Trypanosoma cruzi P21 recombinant protein modulates Toxoplasma gondii infection in different experimental models of the human maternal-fetal interface.

Introduction: Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease that affects about one-third of the human population. Most infected individuals are asymptomatic, but severe cases can occur such as in congenital transmission, which can be aggravated in individuals infected with other pathogens, such as HIV-positive pregnant women. However, it is unknown whether infection by other pathogens, such as Trypanosoma cruzi, the etiologic agent of Chagas disease, as well as one of its proteins, P21, could aggravate T. gondii infection. Methods: In this sense, we aimed to investigate the impact of T. cruzi and recombinant P21 (rP21) on T. gondii infection in BeWo cells and human placental explants. Results: Our results showed that T. cruzi infection, as well as rP21, increases invasion and decreases intracellular proliferation of T. gondii in BeWo cells. The increase in invasion promoted by rP21 is dependent on its binding to CXCR4 and the actin cytoskeleton polymerization, while the decrease in proliferation is due to an arrest in the S/M phase in the parasite cell cycle, as well as interleukin (IL)-6 upregulation and IL-8 downmodulation. On the other hand, in human placental villi, rP21 can either increase or decrease T. gondii proliferation, whereas T. cruzi infection increases T. gondii proliferation. This increase can be explained by the induction of an anti-inflammatory environment through an increase in IL-4 and a decrease in IL-6, IL-8, macrophage migration inhibitory factor (MIF), and tumor necrosis factor (TNF)-α production. Discussion: In conclusion, in situations of coinfection, the presence of T. cruzi may favor the congenital transmission of T. gondii, highlighting the importance of neonatal screening for both diseases, as well as the importance of studies with P21 as a future therapeutic target for the treatment of Chagas disease, since it can also favor T. gondii infection.

Encapsulation of benznidazole in nanostructured lipid carriers and increased trypanocidal activity in a resistant Trypanosoma cruzi strain

Abstract Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi, whose treatment has remained unsatisfactory for over 50 years, given that it is limited to two drugs. Benznidazole (BZN) is an efficient antichagasic drug used as the first choice, although its poor water-solubility, irregular oral absorption, low efficacy in the chronic phase, and various associated adverse effects are limiting factors for treatment. Incorporating drugs with such characteristics into nanostructured lipid carriers (NLC) is a promising alternative to overcome these limiting obstacles, enhancing drug efficacy and bioavailability while reducing toxicity. Therefore, this study proposed NLC-BZN formulations in different compositions prepared by hot-melt homogenization followed by ultrasound, and the optimized formulation was characterized by FTIR, DRX, DSC, and thermogravimetry. Biological activities included in vitro membrane toxicity (red blood cells), fibroblast cell cytotoxicity, and trypanocidal activity against epimastigotes of the Colombian strain of T. cruzi. The optimized NLC-BZN had a small size (110 nm), negative zeta potential (-18.0 mV), and high encapsulation (1.64% of drug loading), as shown by infrared spectroscopy, X-ray diffraction, and thermal analysis. The NLC-BZN also promoted lower in vitro membrane toxicity (<3% hemolysis), and 50% cytotoxic concentration (CC50) for NLC-BZN in L929 fibroblast cells (110.7 µg/mL) was twice the value as the free BZN (51.3 µg/mL). Our findings showed that the NLC-BZN had higher trypanocidal activity than free BZN against the epimastigotes of the resistant Colombian strain, and this novel NLC-BZN formulation proved to be a promising tool in treating Chagas disease and considered suitable for oral and parenteral administration

Axl receptor induces efferocytosis, dampens M1 macrophage responses and promotes heart pathology in Trypanosoma cruzi infection.

Adaptive immunity controls Trypanosoma cruzi infection, but the protozoan parasite persists and causes Chagas disease. T cells undergo apoptosis, and the efferocytosis of apoptotic cells might suppress macrophages and exacerbate parasite infection. Nonetheless, the receptors involved in the efferocytosis of apoptotic lymphocytes during infection remain unknow. Macrophages phagocytose apoptotic cells by using the TAM (Tyro3, Axl, Mer) family of receptors. To address how the efferocytosis of apoptotic cells affects macrophage-mediated immunity, we employ here Axl receptor- and Mer receptor-deficient mouse strains. In bone marrow-derived macrophages (BMDMs), both Axl and Mer receptors play a role in the efferocytosis of proapoptotic T cells from T. cruzi-infected mice. Moreover, treatment with a TAM receptor inhibitor blocks efferocytosis and upregulates M1 hallmarks induced by immune T cells from infected mice. Remarkably, the use of Axl-/- but not Mer-/- macrophages increases T-cell-induced M1 responses, such as nitric oxide production and control of parasite infection. Furthermore, infected Axl-/- mice show reduced peak parasitemia, defective efferocytosis, improved M1 responses, and ameliorated cardiac inflammation and fibrosis. Therefore, Axl induces efferocytosis, disrupts M1 responses, and promotes parasite infection and pathology in experimental Chagas disease. Axl stands as a potential host-direct target for switching macrophage phenotypes in infectious diseases.

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

Planejamento, síntese e avaliação biológica de candidatos a fármacos: busca de inibidores epigenéticos da Sirtuína 2 Sir2 / Design, synthesis and biological evaluation of drug candidates: search for Sirtuin 2 (Sir 2) epigenetic inhibitors

As doenças negligenciadas são causadas por agentes infecciosos e parasitários, como vírus, bactérias, protozoários e helmintos. Essas doenças são prevalentes em populações de baixa renda que vivem em países em desenvolvimento e são responsáveis por incapacitar e levar milhares de pessoas à morte. Este nome se dá pois, apesar de sua grande relevância médica, recebem pouca atenção dos governos e indústrias farmacêuticas. Dentre essas doenças podemos destacar a Doença de Chagas, doença infecciosa causada pelo parasita hemoflagelado Trypanosoma cruzi. Endêmica em 21 países, com 6 a 7 milhões de pessoas infectadas resultando em 7500 mortes por ano. A quimioterapia disponível contra essa parasitose é baseada em apenas dois medicamentos, o benznidazol e o nifurtimox, ativos principalmente na fase aguda da doença e com efeitos adversos graves que comprometem a adesão ao tratamento e, além disso, apesar dos enormes esforços na pesquisa de novos agentes antichagásicos em nível nacional e internacional, na maioria realizada academicamente, ainda não foram encontradas alternativas terapêuticas para a doença, persistindo, assim, a necessidade de descoberta e desenvolvimento de novos fármacos. O início de um planejamento de um novo fármaco se dá pela definição de um alvo bioquímico a ser utilizado na busca de moléculas que possam exercer a função de inibidores ou moduladores, conforme a atividade biológica desejada. Neste sentido, as sirtuínas 2 (Sir2) são enzimas que se mostraram essenciais para o crescimento in vitro do T. cruzi em suas formas amastigota e epimastigota. No caso de tripanossomatídeos, em geral, a superexpressão de Sir2 está relacionada à sobrevivência de formas amastigotas. Assim, essas evidências indicam que a Sir2 de tripanosomatídeos tem grande potencial como alvo biológico na busca e desenvolvimento de novos fármacos antichagásicos. O objetivo principal deste projeto foi identificar moléculas que apresentaram atividade inibitória para a sirtuína 2 de T. cruzi por meio da utilização da estratégia de Planejamento de Fármacos Baseada no Ligante - Ligand Based Drug Design (LBDD) e o desenvolvimento de análogos dos inibidores da Sir2. A modificação molecular está entre algumas das técnicas tradicionais usadas no desenvolvimento racional de um fármaco, e é usada principalmente no desenvolvimento de análogos, e busca melhorar as propriedades farmacocinéticas e/ou farmacodinâmicas de um protótipo, obter propriedades de interação semelhantes ao alvo e, em alguns casos, revelar uma atividade biológica. Com este intuito, análogos do sirtinol e da salermida foram sintetizados e uma nova rota sintética utilizando o microrreator em fluxo contínuo foi desenvolvida e apresentou rendimento superior quando comparado à síntese em bancada. A partir desta metodologia foram obtidos 20 compostos. Os ensaios in vitro contra formas amastigotas do T. cruzi indicaram que 8 compostos inibiram a atividade parasitária em mais de 50%, na dose de 10 µM, sendo que alguns destes apresentaram maior inibição parasitária quando comparados ao benznidazol, o fármaco de referência e único disponível no Brasil. Com estes resultados preliminares, novos ensaios estão sendo realizados para identificar potência e mecanismo de ação destes candidatos a agentes tripanomicidas

Neglected diseases are caused by infectious and parasitic agents such as viruses, bacteria, protozoa and helminths. These diseases are prevalent in low-income populations living in developing countries and are responsible for disabling and killing thousands of people. They get this name because, despite their great medical relevance, they end up receiving little attention from governments and pharmaceutical industries. Among these diseases, we can highlight Chagas disease, an infectious endemic disease caused by the hemoflagellate parasite Trypanosoma cruzi. This disease is endemic in 21 countries, with 6 to 7 million people infected resulting in 7,500 deaths per year. Chemotherapy is based on just two drugs, benznidazole and nifurtimox, which are mainly active in the acute phase of the disease. These drugs have adverse effects that compromise adherence, even more, considering that they are not effective from the point of view of the chronic phase of the disease. Despite the enormous efforts in researching new anti-chagasic agents at the national and international level, and mostly carried out academically, therapeutic alternatives for the disease have not yet been found, thus, the need for the discovery and development of new drugs persists. Sirtuins 2 (Sir2) are enzymes that have been shown to be essential for the in vitro growth of T. cruzi in its amastigote and epimastigote forms. In the case of trypanosomatids in general, Sir2 overexpression is related to the survival of amastigote forms. Sir2 inhibitors, such as sirtinol, have shown efficacy in leishmanicides. Thus, these evidences indicate that Sir2 from trypanosomatids can be considered as a biological target in the search and development of new anti-chagasic drugs. The beginning of a new drug planning study is the definition of a biochemical target to be used in the search for molecules that can play the role of inhibitors or modulators, according to the desired biological activity. The main objective of this project was to identify molecules that presented inhibitory activity to sirtuin 2 of T. cruzi using the Ligand Based Drug Design (LBDD) strategy of planning and the development of analogues of Sir2 inhibitors. Molecular modification is a traditional technique used in the rational development of a drug, as well as the use of natural products, combinatorial chemistry, high-throughput screening (HTS), among others. Mainly used in the development of analogues, molecular modification is applied for different purposes, among them, it seeks to improve the pharmacokinetic and/or pharmacodynamic properties of a prototype, obtain target-like interaction properties and, in some cases, reveal an activity biological. For this purpose, analogues of sirtinol and salermide were synthesized and a new synthetic route using the microreactor in continuous flow was developed and presented superior yield when compared to benchtop synthesis. From this methodology, 20 compounds were obtained. in vitro assays against amastigote forms of T. cruzi indicated that 8 compounds inhibited parasitic activity by more than 50% at a dose of 10 µM, and some of these showed greater parasitic inhibition when compared to benznidazole, the reference drug, and only available in Brazil. With these preliminary results, new assays are being carried out to identify the potency and mechanism of action of these candidate trypanocidal agents

Análise histopatológica dos efeitos do bioterápico 200dh em camundongos infectados por Trypanosoma Cruzi / Histopathological analysis of the effects of 200dh biotherapeutic in mice infected with Trypanosoma Cruzi

O Trypanosoma cruzi é um protozoário que instaura uma infecção grave em seu hospedeiro vertebrado, podendo causar danos irreversíveis, principalmente em suas células musculares, conhecida como a doença tripanossomíase ou Doença de Chagas. dicação disponível no mercado, que combate esse parasito, não é completamente eficiente e produz danos indesejáveis e irreversíveis, fazendo-se necessário a busca por outros campos da medicina, em termos de medicação, para combater essa doença. Nesse sentido, pesquisas com medicamentos homeopáticos têm se revelado promissoras no combate ao parasito, sobretudo as formas amastigotas, que são mais difíceis de serem exterminadas. Os bioterápicos são os homeopáticos que obtiveram maior sucesso em relação à infecção por T. cruzi, uma vez que esses medicamentos possuem seu princípio ativo retirado do próprio parasito, suas toxinas, parte de membros, realizando uma terapia conhecida como isoterapia ­ cura pelo igual. Resultados encontrados indicam que o bioterápico 200dH atua coagindo o sistema imune a combater o T. cruzi em cobaias contaminadas, fomentando uma resposta imune celular e humoral mais eficiente do que a fisiológica. Acredita-se que esse medicamento atue estimulando as células de defesa, que passarão a responder de forma antígeno- -específico, favorecendo o combate de amastigotas. Esse processo, provavelmente, é iniciado pela estimulação do macrófago, que por sua vez, de acordo com os resultados encontrados, inicia uma cascata inflamatória, com predominância da via Th1, fomentando a produção de IL4, IL-10 e interferon, auxiliando no combate as amastigotas.

The trypanosoma cruzi is a protozoan that causes a serious infeccion in the vertebrate host, being capable of causing irreversible damages mainly in the muscle cells, also known as trypanosomiasis disease or Chagas Disease. The commercially available drug medication which fights this parasite is not completely efficient and causes undesirable and irreversible damages making it necessary to search for other fields of medicine in terms of medication to combat this disease. Therefore research with homeopathic medicines has being promising in the fight against the parasite, especially the amastigote forms, which are more difficult to be exterminated. Biotherapics are the homeopathic ones that have been more successful in relation to T. cruzi infection, since these drugs have their active principle removed from the parasite itself, its toxins, part of its members, performing a therapy known as isotherapy - cure by the same. The results indicates that the 200dH biotherapic acts by coercing the immune system to fight T. cruzi in contaminated guinea pigs, promoting a cellular and humoral imune response more efficient than the physiological one. It is believed that this medicine works by stimulating the defense cells which will respond in an antigen-specific manner favoring the fight against amastigotes. This process is probably initiated by the stimulation of the macrophage which in turn, according to the remains found, initiates an inflammatory cascade with predominance of the Th1 pathway, promoting the production of IL-4e IL-5 and interferon helping to combat amastigotes

4-Chlorophenylthioacetone-derived thiosemicarbazones as potent antitrypanosomal drug candidates: Investigations on the mode of action.

Chagas disease (ChD), caused by Trypanosoma cruzi, remains a challenge for the medical and scientific fields due to the inefficiency of the therapeutic approaches available for its treatment. Thiosemicarbazones and hydrazones present a wide spectrum of bioactivities and are considered a platform for the design of new anti-T. cruzi drug candidates. Herein, the potential antichagasic activities of [(E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-hydrazinecarbothioamides] (C1, C3), [(E)-N'-(1-((4-chlorophenyl)thio)propan-2-ylidene)benzohydrazide] (C2), [(E)-2-(1-(4-, and [(E)-2-(1-((4-chlorophenyl)thio)propan-2-ylidene)hydrazinecarboxamide] (C4) were investigated. Macrophages (MOs) from C57BL/6 mice stimulated with C1 and C3, but not with C2 and C4, reduced amastigote replication and trypomastigote release, independent of nitric oxide (NO) and reactive oxygen species production and indoleamine 2,3-dioxygenase activity. C3, but not C1, reduced parasite uptake by MOs and potentiated TNF production. In cardiomyocytes, C3 reduced trypomastigote release independently of NO, TNF, and IL-6 production. C1 and C3 were non-toxic to the host cells. A reduction of parasite release was found during infection of MOs with trypomastigotes pre-incubated with C1 or C3 and MOs pre-stimulated with compounds before infection. Moreover, C1 and C3 acted directly on trypomastigotes, killing them faster than Benznidazole, and inhibited T. cruzi proliferation at various stages of its intracellular cycle. Mechanistically, C1 and C3 inhibit parasite duplication, and this process cannot be reversed by inhibiting the DNA damage response. In vivo, C1 and C3 attenuated parasitemia in T. cruzi-infected mice. Moreover, C3 loaded in a lipid nanocarrier system (nanoemulsion) maintained anti-T. cruzi activity in vivo. Collectively, these data suggest that C1 and C3 are candidates for the treatment of ChD and present activity in both the host and parasite cells.

Measurement of multiple cytokines for discrimination and risk stratification in patients with Chagas' disease and idiopathic dilated cardiomyopathy.

Chagas' disease (CD), caused by the hemoflagellate protozoan, Trypanosoma cruzi, is endemic in most countries of Latin America. Heart failure (HF) is often a late manifestation of chronic CD, and is associated with high morbidity and mortality. Inflammatory processes mediated by cytokines play a key role in the pathogenesis and progression of CD. Keeping in view the inflammatory nature of CD, this study investigated the possible role of 21 different inflammatory cytokines as biomarkers for prediction and prognosis of CD. The plasma concentration of these cytokines was measured in a group of patients with CD (n = 94), and then compared with those measured in patients with dilated cardiomyopathy (DCM) from idiopathic causes (n = 48), and with control subjects (n = 25). Monovariately, plasma levels of cytokines such as stem cell growth factor beta (SCGF beta), hepatocyte growth factor (HGF), monokine induced by interferon gamma (CXCL9), and macrophage inhibitory factor (MIF) were significantly increased in CD patients with advanced HF compared to control group. None of the cytokines could demonstrate any prognostic potency in CD patients, and only MIF and stromal derived factor-1 alpha (CXCL12) showed significance in predicting mortality and necessity for heart transplant in DCM patients. However, multivariate analysis prognosticated a large proportion of CD and DCM patients. In CD patients, HGF and Interleukin-12p40 (IL-12p40) together separated 81.9% of 3-year survivors from the deceased, while in DCM patients, CXCL12, stem cell factor (SCF), and CXCL9 together discriminated 77.1% of survivors from the deceased. The significant increase in plasma concentrations of cytokines such as HGF and CXCL9 in CD patients, and the ability of these cytokines to prognosticate a large proportion of CD and DCM patients multivariately, encourages further studies to clarify the diagnostic and prognostic potential of cytokines in such patients.

Induction of autophagy increases the proteolytic activity of reservosomes during Trypanosoma cruzi metacyclogenesis.

Cruzipain, the major cysteine protease of the pathogenic protozoa Trypanosoma cruzi, is an important virulence factor that plays a key role in the parasite nutrition, differentiation and host cell infection. Cruzipain is synthesized as a zymogen, matured, and delivered to reservosomes. These organelles that store proteins and lipids ingested by endocytosis undergo a dramatic decrease in number during the metacyclogenesis of T. cruzi. Autophagy is a process that digests the own cell components to supply energy under starvation or different stress situations. This pathway is important during cell growth, differentiation and death. Previously, we showed that the autophagy pathway of T. cruzi is induced during metacyclogenesis. This work aimed to evaluate the participation of macroautophagy/autophagy in the distribution and function of reservosomes and cruzipain during this process. We found that parasite starvation promotes the cruzipain delivery to reservosomes. Enhanced autophagy increases acidity and hydrolytic activity in these compartments resulting in cruzipain enzymatic activation and self- processing. Inhibition of autophagy similarly impairs cruzipain traffic and activity than protease inhibitors, whereas mutant parasites that exhibit increased basal autophagy, also display increased cruzipain processing under control conditions. Further experiments showed that autophagy induced cruzipain activation and self-processing promote T. cruzi differentiation and host cell infection. These findings highlight the key role of T. cruzi autophagy in these processes and reveal a potential new target for Chagas disease therapy.Abbreviations: Baf: bafilomycin A1; CTE: C-terminal extension; Cz: cruzipain; IIF: indirect immunofluorescence; K777: vinyl sulfone with specific Cz inhibitory activity; Prot Inh: broad-spectrum protease inhibitor; Spa1: spautin-1; Wort: wortmannin.

Complexos imínicos de cobre com atividade antiparasitária frente à doença de chagas / Imine copper complexes with antiparasitic activity against Chagas disease

Neste trabalho foram sintetizados e caracterizados três complexos de cobre com ligantes imínicos, com o objetivo de avaliar sua atividade tripanocida. Esses complexos foram caracterizados por diversas técnicas espectroscópicas, como UV-Vis, Infravermelho e EPR, além de análise elementar e espectrometria de massa. Juntamente com outros complexos similares previamente sintetizados pelo nosso grupo, tiveram suas atividades avaliadas frente à forma tripomastigota do parasita T. cruzi, responsável pela fase aguda da doença de Chagas, por ensaios de viabilidade celular, com determinação do valor de seus IC50, concentração em que observamos a morte de 50% da cultura celular, pela metodologia denominada MTT. Todos os complexos mostraram-se eficientes frente a tripomastigotas, apresentando valores de IC50 abaixo de 10 µM, com quatro deles obtendo índice de seletividade maior que 10, fator importante para definir agentes promissores antichagásicos. Complexos selecionados também tiveram sua atividade verificada frente à forma amastigota do parasita, responsável pela fase crônica da doença, utilizando método de imageamento por microscópio de fluorescência e contagem celular. Estudos de inibição da cruzaína, uma cisteíno-protease importante para o metabolismo do parasita foram conduzidos em colaboração com o laboratório do Prof. Wagner Alves de Souza Júdice, da Universidade de Mogi das Cruzes. Quatro dos compostos testados apresentaram atividade inibitória frente a cruzaína, sendo dois de cobre, um de zinco e um ligante livre. Os estudos também permitiram diferenciar os mecanismos de inibição dos compostos, com os complexos de cobre apresentando um mecanismo de inibição clássico e o composto de zinco e o ligante livre apresentando o mecanismo de inibição competitiva parabólica com cooperatividade

In this work, three copper complexes with iminic ligands were synthesized and characterized, with the objective of evaluating their trypanocidal activity. These complexes were characterized by several spectroscopic techniques, such as UV-Vis, Infrared and EPR, in addition to elementary analysis and mass spectrometry. Together with other similar complexes previously synthesized by our group, their activities were evaluated against the trypomastigote form of the parasite T. cruzi, responsible for the acute phase of Chagas disease, by cell viability tests, with determination of the value of their IC50, concentration in that we observed the death of 50% of the cell culture, by the methodology called MTT, all presenting IC50 values below 10 µM, with four of them obtaining a selectivity index greater than 10, important factor for defining promising antichagasic agents. Selected complexes also had their activity verified against the amastigote form of the parasite, responsible for the chronic phase of the disease, using a fluorescence microscope and cell counting imaging method. Inhibition studies of cruzain, a cysteine protease important for the metabolism of the parasite, were conducted in collaboration with the laboratory of Professor Wagner Alves de Souza Júdice at the University of Mogi das Cruzes. Four of the tested compounds showed inhibitory activity against cruzain, two of copper, one of zinc and a free ligand. The studies also allowed to differentiate the mechanisms of inhibition of the compounds, with the copper complexes presenting a classic inhibition mechanism and the zinc compound and the free ligand presenting the competitive parabolic inhibition mechanism with cooperativity

Dual and Opposite Roles of Reactive Oxygen Species (ROS) in Chagas Disease: Beneficial on the Pathogen and Harmful on the Host.

Chagas disease is a neglected tropical disease, which affects an estimate of 6-7 million people worldwide. Chagas disease is caused by Trypanosoma cruzi, which is a eukaryotic flagellate unicellular organism. At the primary infection sites, these parasites are phagocytized by macrophages, which produce reactive oxygen species (ROS) in response to the infection with T. cruzi. The ROS produce damage to the host tissues; however, macrophage-produced ROS is also used as a signal for T. cruzi proliferation. At the later stages of infection, mitochondrial ROS is produced by the infected cardiomyocytes that contribute to the oxidative damage, which persists at the chronic stage of the disease. The oxidative damage leads to a functional impairment of the heart. In this review article, we will discuss the mechanisms by which T. cruzi is able to deal with the oxidative stress and how this helps the parasite growth at the acute phase of infection and how the oxidative stress affects the cardiomyopathy at the chronic stage of the Chagas disease. We will describe the mechanisms used by the parasite to deal with ROS and reactive nitrogen species (RNS) through the trypanothione and the mechanisms used to repair the damaged DNA. Also, a description of the events produced by ROS at the acute and chronic stages of the disease is presented. Lastly, we discuss the benefits of ROS for T. cruzi growth and proliferation and the possible mechanisms involved in this phenomenon. Hypothesis is put forward to explain the molecular mechanisms by which ROS triggers parasite growth and proliferation and how ROS is able to produce a long persisting damage on cardiomyocytes even in the absence of the parasite.

Identification of Hub Genes and Key Pathways Associated with Peripheral T-cell Lymphoma.

Peripheral T-cell lymphoma (PTCL) is a very aggressive and heterogeneous hematological malignancy and has no effective targeted therapy. The molecular pathogenesis of PTCL remains unknown. In this study, we chose the gene expression profile of GSE6338 from the Gene Expression Omnibus (GEO) database to identify hub genes and key pathways and explore possible molecular pathogenesis of PTCL by bioinformatic analysis. Differentially expressed genes (DEGs) between PTCL and normal T cells were selected using GEO2R tool. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID). Moreover, the Search Tool for the Retrieval of Interacting Genes (STRING) and Molecular Complex Detection (MCODE) were utilized to construct protein-protein interaction (PPI) network and perform module analysis of these DEGs. A total of 518 DEGs were identified, including 413 down-regulated and 105 up-regulated genes. The down-regulated genes were enriched in osteoclast differentiation, Chagas disease and mitogen-activated protein kinase (MAPK) signaling pathway. The up-regulated genes were mainly associated with extracellular matrix (ECM)-receptor interaction, focal adhesion and pertussis. Four important modules were detected from the PPI network by using MCODE software. Fifteen hub genes with a high degree of connectivity were selected. Our study identified DEGs, hub genes and pathways associated with PTCL by bioinformatic analysis. Results provide a basis for further study on the pathogenesis of PTCL.

The role of fat on cardiomyopathy outcome in mouse models of chronic Trypanosoma cruzi infection.

The underlying pathogenic mechanisms of cardiomyopathy in Chagas disease are still unsolved. In order to better clarify the role of fat on the evolution of cardiomyopathy, the present study employed three murine models of chronic Trypanosoma cruzi infection: (1) aP2-RIDα/ß transgenic mice (RID mice; an adipose tissue model which express a gain-of-function potent anti-inflammatory activity), (2) allograft inflammatory factor-1 knockout mice (Aif1-/-), and (3) a Swiss outbred mice. RID mice and non-transgenic mice (wild type, WT) were infected with blood trypomastigotes of Brazil strain. During the acute stage of infection, RID mice had lower parasitemia, lower heart inflammation, and a decrease in the relative distribution of parasite load from cardiac muscle tissue toward epididymal fat. Nevertheless, comparable profiles of myocardial inflammatory infiltrates and relative distribution of parasite load were observed among RID and WT at the chronic stage of infection. Aif1-/- and Aif1+/+ mice were infected with bloodstream trypomastigotes of Tulahuen strain and fed with high-fat diet (HFD) or regular diet (RD). Interestingly, Aif1+/+ HFD infected mice showed the highest mortality. Swiss mice infected with blood trypomastigotes of Berenice-78 strain on a HFD had higher levels of TNFα and more inflammation in their heart tissue than infected mice fed a RD. These various murine models implicate adipocytes in the pathogenesis of chronic Chagas disease and suggest that HFD can lead to a significant increase in the severity of parasite-induced chronic cardiac damage. Furthermore, these data implicate adipocyte TLR4-, TNFα-, and IL-1ß-mediated signaling in pro-inflammatory pathways and Aif-1 gene expression in the development of chronic Chagas disease.

Trypanosoma cruzi Calreticulin: Immune Evasion, Infectivity, and Tumorigenesis.

To successfully infect, Trypanosoma cruzi evades and modulates the host immune response. T. cruzi calreticulin (TcCalr) is a multifunctional, endoplasmic reticulum (ER)-resident chaperone that, translocated to the external microenvironment, mediates crucial host-parasite interactions. TcCalr binds and inactivates C1 and mannose-binding lectin (MBL)/ficolins, important pattern- recognition receptors (PRRs) of the complement system. Using an apoptotic mimicry strategy, the C1-TcCalr association facilitates the infection of target cells. T. cruzi infection also seems to confer protection against tumorigenesis. Thus, recombinant TcCalr has important antiangiogenic properties, detected in vitro, ex vivo, and in ovum, most likely contributing at least in part, to its antitumor properties. Consequently, TcCalr is useful for investigating key issues of host-parasite interactions and possible new immunological/pharmacological interventions in the areas of Chagas' disease and experimental cancer.

Although with intact mucosa at colonoscopy, chagasic megacolons have an overexpression of Gal-3.

OBJECTIVE: To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. METHODS: Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. RESULTS: Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. CONCLUSION: The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.

The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro.

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.

Although with intact mucosa at colonoscopy, chagasic megacolons have an overexpression of Gal-3 / Embora com mucosa íntegra à colonoscopia, megacólons chagásicos apresentam superexpressão de Gal-3

ABSTRACT Objective To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. Methods Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. Results Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. Conclusion The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.

RESUMO Objetivo Avaliar a densidade de células imunomarcadas por anti-galectina-3, a percentagem de colágeno, a densidade de mastócitos e a presença de processos patológicos na musculatura intestinal de pacientes biopsiados. Métodos Foram selecionados 35 pacientes submetidos à biópsia de intestino entre 1997 a 2015. Os pacientes foram divididos em três grupos: chagásicos com lesão de mucosa (n=13), chagásicos com mucosa íntegra (n=12) e não chagásicos sem lesão de mucosa (n=10). Foram realizados processamento histológico dos fragmentos biopsiados e imunohistoquímica para galectina-3. Cortes adicionais foram corados por hematoxilina e eosina, para avaliar os processos patológicos gerais, pelo picrosírius, para avaliação do colágeno, e pelo azul de toluidina, para avaliar a densidade de mastócitos. Resultados Os pacientes do grupo chagásicos com lesão de mucosa apresentaram frequência significativamente maior de ganglionite e miosite quando comparados aos dos grupos chagásico com mucosa íntegra e não chagásicos. A densidade das células imunomarcadas por anti-galectina-3 foi significativamente maior no grupo chagásicos com mucosa íntegra quando comparada ao grupo não chagásico. O grupo de chagásicos com mucosa íntegra apresentou maior percentagem de colágeno em relação aos grupos chagásicos com mucosa lesada e ao grupo de não chagásicos, com diferença significativa. Não houve diferença significativa com relação à densidade de mastócitos entre os três grupos. Conclusão A maior densidade de células imunomarcadas por anti-galectina-3 nos pacientes do grupo chagásico com mucosa íntegra sugere a necessidade de maior atenção na avaliação clínica desses pacientes, uma vez que essa proteína está associada com transformação e progressão neoplásica.

Effects of ghrelin supplementation on the acute phase of Chagas disease in rats.

BACKGROUND: Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic to subtropical and tropical Americas. The disease treatment remains partially ineffective, involving therapies directed to the parasite as well as palliative strategies for the clinical manifestations. Therefore, novel candidates for disease control are necessary. Additionally, strategies based on parasite inhibition via specific targets and application of compounds which improve the immune response against the disease is welcomed. Ghrelin is a peptide hormone pointed as a substance with important cardioprotective, vasodilatory, anti-apoptotic, anti-oxidative and immune modulatory functions. The aims of this study were to evaluate the immunomodulatory effects of ghrelin in male Wistar rats infected with the Y strain of T. cruzi. METHODS: In order to delineate an immune response against T. cruzi mediated by ghrelin, we evaluated the following parameters: quantification of blood and cardiac parasites; analysis of cell markers (CD3+, CD8+, NK, NKT, CD45RA+, macrophage and RT1B+); nitric oxide (NO) production; lymphoproliferation assays; splenocyte apoptosis; and INF-γ, IL-12 and IL-6 quantification in sera. RESULTS: The animals infected with T. cruzi and supplemented with ghrelin demonstrated an upregulated pattern in macrophage and NO production, whereas an anti-inflammatory response was observed in T cells and cytokines. The low response against T. cruzi mediated by T cells probably contributed to a higher colonization of the cardiac tissue, when compared to infected groups. On the other side, the peptide decreased the inflammatory infiltration in cardiac tissue infected with T. cruzi. CONCLUSIONS: Ghrelin demonstrated a dual function in animals infected with T. cruzi. Further studies, especially related to the decrease of cardiac tissue inflammation, are needed in order to determine the advantages of ghrelin supplementation in Chagas disease, mostly for populations from endemic areas.