Successful Aspects of the Coadministration of Sterol 14α-Demethylase Inhibitor VFV and Benznidazole in Experimental Mouse Models of Chagas Disease Caused by the Drug-Resistant Strain of Trypanosoma cruzi.

Up to now, no vaccines are available for Chagas disease, and the current therapy is largely unsatisfactory. Novel imidazole-based scaffolds of protozoan sterol 14α-demethylase (CYP51) inhibitors have demonstrated potent antiparasitic activity with no acute toxicity. Presently our aim was to investigate the effectiveness of the experimental 14α-demethylase inhibitor VFV in the mouse models of Trypanosoma cruzi infection using a naturally drug-resistant Colombiana strain, under monotherapy and in association with the reference drug, benznidazole (Bz). The treatment with VFV resulted in complete parasitemia suppression and 100% animal survival when administered orally (given in 10% DMSO plus 5% Arabic gum) at 25 mg/kg (bid) for 60 days. However, as parasite relapse was found using VFV alone under this treatment scheme, the coadministration of VFV with Bz was assayed giving simultaneously (for 60 days, bid) by oral route, under two different drug vehicles (10% DMSO plus 5% Gum Arabic with or without 3% Tween 80). All tested mice groups resulted in >99.9% of parasitemia decrease and 100% animal survival. qPCR analysis performed on cyclophosphamide immunosuppressed mice revealed that, although presenting lack of cure, VFV given as monotherapy was 14-fold more active than Bz, and the coadministration of Bz plus VFV (given simultaneously, using 10% DMSO plus 5% Gum Arabic as vehicle) resulted in 106-fold lower blood parasitism as compared to the monotherapy of Bz. Another interesting finding was the parasitological cure in 70% of the animals treated with Bz and VFV when the coadministration was given using the VFV suspension in 10% DMSO + Arabic gum + Tween 80 (a formulation that we have found to provide a better pharmacokinetics), even after immunosuppression using cyclophosphamide cycles, supporting the promising aspect of the drug coadministration in improving the efficacy of therapeutic arsenal against T. cruzi.

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection.

BACKGROUND: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. METHODS: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. RESULTS: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 µL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p = 0.02) in TNF-alpha levels than infected and untreated mice. CONCLUSIONS: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.

Resveratrol prevents p53 aggregation in vitro and in breast cancer cells.

One potential target for cancer therapeutics is the tumor suppressor p53, which is mutated in more than 50% of malignant tumors. Loss of function (LoF), dominant negative (DN) and gain of function (GoF) mutations in p53 are associated with amyloid aggregation. We tested the potential of resveratrol, a naturally occurring polyphenol, to interact and prevent the aggregation of wild-type and mutant p53 in vitro using fluorescence spectroscopy techniques and in human breast cancer cells (MDA-MB-231, HCC-70 and MCF-7) using immunofluorescence co-localization assays. Based on our data, an interaction occurs between resveratrol and the wild-type p53 core domain (p53C). In addition, resveratrol and its derivatives pterostilbene and piceatannol inhibit mutant p53C aggregation in vitro. Additionally, resveratrol reduces mutant p53 protein aggregation in MDA-MB-231 and HCC-70 cells but not in the wild-type p53 cell line MCF-7. To verify the effects of resveratrol on tumorigenicity, cell proliferation and cell migration assays were performed using MDA-MB-231 cells. Resveratrol significantly reduced the proliferative and migratory capabilities of these cells. Our study provides evidence that resveratrol directly modulates p53, enhancing our understanding of the mechanisms involved in p53 aggregation and its potential as a therapeutic strategy for cancer treatment.

Trypanosoma cruzi antigens induce inflammatory angiogenesis in a mouse subcutaneous sponge model.

Acute inflammation and angiogenesis are persistent features of several pathological conditions induced by biological agents leading to the resolution of local and systemic events. Glycoproteins derived from the protozoan Trypanosoma cruzi are suggested to mediate angiogenesis induced by inflammatory cells with still undescribed mechanisms. In this study, we investigated the effects of total antigen from trypomastigote forms of T. cruzi (Y strain), inoculated in sponges 24h after implantation in mice, on angiogenesis, inflammatory cell pattern and endogenous production of inflammatory and angiogenic mediators on days 1, 4, 7 and 14 post-implant. There was an increase in hemoglobin content and in the number of blood vessels associated with T. cruzi antigen stimuli on the 14th day, assessed by the hemoglobin of the implants and by morphometric analysis. However, these antigens were not able to increase type I collagen content on the 14th day. Parasite antigens also induced high production of vascular endothelial growth factor (VEGF) and inflammatory mediators TNF-alpha, CCL2 and CCL5 on the 7th day in sponges when compared to the unstimulated group. Neutrophils and macrophages were determined by measuring myeloperoxidase (MPO) and N-acetyl-ß-d-glucosaminidase (NAG) enzyme activities, respectively. Only NAG was increased after stimulation with antigens, starting from day 4 and peaking at day 7. Together, these data showed that antigens from the Y strain of T. cruzi are able to promote inflammatory neovascularization probably induced by macrophage-induced angiogenic mediators in T. cruzi antigen-stimulated sponges in Swiss mice.