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Signaling events in the interaction of Paracoccidioides brasiliensis with epithelial and mononuclear cells involved in immune response

Grant number: 02/07306-3
Support Opportunities:Research Projects - Thematic Grants
Duration: July 01, 2003 - August 31, 2009
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Maria José Soares Mendes Giannini
Grantee:Maria José Soares Mendes Giannini
Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
Pesquisadores principais:
Gil Benard


Paracoccidoidomycosis (PCM) is an endemic deep mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the leading endemic deep mycosis in Latin America, specially Brazil. It is generally accepted that the infection is acquired through the respiratory route, by inhalation of conidia produced by the mycelial phase of the fungus. These conidia invade alveoli and terminal bronchi, where they transform into the yeast form. The ability of P. brasiliensis in causing not only human disease, but also to cause a mycosis with a wide range of clinical manifestations, from mild, localized disease to severe, disseminated disease, occasionally life-threatening, probably depends on the fungus virulence, its capacity of interact with the superficial structures of the host and the capability to invade and the immune response of the latter. With the purpose of evaluate the steps involved from the initial contact of P. brasiliensis with the host to the adhesion and invasion processes, which ultimately result in the host's cell penetration, our group has developed interesting models of in vitro cell cultures. By this mean, we demonstrated that not only P. brasiliensis has the capacity of invading epithelial cell tines of human and animal origin, but also, and for the first time, that the degree of virulence correlated with the adhesion capacity of the fungal isolates studied. On the other hand, we verified that adhesion to components of the extracellular matrix, such as type I and IV collagen, laminin, fibronectin and entactin, was differential for isolates with different degrees of virulence. The immunodominant antigen of the fungus, a 43kDa glycoprotein (gp43), was also found to behave as an adhesion component to laminin and fibronectin (data not published), among other components that still need to be better characterized. We further verified that, among the components of the cytoskeleton, P. brasiliensis probably utilizes actin and tubulin to invade cells, and the protein cytokeratin seems to play an important role in this process. Finally, we observed in this same model of cell cultures that P. brasiliensis is able to induce apoptosis of epithelial cells. On the other hand, our group has demonstrated in previous studies the existence of suppression of the antigen-specific cellular immune response of PCM patients with the different forms of the disease. A clear evidence for this suppression was the decrease in the lymphocyte proliferative response to gp43, particularly in the severe forms. We demonstrated that this immunosuppression was associated with exacerbated Th-2 immune responses concomitant to down regulation of Th-1 responses, i.e., low levels of IL-2 and lFN-y and sustained levels of IL-10 in the response to gp43 by one side, and high titers of IgE and IgG4 anti-gp43 antibodies by the other. We further demonstrated that this immune imbalance also correlated with the severity of the clinical manifestations. Subsequently, we isolated monocytes from PCM patients and found that such cell type was responsible for a major proportion of the early high IL-10 production, thus probably acting as an important suppressor factor of the Th-1 response of the patients. More recently we have documented the co-participation of the deficient IL-12 production of mononuclear cells of patients in the suppressor mechanism. In vitro IL-12 addition to the cultures, in association with neutralization of endogenously secreted IL-10 by a monoclonal antibody, resulted in restoration of some Th-1 parameters of the patients' immune response. In parallel, we also observed that apoptosis of gp43 responding T -cells of patients could be an additional, alternative mechanism for the antigen-specific Th-1 hyporesponsiveness. However, the involvement of costimulatory molecules in the induction of anergy, as well as the participation of intracellular transducing signaling, illustrated by activation of Stats (signal transducer and activator of transcription) and caspases, in this hyporeactivity, has not been investigated yet. Based on the evidence that the result of the interaction of P. brasiliensis with epithelial and mononuclear cells of the human host seems to be determinant to the outcome of the infection and to the variability in the clinical presentation, the present project intends to pursue the previous collaborative efforts of our laboratories by focusing in the interaction of P. brasiliensis with the above mentioned cellular components. We propose to genotypically characterize isolates of the fungus that vary in their epithelial cell adhesion ability, isolate and characterize the putative adhesins involved in this process, as well as the extra-cellular matrix components (fibronectin and collagen) receptors, and to evaluate the fungal components (e.g., gp43) that can modulate, through signaling events, the epithelial cell invading processo In parallel, we will study the participation of costimulatory molecules and the molecular aspects (Stats and caspases activation) related to PCM patients' vs. cured/sensitized individuaIs' lymphocyte proliferation and cytokine production after challenge with gp43, and to the apoptosis phenomena. We will also verify the role of lL-12 and costimulatory molecules in the induction/protection of/from apoptosis as a possible mechanism associated with the immunological imbalance of PCM. The simultaneous study of the components of the fungus and host cells signaling and costimulatory pathways will provide a better understanding of the mechanisms involved both in the pathogenicity of P. brasiliensis and in the suppression of the Th-l response found in the patients, as compared to the healthy exposed individuaIs or cured patients. As a consequence, we will be able to conjunctly evaluate a higher range of cellular events associated with the pathogenesis of paracoccidioidomycosis. (AU)

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Scientific publications (4)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MONTEIRO DA SILVA, J. L.; ANDREOTTI, P. F.; BENARD, G.; SOARES, C. P.; MIRANDA, E. T.; MENDES-GIANNINI, M. J. S.. Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis. ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, v. 92, n. 1, p. 129-135, . (02/07306-3)
CACERE, CAMILA R.; MENDES-GIANNINI, MARIA J. S.; DO VALLE, ANTONIO CARLOS F.; DUARTE, ALBERTO J. S.; BENARD, GIL. Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis. Clinical and Vaccine Immunology, v. 16, n. 6, p. 953-955, . (02/07306-3)
BENARD, GIL; KAVAKAMA, JORGE; MENDES GIANNINI, MARIA J. S.; KONO, ADRIANA; DUARTE, ALBERTO J. S.; SHIKANAI-YASUDA, MARIA A.. Contribution to the natural history of paracoccidioidomycosis: identification of the primary pulmonary infection in the severe acute form of the disease - a case report. Clinical Infectious Diseases, v. 40, n. 1, p. e1-e4, . (02/07306-3)
CACERE, CAMI A. R.; MENDES-GIANNINI, MARIA J. S.; FONTES, COR J.; KONO, ADRIANA; DUARTE, ALBERTO J. S.; BENARD, GIL. Altered expression of the costimulatory molecules CD80, CD86, CD152, PD-1 and ICOS on T-cells from paracoccidioidomycosis patients: Lack of correlation with T-cell hyporesponsiveness. Clinical Immunology, v. 129, n. 2, p. 341-349, . (02/07306-3)

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