Phenotypic characterization of Peripheral B cells in Mycobacterium tuberculosis infection and disease in Addis Ababa, Ethiopia.

BACKGROUND: Mortality and morbidity from tuberculosis (TB) remain one of the most important public health issues. Although cell-mediated immunity is the main immune response against Mycobacterium tuberculosis (MTB), the role of B-cells during MTB infection and disease is unclear. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from treatment naïve Pulmonary TB patients (TB, n = 16), latent TB-infected participants (LTBI, n = 17), and healthy controls (HC, n = 19). PBMCs were stained with various fluorescently labeled antibodies to define B-cell subsets using multicolor flow cytometry. RESULTS: Atypical memory B cells (CD19+CD27-CD21-) and circulating marginal zone B-cells (CD19+CD27+CD21+IgM+IgD+CD23-) were significantly higher in active TB when compared to LTBI and HC. CD5+ regulatory B cells (Breg, CD19+CD24hiCD38hiCD5+) and resting B-cells (CD19+CD27+CD21+) in Active TB patients were significantly lower compared to HC and LTBI. Overall, there were no differences in B cell percentages (CD19+), naïve B cells (CD19+CD27-CD21+), Breg (CD19+CD24hiCD38hi), and activated memory B cells (CD19+CD27+CD21-) among the three study groups. CONCLUSIONS: These results indicated that multiple subsets of B cells were associated with TB infection and disease. It will be useful to examine these cell populations for their potential use as biomarkers for TB disease and LTBI.

Evolution of long-term vaccine-induced and hybrid immunity in healthcare workers after different COVID-19 vaccine regimens.

BACKGROUND: Both infection and vaccination, alone or in combination, generate antibody and T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the maintenance of such responses-and hence protection from disease-requires careful characterization. In a large prospective study of UK healthcare workers (HCWs) (Protective Immunity from T Cells in Healthcare Workers [PITCH], within the larger SARS-CoV-2 Immunity and Reinfection Evaluation [SIREN] study), we previously observed that prior infection strongly affected subsequent cellular and humoral immunity induced after long and short dosing intervals of BNT162b2 (Pfizer/BioNTech) vaccination. METHODS: Here, we report longer follow-up of 684 HCWs in this cohort over 6-9 months following two doses of BNT162b2 or AZD1222 (Oxford/AstraZeneca) vaccination and up to 6 months following a subsequent mRNA booster vaccination. FINDINGS: We make three observations: first, the dynamics of humoral and cellular responses differ; binding and neutralizing antibodies declined, whereas T and memory B cell responses were maintained after the second vaccine dose. Second, vaccine boosting restored immunoglobulin (Ig) G levels; broadened neutralizing activity against variants of concern, including Omicron BA.1, BA.2, and BA.5; and boosted T cell responses above the 6-month level after dose 2. Third, prior infection maintained its impact driving larger and broader T cell responses compared with never-infected people, a feature maintained until 6 months after the third dose. CONCLUSIONS: Broadly cross-reactive T cell responses are well maintained over time-especially in those with combined vaccine and infection-induced immunity ("hybrid" immunity)-and may contribute to continued protection against severe disease. FUNDING: Department for Health and Social Care, Medical Research Council.

Composition and capacity of Institutional Review Boards, and challenges experienced by members in ethics review processes in Addis Ababa, Ethiopia: An exploratory qualitative study.

Few studies in sub-Saharan Africa evaluate Institutional Review Boards (IRBs) capacity. The study aims to explore the composition of IRBs, training, and challenges experienced in the ethics review processes by members of research institutions and universities in Addis Ababa, Ethiopia. Our findings indicate that most IRBs members were trained on research ethics and good clinical practice. However, majority perceived the trainings as basic. IRB members faced several challenges including: investigators wanting rapid review; time pressure; investigators not following checklists; limited expertise in reviewing clinical trials, studies on genetics, and traditional medicine; lack of IRB offices for administrative work; competing tasks; limited staffing and the lack of a standardized review system. There is need for advanced training on research ethics to meet the evolving research needs. In addition, investments in IRBs are needed in terms of funding, and physical and human resources in Addis Ababa and Ethiopia in general.

Elevated KIR expression and diminished intensity of CD7 on NK cell subsets among treatment naïve HIV infected Ethiopians.

Natural killer (NK) cells are crucial effector cells of the innate immune response to viral infections, including HIV, through cytolytic activity and the production of cytokines with anti-HIV activities. We recruited 15 treatment naïve HIV patients and 16 healthy controls (HC) to assess NK cell subsets or expression of multiple markers by flow cytometry. The frequency of circulating CD56brightCD16-ve and CD56dimCD16bright NK cell subsets was significantly lower among the HIV group than in HC. The CD56-veCD16bright subset was higher in HIV patients, but this was only apparent when gated among total NK cells, not total lymphocytes. NK cells among HIV participants also showed a lower and higher frequency of CD8 and HLA-DR expressing cells, respectively. In addition, CD7 median fluorescent intensity and CD2+CD7- frequencies were significantly lower in HIV patients. A distinct population of KIR3DL1/S1 cells was unexpectedly higher among CD56brightCD16-ve NK cells in HIV patients. In conclusion, this study in the Ethiopian setting confirms many previous findings, but the down-regulation of CD7 and enhanced KIR3DL1/S1 within the CD56bright subsets have not been widely reported among HIV patients and merit further research.

Alteration of Endocrine Hormones and Antibody Responses in Different Spectrum of Tuberculosis Disease.

Effective control of Mycobacterium tuberculosis (Mtb) infection is mediated by multifaceted factors that involve both the endocrine and immune system. Profiling hormones and antibodies in different stages of TB provides insight in the pathogenesis of the disease. In this study, we profiled endocrine hormones (dehydroepiandrosterone (DHEA), cortisol, testosterone, estradiol, growth hormone and leptins) and Mtb strain H37RV lipoarabinomannan (LAM)-specific antibody levels in plasma samples, collected from pulmonary TB (PTB) patients, TB lymphadenitis (TBLN) patients and latently infected (QFT-positive) or uninfected (QFT-negative) apparently healthy individuals using ELISA. Plasma levels of leptin and DHEA were significantly low in PTB and TBLN patients compared to healthy controls (P<0.0001 and P=0.02, respectively), whereas these levels significantly increased following anti-TB treatment (P=0.002 and P=0.0001, respectively) among TB patients. The levels of estradiol and testosterone significantly improved following anti-TB treatment (P=0.03 and P=0.0003, respectively), whereas cortisol and growth hormones declined significantly (P <0.05). Similarly, LAM-specific IgG, IgM and IgA were significantly higher in PTB patients compared to other groups, whereas levels of IgG1 subtype were significantly higher among LTBI groups compared to both TB patients and QFT-negative individuals (P<0.0001). Overall, we observed significantly variable levels of endocrine hormones as well as immunoglobulins across the spectrum of TB illness and such profiling has a significant contribution in selection of effective biomarkers that have roles in TB treatment monitoring or diagnostics. Although this study did not show a functional association between hormones and antibodies, alterations in the levels of these biomarkers suggest the key roles these markers play in TB pathogenesis.

Immunosuppressive Features of the Microenvironment in Lymph Nodes Granulomas from Tuberculosis and HIV-Co-Infected Patients.

Tuberculosis (TB) and HIV co-infection claims many lives every year. This study assessed immune responses in Mycobacterium tuberculosis-infected lymph node tissues from HIV-negative and HIV-positive patients compared with the peripheral circulation with a focus on myeloid cells and the cell-signaling enzymes, inducible nitric oxide synthase, and arginase (Arg)-1. Methods included immunohistochemistry or confocal microscopy and computerized image analyses, quantitative real-time PCR, multiplex Luminex, and flow cytometry. These findings indicate enhanced chronic inflammation and immune activation in TB/HIV co-infection but also enhanced immunosuppressive responses. Poorly formed necrotic TB granulomas with a high expression of M. tuberculosis antigens were elevated in TB/HIV-co-infected lymph nodes, and inducible nitric oxide synthase and Arg-1 expression was significantly higher in TB/HIV-co-infected compared with HIV-negative TB or control tissues. High Arg-1 expression was found in myeloid cells with a phenotype characteristic of myeloid-derived suppressor cells (MDCS) that were particularly abundant in TB/HIV-co-infected tissues. Accordingly, Lin-/HLA-DRlow/int/CD33+/CD11b+/CD15+ granulocytic myeloid-derived suppressor cells were significantly elevated in blood samples from TB/HIV-co-infected patients. CD15+ myeloid-derived suppressor cells correlated with plasma HIV viral load and M. tuberculosis antigen load in tissue but were inversely associated with peripheral CD4 T-cells counts. Enhanced chronic inflammation driven by M. tuberculosis and HIV co-infection may promote Arg-1-expressing MDSCs at the site of infection thereby advancing TB disease progression.

Latent tuberculosis infection and associated risk indicators in pastoral communities in southern Ethiopia: a community based cross-sectional study.

BACKGROUND: Research pertaining to the community-based prevalence of latent tuberculosis infection (LTBI) is important to understand the magnitude of this infection. This study was conducted to estimate LTBI prevalence and to identify associated risk factors in the Omo Zone of Southern Ethiopia. METHODS: A community-based cross-sectional study was conducted in six South Omo districts from May 2015 to February 2016. The sample size was allocated to the study districts proportional to their population sizes. Participants were selected using a multi-stage sampling approach. A total of 497 adult pastoralists were recruited. Blood samples were collected from the study participants and screened for LTBI using a U.S. Food and Drug Administration approved interferon-gamma release assay (IGRA). Logistic regression was used to model the likelihood of LTBI occurrence and to identify risk factors associated with LTBI. RESULTS: The prevalence of LTBI was 50.5% (95% CI: 46%, 55%) with no significant gender difference (49.8% among males versus 51.2% among females; Chi-square (χ2) = 0.10; P = 0.41) and marginally non-significant increasing trends with age (44.6% among those below 24 years and 59.7% in the age range of 45-64 years; χ2 = 6.91; P = 0.075). Being residence of the Dasanech District (adjusted odds ratio, AOR = 2.62, 95% CI: 1.30, 5.28; P = 0.007) and having a habit of eating raw meat (AOR = 2.89, 95% CI: 1.09, 7.66; P = 0.033) were significantly associated with an increased odds of being positive for LTBI. A large family size (size of 5 to 10) has significant protective effect against associated a reduced odds of being positive for LTBI compared to a family size of below 5 (AOR = 0.65, 95% CI: 0.42, 0.99; P = 0.045). CONCLUSIONS: A high prevalence of LTBI in the South Omo Zone raises the concern that elimination of TB in the pastoral communities of the region might be difficult. Screening for and testing individuals infected with TB, independent of symptoms, may be an effective way to minimize the risk of disease spread.

A phase I, open-label trial on the safety and immunogenicity of the adjuvanted tuberculosis subunit vaccine H1/IC31® in people living in a TB-endemic area.

BACKGROUND: H1/IC31® is a tuberculosis (TB) subunit vaccine candidate consisting of the fusion protein of Ag85B and ESAT-6 (H1) formulated with the IC31® adjuvant. Previous trials have reported on the H1/IC31® vaccine in M. tuberculosis (Mtb)-naïve, BCG-vaccinated and previously Mtb-infected individuals. In this trial, conducted between December 2008 and April 2010, the safety and immunogenicity of H1/IC31® was assessed in participants living in Ethiopia - a highly TB-endemic area. METHODS: Healthy male participants aged 18-25 years were recruited into four groups. Participants in group 1 (N = 12) and group 2 (N = 12) were Tuberculin Skin Test (TST) negative and QuantiFERON-TB Gold in-tube test (QFT) negative (Mtb-naïve groups), participants in group 3 (N = 3) were TST positive and QFT negative (BCG group), and participants in group 4 (N = 12) were both TST and QFT positive (Mtb-infected group). H1 vaccine alone (group 1) or H1 formulated with the adjuvant IC31® (groups 2, 3 and 4) was administered intramuscularly on day 0 and day 56. Safety and immunogenicity parameters were evaluated for up to 32 weeks after day 0. RESULTS: The H1/IC31®vaccine was safe and generally well tolerated. There was little difference among the four groups, with a tendency towards a higher incidence of adverse events in Mtb-infected compared to Mtb-naïve participants. Two serious adverse events were reported in the Mtb-infected group where a relationship to the vaccine could not be excluded. In both cases the participants recovered without sequelae within 72 h. Immunogenicity assays, evaluated in the 29 participants who received both vaccinations, showed a stronger response to TB antigens in the Mtb-naïve group vaccinated with the adjuvant. CONCLUSION: The trial confirmed the need for an adjuvant for the vaccine to be immunogenic and highlighted the importance of early phase testing of a novel TB vaccine candidate in TB-endemic areas. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT01049282. Retrospectively registered on 14 January 2010.

Ex-vivo characterization of regulatory T cells in pulmonary tuberculosis patients, latently infected persons, and healthy endemic controls.

BACKGROUND: Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. METHOD: We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. RESULTS: The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Progression of clinical tuberculosis is associated with a Th2 immune response signature in combination with elevated levels of SOCS3.

In this study, we explored the local cytokine/chemokine profiles in patients with active pulmonary or pleural tuberculosis (TB) using multiplex protein analysis of bronchoalveolar lavage and pleural fluid samples. Despite increased pro-inflammation compared to the uninfected controls; there was no up-regulation of IFN-γ or the T cell chemoattractant CCL5 in the lung of patients with pulmonary TB. Instead, elevated levels of IL-4 and CCL4 were associated with high mycobacteria-specific IgG titres as well as SOCS3 (suppressors of cytokine signaling) mRNA and progression of moderate-to-severe disease. Contrary, IL-4, CCL4 and SOCS3 remained low in patients with extrapulmonary pleural TB, while IFN-γ, CCL5 and SOCS1 were up-regulated. Both SOCS molecules were induced in human macrophages infected with Mycobacterium tuberculosis in vitro. The Th2 immune response signature found in patients with progressive pulmonary TB could result from inappropriate cytokine/chemokine responses and excessive SOCS3 expression that may represent potential targets for clinical TB management.

Parasitic infection may be associated with discordant responses to QuantiFERON and tuberculin skin test in apparently healthy children and adolescents in a tuberculosis endemic setting, Ethiopia.

BACKGROUND: M. tuberculosis remains one of the world's deadliest pathogens in part because of its ability to establish persistent, latent infections, which can later reactivate to cause disease. In regions of the globe where disease is endemic, as much as 50% of the population is thought to be latently infected, complicating diagnosis and tuberculosis control. The tools most commonly used for diagnosis of latent M. tuberculosis infection are the tuberculin skin test and the newer interferon-gamma release assays, both of which rely on an antigen-specific memory response as an indicator of infection. It is clear that the two tests, do not always give concordant results, but the factors leading to this are only partially understood. METHODS: In this study we examined 245 healthy school children aged from 12 to 20 years from Addis Ababa, a tuberculosis-endemic region, characterised them with regard to response in the tuberculin skin test and QuantIFERON™ test and assessed factors that might contribute to discordant responses. RESULTS: Although concordance between the tests was generally fair (90% concordance), there was a subset of children who had a positive QuantIFERON™ result but a negative tuberculin skin test. After analysis of multiple parameters the data suggest that discordance was most strongly associated with the presence of parasites in the stool. CONCLUSIONS: Parasitic gut infections are frequent in most regions where M. tuberculosis is endemic. This study, while preliminary, suggests that the tuberculin skin test should be interpreted with caution where this may be the case.

Development of a proof of concept immunochromatographic lateral flow assay for point of care diagnosis of Mycobacterium tuberculosis.

BACKGROUND: Despite major public health initiatives and the existence of efficacious treatment regimes, tuberculosis (TB) remains a threat, particularly in resource-limited settings. A significant part of the problem is the difficulty of rapidly identifying infected individuals, and as a result, there has been renewed interest in developing better diagnostics for infection or disease caused by Mycobacterium tuberculosis. Many of the existing tools, however, have limitations such as poor sensitivity or specificity, or the need for well-equipped laboratories to function effectively. Serodiagnostic approaches in particular have long drawn attention, due to their potential utility in large field studies, particularly in resource-poor settings. Unfortunately none of the serodiagnostic approaches have so far proven useful under field conditions. RESULTS: We screened a large panel of antigens with serodiagnostic potential by ELISA and selected a subpanel that was strongly and broadly recognised by TB patients, but not by controls. These antigens were then formulated into a simple immuno-chromatographic lateral flow assay format, suitable for field use, and tested against panels of plasma and blood samples from individuals with different clinical status (confirmed TB patients, household contacts, and apparently healthy community controls), recruited from Ethiopia (a highly TB-endemic country) and Turkey (a TB meso-endemic country). While specificity was good (97-100% in non TB-endemic controls), the sensitivity was not as high as expected (46-54% in pulmonary TB, 25-29% in extra-pulmonary TB). CONCLUSIONS: Though below the level of sensitivity the consortium had set for commercial development, the assay specifically identified M. tuberculosis-infected individuals, and provides a valuable proof of concept.

BCG-specific IgG-secreting peripheral plasmablasts as a potential biomarker of active tuberculosis in HIV negative and HIV positive patients.

BACKGROUND: Diagnosis of active tuberculosis (TB) among sputum-negative cases, patients with HIV infection and extra-pulmonary TB is difficult. In this study, assessment of BCG-specific IgG-secreting peripheral plasmablasts, was used to identify active TB in these high-risk groups. METHODS: Peripheral blood mononuclear cells were isolated from patients with TB and controls and cultured in vitro using an assay called Antibodies in Lymphocyte Supernatant, which measures spontaneous IgG antibody release from migratory plasmablasts. A BCG-specific ELISA and flow cytometry were used to quantify in vivo activated plasmablasts in blood samples from Ethiopian subjects who were HIV negative or HIV positive. Patients diagnosed with different clinical forms of sputum-negative active TB or other diseases (n=96) were compared with asymptomatic individuals including latent TB and non-TB controls (n=85). Immunodiagnosis of TB also included the tuberculin skin test and the interferon (IFN)-γ release assay, QuantiFERON. RESULTS: This study demonstrated that circulating IgG+ plasmablasts and spontaneous secretion of BCG-specific IgG antibodies were significantly higher in patients with active TB compared with latent TB cases and non-TB controls. BCG-specific IgG titres were particularly high among patients coinfected with TB and HIV with CD4 T-cell counts <200 cells/ml who produced low levels of Mycobacterium tuberculosis-specific IFNγ in vitro. CONCLUSIONS: These results suggest that BCG-specific IgG-secreting peripheral plasmablasts could be successfully used as a host-specific biomarker to improve diagnosis of active TB, particularly in people who are HIV positive, and facilitate administration of effective treatment to patients. Elevated IgG responses were associated with impaired peripheral T-cell responses, including reduced T-cell numbers and low M tuberculosis-specific IFNγ production.

Diagnosis of latent tuberculosis infection in healthy young adults in a country with high tuberculosis burden and BCG vaccination at birth.

BACKGROUND: One third of the world's population is thought to have latent tuberculosis infection (LTBI) with the potential for subsequent reactivation of disease. To better characterize this important population, studies comparing Tuberculin Skin Test (TST) and the new interferon-γ release assays including QuantiFERON®-TB Gold In-Tube (QFT-GIT) have been conducted in different parts of the world, but most of these have been in countries with a low incidence of tuberculosis (TB). The aim of this study was therefore to evaluate the use of QFT-GIT assay as compared with TST in the diagnosis of LTBI in Ethiopia, a country with a high burden of TB and routine BCG vaccination at birth. METHODS: Healthy medical and paramedical male students at the Faculty of Medicine, Addis Ababa University, Ethiopia were enrolled into the study from December 2008 to February 2009. The TST and QFTG-IT assay were performed using standard methods. RESULTS: The mean age of the study participants was 20.9 years. From a total of 107 study participants, 46.7% (95%CI: 37.0% to 56.6%) had a positive TST result (TST≥10 mm), 43.9% (95%CI: 34.3% to 53.9%) had a positive QFT-GIT assay result and 44.9% (95%CI: 35.2% to 54.8%) had BCG scar. There was strong agreement between TST (TST ≥10mm) and QFT-GIT assay (Kappa = 0.83, p value = 0.000). CONCLUSION: The TST and QFT-GIT assay show similar efficacy for the diagnosis of LTBI in healthy young adults residing in Ethiopia, a country with high TB incidence.

Modulation of cell death by M. tuberculosis as a strategy for pathogen survival.

It has been clearly demonstrated that in vitro, virulent M. tuberculosis can favor necrosis over apoptosis in infected macrophages, and this has been suggested as a mechanism for evading the host immune response. We recently reported that an effect consistent with this hypothesis could be observed in cells from the blood of TB patients, and in this paper, we review what is known about evasion strategies employed by M. tuberculosis and in particular consider the possible interaction of the apoptosis-inhibiting effects of M. tuberculosis infection with another factor (IL-4) whose expression is thought to play a role in the failure to control M. tuberculosis infection. It has been noted that IL-4 may exacerbate TNF-α-induced pathology, though the mechanism remains unexplained. Since pathology in TB typically involves inflammatory aggregates around infected cells, where TNF-α plays an important role, we predicted that IL-4 would inhibit the ability of cells to remove M. tuberculosis by apoptosis of infected cells, through the extrinsic pathway, which is activated by TNF-α. Infection of human monocytic cells with mycobacteria in vitro, in the presence of IL-4, appears to promote necrosis over apoptosis in infected cells-a finding consistent with its suggested role as a factor in pathology during M. tuberculosis infection.

Immunogenicity of Mycobacterium leprae unique antigens in leprosy endemic populations in Asia and Africa.

Ongoing transmission of leprosy is evident from the stable disease incidence in high burden areas. Tools for early detection of Mycobacterium leprae (M. leprae) infection, particularly in sub-clinically infected individuals, are urgently required to reduce transmission. Following the sequencing of the M. leprae genome, many M. leprae-unique candidate proteins have been identified, several of which have been tested for induction of M. leprae specific T cell responses in different leprosy endemic areas. In this study, 21 M. leprae-unique proteins and 10 peptide pools covering the complete sequence of five M. leprae-unique proteins (ML0576, ML1989, ML1990, ML2283, and ML2567) were evaluated in 160 individuals in Nepal and Ethiopia. These included: tuberculoid and borderline tuberculoid (TT/BT), borderline borderline and borderline lepromatous (BB/BL) leprosy patients; healthy household contacts (HHC); tuberculosis (TB) patients and endemic controls (EC). Immunogenicity of the proteins was determined by IFN-gamma secretion via stimulation of PBMC in 6 days lymphocyte stimulation tests (LST) or in whole blood assays (WBA). In LST, BB/BL patients (40%) responded to ML0573 and ML1601 whereas ML1604 was most immunogenic in TT/BT (35%) and HHC (36%). Additionally, significant numbers of EC displayed IFN-gamma production in response to ML0573 (54%), ML1601 (50%) and ML1604 (54%). TB patients on the other hand, hardly responded to any of the proteins except for ML1989. Comparison of IFN-gamma responses to ML0121, ML0141 and ML0188 for TT/BT patients showed specific increase in diluted 6 days WBA compared to the undiluted 24 hours WBA, whereas EC showed a reduced response in the diluted WBA, which may indicate detection of disease-specific responses in the 6 days WBA. In summary, identification of multiple M. leprae proteins inducing M. leprae-specific T cell responses in groups at high risk of developing leprosy may contribute to improve early detection for M. leprae infection.

Expression of apoptosis-related genes in an Ethiopian cohort study correlates with tuberculosis clinical status.

Mycobacterium tuberculosis remains one of the world's deadliest pathogens in part because of its ability to persist in the face of an active immune response. It has been suggested that apoptosis of infected macrophages is one way in which the host deals with intracellular pathogens and that M. tuberculosis can inhibit this process. To assess the relevance of this process for human disease, we compared the expression of multiple genes involved in the activation of the extrinsic ("death receptor initiated") pathway of apoptosis in 29 tuberculosis patients, 70 tuberculosis contacts and 27 community controls from Ethiopia. We found that there is a strong upregulation of genes for factors that promote apoptosis in PBMC from individuals with active disease, including TNF-alpha and its receptors, Fas and FasL and pro-Caspase 8. The anti-apoptotic factor FLIP, however, was also upregulated. A possible explanation for this dichotomy was given by fractionation of PBMC using CD14, which suggests that macrophage/monocytes may regulate several key molecules differently from non-monocytic cells (especially TNF-alpha and its receptors, a finding confirmed by protein ELISA) potentially reducing the sensitivity to apoptotic death of monocyte/macrophages--the primary host cell for M. tuberculosis. This may represent an important survival strategy for the pathogen.

From genome-based in silico predictions to ex vivo verification of leprosy diagnosis.

The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to >or=1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283- and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses.