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- ItemTargeting of myeloid-derived suppressor cells by all-trans retinoic acid as host-directed therapy for human tuberculosis(Elsevier Inc., 2021-06) Leukes, Vinzeigh N.; Dorhoi, Anca; Malherbe, Stephanus T.; Maasdorp, Elizna; Khoury, Justine; McAnda, Shirley; Walzl, Gerhard; Du Plessis, NelitaConventional anti-tuberculosis (TB) therapies comprise lengthy antibiotic treatment regimens, exacerbated by multi-drug resistant and extensively drug resistant mycobacterial strains. We assessed the ability of all-trans retinoic acid (ATRA), as repurposed compound serving as host-directed therapy (HDT), to counteract the suppressive effects of myeloid-derived suppressor cells (MDSCs) obtained from active TB cases (untreated or during week one of treatment) on T-cell responsiveness. We show for the first time that MDSCs suppress non-specific T-cell activation and production of interleukin (IL)-2, IL-4, IL-13 and GM-CSF via contact-dependent mechanisms. ATRA treatment decreases MDSC frequency, but fails to mature MDSCs to non-suppressive, terminally differentiated myeloid cells and does not restore T-cell function or cytokine production in the presence of MDSCs. The impact of ATRA treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.
- ItemImplementing a pipeline for analysing single-cell RNA sequencing data(Stellenbosch : Stellenbosch University, 2023-03) Ahiavi, Kwame; Tromp, Gerard; van der Spuy, Gian; Maasdorp, Elizna; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Single-cell RNA sequencing (scRNA-seq) has permitted the dissection of gene expression at single-cell resolution and provides novel insights into the composition of apparently homogeneous cell types and transitions between cell states — thereby deepening our understanding of the cell as a functional unit. The data generated by scRNA-seq are characterised by sparsity, heterogeneity, and high-dimensionality as well as large scale. As a result of biological and technical limitations, scRNA-seq data are “noisier” and more complex than their bulk RNA-seq counterparts. Thus, analysing scRNA-seq data demands new statistical and computational methods. Analytical algorithms employed in scRNA-seq pipelines are prone to producing different results depending on the state at the start of the analysis and the number of iterations of computation, complicating reproducibility. I developed a highly robust, scalable, and reproducible analysis pipeline for scRNA-seq data, implemented in Nextflow — a workflow management system that complies with current best practices in bioinformatics. The pipeline implements pre-processing and comprehensive downstream analyses for scRNA-seq data. With the publicly available datasets used for testing, the pipeline identified cell types and differentially expressed genes that enabled the identification of cell subtypes. Trajectory inference also showed the differentiation trajectory of cells, identifying subclusters within cells. In addition, the pipeline documents all steps and transformations, records software packages and versions, and incorporates ontological metadata annotation. Containerisation of pipeline processes ensures that software dependencies are satisfied — contributing to consistent, robust, and reproducible science.
- ItemPipeline and tools for the analysis of multiplexed ELISA data(Stellenbosch : Stellenbosch University, 2023-03) Asimeng, Jesse; Tromp, Gerard; Maasdorp, Elizna; Gian, van der Spuy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: A cornerstone of scientific progress is independent data verification. It is, therefore, necessary to develop robust analysis pipelines that can ensure reproducible and verifiable analyses. The pipeline should also record all steps and software that generated the results. The analysis of multiplexed ELISA data (Luminex data) can be challenging due to its complexity and variability. In particular, the data preprocessing stage has many steps and is often ad hoc, leading to inconsistency, non-standard approaches and lack of reproducibility. An existing in-house data reprocessing pipeline, the Luminex Pipeline, addresses some of the aforementioned challenges. However, there remains substantial work to extend its utility, robustness, and overall reproducibility. Thus, in this work, I improved the summary statistic reports by using Rmarkdown and implemented unit testing of pipeline components using the R Testthat package. Unit testing ensured the greater robustness of the code, which was compiled into an R package. The pipeline execution was also automated by using the Nextflow workflow management system. Finally, I deployed the pipeline in a Singularity container for execution on any platform including high-performance computing clusters.
- ItemAnalysis 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.
- ItemEvaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads.(Stellenbosch : Stellenbosch University, 2023-03) Ymele Soko, Vivette; Mavumengwana, Vuyo; Loxton, Andre G.; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Apoptosis is a natural immune protective mechanism that allows the host to clean up nonfunctional cells. Not surprisingly, blocking apoptosis is presented as one of the strategies Mycobacterium tuberculosis (M.tb) uses to avoid host immune defence. In the absence of apoptosis, infected cells are taken captive to support the growth of their invader and eventually progress to an uncontrolled death (necrosis), which in the case of M.tb infection is called caseous necrosis and is characteristic of pathology observed in tuberculosis patients. In this project, host-directed therapy (HDT) was exploited and involved using pro-apoptotic drugs to induce and restore the normal process of controlled cell death (apoptosis) in infected macrophages. The advantage of this strategy is that, unlike current tuberculosis (TB) therapy which is not specific to certain forms of bacteria. Including dormant and multi-resistant M.tb, HDT could affect all forms of bacteria because it does not directly target the pathogen. This project used Mycobacterium smegmatis mc2 155 (M. smegmatis)-infected macrophages as a model to assess the efficacy of selected apoptotic inducer (API) drugs in controlling the intracellular growth of M. smegmatis. The model has been structured so that its product can be cultured on agar plates to evaluate the growth of bacteria representative of the inhibition or growth of intracellular bacteria. Preliminary tests were performed to evaluate the antimicrobial susceptibility of mycobacterium against the selected (API) drugs. Amongst the 12 drugs that were assessed, cepharanthine, CP31398 dihydrochloride hydrate, marinopyrrole A, and nutlin-3a showed activity against M. tuberculosis and M. smegmatis mc2 155. These four drugs have a minimum inhibitory concentration (MIC) of 3.1 - 6.2 µg/mL, 6.2 – 12.5 µg/mL, 25 – 50 µg/mL and 50–100 µg/mL, respectively, against M. smegmatis mc2 155. Also, when tested against M.tb H37Rv, the MIC value of these drugs was one-fold lower than their respective MIC values observed on M. smegmatis. Furthermore, the minimum bactericidal concentration (MBC) test revealed that cepharanthine or CP-31398 dihydrochloride hydrate could kill M. smegmatis at 12.5 µg/mL or 25 µg/mL, respectively. Ex-vivo treatment of M. smegmatis-infected macrophages with tolerated concentrations of API drugs indicated there was some limitations in this host model due to spontaneous inhibition of M. smegmatis growth in THP-1 macrophages. Nonetheless, it was possible to test the efficacy of drugs intended for HDT using the difference in the relative survival of intracellular bacteria as indicators. Cepharanthine and CP-31398 dihydrochloride hydrate inhibited the intracellular growth of the bacteria after 12hrs treatments at a concentration representing half of their MIC value or one-quarter of their MBC value. Higher concentrations of these drugs, for instance, 6.2 µg/mL of cepharanthine; or 9.2 µg/mL of CP-31398 dihydrochloride hydrate, resulted in the absence of growth of intracellular bacteria after only 6 hrs treatment. Multi-cytokines analysis on the cell culture supernatants sampled during the treatment of macrophages indicated that mild expression of IFN-gamma, TNF-alpha, IL-1beta, IL-5, combined with the inhibition of IL-6 and IL-22 was required to eliminate intracellular M. smegmatis mc2 155. However, high levels of TNF-alpha, IL-1beta, IL-6, and IL-22 coupled with the absence of expression of IFN-gamma and IL-5 correlated with a high burden of intracellular bacteria.