Globally, more than 10 million people developed active tuberculosis (TB), with 1.4 million deaths in 2020. In addition, the emergence of drug-resistant strains in many regions of the world threatens national TB control programs. This requires an understanding of host-pathogen interactions and finding novel treatments including host-directed therapies (HDTs) is of utter importance to tackle the TB epidemic. Mycobacterium tuberculosis (Mtb), the causative agent for TB, mainly infects the lungs causing inflammatory processes leading to immune activation and the development and formation of granulomas. During TB disease progression, the mononuclear inflammatory cell infiltrates which form the central structure of granulomas undergo cellular changes to form epithelioid cells, multinucleated giant cells and foamy macrophages. Granulomas further contain neutrophils, NK cells, dendritic cells and an outer layer composed of T and B lymphocytes and fibroblasts. This complex granulomatous host response can be modulated by Mtb to induce pathological changes damaging host lung tissues ultimately benefiting the persistence and survival of Mtb within host macrophages. The development of cavities is likely to enhance inter-host transmission and caseum could facilitate the dissemination of Mtb to other organs inducing disease progression. This review explores host targets and molecular pathways in the inflammatory granuloma host immune response that may be beneficial as target candidates for HDTs against TB.
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.
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.
IntroductionStatins, also known as 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, are lipid-lowering agents that are central in preventing or reducing the complications of atherosclerotic cardiovascular disease. Because statins have anti-inflammatory properties, there is considerable interest in their therapeutic potential in other chronic inflammatory conditions. We aim to identify the statin with the greatest ability to reduce systemic inflammation, independent of the underlying disease entity.Methods and analysisWe aim to conduct a comprehensive search of published and peer-reviewed randomised controlled clinical trials, with at least one intervention arm of a Food & Drug Administration-licensed or European Medicines Agency-licensed statin and a minimum treatment duration of 12 weeks. Our objective is to investigate the effect of statins (atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin) on lipid profile, particularly, cholesterol low-density lipoprotein and inflammation markers such as high-sensitive C reactive protein (hsCRP), CRP, tumour necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, soluble cluster of differentiation 14 (sCD14) or sCD16 in adults, published in the last 20 years (between January 1999 and December 2019). We aim to identify the most potent statin to reduce systemic inflammation and optimal dosing. The following databases will be searched: Medline, Scopus, Web of Science and Cochrane Library of Systematic Reviews. The risk of bias of included studies will be assessed by Cochrane Risk of Bias Tool and Quality Assessment Tool for Quantitative Studies. The quality of studies will be assessed, to show uncertainty, by the Jadad Score. If sufficient evidence is identified, a meta-analysis will be conducted with risk ratios or ORs with 95% CIs in addition to mean differences.Ethics and disseminationEthics approval is not required as no primary data will be collected. Results will be presented at conferences and published in a peer-reviewed journal.PROSPERO registration numberCRD42020169919
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.
Background Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of mortality worldwide. Even though treatment is readily available the emergence of drug resistance amongst Mtb strains highlights the need for new advances in the TB field such as host-directed therapies (HDT). Recent studies have highlighted the importance of BiP in cells, which can become a target in many diagnostic settings as it has been implicated in conditions including arthritis, cancer, bacterial infection and autoimmune diseases. In our studies, we are aiming to identify expression differences of BiP in different Mtb infection stages to help us understand the change of function in immune cells in relation to infection stress.MethodBiP secretion levels were assessed in plasma samples using ELISA technique. This included participants at TB diagnosis (TBDx), TB treatment group (Week 1, Month 2 and Month 6) and healthy (unexposed) participants. BiP concentration results were analysed using GraphPad Prism 7.ResultsSecretion of BiP was comparable between newly diagnosed untreated TB cases and healthy unexposed controls, with levels obtained in healthy group (42.64 µg/ml) and in TBDx (40.88 µg/ml). Highest levels of plasma BiP during treated TB was observed by Week 1 (mean 68.57 µg/ml) and declined by Month 2 with 60.92 µg/ml and Month 6 with 51.40 µg/ml.ConclusionDetection of BiP in plasma samples indicated metabolic change in immune cells due to stress posed onto cells by Mtb burden. This is due to the amount of protein product required by the immune system to mitigate the spread of the pathogen. Even though not significant, we observed a decrease in the mean levels of BiP over the course of TB treatment which correlates with a reduction in the accumulation of unfolded polypeptides in the endoplasmatic reticulum. This observation requires further testing in larger prospective studies.
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