Anticorpos líticos induzidos por infecção pelo Trypanosoma cruzi reconhecem epitopos presentes nas formas tripomastigotas e epimastigotas do parasita

Sera of Chaga's disease patients containing anti-T. cruzi lytic antibodies were submitted to affinity chromatography using Sepharose 4B conjugated with antigen extracted from epimasiigote or trypomasiigote forms of the parasite. Epimastigotes were obtained from culture at the exponential growth phase and the trypomastigotes from blood of infected and immunosuppressed mice. Antigen of both parasite forms was obtained by sonication of the parasites followed by centrifugation. Both antigens were then conjugated to activated Sepharose 4B. Affinity chromatography was performed by passing sera from chagasic patients through an immunoadsorbent column containing either epimasiigote or trypomasiigote antigens. Antibodies bound to the column were eluted with cold 0,2 M glycine buffer pH 2,8. The eluted antibodies were analysed regarding their isotype and lytic activity. The results showed that anti-T. cruzi lytic antibodies present in sera from chagasic patients are mainly located in the IgG isotype and recognize epitopes present in both trypomasiigote and epimastigote forms. A brief report of this work has already been published12.Soro de pacientes com doença de Chagas na fase crônica foram submetidos a cromatografia de afinidade com Sepharose 4B conjugada com um extrato antigênico obtido de formas epimastigotas ou tripomastigotas de T. cruzi: os epimastigotas foram obtidos de cultura na fase exponencial de crescimento e os tripomastigotas de sangue de camundongos infectados e imunossuprimidos. Os antígenos de ambas formas parasitárias foram obtidos por tratamento dos parasitas por ultra-som, seguido de centrifugação. A cromatografia de afinidade foi feita passando-se os soros chagásicos através de uma coluna de imunoadsorvente contendo antígenos de epimastigotas ou tripomastigotas. Os anticorpos foram eluídos da coluna com tampão glicina 0,2 M pH 2,8 a 4°C. Os anticorpos eluidos foram analisados quanto ao seu isotipo e atividade lítica. Os resultados mostraram que os anticorpos anti-T. cruzi com atividade lítica presentes em soros chagásicos estão localizados no isotipo IgG e reconhecem epitopos presentes tanto nos tripomastigotas quanto nos epimastigotas

A rapid and efficient purification method for horse IgG(T) using a rat monoclonal antibody

1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT-containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened by ELISA using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG(T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 10(10) M(-1). 3. Ascites was induced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T)

Modulation of parasitemia and antibody responce to Trypanosoma cruzy by cyclophosphamide in Calomys callosus (Rodentia, Cricetidae) Modulação da parasitemia e da resposta de anticorpos ao Trypanosoma cruzi pela ciclofosfamida em Calomys callosus (Rodentia, Cricetidae)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan. Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. calossus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10³ T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callossus to T. cruzi.<br>Calomys-callosus, roedor silvestre, que já foi encontrado naturalmente infectado pelo Trypanosoma cruzi, tem baixa suscetibilidade à infecção experimental por este protozoário. Foram feitos experimentos para avaliar a contribuição da resposta imune a essa baixa suscetibilidade. Animais foram submetidos a injeção de doses altas (200 mg/kg peso corporal) ou doses baixas (20 mg/kg peso corporal) de ciclofosfamida nos dias -1 ou -1 e +5, e inoculados com 4 x 10³ T. cruzi no dia O. Observou-se a curva de parasitemia, mortalidade e resposta de anticorpos medida por aglutinação direta de tripomastigotas. Doses de 200 mg resultaram em parasitemia e mortalidade mais elevada e supressão da resposta de anticorpos. Uma dose de 20 mg aumentou os níveis de anticorpos no 20º dia após a infecção, enquanto a administração de uma segunda dose não alterou significativamente a produção de anticorpos. Os níveis de parasitemia não diminuíram, mas pelo contrário, elevaram-se em relação aos animais testemunhos, em ambos os grupos. A transferência passiva de soro anti-T. cruzi de C. callosus resultou em parasitemia e mortalidade mais baixa nos animais imunossuprimidos. Estes resultados indicam que a resposta imune é um importante fator na resistência de C. callosus à infecção por T. cruzi