Multitarget anti-parasitic activities of isoquinoline alkaloids isolated from Hippeastrum aulicum (Amaryllidaceae).

BACKGROUND: Chagas disease and leishmaniasis affect a significant portion of the Latin American population and still lack efficient treatments. In this context, natural products emerge as promising compounds for developing more effective therapies, aiming to mitigate side effects and drug resistance. Notably, species from the Amaryllidaceae family emerge as potential reservoirs of antiparasitic agents due to the presence of diverse biologically active alkaloids. PURPOSE: To assess the anti-Trypanosoma cruzi and anti-Leishmania infantum activity of five isolated alkaloids from Hippeastrum aulicum Herb. (Amaryllidaceae) against different life stages of the parasites using in silico and in vitro assays. Furthermore, molecular docking was employed to evaluate the interaction of the most active alkaloids. METHODS: Five natural isoquinoline alkaloids isolated in suitable quantities for in vitro testing underwent preliminary in silico analysis to predict their potential efficacy against Trypanosoma cruzi (amastigote and trypomastigote forms) and Leishmania infantum (amastigote and promastigote forms). The in vitro antiparasitic activity and mammalian cytotoxicity were investigated with a subsequent comparison of both analysis (in silico and in vitro) findings. Additionally, this study employed the molecular docking technique, utilizing cruzain (T. cruzi) and sterol 14α-demethylase (CYP51, L. infantum) as crucial biological targets for parasite survival, specifically focusing on compounds that exhibited promising activities against both parasites. RESULTS: Through computational techniques, it was identified that the alkaloids haemanthamine (1) and lycorine (8) were the most active against T. cruzi (amastigote and trypomastigote) and L. infantum (amastigote and promastigote), while also revealing unprecedented activity of alkaloid 7­methoxy-O-methyllycorenine (6). The in vitro analysis confirmed the in silico tests, in which compound 1 presented the best activities against the promastigote and amastigote forms of L. infantum with half-maximal inhibitory concentration (IC50) 0.6 µM and 1.78 µM, respectively. Compound 8 exhibited significant activity against the amastigote form of T. cruzi (IC50 7.70 µM), and compound 6 demonstrated activity against the trypomastigote forms of T. cruzi and amastigote of L. infantum, with IC50 values of 89.55 and 86.12 µM, respectively. Molecular docking analyses indicated that alkaloids 1 and 8 exhibited superior interaction energies compared to the inhibitors. CONCLUSION: The hitherto unreported potential of compound 6 against T. cruzi trypomastigotes and L. infantum amastigotes is now brought to the forefront. Furthermore, the acquired dataset signifies that the isolated alkaloids 1 and 8 from H. aulicum might serve as prototypes for subsequent structural refinements aimed at the exploration of novel leads against both T. cruzi and L. infantum parasites.

Oral Chagas Disease in Colombia-Confirmed and Suspected Routes of Transmission.

Chagas disease (CD) remains endemic throughout many regions of Colombia despite implementing decades of vector control strategies in several departments. Some regions have had a significant decrease in vectorial transmission, but the oral ingestion of Trypanosoma cruzi through consumption of contaminated food and drink products is increasingly described. This form of transmission has important public health relevance in Colombia due to an increase in reported acute CD cases and clinical manifestations that often lead to significant morbidity and mortality. Oral CD in Colombia has been associated with the consumption of contaminated fruit juices, such as palm wine, sugar cane, or tangerine juice and water for consumption, or contaminated surfaces where food has been prepared. Another interesting route of oral transmission includes ingestion of unbeknownst infected armadillos' blood, which is related to a traditional medicine practice in Colombia. Some earlier reports have also implemented consumption of infected bush meat as a source, but this is still being debated. Within the Amazon Basin, oral transmission is now considered the principal cause of acute CD in these regions. Furthermore, new cases of acute CD are now being seen in departments where CD has not been documented, and triatomine vectors are not naturally found, thus raising suspicion for oral transmission. The oral CD could also be considered a food-borne zoonosis, and odoriferous didelphid secretions have been implemented in contaminating the human dwelling environment, increasing the risk of consumption of infectious metacyclic trypomastigotes. In this article, we will discuss the complex transmission dynamics of oral CD in Colombia and further examine the unique clinical manifestations of this route of infection. New insights into the oral transmission of Trypanosoma cruzi are being discovered in Colombia, which can help bring increased awareness and a better understanding of this neglected tropical disease to reduce the burden of CD throughout Latin America.

Multitarget anti-parasitic activities of isoquinoline alkaloids isolated from Hippeastrum aulicum (Amaryllidaceae)

Background Chagas disease and leishmaniasis affect a significant portion of the Latin American population and still lack efficient treatments. In this context, natural products emerge as promising compounds for developing more effective therapies, aiming to mitigate side effects and drug resistance. Notably, species from the Amaryllidaceae family emerge as potential reservoirs of antiparasitic agents due to the presence of diverse biologically active alkaloids. Purpose To assess the anti-Trypanosoma cruzi and anti-Leishmania infantum activity of five isolated alkaloids from Hippeastrum aulicum Herb. (Amaryllidaceae) against different life stages of the parasites using in silico and in vitro assays. Furthermore, molecular docking was employed to evaluate the interaction of the most active alkaloids. Methods Five natural isoquinoline alkaloids isolated in suitable quantities for in vitro testing underwent preliminary in silico analysis to predict their potential efficacy against Trypanosoma cruzi (amastigote and trypomastigote forms) and Leishmania infantum (amastigote and promastigote forms). The in vitro antiparasitic activity and mammalian cytotoxicity were investigated with a subsequent comparison of both analysis (in silico and in vitro) findings. Additionally, this study employed the molecular docking technique, utilizing cruzain (T. cruzi) and sterol 14α-demethylase (CYP51, L. infantum) as crucial biological targets for parasite survival, specifically focusing on compounds that exhibited promising activities against both parasites. Results Through computational techniques, it was identified that the alkaloids haemanthamine (1) and lycorine (8) were the most active against T. cruzi (amastigote and trypomastigote) and L. infantum (amastigote and promastigote), while also revealing unprecedented activity of alkaloid 7‑methoxy-O-methyllycorenine (6). The in vitro analysis confirmed the in silico tests, in which compound 1 presented the best activities against the promastigote and amastigote forms of L. infantum with half-maximal inhibitory concentration (IC50) 0.6 µM and 1.78 µM, respectively. Compound 8 exhibited significant activity against the amastigote form of T. cruzi (IC50 7.70 µM), and compound 6 demonstrated activity against the trypomastigote forms of T. cruzi and amastigote of L. infantum, with IC50 values of 89.55 and 86.12 µM, respectively. Molecular docking analyses indicated that alkaloids 1 and 8 exhibited superior interaction energies compared to the inhibitors. Conclusion The hitherto unreported potential of compound 6 against T. cruzi trypomastigotes and L. infantum amastigotes is now brought to the forefront. Furthermore, the acquired dataset signifies that the isolated alkaloids 1 and 8 from H. aulicum might serve as prototypes for subsequent structural refinements aimed at the exploration of novel leads against both T. cruzi and L. infantum parasites.

Bioactivity-guided isolation of trypanocidal coumarins and dihydro-pyranochromones from selected Apiaceae plant species.

Bioactivity-guided isolation of natural products from plant matrices is widely used in drug discovery. Here, this strategy was applied to identify trypanocidal coumarins effective against the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease (American trypanosomiasis). Previously, phylogenetic relationships of trypanocidal activity revealed a coumarin-associated antichagasic hotspot in the Apiaceae. In continuation, a total of 35 ethyl acetate extracts of different Apiaceae species were profiled for selective cytotoxicity against T. cruzi epimastigotes over host CHO-K1 and RAW264.7 cells at 10 µg/mL. A flow cytometry-based T. cruzi trypomastigote cellular infection assay was employed to measure toxicity against the intracellular amastigote stage. Among the tested extracts, Seseli andronakii aerial parts, Portenschlagiella ramosissima and Angelica archangelica subsp. litoralis roots exhibited selective trypanocidal activity and were subjected to bioactivity-guided fractionation and isolation by countercurrent chromatography. The khellactone ester isosamidin isolated from the aerial parts of S. andronakii emerged as a selective trypanocidal molecule (selectivity index ∼9) and inhibited amastigote replication in CHO-K1 cells, though it was significantly less potent than benznidazole. The khellactone ester praeruptorin B and the linear dihydropyranochromones 3'-O-acetylhamaudol and ledebouriellol isolated from the roots of P. ramosissima were more potent and efficiently inhibited the intracellular amastigote replication at < 10 µM. The furanocoumarins imperatorin, isoimperatorin and phellopterin from A. archangelica inhibited T. cruzi replication in host cells only in combination, indicative of superadditive effects, while alloimperatorin was more active in fractions. Our study reports preliminary structure-activity relationships of trypanocidal coumarins and shows that pyranocoumarins and dihydropyranochromones are potential chemical scaffolds for antichagasic drug discovery.

Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations.

BACKGROUND: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD. METHODS: All complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c-2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis. RESULTS: The analytical pipeline with an adjusted P-value of < 0.05 and fold-change > 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation-reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes. CONCLUSIONS: The transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD.

Protective effects of Theracurmin treatment during experimental infection of the Colombian strain of Trypanosoma cruzi at the testicular site.

Introduction: Chagas' disease is a tropical neglected illness caused by Trypanosoma cruzi and remains one of the most significant causes of morbidity and mortality in South and Central Americas. The disease is caused by a moderate to intense and persistent inflammatory response characterized by local upregulated expression and production of inflammatory mediators that favors the activation and recruitment of distinct cells of the immune system into different tissues to eliminate the parasites. Theracurmin is a curcumin's derived formulation of nanoparticles. Its anti-inflammatory properties make this bioactive compound a mitigating factor in pathological cases after an overwhelming inflammatory response. Methods: Our research focused on the testicular investigation in 28 mice infected by 103 trypomastigote forms of Colombian strain of T. cruzi and preventively treated with Theracurmin. The mice were treated with 30 mg/Kg of Theracurmin during the period of 30 days. At the 30th day post infection animals were euthanized, and its testicles were collected to morphological and immunological assays. Results: The animals infected and treated with Theracurmin presented a reduction in the testicular levels of IL-15 and IL-6. The volume density (%) of the tunica propria was also higher in all infected animals, but Theracurmin decreased this parameter in the treated animals. In the intertubular area, the percentage of some intertubular components was decreased in the infected animals such as the percentage and volume of Leydig cells, connective tissue, and macrophages. Discussion: Furthermore, our data pointed to the daily use of Theracurmin in the diet as a protective element of the testicular function.

Effectiveness of the repurposed drug isotretinoin in an experimental murine model of Chagas disease.

Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.

Candimine from Hippeastrum escoipense (Amaryllidaceae): Anti-Trypanosoma cruzi activity and synergistic effect with benznidazole.

BACKGROUND: Chagas disease (CD), caused by Trypanosoma cruzi, represents a health threat to around 20 million people worldwide. Side effects of benznidazole (Bzn) cause 15-20% of patients to discontinue their treatment. Evidence has increased in favor of the use of drug combinations to improve the efficacy and tolerance of the treatment. Natural products are well known to provide structures that could serve as new drugs or scaffolds for CD treatment. Spp of the Amaryllidoideae sub family of Amaryllidaceae family are known by their bioactives alkaloids, which have been reported by their antiparasitic activities. PURPOSE: To evaluate the anti-T. cruzi activity of the isolated alkaloid candimine (Cnd) from Hippeastrum escoipense Slanis & Huaylla; and to assess the combination effect between Cnd and Bzn against different life stages of T. cruzi parasites. METHODS: The chemical profile of H. escoipense alkaloids extract (AE-H. escoipense), including quantitation of Cnd was performed through GC/MS and UPLC-MS/MS techniques. Subsequently, Cnd was isolated using Shephadex LH-20. Then, the AE-H. escoipense and Cnd were tested against T. cruzi, (epimastigotes, trypomastigotes, and amastigotes) by in vitro proliferation and viability assays. The cytotoxicity was evaluated against Vero and HepG2 mammalian cells. The ultrastructural analysis was perform by transmission electron microscopy (TEM) and mitochondrial activity was carried out by MTT assay. Drug combination assay between Cnd and Bzn was evaluated using the Chou-Talalay method. RESULTS: The AE-H. escoipense and Cnd showed high and specific anti-T. cruzi activity, comparable to Bzn. Cnd induces ultrastructural changes in T. cruzi, such as vacuolization, membrane blebs, and increased mitochondrial activity. Regarding the interaction between Cnd and Bzn, it generates synergism in the combinations of 0.25×IC50 in epimastigotes, 2×IC50 in trypomastigotes+amastigotes, and 0.25, 2, and 4×IC50 in amastigotes. CONCLUSION: The synergism between Cnd and Bzn indicates that the combination at the concentration of 4×IC50 could be useful as an effective new therapy against CD in the chronic stage. Thus, Cnd isolated from the leaves of H. escoipense emerges as potential candidate for the development of a new drug for the treatment of CD.

Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents.

BACKGROUND: Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo. METHODS: As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging. RESULTS: As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model. CONCLUSIONS: For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Synthetic Analogues of Gibbilimbol B Induce Bioenergetic Damage and Calcium Imbalance in Trypanosoma cruzi.

Chagas disease is an endemic tropical disease caused by the protozoan Trypanosoma cruzi, which affects around 7 million people worldwide, mostly in development countries. The treatment relies on only two available drugs, with severe adverse effects and a limited efficacy. Therefore, the search for new therapies is a legitimate need. Within this context, our group reported the anti-Trypanosoma cruzi activity of gibbilimbol B, a natural alkylphenol isolated from the plant Piper malacophyllum. Two synthetic derivatives, LINS03018 (1) and LINS03024 (2), demonstrated a higher antiparasitic potency and were selected for mechanism of action investigations. Our studies revealed no alterations in the plasma membrane potential, but a rapid alkalinization of the acidocalcisomes. Nevertheless, compound 1 exhibit a pronounced effect in the bioenergetics metabolism, with a mitochondrial impairment and consequent decrease in ATP and reactive oxygen species (ROS) levels. Compound 2 only depolarized the mitochondrial membrane potential, with no interferences in the respiratory chain. Additionally, no macrophages response of nitric oxide (NO) was observed in both compounds. Noteworthy, simple structure modifications in these derivatives induced significant differences in their lethal effects. Thus, this work reinforces the importance of the mechanism of action investigations at the early phases of drug discovery and support further developments of the series.

Prevalence of digestive disorders associated with imported Chagas disease (PADChI study): an observational study.

BACKGROUND: Chagas disease (CD) is a parasitic disease caused by Trypanosoma cruzi, in which up to 10-20% of those affected may suffer digestive disorders. Multiple studies have been carried out on CD in non-endemic countries, mainly related to cardiological involvement. However, digestive disorders have not been analyzed in such depth. The objective of the study was to determine the prevalence of digestive disorders in imported CD at the time of first care. METHODS: An observational cross-sectional descriptive analysis of imported CD was performed. Chagasic structural damage and infectious digestive comorbidity were evaluated. The association between Chagasic structural damage and heart disease in Chagas patients was also investigated. RESULTS: After reviewing a total of 1,216 medical records, those of 464 patients were selected for analysis. Globally, the prevalence of digestive disorders in imported Chagas was 57.76%, 95% CI (53.25-62.27). The prevalence of comorbidity of infectious diseases was 40.73% CI 95% (36.25-45.22). Colonic abnormalities were found in 84 of 378 barium enema patients. CD-related esophageal abnormalities were present in 63 of 380 patients studied with esophagogram. CONCLUSIONS: The prevalence of digestive disorders associated with CD is high, so the presence of infectious diseases (mainly parasitic and H. pylori infection) should be ruled out. It is important to exclude structural involvement in all symptomatic patients, and asymptomatic patients should also be considered and offered.

Evaluation of Anti-Trypanosoma cruzi Activity of Chemical Constituents from Baccharis sphenophylla Isolated Using High-Performance Countercurrent Chromatography.

Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene - sphenophyllol (1) - as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O-caffeoylquinic acids (6-8). Compounds 1-8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC50 values of 20.1 µM and 2.9 µM, respectively. The mixture of chlorogenic acids 6-8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC50 values of 24.9, 12.8, and 2.7 µM, respectively. Nonetheless, tested compounds 1-8 displayed no cytotoxicity against mammalian cells (CC50 > 200 µM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases.

Impact of Lipid Matrix Composition on Activity of Membranotropic Enzymes Galactonolactone Oxidase from Trypanosoma cruzi and L-Galactono-1,4-Lactone Dehydrogenase from Arabidopsis thaliana in the System of Reverse Micelles.

The study of many membrane enzymes in an aqueous medium is difficult due to the loss of their catalytic activity, which makes it necessary to use membrane-like systems, such as reverse micelles of surfactants in nonpolar organic solvents. However, it should be taken into account that the micelles are a simplified model of natural membranes, since membranes contain many different components, a significant part of which are phospholipids. In this work, we studied impact of the main phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), on activity of the membrane enzymes using galactonolactone oxidase from Trypanosoma cruzi (TcGAL) and L-galactono-1,4-lactone dehydrogenase from Arabidopsis thaliana (AtGALDH) as examples. Effect of the structure (and charge) of the micelle-forming surfactant itself on the activity of both enzymes has been studied using an anionic surfactant (AOT), a neutral surfactant (Brij-96), and a mixture of cationic and anionic surfactants (CTAB and AOT) as examples. The pronounced effect of addition of PC and PE lipids on the activity of AtGALDH and TcGAL has been detected, which manifests as increase in catalytic activity and significant change in the activity profile. This can be explained by formation of the tetrameric form of enzymes and/or protein-lipid complexes. By varying composition and structure of the micelle-forming surfactants (AOT, CTAB, and Brij-96) it has been possible to change catalytic properties of the enzyme due to effect of the surfactant on the micelle size, lipid mobility, charge, and rigidity of the matrix itself.

Rattus norvegicus infectados com Trypanosoma cruzi: efeitos clínicos do tratamento com Lycopodium clavatun e phosphorus ultradiluídos / Rattus norvegicus infected with Trypanosoma cruzi: clinical effects of treatment with highly diluted Lycopodium clavatun and phosphorus / Rattus norvegicus infectado con Trypanosoma cruzi: efectos clínicos del tratamiento con Clavatun licopodio altamente diluido y fósforo

A doença de Chagas causada pelo parasita Trypanosoma cruzi acomete milhões de pessoas no mundo e não conta com um medicamento de ação efetiva para o seu tratamento etiológico. As drogas disponíveis, o nifurtimox e o benznidazol possuem índices de cura baixos com efeitos colaterais e toxidade que dificultam a adesão dos pacientes à terapia. Este fato impulsiona a busca por alternativas de tratamento que sejam mais efetivas e menos agressivas. Sendo assim, este trabalho teve como objetivo a avaliação dos efeitos clínicos apresentados por Rattus norvergicus infectados por T. cruzi e tratados com soluções ultradiluídas de Lycopodium clavatum ou Phosphorus. O estudo envolveu 93 ratos com quarenta e cinco dias de idade infectados intraperitonealmente com 5x106 formas tripomastigotas sanguíneos da cepa Y de T. cruzi, distribuídos nos grupos: Sadio SD (n=13) - controle não infectado e não tratado, grupo CI (n=27) - controle infectado e tratado com solução hidroalccólica 7% (etanol ­ água), grupo LY diluição 1:1x1026 (n=27) - infectado e tratado com Lycopodium, grupo PH diluição 1:1x1026 (n=26) - infectado e tratado com Phosphorum. Os animais foram avaliados clinicamente através dos parâmetros peso, temperatura, consumo de água e ração, quantidade de excretas, diâmetro e comprimento intestinal, aspecto da pelagem e consistência das fezes. Este estudo mostrou que os parâmetros utilizados foram importantes para a definição clínica da infecção de Rattus novergicus, linhagem Wistar pelo T. cruzi. Mostrou que os medicamentos LY e PH apresentam efeitos benéficos na evolução da clínica dos animais tratados. A utilização de Lycopodium clavatum e Phosphorus diluídos na proporção de 1:1x1026, apresentaram efeitos diferentes. Oito e seis parâmetros de quatorze analisados mostraram efeitos positivos para LY e PH, respectivamente. Os parâmetros consumo de água e ração, quantidade de excretas, diarreia, alopecia difusa e comprimento intestinal apresentaram diferenças significativas em relação ao controle infectado mostrando que mais estudos são necessários com o uso de medicamentos ultradiluídos na infecção pelo T. cruzi.

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people worldwide and does not have an effective drug for its etiological treatment. The available drugs, nifurtimox and benznidazole, have low cure rates with side effects and toxicity that make it difficult for patients to adhere to therapy. This fact drives the search for treatment alternatives that are more effective and less aggressive. Therefore, this work aimed to evaluate the clinical effects presented by Rattus norvergicus infected by T. cruzi and treated with ultradiluted solutions of Lycopodium clavatum or Phosphorus. The study involved 93 forty five day old rats intraperitoneally infected with 5x106 blood trypomastigotes forms of the Y strain of T. cruzi, distributed in the following groups: Healthy SD (n=13) - non-infected and untreated control, CI group (n =27) - infected control and treated with 7% hydroalcoholic solution (ethanol ­ water), LY group dilution 1:1x1026 (n=27) - infected and treated with Lycopodium, PH group dilution 1:1x1026 (n=26) - infected and treated with Phosphorum. The animals were clinically evaluated through the parameters weight, temperature, water and feed consumption, amount of excreta, intestinal diameter and length, coat appearance and feces consistency. This study showed that the parameters used were important for the clinical definition of infection of Rattus novergicus, Wistar lineage by T. cruzi. It showed that LY and PH drugs have beneficial effects on the clinical evolution of treated animals. The use of Lycopodium clavatum and Phosphorus diluted in the ratio of 1:1x1026, showed different effects. Eight and six parameters out of fourteen analyzed showed positive effects for LY and PH, respectively. The parameters water and feed consumption, amount of excreta, diarrhea, diffuse alopecia and intestinal length showed significant differences in relation to the infected control, showing that more studies are needed with the use of ultradiluted drugs in T. cruzi infection.

La enfermedad de Chagas causada por el parásito Trypanosoma cruzi afecta a millones de personas en todo el mundo y no cuenta con un fármaco eficaz para su tratamiento etiológico. Los fármacos disponibles, nifurtimox y benznidazol, presentan bajas tasas de curación con efectos secundarios y toxicidad que dificultan la adherencia terapéutica de los pacientes. Este hecho impulsa la búsqueda de alternativas de tratamiento más eficaces y menos agresivas. Por lo tanto, este trabajo tuvo como objetivo evaluar los efectos clínicos presentados por Rattus norvergicus infectados por T. cruzi y tratados con soluciones ultradiluidas de Lycopodium clavatum o Fósforo. En el estudio participaron 93 ratas de cuarenta y cinco días de edad infectadas intraperitonealmente con 5x106 formas tripomastigotes sanguíneas de la cepa Y de T. cruzi, distribuidos en los siguientes grupos: SD sano (n=13) - control no infectado y no tratado, grupo CI (n =27) - control infectado y tratado con solución hidroalcohólica al 7% (etanol - agua), grupo LY dilución 1:1x1026 (n=27) - infectado y tratado con Lycopodium, grupo PH dilución 1:1x1026 (n=26) - infectado y tratado con Phosphorum. Los animales fueron evaluados clínicamente mediante los parámetros peso, temperatura, consumo de agua y pienso, cantidad de excrementos, diámetro y longitud intestinal, aspecto del pelaje y consistencia de las heces. Este estudio demostró que los parámetros utilizados eran importantes para la definición clínica de la infección de Rattus novergicus, linaje Wistar por T. cruzi. Demostró que los fármacos LY y PH tienen efectos beneficiosos en la evolución clínica de los animales tratados. El uso de Lycopodium clavatum y Phosphorus diluidos en la proporción de 1:1x1026, mostró efectos diferentes. Ocho y seis parámetros de los catorce analizados mostraron efectos positivos para LY y PH, respectivamente. Los parámetros consumo de agua y pienso, cantidad de excretas, diarrea, alopecia difusa y longitud intestinal mostraron diferencias significativas en relación al control infectado, mostrando que son necesarios más estudios con el uso de fármacos ultradiluidos en la infección por

Anti-protozoal activity and metabolomic analyses of Cichorium intybus L. against Trypanosoma cruzi.

Chagas disease, caused by the protozoa Trypanosoma cruzi, is a potentially life-threatening parasitic zoonosis infecting 6-7 million people worldwide, mainly in Latin America. Due to the limited numbers of drugs available against this neglected disease and their frequent adverse effects, novel anti-chagasic agents are urgently needed. Cichorium intybus L. (chicory) is a bioactive plant with potent activity against parasitic nematodes, but its effects on protozoans are poorly known and no studies have explored its trypanocidal potential. Here, we investigated the activity of C. intybus against extracellular and intracellular stages of T. cruzi, including the prediction of trypanocidal compounds by metabolomic analyses and bioactivity-based molecular networking. Purified C. intybus extracts were prepared from leaves and roots of five C. intybus cultivars (cv. 'Benulite', 'Goldine', 'Larigot', 'Maestoso' and 'Spadona'). All C. intybus extracts induced concentration-dependent effects against T. cruzi trypomastigotes. C. intybus leaf extracts had higher trypanocidal selectivity and lower cytotoxicity on mammalian cells than root extracts. The leaf extract of C. intybus cv. Goldine also significantly reduced the number of mammalian cells infected with T. cruzi amastigotes. Metabolomic and bioactivity-based molecular networking analyses revealed 11 compounds in C. intybus leaves strongly linked with activity against trypomastigotes, including the sesquiterpene lactone lactucin, and flavonoid- and fatty acid-derivatives. Furthermore, seven distinct C. intybus molecules (including two sesquiterpene lactone-derivatives) were predicted to be involved in reducing the number of mammalian cells infected with amastigotes. This is the first report of the anti-protozoal activity of C. intybus against trypanosomatid parasites and expands our understanding of the anti-parasitic effects of this plant and its bioactive metabolites. Further studies to elucidate the anti-protozoal compound(s) in C. intybus and their mode(s) of action will improve our knowledge of using this bioactive plant as a promising source of novel broad-spectrum anti-parasitic compounds with associated health benefits and biomedical potential.

Melatonin regulates antioxidant defense and inflammatory response by activating Nrf2-dependent mechanisms and inhibiting NFkappaB expression in middle-aged T. cruzi infected rats.

Oxidative stress with higher levels of leptin and inflammatory response are key processes related to pathogenesis of both T. cruzi infection and aging. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of several genes implicated in the oxidative stress response in many pathological conditions. Melatonin is a pleiotropic hormone with, antioxidant, anti-inflammatory and anti-aging actions. Then, we hypothesized that Nrf2 response is impaired during the acute T. cruzi (9 days) infection and that melatonin rescues Nrf2 responses. Young (5 weeks-old) and middle-aged (18 months-old) male Wistar rats were infected with T. cruzi. Nrf2 translocation and markers of inflammation and oxidative stress were analyzed in blood and spleen. Increased apoptosis levels and oxidative stress indicators were observed in the rat spleen during T. cruzi infection. These responses were accompanied by decreased Nrf2 expression and increased expression of nuclear factor kappa B (NFκB). Melatonin (5 mg/kg/day; p.o. gavage) attenuated the superoxide anion (O2-) and hydrogen peroxide (H2O2) production induced by T. cruzi infection. Increased expressions of catalase and superoxide dismutase (SOD) were detected in the spleen of melatonin-treated rats infected with T. cruzi. Melatonin treatment inhibited the spleen NF-κB activation and downregulates the levels of circulating interleukin (IL)-4, IL-10 and tumor necrosis factor (TNF)-α in T. cruzi middle-aged infected rats. Increased levels of the chemokine CXCL1 in middle-aged control rats was observed, confirming that aging alters the production of this chemokine. In T. cruzi infected young animals, CXCL1 was up-regulated when compared to non-infected young ones. For young or middle-aged animals, melatonin treatment had no significant effect on CXCL1 levels. Our findings demonstrate an important role for Nrf2/NF-kB regulation as a possible mechanism by which melatonin attenuates oxidative stress, and provide new insights for further studies of this indoleamine as a therapeutic co-adjuvant agent against T. cruzi infection.

Potential of sulfur-selenium isosteric replacement as a strategy for the development of new anti-chagasic drugs.

Current treatment for Chagas disease is based on only two drugs: benznidazole and nifurtimox. Compounds containing sulfur (S) in their structure have shown promising results in vitro and in vivo against Trypanosoma cruzi, the parasite causing Chagas disease. Notably, some reports show that the isosteric replacement of S by selenium (Se) could be an interesting strategy for the development of new compounds for the treatment of Chagas disease. To date, the activity against T. cruzi of three Se- containing groups has been compared with their S counterparts: selenosemicarbazones, selenoquinones, and selenocyanates. More studies are needed to confirm the positive results of Se compounds. Therefore, we have investigated S compounds described in the literature tested against T. cruzi. We focused on those tested in vivo that allowed isosteric replacement to propose their Se counterparts as promising compounds for the future development of new drugs against Chagas disease.

Insights from the use of erythropoietin in experimental Chagas disease.

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

Resveratrol and Curcumin for Chagas Disease Treatment-A Systematic Review.

Chagas disease (CD) is a neglected protozoan infection caused by Trypanosoma cruzi, which affects about 7 million people worldwide. There are two available drugs in therapeutics, however, they lack effectiveness for the chronic stage-characterized mainly by cardiac (i.e., cardiomyopathy) and digestive manifestations (i.e., megaesophagus, megacolon). Due to the involvement of the immuno-inflammatory pathways in the disease's progress, compounds exhibiting antioxidant and anti-inflammatory activity seem to be effective for controlling some clinical manifestations, mainly in the chronic phase. Resveratrol (RVT) and curcumin (CUR) are natural compounds with potent antioxidant and anti-inflammatory properties and their cardioprotective effect have been proposed to have benefits to treat CD. Such effects could decrease or block the progression of the disease's severity. The purpose of this systematic review is to analyze the effectiveness of RVT and CUR in animal and clinical research for the treatment of CD. The study was performed according to PRISMA guidelines and it was registered on PROSPERO (CDR42021293495). The results did not find any clinical study, and the animal research was analyzed according to the SYRCLES risk of bias tools and ARRIVE 2.0 guidelines. We found 9 eligible reports in this study. We also discuss the potential RVT and CUR derivatives for the treatment of CD as well.

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.