Ação in vitro da neuwiedase sobre a infecção por T. gondii em fibroblastos humanos e na produção de mediadores inflamatórios por células mononucleares do sangue periférico humano

Abstract

CHAPTER II: The infection for T. gondii affects more than a third of the world population, affecting mainly imunocompromised individuals. Therapies in use are limited because of parasite resistance and side effects. Then, the search for new drugs to control infection is extremly important. The effects of neuwiedase, an isolated metaloprotease from Bothrops pauloensis venom, on the in vitro invasion and proliferation of the T. gondii in human fibroblastos.was investigated The treatment was done on cells previously infected and on the parasite before the infection, and results showed infection inhibition levels of 71% and 61%, respectively. The mechanisms involved in neuwiedase action need to be better investigated, but the capacity of enzyme to degraded some extracelular matrix components such as laminin, fibronectin and collagen type I can be important in the reduction of host cells invasion by T. gondii . CHAPTER III: The release of pro-inflammatory cytokines and chemokine (IL-12, TNF-α and IL-8) and anti-inflammatory cytokine (IL-10) from human peripheral blood mononuclear cells (PBMC) was evaluated after exposure to neuwiedase, a Zn2+ dependent metaloprotease isolated from Bothrops pauloensis venom. Initially, using the MTT metabolization assay, we show that in all concentrations tested (3, 6 e 12 μg/mL), neuwiedase did not affect PBMCs viability. Suppernatants of PBMC culture in presence of neuwiedase were collected and cytokine and chemokine levels were determined by sandwich ELISA. Under neuwiedase stimulation, PBMCs significantly increased the production of IL-12p40, IL-8 and TNF-α but not IL-10 production compared cells to not stimulated with neuwiedase. This study confirms the inflammatory properties of neuwiedase involving the liberation of proinflammatory mediators, probably contributing to the severe local damage induced by the Bothrops pauloensis venon. CHAPTER III: The release of pro-inflammatory cytokines and chemokine (IL-12, TNF-α and IL-8) and anti-inflammatory cytokine (IL-10) from human peripheral blood mononuclear cells (PBMC) was evaluated after exposure to neuwiedase, a Zn2+ dependent metaloprotease isolated from Bothrops pauloensis venom. Initially, using the MTT metabolization assay, we show that in all concentrations tested (3, 6 e 12 μg/mL), neuwiedase did not affect PBMCs viability. Suppernatants of PBMC culture in presence of neuwiedase were collected and cytokine and chemokine levels were determined by sandwich ELISA. Under neuwiedase stimulation, PBMCs significantly increased the production of IL-12p40, IL-8 and TNF-α but not IL-10 production compared cells to not stimulated with neuwiedase. This study confirms the inflammatory properties of neuwiedase involving the liberation of proinflammatory mediators, probably contributing to the severe local damage induced by the Bothrops pauloensis venon.