RESUMO
Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach's myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development.
Assuntos
Doença de Chagas , Modelos Animais de Doenças , Células Intersticiais de Cajal , Megacolo , Animais , Células Intersticiais de Cajal/patologia , Doença de Chagas/patologia , Doença de Chagas/fisiopatologia , Megacolo/parasitologia , Megacolo/patologia , Megacolo/fisiopatologia , Camundongos , Citometria de Fluxo , Masculino , Trypanosoma cruzi/fisiologiaRESUMO
Benznidazole (BZ) tablets are the currently prescribed treatment for Chagas disease. However, BZ presents limited efficacy and a prolonged treatment regimen with dose-dependent side effects. The design and development of new BZ subcutaneous (SC) implants based on the biodegradable poly-É-caprolactone (PCL) is proposed in this study for a controlled release of BZ and to improve patient compliance. The BZ-PCL implants were characterized by X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy, which indicated that BZ remains in its crystalline state dispersed in the polymer matrix with no polymorphic transitions. BZ-PCL implants, even at the highest doses, induce no alteration of the levels of hepatic enzymes in treated animals. BZ release from implants to blood was monitored in plasma during and after treatment in healthy and infected animals. Implants at equivalent oral doses increase the body's exposure to BZ in the first days compared with oral therapy, exhibiting a safe profile and allowing sustained BZ concentrations in plasma to induce a cure of all mice in the experimental model of acute infection by the Y strain of T. cruzi. BZ-PCL implants have the same efficacy as 40 daily oral doses of BZ. Biodegradable BZ implants are a promising option to reduce failures related to poor adherence to treatment, with more comfort for patients, and with sustained BZ plasma concentration in the blood. These results are relevant for optimizing human Chagas disease treatment regimens.
RESUMO
Introduction: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi. Although endemic mainly in Latin America, CD has become a global public health problem due to migration of infected individuals to non-endemic regions. Despite progress made in drug development, preclinical assays for drug discovery are required to accelerate the development of new drugs with reduced side effects, which are much needed for human treatment. Methods: We used a cure model of infected mice treated with Fexinidazole (FZ) to further validate a novel Enzyme Linked Aptamer (ELA) assay that detects parasite biomarkers circulating in the blood of infected animals. Results: The ELA assay showed cure by FZ in ~71% and ~77% of mice infected with the VL-10 and Colombiana strains of T. cruzi, respectively. The ELA assay also revealed superior treatment efficacy of FZ compared to Benznidazole prior to immunosuppression treatment. Discussion: Our study supports the use of ELA assay as an alternative to traditional serology or blood PCR to assess the efficacy of antichagasic drugs during their preclinical phase of development. Further, the combination of high sensitivity and ease of use make this parasite antigen detection assay an attractive new tool to facilitate the development of much needed new therapies for CD.
Assuntos
Doença de Chagas , Nitroimidazóis , Trypanosoma cruzi , Humanos , Animais , Camundongos , Doença de Chagas/diagnóstico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Resultado do Tratamento , Testes Imunológicos , Oligonucleotídeos/farmacologiaRESUMO
BACKGROUND: The emergence of Trypanosoma cruzi infection via oral transmission has a habitual character in its primitive endemic cycle. Recent findings revealed the first death by oral transmission of T. cruzi in the state of Espírito Santo, Brazil, in 2012, which was recorded in the rural area of Guarapari. This study evaluated the characteristics related to the occurrence of natural T. cruzi infection among dogs from the rural areas of Alegre and Iconha, municipalities of Espírito Santo. METHODS: Logistic regression analysis of factors contributing to serological detection of T. cruzi in dogs was performed in environments where Espírito Santo's Department of Health Surveillance had previously notified triatomines positive for Trypanosoma spp. from 2014 to 2017. RESULTS: A total of 36 dogs were analyzed, of which 10 (27.77%) tested positive, one was borderline (2.79%), and 25 tested negative (69.44%) for T. cruzi infection. São Caetano, a district from the Iconha municipality, presented a 25 times greater chance for the detection of positive tests (OR:25; 95% CI; 2.37->100). Dogs with updated mandatory vaccination presented with a lower risk of positive serodiagnosis (OR:0.12; 95% CI: 0.02-0.63). CONCLUSIONS: Our results highlight for the first time the occurrence of natural T. cruzi canine infection, detected in the municipality of Iconha, mainly among dogs with un-updated mandatory vaccines in the district of São Caetano.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Cães , Brasil/epidemiologia , Insetos Vetores , Doença de Chagas/diagnóstico , Doença de Chagas/epidemiologia , Doença de Chagas/veterináriaRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, is an important public health problem in Latin America. Nanoencapsulation of anti-T. cruzi drugs has significantly improved their efficacy and reduced cardiotoxicity. Thus, we investigated the in vitro interaction of polyethylene glycol-block-poly(D,L-lactide) nanocapsules (PEG-PLA) with trypomastigotes and with intracellular amastigotes of the Y strain in cardiomyoblasts, which are the infective forms of T. cruzi, using fluorescence and confocal microscopy. Fluorescently labeled nanocapsules (NCs) were internalized by non-infected H9c2 cells toward the perinuclear region. The NCs did not induce significant cytotoxicity in the H9c2 cells, even at the highest concentrations and interacted equally with infected and non-infected cells. In infected cardiomyocytes, NCs were distributed in the cytoplasm and located near intracellular amastigote forms. PEG-PLA NCs and trypomastigote form interactions also occurred. Altogether, this study contributes to the development of engineered polymeric nanocarriers as a platform to encapsulate drugs and to improve their uptake by different intra- and extracellular forms of T. cruzi, paving the way to find new therapeutic strategies to fight the causative agent of Chagas disease.
Assuntos
Doença de Chagas , Nanocápsulas , Trypanosoma cruzi , Doença de Chagas/tratamento farmacológico , Humanos , Poliésteres , PolietilenoglicóisRESUMO
ABSTRACT Background: The emergence of Trypanosoma cruzi infection via oral transmission has a habitual character in its primitive endemic cycle. Recent findings revealed the first death by oral transmission of T. cruzi in the state of Espírito Santo, Brazil, in 2012, which was recorded in the rural area of Guarapari. This study evaluated the characteristics related to the occurrence of natural T. cruzi infection among dogs from the rural areas of Alegre and Iconha, municipalities of Espírito Santo. Methods: Logistic regression analysis of factors contributing to serological detection of T. cruzi in dogs was performed in environments where Espírito Santo's Department of Health Surveillance had previously notified triatomines positive for Trypanosoma spp. from 2014 to 2017. Results: A total of 36 dogs were analyzed, of which 10 (27.77%) tested positive, one was borderline (2.79%), and 25 tested negative (69.44%) for T. cruzi infection. São Caetano, a district from the Iconha municipality, presented a 25 times greater chance for the detection of positive tests (OR:25; 95% CI; 2.37->100). Dogs with updated mandatory vaccination presented with a lower risk of positive serodiagnosis (OR:0.12; 95% CI: 0.02-0.63). Conclusions: Our results highlight for the first time the occurrence of natural T. cruzi canine infection, detected in the municipality of Iconha, mainly among dogs with un-updated mandatory vaccines in the district of São Caetano.
RESUMO
We investigated the in vitro activity and selectivity, and in vivo efficacy of ravuconazole (RAV) in self-nanoemulsifying delivery system (SNEDDS) against Trypanosoma cruzi. Novel formulations of this poorly soluble C14-α-demethylase inhibitor may improve its efficacy in the experimental treatment. In vitro activity was determined in infected cardiomyocytes and efficacy in vivo evaluated in terms of parasitological cure induced in Y and Colombian strains of T. cruzi-infected mice. In vitro RAV-SNEDDS exhibited significantly higher potency of 1.9-fold at the IC50 level and 2-fold at IC90 level than free-RAV. No difference in activity with Colombian strain was observed in vitro. Oral treatment with a daily dose of 20 mg/kg for 30 days resulted in 70% of cure for RAV-SNEDDS versus 40% for free-RAV and 50% for 100 mg/kg benznidazole in acute infection (T. cruzi Y strain). Long-term treatment efficacy (40 days) was able to cure 100% of Y strain-infected animals with both RAV preparations. Longer treatment time was also efficient to increase the cure rate with benznidazole (Y and Colombian strains). RAV-SNEDDS shows greater efficacy in a shorter time treatment regimen, it is safe and could be a promising formulation to be evaluated in other pre-clinical models to treat T. cruzi and fungi infections.
Assuntos
Doença de Chagas/tratamento farmacológico , Tiazóis/administração & dosagem , Triazóis/administração & dosagem , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/parasitologia , Emulsões , Feminino , Células Hep G2 , Humanos , Concentração Inibidora 50 , Camundongos , Miócitos Cardíacos , Nanoestruturas , Ratos , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Tiazóis/toxicidade , Triazóis/farmacologia , Triazóis/uso terapêutico , Triazóis/toxicidadeRESUMO
Drug combinations have been evaluated for Chagas disease in an attempt to improve efficacy and safety. In this line, the objective of this work is to assess the effects of treatment with nitro drugs combinations using benznidazole (BZ) or nifurtimox (NFX) plus the sulfone metabolite of fexinidazole (fex-SFN) in vitro and in vivo on Trypanosoma cruzi infection. The in vitro interaction of fex-SFN and BZ or NFX against infected H9c2 cells by the Y strain was classified as an additive (0.5⩾ΣFIC<4), suggesting the possibility of a dose reduction in the in vivo T. cruzi infection. Next, the effect of combining suboptimal doses was assessed in an acute model of murine T. cruzi infection. Drug combinations led to a faster suppression of parasitemia than monotherapies. Also, the associations led to higher cure levels than those in the reference treatment BZ 100 mg day−1 (57.1%) (i.e. 83.3% with BZ/fex-SFN and 75% with NFX/fex-SFN). Importantly, toxic effects resulting from the associations were not observed, according to weight gain and hepatic enzyme levels in the serum of experimental animals. Taken together, this study is a starting point to explore the potential effects of nitro drugs combinations in preclinical models of kinetoplastid-related infections.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitrocompostos/uso terapêutico , Animais , Quimioterapia Combinada , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Doenças Negligenciadas/tratamento farmacológico , Nifurtimox/efeitos adversos , Nifurtimox/uso terapêutico , Nitrocompostos/efeitos adversos , Nitroimidazóis/efeitos adversos , Nitroimidazóis/metabolismo , Nitroimidazóis/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Sulfonas/efeitos adversos , Sulfonas/uso terapêuticoRESUMO
Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi. Currently, only nitroheterocyclic nifurtimox (NFX) and benznidazole (BNZ) are available for the treatment of Chagas disease, with limitations such as variable efficacy, long treatment regimens and toxicity. Different strategies have been used to discover new active molecules for the treatment of Chagas disease. Target-based and phenotypic screening led to thousands of compounds with anti-T. cruzi activity, notably the nitroheterocyclic compounds, fexinidazole and its metabolites. In addition, drug repurposing, drug combinations, re-dosing regimens and the development of new formulations have been evaluated. The CYP51 antifungal azoles, as posaconazole, ravuconazole and its prodrug fosravuconazole presented promising results in experimental Chagas disease. Drug combinations of nitroheterocyclic and azoles were able to induce cure in murine infection. New treatment schemes using BNZ showed efficacy in the experimental chronic stage, including against dormant forms of T. cruzi. And finally, sesquiterpene lactone formulated in nanocarriers displayed outstanding efficacy against different strains of T. cruzi, susceptible or resistant to BNZ, the reference drug. These pre-clinical results are encouraging and provide interesting evidence to improve the treatment of patients with Chagas disease.
RESUMO
Chagas disease, caused by Trypanosoma cruzi, is a major neglected tropical disease that occurs mainly as chronic infection and systemic infection. Currently, there is no suitable and effective drug to treat this parasitic disease. Administration of nutrients with immunomodulatory properties, such as arginine and nitric oxide radicals, may be helpful as antiparasitic therapy. In this study, we evaluated the effects of arginine supplementation during the acute phase of infection under the development of chronic Chagas' heart disease in Swiss mice inoculated with the Berenice-78 strain of T. cruzi. The effectiveness of arginine was determined by daily detection of the parasite in the blood and long-term serum levels of nitric oxide and tumor necrosis factor-alpha, in addition to evaluation of heart tissue damage. Arginine could flatten parasitemia and prevent elevation of tumor necrosis factor-alpha in T. cruzi-infected mice. Regarding chronic inflammatory myocardial derangements, similar findings were verified among T. cruzi-infected groups. Arginine promoted collagenogenesis in the heart muscle tissue of T. cruzi-infected arginine-supplemented group. These data show the paradoxical benefits of arginine in improving the outcome of Chagas chronic cardiomyopathy.
Assuntos
Arginina/metabolismo , Cardiomiopatia Chagásica/patologia , Colágeno/fisiologia , Coração/parasitologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Ração Animal/análise , Animais , Arginina/administração & dosagem , Arginina/farmacologia , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/parasitologia , Dieta , Suplementos Nutricionais/análise , Coração/efeitos dos fármacos , Camundongos , Tripanossomicidas/administração & dosagem , Tripanossomicidas/metabolismoRESUMO
There is a growing consensus that the balance between the persistence of infection and the host immune response is crucial for chronification of Chagas heart disease. Extrapolation for chagasic megacolon is hampered because research in humans and animal models that reproduce intestinal pathology is lacking. The parasite-host relationship and its consequence to the disease are not well-known. Our model describes the temporal changes in the mice intestine wall throughout the infection, parasitism, and the development of megacolon. It also presents the consequence of the infection of primary myenteric neurons in culture with Trypanosoma cruzi (T. cruzi). Oxidative neuronal damage, involving reactive nitrogen species induced by parasite infection and cytokine production, results in the denervation of the myenteric ganglia in the acute phase. The long-term inflammation induced by the parasite's DNA causes intramuscular axonal damage, smooth muscle hypertrophy, and inconsistent innervation, affecting contractility. Acute phase neuronal loss may be irreversible. However, the dynamics of the damages revealed herein indicate that neuroprotection interventions in acute and chronic phases may help to eradicate the parasite and control the inflammatory-induced increase of the intestinal wall thickness and axonal loss. Our model is a powerful approach to integrate the acute and chronic events triggered by T. cruzi, leading to megacolon.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Intestinos , Plexo Mientérico , NeurôniosRESUMO
Mining existing agents that enhance the therapeutic potential of ergosterol biosynthesis inhibitors (EBI) is a promising approach to improve Chagas disease chemotherapy. In this study, we evaluated the effect of ravuconazole, an EBI, combined with amlodipine, a calcium channel blocker, upon Trypanosoma cruzi experimental infection. In vitro assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibitory concentration [ΣFIC] > 0.5) of the combined treatment without additional toxicity to host cells. In vivo experiments, using a murine model of the T. cruzi Y strain in a short-term protocol, demonstrated that amlodipine, although lacking trypanocidal activity, dramatically increased the antiparasitic activity of underdosing ravuconazole regimens. Additional analysis using long-term treatment (20 days) showed that parasitemia relapse until 60 days after treatment was significatively lower in mice treated with the combination (4 out of 14 mice) than ravuconazole monotherapy (10 out of 14 mice), even in the presence of immunosuppressant pressure. Furthermore, the combined therapy was well tolerated and protected the mice from mortality. The treatments also impacted on the cellular and humoral immune response of infected animals, inducing a reduction of serum cytokine levels in all ravuconazole-treated mice. Our findings demonstrate that amlodipine is efficacious in enhancing the antiparasitic activity of ravuconazole in an experimental model of T. cruzi infection and indicates a potential strategy to be explored in Chagas disease treatment.
Assuntos
Doença de Chagas , Nitroimidazóis , Tripanossomicidas , Trypanosoma cruzi , Anlodipino/farmacologia , Anlodipino/uso terapêutico , Animais , Doença de Chagas/tratamento farmacológico , Camundongos , Nitroimidazóis/uso terapêutico , Parasitemia/tratamento farmacológico , Tiazóis , Triazóis , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêuticoRESUMO
The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.
Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologiaRESUMO
Combination therapy has been proposed as an alternative therapeutic approach for the treatment of Chagas disease. In this study, we evaluated the effect of treatment with benznidazole combined with E1224 (ravuconazole prodrug) in an experimental murine model of acute infection. The first set of experiments assessed the range of E1224 doses required to induce parasitological cure using Trypanosoma cruzi strains with different susceptibilities to benznidazole (Y and Colombian). All E1224 doses were effective in suppressing the parasitemia and preventing death; however, parasitological cure was observed only in mice infected with Y strain. Considering these results, we evaluated the effect of combined treatment against Colombian, a multidrug-resistant T. cruzi strain. After exclusion of antagonistic effects using in vitro assays, infected mice were treated with E1224 and benznidazole in monotherapy or in combination at day 4 or 10 postinoculation. All treatments were well tolerated and effective in suppressing parasitemia; however, parasitological and PCR assays indicated no cure among mice treated with monotherapies. Intriguingly, the outcome of combination therapy was dependent on treatment onset. Early treatment using optimal doses of E1224-benznidazole induced a 100% cure rate, but this association could not eliminate a well-established infection. The beneficial effect of combination therapy was evidenced by further reductions of the patent parasitemia period in the group receiving combined therapy compared with monotherapies. Our results demonstrated a positive interaction between E1224 and benznidazole against murine T. cruzi infection using a multidrug-resistant strain and highlighted the importance of a stringent experimental model in the evaluation of new therapies.
Assuntos
Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Terapia Combinada , Modelos Animais de Doenças , Interações Medicamentosas , Feminino , Camundongos , Testes de Sensibilidade Microbiana , Tiazóis/farmacologia , Triazóis/farmacologiaRESUMO
Self-emulsifying drug delivery systems (SEDDSs) are lipid-based anhydrous formulations composed of an isotropic mixture of oil, surfactant, and cosurfactants usually presented in gelatin capsules. Ravuconazole (Biopharmaceutics Classification System [BCS] Class II) is a poorly water-soluble drug, and a SEDDS type IIIA was designed to deliver it in a predissolved state, improving dissolution in gastrointestinal fluids. After emulsification, the droplets had mean hydrodynamic diameters <250 nm, zeta potential values in the range of -45 mV to -57 mV, and showed no signs of ravuconazole precipitation. Asymmetric flow field-flow fractionation with dynamic and multiangle laser light scattering was used to characterize these formulations in terms of size distribution and homogeneity. The fractograms obtained at 37°C showed a polydisperse profile for all blank and ravuconazole-SEDDS formulations but no large aggregates. SEDDS increased ravuconazole in vitro dissolution extent and rate (20%) compared to free drug (3%) in 6 h. The in vivo toxicity of blank SEDDS comprising Labrasol® surfactant in different concentrations and preliminary safety tests in repeated-dose oral administration (20 days) showed a dose-dependent Labrasol toxicity in healthy mice. Ravuconazole-SEDDS at low surfactant content (10%, v/v) in Trypanosoma cruzi-infected mice was safe during the 20-day treatment. The anti-T. cruzi activity of free ravuconazole, ravuconazole-SEDDS and each excipient were evaluated in vitro at equivalent ravuconazole concentrations needed to inhibit 50% or 90% (IC50 and IC90), respectively of the intracellular amastigote form of the parasite in a cardiomyocyte cell line. The results showed a clear improvement of the ravuconazole anti-T. cruzi activity when associated with SEDDS. Based on our results, the repurposing of ravuconazole in SEDDS dosage form is a strategy that deserves further in vivo investigation in preclinical studies for the treatment of human T. cruzi infections.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Emulsões/administração & dosagem , Tiazóis/administração & dosagem , Tiazóis/toxicidade , Triazóis/administração & dosagem , Triazóis/toxicidade , Trypanosoma cruzi/efeitos dos fármacos , Administração Oral , Animais , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Emulsões/química , Emulsões/farmacologia , Excipientes/química , Feminino , Glicerídeos/química , Lipídeos/química , Masculino , Camundongos , Solubilidade , Tensoativos/química , Tiazóis/química , Triazóis/química , Trypanosoma cruzi/patogenicidadeRESUMO
We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.
Assuntos
Doença de Chagas/patologia , Modelos Animais de Doenças , Sistema Nervoso Entérico/patologia , Megacolo/patologia , Neurônios/patologia , Animais , Doença de Chagas/complicações , Denervação , Feminino , Megacolo/complicações , Camundongos , Músculo Liso/inervaçãoRESUMO
Trypanosoma cruzi is the etiological agent of American trypanosomiasis (Chagas' disease), which affects 6-7 million people worldwide, mainly in Latin America. It presents great genetic and biological variability that plays an important role in the clinical and epidemiological features of the disease. Our working hypothesis is that the genetic diversity of T. cruzi has an important impact on detection of the parasite using diagnostic techniques. The present study evaluated the diagnostic performance of parasitological, molecular, and serological techniques for detecting 27 strains of T. cruzi that belonged to discrete typing units (DTUs) TcI (11 strains), TcII (four strains), and TcIV (12 strains) that were obtained from different hosts in the states of Amazonas and Paraná, Brazil. Blood samples were taken from experimentally infected mice and analyzed by fresh blood examination, hemoculture in Liver Infusion Tryptose (LIT) medium, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction presented the best detection of TcI, with 80.4% positivity. For all of the detection methods, the animals that were inoculated with TcII presented the highest positivity rates (94.1-100%). ELISA that was performed 7 months after inoculation presented a higher detection ability (95.4%) for TcIV. Intra-DTU comparisons showed that the reproducibility of the majority of the results that were obtained with the different methods was weak for TcI and good for TcII and TcIV. Our data indicate that the detection capability of different techniques varies with the DTUs of the parasites in mammalian blood. The implications of these findings with regard to the diagnosis of human T. cruzi infection are discussed.
Assuntos
Doença de Chagas/parasitologia , Parasitemia/parasitologia , Trypanosoma cruzi/isolamento & purificação , Animais , Brasil , Doença de Chagas/diagnóstico , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Camundongos , Parasitemia/diagnóstico , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
Chagas disease, which is caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.
Assuntos
Regulação para Baixo , MicroRNAs/fisiologia , Miócitos Cardíacos/parasitologia , PTEN Fosfo-Hidrolase/metabolismo , Biossíntese de Proteínas , Trypanosoma cruzi/metabolismo , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular , Formazans , Genes Reporter , Miócitos Cardíacos/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , Trypanosoma cruzi/classificaçãoRESUMO
Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.
Assuntos
Animais , Ratos , Regulação para Baixo , MicroRNAs/fisiologia , Miócitos Cardíacos/parasitologia , Biossíntese de Proteínas , PTEN Fosfo-Hidrolase/metabolismo , Trypanosoma cruzi/metabolismo , Western Blotting , Linhagem Celular , Sobrevivência Celular , Formazans , Genes Reporter , Miócitos Cardíacos/metabolismo , Fosforilação , PTEN Fosfo-Hidrolase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/metabolismo , Sais de Tetrazólio , Trypanosoma cruzi/classificaçãoRESUMO
The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.