CONICET | Buscador de Institutos y Recursos Humanos

Human echinococcosis is a zoonotic cestode disease caused by Echinococcus sp. larval stage (protoescoleces- PTS- and cysts or metacestode-MTC-). These helminth parasites lack digestive and excretory system but they have developed active endocytic-exocytic cellular processes to regulate metabolite uptake and excretion. In the present work, we analyzed the cestode extracellular vesicles (EVs) production and its interaction with host cells. Loperamide (Lp) a calcium channel blocker, reduced PTS and MTC viability in a dose-dependent manner showing a significant effect with 10 μM and 25 μM after 24 h of treatment, respectively. Additionaly, using the Fluo3-AM fluorescent probe, a cytosolic calcium level increment (which was considered as an exocytosis stimulus) was determined in Lp-treated parasites in comparison to the controls. Moreover, the acetylcholinesterase activity in culture supernatants was determined as an EVs-release indicator, revealing a higher activity in presence of Lp. In addition, EVs were purified from parasite-culture medium through several centrifugation and ultracentrifugation steps and it was shown that Lp-treatment provoked higher density of vesicles than the control condition. On the other hand, transmission electron microscopy enabled the vesicles morphological characterization and the identification of abundant exosomes. Finally, the EVs-host cell interaction was examined. Purified exosomes were labeled with PKH26 red fluorescent labeling and the macrophages uptake and interaction with hepatic cells were analyzed by confocal microscopy. These results show that Echinococcus granulosus possesses a great vesicular traffic which could be involved in the host immune response.

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