Medical Microbiology

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Browsing Medical Microbiology by browse.metadata.advisor "Glashoff, Richard Helmuth"

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    Characterisation of follicular helper T (Tfh) cells in early treated HIV-infected children: relationship to immune activation and inflammation.
    (Stellenbosch : Stellenbosch University, 2022-11-24) Olifant, Paulina; Glashoff, Richard Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
    ENGLISH ABSTRACT: Background: South Africa has a high burden of Human Immunodefiency Virus (HIV) infection. Babies of HIV-positive pregnant women can become HIV-infected or exposed through vertical transmission. Follicular helper T (Tfh) cells have been of particular interest in HIV infection due to their preferential expansion, contribution to the HIV reservoir and dysregulation within HIV-infected individuals. The aim of this study was to investigate the Tfh cell population within children from the Children with HIV Early Antiretroviral Therapy (CHER) trial who started treatment within the first six months of life to determine whether the numbers of these cells is altered as compared to uninfected children and also whether persistent immune activation and inflammation in these children is associated with Tfh cell dysregulation. Methodology: This retrospective cross-sectional observational study consisted of three groups, i.e., early antiretroviral-treated HIT (HIV Infected Treated), HEU (HIV Exposed Uninfected) and HUU (HIV Uninfected Unexposed), of children. Cryopreserved peripheral mononuclear blood cells (PBMCs) were stained with an 11-colour antibody panel designed and optimised for phenotypic identification and quantification of T cell populations using flow cytometry. Tfh cells populations were characterised as CD4+CXCR5+PD-1+ with/without ICOS+. CD4, CD8 and Treg cells were defined as follicular/ follicular-homing based on CXCR5+ expression and activated based on CD38+ and/or CD69+ expression. Secondary data of clinical parameters and inflammatory cytokines for each group were evaluated. Statistical comparisons between groups were made using the Mann-Whitney test to identify significant differences. Significant correlations between Tfh cells and clinical parameters, other T cell populations and inflammatory cytokines were identified using Spearman’s rank order test. Results: Phenotypic results generally indicated significantly increased proportions of CD38+ subsets in HIT group and CD69+ subsets in HEU group. Although there was no significant difference in median CD4+CXCR5+PD-1+ Tfh cell percentage between groups, the ICOS-expressing subset namely CD4+CXCR5+PD-1+ICOS+ Tfh cells was significantly higher in the HIT (33.6% vs 19.2%; p = 0.016) and HEU group (31.6% vs 19.2%; p = 0.006) compared to the HUU group. In the HIT group, CD4+CXCR5+PD-1+ Tfh cells shared significant negative correlations with a majority of activated T cell subsets. A significant positive correlation between CD4+CXCR5+PD-1+ Tfh and CD8+PD-1+ Tc cells, general indicator of immune exhaustion, was demonstrated. Lastly, the HIT group showed the highest level of INF-α and hsCRP inflammatory cytokines and levels of IL-1β and hsCRP significantly correlated with CD4+CXCR5+PD-1+ICOS+ Tfh cells within this group. Conclusions: Overall levels of immune activation were significantly higher in HIT and HEU groups. Several activated T cell subsets inversely correlated with CD4+CXCR5+PD-1+ Tfh cells, suggesting high levels of immune activation can lead to decreased proportions of circulating Tfh cells. Even though no significant difference in the proportion of CD4+CXCR5+PD-1+ Tfh cells was found between groups, the ICOS+ subset was significantly expanded in HIT and HEU children in comparison to HUU children. The significant positive correlation between IL-1β and ICOS-expressing Tfh cells, within the entire study population and HIT group, suggested that increased inflammation resulted in an Tfh cell increase.
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    Phenotypic and functional immune cell profiling of patients with primary immunodeficiency associated with mycobacterial infections in a tuberculosis endemic region
    (Stellenbosch : Stellenbosch University, 2022-12) Van Coller, Ansia; Glashoff, Richard Helmuth; Glanzmann, Brigitte; Esser, Monika; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.
    ENGLISH ABSTRACT: Background: Inborn errors of immunity (IEI) relating to increased susceptibility to severe, persistent, unusual and/or recurrent (SPUR) mycobacterial infections are of particular concern in countries such as South Africa that are hyperendemic for tuberculosis (TB). Mendelian susceptibility to mycobacterial disease (MSMD), the principal IEI relating to SPUR mycobacterial infections, was originally defined to encompass only weakly pathogenic mycobacteria such as the Bacillus Calmette–Guérin (BCG) vaccine. However, more recent studies have shown that South African MSMD patients are also likely to present with SPUR TB. There are 15 known MSMD-associated genes, which all fall within the Interleukin-12-Interferon-γ (IL-12-IFN-γ) immunological pathway, and mutations in these genes have been described to result in either reduced IFN-γ production or a lack of immune response to IFN-γ. The aim of this study was to evaluate immune dysfunction in patients that present with suspected MSMD using genetic sequencing and in vitro functional profiling assays. Additionally, T-bet, a common transcription factor that is also integral to the IL-12-IFN-γ pathway, was investigated as a potential proxy marker for MSMD. Methodology: Blood was collected from 18 patients presenting with SPUR TB for genetic sequencing and functional immune profiling. Whole genome Sequencing (WGS) was performed to identify candidate disease-associated variants. The immune profiling assays included assessment of the IL-12-IFN-γ pathway through flow cytometric detection of cytokine receptors and intracellular signalling, as well as assessment of immune cell subset distributions and Luminex®-based detection of cytokine/chemokine readouts following stimulation of patient cells with Phytohemagglutinin (PHA), BCG, and IL-12/IFN-γ. T-bet expression was measured by means of intracellular flow cytometry. Results: Through WGS, likely disease-associated novel variants were identified in 82% of the 11 patients that were successfully sequenced and 89% of the identified variants were in known MSMD-associated genes. All patients had some degree of impairment in the IL-12-IFN-γ pathway, and these readouts corresponded with the WGS findings. Further immune investigations revealed that the overall patient group had significantly reduced levels of circulating CD16+ monocytes and lymphocytes as well as reduced levels of inflammatory cytokine/chemokine production following PHA or BCG stimulation. All patients also had aberrant T-bet expression, with reduced expression in CD16+ monocytes and natural killer (NK) cells being the most prominent. There were also very strong correlations between the components of the IL-12-IFN-γ pathway, T-bet expression, CD16-expressing cells, and various cytokines/chemokines. Conclusions: The in vitro functional assessment revealed disruptions in the IL-12-IFN-γ pathway of all patients, and a lack of key immune cell subsets and the cytokines/chemokines that are typically expressed by these cells, supporting the clinical diagnosis of MSMD in these patients. While there were some commonalities for the overall patient group, each individual had a very unique phenotype, emphasising the importance of in vitro assessment in all individuals with suspected MSMD – patients with the same or different variants in the same gene had different functional readouts. T-bet was demonstrated to be a promising proxy marker for MSMD as it correlated well with the immunological deficits observed in the patients and will allow for easier detection of potential MSMD in patients with SPUR TB, which may aid in the estimation of the true prevalence of MSMD in future studies.