings of the current study suggest that monocyte functionality may be retained during HIV-infection and that CD14+CD16+ monocytes play a vital role during HIV-infection evidenced by their preferential expansion and priming for GALT migration. The production of IL-10 by this subset further highlights their importance and emphasizes the need for future studies on the role of these cells in chronic stable HIV-1 infection and whilst disease progresses.
- ItemPhenotypic and functional characterization of cytotoxic T lymphocytes in HIV-1 infected South African adults(Stellenbosch : University of Stellenbosch, 2010-12) Pillay, Santhoshan Thiagaraj; Glashoff, Richard; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: In just 25 years since the first reported cases in 1981, the number of Human Immunodeficiency virus (HIV) infected people has risen to 65 million, and over 25 million have died of acquired immunodeficiency syndrome (AIDS). Sub-Saharan Africa accounts for 67% of all people living with HIV and 72% of deaths in this region were AIDS related. Tuberculosis (TB) is one of the most common opportunistic infections in AIDS patients, particularly in developing countries, where 60 - 70% of TB cases occur in HIV-1-infected persons. HIV-1 is a high risk factor for the development of TB, the reactivation of a latent Mycobacterium tuberculosis infection and also progressive TB. CD8+ Cytotoxic T Lymphocytes (CTL) are pivotal in the host immune response to HIV infection. CTL are associated with resolution of acute infection and with reduction in viral load. Studies in macaques and humans indicate the importance of CTL in the control of HIV infection, where reduction in CD8+ T cell number has been correlated with progression to AIDS. The current study was a cross-sectional descriptive study of CD8+ T cells of HIV+ adult South Africans with and without TB co-infection (TB disease). The cohort consisted of anti-retroviral therapy (ART) naive patients and all CTL analyses were carried out on peripheral blood mononuclear cells (PBMCs). A total of 60 South African adults from the Western Cape were utilized in this study, including 15 healthy controls; 30 HIV+TB-individuals and 15 HIV+TB+ individuals. Expression of phenotypic, activation and functional markers were investigated by flow cytometry with the use of fluorochomeconjugated antibodies. The markers examined included the novel activation marker CD137, the CTL associated markers Perforin, Granzyme A, CD107a/b, Fas (CD95), and FasL (CD95L), intracellular cytokines IFN-y and TNF-a and the chronic HIV CTL dysfunction marker PD-1. HIV infection alone was associated with increased baseline expression of TNF-a, Perforin, Granzyme A, PD-1, Fas (CD95), and FasL (CD95L), but not CD137(4-1BB) or IFN-y as compared to uninfected controls. TB co-infection resulted in further increased baseline expression of TNF-a, perforin, PD-1, FasL (CD95L), as well as increased IFN-y. HIV-1 antigen (gag)-specific stimulation in vitro indicated that in HIV infection was associated with antigen-specific upregulation of activation and cytotoxicity markers CD137, IFN-y, TNF-a, Fas, FasL and CD107a/b. In TB co-infection a reduction in antigen-specific degranulation (CD107a/b up-regulation) and also Fas and FasL expression was observed. TB co-infection (in the form of active pulmonary TB) reduced antigen-specific CTL functional activity, but simultaneously there was an association with increased baseline PD-1 expression and also cytolytic marker expression (Fas, FasL, TNF-a). These cytolytic markers could be involved in non-antigen-specific bystander target cell death. The expression of the co-stimulatory molecule CD137 appeared to correlate with interferon-y production and levels of degranulation, confirming its usefulness as a putative surrogate marker of functional responsiveness. These data indicate that in addition to impacting on CD4 T cell function, TB co-infection leads to higher baseline expression of CTL-associated markers, but to dysfunctional antigen-specific CTL responses.
- ItemT lymphocyte inhibitory/exhaustion marker expression in chronic HIV-1 infection and the impact of TB co-infection(Stellenbosch : Stellenbosch University, 2016-03) Golden, Maxine; Glashoff, Richard; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Introduction Chronic HIV-1 infection is driven by inflammation and immune activation which ultimately induces T lymphocyte exhaustion. Co-infection with TB is problematic as the HIV-impaired immune system is unable to effectively contain the TB, and in turn the TB disease promotes further immune activation and exhaustion through additional antigenic burden. In the current two component study, a total of 101 HIV-1 infected, 25 HIV-1-TB co-infected and 52 uninfected control individuals were included from the Cape Town peri-urban region. A cross sectional investigation of expression of markers of immune activation (CD38 and HLA-DR), exhaustion/inhibition (PD-1, Tim-3, LAG-3, 2B4) and apoptosis (CD95) was investigated on both CD4+ and CD8+ T cells. Functional proliferative responsiveness of T cells was also assessed. Materials and Methods Flow cytometry (both 4–colour and 10-colour) was used to determine expression of phenotypic markers using both fresh whole blood (pilot study) and PBMC (main study). CD4+ and CD8+ T lymphocyte populations of all study groups were stained for all the markers and evaluated for positive expression or co-expression. A functional proliferation assay (αCD3 and αCD28 stimulation) was conducted using CFSE-staining on the HIV-1+ samples. Impact of blocking Tim-3 and PD-1 pathways was evaluated. Results Chronic HIV-1 infection was accompanied by a significant increase in CD38 (p<0.0001), CD95 (p<0.01), PD-1 (p<0.01) and Tim-3 (p<0.01) expression on CD8+ T cells (pilot study). TB co-infection led to significantly elevated expression of CD38, CD95 and Tim-3, but not PD-1 (all p<0.05). CD4+ T cells displayed decreased expression of all of these markers in the infected groups, except for Tim-3, which was consistently <5%. In the main study CD8+ T cell-associated CD38, CD95, and PD-1 displayed a similar trend, with significant higher expression in the infected groups (p<0.0001, p<0.05, and p<0.0001, respectively), In contrast to the pilot study, Tim-3 expression was consistently <10%, with no difference between the groups. The novel marker 2B4 showed high level baseline expression (median 59.2%) which was significantly increased in the HIV and HIV/TB groups (69.6% and 75.1% respectively, p=0.025). LAG-3 was poorly expressed. Co-expression of PD-1 and 2B4 as well as CD95 and CD38 was also significantly increased (p<0.0001 for both). Discussion Increased immune activation and exhaustion was evident in the infected groups in both studies. PD-1 and 2B4 were both strongly expressed on CD8+ T cells and were significantly up-regulated in infected groups. PD-1 correlated positively with Tim-3 and LAG-3, and 2B4 with LAG-3, (both p<0.01) indicating that they are useful as biomarkers of exhaustion. Although significantly elevated exhaustion markers were observed in the TB co-infected setting in the pilot study, this did not reflect in the main study (except for CD95, 2B4). This suggests that immune dysfunction is mainly driven by HIV-1 infection alone. Short-term anti-TB therapy may also have had a restorative impact on the exhaustive marker expression. Differences in expression patterns using fresh whole blood vs. PBMC (especially Tim-3) warrant further investigation. Blocking Tim-3 and PD-1 expression on CD4+ and CD8+ T cells did not appear to have beneficial effects on T cell proliferation.