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INTRODUCTION: Upon infection, Trypanosoma cruzi, a protozoan parasite, crosses the placental barrier and causes congenital Chagas disease. Ex vivo infection of human placental explants (HPEs) with the parasite induces apoptotic cell death. This cellular process involves changes in gene expression, which are partially regulated by miRNAs. In this study, we investigated the role of miR-512-3p, a highly expressed miRNA in the placenta, in parasite-induced apoptosis. METHODS: HPE cells were transfected with antagomirs or mimics of miR-512-3p and subsequently challenged with the parasite. The expression levels of miR-512-3p, caspase 3, caspase 8, and Livin were measured using RT-qPCR, and apoptotic cell death was analyzed based on caspase activity and DNA fragmentation assays. RESULTS: Targeted inhibition of miR-512-3p effectively prevented parasite-induced expression and enzymatic activity of caspase 3 and caspase 8. However, it did not completely prevent DNA fragmentation, indicating the involvement of other factors in this process. Furthermore, the findings suggest that Livin may be regulated by miR-512-3p. DISCUSSION: Our findings suggest that miR-512-3p modulates parasite-induced apoptosis in the trophoblast. By understanding the mechanisms involved in this process, we can gain insights into the pathogenesis of congenital Chagas disease and develop targeted therapeutic strategies.
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Doença de Chagas , MicroRNAs , Trypanosoma cruzi , Humanos , Gravidez , Feminino , Placenta/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Caspase 3/metabolismo , Caspase 8 , Doença de Chagas/genética , Apoptose/genéticaRESUMO
Cardiac fibroblasts (CFs) activation is a common response to most pathological conditions affecting the heart, characterized by increased cellular secretory capacity and increased expression of fibrotic markers, such as collagen I and smooth muscle actin type alpha (α-SMA). Fibrotic activation of CFs induces the increase in tissue protein content, with the consequent tissue stiffness, diastolic dysfunction, and heart failure. Therefore, the search for new mechanisms of CFs activation is important to find novel treatments for cardiac diseases characterized by fibrosis. In this regard, TGF-ß1, a cytokine with proinflammatory and fibrotic properties, is crucial in the CFs activation and the development of fibrotic diseases, whereas its molecular targets are not completely known. Serum and glucocorticoid-regulated kinase (SGK1) is a protein involved in various pathophysiological phenomena, especially cardiac and renal diseases that curse with fibrosis. Additionally, SGK1 phosphorylates and regulates the activity and expression of several targets, highlighting FoxO3a for its role in the regulation of oxidative stress and CFs activation induced by TGF-ß1. However, the regulation of SGK1 by TGF-ß1 and its role in CFs activation have not been studied. In this work, we evaluate the role of SGK1 in CFs isolated from neonatal Sprague-Dawley rats. The participation of SGK1 in the fibrotic activation of CFs induced by TGF-ß1 was analyzed, using an inhibitor or siRNA of SGK1. In addition, the role of SGK1 on the regulation of FoxO3a and oxidative stress induced by TGF-ß1 was analyzed. Our results indicate that TGF-ß1 increased both the activity and expression of SGK1 in CFs, requiring the activation of MAPKs, ERK1/2, p38 and JNK, while inhibition and silencing of SGK1 prevented TGF-ß1-induced fibrotic activation of CFs. In addition, SGK1 inhibition prevented FoxO3a inactivation and expression reduction, catalase and SOD2 expression decrease, and the increase of oxidative stress induced by TGF-ß1. Taken together, our results position SGK1 as an important regulator of CFs activation driven by TGF-ß1, at least in part, through the regulation of FoxO3a and oxidative stress.
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Miocárdio , Fator de Crescimento Transformador beta1 , Ratos , Animais , Ratos Sprague-Dawley , Miocárdio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Estresse Oxidativo , Fibroblastos/metabolismo , FibroseRESUMO
Cardiac cells respond to various pathophysiological stimuli, synthesizing inflammatory molecules that allow tissue repair and proper functioning of the heart; however, perpetuation of the inflammatory response can lead to cardiac fibrosis and heart dysfunction. High concentration of glucose (HG) induces an inflammatory and fibrotic response in the heart. Cardiac fibroblasts (CFs) are resident cells of the heart that respond to deleterious stimuli, increasing the synthesis and secretion of both fibrotic and proinflammatory molecules. The molecular mechanisms that regulate inflammation in CFs are unknown, thus, it is important to find new targets that allow improving treatments for HG-induced cardiac dysfunction. NFκB is the master regulator of inflammation, while FoxO1 is a new participant in the inflammatory response, including inflammation induced by HG; however, its role in the inflammatory response of CFs is unknown. The inflammation resolution is essential for an effective tissue repair and recovery of the organ function. Lipoxin A4 (LXA4) is an anti-inflammatory agent with cytoprotective effects, while its cardioprotective effects have not been fully studied. Thus, in this study, we analyze the role of p65/NFκB, and FoxO1 in CFs inflammation induced by HG, evaluating the anti-inflammatory properties of LXA4. Our results demonstrated that HG induces the inflammatory response in CFs, using an in vitro and ex vivo model, while FoxO1 inhibition and silencing prevented HG effects. Additionally, LXA4 inhibited the activation of FoxO1 and p65/NFκB, and inflammation of CFs induced by HG. Therefore, our results suggest that FoxO1 and LXA4 could be novel drug targets for the treatment of HG-induced inflammatory and fibrotic disorders in the heart.
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Lipoxinas , Humanos , Lipoxinas/farmacologia , NF-kappa B , Inflamação/tratamento farmacológico , Fibrose , Glucose/toxicidade , Fibroblastos , Proteína Forkhead Box O1RESUMO
Trypanosoma cruzi and Toxoplasma gondii are two zoonotic parasites that constitute significant human and animal health threats, causing a significant economic burden worldwide. Both parasites can be transmitted congenitally, but transmission rates for T. gondii are high, contrary to what has been observed for T. cruzi. The probability of congenital transmission depends on complex interactions between the pathogen and the host, including the modulation of host cell gene expression by miRNAs. During ex vivo infection of canine and ovine placental explants, we evaluated the expression of 3 miRNAs (miR-30e-3p, miR-3074-5p, and miR-127-3p) previously associated with parasitic and placental diseases and modulated by both parasites. In addition, we identified the possible target genes of the miRNAs by using computational prediction tools and performed GO and KEGG enrichment analyses to identify the biological functions and associated pathologies. The three miRNAs are differentially expressed in the canine and ovine placenta in response to T. cruzi and T. gondii. We conclude that the observed differential expression and associated functions might explain, at least partially, the differences in transmission rates and susceptibility to parasite infection in different species.
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Doença de Chagas , MicroRNAs , Toxoplasma , Trypanosoma cruzi , Animais , Doença de Chagas/veterinária , Cães , Feminino , Humanos , MicroRNAs/genética , Placenta/parasitologia , Gravidez , Ovinos , Toxoplasma/genética , Trypanosoma cruzi/genéticaRESUMO
MicroRNAs (miRNAs) are a group of small non-coding RNAs present in a wide diversity of organisms. MiRNAs regulate gene expression at a post-transcriptional level through their interaction with the 3' untranslated regions of target mRNAs, inducing translational inhibition or mRNA destabilization and degradation. Thus, miRNAs regulate key biological processes, such as cell death, signal transduction, development, cellular proliferation and differentiation. The dysregulation of miRNAs biogenesis and function is related to the pathogenesis of diseases, including parasite infection. Moreover, during host-parasite interactions, parasites and host miRNAs determine the probability of infection and progression of the disease. The present review is focused on the possible role of miRNAs in the pathogenesis of diseases of clinical interest caused by parasitic protists. In addition, the potential role of miRNAs as targets for the design of drugs and diagnostic and prognostic markers of parasitic diseases is also discussed.
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MicroRNAs , Parasitos , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , MicroRNAs/metabolismo , Parasitos/genética , Parasitos/metabolismoRESUMO
Congenital Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for 22.5% of new cases each year. However, placental transmission occurs in only 5% of infected mothers and it has been proposed that the epithelial turnover of the trophoblast can be considered a local placental defense against the parasite. Thus, Trypanosoma cruzi induces cellular proliferation, differentiation, and apoptotic cell death in the trophoblast, which are regulated, among other mechanisms, by small non-coding RNAs such as microRNAs. On the other hand, ex vivo infection of human placental explants induces a specific microRNA profile that includes microRNAs related to trophoblast differentiation such as miR-512-3p miR-515-5p, codified at the chromosome 19 microRNA cluster. Here we determined the expression validated target genes of miR-512-3p and miR-515-5p, specifically human glial cells missing 1 transcription factor and cellular FLICE-like inhibitory protein, as well as the expression of the main trophoblast differentiation marker human chorionic gonadotrophin during ex vivo infection of human placental explants, and examined how the inhibition or overexpression of both microRNAs affects parasite infection. We conclude that Trypanosoma cruzi-induced trophoblast epithelial turnover, particularly trophoblast differentiation, is at least partially mediated by placenta-specific miR-512-3p and miR-515-5p and that both miRNAs mediate placental susceptibility to ex vivo infection of human placental explants. Knowledge about the role of parasite-modulated microRNAs in the placenta might enable their use as biomarkers, as prognostic and therapeutic tools for congenital Chagas disease in the future.
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microRNAs (miRNAs) are a group of small non-coding RNAs that regulate gene expression post-transcriptionally through their interaction with the 3' untranslated regions (3' UTR) of target mRNAs, affecting their stability and/or translation. Therefore, miRNAs regulate biological processes such as signal transduction, cell death, autophagy, metabolism, development, cellular proliferation, and differentiation. Dysregulated expression of microRNAs is associated with infectious diseases, where miRNAs modulate important aspects of the parasite-host interaction. Helminths are parasitic worms that cause various neglected tropical diseases affecting millions worldwide. These parasites have sophisticated mechanisms that give them a surprising immunomodulatory capacity favoring parasite persistence and establishment of infection. In this review, we analyze miRNAs in infections caused by helminths, emphasizing their role in immune regulation and its implication in diagnosis, prognosis, and the development of therapeutic strategies.
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Introduction: Chronic Chagasic cardiomyopathy (CCC), caused by the protozoan Trypanosoma cruzi, is the most severe manifestation of Chagas disease.CCC is characterized by cardiac inflammation and fibrosis caused by a persistent inflammatory response. Following infection, macrophages secrete inflammatory mediators such as IL-1ß, IL-6, and TNF-α to control parasitemia. Although this response contains parasite infection, it causes damage to the heart tissue. Thus, the use of immunomodulators is a rational alternative to CCC. Rho-associated kinase (ROCK) 1 and 2 are RhoA-activated serine/threonine kinases that regulate the actomyosin cytoskeleton. Both ROCKs have been implicated in the polarization of macrophages towards an M1 (pro-inflammatory) phenotype. Statins are FDA-approved lipid-lowering drugs that reduce RhoA signaling by inhibiting geranylgeranyl pyrophosphate (GGPP) synthesis. This work aims to identify the effect of statins on U937 macrophage polarization and cardiac tissue inflammation and its relationship with ROCK activity during T. cruzi infection. Methods: PMA-induced, wild-type, GFP-, CA-ROCK1- and CA-ROCK2-expressing U937 macrophages were incubated with atorvastatin, or the inhibitors Y-27632, JSH-23, TAK-242, or C3 exoenzyme incubated with or without T. cruzi trypomastigotes for 30 min to evaluate the activity of ROCK and the M1 and M2 cytokine expression and secretion profiling. Also, ROCK activity was determined in T. cruzi-infected, BALB/c mice hearts. Results: In this study, we demonstrate for the first time in macrophages that incubation with T. cruzi leads to ROCK activation via the TLR4 pathway, which triggers NF-κB activation. Inhibition of ROCKs by Y-27632 prevents NF-κB activation and the expression and secretion of M1 markers, as does treatment with atorvastatin. Furthermore, we show that the effect of atorvastatin on the NF-kB pathway and cytokine secretion is mediated by ROCK. Finally, statin treatment decreased ROCK activation and expression, and the pro-inflammatory cytokine production, promoting anti-inflammatory cytokine expression in chronic chagasic mice hearts. Conclusion: These results suggest that the statin modulation of the inflammatory response due to ROCK inhibition is a potential pharmacological strategy to prevent cardiac inflammation in CCC.
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Cardiomiopatias , Doença de Chagas , Inibidores de Hidroximetilglutaril-CoA Redutases , Trypanosoma cruzi , Humanos , Animais , Camundongos , Trypanosoma cruzi/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Quinases Associadas a rho/metabolismo , NF-kappa B/metabolismo , Atorvastatina/farmacologia , Células U937 , Macrófagos/metabolismo , Doença de Chagas/genética , Citocinas/metabolismo , Cardiomiopatias/metabolismo , Inflamação/metabolismoRESUMO
Chagas disease and toxoplasmosis, caused by Trypanosoma cruzi and Toxoplasma gondii, respectively, are important zoonotic diseases affecting humans, companion animals, and livestock, responsible for major health and economic burden. Both parasites can be transmitted vertically in different mammalian species through the placenta. Of note, the transmission rate of T. cruzi is low in dogs, whereas that of T. gondii is high in sheep. The probability of congenital infection depends on complex parasite-host interactions; parasite factors, maternal and fetal immune responses and placental responses all have a role in infection establishment. Since the innate immune response is regulated, at least partially, by NF-κB signaling pathways, our main objective was to determine the effect of ex vivo infection of canine (CPE) and ovine (OPE) placental explants with both parasites, on the activation of canonical and non-canonical NF-κB pathways and its relation to infection. Here, we show that T. cruzi activates both the NF-κB canonical and non-canonical pathways in CPE and OPE, unlike T. gondii, that activates only the canonical pathway in CPE and has no effect on the non-canonical pathway in both explants. Moreover, the inhibition of either or both NF-κB pathways increases the DNA load of T. cruzi in both explants, modulates, on the other hand, T. gondii infection in a differential fashion. Overall, we conclude that the differential modulation of the NF-κB pathways by both pathogens in placental explants might explain, at least partially, the differences in transmission rates of T. cruzi and T. gondii in different mammalian species.
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Cães/metabolismo , Placenta/parasitologia , Ovinos/metabolismo , Transdução de Sinais/imunologia , Toxoplasma/fisiologia , Trypanosoma cruzi/fisiologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Isoquinolinas/farmacologia , NF-kappa B/metabolismo , Nitrilas/farmacologia , Placenta/imunologia , Placenta/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sulfonas/farmacologia , Técnicas de Cultura de Tecidos , Toxoplasma/imunologia , Trypanosoma cruzi/imunologiaRESUMO
Trypanosoma cruzi and Toxoplasma gondii are two parasites than can be transmitted from mother to child through the placenta. However, congenital transmission rates are low for T. cruzi and high for T. gondii. Infection success or failure depends on complex parasite-host interactions in which parasites can alter host gene expression by modulating non-coding RNAs such as miRNAs. As of yet, there are no reports on altered miRNA expression in placental tissue in response to either parasite. Therefore, we infected human placental explants ex vivo by cultivation with either T. cruzi or T. gondii for 2 h. We then analyzed the miRNA expression profiles of both types of infected tissue by miRNA sequencing and quantitative PCR, sequence-based miRNA target prediction, pathway functional enrichment, and upstream regulator analysis of differentially expressed genes targeted by differentially expressed miRNAs. Both parasites induced specific miRNA profiles. GO analysis revealed that the in silico predicted targets of the differentially expressed miRNAs regulated different cellular processes involved in development and immunity, and most of the identified KEGG pathways were related to chronic diseases and infection. Considering that the differentially expressed miRNAs identified here modulated crucial host cellular targets that participate in determining the success of infection, these miRNAs might explain the differing congenital transmission rates between the two parasites. Molecules of the different pathways that are regulated by miRNAs and modulated during infection, as well as the miRNAs themselves, may be potential targets for the therapeutic control of either congenital Chagas disease or toxoplasmosis.
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Doença de Chagas , Regulação da Expressão Gênica/imunologia , MicroRNAs/imunologia , Placenta , Toxoplasma/imunologia , Toxoplasmose , Trypanosoma cruzi/imunologia , Doença de Chagas/imunologia , Doença de Chagas/patologia , Feminino , Humanos , Placenta/imunologia , Placenta/parasitologia , Placenta/patologia , Gravidez , Toxoplasmose/imunologia , Toxoplasmose/patologiaRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, endemic in Latin America but distributed worldwide because of migration. Without appropriate treatment, the disease progresses from an acute asymptomatic phase to a chronic, progressive inflammatory cardiomyopathy causing heart failure and death. Despite specific trypanocidal therapy, heart damage progression cannot be stopped or reversed. Statins, as part of their pleiotropic actions, can modulate chagasic myocarditis by inducing the production of 15-epi-lipoxin A4 (15-epi-LXA4), a proresolution lipid mediator in inflammation. Furthermore, several reports suggest that simvastatin activates the Notch pathway after stroke in cerebral endothelial cells, enhancing blood flow by promoting angiogenesis. Thus, statins are an attractive therapeutic strategy for modulating the Notch pathway to reverse the chronic heart damage induced by T. cruzi BALB/c mice chronically infected with T. cruzi were treated with 1 mg/kg/day simvastatin or 25 µg/kg/day 15-epi-LXA4 for 20 days. During the treatment period, cardiac function was evaluated by echocardiography. At 80 days postinfection, the heart tissues were assessed for Notch 1 activity. T. cruzi infection activated the Notch 1 pathway, and simvastatin (but not 15-epi-lipoxin A4) produced a further increase in that activity, correlating with improvement in the ejection fraction and histopathologic findings typical of T. cruzi infection, including improvements in inflammation and fibrosis. Moreover, simvastatin increased the number of isolectin B4-positive cells, suggesting active angiogenesis in the chronically infected hearts without alteration of the parasitic load. Simvastatin, probably acting through the Notch 1 pathway, decreases inflammation, improving cardiac function in mice chronically infected with T. cruzi.
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Cardiomiopatia Chagásica , Doença de Chagas , Trypanosoma cruzi , Animais , Cardiomiopatia Chagásica/tratamento farmacológico , Células Endoteliais , Camundongos , Camundongos Endogâmicos BALB C , Sinvastatina/farmacologia , Sinvastatina/uso terapêuticoRESUMO
INTRODUCTION: Colorectal cancer (CRC) is a critical health issue worldwide. The high rate of liver and lung metastasis associated with CRC creates a significant barrier to effective and efficient therapy. Tumour cells, including CRC cells, have metabolic alterations, such as high levels of glycolytic activity, increased cell proliferation and invasiveness, and chemo- and radio-resistance. However, the abnormally elevated mitochondrial transmembrane potential of these cells also provides an opportunity to develop drugs that selectively target the mitochondrial functions of tumour cells. METHODS: In this work, we used a new batch of benzoic acid esters with cytotoxic activities attached to the triphenylphosphonium group as a vehicle to target tumour mitochondria and improve their activity. We evaluated the cytotoxicity, selectivity, and mechanism of action of these derivatives, including the effects on energy stress-induced apoptosis and metabolic behaviour in the human CRC cell lines HCT-15 and COLO-205. RESULTS: The benzoic acid derivatives selectively targeted the tumour cells with high potency and efficacy. The derivatives induced the uncoupling of the oxidative phosphorylation system, decreased the transmembrane potential, and reduced ATP levels while increasing AMPK activation, thereby triggering tumour cell apoptosis in both tumour cell lines tested. CONCLUSION: The benzoic acid derivatives studied here are promising candidates for assessing in vivo models of CRC, despite the diverse metabolic characteristics of these tumour cells.
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Antineoplásicos/farmacologia , Benzoatos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Compostos Organofosforados/farmacologia , Trifosfato de Adenosina/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Oxigênio/metabolismoRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, and Toxoplasma gondii, which is responsible for Toxoplasmosis, are two parasites that cause significant protozoan zoonoses and consequently important economic losses in human, companion animals and livestock. For the congenital transmission to occur, both parasites must cross the barrier present in the mammalian placenta, which differs between species. Particularly, hemochorial, endotheliochorial and epitheliochorial placental barriers are present, respectively, in human, dog and sheep. The type of placental barrier has been associated with the probability of transmission of pathogens. In this study, we used experimental placental ex vivo infection models of T. cruzi and T. gondii in the above-mentioned mammals in order to study tissue alterations and to compare infection efficiency. Here, we infected placental term explants from human, dog and sheep and analyzed tissue damage by standard histological and histochemical methods. Comparative infection efficiency was determined by quantitative PCR. Both parasites are able to infect the different placental explants; however, more T. gondii parasites were detected, and T. gondii causes a more severe tissue damage in human and canine explants than T. cruzi. The histopathological changes observed in ovine placenta explants were similar in presence of both parasites. We conclude that the infection efficiency of T. gondii is higher, compared to T. cruzi, during the ex vivo infection of human, canine and ovine placental explants. In addition, the ex vivo infection of mammalian placental explants constitutes an interesting experimental approach to study part of the infection mechanisms as well as host responses during congenital infection of both parasites.
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Doença de Chagas/patologia , Placenta/patologia , Placenta/parasitologia , Toxoplasmose Animal/patologia , Toxoplasmose/patologia , Animais , Doença de Chagas/veterinária , Cães/parasitologia , Feminino , Humanos , Técnicas In Vitro , Gravidez , Ovinos/parasitologia , Toxoplasma/patogenicidade , Trypanosoma cruzi/patogenicidadeRESUMO
Chagas disease is a vector-borne disease caused by the protozoan parasite Trypanosoma cruzi. Current therapy involves benznidazole. Benznidazole and other drugs can modify gene expression patterns, improving the response to the inflammatory influx induced by T. cruzi and decreasing the endothelial activation or immune cell recruitment, among other effects. Here, we performed a microarray analysis of human umbilical vein endothelial cells (HUVECs) treated with benznidazole and the anti-inflammatory drugs acetylsalicylic acid or simvastatin and infected with T. cruzi. Parasitic infection produces differential expression of a set of genes in HUVECs treated with benznidazole alone or a combination with simvastatin or acetylsalicylic acid. The differentially expressed genes were involved in inflammation, adhesion, cardiac function, and remodeling. Notch1 and high mobility group B1 were genes of interest in this analysis due to their importance in placental development, cardiac development, and inflammation. Quantitative polymerase chain reaction confirmation of these two genes indicated that both are upregulated in the presence of benznidazole.
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Aspirina/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/metabolismo , Células Endoteliais da Veia Umbilical Humana/parasitologia , Nitroimidazóis/farmacologia , Receptor Notch1/metabolismo , Sinvastatina/farmacologia , Células Cultivadas , Doença de Chagas/tratamento farmacológico , Humanos , Trypanosoma cruziRESUMO
Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) are the causative agents of Chagas disease and Toxoplasmosis. T. cruzi and T. gondii present, respectively, low and high congenital transmission rates and induce a distinctive cytokine/chemokine profile in ex vivo infected human placental explants (HPE). Since the innate immune response is regulated, at least partially, by NF-κB signaling pathways, our main objective was to determine the effect of ex vivo infection with both parasites on the activation of canonical and non-canonical NF-κB pathways and its relation to parasite infection. T. cruzi activates both, the canonical and non-canonical pathways of NF-κB, unlike T. gondii, which has no effect on the canonical pathway and inhibits the non-canonical pathway. The inhibition of both pathways of NF-κB increases the DNA load of T. cruzi and T. gondii in HPE. Therefore, the differential modulation of NF-κB signal transduction pathways by both parasites might explain, at least partially, the low and high congenital transmission rates of T. cruzi and T. gondii.
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Doença de Chagas/imunologia , NF-kappa B/fisiologia , Placenta/parasitologia , Transdução de Sinais/fisiologia , Toxoplasmose/imunologia , Animais , Doença de Chagas/transmissão , Chlorocebus aethiops , Feminino , Humanos , Imunidade Inata , Gravidez , Toxoplasmose/transmissão , Trypanosoma cruzi/imunologia , Células VeroRESUMO
In the search for a more effective chemotherapy for the treatment of Chagas' disease and human African trypanosomiasis, caused by Trypanosoma cruzi and Trypanosoma brucei parasites, respectively, the use of organometallic compounds may be a promising strategy. In this work, eight new heterobimetallic compounds are described including four 5-nitrofuryl containing thiosemicarbazones as bioactive ligands (HL1-HL4) and dppf = 1,1'-bis(diphenylphosphino) ferrocene as an organometallic co-ligand. Complexes of the formula [MII(L)(dppf)](PF6) with M = Pd or Pt were synthesized and fully characterized in the solid state and in solution, including the determination of the molecular structure of four of them by single crystal X-ray diffraction methods. Most compounds showed activity in the low micromolar or submicromolar range against both parasites, with the platinum compounds being more active than the palladium analogues. Activity was significantly increased by generation of the M-dppf compounds (3-24 fold increase with respect to free ligands HL for T. cruzi and up to 99 fold increase with respect to HL for T. brucei). The inclusion of the organometallic co-ligand also led to lower toxicity in mammalian cells and higher selectivity towards both parasites when compared to the free HL compounds. The complexes interact with DNA and affect the redox metabolism of the parasites. Furthermore, the most active and selective compound of the new series showed no in vivo toxicity in zebrafish embryos.
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Compostos Ferrosos/química , Metalocenos/química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Paládio/química , Platina/química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Células A549 , Animais , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Compostos Organometálicos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Soroalbumina Bovina/metabolismo , Tripanossomicidas/metabolismo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
BACKGROUND: Neglected diseases are becoming more prevalent due to globalization. This has inspired active research in the development of new drugs for the treatment of parasitic diseases such as Chagas disease. OBJECTIVES: With the aim of finding new trypanocidal agents, we report the in vitro evaluation of a new series of 3-amidocoumarins with or without hydroxyl substituents at position 4 of the coumarin ring. METHODS: Electrochemical and biological assays were performed in order to assess the antioxidant and trypanocidal potential of these compounds and to better understand the mechanisms involved in their activity. RESULTS: Most of the studied compounds showed high trypanocidal activity against both epimastigote and trypomastigote forms, with IC50 values in the low micromolar range. Some of them have greater activity and selectivity than the reference compound, nifurtimox. CONCLUSION: Compound 2 is the most active of this series, being also non-cytotoxic against murine RAW 264.7 macrophages. Electrochemical and radical scavenging experiments were carried out, providing new information about the profile of the best derivatives, and the potential therapeutic application of the new 3-amidocoumarins.
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Amidas/farmacologia , Antioxidantes/farmacologia , Cumarínicos/farmacologia , Tripanossomicidas/farmacologia , Amidas/síntese química , Amidas/química , Amidas/toxicidade , Animais , Antioxidantes/síntese química , Antioxidantes/química , Antioxidantes/toxicidade , Cromanos/farmacologia , Cumarínicos/síntese química , Cumarínicos/química , Cumarínicos/toxicidade , Técnicas Eletroquímicas , Radicais Livres/química , Camundongos , Modelos Químicos , Estrutura Molecular , Nifurtimox/farmacologia , Testes de Sensibilidade Parasitária , Células RAW 264.7 , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidadeRESUMO
The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host-parasite interaction.
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Doença de Chagas/imunologia , NADPH Desidrogenase/imunologia , Prostaglandina-Endoperóxido Sintases/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/genética , Doença de Chagas/patologia , Chlorocebus aethiops , Células HeLa , Humanos , NADPH Desidrogenase/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Células VeroRESUMO
Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase ß participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase ß To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase ß which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase ß were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase ß mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase ß in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.
Assuntos
DNA Polimerase beta/biossíntese , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Northern Blotting , Western Blotting , DNA Polimerase beta/metabolismo , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Peróxidos/toxicidade , Fosforilação , Proteoma/análise , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/efeitos dos fármacosRESUMO
To face the high costs of developing new drugs, researchers in both industry and academy are looking for ways to repurpose old drugs for new uses. In this sense, bisphosphonates that are clinically used for bone diseases have been studied as agents against Trypanosoma cruzi, causative parasite of Chagas disease. In this work, the development of first row transition metal complexes (M = Co2+, Mn2+, Ni2+) with the bisphosphonate ibandronate (iba, H4iba representing the neutral form) is presented. The in-solution behavior of the systems containing iba and the selected 3d metal ions was studied by potentiometry. Mononuclear complexes [M(Hxiba)](2-x)- (x = 0-3) and [M(Hiba)2]4- together with the formation of the neutral polynuclear species [M2iba] and [M3(Hiba)2] were detected for all studied systems. In the solid state, complexes of the formula [M3(Hiba)2(H2O)4]·6H2O were obtained and characterized. All obtained complexes, forming [M(Hiba)]- species under the conditions of the biological studies, were more active against the amastigote form of T. cruzi than the free iba, showing no toxicity in mammalian Vero cells. In addition, the same complexes were selective inhibitors of the parasitic farnesyl diphosphate synthase (FPPS) enzyme showing poor inhibition of the human one. However, the increase of the anti-T. cruzi activity upon coordination could not be explained neither through the inhibition of TcFPPS nor through the inhibition of TcSPPS (T. cruzi solanesyl-diphosphate synthase). The ability of the obtained metal complexes of catalyzing the generation of free radical species in the parasite could explain the observed anti-T. cruzi activity.