03430nas a2200421 4500000000100000008004100001260001300042653002400055653002300079653001400102653001100116653002100127653001900148653001200167653002600179653002800205653002500233653001300258653002500271653003200296653001800328100001200346700002300358700001800381700001700399700001600416700002000432700001400452700001200466700001600478245011500494856007200609300001300681490000700694050001500701520227800716022001402994 2010 d c2010 Jun10aAmino Acid Sequence10aBacterial Proteins10aCytokines10aHumans10aInterferon-gamma10aInterleukin-1210aleprosy10aLymphocyte Activation10aMolecular Sequence Data10aMycobacterium leprae10aPeptides10aRecombinant Proteins10aSensitivity and Specificity10aT-Lymphocytes1 aGeluk A1 aPloeg-van Schip JJ1 aMeijgaarden K1 aCommandeur S1 aDrijfhout J1 aBenckhuijsen WE1 aFranken K1 aNaafs B1 aOttenhoff T00aEnhancing sensitivity of detection of immune responses to Mycobacterium leprae peptides in whole-blood assays. uhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884434/pdf/0046-10.pdf a993-10040 v17 aGELUK 20103 a

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-gamma) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-gamma than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-gamma production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-gamma production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-gamma in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-gamma was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1beta (MIP-1beta) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-gamma-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.

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