02513nas a2200385 4500000000100000008004100001260001300042653003100055653003100086653001400117653001400131653001900145653001100164653002100175653001900196653001800215653001200233653002500245653001500270653002800285653003200313100001600345700001400361700001600375700001700391700001400408700001400422700001600436245010800452300001200560490000700572050001400579520152000593022001402113 2004 d c2004 Aug10aCD4-Positive T-Lymphocytes10aCD8-Positive T-Lymphocytes10aCytokines10aCytoplasm10aFlow Cytometry10aHumans10aInterferon-gamma10aInterleukin-1010aInterleukin-410aleprosy10aMycobacterium leprae10aTuberculin10aTuberculosis, Pulmonary10aTumor Necrosis Factor-alpha1 aAntas P R Z1 aSales J S1 aPereira K C1 aOliveira E B1 aCunha K S1 aSarno E N1 aSampaio E P00aPatterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections. a1119-290 v37 aANTAS20043 a
Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.
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