Evaluation of the anti-Trypanosoma cruzi activity in vitro and in vivo of silibinin and silibinin in association to benznidazole.

Chagas disease (CD) is endemic in Latin America. Drugs available for its treatment are benznidazole (BZ)/nifurtimox (NF), both with low efficacy in the late infection and responsible for several side effects. Studies of new drugs for CD among natural products, and using drug combinations with BZ/NF are recommended. Silibinin (SLB) is a natural compound that inhibits the efflux pump (Pgp) of drugs in host cell membranes, causes death of trypanosomatids, has anti-inflammatory activity, and was never assayed against T. cruzi. Here, in vitro and in vivo activities of SLB, SLB+BZ, and BZ against T. cruzi Y strain were evaluated. Cytotoxicity of SLB in VERO cells by the MTT method revealed IC50 of 250.22 µM. The trypanocidal activity evaluated by resazurin method in epimastigotes showed that SLB 25 µM inhibited parasite growth. SLB IC50 and selectivity index (SI) for amastigote were 79.81 µM and 3.13, respectively. SLB100+BZ10 showed higher parasite inhibition (91.44%) than SLB or BZ. Swiss mice infected with Y strain were treated with SLB, SLB+BZ, and BZ. Parasitemia was evaluated daily and 90, 180, and 240 days after treatment in surviving animals by hemoculture, blood qPCR, and after euthanasia, by qPCR in heart tissue. SLB monotherapy was not able to control the parasitemia/mortality of the animals. Parasitological negativation of 85.7-100% was observed in the experimental groups treated with SLB+BZ. Although SLB had shown activity against T. cruzi in vitro, it was not active in mice. Thus, the results of the therapeutic effect observed with SLB+BZ may be interpreted as a result from BZ action.

Evaluation of parasite and host genetics in two generations of a family with Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered to be a multifactorial disease associated with host and parasite genetics, which influence clinical aspects of the disease and other host conditions. In order to understand better the evolution of the disease, this study intended to evaluation of parasite and host genetics in two generations of a family with Chagas disease from the Alto Paranaiba region, Minas Gerais, Brazil, comprising a mother and her five daughters. Several features were evaluated, including the characterization of T. cruzi directly from the blood of patients, host polymorphisms of genes related to cardiomyopathy (TNF, WISP1, CCR5, and TGF-ß1) and clinical aspects of the patients. To verify the intraspecific variability of the parasite, the characterization was done directly from human blood using the PCR-LSSP technique and analyzed based on Dice coefficient and unweighted pair group analysis (UPGMA). The host polymorphism was evaluated by PCR-RFLP. The global results showed low variability of the parasites characterized from blood of patients, through Shannon index (0.492) and mean heterozygosity value per locus (0.322). All six patients presented the same genetic polymorphism profile for TNF, WISP1, and TGF-ß1, and only one patient was homozygous to CCR5, which suggests that there is no association between the clinical aspects of the patients and their genetic profiles. In conclusion, the findings confirm that the understanding of the clinical evolution of Chagas disease goes beyond the genetic aspects of the parasite and the host.

Priming astrocytes with TNF enhances their susceptibility to Trypanosoma cruzi infection and creates a self-sustaining inflammatory milieu.

BACKGROUND: In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. METHODS: Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. RESULTS: rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain. CONCLUSIONS: Our data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.

A Mini-Review of Diagnostic Methods for the Antigen and Antibody Detection of Rocky Mountain and Brazilian Spotted Fever.

Rocky Mountain or Brazilian spotted fever, caused by Rickettsia rickettsii, is a fulminant, seasonal, and neglected disease that occurs in focal points of North America and South America. Its rapid detection is essential for the better prognosis and survival rate of infected individuals. However, disease diagnosis still faces challenges as the accuracy of many of the available laboratory tests fluctuates. This review aimed to analyze methods for antibody or antigen detection, their gaps, and their evolution over time. A search was conducted to find all studies in the Pubmed database that described the antibody or antigen detection of R. rickettsii infections. Initially, a total of 403 articles were screened. Of these articles, only 17 fulfilled the pre-established inclusion criteria and were selected. Among the different methods applied, the IFA technique was the one most frequently found in the studies. However, it presented varied results such as a low specificity when using the indirect method. Other techniques, such as ELISA and immunohistochemistry, were also found, although in smaller numbers and with their own limitations. Although some studies showed promising results, there is a pressing need to find new techniques to develop a rapid and effective diagnosis of R. rickettssi infection.

Trypanosoma cruzi-induced depressive-like behavior is independent of meningoencephalitis but responsive to parasiticide and TNF-targeted therapeutic interventions.

Inflammatory cytokines and microbe-borne immunostimulators have emerged as triggers of depressive behavior. Behavioral alterations affect patients chronically infected by the parasite Trypanosoma cruzi. We have previously shown that C3H/He mice present acute phase-restricted meningoencephalitis with persistent central nervous system (CNS) parasitism, whereas C57BL/6 mice are resistant to T. cruzi-induced CNS inflammation. In the present study, we investigated whether depression is a long-term consequence of acute CNS inflammation and a contribution of the parasite strain that infects the host. C3H/He and C57BL/6 mice were infected with the Colombian (type I) and Y (type II) T. cruzi strains. Forced-swim and tail-suspension tests were used to assess depressive-like behavior. Independent of the mouse lineage, the Colombian-infected mice showed significant increases in immobility times during the acute and chronic phases of infection. Therefore, T. cruzi-induced depression is independent of active or prior CNS inflammation. Furthermore, chronic depressive-like behavior was triggered only by the type I Colombian T. cruzi strain. Acute and chronic T. cruzi infection increased indoleamine 2,3-dioxygenase (IDO) expression in the CNS. Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine abrogated the T. cruzi-induced depressive-like behavior. Moreover, treatment with the parasiticide drug benznidazole abrogated depression. Chronic T. cruzi infection of C57BL/6 mice increased tumor necrosis factor (TNF) expression systemically but not in the CNS. Importantly, TNF modulators (anti-TNF and pentoxifylline) reduced immobility. Therefore, direct or indirect parasite-induced immune dysregulation may contribute to chronic depressive disorder in T. cruzi infection, which opens a new therapeutic pathway to be explored.

Short-term therapy with simvastatin reduces inflammatory mediators and heart inflammation during the acute phase of experimental Chagas disease.

Trypanosoma cruzi infection induces progressive cardiac inflammation that leads to fibrosis and modifications in the heart architecture and functionality. Statins, such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, have been studied due to their pleiotropic roles in modulating the inflammatory response. Our goal was to evaluate the effects of simvastatin on the cardiac inflammatory process using a cardiotropic strain of T. cruzi in a murine model of Chagas cardiomyopathy. C57BL/6 mice were infected with 500 trypomastigotes of the Colombian strain of T. cruzi and treated with an oral dose of simvastatin (20 mg/Kg/day) for one month and inflammatory and morphometric parameters were subsequently evaluated in the serum and in the heart, respectively. Simvastatin reduced the total cholesterol and inflammatory mediators (interferon-gamma, tumour necrosis factor-alpha, CCL2 and CCL5) in the serum and in the heart tissue at 30 days post-infection. Additionally, a proportional reduction in heart weight and inflammatory infiltration was observed. Simvastatin also reduced epimastigote proliferation in a dose-dependent manner in vitro and was able to reduce blood trypomastigotes and heart amastigote nests during the acute phase of Chagas disease in vivo. Based on these data, we conclude that simvastatin exerts a modulatory effect on the inflammatory mediators that are elicited by the Colombian strain of T. cruzi and ameliorates the heart damage that is observed in a murine model of Chagas disease.

[Spatial and temporal patterns of mortality among motorcyclists in a State of the Brazilian Northeast in the 21st century]. / Padrões espaciais e temporais da mortalidade de motociclistas em estado do nordeste brasileiro no século XXI.

The scope of this work was to analyze the trend and distribution of mortality among motorcyclists in traffic accidents in the State of Alagoas. It involved an ecological study relating to all deaths resulting from motorcycle accidents in the state in the period from 2001 to 2015. Mortality data were obtained from the Mortality Information System (MIS). Mortality rates were calculated and stratified by gender. The joinpoint regression model was used for trend analysis and the Annual Percentage Variation (APV) was calculated with a significance rate of 5%. For the spatial analysis, local empirical Bayesian modeling and Moran statistics and spatial scanning statistics were applied. There were 1,458 deaths of motorcyclists in the period studied, 91.3% of which were men. Three temporal behaviors were observed in this population group: growth (2001-2005), stationary pattern (2005-2013) and decline from 2013 onwards. The highest rates were observed in the 'agreste' and 'sertão' regions of the state of Alagoas. Five spatial clusters were revealed with relation to general and male mortality, all located in the 'agreste' and 'sertão' hinterlands of Alagoas. The modeling showed a reduction of mortality from 2013 onwards and the spatial analysis revealed that the problem is more acute in the interior of the state.

Este trabalho objetivou analisar a tendência e a distribuição espacial da mortalidade de motociclistas em acidentes de transporte no estado de Alagoas. Trata-se de um estudo ecológico referente a todos os óbitos decorrentes de acidentes motociclísticos no estado no período 2001-2015. Os dados de mortalidade foram obtidos do Sistema de Informações sobre Mortalidade (SIM). As taxas de mortalidade foram calculadas e estratificadas por sexo. Para a análise de tendência, foi empregado o modelo de regressão por pontos de inflexão. Calculou-se a Variação Percentual Anual (VPA). Significância de 5%. Para a análise espacial, aplicou-se modelagem bayesiana empírica local, estatística de Moran e estatística de varredura espacial. Foram registrados 1.458 óbitos de motociclistas no período estudado, sendo 91,3% homens. Três comportamentos temporais foram observados nessa população: crescimento (2001-2005), padrão estacionário (2005-2013) e declínio a partir de 2013. As maiores taxas foram observadas no agreste e sertão. Cinco aglomerados espaciais foram evidenciados no que se refere à mortalidade geral e masculina, todos situados no agreste e sertão alagoanos. A modelagem mostrou redução da mortalidade a partir de 2013 e a análise espacial evidenciou que o problema é mais grave no interior do estado.

Padrões espaciais e temporais da mortalidade de motociclistas em estado do nordeste brasileiro no século XXI / Spatial and temporal patterns of mortality among motorcyclists in a State of the Brazilian Northeast in the 21st century

Resumo Este trabalho objetivou analisar a tendência e a distribuição espacial da mortalidade de motociclistas em acidentes de transporte no estado de Alagoas. Trata-se de um estudo ecológico referente a todos os óbitos decorrentes de acidentes motociclísticos no estado no período 2001-2015. Os dados de mortalidade foram obtidos do Sistema de Informações sobre Mortalidade (SIM). As taxas de mortalidade foram calculadas e estratificadas por sexo. Para a análise de tendência, foi empregado o modelo de regressão por pontos de inflexão. Calculou-se a Variação Percentual Anual (VPA). Significância de 5%. Para a análise espacial, aplicou-se modelagem bayesiana empírica local, estatística de Moran e estatística de varredura espacial. Foram registrados 1.458 óbitos de motociclistas no período estudado, sendo 91,3% homens. Três comportamentos temporais foram observados nessa população: crescimento (2001-2005), padrão estacionário (2005-2013) e declínio a partir de 2013. As maiores taxas foram observadas no agreste e sertão. Cinco aglomerados espaciais foram evidenciados no que se refere à mortalidade geral e masculina, todos situados no agreste e sertão alagoanos. A modelagem mostrou redução da mortalidade a partir de 2013 e a análise espacial evidenciou que o problema é mais grave no interior do estado.

Abstract The scope of this work was to analyze the trend and distribution of mortality among motorcyclists in traffic accidents in the State of Alagoas. It involved an ecological study relating to all deaths resulting from motorcycle accidents in the state in the period from 2001 to 2015. Mortality data were obtained from the Mortality Information System (MIS). Mortality rates were calculated and stratified by gender. The joinpoint regression model was used for trend analysis and the Annual Percentage Variation (APV) was calculated with a significance rate of 5%. For the spatial analysis, local empirical Bayesian modeling and Moran statistics and spatial scanning statistics were applied. There were 1,458 deaths of motorcyclists in the period studied, 91.3% of which were men. Three temporal behaviors were observed in this population group: growth (2001-2005), stationary pattern (2005-2013) and decline from 2013 onwards. The highest rates were observed in the 'agreste' and 'sertão' regions of the state of Alagoas. Five spatial clusters were revealed with relation to general and male mortality, all located in the 'agreste' and 'sertão' hinterlands of Alagoas. The modeling showed a reduction of mortality from 2013 onwards and the spatial analysis revealed that the problem is more acute in the interior of the state.

Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.

Apoio ao processo de avaliação do serviço de abastecimento de água no Rio Grande do Norte: uma abordagem multicritério / Support to the assessment process of the water supply service in Rio Grande do Norte State: a multicriteria approach

RESUMO O abastecimento de água e o saneamento são dois serviços essenciais para a sociedade, ainda mais no Brasil, onde há, historicamente, um déficit desses serviços básicos. Além disso, também se sabe que tais serviços envolvem muitos atores, que muitas vezes possuem perspectivas conflitantes, características de problemas multicriteriais. Diante disso, o presente estudo teve como objetivo avaliar o serviço de abastecimento de água de oito municípios do Estado do Rio Grande do Norte (RN). Para tanto, foram selecionados 11 critérios, levando-se em consideração aspectos operacionais, financeiros e de qualidade da água, e foi adotado o método de apoio multicritério à decisão TOPSIS. Também foi utilizado o procedimento da entropia para a obtenção dos pesos de cada critério. Os resultados apontaram o município de Lagoa Nova como a alternativa com melhor serviço de abastecimento de água entre as opções estudadas. Em contrapartida, Santana do Matos foi o município com a pior avaliação do conjunto de alternativas estudadas.

ABSTRACT Water supply and sewage are two essential services to society, especially in Brazil, where historically there has been a deficit of these crucial services. Moreover, it is also known that such services involve many actors who often have conflicting perspectives, characteristics of multicriteria problems. Thus, this paper aimed to evaluate the water supply service in eight cities of Rio Grande do Norte State, Brazil. Eleven criteria were selected, considering operational, financial and water quality aspects. We adopted the TOPSIS method to support the multicriteria decision. In addition, the entropy method was used to obtain the weights for each criterion. The results showed the city of Lagoa Nova as the alternative with the best water supply service of the alternatives studied. In contrast, Santana do Matos was the city with the worst water supply assessment of all the studied alternatives.

Absence of detection of Xanthomonas axonopodis Pv. Malvacearum in cotton seeds used in the northeast region of Mato Grosso do Sul in a three-based crop season study / Ausência de detecção de Xanthomonas axonopodis Pv. Malvacearum em sementes de algodoeiro usadas na região nordeste de Mato Grosso Do Sul, em um estudo de três safras agrícolas

The aim of this work was to evaluate the presence of the causal agent of angular leaf spot, Xanthomonas axonopodis pv. malvacearum, in 38 cotton seeds samples with or without linter used in the northeast region of Mato Grosso do Sul, Brazil, during the crop seasons of 2008/09, 2009/10 e 2010/11. It was used the semi selective culture media MSSXAN (beef extract - 3 g, peptone - 5 g, soluble starch - 10 g, sucrose - 5g; Tween 80 - 10 mL; CaCl2 - 0.25 g; crystal violet solution at 1% - 150 mL; cephalexin - 50 mg; chlorothalonil - 10 mg; methyl thyophanate - 10 mg; agar ­ 15 g; sterile distilled water - 1000 ml) for the isolation of the bacteria and the suspect colonies of being X. axonopodis pv. malvacearum were purified and submitted to the Gram stain, potassium hydroxide and pathogenicity tests in the leaves of cotton plants of the susceptible genotype NC 53345. The presence of X. a. pv. malvacearum was not detected in the evaluated seed samples.

O objetivo deste trabalho foi avaliar a presença do agente causal da mancha-angular, Xanthomonas axonopodis pv. malvacearum, em 38 amostras de sementes de algodoeiro com ou sem línter utilizadas na região nordeste de Mato Grosso do Sul nas safras 2008/09, 2009/10 e 2010/11. Foi empregado o meio de cultura semi-seletivo MSSXAN (extrato de carne - 3 g; peptona - 5 g; amido solúvel - 10 g; sacarose - 5g; Tween 80 - 10 mL; CaCl2 - 0,25 g; solução de cristal violeta a 1 % - 150 µL; cefalexina - 50 mg; clorothalonil - 10 mg; tiofanato metílico - 10 mg; ágar - 15 g; água destilada esterilizada - 1.000 mL) para o isolamento da bactéria nas sementes dos diferentes lotes e colônias suspeitas de serem X. axonopodis pv. malvacearum foram purificadas e posteriormente submetidas à coloração diferencial de Gram, teste de KOH e patogenicidade em folhas de plântulas suscetíveis de algodoeiro, genótipo NC 53345. Não foi constatada a presença de X. a. pv. malvacearum nas amostras de sementes analisadas.

Short-term therapy with simvastatin reduces inflammatory mediators and heart inflammation during the acute phase of experimental Chagas disease

Trypanosoma cruzi infection induces progressive cardiac inflammation that leads to fibrosis and modifications in the heart architecture and functionality. Statins, such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, have been studied due to their pleiotropic roles in modulating the inflammatory response. Our goal was to evaluate the effects of simvastatin on the cardiac inflammatory process using a cardiotropic strain of T. cruzi in a murine model of Chagas cardiomyopathy. C57BL/6 mice were infected with 500 trypomastigotes of the Colombian strain of T. cruzi and treated with an oral dose of simvastatin (20 mg/Kg/day) for one month and inflammatory and morphometric parameters were subsequently evaluated in the serum and in the heart, respectively. Simvastatin reduced the total cholesterol and inflammatory mediators (interferon-gamma, tumour necrosis factor-alpha, CCL2 and CCL5) in the serum and in the heart tissue at 30 days post-infection. Additionally, a proportional reduction in heart weight and inflammatory infiltration was observed. Simvastatin also reduced epimastigote proliferation in a dose-dependent manner in vitro and was able to reduce blood trypomastigotes and heart amastigote nests during the acute phase of Chagas disease in vivo. Based on these data, we conclude that simvastatin exerts a modulatory effect on the inflammatory mediators that are elicited by the Colombian strain of T. cruzi and ameliorates the heart damage that is observed in a murine model of Chagas disease.