7-Aryl-7-deazapurine 3 '-deoxyribonucleoside derivative as a novel lead for Chagas' disease therapy : in vitro and in vivo pharmacology

Background The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas' disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside 'hit' led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3 '-deoxy-beta-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. Objectives To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. Results Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. Conclusions Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance

Ischaemia alters the effects of cardiomyocyte-derived extracellular vesicles on macrophage activation

We thank Dr Nuno Alves (Cardiology Department, CHUC‐HG) who performed the collection of human blood samples and Doctor Francisco Caramelo (iCBR/FMUC) for helping with the statistical analysis. This work was supported by the European Regional Development Fund (ERDF) through the Operational Program for Competitiveness Factors (COMPETE) [under the projects PAC “NETDIAMOND” POCI‐01‐0145‐FEDER‐016385; HealthyAging2020 CENTRO‐01‐0145‐ FEDER‐000012‐N2323; POCI‐01‐0145‐FEDER‐007440, CENTRO‐01‐ 0145‐FEDER‐032179, CENTRO‐01‐0145‐FEDER‐032414 and FCT‐ UID/NEU/04539/2013 to CNC.IBILI]. TMM was supported by PD/ BD/106043/2015 and TRR by PD/BD/52294/2013 from Fundação para a Ciência e a Tecnologia (FCT). JS was supported by Horizon2020 ERC‐2016‐COG EVICARE (725229).Myocardial ischaemia is associated with an exacerbated inflammatory response, as well as with a deregulation of intercellular communication systems. Macrophages have been implicated in the maintenance of heart homeostasis and in the progression and resolution of the ischaemic injury. Nevertheless, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages remain largely underexplored. Extracellular vesicles (EVs) have emerged as key players of cell-cell communication in cardiac health and disease. Hence, the main objective of this study was to characterize the impact of cardiomyocyte-derived EVs upon macrophage activation. Results obtained demonstrate that EVs released by H9c2 cells induced a pro-inflammatory profile in macrophages, via p38MAPK activation and increased expression of iNOS, IL-1β and IL-6, being these effects less pronounced with ischaemic EVs. EVs derived from neonatal cardiomyocytes, maintained either in control or ischaemia, induced a similar pattern of p38MAPK activation, expression of iNOS, IL-1β, IL-6, IL-10 and TNFα. Importantly, adhesion of macrophages to fibronectin was enhanced by EVs released by cardiomyocytes under ischaemia, whereas phagocytic capacity and adhesion to cardiomyocytes were higher in macrophages incubated with control EVs. Additionally, serum-circulating EVs isolated from human controls or acute myocardial infarction patients induce macrophage activation. According to our model, in basal conditions, cardiomyocyte-derived EVs maintain a macrophage profile that ensure heart homeostasis, whereas during ischaemia, this crosstalk is affected, likely impacting healing and post-infarction remodelling.publishersversionpublishe