Browsing by Author "Smith, Bronwyn Kerry"

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    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.