Change of Th0 to Th1 cell-cytokine profile following tuberculosis 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)-gamma + interleukin (IL)-4) secreting profile. In contrast, the majority of postchemotherapy CD4 T-cell clones originated from blood and were of the IFN-gamma 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.

Broad clonal heterogeneity of antigen-specific CD4+ T-cells localizing at the site of disease during tuberculosis.

The repertoire of CD4+ T-lymphocytes was investigated in six patients affected by tuberculosis, who had a negative PPD skin test 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+ T-cell lines from the site of disease responded to PPD, and to the 16- and 38-kDa proteins, and derived epitopes in vitro. The repertoire of CD4+ T-cells accumulating at the site of disease was found to be widely heterogeneous as demonstrated by the finding that at least seven different peptides from the 16- and 38-kDa proteins were recognized by every patient. These results indicate that CD4+ T-cells localized at the site of disease in tuberculosis recognize a vast array of M. tuberculosis epitopes.

Sequestration of T lymphocytes to body fluids in tuberculosis: reversal of anergy following chemotherapy.

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.

Human T cell responses to peptide epitopes of the 16-kD antigen in tuberculosis.

The 16-kD protein constituent of the 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 the entire sequence of this protein. Four of the 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 17-27% 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 sufficient 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).

Genetically permissive recognition of adjacent epitopes from the 19-kDa antigen of Mycobacterium tuberculosis by human and murine T cells.

The specificity of the T cell-immune repertoire at the level of individual antigenic determinants could play a fundamental role in the immunopathogenesis of tuberculous infections. Therefore, we analyzed the immunogenicity, genetic restriction, and epitope core structure of two adjacent, yet distinct, immunodominant T cell determinants from the 19-kDa Ag of Mycobacterium tuberculosis. After immunization with two peptides comprising residues 45 to 64 and 61 to 80, vigorous in vitro proliferative responses to the homologous peptide were elicited in five different strains of C57BL/10 mice (H-2b,k,d,s,f), indicating that both epitopes were recognized in a genetically permissive manner. When immunized with intact 19-kDa protein, lymph node cells from the same mouse strains responded to both peptides, with the exception of H-2b mice, which did not respond to p45-64. Delayed-type hypersensitivity responses in C57BL/10 (H-2b) mice were elicited by p61-80 only, whereas in H-2d mice both peptides were delayed-type hypersensitivity negative, despite eliciting pronounced proliferative responses. Analysis of the proliferative responses of human PBMC in purified protein derivative-positive healthy subjects and tuberculosis patients revealed significant differences in the antigenicity to the two peptides. All purified protein derivative-positive healthy and diseased individuals manifested strong responses to p45-64, indicating HLA permissive recognition. In contrast, pronounced responses to p61-80 were detected only in patients with lymphatic tuberculosis. Epitope core structures, composed of 6 or 7 residues within each peptide, have been mapped with peptides of overlapping sequence. Significantly, for both epitopes, the core sequences recognized by both human and murine T cells were almost identical. We conclude that despite many similarities between murine and human T cell epitope recognition, distinct differences in the responsiveness of the infected host could play a role in pathogenesis. Furthermore, the genetically permissive nature of the identified epitopes is a potentially important attribute for the development of peptide-based diagnostic reagents and vaccines.

T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38-kDa antigen in patients with active disease.

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.

Mutagenic epoxide impurities discovered in two new beta-adrenergic blocking agents.

Preparations of two new beta-adrenergic blocking drugs, Zami 1305 [1-(2-nitro-3-methyl-phenoxy)-3-tert-butylaminopropan-2-ol] and Zami 1327 [1-(6-nitro-3-methyl-phenoxy)-3-tert-butylaminopropan-2-ol], were found to be contaminated by expoxides which are direct acting mutagens on TA 100 and TA 1535 in the Salmonella/microsome mutagenicity test. Because of the suggested correlation between mutagenicity and carcinogenicity of a chemical [10,11], beta-adrenergic blocking agents contaminated by mutagenic expoxide impurities may be a health hazard.