Medical Microbiology
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Browsing Medical Microbiology by browse.metadata.advisor "Esser, Monika"
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- ItemDevelopment of functional immune readouts for the confirmation of mendelian susceptibility to mycobacterial disease and related primary immunodeficiencies(Stellenbosch : Stellenbosch University, 2019-04) Van Coller, Ansia; Glashoff, Richard; Esser, Monika; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.Background: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a primary immunodeficiency (PID) characterised by a predisposition to infection by weakly-pathogenic mycobacteria. In countries with a high prevalence of tuberculosis, individuals with MSMD are also prone to severe, persistent, unusual or recurrent infections by pathogenic Mycobacterium tuberculosis. Several MSMD-associated genes have been described, including IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1, NEMO, ISG15, IRF8, TYK2, and CYBB, many resulting in a disruption of IL-12 and IFN-γ cytokine axis, which is essential for control of mycobacterial infections. This genetic heterogeneity results in many distinct disorders, which vary in their mode of inheritance and clinical presentation. An accurate molecular diagnosis, confirmed by immune functional studies, is essential to ensure that the patient receives optimal treatment and prophylaxis for infections. The aim of this study was to implement and optimise a set of immune phenotyping and functional validation tests for the key pathway, the IFN-γ and IL-12 cytokine axis, involved in MSMD, and to use these assays to assess immune function in a cohort of suspected MSMD patients. Methodology: Blood was collected from 17 participants with MSMD-like clinical phenotypes. DNA was extracted and PBMCs were isolated from the patients’ blood. Whole exome sequencing (WES) was performed and the resulting data was processed using an in-house bioinformatics pipeline, TAPER™. A set of flow cytometry and ELISA-based functional assays were implemented and optimised to assess the integrity of the IL-12-IFN-γ pathway. IFN-γR1 and IL-12Rβ1 expression were assessed by means of standard surface flow cytometry, and IFN-γ and IL-12 signalling was assessed by the detection of pSTAT1 and pSTAT4 respectively through intracellular phospho-specific flow cytometry. IFN-γ-induced IL-12 production as well as IL-12-induced IFN-γ production was also assessed by ELISA after 48-hour in vitro stimulation. The functional and genetic data were then reconciled in order to confirm the extent of functional impairment associated with each genetic variant. Results: Plausible disease-causing variants were identified through genetic investigations for 11 of the 17 participants. Variants in MSMD-associated genes were found in 8 of these patients, although only one of the identified variants, IFNGR1 (c.818del4), has been described before. Variants in genes not previously associated with MSMD were also found, including variants in IKZF1, NOD2, IRAK1, IKBKB, and NFKB2. All the functional assays were optimised and the combination of the three assays for the assessment of the integrity of the IL-12-IFN-γ pathway was successful in identifying immune deficits in essentially all of the participants included in this study. Conclusions: The current study led to the implementation of functional immune readouts that allowed for the evaluation of the functional impact of both novel and previously described genetic variants on the IL-12-IFN-γ pathway. The results generated from the functional assays were highly variable and often defects within the same gene lead to different phenotypes, which emphasises the importance of in vitro functional confirmation of all PIDs. Hence it would be beneficial to apply these assays routinely for patients with suspected PID relating to mycobacterial susceptibility. A molecular diagnosis with confirmed functional impairment paves the way for targeted treatment and improved disease management options for these patients.
- ItemLongitudinal investigation of vaccine specific antibody levels and cellular markers of adaptive immune responses in HIV Exposed Uninfected (HEU) and Unexposed (UE) infants(Stellenbosch : Stellenbosch University, 2012-03) Naidoo, Shalena; De Beer, Corena; Esser, Monika; Ipp, Hayley; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.ENGLISH ABSTRACT: Background: In South Africa alone, 30% of women of child-bearing age are infected with HIV. With the increasing focus and success of prevention of mother-to-child transmission (PMTCT) programmes, an estimated 300 000 infants are born exposed to HIV every year. The underlying impact of in utero HIV exposure on infant immune health has not been extensively characterised. Clinical follow-up of these HIV-exposed uninfected (HEU) infants reveals increased infectious morbidity and mortality compared to their unexposed (UE) counterparts. Objectives: (i) To evaluate and characterise adaptive immune properties by measuring vaccine-specific antibody levels in children from 2 weeks to 2 years of age in the presence and absence of maternal HIV infection. (ii) To investigate specific cellular markers of immune activation, immune regulation, apoptosis and B cell memory on T and B cell populations in HEU and UE children measured at 18 and 24 months of age. Methods: This sub-investigation formed part of a collaborative pilot study between the universities of British Columbia (Vancouver, Canada) and Stellenbosch. A total of 95 HIV-positive and HIV-negative mothers were recruited after delivery at Tygerberg Hospital, and signed informed consent for their infants to be included in the study. Of these infants, only 27 HEU and 30 UE infants were eventually enrolled and followed up at various time points, starting at two weeks of age. Four of these infants were confirmed to be HIV-positive at 2 weeks and clinically followed up according to the protocol, but were excluded from statistical data analyses. Blood was collected at 2, 6 and 12 weeks and again at 6, 12, 18 and 24 months of age. Quantitative IgG-specific antibodies to Haemophilus influenzae B (Hib), Bordetella pertussis, tetanus and pneumococcus were measured at each time point, using commercially available ELISA (Enzyme-Linked ImmunoSorbent) kits. Cellular markers of immune activation, immune regulation, apoptosis and memory were measured in various populations of T and B cells at 18 and 24 months only, by using four-colour flow cytometry and validated whole-blood staining methods. In addition, a functional assay was developed to evaluate cell susceptibility to apoptosis (spontaneously) by measuring the expression of Annexin V on both CD4+ T and CD20+ B cells after 16 and 24-hour incubation periods. The statistical analysis of the antibody data was conducted by repeated-measures ANOVA (i.e. analysis of variance), using a mixed-model approach. Differences in the expression of the two groups’ cellular markers were compared by employing one-way ANOVA. An F test p value (which assumes normality) was reported, while the non-parametric Mann-Whitney U test served as confirmatory tool. Repeated-measures ANOVA was used for the evaluation of the functional spontaneous apoptosis assay at three time points (ex vivo, 16 and 24 hours) on the 18-month samples, while one-way ANOVA was used for the 24-month samples. Results: The HEU group (n = 23) displayed significantly lower levels of antibodies to pertussis (20.80 vs 28.01 Food and Drug Administration [FDA] U/ml; p = 0.0237), tetanus (0.08 vs 0.53 IU/ml; p < 0.001) and pneumococcus (31.67 vs 80.77 mg/l; p = 0.003) than the UE group (n = 23) at 2 weeks of age. No statistical differences were noted for Hib antibody levels between the two groups at this time point. At 6 weeks of age, HEU infants displayed lower mean levels of all antibodies measured; however, these differences did not reach statistical significance. Following vaccination, compared to UE controls, the HEU group presented with statistically significantly higher antibody levels to pertussis at 6 months (155.49 vs 63.729 FDA U/ml; p = 0.0013), 12 months (26.54 vs 8.50 FDA U/ml; p < 0.001) and 18 months of age (1658.94 vs 793.03 FDA U/ml; p = 0.0362). A significant difference in tetanus antibody levels between the two groups was only evident at 24 months, with the HEU group displaying higher levels (3.28 vs 1.70 IU/ml; p = 0.018) than the UE group. No differences were observed between the two groups following vaccination for Hib. At 18 and 24 months, the HEU group showed increased expression of cellular markers of immune activation (CD69 and CD40L) on CD4+ T cells compared to UE controls. The two groups showed similar expression of the cellular marker of activation CD38 on CD8+ T cells. The HEU group displayed significantly higher levels of CD127, the interleukin (IL) 7 receptor, on CD4+ T cells compared to UE controls at 18 months of age. The HEU group also showed increased expression of cellular markers of apoptosis on both CD4+ T and CD8+ T cells. No statistical significance was noted for the expression of Fas on CD4+ T cells at 18 and 24 months of age. However, at 24 months, the HEU group showed significantly increased expression of FasL on both CD4+ T and CD8+ T cells. During cell culture experiments, the HEU group displayed increased susceptibility to spontaneous apoptosis shown by increased Annexin V expression on CD4+ T cells after a 16-hour incubation period at both 18 and 24 months. At 18 and 24 months, no difference was noted in the two groups’ immune regulation as measured by the expression of CTLA-4. The HEU group displayed increased levels of the cellular markers of immune activation CD80 on CD20+ B cells at 18 and 24 months of age. The HEU group also showed significantly increased levels of CD69 on CD19+ B cells at 24 months. No statistical significance was reached for the expression of CD62L and CD10 at either 18 or 24 months. Although the HEU group displayed increased levels of apoptosis (Fas) on CD20+ B cells, no statistical significance was reached at 18 or 24 months of age. In addition, the HEU group showed no difference in the expression of programmed death 1 (PD-1) at 18 and 24 months. HEU and UE groups showed similar expression of Annexin V after 16 hours of incubation in the 18 and 24-month samples. The expression of the biomarker of B cell memory CD27 on CD20+ B and CD19+ B cells was comparable between the two groups at both time points. Conclusion: At 2 and 6 weeks, lower mean antibody responses in HEU infants suggest poor placental transfer due to maternal HIV infection, while increased responses to specific antibodies may reflect an exaggerated immune response to immunisation. These robust responses may be due to the lack of competition with maternal antibodies, or may be ascribed to indirect stimulation of B cells via the activation of T cells. A hyper-inflammatory state is an imminent danger, with increased expression of cellular markers of immune activation and apoptosis that may be consistent with early HIV exposure that persists following infancy. These observations may serve as contributing factors to the extensively documented increased susceptibility to infections in the HEU population. Although these findings are consistent with a primed immune system, larger studies are required to confirm these observations in relation to clinical outcomes and to assess further whether these differences persist in later years.
- ItemSequence-based molecular diagnosis of X-linked agammaglobulinemia in South African individuals(Stellenbosch : University of Stellenbosch, 2011-03-04) Leo, Melanie Joy; Esser, Monika; Scholtz, Charlotte; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting.