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Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T.cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosomacruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T.cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).

Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the

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-β-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.

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-β-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.

Phenotypic assay against Leishmania amazonensis in vitro and in vivo led to identification of an adamantyl-based phenyl sulfonyl acetamide (compound 1) as a promising antileishmanial agent. Compound 1 inhibited the growth of intracellular forms of L. amazonensis (50% inhibitory concentration [IC 50 ] = 4 μM) and exhibited low toxicity to host cells, with a selectivity index (SI) of >125. However, in a cutaneous leishmaniasis (CL) mouse model, compound 1 did not reduce lesions and parasite load when administered as monotherapy or when given simultaneously with a suboptimal dose of miltefosine.

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

TGF-β involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-β signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TβR1/ALK5, on cardiac function in an experimental model of chronic Chagas’ heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-β signaling pathway reduced TGF-β/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas’ heart disease by TGF-β inhibitors., Author summary TGF-β is a key molecule in many physiological processes as well as pathologies. We have previously described the role of TGF-β in Chagas disease, caused by the eukaryotic protozoan parasite Trypanosoma cruzi. Besides the high disease burden in many countries, one of the severe aspects of Chagas disease is the chronic heart condition developed by patients, for which there is no specific treatment. In search for a better treatment, we have investigated the potential of the TGF-β signaling blocker, GW788388, on disease hallmarks in a mouse model of chronic Chagas’ heart disease. Oral administration of GW788388 produces a global reversion of cardiac damage, and, remarkably, reduces fibrosis, one of the most important death-associated features in chronic Chagas’ heart disease.

Background Caused by Mycobacterium tuberculosis, tuberculosis (TB) is an extremely contagious disease predominantly affecting the lungs. TB is found worldwide and has a major impact on public health safety primarily due to its high mortality rate. Applied for over a hundred years as a preventive measure, Mycobacterium bovis BCG remains the only available TB vaccine. Only one seminal study about the apoptotic pathways induced by this vaccine in the monocytic lineage of the host cell has found the effects of BCG on regulation of apoptosis. The aim of this study was to explore beyond that pioneer study the pathway related to the in vitro cell-death pattern and the inflammatory response to the BCG vaccine in human monocytes. Methods Cohorts of HIV-negative volunteers were enrolled: adult Healthy Donors (HD) and neonates’ Umbilical Cord Blood (UCB) individuals. Host mononuclear cells were infected with the M. bovis Moreau strain of BCG vaccine at 16, 24, 48, and 72 h. The Real-Time RT-PCR for TRADD, Bcl-2, and Caspases-1 and -3 were performed, and supernatants were assayed in parallel for Caspase-1, NLRP3, HO-1, and IL-1β levels whereas caspases were assessed intracellularly. The effect of a BCG infection in monocytes was characterized via a metabolic activity assay by LDH release profiles. Results Overall, the BCG vaccine induced significantly higher Caspase-1 and Bcl-2 mRNA levels in both the HD and UCB groups (p-value ≤0.05). In addition, a significant increase solely in Caspase-1 protein levels was also noted in both HD and UCB (p-value ≤0.05) notwithstanding the absence of any damaged cell membranes. Conclusions Our data directly corroborate other findings showing that BCG Moreau led to an increased secretion of IL-1β but not IL-18, two Caspase-1-activated cytokines, and are also in support of the model that the BCG Moreau infection of human mononuclear cells may induce a cell-death pattern involving Caspase-1 activation.

Background: Chagas disease (CD) has high morbimortality and the available trypanocidal treatment, including benznidazole (BZ), has limited efficacy in chronic patients. Furthermore, BZ causes adverse effects (AE) that prevent treatment completion in up to 30% of patients. The use of repositioned drugs or drug combination may provide an effective trypanocidal treatment. Disulfiram (DF) may enhance BZ activity and decrease BZ related AE. This study aims to assess the safety of a new combination of drugs for CD therapy, assuming BZ as the drug of choice plus DF as repositioned drug. Methods: This single-centre, open-label, phase I/II clinical trial was designed to evaluate the safety of the combined use of BZ plus DF for CD therapy. Participants are adults with indeterminate form of chronic CD, both sexes, aged from 18 to 70 years old and Trypanosoma cruzi polymerase chain reaction-positive. The primary outcome will be the occurrence of serious AE. The secondary outcome will be post-treatment Trypanosoma cruzi polymerase chain reaction negativization. Six groups of 9 patients will be sequentially tested. The first group will be allocated to receive BZ 100mg/d + DF 250mg/d for 60 days. Upon safety confirmation (

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.