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Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus.

ABSTRACT: BACKGROUND: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. METHODS: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-? production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. RESULTS: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-? and IFN-?) in T cells from non-human primates (NHPs) after BCG vaccination. CONCLUSIONS: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

SUBMITTER: Ahmed RK

PROVIDER: S-EPMC3305616 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus.

Ahmed Raija K RK Rohava Zoyia Z Balaji Kithiganahalli N KN Hoffner Sven E SE Gaines Hans H Magalhaes Isabelle I Zumla Alimuddin A Skrahina Alena A Maeurer Markus J MJ

BMC infectious diseases 20120215

<h4>Background</h4>Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines.<h4>Methods</h4>Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.t ...[more]

PMID: 22336002

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Project description:A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

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Dataset Information

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus.

Publications

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus.

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