It is generally accepted that both host protection and pathogenic reactions in tuberculosis are mediated by T lymphocytes. However, little is known about the structures and discreet functions of epitopes stimulating the immune response. In this study, proliferative responses of blood T lymphocytes to synthetic peptides derived from the sequence of the 38-kDa antigen from Mycobacterium tuberculosis have been investigated in 41 healthy individuals and in 36 patients with active tuberculosis. Of the healthy purified protein derivative (PPD)-positive donors, 90% responded to a permissively recognized peptide, 38.G (residues 350-359), located at the carboxy terminus of the molecule. Four other permissively recognized epitopes of this molecule (38.A, 38.I, 38.E, 38.K) were stimulatory for more than 50% of healthy PPD-positive individuals. Patients with lymphatic tuberculosis responded to these peptides in a similar manner. In contrast, we observed a selective anergy to stimulation with peptide 38.G in the majority of patients with pulmonary (11% responders) and nonlymphatic extrapulmonary tuberculosis (25% responders). The lack of responsiveness to 38.G was epitope specific since the degree of responsiveness to the other four permissively recognized peptide epitopes was similar for patients and PPD-positive controls. Using the PEPSCAN technology and truncated peptides, the core epitope of 38.G was localized to a peptide 10 amino acids long (HFQPLPPAVV). This minimal structure was capable of inducing a proliferative response in all healthy 38.G responders tested. The mechanisms influencing this epitope-specific anergy in patients could give new insights into the immunopathogenesis of tuberculosis.

The specificity of CD4 T lymphocytes was investigated in 6 patients affected by tuberculosis who had negative tuberculin purified protein derivative (PPD) skin tests at diagnosis. Polyclonal CD4 T cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4 cell lines from the disease site responded to PPD and to the 16- and 38-kDa proteins and derived epitopes in vitro. Four months after chemotherapy, the patients became responsive to PPD. The proliferative response to PPD and to the 16- or 38-kDa proteins and their derived peptides decreased in CD4 T cell lines from the disease site and increased in lines from the peripheral blood. These results indicate that CD4 T cells recognizing a vast array of M. tuberculosis epitopes are compartmentalized at the site of disease in anergic patients but appear in peripheral blood after chemotherapy.

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16‐kDa antigen‐reactive CD4 T‐cell clones prior to therapy resided mostly in disease‐associated body fluids and were of the Th0 (interferon (IFN)‐γ + interleukin (IL)‐4) secreting profile. In contrast, the majority of postchemotherapy CD4 T‐cell clones originated from blood and were of the IFN‐γ secreting Th1 type. However, the recognition of several peptides derived from the 16‐kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%).

SUMMARYThe 16-k.D protein constituent ofthe Mycobacterium tuberculosis complex has been known mainly for its prominent serological immunogenicity and species specificity in tuberculous infection. In this study, we evaluated the T cell Immune repertoire in 27 sensitized healthy subjects and 46 patients with active tuberculosis using 14 overlapping 20mer peptides spanning ihe entire sequence of this protein. Eour ofthe tested peptides individually stimulated proliferation of blood mononuclear cells from more than 50% of healthy controls. Tuberculosis patients reacted to a narrower peptide range and with a \1-Tl% lower rate of responses to the four most immunogenic peptides. but these differences do not distinguish in any simple way between the T cell repertoire of patients and sensitized healthy subjects. The most immunogenic peptide (91-110) was recognized by 67% of healthy subjects and by 50% of tuberculosis patients. Importantly, several non-responders to this peptide were stimulated with the other three most permissive peptides with sequences of 111-130, 71-91 and 21-40. resulting in an overall response rate to at least one of these four peptides of 93% in healthy controls and 74% in tuberculosis patients. In view of this additive effect between the most immunogenic peptides, their combined use may achieve suificient sensitivity in a test aimed at the specific discrimination between infected and non-infected healthy subjects. The major interest in testing with these peptides rests in their species specificity, which is not achieved using purified protein derivative (PPD).