Leprosy is an infectious disease caused by Mycobacterium leprae. Tumor necrosis factor (TNF) plays a key role in the host response. Some association studies have implicated the single nucleotide polymorphism TNF -308G>A in leprosy susceptibility, but these results are still controversial. We first conducted 4 association studies (2639 individuals) that showed a protective effect of the -308A allele (odds ratio [OR] = 0.77; P = .005). Next, results of a meta-analysis reinforced this association after inclusion of our new data (OR = 0.74; P = .04). Furthermore, a subgroup analysis including only Brazilian studies suggested that the association is specific to this population (OR = 0.63; P = .005). Finally, functional analyses using whole blood cultures showed that patients carrying the -308A allele produced higher TNF levels after lipopolysaccharide (LPS) (6 hours) and M. leprae (3 hours) stimulation. These results reinforce the association between TNF and leprosy and suggest the -308A allele as a marker of disease resistance, especially among Brazilians.

Several antigens of Mycobacterium tuberculosis have been identified and specificity to one or multiple antigens could determine the distinction between protective and pathogenic host reaction. Therefore T cell immune response to combinations 38 kDa/CFP‐10, 38 kDa/MPT‐64, ESAT‐6/MPT‐64 and ESAT‐6/CFP‐10 (each related to a single protein of Mycobacterium tuberculosis) in individuals from tuberculosis endemic areas have been examined. ELISA was used to detect IFN‐γ production in PBMC priming with single proteins and combinations in a panel of 105 individuals: 38 tuberculosis patients (6 untreated and 32 treated) and 67 healthy controls with tuberculin skin test positive or negative (TST). Brazilian TB patients highly recognized ESAT‐6 (66%), but combinations improved response in the following order: ESAT‐6/MPT‐64 (89%) > ESAT‐6/CFP‐10 (73%) > 38 kDa/CFP‐10 (70%), the last combination showing the highest specificity (TST+=42% and TST–=83%). Average IFN‐γ production in TB patients was significantly higher for 38 kDa/CFP‐10 (P=0.012) and 38 kDa/MPT‐64 (P<0.035), when compared to single antigens. None of the combinations was able to discriminate TB patients from TST+ controls; however, 38 kDa/CFP‐10 displayed a borderline significance (P=0.053). Similar to the ESAT‐6/CFP‐10 combination, IFN‐γ response to 38 kDa/CFP‐10 showed an increased tendency in treated patients, although not significant (P=0.16). We demonstrated for the first time that 38 kDa/CFP‐10 had prediction sensitivity for TB patients similar to the ESAT‐6/CFP‐10 combination and also significant response improvement related to the single proteins with more selective reactivity among TST‐positive individuals, which could be of potential interest for diagnostic evaluation for tuberculosis infection.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n ‫؍‬ 59) and healthy leprosy contacts (n ‫؍‬ 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-␥) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had >5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of >90% (percentage of United Kingdom donors who were nonresponders for IFN-␥ secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.The completion of the sequencing of the genome of Mycobacterium tuberculosis (7) and the availability of almost 98% of the genome sequence of Mycobacterium leprae (http://www .sanger.ac.uk) provide a unique opportunity to identify specific antigens within these pathogens, which could be used as diagnostic tools. One approach, which has been used previously to develop M. tuberculosis-specific diagnostic antigens, is to identify genes present in M. tuberculosis which have been deleted from Mycobacterium bovis BCG (20) Previous studies of the human T-cell response in leprosy patients have identified a number of antigens that induce T-cell responses, measured by lymphocyte proliferation or gamma interferon (IFN-␥) secretion, in patients with tuberculoid leprosy. Such antigens include the M. leprae 70-kDa, 65-kDa, 45-kDa, 35-kDa, 18-kDa, and 10-kDa antigens (1,2,6,12,16,29,31). The members of the heat shock family of proteins are highly conserved, and homologues of the M. leprae 70-kDa, 65-kDa, and 10-kDa antigens show over 90% homology between M. leprae and M. tuberculosis. It is the...

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-γ (IFN-γ)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-γ human T cell immune responses, to four recombinant proteins NarL, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) Mycobacterium tuberculosis is an extremely successful pathogen infecting a third of the world's population, the cases being concentrated in developing countries (WHO 2002, Guzmán et al. 2003. Koch bacillus, which is transmitted by respiratory route from person to person, invades macrophage, multiply and can remain latent within the lung granuloma for years. Disease control is based on early identification of infected individuals followed by appropriate treatment. Disease progression prevention through inducement of protective immunity may be achieved through vaccination; however the current available Bacillus Calmette-Guérin (BCG) efficacy is controversial (Colditz et al. 1994, Fine 1995 recognition. However, little is known about the reactivity to these antigens in patients from Rio de Janeiro where are described the highest rates of tuberculosis (TB) incidence in Brazil (Hijjar 2005). The 16 kDa molecule is a polypeptide belonging to the α-cristallin family of low molecular weight, heat shock proteins, of which the coding gene (Rv2031c) has been found exclusively in the M. tuberculosis complex (Yuan et al. 1998). It has been reported as dominant protein produced in the static growth phase or under oxygen deprivation and required for bacterial replication inside macrophages (Yuan et al. 1998, Agrewala & Wilkinson 1998. MBP-3 is a 23 kDa maltose binding protein found as residue in the purification process of the MPT-64 antigen. The MT-10.3 or TB10.3 (Rv3019c) antigen, together with TB12.9 and TB10.4, comprise one subfamily within the esat-6 gene family and seem to be expressed during the TB infection (Skjot et al. 2002, Demissie et al. 2006. Two regulatory system components are ubiquitously distributed among bacteria and plants and are involved in the organisms' virulence (Urao et al. 2000). Nar L is the product of the response regulator narL gene of M. tuberculosis regulatory system (Parish et al. 2003).In the present study, we evaluated the production of interferon-γ (IFN-γ) and the proliferative response of peripheral blood mononuclear cells (PBMC), from TB patients from Rio de Janeiro, stimulated with the 858 858 858 858 858Immune response to mycobacterial antigens • Ricardo Candido Oliveira Tavares et al.following M. tuberculosis recombinant proteins: 16kDa, MBP-3, MT-10.3, and NarL. MATERIALS AND METHODSStudy subject -TB diagnosis was performed at district Hospital Souza Aguiar (H...