The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-␥), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-␥ secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-␥ in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-␥ in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-␥ production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.Tuberculosis (TB) remains a major public health problem in the 21st century. A third of the world's population is infected with Mycobacterium tuberculosis, and 5 to 10% of the infected population will develop the disease during their lifetime. TB is responsible for more than 2 million deaths per year worldwide (9). The situation is exacerbated by coinfections with human immunodeficiency virus (HIV) and the emergence of multidrug-resistant strains of M. tuberculosis. The Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine available against TB, and yet its efficacy is controversial. BCG has shown extremely variable levels of protection in different populations (12), ranging from 0% (38) in the study of Chingleput, South India, to 80% in The British MRC BCG Trial, Great Britain (37). A cell-mediated immune (CMI) response of the Th1 type, characterized by elevated production of gamma interferon (IFN-␥) and interleukin-12 (IL-12), is essential to mount a protective immunity against M. tuberculosis (8,13,26). Besides, a basic principle for selecting novel antigen candidates for subunit vaccine design is their capacity to induce a protective Th1 response. The definition of new antigens to be applied in an immune-based diagnostic assay for early detection of TB also has a high priority. Purified protein derivative (PPD) from M. tuberculosis is a mixture of complex antigens that has been long used as a skin...

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Summary Previous studies have demonstrated that cells from both multi-drugresistant tuberculosis (MDR-TB

Using a short-term bulk culture protocol designed for an intracellularstaining 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 cytokineproducing 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-γ-, TNF-α-, 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-α was the first (8 h) to be produced, followed by IFN-γ (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-α than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-γ. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations.

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation There has been a concentrated effort over the last few years to characterize specific Mtb molecules for inclusion in a novel TB vaccine and development of more efficacious diagnostic tools. Up to now, evaluations of the functional T cell response in vitro have been based on the secretion of cytokines (mainly IFNγ) and on the expression of activation molecules on the cell surface. Nevertheless, very little information on the phenotypic changes present in the antigen-specific stimulated cells has been made available (Hviid et al. 1993). Based on the recognition of Mtb Ags, the objective of the current study was to evaluate the kinetic expression of early activation molecules (CD69 and CD25) in response to PPD (Statens Serum Institute, Denmark) and the Mtb recombinant proteins Ag85B and ferritin (10 µg/ml) in primary peripheral