Tridimensional ultrastructure and glycolipid pattern studies of Trypanosoma dionisii.

Trypanosoma (Schizotrypanum) dionisii is a non-pathogenic bat trypanosome closely related to Trypanosoma cruzi, the etiological agent of Chaga's disease. Both kinetoplastids present similar morphological stages and are able to infect mammalian cells in culture. In the present study we examined 3D ultrastructure aspects of the two species by serial sectioning epimastigote and trypomastigote forms, and identified common carbohydrate epitopes expressed in T. dionisii, T. cruzi and Leishmania major. A major difference in 3D morphology was that T. dionisii epimastigote forms present larger multivesicular structures, restricted to the parasite posterior region. These structures could be related to T. cruzi reservosomes and are also rich in cruzipain, the major cysteine-proteinase of T. cruzi. We analyzed the reactivity of two monoclonal antibodies: MEST-1 directed to galactofuranose residues of glycolipids purified from Paracoccidioides brasiliensis, and BST-1 directed to glycolipids purified from T. cruzi epimastigotes. Both antibodies were reactive with T. dionisii epimastigotes by indirect immunofluorescense, but we noted differences in the location and intensity of the epitopes, when compared to T. cruzi. In summary, despite similar features in cellular structure and life cycle of T. dionisii and T. cruzi, we observed a unique morphological characteristic in T. dionisii that deserves to be explored.

Expression of antibodies directed to Paracoccidioides brasiliensis glycosphingolipids during the course of paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The immunoglobulin classes and isotypes of antibodies directed to acidic glycosphingolipids (GSLs) and glucosylceramide of P. brasiliensis were determined by enzyme-linked immunosorbent assay of sera from 31 PCM patients. The reactivities of 38 serum samples were analyzed by considering the stage of treatment: before antifungal treatment (n = 10), during 1 to 4 months of treatment (T1-4; n = 9), during 5 to 12 months of treatment (T5-12; n = 9), and posttreatment (PT; n = 10). Sera from healthy subjects (n = 12) were used as controls. Only the GSL Pb-1 antigen, which presents the carbohydrate structure Galfbeta1-6(Manalpha1-3)Manbeta1, was reactive with the PCM patient sera. The PCM patient sera did not react with Pb-2, which lacks the Galf residue and which is considered the biosynthetic precursor of Pb-1, indicating that the Galf residue is essential for antibody reactivity. The Pb-1 glycolipid from nontreated patients elicited a primary immune response with immunoglobulin M (IgM) production and subsequent switching to IgG1 production. The IgG1 titer increased after the start of antifungal treatment (T1-4 group), and general decreases in the anti-Pb-1 antibody titers were observed after 5 months of treatment (T5-12 and PT groups). The Pb-1 antigen, an acidic GSL with terminal Galf residue, has potential application as an elicitor of the host immune response in patients with PCM.

Análise de anticorpos anti-glicolipídeos em soros de pacientes com doença de chagas e paracoccidioidomicose do anticorpo monoclonal BST-1 dirigido a resíduos de bata-D-galactofuranose de Trypanosoma cruzi / Analysis of anti-glycolipidis antibodies in sera of patients with Chagas disease and paracoccidioidomycosis: study and properties of monoclonal antibody BST-1 direct to beta-D-galactofuranosyl residues or Trypanosoma cruzi

Nesta tese analisamos anticorpos presentes em soros de pacientes com paracoccidioidomicose (PCM) e doença de Chagas reativos com glicolipídeos contendo ou não resíduos de galactofuranose (Galf). Por EL1SA, verificamos que 100 por cento dos soros de pacientes com PCM foram reativos com o glicolipídeo Pb-1, isolado de Paracoccidioides brasiliensis, cuja estrutura é: Gal~l 6 (Manpαl-3) Manpαl-2Ins-P-Cer , mas não reconheceram os glicolipídeos Pb-2 e glucosilceramida. Soros de pacientes com PCM antes e durante o tratamento com antifúngicos foram testados quanto a reatividade com o antígeno Pb-1. Foram verificados altos títulos de anticorpos anti-Pb-1 em pacientes antes do tratamento, e tratados por um período de 1 a 4 meses. 19G (1gG1) , 1gM e 1gA foram as principais imunoglobulinas de imunoglobulinas dirigidas ao antígeno Pb-1. Soros de pacientes com doença de Chagas foram altamente reativos com glicolipídeos de epimastigotas de T. cruzi da cepa CL, apresentando menor afinidade por glicolipídeos purificados de epimastigotas da cepa Tulahuen. 1gG1, 1gM e1gA foram as principais imunoglobulinas presentes em soros de pacientes com doença de Chagas dirigidas aos glicolipídeos de epimastigotas. Com o propósito de se obter uma ferramenta importante para avaliar o papel dos glicolipídeos contendo resíduos de Galf na patogenia da doença de Chagas, foi produzido um anticorpo monoclonal (mAb) , denominado de BST-1, dirigido a glicolipídeos de epimastigotas da cepa CL. Por EL1SA, foi verificado que o mAb BST-1 é reativo com glicolipídeos majoritários expressos em epimastigotas das cepas CL e Y, reconhecendo um componente minoritário da cepa G e não reconhecendo glicolipídeos da cepa Tulahuen...