Doctoral Degrees (Medical Virology)
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Browsing Doctoral Degrees (Medical Virology) by browse.metadata.advisor "Glashoff, Richard H."
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- ItemBlood dendritic cells in chronically HIV-1 infected individuals in South Africa: Phenotype, function and immune modulation(Stellenbosch : Stellenbosch University, 2016-12) De Swardt, Dalene; Glashoff, Richard H.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical VirologyENGLISH ABSTRACT :HIV-1 infection detrimentally affects CD4 T lymphocytes as well as the blood plasmacytoid (pDC) and myeloid dendritic cell (mDC) compartment. DCs act as innate sensors and as initiators and directors of antigen-specific immune responses. Whereas, mDCs have the unique ability to prime naïve T-lymphocytes and activate adaptive immune responses, pDCs are primary producers of type 1 interferons (IFNs), playing a pivotal role in anti-viral immunity. In the current study both pDCs and mDCs from chronically HIV-1 infected South African individuals (on or naïve for ARV therapy) as well as with and without concurrent TB disease, were compared to matched uninfected controls. Similar to CD4 T lymphocytes, bothpDCs and mDCs, were significantly depleted during HIV-1 infection, (reduction of pDC, mDC and CD4 T lymphocyte was 63% (p 0.001), 80% (p 0.001) and 31% (p 0.01), respectively). In parallel, significantly higher levels of generalised immune activation and exhaustion (CD38+CD8+, PD-1+CD8+ and CD38+PD-1+CD8+ T lymphocytes) were observed. ARV treatment ( 1 year) did not result in DC number recovery despite a significant increase of CD4 T lymphocytes numbers (CD4 T lymphocyte number gain of 89% (p 0.01), it fell short of full recovery).TB co-infection did not exacerbate number loss. Phenotypic characterisation of DC populations in circulation during HIV-1 infection may indicate the underlying reasons for the loss from circulation. Phenotypic profiling by multiparameter flow cytometry included: markers of activation (CD86, CD80 and CD62L), maturation (CD83), apoptosis (TNF-R2, FAS, FASL and TRAIL R1-R4) and chemotaxis (CCR5, CCR7, CCR9 and CXCR6). HIV-infection was associated with a significantly higher percentage of CD86+mDCs which may be indicative of early maturation or transition to secondary lymphoid tissue. The frequency of the CD86+mDCs subset normalised upon ARV therapy. Also, HIV-1 infection influenced the distribution of TNF-R2+pDCs and TNF-R2+mDCs. Increased TNF-R2 expression in both subsets, may attest to enhanced survival function. Functionally, DCs of HIV-1 infected individuals were reactive to TLR-L stimulation and in some cases showed enhanced responses compared to uninfected individuals. A significantly higher frequency of TNF-R2+pDCs, IFN- +pDCs, and TNF +mDCs was observed in whole blood TLR cultures of HIV-1 infected individuals (TNF-R2+pDCs: LPS (p = 0.002) and R848 (p = 0.01), IFN- +pDCs: R848 (p = 0.04), TNF +mDCs: LPS (p = 0.003))s.In addition, whole blood TLR cultures of the ARV treated study group generally showed normalisation of the responses, however; in certain cases ARV therapy reduced responsiveness to levels significantly lower than the control study group (i.e.TNF-R2+pDCs and TNF-R2+mDCs in CpG ODN stimulation). In contrast, a significantly lower frequency of IL12p40+mDCs was observed during HIV-1 infection (p = 0.02). TLR-L cultures of the ARV treated study groups showed normalisation of IL12p40+mDCsfrequency. Notably, treatment with the immunomodulating peptide VIP induced a decline in IL12p40+mDCs to levels lower than the control study group.The frequency of TNF +pDCs in TLR-L whole blood cultures was similar between the healthy, untreated and treated HIV-1 infected study groups, however, significantly reduced frequencies were observed in these study groups upon VIP treatment. These data indicate unique phenotypic and functional changes in DC subsets in chronic HIV- 1 infection which may provide potential targets for immunotherapeutic intervention.
- ItemGenetic aspects of HIV-1 risk in an African setting(Stellenbosch : Stellenbosch University, 2006-12) Petersen, Desiree C.; Hayes, Vanessa M.; Dean, Michael; Janse van Rensburg, Estrelita; Glashoff, Richard H.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.Host susceptibility to human immunodeficiency virus-1 (HIV-1) infection and disease progression to acquired immunodeficiency syndrome (AIDS) varies widely amongst individuals. This observation led to the identification of host genetic factors playing a vital role in HIV-1 pathogenesis. Previous studies mainly focusing on Caucasian-based populations have indicated possible associations between genetic variants and host susceptibility to HIV-1/AIDS. The limited studies performed on African-based populations have emphasised the need for extensive investigation of both previously reported and particularly novel genetic variants within the older and genetically diverse Sub-Saharan African populations. In this study, the case-control samples were represented by African individuals of Xhosa descent, all residing in the Western Cape Province of South Africa. This included 257 HIV-1 seropositive patients and 110 population-matched HIV-1 seronegative controls. Mutational screening was performed in a subset of individuals for the entire coding regions of the CC chemokine receptor 5 (CCR5) and CC chemokine receptor 2 (CCR2) genes, and the 3’ untranslated region of the CXC chemokine ligand (CXCL12) gene, as previously reported (Petersen, 2002). Further analysis of these genes in a larger study sample involved the genotyping of previously identified mutations and single nucleotide polymorphisms (SNPs), which forms part of the present study. In addition, mutational screening was performed for the entire coding region of the CXC chemokine receptor 4 (CXCR4) gene, partial coding region of the mannose binding lectin (MBL) gene, and the promoter regions of interleukin 4 (IL4), interleukin 10 (IL10) and the solute carrier 11A1 (SLC11A1) genes. This was followed by genotyping of SNPs occurring in CCR5, CCR2, CXCL12, MBL, IL4, IL10, CX3C chemokine receptor 1 (CX3CR1), CC chemokine ligand 5 (CCL5) and tumour necrosis factor alpha (TNFα) genes. Significant associations were observed with HIV-1 susceptibility in the Xhosa population of South Africa. These included the CCR5-2733A>G, CX3CR1V249I, IL10-819C>T and IL10-592C>A SNPs being associated with a reduced risk for HIV-1 infection, while the CCR5-2135C>T and SDF1-3’G>A (CXCL12-3’G>A) SNPs were associated with increased susceptibility to HIV-1 infection. Furthermore, certain haplotypes for IL4 and IL10 showed association with reduced risk for HIV-1 infection. This included the identification of a novel IL4 haplotype restricted to the HIV-1 seronegative control group. This study emphasises the importance of considering genetic diversity across all populations, as certain HIV-1/AIDS associations appear to be restricted to specific ethnic groups. These findings have also provided an understanding for further elucidating the functional roles of genetic variants in determining HIV-1/AIDS susceptibility. Ultimately, such genetic association studies will contribute to establishing HIV-1/AIDS risk profiles for African-based populations from pandemic-stricken Sub-Saharan Africa.
- ItemMutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains(Stellenbosch : University of Stellenbosch, 2011-03) Romani, Bizhan; Engelbrecht, Susan; Glashoff, Richard H.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical VirologyENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations.