Inhibition of Brazilian ZIKV strain replication in primary human placental chorionic cells and cervical cells treated with nitazoxanide.

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.

Cellular growth and mitochondrial ultrastructure of leishmania (Viannia) braziliensis promastigotes are affected by the iron chelator 2,2-dipyridyl.

BACKGROUND: Iron is an essential element for the survival of microorganisms in vitro and in vivo, acting as a cofactor of several enzymes and playing a critical role in host-parasite relationships. Leishmania (Viannia) braziliensis is a parasite that is widespread in the new world and considered the major etiological agent of American tegumentary leishmaniasis. Although iron depletion leads to promastigote and amastigote growth inhibition, little is known about the role of iron in the biology of Leishmania. Furthermore, there are no reports regarding the importance of iron for L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the effect of iron on the growth, ultrastructure and protein expression of L. (V.) braziliensis was analyzed by the use of the chelator 2,2-dipyridyl. Treatment with 2,2-dipyridyl affected parasites' growth in a dose- and time-dependent manner. Multiplication of the parasites was recovered after reinoculation in fresh culture medium. Ultrastructural analysis of treated promastigotes revealed marked mitochondrial swelling with loss of cristae and matrix and the presence of concentric membranar structures inside the organelle. Iron depletion also induced Golgi disruption and intense cytoplasmic vacuolization. Fluorescence-activated cell sorting analysis of tetramethylrhodamine ester-stained parasites showed that 2,2-dipyridyl collapsed the mitochondrial membrane potential. The incubation of parasites with propidium iodide demonstrated that disruption of mitochondrial membrane potential was not associated with plasma membrane permeabilization. TUNEL assays indicated no DNA fragmentation in chelator-treated promastigotes. In addition, two-dimensional electrophoresis showed that treatment with the iron chelator induced up- or down-regulation of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications, without altering their mRNA levels. CONCLUSIONS: Iron chelation leads to a multifactorial response that results in cellular collapse, starting with the interruption of cell proliferation and culminating in marked mitochondrial impairment in some parasites and their subsequent cell death, whereas others may survive and resume proliferating.

Oral administration of GW788388, an inhibitor of transforming growth factor beta signaling, prevents heart fibrosis in Chagas disease.

BACKGROUND: Chagas disease induced by Trypanosoma cruzi (T. cruzi) infection is a major cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. Transforming Growth Factor beta (TGFß) has been involved in several regulatory steps of T. cruzi invasion and in host tissue fibrosis. GW788388 is a new TGFß type I and type II receptor kinase inhibitor that can be orally administered. In the present work, we studied its effects in vivo during the acute phase of experimental Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Male Swiss mice were infected intraperitoneally with 10(4) trypomastigotes of T. cruzi (Y strain) and evaluated clinically. We found that this compound given once 3 days post infection (dpi) significantly decreased parasitemia, increased survival, improved cardiac electrical conduction as measured by PR interval in electrocardiography, and restored connexin43 expression. We could further show that cardiac fibrosis development, evaluated by collagen type I and fibronectin expression, could be inhibited by this compound. Interestingly, we further demonstrated that administration of GW788388 at the end of the acute phase (20 dpi) still significantly increased survival and decreased cardiac fibrosis (evaluated by Masson's trichrome staining and collagen type I expression), in a stage when parasite growth is no more central to this event. CONCLUSION/SIGNIFICANCE: This work confirms that inhibition of TGFß signaling pathway can be considered as a potential alternative strategy for the treatment of the symptomatic cardiomyopathy found in the acute and chronic phases of Chagas disease.

Experimental Chemotherapy for Chagas Disease: A Morphological, Biochemical, and Proteomic Overview of Potential Trypanosoma cruzi Targets of Amidines Derivatives and Naphthoquinones.

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately eight million individuals in Latin America and is emerging in nonendemic areas due to the globalisation of immigration and nonvectorial transmission routes. Although CD represents an important public health problem, resulting in high morbidity and considerable mortality rates, few investments have been allocated towards developing novel anti-T. cruzi agents. The available therapy for CD is based on two nitro derivatives (benznidazole (Bz) and nifurtimox (Nf)) developed more than four decades ago. Both are far from ideal due to substantial secondary side effects, limited efficacy against different parasite isolates, long-term therapy, and their well-known poor activity in the late chronic phase. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients. Although several classes of natural and synthetic compounds have been reported to act in vitro and in vivo on T. cruzi, since the introduction of Bz and Nf, only a few drugs, such as allopurinol and a few sterol inhibitors, have moved to clinical trials. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity. In addition, a large number of in vitro studies have been conducted using only epimastigotes and trypomastigotes instead of evaluating compounds' activities against intracellular amastigotes, which are the reproductive forms in the vertebrate host and are thus an important determinant in the selection and identification of effective compounds for further in vivo analysis. In addition, due to pharmacokinetics and absorption, distribution, metabolism, and excretion characteristics, several compounds that were promising in vitro have not been as effective as Nf or Bz in animal models of T. cruzi infection. In the last two decades, our team has collaborated with different medicinal chemistry groups to develop preclinical studies for CD and investigate the in vitro and in vivo efficacy, toxicity, selectivity, and parasite targets of different classes of natural and synthetic compounds. Some of these results will be briefly presented, focusing primarily on diamidines and related compounds and naphthoquinone derivatives that showed the most promising efficacy against T. cruzi.

Pharmacological inhibition of transforming growth factor beta signaling decreases infection and prevents heart damage in acute Chagas' disease.

Chagas' disease induced by Trypanosoma cruzi infection is an important cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. We previously reported that transforming growth factor beta (TGF-beta) is implicated in several regulatory aspects of T. cruzi invasion and growth and in host tissue fibrosis. This prompted us to evaluate the therapeutic action of an inhibitor of TGF-beta signaling (SB-431542) administered during the acute phase of experimental Chagas' disease. Male Swiss mice were infected intraperitoneally with 10(4) trypomastigotes of T. cruzi (Y strain) and evaluated clinically for the following 30 days. SB-431542 treatment significantly reduced mortality and decreased parasitemia. Electrocardiography showed that SB-431542 treatment was effective in protecting the cardiac conduction system. By 14 day postinfection, enzymatic biomarkers of tissue damage indicated that muscle injury was decreased by SB-431542 treatment, with significantly lower blood levels of aspartate aminotransferase and creatine kinase. In conclusion, inhibition of TGF-beta signaling in vivo appears to potently decrease T. cruzi infection and to prevent heart damage in a preclinical mouse model. This suggests that this class of molecules may represent a new therapeutic agent for acute and chronic Chagas' disease that warrants further clinical exploration.

Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies.

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

Electrocardiographic findings in acutely and chronically T. cruzi-infected mice treated by a phenyl-substituted analogue of furamidine DB569.

Aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. Recently we reported that a N-phenyl-substituted analogue of furamidine (DB569) exerts a micromolar trypanocidal activity against Trypanosoma cruzi in vitro. Since DB569 also reduces the cardiac parasitism and increases the survival rates of T. cruzi-infected mice, our present aim was to analyze the potential protection of DB569 in the development of altered cardiac electrical conduction system during acute and chronic T. cruzi infection. In our experimental model of acute infection (Swiss mice inoculated with Y strain of T. cruzi), the prevailing disorder observed in electrocardiogram (ECG) analyses was sinus bradycardia. This ECG alteration was reverted in acutely infected mice treated with DB569. Interestingly, the DB569 treatment reduced significantly the numbers of CD8(+) T cells in the cardiac infiltration. In addition, the noticed protection of DB569 in the ECG findings of acutely-infected animals was further extended to the chronic infection. Our data suggest that the reversion to and further maintenance of normal ECG profile in the DB569-treated infected animals may be associated with the reduced cardiac CD8(+) lymphocyte infiltration and parasitism that might be ultimately contributing to their increased survival rates.

Trypanocidal activity of the phenyl-substituted analogue of furamidine DB569 against Trypanosoma cruzi infection in vivo.

OBJECTIVES: Aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. One such compound is furamidine (DB75) and we recently reported that one of its analogues, an N-phenyl analogue (DB569), exhibits higher trypanocidal dose and time-dependent effects against different forms of Trypanosoma cruzi as compared with DB75. Our present aim was to investigate the efficacy of DB569 in a T. cruzi mouse model. METHODS: The trypanocidal activity of the compound was evaluated by clinical, parasitological, histopathological and biochemical investigations. RESULTS: Treatment with DB569 significantly reduced cardiac parasitism, partially increased the survival rates of mice and lowered the levels of alanine aminotransferase and creatinine indicating a protective role against renal and hepatic lesions caused by the parasite infection. CONCLUSIONS: Altogether, the data support the potential effect of this class of compounds against T. cruzi and motivate the screening of new diamidines for efficacy against Chagas' disease.