Triagem biológica de inibidores de sirtuína 2 (TcSir2rp1) candidatos a antichagásicos / Biological screening of sirtuin 2 inhibitors (TcSir2rp1) antichagasic candidates

A doença de Chagas é causada pelo Trypanosoma cruzi, e atualmente, acomete entre 6 a 7 milhões de pessoas em todo o mundo. A quimioterapia disponível para seu o tratamento se baseia apenas em dois fármacos, nifurtimox e benznidazol, com mais de 50 anos de descoberto. Estes fármacos apresentam eficácia limitada, pois são pouco efetivos na fase crônica e apresentam alta toxicidade, resultando em efeitos adversos graves. Esse panorama mostra a necessidade de novas abordagens terapêuticas contra essa doença. Nesse sentido, a inibição de vias bioquímicas essencias para o parasita se mostram como uma boa sugestão para identificação de compostos promissores candidatos a novos agentes quimioterápicos. A sirtuína 2 (Sir2) são enzimas reguladoras que participam de mecanismos epigenéticos em tripanossomatídeos, e no T. cruzi possuem um papel fundamental em todos os seus estágios evolutivos, devido a este fato, se apresentam como um alvo promissor na busca por novos fármacos contra a doença de Chagas. Neste sentido propomos a busca de inibidores da Sir2 proteína 1 do T. cruzi (TcSir2rp1) que é geneticamente validada como alvo farmacológico, por meio da estratégia de triagem biológica. Realizou-se a expressão da enzima recombinante por biologia molecular em um sistema de transformação utilizando cepa de Escherichia coli Artic Express (DE3). Foi feita a purificação e a confirmação da obtenção da proteína recombinante se deu por gel SDS-PAGE. Após a obtenção da enzima os parâmetros cinéticos foram determinados por experimentos de fluorimetria. A triagem foi realizada para um conjunto de 82 compostos, previamente sintetizados pelo nosso grupo de pesquisa, como inibidores da TcSir2p1 em dose única de 100 µM. Os ensaios foram realizados em triplicata e em experimentos independentes. Dentre os 82 compostos testados, 20 apresentaram inibições maior que 50% contra a enzima TcSir2rp1, na dose de 100 µM. Dentre estes, se destacaram 3 compostos derivados de chalconas, para os quais foi determinada a potência. O composto 1 foi o que mais potente, apresentando valor de IC50 de 11,65 µM, já os compostos 3 e 5 foram menos potentes (IC50= 38,50 µM e 19,85 µM, respectivamente). Diante dos resultados obtidos, pode-se concluir que a estratégia de triagem biológica é promissora na identificação de inibidores da TcSir2p1 candidatos a agentes anti- T. cruzi

Chagas disease is caused by Trypanosoma cruzi, and currently affects 6 to 7 million people worldwide. The chemotherapy available for its treatment is based on only two drugs, nifurtimox and benznidazole, with more than 50 years of discovery. These drugs have limited efficacy, as they are ineffective in the chronic phase and have high toxicity, resulting in serious adverse effects. This panorama shows the need for new therapeutic approaches against this disease. In this sense, the inhibition of essential biochemical pathways for the parasite proves to be a good suggestion for the identification of promising compounds candidates for new chemotherapeutic agents. Sirtuin 2 (Sir2) are regulatory enzymes that participate in epigenetic mechanisms in trypanosomatids, and in T. cruzi they have a fundamental role in all their evolutionary stages, due to this fact, they present themselves as a promising target in the search for new drugs against Chagas disease. In this sense, we propose the search for inhibitors of Sir2 protein 1 of T. cruzi (TcSir2rp1) which is genetically validated as a pharmacological target, through the biological screening strategy. The expression of the recombinant enzyme was performed by molecular biology in a transformation system using strain of Escherichia coli Artic Express (DE3). Purification was performed and confirmation of obtaining the recombinant protein was performed by SDS-PAGE gel. After obtaining the enzyme, the kinetic parameters were determined by fluorimetry experiments. Screening was performed for a set of 82 compounds, previously synthesized by our research group, as TcSir2p1 inhibitors in a single dose of 100 µM. Assays were performed in triplicate and in independent experiments. Among the 82 compounds tested, 20 showed inhibitions greater than 50% against the enzyme TcSir2rp1, at a dose of 100 µM. Among these, 3 compounds derived from chalcones stood out, for which the potency was determined. Compound 1 was the most potent, with an IC50 value of 11.65 µM, while compounds 3 and 5 were less potent (IC50= 38.50 µM and 19.88 µM, respectively). In view of the results obtained, it can be concluded that the biological screening strategy is promising in the identification of TcSir2p1 inhibitors candidates for anti-T. cruzi agents

Detection of Trypanosoma cruzi strains circulating in Córdoba department (Colombia) isolated from triatomines (Hemiptera: Reduviidae) collected by the community / Detección y caracterización molecular de cepas de Trypanosoma cruzi aisladas de triatominos recolectados por la comunidad en el departamento de Córdoba, Colombia

Abstract Introduction: From 2011 to 2016, 24 cases of Chagas disease were reported in Córdoba according to the national public health surveillance system (Sistema Nacional de Vigilancia en Salud Pública, Sivigila), but the information regarding Trypanosoma cruzi circulating strains and infection rates are unknown. Objectives: To establish the triatomine species with which people come in contact and recognize as Chagas disease vectors, as well as to assess the infection with trypanosomes and make an exploratory approach to host feeding preferences with the participation of the local community. Materials and methods: Triatomines sampling was conducted in 12 municipalities between 2011 and 2016; T. cruzi infection was established by k-PCR, SAT-PCR, while strain genotyping was done by mini-exon and SL-IR (spliced-leader intergenic region) sequence characterization. We also screened for blood sources. Results: Local community members collected the majority of triatomines and we identified three species: Rhodnius pallescens, Panstrongylus geniculatus, and Eratyrus cuspidatus. The overall T. cruzi infection rate in collected triatomines was 66.6% and we detected the TcIDOM and TcI sylvatic strains. Community-based insect collection allowed reporting the presence of P. geniculatus in two new disperse rural settlements, T. cruzi infection of P. geniculatus in Córdoba, and the first report of triatomines infected with T. cruzi in Montería municipality. Conclusions: These results revealed the presence of triatomines infected with T. cruzi inside dwellings in five municipalities of Córdoba. The dominant circulating T. cruzi strain was TcIDOM, a genotype associated with human Chagas disease and cardiomyopathies in Colombia. Our results highlight the importance of local community participation in entomological surveillance tasks.

Resumen Introducción. Entre el 2011 y el 2016, se reportaron 24 casos de enfermedad de Chagas en Córdoba, según el Sistema Nacional de Vigilancia en Salud Pública (Sivigila), pero la información sobre las unidades discretas de tipificación de Trypanosoma cruzi circulantes y las tasas de infección se desconoce. Objetivos. Identificar las especies de triatominos con las cuales las personas entran en contacto y que reconocen como vectores de la enfermedad de Chagas, así como establecer la infección por tripanosomas y explorar posibles fuentes de alimentación de los triatominos con la participación de la comunidad. Materiales y métodos. El muestreo de triatominos se hizo en 12 municipios entre el 2011 y el 2016. T. cruzi se detectó mediante las técnicas de kinetic-polymerase chain reaction (k-PCR) y serial amplification of targets-polymerase chain reaction (SAT-PCR), en tanto que la genotipificación de las cepas se logró mediante la caracterización de secuencias de genes miniexon y de la región intergénica SL-IR (Spliced-Leader Intergenic Region). Se evaluaron, asimismo, las fuentes de alimento. Resultados. La mayoría de los triatominos fue recolectada por miembros de la comunidad y se identificaron tres especies: Rhodnius pallescens, Panstrongylus geniculatus y Eratyrus cuspidatus. La tasa de infección general por T. cruzi fue de 66,6 % y se detectaron las cepas TcIDOM y TcI sylvatic. La participación de la comunidad permitió reportar la presencia de P. geniculatus en dos nuevas localidades, la infección con T. cruzi de P. geniculatus en Córdoba y reportar por primera vez triatominos infectados con T. cruzi en Montería. Conclusiones. Se demostró la presencia de triatominos infectados con T. cruzi dentro de las viviendas en cinco municipalidades. La cepa circulante dominante fue T. cruzi TcIDOM, asociada con la enfermedad de Chagas y con cardiomiopatías en Colombia. Los resultados resaltan la importancia de vincular a miembros de la comunidad en la vigilancia entomológica.

Aplicação de planejamento baseado na estrutura do receptor na busca de inibidores de cisteíno-proteases parasitárias (cruzaína (T. cruzi) e PCB (Leishmanioses) / Structure-based virtual screening in the search of parasitic cysteine-proteases inhibitors

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente

Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally

Detección de unidades discretas de tipificación de Trypanosoma cruzi en triatominos recolectados en diferentes regiones naturales de Perú / Identifying Trypanosoma cruzi discreet typing units in triatomines collected in different natural regions of Perú

Resumen Introducción. Trypanosoma cruzi se ha dividido en seis unidades taxonómicas discretas (Discreet Typing Units, DTU) denominadas TcI, TcII, TcIII, TcIV, TcV y TcVI. Aún se desconocen los factores determinantes de la dinámica de la transmisión vectorial de los genotipos de T. cruzi en las diferentes regiones geográficas de distribución de la enfermedad de Chagas en Perú. Objetivo. Detectar y tipificar las unidades taxonómicas discretas de T. cruzi en las heces de siete especies de triatominos (Panstrongylus chinai, P. geniculatus, P. herreri, Rhodnius robustus, R. pictipes, Triatoma carrioni y T. infestans), capturados en ocho departamentos de diferentes regiones naturales de Perú. Materiales y métodos. Se examinaron 197 insectos para la detección de tripanosomas. Se extrajo el ADN del contenido intestinal de cada insecto y se amplificó mediante reacción en cadena de la polimerasa (PCR) de los genes kDNA, SL-IR, 24Sa rRNA y 18Sa RNA para detectar las DTU de T. cruzi. Resultados. Se detectaron cinco infecciones con T. rangeli y 113 con T. cruzi. De estas últimas, fue posible identificar 95 de TcI (dos en P. chinai, una en P. geniculatus, 68 en P. herreri, cuatro en R. pictipes, siete en R. robustus, una en T. carrioni, y 12 en T. infestans); cinco de TcII (cuatro en P. herreri, una en T. infestans); cuatro de TcIII (tres en P. herreri, una en R. robustus) y cuatro infecciones de TcIV en P. herreri. Conclusión. Este es el primer trabajo de caracterización a gran escala de T. cruzi en el intestino de vectores de importancia epidemiológica en Perú, orientado a generar información básica que permita entender la dinámica de la transmisión vectorial de T. cruzi en esta región del continente.

Abstract Introduction: Trypanosoma cruzi has been divided by international consensus into six discrete typing units (DTU): TcI, TcII, TcIII, TcIV, TcV y TcVI. The factors determining the dynamics of T. cruzi genotypes vector transmission of Chagas' disease in the different geographical regions of Perú are still unknown. Objective: To detect and type T. cruzi DTUs from the faeces of seven species of triatomines (Panstrongylus chinai, P. geniculatus, P. herreri, Rhodnius robustus, R. pictipes, Triatoma carrioni and T. infestans) captured in eight departments from different natural regions of Perú. Materials and methods: We examined 197 insects for detecting trypanosomes. DNA was extracted from each insect intestinal contents and PCR amplification of kDNA, SL-IR, 24Sa rRNA and 18Sa RNAwas performed for detecting T. cruzi DTUs. Results: Five T. rangeli and 113 T. cruzi infections were detected; 95 of the latter were identified as TcI (two in P. chinai, one in P. geniculatus, 68 in P. herreri, four in R. pictipes, seven in R. robustus, one in T. carrioni, 12 in T. infestans), five as TcII (four in P. herreri, one in T. infestans), four as TcIII (three in P. herreri, one in R. robustus) and four TcIV infections in P. herreri. Conclusions: This is the first study which has attempted a large-scale characterization of T. cruzi found in the intestine of epidemiologically important vectors in Perú, thus providing basic information that will facilitate a better understanding of the dynamics of T. cruzi vector transmission in Perú.

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

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.

Evolution of Trypanosoma cruzi: clarifying hybridisations, mitochondrial introgressions and phylogenetic relationships between major lineages

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIVS and TcIVN). In addition, we collected evidence that TcIII received TcIVS kDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIVS in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIVS kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.

Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity

This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.

Trypanosoma cruzi strains from triatomine collected in Bahia and Rio Grande do Sul, Brazil / Trypanosoma cruzi isolado de triatomíneos coletados na Bahia e Rio Grande do Sul

OBJECTIVE Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. METHODS First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. RESULTS Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. CONCLUSIONS The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship. .

OBJETIVO Isolar cepas de Trypanosoma cruzi em triatomíneos capturados nos ambientes domiciliar, peridomiciliar e silvestre da Bahia e do Rio Grande do Sul. MÉTODOS Os triatomíneos capturados nos estados da Bahia e Rio Grande do Sul foram identificados por meio de chaves entomológicas. O conteúdo intestinal foi examinado por compressão abdominal e as amostras que continham formas de Trypanosomatidae foram inoculadas em meio de cultura Liver Infusion Tryptose e em camundongos Swiss. RESULTADOS Foram identificadas seis espécies de triatomíneos nas coletas realizadas em municípios do Estado da Bahia: Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) e T. sordida (460), e duas no Estado do Rio Grande do Sul: T. circummaculata (11) e T. rubrovaria (115). Dos exemplares examinados, T. cruzi foi isolado de 28 triatomíneos pertencentes a quatro espécies: T. melanocephala (uma), T. lenti (uma), T. rubrovaria (16) e T. sordida (10). O índice de infecção natural de triatomíneos por T. cruzi foi de 6,4%. CONCLUSÕES O isolamento de cepas de T. cruzi em triatomíneos encontrados no intra e peridomicílio reflete o potencial risco de transmissão da doença de Chagas nos municípios estudados. .

Myenteric plexus is differentially affected by infection with distinct Trypanosoma cruzi strains in Beagle dogs

Chagasic megaoesophagus and megacolon are characterised by motor abnormalities related to enteric nervous system lesions and their development seems to be related to geographic distribution of distinct Trypanosoma cruzi subpopulations. Beagle dogs were infected with Y or Berenice-78 (Be-78) T. cruzi strains and necropsied during the acute or chronic phase of experimental disease for post mortem histopathological evaluation of the oesophagus and colon. Both strains infected the oesophagus and colon and caused an inflammatory response during the acute phase. In the chronic phase, inflammatory process was observed exclusively in the Be-78 infected animals, possibly due to a parasitism persistent only in this group. Myenteric denervation occurred during the acute phase of infection for both strains, but persisted chronically only in Be-78 infected animals. Glial cell involvement occurred earlier in animals infected with the Y strain, while animals infected with the Be-78 strain showed reduced glial fibrillary acidic protein immunoreactive area of enteric glial cells in the chronic phase. These results suggest that although both strains cause lesions in the digestive tract, the Y strain is associated with early control of the lesion, while the Be-78 strain results in progressive gut lesions in this model.

Aspectos biológicos e potencial vetorial de Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira e Barata, 2002 (Hemiptera: Reduviidae: Triatominae) em condições de laboratório / Biological aspects and vectorial potential of triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata, 2002 (hemiptera: reduviidae: triatominae) in laboratory conditions

No presente estudo avaliaram-se diferentes parâmetros biológicos de T. sherlocki para inferir sua capacidade vetorial. Para isso, foram efetuadas coletas no ambiente silvestre e domiciliar em 2009, quando foi feita a pesquisa para presença de T. cruzi-like, e em 2010, para o estabelecimento de colônias mantidas em condições controladas de temperatura (24,6 ± 1,3 ºC) e umidade relativa (71,6 porcento ± 6,3) (não controlada). Tais colônias deram suporte aos estudos do ciclo biológico, do comportamento alimentar e de defecação e da resistência ao jejum. A taxa de infecção natural foi obtida pelo exame microscópico de fezes dos triatomíneos. Para o estudo do ciclo biológico e do comportamento alimentar e de defecação, foram selecionados aleatoriamente 123 ovos a partir de trinta casais mantidos juntos. Assim, acompanhou-se o desenvolvimento de ovo-adulto e o seu comportamento de alimentação e de defecação após a alimentação até 10 min. Os insetos foram alimentados semanalmente em camundongos Mus musculus. A resistência ao jejum foi estudada através da seleção aleatória de um grupo de 50 ovos e 50 ninfas de 2º a 5º estádio, perfazendo um total de 350 espécimes, os quais foram alimentados semanalmente em camundongos e observados diariamente para registrar a eclosão ou ecdise...

The insects were fed weekly on mice Mus musculus. The resistance to starvation was studied by randomlyselecting a group of 50 eggs and 50 nymphs of 2nd to 5th instar, which were fed weekly on mice and observed daily to record hatching or moulting. Thereafter, each triatomine wasobserved individually for the record of the period of resistance to starvation unto death. During field sampling, 471 specimens were collected, 170 in 2009 and 301 in 2010. Of the 170 specimens collected in 2009, 145 were examined for the presence of T. cruzi-like, ofwhich 20 percent were positive. Regarding the biological cycle, T. sherlocki showed averagedevelopment time from egg to adult of 325.0 ± 40.0 days, indicating that this species has one generation per year. The number of blood meal ranged from 1 to 11 depending on the instar ofdevelopment. This feature increases the vector-host contact, increasing the likelihood of acquisition or transmission of T. cruzi. The overall mortality rate was low (6.5 percent) compared toother species of triatomines, showing that T. sherlocki has well adapted to laboratory conditions. Among the main results obtained in the study of feeding behavior and defecation highlights the short time interval between feeding and defecation of the early nymphal instarof T. sherlocki, average time of 1.38 minutes for the 1st instar and 2.15 minutes for the 2nd instar, and the 63.2 percent (n = 114) of the 1st nymphal instar and 56.7 percent (n = 60) of the 2nd nymphal instar defecated until 1 minute after the 1st and 2nd feeds, respectively. The nymphs of remaining instars took longer to defecate, but had an average time of defecation afterfeeding up to 10 min lower to 4.00 min...

Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model / Caracterização molecular de cepas mexicanas de Trypanosoma cruzi e seu comportamento no modelo experimental do camundongo

INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA). RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100 percent of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

INTRODUÇÃO: Durante muito tempo, foi questionada a importância da doença de Chagas no México onde muitos a consideravam um padecimento exótico. Considerando a grande diversidade genética existente, entre os isolados de Trypanosoma cruzi, a importância da caracterização biológica desses e o escasso número de informações sobre os aspectos clínicos e biológicos da doença de Chagas no México, o objetivo deste trabalho foi realizar a caracterização biológica e molecular de isolados de Trypanosoma cruzi originários de diferentes áreas endêmicas deste país, principalmente do Estado de Jalisco. MÉTODOS: Oito cepas mexicanas de Trypanosoma cruzi foram caracterizadas biologicamente e geneticamente (PCR específica para Trypanosoma cruzi, PCR-multiplex, amplificação do espaço não transcrito dos genes do mini-exon, amplificação das regiões polimórficas do gene do mini-exon, classificação pela amplificação de regiões intergênicas dos genes do spliced leader, RAPD - random amplified polymorphic DNA). RESULTADOS: Foram observados dois tipos de parasitemia: patente com picos máximos de parasitemia entre 4,6x10(6) e 10(7) parasitas/mL e subpatente. Além disso, todos os isolados foram capazes de infectar 100 por cento dos animais. Observou-se tropismo predominante pelo músculo estriado (cardíaco e esquelético). As técnicas de PCR do gene do mini-éxon classificaram as oito cepas como TcI e a técnica de RAPD mostrou variação intra-especifica das mesmas, separando as cepas isoladas de humanos daquelas de triatomíneos e por origem geográfica. CONCLUSÕES: As cepas mexicanas de Trypanosoma cruzi são miotrópicas e correspondem ao TcI.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

Induction of phagocytic activity and nitric-oxide production in natural populations of Trypanosoma Cruzi I and II from the state of Paraná, Brazil / Indução da atividade fagocitária e produção de óxido nítrico numa população natural de Trypanosoma cruzi I e II do Estado do Paraná, Brasil

Twelve strains of Trypanosoma cruzi isolated from wild reservoirs, triatomines, and chronic chagasic patients in the state of Paraná, southern Brazil, and classified as T. cruzi I and II, were used to test the correlation between genetic and biological diversity. The Phagocytic Index (PI) and nitric-oxide (NO) production in vitro were used as biological parameters. The PI of the T. cruzi I and II strains did not differ significantly, nor did the PI of the T. cruzi strains isolated from humans, triatomines, or wild reservoirs. There was a statistical difference in the inhibition of NO production between T. cruzi I and II and between parasites isolated from humans and the strains isolated from triatomines and wild reservoirs, but there was no correlation between genetics and biology when the strains were analyzed independently of the lineages or hosts from which the strains were isolated. There were significant correlations for Randomly Amplified Polymorphic Deoxyribonucleic acid (RAPD) and biological parameters for T. cruzi I and II, and for humans or wild reservoirs when the lineages or hosts were considered individually.

Doze cepas de Trypanosoma cruzi isoladas de reservatórios silvestres, triatomíneos e de pacientes chagásicos crônicos do Estado do Paraná, Brasil, classificadas como Tc I e II foram usadas para avaliar a correlação entre genética e diversidade biológica. Índice fagocítico (IF) e produção de óxido nítrico (ON) in vitro foram os parâmetros biológicos utilizados. O IF de cepas T. cruzi I e II não diferiram significativamente assim como o IF de cepas isoladas de humanos, triatomíneos ou de reservatórios silvestres. Há diferença estatística na inibição da produção de ON entre T. cruzi I e II e entre parasitos isolados de humanos e de cepas isoladas de triatomíneos e reservatórios silvestres, mas não foi observada correlação entre genética e biologia quando as cepas foram analisadas independentemente da linhagem ou hospedeiros das quais elas foram isoladas. Observou-se correlação significativa para amplificação aleatória do DNA polimórfico e parâmetros biológicos de Tc I ou II e para os seres humanos ou reservatório silvestre quando linhagens ou hospedeiros são consideradas separadamente.

A própolis no combate a tripanossomatídeos de importância médica: uma perspectiva terapêutica para doença de chagas e leishmaniose / Propolis activity against trypanosomatids of medical relevance: a therapeutic perspective for chagas disease and leishmaniasis

Protozoonoses tais como leishmaniose e doença de Chagas, consideradas doenças negligenciadas, afetam milhões de pessoas em todo o mundo, por isso são constantes as pesquisas para o desenvolvimento de terapias mais eficazes. O interesse pela fitoterapia como fonte alternativa para o tratamento de inúmeras enfermidades tem crescido e merece destaque o uso da própolis, amplamente utilizada na prática médica popular. São atribuídas à própolis, principalmente às variedades encontradas no Brasil, diversas propriedades farmacológicas por sua complexa composição. Estudos in vivo e in vitro com extratos de própolis contra Trypanosoma cruzi e Leishmania vêm sendo realizados nos últimos anos com resultados promissores, embora sejam necessários estudos complementares. Este trabalho traz uma revisão sobre os aspectos clínicos e epidemiológicos da leishmaniose e doença de Chagas, assim como a aplicabilidade da própolis como um recurso terapêutico eficaz e seguro para o tratamento dessas protozoonoses.

American tripanosomiasis: a study on the prevalence of Trypanosoma cruzi and Trypanosoma cruzi-like organisms in wild rodents in San Luis province, Argentina / Tripanosomiasis americana: um estudo sobre a prevalência do Trypanosoma cruzi e Trypanosoma cruzi-like em roedores silvestres da provincia de San Luis, Argentina

INTRODUCTION: Chagas disease is caused by Trypanosoma cruzi. Wild and perianthropic mammals maintain the infection/transmission cycle, both in their natural habitat and in the peridomestic area. The aim of this paper was to present the results from a study on wild rodents in the central and northern regions of San Luis province, Argentina, in order to evaluate the prevalence of this infection. METHODS: Sherman traps were set up in capture areas located between latitudes 32º and 33º S, and longitudes 65º and 66º W. The captured rodents were taxonomically identified and hemoflagellates were isolated. Morphological, biometric and molecular studies and in vitro cultures were performed. Infection of laboratory animals and histological examination of the cardiac muscle and inoculation area were also carried out. Parasites were detected in circulating blood in Calomys musculinus, Graomys griseoflavus, Phyllotis darwini and Akodon molinae. The parasites were identified using biological criteria. Molecular PCR studies were performed on some isolates, which confirmed the characterization of these hemoflagellates as Trypanosoma cruzi. RESULTS AND CONCLUSIONS: Forty-four percent of the 25 isolates were identified as Trypanosoma cruzi, and the remaining 56 percent as Trypanosoma cruzi-like. These findings provide evidence that wild rats infected with Trypanosoma cruzi and Trypanosoma cruzi-like organisms are important in areas of low endemicity.

INTRODUÇÃO: A doença de Chagas é causada pelo Trypanosoma cruzi e os mamíferos periantrópicos e silvestres mantêm o ciclo de infecção/transmissão, tanto no ambiente natural, como no peridomicílio. O objetivo deste trabalho foi mostrar os resultados de um estudo de roedores silvestres do centro e norte da Província de San Luis, Argentina, para avaliar a prevalência da infecção. MÉTODOS: Estabeleceram-se lugares de caça com armadilhas tipo Sherman entre os 32º - 33º de latitude S e 65º - 66º de longitude W. Identificou-se taxonomicamente os roedores, isolou-se os hemoflagelados e fizeram-se estudos morfológicos, biométricos, moleculares, cultivo in vitro, infecção a animais de laboratório, histologia de músculo cardíaco e de zona de inoculação. Observou-se parasitas em sangue circulante: Calomys musculinus, Graomys griseoflavus, Phyllotis darwini e Akodon molinae. A identificação dos parasitas foi feita utilizando critérios biológicos e, em alguns, realizou estudos moleculares por PCR que confirmaram a caracterização desses hemoflagelados como Trypanosoma cruzi. RESULTADOS E CONCLUSÕES: Dos 25 isolados, 44 por cento foram identificados como Trypanosoma cruzi e 56 por cento como Trypanosoma cruzi like. Este achado nos induz a considerar a importância dos ratos do mato infectados com Trypanosoma cruzi y Trypanosoma cruzi like, em área de baixa endemicidade.

Trypanosoma cruzi: parasite antigens sequestered in heart interstitial dendritic cells are related to persisting myocarditis in benznidazole-treated mice

We investigated whether sequestered Trypanosoma cruzi antigens found in heart interstitial dendritic cells (IDCs) contribute to the residual myocarditis found in mice following treatment with benznidazole, a specific chemotherapeutic drug. IDCs are antigen-presenting cells that are MHC-II-receptor dependent. Swiss mice were divided into two experimental groups: the 1st group was infected with the Colombian strain of T. cruzi, which is resistant to treatment with benznidazole, and the 2nd group was infected with clone 21SF-C 3, which has a medium susceptibility to the drug. Treatment of the Colombian strain group started on the 120th day post-infection and for the 21SF-C3 strain group treatment was started on the 90th day. In both groups, treatment lasted for 90 days. The animals were sacrificed either 150 or 200 days post-treatment. The myocardium was analysed by immunohistochemistry using anti-MAC3, 33D1, CD11b and CD11c monoclonal antibodies for IDCs or anti-T. cruzi purified antibodies. Parasite antigens were expressed on the IDC membranes in both treated and untreated mice. Myocarditis subsided following treatment, evidenced by both histological and morphometrical evaluation. A reduction in the number of IDCs carrying T. cruzi antigens in the treated group indicates that the elimination of parasites influences antigen presentation with concomitant decreases in inflammation. There is a correlation between the presence of T. cruzi antigens in these cells and the chronic focal, residual myocarditis seen in treated mice.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

á-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an á-N-acetylglucosamine (á-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.