Molecular Biology and Human Genetics

Permanent URI for this community

https://scholar.sun.ac.za/handle/10019.1/140

Browse

Now showing 1 - 20 of 280

2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria
(Stellenbosch : Stellenbosch University, 2019-12) Mishra, Abhilasha Madhvi; Baker, Bienyameen; Leisching, Gina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background:Tuberculosis (TB) is a major cause of infection-related mortalityworldwide. In 2017 an estimated 1.3 million people who were HIV-negative died of TB. An estimated 5-10% of infected individual develop active TB during their lifetime, while the remaining90% (of infected population) successfully control the bacteria. Also, some of the close household contacts of TB patients remain uninfected and healthy. Studying host immune response towards Mycobacterium tuberculosis(M. tb) can unfold the reason behind this enigma. Methods:We conducted a detailed investigation of in vitrohost response from human monocyte derived macrophages(hMDMs)towards different strains of mycobacteria(grown in detergent-freemedia), i.e. pathogenic (M. tbR179) andnon-pathogenic (M. smegmatisand M. bovisBCG). The host response was measured post-infection (at mRNA and protein levels) using AmpliSeq, quantitative real time polymerase chain reaction (qRT-PCR), multiplex ELISA (Luminex), intracellular mycobacterial survivaland cytotoxicity assay. Biological network analysis (ingenuity pathway analysis IPA) was performed to understand the gene regulatory networkinvolved in the pathophysiology associated with the host-immune system.Based on false discovery rate (FDR) and biological functions, we selected an inter-related gene family of interferon induced protein with tetratricopeptides (IFIT1, IFIT2 andIFIT3) from the list of 19 potential differentially expressed genes(DEGs)for knock-up (vector-based over-expression)/down experiments. This gene family is known to form a protein complex during viral infection to act against the antigen. Studyencompassing their role against bacteria is not well established.Therefore, we performed knocking-up of IFITsvia vector-based transfection and knocking-down via small interferingRNA (siRNA) approach to investigate their effect upon mycobacteria inside the host macrophages. Results:AmpliSeqanalysis found 19 DEGs at 12 hours post-infection across all three strains. We observed lower number of mycobacterial CFUs and higher host response (at both RNA and protein level) in hMDMs infected with M. smegmatisas compared to other two strains. Biological network analysis revealed interferon-interleukin associated signalling pathways as most prominent among the 19 differentially expressed genes.We found a differed host response towardsall three strains, which mayattributeto their pathogenicity. Messenger RNA and protein level comparisons at different time points, depicted strong role of interferon and interleukin associated gene network. This network was able to successfully counter M. smegmatisbut succumb to M. bovisBCG andM. tbR179. Most importantly, across all three strains, intra-cellular bacterial growth and survival measured through colony forming units (CFUs)decreased significantly upon knocking up of IFITs(IFIT1, IFIT2 andIFIT3),while we recordedan increase in CFUs upon knocking down ofIFITsin the host macrophages. Using multiplex ELISA, we found higher expression of key pro-inflammatory cytokines (i.e. IDO1, IFN-γ, IL-6, and IL-23) during knock-up (vector-based over-expression)of IFITsresulting in reduction of mycobacteria. Conclusion:Differentially expressed IFITs showed a strong effect against mycobacteria, which can be used as a promising therapeutic targetadjunct to anti-TB therapy. This knowledge will broaden the scope of host drug targets for resistance free bacteriostatic immuno-therapy.
Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigens
(Stellenbosch : Stellenbosch University, 2002-12) Scriba, Thomas Jens; Van Rensburg, E. Janse; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is overwhelmingly prevalent in Botswana and South Africa and to date no interventions have been successful enough to curb the rapid spread of the virus. A number of HIV-1 vaccine strategies are being developed, however the breadth and efficacy of such candidate vaccines, many of which are based on the HIV-1 structural genes pol, gag and env, have mostly been found to be inadequate. The HIV-1 accessory genes are attractive components of HIV vaccines due to their role in viral pathogenesis, early expression and the high ratio of conserved CTl epitopes. Yet, because of undesirable properties questions regarding their safety as vaccine components are raised. In this study candidate tat, rev and nefmutants were assessed for efficient expression and inactivation of undesirable functionality. / Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat, Rev and Nef proteins were constructed. The coding sequences of the genes were codon-optimised for optimum protein expression and these synthetic genes were constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins were mutated at previously described sites by PCR-based site-directed mutagenesis to render them inactive for their respective functions. Corresponding wild-type Tat, Rev and Nef constructs were also made from viral isolates that were least dissimilar to the respective consensus amino acid sequences. tn vitro expression of the different constructs were assessed in 293 cells by Western blotting with polyclonal mouse sera, which were generated by DNA immunisation with one of the Tat, Rev and Nef constructs. The transactivation activity of Tat variants and Rev-mediated nuclear export activity of RRE-containing transcripts were studied in cotransfection experiments using reporter-gene-based assays while Nef functionality was assessed in a cotransfection assay with subsequent flow cytometric analysis of surface CD4 and MHC-I expression on 293 cells. Sequence analysis of the South African HIV-1 subtype C consensus sequences of Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence that was drawn up from a large number of viruses from southern Africa. These consensus sequences were also closer to individual viral isolate sequences than any individual sequences were, indicating that the use of a consensus sequence may serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not significantly higher than the wild-type constructs, however, the codon-optimised rev mutant exhibited markedly higher expression than the wild-type rev construct. Immunoreactivity of the protein with the mouse sera demonstrates expression and immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent CAT expression in 293T and Hela cells. Yet, this activity was significantly impaired using the single mutation, C3?, or the double mutation, C22C3? Compared to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4 downregulation was completely abrogated in one of the mutants, while both Nef mutants were entirely deficient for MHC-I downregulation. These data demonstrate the high expression levels and impaired functionality of sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens that may be used as single-standing vaccine components or form part of a multicomponent HIV-1 vaccine.
Accuracy and impact of the MTBDRplus v2 and MTBDRsl v2 line probe assays for the detection of first-line and second-line drug resistant tuberculosis
(2023-02) Pillay, Samantha; Theron, Grant; de Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Combating drug-resistant tuberculosis (DR-TB) remains a challenge globally. Treatment success rates are often derailed by under-diagnosis and under-reporting of disease. Patients remain contagious for prolonged periods prior to initiation of appropriate treatment which is further exacerbated by the amplification of drug resistance and poor treatment outcomes. Using current and new diagnostic tools effectively is key to rapid diagnosis of tuberculosis and early detection of drug resistance. Firstly, (chapter 2) line probe assays (LPAs) frequent inability to generate a resistance call in paucibacillary specimens is problematic. We showed that while MTBDRplus and MTBDRsl tests work well on smear-negative specimens for detecting drug resistance, failure rates remained high. We demonstrated with the use of routine key programmatic data how time-to-reporting of results improved with the use of molecular assays and provided evidence on how standard-of-care can be improved in a programmatic context. Secondly, (chapter 3) LPA testing on smear-negative specimens is not always performed causing diagnostic delays and hindering their role as a direct front-line diagnostic tests. Thus, by using Xpertgenerated data we determined the ratio of actionable-to-non-actionable results and the number of missed resistant cases at varying thresholds. We demonstrated that Xpert semiquantitation category is superior to informing reflex LPA testing than smear status. In short, this method provides a framework by which laboratories that currently do not test smear-negative specimens to expand testing. Thirdly (chapter 4) current pathways using Xpert MTB/RIF or Xpert Ultra as frontline tests for diagnosing TB and rifampicin resistance lack further treatment guidance. We did a systematic review and assessed the performance of Xpert MTB/XDR for the detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. Participants consisted of 1228 for pulmonary tuberculosis detection and 1141 for drug resistance. We found Xpert MTB/XDR is unlikely to test positive as a follow-up test for the detection of Mycobacterium tuberculosis in samples that test Xpert Ultra
Admixture mapping of TB susceptibility and the contribution of type 2 diabetes in a South African population
(Stellenbosch : Stellenbosch University, 2022-12) Swart, Yolandi; Moller, Marlo; Uren, Caitlin; Kleynhans, Leanie; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH SUMMARY: Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), co-evolved with humans throughout known history. TB is still an ongoing global health threat and the development of TB is complex depended on multiple factors. Although it was established host genetic factors may play a role in developing active TB, study results mostly differed between ancestry groups. Apart from a severe lack of representation of genomic samples from Sub-Saharan Africa, it is also one of the highest TB burden regions worldwide. In addition, other factors such as type 2 diabetes (T2D) are rising at alarming rates on the African continent due to globalization and Westernised lifestyles. The challenge, however, in sub-Saharan African populations is to account for the vast levels of genetic diversity observed, and in some cases extending to a five-way admixed scenario. Besides the continuous overrepresentation of individuals of European descent in genetic research, the methods and available software tools used in genetic research are developed under the assumptions that individuals are homogenous (Chapter 1). Even though multiple studies inferred ancestry in admixed individuals to elucidate demographic history of a particular region, the frequent lack of admixture-specific methods lead to admixed populations often being excluded from these studies (Chapter 1). Nonetheless, correctly adjusting for population substructure in genetic association studies may lead to the identification of novel ancestry-specific genetic loci associated with a variety of phenotypes. To account for this genetic heterogeneity amongst South African admixed individuals in this thesis, we incorporated local ancestry as the number of inherited alleles (0,1 and/or 2) from each ancestral population (Chapter 2). With the use of additional models (Local Ancestry Allelic Association (LAAA) model), we were able to identify additional African-specific genetic variants associated with TB susceptibility in a five-way admixed South African population. To expand on the application of the LAAA model, simulation studies were conducted to test if the LAAA model will be able to capture the true causal variants when faced with any admixture scenario (Chapter 3). Our results indicated that one should not rely on one genome-wide association (GWAS) model to capture true causal variants. Furthermore, the inclusion of an interaction between the allele (major or minor) and ancestry present at each genomic loci along the genome of an admixed individual are recommended in statistical analysis (Chapters 2 and 3). Finally, cis-eQTL mapping was conducted to close the gap between GWAS and the clinical implementation of genetics in a TB-T2D cohort from South Africa (Chapter 4). Additionally, local ancestry was incorporated in cis-eQTL mapping analysis and enabled us to narrow down a list of most probable ancestry-specific genetic variants influencing the development of TB in T2D patients and healthy controls. Previously identified biological pathways implicated in both TB and T2D were identified and linked with a corresponding ancestry. We conclude with an overview of the findings in this thesis and recommend directions for future research. Including populations of complex ancestry and admixture is challenging but will become necessary to improve the quality of research in sub-Saharan African groups.
Advancing the understanding of the PE/PPE protein families through PPE_MPTR protein expression and analysis of pe/ppe single nucleotide variants in differentially drug sensitive Mycobacterium tuberculosis.
(Stellenbosch : Stellenbosch University, 2022-11) Bartizal, Tom Jack; Sampson, Samantha; Kriel, Nastassja; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of infection and death by a single microorganism. The M. tuberculosis genome contains two prominent gene families, encoding proteins known as the PE (proline-glutamate) and PPE (proline-proline-glutamate) families. Several of these are essential for M. tuberculosis pathogenesis, however, the majority of the PE/PPE proteins, especially those within the PPE_MPTR subfamily are understudied. This is apparent regarding the sub-cellular localisation of PPE_MPTR proteins which may interact at the host-pathogen interface. Some functional aspects have also been overlooked, such as the potential for pe/ppe variants to contribute to drug resistance in M. tuberculosis. These genes are often overlooked due to their polymorphic nature, as well as the challenges associated with short-read sequencing technologies to reliably assemble their highly repetitive and GC-rich sequences. For the first part of our study, we aimed to identify the sub-cellular localisation of select PPE_MPTR proteins (PPE_MPTR10, -24, -40, 42, -53 and -62) by expressing them as fluorescently tagged proteins in Mycobacterium smegmatis. We successfully amplified all six genes from M. tuberculosis H37Rv DNA, and successfully cloned ppe_mptr10 into the pCG expression vector. Unfortunately, no protein expression was detected and we were unable to determine the localisation of PPE_MPTR10 within M. smegmatis. For the second aim of our study, we made use of a newly developed analytical pipeline to screen whole-genome sequencing (WGS) data and identify high-confidence pe/ppe singlenucleotide variants (SNVs) associated with drug resistance profiles. Nine SNVs were predicted to be unique to either drug susceptible, multi- or extensively-drug resistant (DS, MDR or XDR) classes of M. tuberculosis. We aimed to verify these findings by PCR and Sanger sequencing of targeted SNVs in an independent set of clinical M. tuberculosis isolates. The SNVs were determined to be true variants identified by the pipeline present in clinical isolates but absent from the reference M. tuberculosis H37Rv. None of the target variants were found to be unique to the drug resistance class for which they were originally predicted. The successful identification of pe/ppe variants by genomic analysis demonstrates the potential to screen these repetitive GC-rich regions for genetic variants. Future studies will be required to establish the role of PPE_MPTR proteins in M. tuberculosis pathogenesis.
Alternative insulin mitogenic signaling pathways in immature osteoblast cell lines
(Stellenbosch : Stellenbosch University, 2002-03) Langeveldt, Carmen Ronel; Hulley, P. A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line.
Analysis and application of evolutionary markers in the epidemiology of Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2008-12) Van der Spuy, Gian Dreyer; Warren, R. M.; Van Helden, P. D.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.
Analysis of a neurochip array dataset to study Parkinson’s disease in a South African study collection
(Stellenbosch : Stellenbosch University,, 2023-02) Step, Kathryn; Bardien, Soraya; Vorster, Alvera; Müller-Nedebock, Amica; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is an incurable, and complex neurodegenerative disease. Both genetic and environmental factors likely contribute to disease onset. Notably, while several pathogenic variants and susceptibility factors have been described in populations of Asian and European ancestry, such variants have seldom been identified in individuals from sub-Saharan Africa (SSA). This could be due to the limited number of studies investigating the genetic etiology of PD in SSA. To address this knowledge gap, the present study undertook the largest, to date, PD-focused genomewide association study (GWAS), and pathogenic variant screening study in SSA to identify possible susceptibility variants and pathogenic variants in South African PD cases. For this, we used raw genotyping data generated from a large collaborative project known as COmprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson’s Disease (Courage-PD), whose goal was to identify PD-associated variants. The NeuroChip array, used to genotype the study participants, contained a total of 306,670 tagging variants and 179,467 custom content variants, including 349 associated with PD. The South African dataset genotyped on the array comprised 452 cases and 280 controls. We hypothesised that these individuals would harbour susceptibility and pathogenic variants. To test this hypothesis, the NeuroChip genotyping data was analysed using various bioinformatic approaches. The quality control (QC) and association analysis were completed using PLINK, and the results were visualized using R software. After excluding 15 individuals during the QC stage, population stratification analysis identified two ‘broad’ ancestral groups, designated as ‘European’ (n=497) and ‘non-European’ (n=220). For the GWAS, no variants reached the genome-wide significance threshold of 5x10-8 , however, variants were found that met the ‘suggestive significance’ criteria (1x10-5 ). A total of 17 variants of interest were identified in the European ancestral group (in the KHDRBS2, FGF14, and PDXK genes) and 2 variants of interest were identified in the non-European ancestral group (in the SYNPR and PDE10A genes). These variants highlighted possible new PD genes that are plausible candidates, but that will need to be confirmed in future, much larger GWAS. Thereafter, a Polygenic Risk Score (PRS) analysis was performed, using PRSice software, on the European ancestral group where the most predictive PRS explained 4.5% of the phenotypic variation (the phenotype being PD). Furthermore, use of the NeuroChip data as a method of pathogenic variant screening, revealed that all 12 variants detected by our group previously were also detected by the array. Moreover, an additional 16 variants in 14 individuals were prioritized as being potentially pathogenic, and warrant further study. Finally, screening of p.G2019S in the LRRK2 gene, arguably the most prevalent PD pathogenic variant, using high-resolution melt analysis, revealed a relatively low frequency of 1.2% (n= 8/689) in our entire PD study collection. Notably, this variant has not been identified in any PD individuals of African ancestry, to date. Collectively, this study highlights the importance of screening and studying underrepresented populations to uncover additional genetic-related risks for PD development. However, future largescale whole-genome sequencing and association studies, including all South African ancestral groups, will likely be needed to identify the remaining, potentially novel genetic factors contributing to PD in our local populations.
Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Celia; Bardien, Soraya; Loos, Ben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental and genetic factors. To date, a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study, and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst pharmacological and surgical interventions are available for PD, the current options exhibit adverse side effects with long term treatment. There is a great need to develop new treatments with i. less side effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One molecule is curcumin, the core component of the curry spice turmeric, which is well known for its antioxidant and anti-inflammatory properties and has already been studied for its possible neuroprotective role in Alzheimer’s disease. The aim of the present study was to create a cellular model of PD by decreasing the expression of PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained from the original cellular model. PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter, PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC- 1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV detection, MLPA was performed on 210 South African PD patients and putative mutations were verified by qRTPCR on the Lightcycler 96. PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant changes were observed in the connectivity of the mitochondrial network, but autophagic flux was significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP (p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in cells with decreased PINK1 expression. Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD patients, and fibroblasts were therefore not obtained. A number of false positive mutations were identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4. In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary regimen in most populations worldwide, further studies on elucidating its biochemical and cellular properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic agents is currently a topical and fast-growing area of research, and holds much promise for clinical application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and PD.
Analysis of ex vivo host biomarkers in sputum samples for diagnosis of pulmonary tuberculosis
(Stellenbosch : Stellenbosch University, 2019-12) Mendy, Joseph F.; Chegou, Novel N.; Sutherland, Jayne; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background Despite all the interventions deployed to control tuberculosis (TB), the disease still continues to be the principal cause of death from a single infectious agent in resource constrained settings. An estimated 60% of suspected TB patients do not have access to TB diagnostic tests. With the limitations of the current diagnostic tests and the importance of early diagnosis and initiation of treatment, biomarker diagnosis of TB would be an optimal option. For biomarkers are indicators of immune activity and state. Therefore, host or pathogen biomarker of TB disease would be ideal. Hence, the aim of this project was to profile a broad array of host markers for development of optimal signatures for detection of pulmonary tuberculosis from other respiratory disorders using ex vivo sputum samples Methods We recruited patients who were seeking medical attention at the MRCG at LSHTM outpatients department and Tuberculosis clinic with symptoms suggestive of TB, prior to clinical or microbiological diagnosis. All age groups were recruited. Sputa were collected at baseline from all participants and at 1 and 2 months from the confirmed TB cases. The sputa were digested with Sputolysin and the supernatant analysed using Luminex arrays while RNA extracted from the pellet were analysed with RT-qPCR. Statistical analyses and graphs were generated using R programming Language and GraphPad Prism, with a q value ≤ 0.05 considered significant. A receiver operating curve (ROC) was used to assess the diagnostic performance of individual and combination markers. Results Confirmed TB (428) and ORD (313) patients were analysed, 70 markers were assessed for diagnostic potential and treatment response. Of these, 37 were significantly different between TB and ORD. The best single marker was MMP-2 with an AUC of 0.73. An eight-marker signature (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES and VEGF) was able to diagnose smear and culture positive TB from ORD with an AUC of 0.77, sensitivity of 78% and specificity of 70%, while a threemarker signature (IL-1β, IL-7 and VEGF) classified smear negative but culture positive TB from ORD with an AUC of 0.74, sensitivity of 86% and specificity of 60%. Among children who had TB, a fourmarker signature (FGF, IL-4, MIP-1a and RANTES) differentiated those with TB from ORD, with an AUC of 0.87, sensitivity of 82% and specificity of 87% and a five-marker signature consisting of BAFF, C3L1, IL-22, MMP-3 and sTNFR1 was able to discriminate TB and HIV co-infected from ORD with an AUC of 0.90, sensitivity of 88% and specificity of 85%. We also found a four-marker signature consisting of EGF, IL-15, MIP-1β and TNF-β that could predict slow versus fast treatment responders at baseline with an AUC of 0.74, sensitivity of 75% and specificity of 80%. Conclusion We have discovered novel sputum host biomarkers and biosignatures for screening of tuberculosis and treatment response. The data is promising for potential translation into a user friendly device as a rapid screening test for pulmonary TB. However, this markers and signatures require further investigations to authenticate their usefulness.
Analysis of host determining factors in susceptibility to tuberculosis in the South African coloured population
(Stellenbosch : University of Stellenbosch, 2009-12) De Wit, Erika; Hoal, Eileen; Van Helden, Paul; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) still represents a global threat due to its devastating effect on health and the subsequent high mortality rate. Previous studies have indicated that host genetic factors are implicated in host susceptibility to TB. Since TB is a complex disease, it can be assumed that susceptibility to M. tuberculosis has multiple genetic causative factors (as well as environmental causes). The current study focussed on a number of South African Coloured (SAC) individuals, some of whom were TB cases and others controls. Population substructure was tested in the admixed SAC population as it can be a strong confounding factor for association studies. Our results using the programme STRUCTURE indicated no population substructure in the SAC population. We further investigated the population structure of the SAC group using Affymetrix 500k SNP chip data which showed that the SAC population group has 4 major ancestral components: the Khoesan, European, African and Asian (Indian). A number of candidate polymorphisms in eight genes, previously indicated to play an important role in TB susceptibility, were tested in case-control associations studies. We found statistically significant associations between IFNGR1, IL-8, IL-1Ra and NRAMP1 polymorphisms and TB susceptibility in the SAC population. It has become increasingly evident that gene-gene interactions play a far more important part in an individual’s susceptibility to a complex disease than single polymorphisms would on their own. The importance of epistasis was clearly identifiable in this study with only four associations found between the individual variants and TB susceptibility, but eight instances of statistically significant gene-gene interactions. A combined data set consisting of 106 variants constructed from our database and also used for gene-gene interaction analysis yielded numerous statistically significant interactions. The interaction between the genotype of the human host and the bacterial strain genotype was also investigated and yielded interesting results. Owing to various polymorphisms in several cytokine genes, the protein levels of the main modulators of the immune system, cytokines and chemokines, are changed in several diseases such as infectious diseases and may affect susceptibility or resistance to TB. The functional polymorphisms or haplotype patterns in some of these cytokine genes might be vital for protective immune responses and may serve as biomarkers of protection or susceptibility to TB. The present study investigated 18 cytokines including pro-inflammatory, anti-inflammatory and chemokine factors in healthy (mantoux positive or negative) children using the Linco-plex immunoassay, and investigated potential interactions. The basic research will one day contribute to personalised genetics which may benefit infectious diseases such as TB. If individuals can be identified as potentially more vulnerable, they may require different vaccination strategies, a higher index of suspicion if exposed to TB, and prophylactic treatment.
Analyzing genetic factors contributing to dysmotility in Hirschsprung’s disease and African Degenerative Leiomyopathy in a South African population
(Stellenbosch : Stellenbosch University, 2019-03) Maluleke, Twananani; Moore, Samuel; Kinnear, Craig; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Introduction Hirschsprung’s disease (HSCR) and African degenerative leiomyopathy (ADL) are rare gastrointestinal disorders affecting neonates and young children. HSCR is characterised by the absence of intrinsic ganglion cells in the distal segment of the intestine; its aetiology has been linked to cellular and molecular mechanisms associated with the enteric nervous system (ENS) development, ADL on the other hand is a distinctive form of visceral myopathy (VSCM) of uncertain aetiology affecting enteric smooth muscles (ESM) of the distal intestine. Gut motility is a result of highly coordinated contractions by muscle layers, neural network and pacemaker intestinal cells of Cajal whose development are controlled by genetic factors. The aetiology of HSCR has been associated with 15 genes linked to ENS development meanwhile ADL has been linked to environmental factors. Actin gamma 2 (ACTG2) is a gene that encodes the ACTG2 protein which is involved in ESM development. Studying the ACTG2 in HSCR patients may ascertain whether individuals affected by HSCR also display muscular dysfunction thereby providing a possible factor in the recurrence of dysmotility post-surgical resection. Additionally, ACTG2 has been identified as the genetic factor in VSCM pathology; therefore, the study may provide novel information regarding the genetic factors of ADL. Aim This project aims to study the genes associated with the development of enteric nervous system (RET, NRG1, SOX10, EDNRB) and smooth muscle cells (ACTG2) that contribute to HSCR and ADL in the South African neonate population. Methods Seventeen whole blood samples were collected from HSCR participants after informed consent; of which only 14 samples were included for genotyping and 9 samples were selected for RNA analysis based on the quality of extracted DNA and RNA respectively. Five whole blood samples were also collected from ADL patients after informed consent. RNA samples from the HSCR cohort were reverse transcribed and quantitative polymerase chain reaction was performed. DNA samples from HSCR and ADL samples were screened for variants in the ACTG2 exons through bidirectional Sanger sequencing. Novel variants were analysed in silico to ascertain their pathogenicity. Results and Discussion In both HSCR and ADL cohorts the variant K119E/R was observed in 64% (9/14) and 60% (3/5) of the study population respectively; K119E/R is likely to function as a disease modifier as it was also observed in the control samples six out nine individuals. Variants S345L and W357G in exon 10 with probable significant effect in the pathogenesis of ESM were identified in the HSCR cohort only. The ADL cohort had polymorphic intronic variants predicted to shift the exonic splice sites namely g>c -IVS12 exon 3 and c>t -IVS3 exon 5. Differential expression of ENS genes EDNRB, RET, SOX10 and NRG1 associated with ENS development in the HSCR cohort was not achieved due to experimental factors. Conclusion ACTG2 encodes an enteric smooth muscle γ-2 actin which plays a pivotal role in the contractile proteins of ESM, thus the data suggests that a muscular component may exist in HSCR aetiology that should be investigated further in vitro and provides further insights into genetic factors that may contribute to ADL pathogenesis.
Antimycobacterial activity of ascidian fungal symbionts
(Stellenbosch : Stellenbosch University, 2022-12) Tapfuma, Kudzanai Ian; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gerhard; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which primarily affects the lungs. Treatment of TB is complicated because the causative agent, Mycobacterium tuberculosis, is an intracellular pathogen which infects and kills cells of the innate immune system, while exhibiting intrinsic and extrinsic resistance to many of the currently available antimicrobial agents. A sizeable percentage of TB patients in the world-population are infected by M. tuberculosis strains which are resistant to currently utilized first- and second-line anti-TB drugs. Drug-discovery studies bioprospecting for compounds with novel anti-TB activities are therefore essential in order to control the spread of TB and to prevent a catastrophic pandemic. In this study, extracts from marine fungi were considered for antimycobacterial activity bioprospecting as they are largely underexplored. A total of 46 cultivable fungi were isolated from ascidians and 32 of these fungal isolates were sequenced and consequently identified. Among these fungi, the methanol crude extract from Clonostachys rogersoniana MGK33 was found to possess the highest antimycobacterial activity with minimum inhibitory concentrations of 0.125 and 0.200 µg/mL against Mycobacterium smegmatis mc2 155 and M. tuberculosis H37Rv, respectively. Untargeted metabolite profiling of the crude extract from C. rogersoniana MGK33 revealed the presence of bionectin F (among other compounds) which has previously been shown to possess antimicrobial activity in other studies. In silico molecular docking and simulation experiments in this study showed that bionectin F is a potential inhibitor of M. tuberculosis β-ketoacyl-ACP reductase (MabA). An attempt was then made to generate novel agents that would be composed of nanoparticles surface functionalized with the bioactive fungal extract from C. rogersoniana MGK33. In particular, mono-metallic SPIONs were synthesized using the co-precipitation method and then surface modified to produce bi-metallic superparamagnetic iron oxide nanoparticles (SPIONs) using nickel, zinc, gold, copper and silver, to produce Ni-SPIONs, Zn-SPIONs, Au-SPIONs, Cu-SPIONs and Ag-SPIONs. Functionalization was then performed using the MGK33 extract to produce Ni-SPIONs@MGK33, Zn-SPIONs@MGK33, Au-SPIONs@MGK33, Cu SPIONs@MGK33 and Ag-SPIONs@MGK33. Among these agents, Cu-SPIONs and Ag SPIONs were found to exhibit the strongest antimycobacterial activity, comparatively stronger than that of the counterparts, Cu-SPIONs@MGK33 and Ag-SPIONs@MGK33. In an experiment involving the treatment of RAW 264.7 macrophage cells infected with M. smegmatis mc2 155, the MGK33 extract exhibited the highest early apoptosis activity (9.61%), followed by Cu-SPIONs@MGK33 (3.34%), both agents tested at 1.96 µg/mL for 24 hours. The MGK33 extract further showed strong antimycobacterial activity against intracellular M. smegmatis mc2 155, compared with the nanoparticles synthesized in this study. Results in this study led to the conclusion that the marine fungus, C. rogersoniana MGK33 is a prolific source of compounds with antimycobacterial and immunomodulatory activity, and that further studies should be done to develop Cu-SPIONs and Ag-SPIONs into lead agents for anti-TB drug development.
The antimycobacterial activity of phytocannabinoids
(Stellenbosch : Stellenbosch University, 2023-02) Williams, Ricquelle Daphne; Mavumengwana, Vuyo; Loxton, Andre; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Tuberculosis (TB) is a deadly and communicable disease that is caused by the bacterium, Mycobacterium tuberculosis (M.tb). M.tb is skilled at manipulating and evading hostdefense mechanisms by alveolar macrophages, allowing its survival and replication intracellularly. Since the current treatment regimen makes use of antibiotics, the everincreasing number of multi-drug resistant (MDR) M.tb strains has resulted in a need for research into alternative options. Although most anti-TB drug discovery strategies target the actual pathogen, slow pace in discovering new antimycobacterials is testament to the need to change strategies. Host directed therapy (HDT) is an intervention where the eradication of the intracellular pathogen is mediated by the host immune response modulated by small molecules. Cannabis sativa L. (C. sativa) is valued for its psychoactive potentials and varied ethnobotanical medicinal properties due to a plethora of bioactive constituents. In addition to the plant’s utility as a treasure trove for medicinal applications, this research study aims to present a case for the use of small molecules derived from C. sativa as an alternative HDT against TB. We aimed to evaluate the antimycobacterial effect of crude extracts of two C. sativa plants, one grown outdoor (C. sativa plant 1 or P1) and one grown under controlled indoor conditions (C. sativa plant 2 or P2) and evaluated their bioactive organic extracts activity in THP-1 macrophages infected with mycobacteria. In addition, we isolated endophytic fungi from C. sativa and evaluated their antimycobacterial activity and whether they produce similar compounds to the host plant. Herein, it was demonstrated that the dichloromethane (DCM) extract of C. sativa plant 1 (DP1) and the methanol and ethyl acetate extracts of C. sativa plant 2 (MP2 and EP2) stimulated THP-1 macrophages in the killing of Mycobacterium smegmatis (M. smegmatis) mc2155 compared to untreated macrophages. In addition, the methanol extract of C. sativa plant 2 (MP2) displayed the best activity with a percentage survival of 14.31% (p = 0.000009) at 6-hours post treatment; 2.63% at 12-hours post treatment (p = 0.0001) and 0% survival at 24-hours post treatment (p = 0.0005). The metabolite profile showed that these three extracts (DP1, MP2 and EP2) share three compounds, cannabinol (CBN), cannabigerol (CBG), and cannabielsoin (CBE), which could be the cause of macrophage stimulation. However, MP2, which showed the best activity, contains cannabidiol (CBD), which could be the cannabinoid causing the increased activity. Although the actual bioactivities of CBN, CBG, CBE and CBD remain speculative until further purification, characterization and reevaluations are carried out, a cautious judgement can be made that these compounds likely play a beneficial role. Fungal endophytes Alternaria alternata (A. alternata), Alternaria infectoria (A. infectoria), Fusarium incarnatum (F. incarnatum), and Fusarium chlamydosporum (F. chlamydosporum) were isolated from surface sterilized buds of C. sativa and identified using molecular and phylogenetic methods. A. alternata showed some compounds (via LC-QTOF-MS) which were previously isolated in C. sativa, with its extracts exhibiting immunomodulatory activity against THP-1 macrophages infected with M. smegmatis mc2155. The percentage survival for treated THP-1 cells was found to be 51.81% at 6 hours (p = 0.0003) compared to 81.91% untreated. Overall, these results establish that cannabinoids are able to influence THP-1 infected cells’ ability to clear mycobacterial infection albeit a low percentage cell survival. To develop specialized HDT strategies targeting macrophages, a deeper comprehension of the mutual interaction between cannabis and immunity is required. Future studies to isolate and characterize compound(s) from the endophytic fungi and plant extracts could result in lead development of naturally sourced drugs for host-directed TB treatment.
Application of Becton Dickinson FACSTM Combinatorial Antibody Profile (FACSTM CAP) technology to the identification of efficiency of tuberculosis therapy
(Stellenbosch : Stellenbosch University, 2015-03) Smith, Bronwyn Kerry; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : Currently the treatment of individuals with active Mycobacterium tuberculosis (Mtb) infection involves a standard six-month multi-drug regimen, impacting negatively on treatment adherence, which in turn fuels multi- and extensive drug resistant TB. However, some patients may not require the full six-month regimen due to less extensive disease or rapid early treatment response. The identification of these patients has been problematic but would allow significant cost savings and may impact positively on treatment adherence if treatment duration could be shortened and if this subgroup constituted a significant portion of patients. The aim of this project was to identify peripheral blood lymphocyte surface markers through a proprietary technology, FACSTM CAP by Becton Dickinson Technologies, to investigate the change in expression during the course of treatment with potential treatment monitoring utility. Peripheral blood mononuclear cells (PBMCs) were isolated from TB patients (n=33), healthy community controls (n=11) and other lung disease controls (OLD, n=9) at diagnosis of disease, week 4 (after commencement of treatment) and week 24 (end of treatment, EOT). Antibodies to 252 surface markers were used to stain PBMCs, the cells were fixed in 2% paraformaldehyde and data acquired on a FACS Calibur flow cytometer. Post-acquisition compensation and analysis was performed using FlowJo software. The analysis was performed by gating on the lymphocytes and overlaying sample plots on isotype controls. Statistics analysis included repeated measures ANOVA, paired t-test and independent t-test. Comparisons were made between the expression levels of patient time points (diagnosis, week 4 and week 24) and participant groups (TB, healthy community controls and OLD controls). Sample wells that provided an uncertain demarcation of the positive and negative expression population were flagged and excluded from analysis. After the application of the Bonferroni correction, results revealed five overall treatment response markers (CD120b, CD126, CD62L, CD48 and CD29) that were significantly different (p-value <0.0002) when comparing expression levels at TB diagnosis and EOT (week 24) samples. A comparison of expression between TB at diagnosis and healthy community controls showed a significant difference for four markers (CD48, CD18, CD126 and fMLPr). Due to the application of the stringent Bonferroni correction, only these few markers were found to be statistically significant therefore all markers with a p-value <0.01 prior to Bonferroni correction, were included for analysis with Ingenuity Pathway Analysis (IPA) and Qlucore Omics Explorer software. IPA identified 23 biological pathways that were associated with two or more markers with significant changes during treatment. The top nine pathways are discussed and included the inflammatory response, cell migration, differentiation and maturation and crosstalk between cells of the innate and adaptive immune responses. In conclusion, this project resulted in the identification of three promising biologically significant surface markers that require further validation as candidates for biomarkers of TB treatment response. Future studies will investigate the most promising markers, including those that showed a trend for differences after the Bonferroni correction, in a candidate biomarker project with a new cohort of TB patients undergoing treatment.
The application of immunological biomarkers and enhanced pathogen detection for the epidemiological characterisation of bovine tuberculosis in African Rhinoceros
(Stellenbosch : Stellenbosch University, 2024-01) Dwyer-Leonard, Rebecca Ann; Miller, Michele Ann; Goosen, Wynand Johan; Witte, Carmel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: African rhinoceros, specifically the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros, are iconic species that are under threat due to poaching for their horns, range/habitat loss, unbalanced genetic/demographic structure, climate change, and infectious diseases, including tuberculosis (TB). Mycobacterium bovis (M. bovis) infection, a cause of TB, has been identified in African rhinoceros populations in Kruger National Park (KNP), South Africa. An interferon-gamma release assay (IGRA) is routinely applied for testing of individuals earmarked for translocation out of the park, and for general surveillance purposes. However, relatively little is understood about the overall susceptibility and pathogenesis of TB in these species, and its impact on affected populations. This study had four broad aims: i.) to collate information on the epidemiology of Mycobacterium tuberculosis complex (MTBC) infections in African rhinoceros, ii.) to determine prevalence and risk factors for M. bovis infection in KNP rhinoceros, iii.) to assess the impact of refrigeration and delayed stimulation of rhinoceros whole blood on mitogen stimulated interferon-gamma (IFN-γ) production, to increase flexibility in implementation of testing, and iv.) to determine whether MTBC can be detected in nasal swabs from rhinoceros with immunological evidence of infection, as an indication of potential infectiousness. Drawing from existing literature on MTBC infections in other species and contexts, a foundational understanding of TB epidemiology in rhinoceros species was developed. In other species, demographic risk factors include sex and age, with males and adults generally being at higher risk than females and younger individuals. Review of limited historical information reflected similar age- and sex-associated patterns for TB in captive African rhinoceros, with more reports of TB disease in black rhinoceros than white rhinoceros. Intra-species transmission of MTBC in rhinoceros was also considered to be a potential source of infection. Free-ranging rhinoceros in bovine TB (bTB) endemic areas may be exposed to MTBC, likely shed by maintenance hosts in KNP such as African buffaloes (Syncerus caffer), greater kudus (Tragelaphus strepsiceros), or warthogs (Phacochoerus africanus), through shared environmental niches, and resources. Based on previous reports, hypotheses were generated then investigated in a population-based study of M. bovis infection in 437 African rhinoceros in KNP. We determined an estimated overall infection prevalence of 15.4% (95% CI: 10.4-21.0%) based on mycobacterial culture and IGRA results for animals sampled between 2016-2020. Notably, a significant spatial clustering of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo herds in the vicinity of the rhinoceros sampling location. Spillover of infection from African buffaloes to white rhinoceros sharing the environment was suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016-2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. Ante-mortem surveillance for M. bovis infection in the Kruger National Park (KNP) rhinoceros population currently relies on results from (QFT)-Mabtech equine interferon-gamma (IFN-γ) release assay (IGRA). However, the requirement for same-day processing of rhinoceros blood samples for the IGRA is a logistical challenge to performing this test, particularly in remote locations. A pilot study showed that relative concentrations of IFN-γ (based on optical density values) in mitogen stimulated whole blood plasma decreased significantly with increased time blood was stored post-collection and prior to QFT stimulation. These findings support a need for same-day processing of rhinoceros blood samples for QFT-IGRA testing, as per the current practice to ensure optimal test performance. It was previously unknown whether M. bovis-infected rhinoceros could shed mycobacteria in respiratory secretions. Previous studies suggested that subclinically M. bovis-infected rhinoceros may pose minimal transmission risk. However, recent advances that have improved detection of MTBC members in paucibacillary samples prompted further investigation of respiratory secretions from rhinoceros with immunological evidence of infection, to elucidate the potential for mycobacterial shedding. A pilot study detected M. bovis in 14/64 (22%; 95% CI: 13-33%) of the IGRA positive rhinoceros, and none in the IGRA negative rhinoceros (n = 11) studied, suggesting that M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment. Overall, these studies address important knowledge gaps related to surveillance and epidemiology of TB in African rhinoceros, specifically, the free-ranging populations in KNP. This has created awareness of the potential threat of this pathogen to the conservation of these species and highlighted important areas for future research that will contribute to understanding the multi-host TB ecosystem in KNP and other complex systems.
Application of spoligotyping in the understanding of the dynamics of Mycobacterium tuberculosis strains in high incidence communities
(University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences., 2007-03) Streicher, Elizabeth Maria; Victor, T. C.; Warren, R. M.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
Tuberculosis (TB) is a global health problem and demands rigorous control management efforts. A dramatic increase in the acquisition and spread of drug resistant TB globally has been observed in recent years. A grim picture has emerged for the control program with the discovery of extreme drug-resistant TB, which is virtually untreatable and is of immense concern for the future of TB control. In the last decade strain-specific genetic markers have been identified to examine the molecular epidemiology and spread of TB, including IS6110 DNA-fingerprinting and spoligotyping. Although spoligotyping has less discriminatory power than the gold standard, IS6110 DNA-fingerprinting, it is simpler, faster and less expensive, as it is PCR-based. Spoligotyping has been applied to enhance our understanding of the dynamics of drug susceptible and drug resistant strains of Mycobacterium tuberculosis in high incidence communities, by studying 3 aspects of the TB epidemic: molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis. By using spoligotyping and other genotypic and phenotypic analysis of drug-resistant M. tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. An emerging drug resistant M. tuberculosis outbreak has been identified, termed DRF150, which has specific genotypic characteristics and is resistant to 5 first-line drugs in 45% of the cases. Inappropriate chemotherapy; poor adherence to treatment and prolonged periods of infectiousness due to the delay in susceptibility testing has led to the development and spread of this drug resistant genotype. The study demonstrates the ability of the spoligotyping technique to accurately determine the pathogenic mechanism of recurrent disease by spoligotyping, making it useful in large-scale intervention studies. Application of spoligotyping and a newly developed PCR-method showed that the occurrence of multiple infections was higher than what was previously assumed and also more frequent in retreatment cases than new cases. These findings have important implications for the understanding of protective immunity, and the development and testing of new vaccines and drugs. Various different molecular markers including spoligotyping has been used to reconstruct the evolutionary history of isolates with less than 6 copies of IS6110 element (termed Low Copy Clade (LCC)), which were previously poor defined. It was also shown that LCC is widely disseminated and play an important role in the global tuberculosis epidemic. Reconstruction of the evolutionary relationship of M. tuberculosis Principal Genetic Group 2 strains, identified previously unknown genetic relationships between strain families and laid the foundation to establish correlations between genotype and phenotype. Spoligotyping signatures, created by evolution of the Direct Repeat region in M. tuberculosis, were identified, which will enable the analysis of the strain population structure in different settings and will also enable the rapid identification of strain families that acquire drug-resistance or escape protective immunity in drug and vaccine trials. This study contributed to our understanding of the molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis in high incidence communities.
Aspects on advanced procedures during endoscopic retrograde cholangiopancreatography for complex hepatobiliary disorders
(Stellenbosch : Stellenbosch University, 2021-03) Lubbe, Jeanne Adele; Moore, Samuel; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Molecular biology and human genetics.
Background: The rapid development in endoscopic technology and associated skills has led to an increase in more advanced procedures being performed during endoscopic retrograde cholangiopancreatography (ERCP). Knowledge is limited regarding clinical value, integration, and outcomes for single operator cholangiopancreatoscopy (SOCP) and endoscopic intervention in the different Bismuth-Corlette (B-C) locations in the hepatic hilum. Objectives: To determine the clinical value of SOCP in the diagnosis and treatment of complex hepatobiliary and pancreatic disease. To describe the nationwide integration of SOCP and the extent to which adverse events are influenced when SOCP is added to ERCP. To compare adverse events and reintervention rates after endoscopic stenting for malignant obstruction in the distal and hilar locations of the biliary tree. To compare outcomes after endoscopic transpapillary (ETP) and percutaneous transhepatic (PTH) stenting in the palliation of malignant hilar obstruction (MHO). Methods: In study I all SOCP procedures performed between March 2007-December 2014 at a tertiary highvolume endoscopy unit were separately graded according to a predefined 4-graded scale estimating therapeutic value and diagnostic yield. Study II was a nationwide case-control study nested within the cohort of ERCP procedures, with- or without SOCP, and registered in the Swedish Registry for Gallstone Surgery and ERCP (GallRiks) between 2007-2012. To assess risk factors for adverse events, multivariate logistic regression was performed, and odds ratios (OR) calculated. The GallRiks registry was also utilised in study III where all patients undergoing endoscopic stenting for malignant biliary obstruction between 2010-2017 (based on International Classification of Diseases (ICD) coding), were included. Kaplan-Meier analysis was employed to calculate stent patency and Cox proportional hazard models to calculate the risk for recurrent biliary obstruction after single metal stent placement. To compare ETP and PTH drainage approaches, a retrospective deconstructed analysis of palliative stenting procedures for MHO at two specialised referral centres over a 5-year period was performed. Within-group analyses were performed to explore outcomes for different B-C types and Kaplan-Meier and restricted mean survival time analyses were performed to calculate and compare duration of therapeutic success. Results: In 365 SOCP procedures, SOCP was found be of pivotal importance in 19% of patients, of great clinical significance in 44%, and did not affect clinical decision-making or alter clinical course in 37% of patients. In study II a learning curve was observed after first introduction of 408 SOCP procedures, and postprocedural adverse events (19.1% vs. 14.0%), pancreatitis (7.4% vs. 3.9%) and cholangitis (4.4% vs. 2.7%) were more prevalent when SOCP was added to ERCP. After multivariate analysis, the risk for postprocedural adverse events remained (OR 1.35, 95% CI [1.04 - 1.74]). In 4623 ERCP procedures performed for stenting of malignant strictures (1364 hilar), adverse events and 6-month reintervention rates were increased after hilar stenting compared to distal stenting (17.2% vs. 12.0%, 73.4% vs. 55.9%). On multivariate analysis the risk for reintervention was three times higher after single metal stent placement in the hilum compared to the distal biliary tree (HR 3.47, 95% CI [2.01-6.00], p<0.001). In 293 patients undergoing palliative stenting for MHO (52.2% ETP, 47.8% PTH), access and bridging success in the ETP and PTH groups were 83.5% vs. 97.2% and 90.2% vs. 84.5%, respectively. Technical and therapeutic success were equivalent between the two groups, but duration of therapeutic success was longer after ETP drainage, with a 3-month gain in duration of therapeutic success after adjustment for B-C type (95% CI [26-160], p=0.006). Cholangitis rates were equivalent (21.4% vs. 24.7%), while pancreatitis was more common in the ETP group and deaths more common in the PTH group. Conclusions: When added to ERCP, SOCP contributes significant clinical value in 64% of cases. However, there is an increased risk of intra- and postprocedural adverse events which, together with a learning curve, suggests that it should likely be performed in specialised high-volume centres. Regarding endoscopic intervention for MHO, stenting in the hepatic hilum compared to the distal biliary tree is associated with more adverse events and decreased stent patency. When comparing palliative ETP with PTH stenting for MHO, both approaches have similar technical and therapeutic success, with ETP drainage being more durable. Future studies should explore the complimentary role of both approaches in specific B-C types.
Bio-profiling of TB patients with and without Type 2 Diabetes before and during anti-tuberculosis drug (ATD) therapy
(Stellenbosch : Stellenbosch University, 2017-03) Selamolela, Mosa; Ronacher, Katharina; Kleynhans, Leanie; Stellenbosch University. Faculty of Medicine and Health Sciences. Biomedical Sciences: Molecular Biology and Human Genetics.
Background Patients with type 2 diabetes (DM2) are three times more likely to acquire active tuberculosis (TB) compared to otherwise healthy people. TB-DM2 comorbidity is characterized by poor TB treatment outcomes, increased risk of failure and relapse. The exact mechanisms of increased susceptibility of diabetics to TB are not well understood, but it is thought that hyperglycaemia is associated with an impairment of the innate and adaptive immune response. Aim To assess soluble (cytokines) and cellular (T-cell subsets) immunological markers of treatment response in TB patients with and without DM2 and their association with glycaemic control. Materials and Methods Serum cytokine concentrations were measured by means of the Luminex assay in 14 TB and 11 TB-DM2 patients at Baseline, Month 2 and Month 6. The HO-1 levels were measured from serum samples by means of ELISA in 40 TB and 20 TB-DM2 patients at Baseline, Week 2, Month 2 and Month 6. The frequency of different T-cell subsets (central memory, naïve, effector memory and terminally differentiated effector memory T cells) was established in 8 healthy controls, 13 DM2, 18 TB and 23 TB-DM2 patients at Baseline, Month 2 and Month 6 using flow cytometry. Results Throughout treatment pro- and anti-inflammatory cytokine concentrations were increased in serum of TB-DM2 patients when compared to TB patients. Fibrinogen and Procalcitonin were higher in TB patients compared to the TB-DM2 patients at the end of treatment. Various cytokines (IL-1β, IL-4, IL-6, IL-7, IL-8, IL-9, G-CSF, GM-CSF, MCP-1(MCAF) and IFN-γ) and growth factors (VEGF and PDGF) exhibited positive correlation at baseline with HbA1c and random blood glucose, respectively. The frequencies of CD4+ naïve T cells increased from Baseline and Month 2 in TB patients. In contrast CD4+ and CD8+ naïve T cells decreased over time in the TB-DM2 patients. CD4+ T CM cells increased over time in TB-DM2 patients. Activated CD8+ naïve T cells increased over time in both TB and TB-DM2 patients while the terminally differentiated effector memory T cells decreased in both groups of study patients over time. No significant changes were observed in any CD8+ T cell subset in both groups of study patients. Conclusion The present study reveals that TB-DM2 patients have altered cytokine production at baseline and throughout treatment which may be linked to chronic inflammation associated with obesity and diabetes. Glycaemic control displays an influence in cytokine production shown in TB-DM2 patients. The frequencies of T cell subsets is altered in TB-DM2 patients and changes throughout treatment. These results show that TB-DM2 patients are characterised by changes in the adaptive immune system, which may contribute to poor treatment outcomes.
Bioinformatics-based strategies to identify PFHBII-causing and HCM main locus and/or HCM modifying mutations
(Stellenbosch : University of Stellenbosch, 2004-12) Yako, Yandiswa; Corfield, Valerie A.; Moolman-Smook, Johanna C.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Progressive familial heart block type II (PFHBII) is an inherited cardiac conduction disorder of unknown aetiology, which has been described in a South African family. The disorder was mapped to a 2.9 centimorgan (cM) locus on chromosome 1q32.2-32.3. Clinically, PFHBII manifests cardiac conduction aberrations, that progress to a disease of the heart muscle, dilated cardiomyopathy (DCM). DCM is also reported as an end phase in hypertrophic cardiomyopathy (HCM), another heart muscle disorder. These cardiomyopathies are genetically heterogeneous with some of the genes reported as causes of both disorders. Therefore, genes identified as causes of HCM and DCM were considered plausible candidates for PFHBII mutation analysis. Additionally, this study provided an opportunity to assess potential modifiers of HCM. HCM exhibits marked phenotypic variability, observed within and between families harbouring the same causative mutation. Genes within the PFHBII locus were selected for PCR-SSCP analysis based on homology to genes previously reported as causing conduction system disorders associated with arrhythmias, DCM and/or HCM. Results were confirmed by direct sequencing and association between the detected variants and HCM parameters was assessed using a quantitative transmission disequilibrium test (QTDT). Eleven plausible candidate genes were selected within the PFHBII locus and two of the genes, PFKFB2 and ATF3, that encode for 6-phosphofructo-2,6-bisphosphatase (PFK-2/FBPase-2) and activating transcription factor 3 (ATF3), respectively, were analysed for PFHBII-causing and HCM main locus and/or HCM modifying mutations. Mutation analysis of PFKFB2 and ATF3 in the PFHBII family revealed no PFHBII causal mutation. PFKFB2 and ATF3 were later localised outside the PFHBII locus, and, therefore, were excluded as PFHBII plausible candidates. Further analysis of the two genes for HCM main locus and/or HCM modifying mutations in the HCM panel identified several sequence variants. QTDT analysis of these variants showed no significant association. Completion of the Human Genome Project (HGP) and annotation of new genes within the PFHBII locus allowed the identification of more PFHBII plausible candidate genes. Identification of causal mutations in plausible PFHBII candidate genes will allow molecular diagnosis of PFHBII pathophysiology. Furthermore, identification of both HCM-modifying and HCM-causing genes will give insight into the phenotypic variability noted among South African HCM-affected individuals and into the molecular cause of the disease among individuals with HCM-like clinical features.

Browse

Browsing Molecular Biology and Human Genetics by browse.metadata.type "Thesis"

Results Per Page

Sort Options