Barbital

Toledo, R. G.1; Gomes, G. I.2; Dias da Silva, W.3; Kipnis, T. L.4

1,2,3,4Universidade Estadual do None Fluminense Darcy Ribeiro - Laboratório de Biologia do Reconhecer

Introduction and objectives: The pathogenicity of Paracoccidioides brasiliensis is influenced by its cell wall hexoses (Sabouraudia, 20:31-40, 1982). These carbohydrates are strong candidates for activation of the complement system (CS) particularly the lectin pathway. It's well described that the fungus is capable of activating the alternative pathway, is opsonized by C3 and C4 fragments and augments its ingestion by murine macrophages (Clin. Immunol. Immunopathol., 12:20-30, 1979; J. Med. Vet. MycoL 30:317-321. 1992; J. Med, Vet. Mycol., 30:481-484, 1992). This study aimed to investigate the activation of the alternative, classical and lectin pathways (AP, CP, LP) activation by the fungus, in vitro. Methods: P. brasiliensis yeast forms at concentrations of 10^sup 5^, 10^sup 6^ or 10^sup 7^ cells/mL were incubated with normal human serum (NHS). The total hemolytic activity was assayed by using sheep red erythrocytes (E^sup s^) pre-sensitized with rabbit antibodies anti-E^sup s^ in GVBS buffer (containing 15 mmol/L CaCl^sub 2^, 0.5 mmol/L MgCl^sub 2^, and 1.8 mmol/L sodium barbital), or antibody non sensitized rabbit red erythrocytes in GVBS-MgEGTA (containing 2 mmol/L MgSO^sub 4^, and 10 mmol/L EGTA), to analyze the functional state of the AP and CP complement activation, respectively. The serum levels of the individual components C3, and C4 were immunochemically quantified by ELISA using specific monoclonal antibodies (mAbs) as probes. The LP activation was analyzed by mannose-binding protein (MBL) consumption using mAbs in ELISA. NHS samples treated with buffer or zymosan were included as controls. Results: We observed that P. brasiliensis activates and significantly reduces the hemolytic activity of NHS when AP and CP were analyzed. The fungal action was dose and time dependent. Levels of C3 and C4 in the respective samples were also significantly reduced when compared to the controls. We observed that samples that activated CS (hemolytic assay and ELISA for C3 and C4) had also consumed MBL. Conclusions: These results indicate that P. brasiliensis yeast forms, of the virulent strain Pb18. activate the AP and LP of CS in NHS samples in vitro, and suggest that these pathways may influence fungi/host cell interactions, in vivo. Since either the CP or the LP lead to C4 consumption, MBL-associated serine proteases (MASPs) consumption are now being measured in order to discriminate the participation of both/one pathways. Financial support: UENF, FAPERJ, CNPq

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