Interesting case from Argentina: Kidney transplant recipient with skin lesions-A Latin American perspective.

This is a case of a kidney transplant recipient who presented with skin lesions, low-grade fevers, and pancytopenia 2 months after his transplant.

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.

Benznidazole, itraconazole, and their combination for the treatment of chronic experimental Chagas disease in dogs.

Treatment for Chagas disease has limited efficacy in the chronic phase. We evaluated benznidazole (BZ) and itraconazole (ITZ) individually and in association in dogs 16 months after infection with a BZ-resistant Trypanosoma cruzi strain. Four study groups (20 animals) were evaluated and treated for 60 days with BZ, ITZ, or BZ + ITZ, and maintained in parallel to control group infected and not treated (INT). All dogs were evaluated in the first, sixth, 12th, 18th and 24th months of study. Polymerase chain reaction (PCR) was negative in 2 of 3 animals in the BZ + ITZ group, 2 of 5 in the BZ group, and 4 of 5 in the ITZ group. Hemoculture performed in the 24th month was negative in all groups. Enzyme-linked immunoassay remained reactive in all treated animals. Echocardiography differentiated treated animals from control animals. Quantitative PCR analysis of cardiac tissue was negative in the BZ + ITZ and BZ groups, positive in 2 of 5 dogs in the ITZ group and in 2 of 3 dogs in the control group, but negative in colon tissue in all groups. Inflammation was significantly reduced in the right atrium and left ventricle of dogs treated with BZ + ITZ and BZ compared with those receiving ITZ alone. Fibrosis was absent in most dogs treated with BZ + ITZ, mild in those treated with BZ or ITZ alone, and intense in the control group. Parasitological and histopathological evaluations showed that BZ + ITZ treatment improved or stabilized the clinical condition of the dogs.

C57BL/6 Mice Pretreated With Alpha-Tocopherol Show a Better Outcome of Trypanosoma cruzi Infection With Less Tissue Inflammation and Fibrosis.

Chagas disease is accompanied by a multisystem inflammatory disorder that follows Trypanosoma cruzi infection. Alpha-tocopherol has been described as an antioxidant and a potential adjuvant to enhance immune responses to vaccines. Therefore, we have evaluated the immune response to T. cruzi infection upon alpha-tocopherol pre-administration. The results show that administration of alpha-tocopherol before the infection results in lower parasitemia and lower mortality of C57BL/6 mice infected with the Tulahuen T. cruzi strain. Alpha-tocopherol administration in normal C57BL/6 mice resulted in higher levels of IFN-γ production by T and NK cells before and after the infection with T. cruzi. More importantly, previous administration of alpha-tocopherol increased the production of IL-10 by T and myeloid suppressor cells and the formation of effector memory T cells while decreasing the expression of PD-1 on T cells. These results suggest that alpha-tocopherol may limit the appearance of dysfunctional T cells during the acute and early chronic phases of T. cruzi infection, contributing to control infection. In addition, alpha-tocopherol could diminish tissue inflammation and fibrosis in late acute disease. These results strongly suggest that alpha-tocopherol may be a helpful agent to be considered in Chagas disease.

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells.

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/ß-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/ß-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/ß-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3ß at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3ß was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of ß-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of ß-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating ß-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a ß-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the ß-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

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

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

Physical Exercise Promotes a Reduction in Cardiac Fibrosis in the Chronic Indeterminate Form of Experimental Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease and a health problem in Latin America. Etiological treatment has limited effectiveness in chronic CD; thus, new therapeutic strategies are required. The practice of physical exercises has been widely advocated to improve the quality of life of CD patients. The most frequent clinical CD manifestation is the chronic indeterminate form (CIF), and the effect of physical exercises on disease progression remains unknown. Here, in a CIF model, we aimed to evaluate the effect of physical exercises on cardiac histological, parasitological, mitochondrial, and oxidative metabolism, electro and echocardiographic profiles, and immunological features. To establish a CIF model, BALB/c and C57BL/6 mice were infected with 100 and 500 trypomastigotes of the Y T. cruzi strain. At 120 days postinfection (dpi), all mouse groups showed normal PR and corrected QT intervals and QRS complexes. Compared to BALB/c mice, C57BL/6 mice showed a lower parasitemia peak, mortality rate, and less intense myocarditis. Thus, C57BL/6 mice infected with 500 parasites were used for subsequent analyses. At 120 dpi, a decrease in cardiac mitochondrial oxygen consumption and an increase in reactive oxygen species (ROS) were detected. When we increased the number of analyzed mice, a reduced heart rate and slightly prolonged corrected QT intervals were detected, at 120 and 150 dpi, which were then normalized at 180 dpi, thus characterizing the CIF. Y-infected mice were subjected to an exercise program on a treadmill for 4 weeks (from 150 to 180 dpi), five times per week in a 30-60-min daily training session. At 180 dpi, no alterations were detected in cardiac mitochondrial and oxidative metabolism, which were not affected by physical exercises, although ROS production increased. At 120 and 180 dpi, comparing infected and non-infected mice, no differences were observed in the levels of plasma cytokines, indicating that a crucial biomarker of the systemic inflammatory profile was absent and not affected by exercise. Compared with sedentary mice, trained Y-infected mice showed similar parasite loads and inflammatory cells but reduced cardiac fibrosis. Therefore, our data show that physical exercises promote beneficial changes that may prevent CD progression.

Chagas disease and SARS-CoV-2 coinfection does not lead to worse in-hospital outcomes.

Chagas disease (CD) continues to be a major public health burden in Latina America. Information on the interplay between COVID-19 and CD is lacking. Our aim was to assess clinical characteristics and in-hospital outcomes of patients with CD and COVID-19, and to compare it to non-CD patients. Consecutive patients with confirmed COVID-19 were included from March to September 2020. Genetic matching for sex, age, hypertension, diabetes mellitus and hospital was performed in a 4:1 ratio. Of the 7018 patients who had confirmed COVID-19, 31 patients with CD and 124 matched controls were included (median age 72 (64-80) years-old, 44.5% were male). At baseline, heart failure (25.8% vs. 9.7%) and atrial fibrillation (29.0% vs. 5.6%) were more frequent in CD patients than in the controls (p < 0.05). C-reactive protein levels were lower in CD patients compared with the controls (55.5 [35.7, 85.0] vs. 94.3 [50.7, 167.5] mg/dL). In-hospital management, outcomes and complications were similar between the groups. In this large Brazilian COVID-19 Registry, CD patients had a higher prevalence of atrial fibrillation and chronic heart failure compared with non-CD controls, with no differences in-hospital outcomes. The lower C-reactive protein levels in CD patients require further investigation.

Diversity and interactions among triatomine bugs, their blood feeding sources, gut microbiota and Trypanosoma cruzi in the Sierra Nevada de Santa Marta in Colombia.

Chagas disease remains a major neglected disease in Colombia. We aimed to characterize Trypanosoma cruzi transmission networks in the Sierra Nevada de Santa Marta (SNSM) region, to shed light on disease ecology and help optimize control strategies. Triatomines were collected in rural communities and analyzed for blood feeding sources, parasite diversity and gut microbiota composition through a metagenomic and deep sequencing approach. Triatoma dimidiata predominated, followed by Rhodnius prolixus, Triatoma maculata, Rhodnius pallescens, Panstrongylus geniculatus and Eratyrus cuspidatus. Twenty-two species were identified as blood sources, resulting in an integrated transmission network with extensive connectivity among sylvatic and domestic host species. Only TcI parasites were detected, predominantly from TcIb but TcIa was also reported. The close relatedness of T. cruzi strains further supported the lack of separate transmission cycles according to habitats or triatomine species. Triatomine microbiota varied according to species, developmental stage and T. cruzi infection. Bacterial families correlated with the presence/absence of T. cruzi were identified. In conclusion, we identified a domestic transmission cycle encompassing multiple vector species and tightly connected with sylvatic hosts in the SNSM region, rather than an isolated domestic transmission cycle. Therefore, integrated interventions targeting all vector species and their contact with humans should be considered.

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.

Immunothrombotic dysregulation in chagas disease and COVID-19: a comparative study of anticoagulation.

Chagas and COVID-19 are diseases caused by Trypanosoma cruzi and SARS-CoV-2, respectively. These diseases present very different etiological agents despite showing similarities such as susceptibility/risk factors, pathogen-associated molecular patterns (PAMPs), recognition of glycosaminoglycans, inflammation, vascular leakage hypercoagulability, microthrombosis, and endotheliopathy; all of which suggest, in part, treatments with similar principles. Here, both diseases are compared, focusing mainly on the characteristics related to dysregulated immunothrombosis. Given the in-depth investigation of molecules and mechanisms related to microthrombosis in COVID-19, it is necessary to reconsider a prompt treatment of Chagas disease with oral anticoagulants.

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.

Ex vivo characterization of Breg cells in patients with chronic Chagas disease.

Despite the growing importance of the regulatory function of B cells in many infectious diseases, their immunosuppressive role remains elusive in chronic Chagas disease (CCD). Here, we studied the proportion of different B cell subsets and their capacity to secrete IL-10 ex vivo in peripheral blood from patients with or without CCD cardiomyopathy. First, we immunophenotyped peripheral blood mononuclear cells from patients according to the expression of markers CD19, CD24, CD38 and CD27 and we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed an increased proportion of naïve B10 cells and a tendency to a higher frequency of transitional B10 cells compared to non-infected donors. Our research demonstrates that transitional B cells are greatly expanded in patients with the cardiac form of CCD and these cells retain the ability to secrete IL-10. These findings provide insight into the phenotypic distribution of regulatory B cells in CCD, an important step towards new strategies to prevent cardiomyopathy associated with T. cruzi infection.

The distribution and chemical coding of enteroendocrine cells in Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is caused by the parasite, Trypanosoma cruzi that causes chronic cardiac and digestive dysfunction. Megacolon, an irreversible dilation of the left colon, is the main feature of the gastrointestinal form of Chagas disease. Patients have severe constipation, a consequence of enteric neuron degeneration associated with chronic inflammation. Dysmotility, infection, neuronal loss and a chronic exacerbated inflammation, all observed in Chagas disease, can affect enteroendocrine cells (EEC) expression, which in turn, could influence the inflammatory process. In this study, we investigated the distribution and chemical coding of EEC in the dilated and non-dilated portion of T. cruzi-induced megacolon and in non-infected individuals (control colon). Using immunohistochemistry, EECs were identified by applying antibodies to chromogranin A (CgA), glucagon-like peptide 1 (GLP-1), 5-hydroxytryptamine (5-HT), peptide YY (PYY) and somatostatin (SST). Greater numbers of EEC expressing GLP-1 and SST occurred in the dilated portion compared to the non-dilated portion of the same patients with Chagas disease and in control colon, but numbers of 5-HT and PYY EEC were not significantly different. However, it was noticeable that EEC in which 5-HT and PYY were co-expressed were common in control colon, but were rare in the non-dilated and absent in the dilated portion of chagasic megacolon. An increase in the number of CgA immunoreactive EEC in chagasic patients reflected the increases in EEC numbers summarised above. Our data suggests that the denervation and associated chronic inflammation are accompanied by changes in the number and coding of EEC that could contribute to disorders of motility and defence in the chagasic megacolon.

Biological and Molecular Effects of Trypanosoma cruzi Residence in a LAMP-Deficient Intracellular Environment.

Trypanosoma cruzi invades non-professional phagocytic cells by subverting their membrane repair process, which is dependent on membrane injury and cell signaling, intracellular calcium increase, and lysosome recruitment. Cells lacking lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) are less permissive to parasite invasion but more prone to parasite intracellular multiplication. Several passages through a different intracellular environment can significantly change T. cruzi's gene expression profile. Here, we evaluated whether one single passage through LAMP-deficient (KO) or wild-type (WT) fibroblasts, thus different intracellular environments, could influence T. cruzi Y strain trypomastigotes' ability to invade L6 myoblasts and WT fibroblasts host cells. Parasites released from LAMP2 KO cells (TcY-L2-/-) showed higher invasion, calcium signaling, and membrane injury rates, for the assays in L6 myoblasts, when compared to those released from WT (TcY-WT) or LAMP1/2 KO cells (TcY-L1/2-/-). On the other hand, TcY-L1/2-/- showed higher invasion, calcium signaling, and cell membrane injury rates, for the assays in WT fibroblasts, compared to TcY-WT and TcY-L1/2-/-. Albeit TcY-WT presented an intermediary invasion and calcium signaling rates, compared to the others, in WT fibroblasts, they induced lower levels of injury, which reinforces that signals mediated by surface membrane protein interactions also have a significant contribution to trigger host cell calcium signals. These results clearly show that parasites released from WT or LAMP KO cells are distinct from each other. Additionally, these parasites' ability to invade the cell may be distinct depending on which cell type they interact with. Since these alterations most likely would reflect differences among parasite surface molecules, we also evaluated their proteome. We identified few protein complexes, membrane, and secreted proteins regulated in our dataset. Among those are some members of MASP, mucins, trans-sialidases, and gp63 proteins family, which are known to play an important role during parasite infection and could correlate to TcY-WT, TcY-L1/2-/-, and TcY-L2-/- biological behavior.

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.