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Pukazhvanthen P.,National Institute for Research in Tuberculosis ICMR | Anbarasu D.,National Institute for Research in Tuberculosis ICMR | Kabeer Basirudeen S.A.,National Institute for Research in Tuberculosis ICMR | Raja A.,National Institute for Research in Tuberculosis ICMR | Singh M.,Lionex Diagnostics and Therapeutics GmbH
Tuberculosis | Year: 2014

Serodiagnostic potential of four recombinant proteins (38 kDa[Rv0934], MPT64[Rv1980c], Adk[Rv0733], and BfrB[Rv3874]) was evaluated in Healthy control subjects (HCS), Healthy household contacts (HHC), Pulmonary tuberculosis patients (PTB), and Human immuno deficiency virus & Tuberculosis co-infected patients (HIV-TB). All the antigens tested individually for the detection of serum IgG by indirect ELISA. All the four antigens have a significantly higher antibody response in PTB compared to healthy controls (P < 0.05). The sensitivity of individual antigens ranged from 20% to 52.5% for the prefixed specificity of 95%. When results of all 4 antigens were combined the sensitivity was increased to 75% and specificity was reduced 89% in HCS. In smear- and culture-positive (S+C+) PTB, four antigen combination gives maximum sensitivity (89.6%) with 89% specificity. In smear negative culture negative (S-C+) PTB, three antigen combination (38 kDa with MPT64 and BfrB) gives maximum sensitivity (69.5%) and specificity (91.6%). In HIV-TB, 4 antigen combinations give the maximum sensitivity of 51.2% with 89% specificity. Combining serology (Four antigen combination) with smear was able to increase the sensitivity from 70% to 92.5% in culture positive PTB. So, we propose that this serology test can be used as adjunct test along with smear for rapid diagnosis of PTB. © 2014 Elsevier Ltd.

Chuquimia O.D.,University of Stockholm | Petursdottir D.H.,University of Stockholm | Rahman M.J.,University of Stockholm | Hartl K.,University of Stockholm | And 2 more authors.
PLoS ONE | Year: 2012

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli. © 2012 Chuquimia et al.

Scholman T.,Saarland University | Straub M.,Albert Ludwigs University of Freiburg | Sotgiu G.,University of Sassari | Elsasser J.,Saarland University | And 5 more authors.
American Journal of Transplantation | Year: 2015

Comparative assessment of the tuberculin skin testing (TST) and commercial IFN-γ release-assays (IGRAs) is hampered by the use of different antigens (tuberculin PPD in TST vs. ESAT-6/CFP-10 in IGRAs). Thus, PPD was used as a common stimulus to compare performance of the TST and three IGRAs in 72 controls, 101 hemodialysis patients and 100 renal transplant recipients. Results of the TST were compared with PPD-induced IFN-γ induction in vitro detected by ELISPOT, ELISA or a flow-cytometric FACS assay. Percentages of positive tests were significantly lower in TST (9.2%) compared to ELISA (55.3%), ELISPOT (45.3%) and FACS (44.9%, p < 0.0001). Agreement between TST and IGRAs was highest for controls (κ = 0.19-0.32) and poor in immunocompromised patients (κ = 0 for transplant patients, κ = 0.06-0.13 for hemodialysis patients). Discrepant results were largely TST negative and IGRA positive. Among IGRAs, agreement was highest between ELISPOT and FACS (κ = 0.61). Unlike TST, all IGRAs were associated with variables of mycobacterial exposure. Among IGRAs, the FACS assay was least affected by the level of immunosuppression. In conclusion, both the percentage of positive results and between-test-agreement were higher with IGRAs as compared to TST. This indicates superiority of IGRAs in detecting a PPD-specific immune response which may also apply for immunity toward Mycobacterium tuberculosis-specific antigens. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Rahman M.J.,University of Stockholm | Degano I.R.,University of Stockholm | Singh M.,Lionex Diagnostics and Therapeutics GmbH | Fernandez C.,University of Stockholm
PLoS ONE | Year: 2010

Background: It has been proposed that the immune system could be primed as early as during the fetal life and this might have an impact on postnatal vaccination. Therefore, we addressed in murine models whether gestational treatment with mycobacterial antigens could induce better immune responses in the postnatal life. Methods/Findings: BALB/c mice were treated subcutaneously (s.c.) at the second week of gestation with antigen (Ag)85A or heparin-binding hemagglutinin (HBHA) in the absence of adjuvant. Following birth, offspring mice were immunized intranasally (i.n.) with the same antigens formulated with the adjuvant cholera toxin (CT) at week 1 and week 4. One week after the last immunization, we assessed antigen-specific recall interferon gamma (IFN-γ) responses by in vitro restimulation of lung-derived lymphocytes. Protection against infection was assessed by challenge with high dose Mycobacterium bovis Bacille Calmette-Gué rin (BCG) given i.n. We found that recall IFN-γ responses were higher in the offspring born to the treated mother compared to the untreated-mother. More importantly, we observed that the offspring born to the treated mother controlled infection better than the offspring born to the untreated mother. Since the gestational treatment was done in absence of adjuvant, essentially there was no antibody production observed in the pregnant mice and therefore no influence of maternal antibodies was expected. We hypothesized that the effect of maternal treatment with antigen on the offspring occurred due to antigen transportation through placenta. To trace the antigens, we conjugated fluorescent nanocrystals with Ag85A (Qdot-ITK-Ag85A). After inoculation in the pregnant mice, Qdot-ITK-Ag85A conjugates were detected in the liver, spleen of pregnant females and in all the fetuses and placentas examined. Conclusion: The fetal immune system could be primed in utero by mycobacterial antigens transported through the placenta. © 2010 Rahman et al.

Hu Y.,St Georges, University of London | Van Der Geize R.,University of Groningen | Besra G.S.,University of Birmingham | Gurcha S.S.,University of Birmingham | And 4 more authors.
Molecular Microbiology | Year: 2010

Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3-ketosteroid 9α-hydroxylase (KSH). Consistent with their predicted roles, the δkshA and δkshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione as primary carbon and energy sources. Interestingly, δkshA and δkshB mutants were also unable to metabolize the steroid substrate 5α-androstane-3,17-dione, whereas wild-type M. tuberculosis H37Rv could. The deletion of either of these genes lead to rapid death of the microorganism in murine infection models and in macrophages, showing that kshA and kshB are essential factors for M. tuberculosis pathogenesis. Penta-acylated trehalose (PAT) biosynthesis was altered in the δkshB mutant, but not the δkshA mutant. The δkshB mutant synthesizes all other types of lipids. The δkshB mutant had a thickened outer layer in its cell wall. KshB thus appears to be involved in multiple processes, probably as a reductase of different oxygenases. We conclude that an impaired 3-ketosteroid 9α-hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants. © 2009 Blackwell Publishing Ltd.

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