Phenotypic analysis of peripheral B cell populations during Mycobacterium tuberculosis infection and disease

Abstract Background Mycobacterium tuberculosis (Mtb) remains an unresolved threat resulting in great annual loss of life. The role of B cells during the protective immunity to Mtb is still unclear. B cells have been described as effector cells in addition to their role as antibody producing cells during disease. Here we aim to identify and characterize the frequency of peripheral B-cell subpopulations during active Tuberculosis and over treatment response. Analysis were done for both class switched (CS) and non-class switched (NCS) phenotypes. Methods We recruited participants with active untreated pulmonary Tuberculosis, other lung diseases and healthy community controls. All groups were followed up for one week from recruitment and the TB cases till the end of treatment (month 6). Results Peripheral blood samples were collected, stained with monoclonal antibodies to CD19 + cells, Immunoglobulin (Ig) M, plasma cells (CD 138 + ), marker of memory (CD27 + ), immune activation (CD23 + ) and acquired on a flow cytometer. Circulating Marginal zone B cells (CD19 + IgM + CD23 − CD27 + ) and memory phenotypes are able to distinguish between TB diagnosis and end of treatment. The frequency of mature B cells from TB cases are lower than that of other-lung diseases at diagnosis. A subpopulation of activated memory B cells (CD19 + IgM + CD23 + CD27 + ) cells are present at the end of TB treatment. Conclusions This study identified distinctive B cell subpopulations present during active TB disease and other lung disease conditions. These cell populations warrants further examination in larger studies as it may be informative as cell markers or as effectors/regulators in TB disease or TB treatment response

Modelling and optimal control of blood glucose levels in the human body

Regulating the blood glucose level is a challenging control problem for the human body. Abnormal blood glucose levels can cause serious health problems over time, including diabetes. Although several mathematical models have been proposed to describe the dynamics of glucose-insulin interaction, none of them have been universally adopted by the research community. In this paper, we consider a dynamic model of the blood glucose regulatory system originally proposed by Liu and Tang in 2008. This model consists of eight state variables naturally divided into three subsystems: the glucagon and insulin transition subsystem, the receptor binding subsystem and the glucosesubsystem. The model contains 36 model parameters, many of which are unknown and difficult to determine accurately. We formulate an optimal parameter selection problem in which optimal values for the model parameters must be selected so that the resulting model best its given experimental data.We demonstrate that this optimal parameter selection problem can be solved readily using the optimal control software MISER 3.3. Using this approach, significant improvements can be made in matching the model to the experimental data. We also investigate the sensitivity of the resulting optimizedmodel with respect to the insulin release rate. Finally, we use MISER 3.3 to determine optimal open loop controls for the optimized model

A semi-automatic technique to quantify complex tuberculous lung lesions on 18F-fluorodeoxyglucose positron emission tomography/computerised tomography images

Background There is a growing interest in the use of 18F-FDG PET-CT to monitor tuberculosis (TB) treatment response. However, TB causes complex and widespread pathology, which is challenging to segment and quantify in a reproducible manner. To address this, we developed a technique to standardise uptake (Z-score), segment and quantify tuberculous lung lesions on PET and CT concurrently, in order to track changes over time. We used open source tools and created a MATLAB script. The technique was optimised on a training set of five pulmonary tuberculosis (PTB) cases after standard TB therapy and 15 control patients with lesion-free lungs. Results We compared the proposed method to a fixed threshold (SUV > 1) and manual segmentation by two readers and piloted the technique successfully on scans of five control patients and five PTB cases (four cured and one failed treatment case), at diagnosis and after 1 and 6 months of treatment. There was a better correlation between the Z-score-based segmentation and manual segmentation than SUV > 1 and manual segmentation in terms of overall spatial overlap (measured in Dice similarity coefficient) and specificity (1 minus false positive volume fraction). However, SUV > 1 segmentation appeared more sensitive. Both the Z-score and SUV > 1 showed very low variability when measuring change over time. In addition, total glycolytic activity, calculated using segmentation by Z-score and lesion-to-background ratio, correlated well with traditional total glycolytic activity calculations. The technique quantified various PET and CT parameters, including the total glycolytic activity index, metabolic lesion volume, lesion volumes at different CT densities and combined PET and CT parameters. The quantified metrics showed a marked decrease in the cured cases, with changes already apparent at month one, but remained largely unchanged in the failed treatment case. Conclusions Our technique is promising to segment and quantify the lung scans of pulmonary tuberculosis patients in a semi-automatic manner, appropriate for measuring treatment response. Further validation is required in larger cohorts

Medroxyprogesterone Acetate Alters Mycobacterium Bovis BCG-Induced Cytokine Production in Peripheral Blood Mononuclear Cells of Contraceptive Users

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

Distinct serum biosignatures are associated with different tuberculosis treatment outcomes.

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-?, 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

A Broad Profile of Co-Dominant Epitopes Shapes the Peripheral Mycobacterium tuberculosis Specific CD8+ T-Cell Immune Response in South African Patients with Active Tuberculosis.

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition

Immunometabolism of Myeloid-Derived Suppressor Cells: Implications for Mycobacterium tuberculosis Infection and Insights from Tumor Biology

The field of immunometabolism seeks to decipher the complex interplay between the immune system and the associated metabolic pathways. The role of small molecules that can target specific metabolic pathways and subsequently alter the immune landscape provides a desirable platform for new therapeutic interventions. Immunotherapeutic targeting of suppressive cell populations, such as myeloid-derived suppressor cells (MDSC), by small molecules has shown promise in pathologies such as cancer and support testing of similar host-directed therapeutic approaches in MDSC-inducing conditions such as tuberculosis (TB). MDSC exhibit a remarkable ability to suppress T-cell responses in those with TB disease. In tumors, MDSC exhibit considerable plasticity and can undergo metabolic reprogramming from glycolysis to fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) to facilitate their immunosuppressive functions. In this review we look at the role of MDSC during M. tb infection and how their metabolic reprogramming aids in the exacerbation of active disease and highlight the possible MDSC-targeted metabolic pathways utilized during M. tb infection, suggesting ways to manipulate these cells in search of novel insights for anti-TB therapies

Isolation of B-cells using Miltenyi MACS bead isolation kits.

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