Potencial terapêutico do fungo agaricus blazei no tratamento de diferentes formas clínicas de leishmanioses e estudo do envolvimento da proteína NLRP12 na infecção por Leishmania chagasi

Abstract

Leishmaniasis is a neglected disease that causes morbidity and mortality in affected patients. Although there are few treatment alternatives, the drugs present toxicity to individuals, reducing the adhesion to treatment which may impair the effectiveness of the products. Seeking to minimize these problems that concern the treatments available for the different clinical forms of leishmaniasis, in the present thesis, an aqueous extract of the Agaricus blazei Murril was prepared and it's leishmanicidal activity was evaluated against different Leishmania species, namely Leishmania amazonensis, L. major and L. chagasi. The extract was effective in reducing the cellular viability of parasites and experiments treating infected macrophages with the extract showed a significant reduction of the intra-macrophages parasite load. Pretreatment of promastigotes with the extract showed a significant reduction of its infective capacity, showing a preventive potential of the fungus extract against Leishmania infection. Another important factor was the absence of cytotoxicity demonstrated by the preparation, both in murine peritoneal macrophages as in human erythrocytes. The therapeutic efficacy of the extract was evaluated in BALB/c mice infected with L. amazonensis by oral treatment, and it was effective in reducing the swelling at the site of infection as well as in the reduction of parasitemia in evaluated organs (paw, spleen and lymph node). Splenocytes from animals treated with the extract showed elevated levels of IFN-gamma and nitric oxide in cultures stimulated ex vivo, and the preparation showed no liver toxicity in the treated animals. Then the fractionation and phytochemical analysis of the extract and five semi-purified fractions were performed. Such products were evaluated for their antileishmanial activity and the fraction Fab 5 was selected as presenting the best results. The therapeutic effectiveness in a model for visceral infection with L. chagasi was also evaluated using aqueous extract or Fab 5 in different treatment regimens, including a chemo-prophylactic regimen. The different types of treatment were effective in reducing the parasitic load in the liver, spleen and lymph node of the animals. The reduction observed in the lymph node and spleen was greater than in the animals treated with the drug used as control, amphotericin B. The chemoprophylaxis using Fab 5 showed better therapeutic efficacy with lower levels of parasite load observed in the spleen and lymph node of the animals. As in the model of cutaneous leishmaniasis, animals infected with L. chagasi and treated with extract and Fa.b 5 showed a higher Th1 response, marked by high IFN-gamma levels and reduced levels of IL-4 in ex vivo stimulated spleen cultures. Thus, the use of A. blazei fungus, in different treatment strategies (conventional treatment as well chemoprophylaxis) was effective in the treatment of both murine cutaneous and visceral models of leishmaniasis. With the purpose of improving the techniques for evaluating the preclinical therapeutic efficacy of drugs in leishmaniasis models, through the acquisition of knowledge in in vivo imaging technique, a project in conjunction with Dr. Mary E. Wilson (University of Iowa, USA). The objective of the project was to evaluate the role of NLRP12 protein in the infection caused by L.i chagasi. In this study, besides verifying the applicability for in vivo imaging in preclinical models of leishmaniasis, we could detect a controlling role in inflammation played by NLRP12, in which could be observed an increase in leukocyte infiltration at the site of inflammation such as neutrophils and inflammatory monocytes in the NLRP12 deficient mice. Furthermore, it could be proved the role of NLRP12 in the dendritic cells migration and also that these cells become non-responsive against chemokines in the absence of this protein. This reduced migration causes changes in the adaptive response due to a reduced antigen presentation in the spleen.