Browsing by Author "Du Plessis, Nelita"

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    Analysis of genetic variants in the 5’ regulatory region of the ALAS1 gene in South African patients with Variegate Porphyria (VP)
    (Stellenbosch : University of Stellenbosch, 2007-03) Du Plessis, Nelita; Warnich, L.; Zaahl, M. G.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    The porphyrias are a group of genetic disorders arising from mutations in either one of the final seven genes encoding the haeme synthesis enzymes. These disease-causing mutations lead to an enzyme deficiency that disrupts normal haeme production, resulting in clinical features due to the subsequent accumulation of porphyrin precursors. Like most of the porphyrias, variegate porphyria (VP) is characterized by high inter- and intra- familial clinical variability, with no apparent genotype-phenotype correlation. The delta-aminolevulinate synthase-1 gene (ALAS1) is an apparent candidate gene to explain the variable clinical expression observed in VP, since it encodes the first and rate-determining enzyme of haeme synthesis. Several studies have defined important regulatory elements for the human-, rat- and chicken ALAS1 gene that regulate expression patterns of this gene. It was hypothesized that in VP individuals, variants within/near critical regulatory sites might alter the transcription rate of this gene, and consequently increase/decrease the amount of haeme precursors accumulating as a result of the defective haeme synthesis enzyme. The aim of this study was to identify genetic variants that could influence gene expression in the proximal promoter area of the ALAS1 gene, as well as the two ALAS1-drug responsive enhancer sequences (ADRES) located further upstream. DNA (2133 bp per patient) of 19 clinically defined VP patients was analysed by polymerase chain reaction (PCR) and semiautomated DNA sequencing. Subsequently, in silico analyses using appropriate software programs, and in vitro studies using the luciferase reporter system, were performed to investigate the functionality of the identified variants on ALAS1 gene transcription...
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    Caveolin-1 controls vesicular TLR2 expression, p38 signaling and T cell suppression in BCG infected Murine Monocytic myeloid-derived suppressor cells
    (Frontiers Media, 2019) John, Vini; Kotze, Leigh A.; Ribechini, Eliana; Walzl, Gerhard; Du Plessis, Nelita; Lutz, Manfred B.
    Monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic MDSCs (G-MDSCs) have been found to be massively induced in TB patients as well in murine Mtb infection models. However, the interaction of mycobacteria with MDSCs and its role in TB infection is not well studied. Here, we investigated the role of Cav-1 for MDSCs infected with Mycobacterium bovis Bacille-Calmette-Guerín (BCG). MDSCs that were generated from murine bone marrow (MDSCs) of wild-type (WT) or Cav1−/− mice upregulated Cav-1, TLR4 and TLR2 expression after BCG infection on the cell surface. However, Cav-1 deficiency resulted in a selective defect of intracellular TLR2 levels predominantly in the M-MDSC subset. Further analysis indicated no difference in the phagocytosis of BCG by M-MDSCs from WT and Cav1−/− mice or caveosome formation, but a reduced capacity to up-regulate surface markers, to secrete various cytokines, to induce iNOS and NO production required for suppression of T cell proliferation, whereas Arg-1 was not affected. Among the signaling pathways affected by Cav-1 deficiency, we found lower phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Together, our findings implicate that (i) Cav-1 is dispensable for the internalization of BCG, (ii) vesicular TLR2 signaling in M-MDSCs is a major signaling pathway induced by BCG, (iii) vesicular TLR2 signals are controlled by Cav-1, (iv) vesicular TLR2/Cav-1 signaling is required for T cell suppressor functions.
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    Distinct serum biosignatures are associated with different tuberculosis treatment outcomes
    (Elsevier, 2019) Ronachera, Katharina; Chegoua, Novel N.; Kleynhansa, Leanie; Siawayac, Joel F. Djoba; Du Plessis, Nelita; Loxton, Andre G.; Maasdorp, Elizna; Tromp, Gerard; Kidd, Martin; Stanleya, Kim; Kriela, Magdalena; Menezesa, Angela; Gutschmidta, Andrea; Van Der Spuya, Gian D.; Warrena, Robin M.; Dietzee, Reynaldo; Okweraf, Alphonse; Thielg, Bonnie; Belisleh, John T.; Cliffi, Jacqueline M.; Boomg, W. Henry; Johnsong, John L.; Van Heldena, Paul D.; Dockrelli, Hazel M.; Walzla, Gerhard
    ENGLISH ABSTRACT: Biomarkers for TB treatment response and outcome are needed. This study characterize changes in immune profiles during TB treatment, define biosignatures associated with treatment outcomes, and explore the feasibility of predictive models for relapse. Seventy-two markers were measured by multiplex cytokine array in serum samples from 78 cured, 12 relapsed and 15 failed treatment patients from South Africa before and during therapy for pulmonary TB. Promising biosignatures were evaluated in a second cohort from Uganda/Brazil consisting of 17 relapse and 23 cured patients. Thirty markers changed significantly with different response patterns during TB treatment in cured patients. The serum biosignature distinguished cured from relapse patients and a combination of two clinical (time to positivity in liquid culture and BMI) and four immunological parameters (TNF-β, sIL-6R, IL-12p40 and IP-10) at diagnosis predicted relapse with a 75% sensitivity (95%CI 0.38–1) and 85% specificity (95%CI 0.75–0.93). This biosignature was validated in an independent Uganda/Brazil cohort correctly classifying relapse patients with 83% (95%CI 0.58–1) sensitivity and 61% (95%CI 0.39–0.83) specificity. A characteristic biosignature with value as predictor of TB relapse was identified. The repeatability and robustness of these biomarkers require further validation in well-characterized cohorts.
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    The functional response of B cells to antigenic stimulation : a preliminary report of latent tuberculosis
    (Public Library of Science, 2016-04) Du Plessis, Willem J.; Kleynhans, Leanie; Du Plessis, Nelita; Stanley, Kim; Malherbe, Stephanus T.; Maasdorp, Elizna; Ronacher, Katharina; Chegou, Novel N.; Walzl, Gerhard; Loxton, Andre G.
    Mycobacterium tuberculosis (M.tb) remains a successful pathogen, causing tuberculosis disease numbers to constantly increase. Although great progress has been made in delineating the disease, the host-pathogen interaction is incompletely described. B cells have shown to function as both effectors and regulators of immunity via non-humoral methods in both innate and adaptive immune settings. Here we assessed specific B cell functional interaction following stimulation with a broad range of antigens within the LTBI milieu. Our results indicate that B cells readily produce pro- and anti-inflammatory cytokines (including IL-1β, IL-10, IL-17, IL-21 and TNF-α) in response to stimulation. TLR4 and TLR9 based stimulations achieved the greatest secreted cytokine-production response and BCG stimulation displayed a clear preference for inducing IL-1β production. We also show that the cytokines produced by B cells are implicated strongly in cell-mediated communication and that plasma (memory) B cells (CD19+CD27+CD138+) is the subset with the greatest contribution to cytokine production. Collectively our data provides insight into B cell responses, where they are implicated in and quantifies responses from specific B cell phenotypes. These findings warrant further functional B cell research with a focus on specific B cell phenotypes under conditions of active TB disease to further our knowledge about the contribution of various cell subsets which could have implications for future vaccine development or refined B cell orientated treatment in the health setting.
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    Heat-killed Mycobacterium tuberculosis prime-boost vaccination induces myeloid-derived suppressor cells with spleen dendritic cell–killing capability
    (American Society for Clinical Investigation, 2019) Ribechini, Eliana; Eckert, Ina; Beilhack, Andreas; Du Plessis, Nelita; Walzl, Gerhard; Schleicher, Ulrike; Ritter, Uwe; Lutz, Manfred B.
    ENGLISH ABSTRACT: Tuberculosis patients and mice infected with live Mycobacterium tuberculosis accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that dead M. tuberculosis vaccines also may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of M. tuberculosis vaccines (heat-killed M. tuberculosis in incomplete Freund’s adjuvant, such as Montanide) but not single or control vaccines without M. tuberculosis strongly expanded CD11b+ myeloid cells in the spleen, leading to T cell suppression of proliferation and killing ex vivo. Dead M. tuberculosis vaccination induced the generation of CD11b+Ly6ChiCD115+ iNOS/Nos2+ monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs were positioned strategically in the splenic bridging channels and then positioned in the white pulp areas. Notably, within 6–24 hours, in a Nos2-dependent fashion, they produced NO to rapidly kill conventional and plasmacytoid DCs while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that M. tuberculosis vaccine induced M-MDSCs do not directly suppress effector T cells in vivo but, instead, indirectly by killing DCs. Collectively, we demonstrate that M. tuberculosis booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs. Our data suggest that formation of MDSCs by M. tuberculosis vaccines should be investigated also in clinical trials.
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    Human monocytic suppressive cells promote replication of mycobacterium tuberculosis and alter stability of in vitro generated granulomas
    (Frontiers Media, 2018) Agrawal, Neha; Streata, Ioana; Pei, Gang; Weiner, January; Kotze, Leigh; Bandermann, Silke; Lozza, Laura; Walzl, Gerhard; Du Plessis, Nelita; Ioana, Mihai; Kaufmann, Stefan H. E.; Dorhoi, Anca
    ENGLISH ABSTRACT: Tuberculosis (TB) has tremendous public health relevance. It most frequently affects the lung and is characterized by the development of unique tissue lesions, termed granulomas. These lesions encompass various immune populations, with macrophages being most extensively investigated. Myeloid derived suppressor cells (MDSCs) have been recently identified in TB patients, both in the circulation and at the site of infection, however their interactions with Mycobacterium tuberculosis (Mtb) and their impact on granulomas remain undefined. We generated human monocytic MDSCs and observed that their suppressive capacities are retained upon Mtb infection. We employed an in vitro granuloma model, which mimics human TB lesions to some extent, with the aim of analyzing the roles of MDSCs within granulomas. MDSCs altered the structure of and affected bacterial containment within granuloma-like structures. These effects were partly controlled through highly abundant secreted IL-10. Compared to macrophages, MDSCs activated primarily the NF-κB and MAPK pathways and the latter largely contributed to the release of IL-10 and replication of bacteria within in vitro generated granulomas. Moreover, MDSCs upregulated PD-L1 and suppressed proliferation of lymphocytes, albeit with negligible effects on Mtb replication. Further comprehensive characterization of MDSCs in TB will contribute to a better understanding of disease pathogenesis and facilitate the design of novel immune-based interventions for this deadly infection.
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    Medroxyprogesterone acetate alters mycobacterium bovis BCG-induced cytokine production in peripheral blood mononuclear cells of contraceptive users
    (Public Library of Science, 2011-09-08) Kleynhans, Leanie; Du Plessis, Nelita; Black, Gillian F.; Loxton, Andre G.; Kidd, Martin; Van Helden, Paul D.; Walzl, Gerhard; Ronacher, Katharina
    Most individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection by a balance of effector and regulatory immune responses. This balance can be influenced by steroid hormones such as glucocorticoids. The widely used contraceptive medroxyprogesterone acetate (MPA) possesses glucocorticoid activity. We investigated the effect of this hormone on immune responses to BCG in household contacts of active TB patients. Multiplex bead array analysis revealed that MPA demonstrated both glucocorticoid and progestogenic properties at saturating and pharmacological concentrations in peripheral blood mononuclear cells (PBMCs) and suppressed antigen specific cytokine production. Furthermore we showed that PBMCs from women using MPA produced significantly lower levels of IL-1α, IL-12p40, IL-10, IL-13 and G-CSF in response to BCG which corresponded with lower numbers of circulating monocytes observed in these women. Our research study is the first to show that MPA impacts on infections outside the genital tract due to a systemic effect on immune function. Therefore MPA use could alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult or adolescent women in the future. © 2011 Kleynhans et al.
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    Monocytic myeloid-derived suppressor cells in chronic infections
    (Frontiers Media, 2018) Dorhoi, Anca; Du Plessis, Nelita
    ENGLISH ABSTRACT: Heterogeneous populations of myeloid regulatory cells (MRC), including monocytes, macrophages, dendritic cells, and neutrophils, are found in cancer and infectious diseases. The inflammatory environment in solid tumors as well as infectious foci with persistent pathogens promotes the development and recruitment of MRC. These cells help to resolve inflammation and establish host immune homeostasis by restricting T lymphocyte function, inducing regulatory T cells and releasing immune suppressive cytokines and enzyme products. Monocytic MRC, also termed monocytic myeloid-derived suppressor cells (M-MDSC), are bona fide phagocytes, capable of pathogen internalization and persistence, while exerting localized suppressive activity. Here, we summarize molecular pathways controlling M-MDSC genesis and functions in microbial-induced non-resolved inflammation and immunopathology. We focus on the roles of M-MDSC in infections, including opportunistic extracellular bacteria and fungi as well as persistent intracellular pathogens, such as mycobacteria and certain viruses. Better understanding of M-MDSC biology in chronic infections and their role in antimicrobial immunity, will advance development of novel, more effective and broad-range anti-infective therapies.
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    Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
    (BioMed Central, 2014-01) Nel, Hendrik J.; Du Plessis, Nelita; Kleynhans, Leanie; Loxton, Andre G.; Van Helden, Paul D.; Walzl, Gerhard
    Background: The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection. Results: We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice. Conclusion: Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.
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    Performance and immune characteristics of bronchoalveolar lavage by research bronchoscopy in pulmonary tuberculosis and other lung diseases in the Western Cape, South Africa
    (BMC (part of Springer Nature), 2019-06-01) Young, Carly; Ahlers, Petri; Hiemstra, Andriette M.; Loxton, Andre G.; Gutschmidt, Andrea; Malherbe, Stephanus T.; Walzl, Gerhard; Du Plessis, Nelita
    Background: Tuberculosis (TB) remains a debilitating, deadly disease that warrants innovative research tools to fully understand the pathogenesis and host immune responses, particularly at the site of infection and disease. In this regard, bronchoscopies with bronchoalveolar lavage (BAL) serve as a valuable technique for site of disease sample retrieval for further clinical- and basic research. Here we investigate the feasibility of research bronchoscopies in a low/middle-income area, where TB remains rife, and assess the value of retrieved BAL cells (BALC) for downstream fluorescent-based cellular evaluations. Methods: Using quantitative and qualitative methods, we evaluate the outcomes, safety, tolerability, participant -perception and -experience, while also providing insight into participant recruitment and screening processes of our study. Using light microscopy differential counting for BALC analysis, we evaluate the cellular composition of BAL fluid (BALF) from TB patients, healthy community controls and patients with other lung diseases. We also use flow cytometry to describe the challenges associated with fluorescence-based phenotypic analysis of autofluorescent BALC. Results: Our findings suggest that research bronchoscopies are safe, acceptable procedures for research participants and are indeed a feasible technique for future study design. We also suggest that the majority of participants are receptive to the proposition of a second research bronchoscopy. This poses an important avenue for research entailing follow-up investigations of the same study participant. Furthermore, our results show that smoking is characterized by retrieval of BALC containing particulate matter, that interferes with fluorescence-based flow cytometry data analysis. Based on light microscopy differential cell counting, our findings suggest that there are differences in the cell yields and cellular composition of the BALF between TB patients, healthy community controls and patients with other lung diseases. We also report on subject characteristics and demographic factors, namely gender and age, that have the potential to affect cell yields and cellular data of BALF. Conclusions: These findings will serve as a valuable reference for appropriate planning and design of studies involving clinical bronchoscopies for TB and lung disease research.
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    Targeting 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, Nelita
    Conventional 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.