Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection. We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P. brasiliensis yeast cell lysate (PbAg). These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system. Our studies showed variation in the cellular responses induced by different antigenic fractions. The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10. The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha. Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form. The findings suggest that P. brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.

Phenotype and cytokine profile of Schistosoma mansoni specific T cell lines and clones derived from schistosomiasis patients with distinct clinical forms.

It is essential to distinguish the role of T lymphocytes on the physiopathology associated to more severe forms of schistosomiasis and on the immunomodulation that evolves in the majority of infected people. In this study, we generated Schistosoma mansoni-specific T cell lines and clones from patients with the acute and chronic (intestinal and hepatosplenic forms) phases of disease, from former ones, and from uninfected individuals sensitized to parasite soluble antigens. T cell lines derived from nontreated acute infected donors were capable of producing IL-4 and IL-5, while cells from treated patients secreted IFN-gamma. Lines from intestinal chronic and antigen-sensitized donors preferentially produced IFN-gamma, while those from hepatosplenic patients secreted all three cytokines. The cytokine analysis of CD4+ T cell clones revealed a Th2/Th0 pattern (clones producing IL-4 and IL-5 and clones producing all three cytokines) for those derived from infected patients, while cells from antigen-sensitized donors exhibited an opposite Th1/Th0 pattern (clones producing IFN-gamma and clones producing all three cytokines). The possible role of these T cell populations on human schistosomiasis mansoni is discussed.

Cytokine profile associated to effector functions of human T cell clones specific for Schistosoma mansoni antigens.

T cell lines and clones specific for Schistosoma mansoni antigens were established to study cellular immunity in human schistosomiasis. Flow cytometric analysis of the clones demonstrated that all of them were of the helper inducer T cell subset (CD3+, CD4+, CD8-), and expressed the alphabeta T cell receptor, besides the IL-2 low affinity receptor CD25. Lymphokine analysis revealed that clones presented Th1, Th2, or either Th0 patterns of secretion. More interestingly, the capability of clones to induce in vitro granuloma reactions seems to be related to the presence of TNF-alpha and the absence of IL-10. In counterpart, IL-10 producer clones did not help in vitro granuloma formation, even in the presence of TNF-alpha.

Characterization of Schistosoma mansoni specific human T-T hybrids.

In this study, we generated antigen-specific T-T hybrids by the fusion of an established T lymphoma, CEM, with T-cell lines derived from Schistosoma mansoni antigen-stimulated peripheral blood mononuclear cells (PBMC) of schistosomiasis patients. These PBMC were capable of in vitro proliferation and granuloma formation upon proper stimulation with antigens derived from different phases of S. mansoni development. Culture supernatants collected from T-T hybrids were responsible for the stimulation or inhibition of antigen-specific responses of other PBMC, on in vitro proliferation and granuloma reaction assays. Clones derived from a T-T hybrid were characterized as CD4+ CD8- HLA-DR- and expressed the cellular markers CD3 and CD25, especially after antigenic stimulation. Their supernatants, as that from the T-T hybrid line, were capable of modulation of in vitro cellular responses, and IL-2, IL-10, and TNF-alpha were detected upon cellular stimulation with antigen or phytohemagglutinin-A (PHA).

Recognition of different Schistosoma mansoni antigens by specific human T cell clones.

Human T lymphocyte clones (TLC) sensitized to Schistosoma mansoni soluble egg antigens (SEA) were developed from chronic schistosomiasis mansoni patients in order to investigate the regulatory mechanisms involved with in vitro granulomatous hypersensitivity to this parasite. All clones studied displayed CD4+ phenotype and required antigen-presenting cells in antigen-driven proliferation and granuloma formation. Each T lymphocyte clone has been shown to proliferate and generate in vitro granulomas in response to SEA, adult worm antigens (SWAP), or cercaria antigens (CAP). In contrast, no proliferation was observed in any of these T cell clones when unrelated S. mansoni antigens were used. Some SEA-generated TLC were not able to proliferate in the presence of SEA; however, S. mansoni SEA-reactive ones were able to recognize epitopes in Schistosoma japonicum SEA, indicating cross-reactivity between these two species. Using IFN-gamma ELISA, it was shown that TLC cells stimulated with SEA can secrete appreciable amount of this lymphokine. Further in vitro studies with these SEA-TLC will help us to understand in more depth the role of regulatory T cells in the human granulomatous hypersensitivity to S. mansoni eggs.

Spiral bacterium associated with gastric, ileal and caecal mucosa of mice.

A spiral shaped bacterium was seen in smears and histological sections (stained by carbolfuchsin) of gastric, ileal and caecal mucosa as well as in stool smears from mice. A significant correlation between the presence of the spiral bacterium and the occurrence of gastritis was observed but the ileal and caecal mucosa seemed unaffected. The bacterium was Gram negative and grew on BHM and Skirrow's medium, under microaerophilic conditions, at 37 degrees C. Its major biochemical characteristics included positive catalase and oxidase reactions and a rapidly positive urease test. There were 2 or 3 spiral turns per cell and a tuft of up to 12 sheathed flagella on each pointed end. Entwined, braided periplasmic fibrils covered the surface of the cell. This spiral bacterium seemed to be part of the normal intestinal flora but was associated with gastritis.