RESUMO
Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve to different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. In the murine model, A/Sn and B10.A isogenic strains of mice are known to be resistant and susceptible, respectively, to this fungal infection. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated the importance of two antigen-presenting cells (APCs), macrophages and B cells, in the development of the immune response to P. brasiliensis. In resistant mice, purified gp43, the main antigenic component of P. brasiliensis, seems to have been preferentially presented by macrophages and stimulated Th1 lymphokine production. On the other hand, in susceptible animals gp43 was distinguishably presented by B cells, which led to stronger activation of Th2 subsets. Moreover, T cells from resistant mice responded as those from susceptible animals when stimulated by gp43 presented by APCs from susceptible mice and vice versa, indicating that there are no significant differences in the T cell repertoires from A/Sn and B10.A mice. When T cells from F1 (A/Sn x B10.A) mice were stimulated by gp43 presented by APCs from A/Sn or B10.A, impaired behavior of B10.A macrophages in activating Th1 cells and a B10.A B cell tendency to stimulate T cells that secrete higher levels of IL-10 were observed. Taken together, our results suggest that APCs may be implicated in the outcome of P. brasiliensis infection.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos de Fungos , Proteínas Fúngicas , Glicoproteínas/imunologia , Oligossacarídeos/imunologia , Paracoccidioides/imunologia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Imunidade Celular , Ativação Linfocitária , Linfocinas/biossíntese , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos A , Paracoccidioidomicose/etiologia , Paracoccidioidomicose/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.
Assuntos
Anticorpos Antifúngicos/farmacologia , Anticorpos Antifúngicos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Fungos/imunologia , Proteínas Fúngicas , Glicoproteínas/imunologia , Laminina/antagonistas & inibidores , Laminina/farmacologia , Oligossacarídeos/imunologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/etiologia , Animais , Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/uso terapêutico , Ligação Competitiva/imunologia , Adesão Celular/efeitos dos fármacos , Cricetinae , Células Epiteliais , Epitélio/metabolismo , Humanos , Laminina/efeitos dos fármacos , Masculino , Paracoccidioides/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a Kd of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.
Assuntos
Glicoproteínas/metabolismo , Laminina/metabolismo , Paracoccidioides/patogenicidade , Adesividade , Animais , Células Cultivadas , Cricetinae , Cães , MasculinoRESUMO
Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.