Potent stimulation of murine B cells to proliferate and to secrete immunoglobulins by a lipoteichoic acid-like molecule produced by Clostridium botulinum C and D.

Bacterial products have served as important immunological tools to study lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW > or = 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 kDa were highly toxic for mice and had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because spleen cells from the LPS non-responder C3H/HeJ mice responded well to the Cb mitogen, and the antibiotic polymyxin B, which is an inhibitor of LPS, had no effect on the Cb-mitogen activity. However, an anti-lipoteichoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent the proliferation of spleen cells induced by the Cb mitogen but had no effect on the LPS or concanavalin A stimulation of these cells. Moreover, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had been conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.

[The pathogenesis of chronic Chagas' myocarditis: the role of autoimmune and microvascular factors]. / Patogênese da miocardite chagásica crônica: o papel de fatores autoimunes e microvasculares.

The pathogenesis of chronic chagasic myocarditis remains incompletely understood. Several hypotheses have been proposed: (1) direct tissue destruction by Trypanosoma cruzi; (2) neurogenic theory; (3) anti-heart immune reactions; and (4) microvascular disease. We present herein a dynamic alternative hypothesis. We believe that the development of myocarditis is related to progressive and additive focal cellular necrosis, and associated reactive and reparative myocardial fibrosis and surrounding myocytes hypertrophy. These processes may be initiated and perpetuated by autoimmune factors and alterations in the myocardial microcirculation. This could imply future therapeutic strategies in the management of chronic chagasic patients to optimize the medical treatment and hopefully improve the prognosis.

A heart-specific CD4+ T-cell line obtained from a chronic chagasic mouse induces carditis in heart-immunized mice and rejection of normal heart transplants in the absence of Trypanosoma cruzi.

To study the role of autoreactive T cells in the pathogenesis of cardiomyopathy in Chagas' disease, we generated a cell line by repeated in vitro antigenic stimulation of purified splenic CD4+ T lymphocytes from a chronically Trypanosoma cruzi-infected mouse. Cells from this line were confirmed to be CD4+ CD8- and proliferated upon stimulation with soluble heart antigens from different animal species, as well as with T. cruzi antigen, in the presence of syngeneic feeder cells. In vitro antigen stimulation of the cell line produced a Th1 cytokine profile, with high levels of IFNgamma and IL-2 and absence of IL-4, IL-5 and IL-10. The cell line also terminated the beating of fetal heart clusters in vitro when cocultured with irradiated syngeneic normal spleen cells. In situ injection of the cell line into well established heart transplants also induced the cessation of heart beating. Finally, adoptive transfer of the cell line to heart-immunized or T. cruzi-infected BALB/c nude mice caused intense heart inflammation.

Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis.

Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains. The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells. In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

Potent stimulation of murine B cells to proliferate and to secrete immunoglobulins by a lipoteichoic acid-like molecule produced by Clostridium botulinum C and D

Bacterial products have served as important immunological tools to study ly,phocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW ò 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 KDa were highly toxic for mice and had no mitogenic activity...