Browsing by Author "Motaung, Bongani"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Effect of binding immunoglobulin protein on induction of regulatory B cells with killer phenotype during inflammation and disease
    (Future Science, 2019-03-05) Motaung, Bongani; Loxton, Andre G.
    Immune responses result from different immune cells acting in synergy to successfully fight infections. This requires a high degree of regulation to prevent excessive production of inflammatory products leading to other disease forms. Regulatory B cells are classified based on surface immunoglobulin expression. These cells are reported to resolve inflammation during chronic or autoimmune diseases. However, during chronic inflammation, their frequencies have been shown to be affected, and they can be induced by exposure to extracellular binding immunoglobulin protein (BiP). This review focuses on the effects on immune cells by extracellular or secreted BiP during various chronic inflammatory responses. For example, cell stress associated with Mycobacterium tuberculosis infection leads to accumulation of unfolded proteins that subsequently activate BiP and its three signal transducers intracellularly. Furthermore, BiP can be translocated from the endoplasmic reticulum to the extracellular environment where it binds immune cells as an autoantigen and leads to functional changes.
  • Thumbnail Image
    Item
    The effect of Binding Immunoglobulin Protein on the induction of regulatory B-cells during Mycobacterium tuberculosis infection
    (Stellenbosch : Stellenbosch University, 2019-03) Motaung, Bongani; Loxton, Andre G.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Regulatory and killer function in B-cells have been described during tuberculosis (TB) disease, and these were shown to regulate inflammation through several mechanisms including both cytokine secretion (IL-10, IL-35, TGF-β, sFas-L and Granzyme-B) and cell surface expression of Fas-L, PD-L1 and FoxP3. During TB treatment, binding immunoglobulin protein (BiP) secretion was assessed through Enzyme Linked ImmunoSorbent (ELISA) assay. This included plasma samples from healthy controls (n=32), TB diagnosis (n=29), week-1 follow up (n=8), month-2 follow up (n=7), and month-6 follow up (n=19) with additional collection from 20 participants (month-6 total (n=39)). Increased detection of BiP in plasma was observed between TB diagnosis and week-1 follow up suggesting a metabolism shift due to cell stress. The effect of extracellular BiP on immune cell responses were determined by stimulating Peripheral Blood Mononuclear Cells (PBMCs) isolated from healthy controls (n=12) and Mycobacterium tuberculosis (M.tb) exposed participants (LTBI) (n=8) with human recombinant BiP at 20 μg/ml. This effect was compared to other antigenic material including Toll-like Receptor-9 agonist (TLR-9a), M.tb H37Rv, Isoniazid (INH), BiP+TLR-9a, pooled Bronchoalveolar Lavage (BAL) fluid at TB diagnosis (TBdx) and month 6 TB treatment (M6) with unstimulated PBMCs as a baseline control. From this, the cytokine profile was established which indicated an elevated level of sFas-L and IL-13 by BiP stimulation. Further, increased frequency of CD19+CD5+ B-cells co-expressing Fas-L and IL-5Rα was greatly induced by BiP in both healthy controls and LTBI participants. Kinase activity was assessed by Luminex multiplex assay between unexposed group, M.tb exposed and stimulation conditions. Taken together, this study shows BiP potential to aid in better M.tb control by upregulating a B-cell population with immune regulatory function through expression of Fas-L. This highlights the potential use of BiP in host directed therapies (HDT) during TB disease to aid in better infection response.
  • Thumbnail Image
    Item
    Isolation of B-cells using Miltenyi MACS bead isolation kits
    (Public Library of Science, 2019-03-20) Moore, Dannielle K.; Motaung, Bongani; du Plessis, Nelita; Shabangu, Ayanda N.; Loxton, Andre G.; SU-IRG Consortium
    This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection—in which B-cells remain “untouched” in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection–in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays.
  • Thumbnail Image
    Item
    The level of the endoplasmic reticulum stress chaperone protein, binding immunoglobulin protein (BiP), decreases following successful tuberculosis treatment
    (Elsevier, 2019) Motaung, Bongani; Walzl, Gerhard; Loxton, Andre G.
    ENGLISH ABSTRACT: An increased Mycobacterium tuberculosis burden inside the host leads to higher demand of response proteins. This in turn results in metabolic shift and cellular stress, which is caused by the accumulation and trafficking of these proteins within the endoplasmic reticulum (ER). To resolve this, cells trigger the unfolded protein response (UPR), which is mainly mediated by binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78) chaperone, and this in turn upregulates its transcription. This chaperone protein facilitates proper protein folding within the ER; however, it can also be passively secreted into the extracellular environment or be expressed on cell surfaces attached to anchor proteins and transmembrane proteins. This notion has been shown in studies on chronic inflammation, including cancer and arthritis, with the detection of BiP-specific antibodies from different sample types. The present study analysed secreted BiP from plasma samples collected from healthy participants and patients with newly diagnosed tuberculosis (TBdx), seen over the course of TB treatment at week 1 (W1), month 2 (M2), and month 6 (M6). The results revealed that during the initial TB disease and treatment period, cells are subjected to stress conditions resulting in metabolic shifts, which lead to the secretion of the intracellular UPR-mediating chaperone protein, BiP. This was indicated by mean differences between TBdx (mean 40.88 ng/ml) and W1 (68.57 ng/ml) in the TB participant groups. However, no difference was observed between the healthy group (mean 42.64 ng/ml) and TBdx group (mean 40.88 ng/ml). Analysis of paired time-point visits revealed increased BiP secretion during early TB treatment. The detection of BiP in plasma samples was found to decrease after successful TB treatment to levels comparable to those in the healthy controls. Evaluation of BiP levels in larger TB treatment studies may lead to the identification of a new target for early TB diagnosis and host-directed immunotherapy.