Assessment of acute toxicity of the ethanolic extract of Lychnophora pinaster (Brazilian arnica)

AbstractSpecies of the Lychnophora genus are plants native to Brazil, popularly known as “Brazilian arnica” and used in folk medicine as alcoholic and hydro-alcoholic preparations for the treatment of bruises, inflammation, pain, rheumatism and insect bites. The present study aimed to evaluate the safety of the use of Lychnophora pinaster Mart., Asteraceae. Acute toxicity of the crude ethanolic extract was evaluated by administration of the extract by oral route to male and female Swiss mice. A single extract dose of 125, 250 or 500mg/kg was administered and the effects on spontaneous locomotor activity, exploratory behavior, muscle strength, body weight, food and water consumption, relative organ weight, histology, as well as hematological and biochemical parameters were evaluated. The three doses administered to the animals did not cause muscle tone alterations, but doses of 250 and 500mg/kg induced a significant inhibition of the spontaneous locomotor activity and exploratory behavior of the animals in open-field test. There was no alteration to hematological parameters and consumption of water and food, body weight variation and organs relative weight. Changes were observed in AST and ALT during assessment of biochemical parameters. The histopathological evaluation showed that the extract provoked cellular alterations, such as vacuolar degeneration and inflammation in kidneys and liver at all doses. Liver morphometric analyses of male and female mice showed that the extract did not have dose-dependent effects. Although females showed a significant increase in inflammatory cells, the effect was not dose-dependent

Benznidazole Treatment: Time- and Dose-Dependence Varies with the Trypanosoma cruzi Strain

Trypanosoma cruzi; Benznidazol; Estratègies terapèutiquesTrypanosoma cruzi; Benznidazol; Estrategias terapéuticasTrypanosoma cruzi; Benznidazole; Therapeutic strategiesAs the development of new drugs for Chagas disease is not a priority due to its neglected disease status, an option for increasing treatment adherence is to explore alternative treatment regimens, which may decrease the incidence of side effects. Therefore, we evaluated the efficacy of different therapeutic schemes with benznidazole (BNZ) on the acute and chronic phases of the disease, using mice infected with strains that have different BNZ susceptibilities. Our results show that the groups of animals infected by VL-10 strain, when treated in the chronic phase with a lower dose of BNZ for a longer period of time (40 mg/kg/day for 40 days) presented better treatment efficacy than with the standard protocol (100 mg/kg/day for 20 days) although the best result in the treatment of the animals infected by the VL-10 strain was with100 mg/kg/day for 40 days. In the acute infection by the Y and VL-10 strains of T. cruzi, the treatment with a standard dose, but with a longer time of treatment (100 mg/kg/day for 40 days) presented the best results. Given these data, our results indicate that for BNZ, the theory of dose and time proportionality does not apply to the phases of infection.Funding was provided by the Universidade Federal de Ouro Preto (UFOP), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), research fellowships from CNPq (Carneiro CM, Correa-Oliveira R), CAPES—Science Without Borders and Senior Research Visitor (Molina I, Correa-Oliveira R), and BERENICE (Collaborative Project supported by the European Commission under the Health Innovation Work Programme of the 7th Framework Programme). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Benznidazole Treatment : Time- and Dose-Dependence Varies with the Trypanosoma cruzi Strain

As the development of new drugs for Chagas disease is not a priority due to its neglected disease status, an option for increasing treatment adherence is to explore alternative treatment regimens, which may decrease the incidence of side effects. Therefore, we evaluated the efficacy of different therapeutic schemes with benznidazole (BNZ) on the acute and chronic phases of the disease, using mice infected with strains that have different BNZ susceptibilities. Our results show that the groups of animals infected by VL-10 strain, when treated in the chronic phase with a lower dose of BNZ for a longer period of time (40 mg/kg/day for 40 days) presented better treatment efficacy than with the standard protocol (100 mg/kg/day for 20 days) although the best result in the treatment of the animals infected by the VL-10 strain was with100 mg/kg/day for 40 days. In the acute infection by the Y and VL-10 strains of T. cruzi, the treatment with a standard dose, but with a longer time of treatment (100 mg/kg/day for 40 days) presented the best results. Given these data, our results indicate that for BNZ, the theory of dose and time proportionality does not apply to the phases of infectio

The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry

Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions

Avaliação do trato digestório de cães Beagle infectados com as cepas Y ou Berenice-78 do Trypanosoma cruzi nas fases aguda ou crônica da doença de chagas experimental.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.O protozoário hemoflagelado Trypanosoma cruzi, agente etiológico da doença de Chagas, apresenta uma ampla variabilidade intraespecífica que determina a existência de subpopulações com características genéticas, morfológicas, moleculares e bioquímicas distintas. Este polimorfismo tem sido associado ao desenvolvimento das diferentes formas clínicas da doença de Chagas e suas implicações epidemiológicas. Neste contexto, a forma digestiva parece estar ausente na América Central, México e Venezuela, porém em países como Brasil e Bolívia pode ocorrer sozinha ou em associação com a forma cardíaca. O megaesôfago e o megacólon, manifestações mais comuns da forma digestiva, são caracterizados por acalásia e obstrução parcial/total do cólon, respectivamente. Achados histopatológicos demonstram o envolvimento do sistema nervoso entérico na patogênese dessas visceromegalias, porém poucos estudos têm abordado qual é o limite de desnervação necessário para as alterações da motilidade. Neste trabalho, cães da raça Beagle foram infectados com as cepas Y ou Berenice-78 do T. cruzi e necropsiados durante as fases aguda (30 dias após infecção-DAI) ou crônica (730 DAI) da doença experimental para avaliação histopatológica do esôfago e cólon. Ambas as cepas foram capazes de infectar o esôfago e o cólon e provocar resposta inflamatória durante a fase aguda. Na fase crônica, a PCR convencional detectou parasitismo tecidual apenas no grupo infectado com a cepa Be-78 o que poderia estar relacionado com a manutenção do processo inflamatório neste grupo. Apenas nos animais infectados com a cepa Y foi observada fibrose na fase crônica. Desnervação dos gânglios mientéricos foi observada na infecção com ambas as cepas durante a fase aguda, porém na fase crônica a desnervação foi persistente apenas nos animais infectados com a cepa Be-78. O comprometimento das células gliais foi precoce nos animais infectados com a cepa Y enquanto os animais infetados com a cepa Be-78 apresentaram redução de células gliais entéricas GFAP-IR apenas na fase crônica. Estes resultados sugerem que embora as duas cepas sejam capazes de provocar lesões no trato digestório, a cepa Y controla precocemente essas alterações, enquanto a cepa Be-78 sustenta as alterações ao longo da infecção experimental, sugerindo o desenvolvimento de lesões digestivas crônicas.The flagellate protozoan Trypanosoma cruzi, etiologic agent of Chagas disease, presents a wide intraspecific variability that determines the existence of subpopulations with distinc genetic, morphological, molecular and biochemical feactures. This polymorphism has been associated with the development of different clinical forms of Chagas disease and its epidemiological implications. In this context, the digestive form seems to be absent in Central America, Mexico and Venezuela, but in countries like Brazil and Bolivia may occur alone or in combination with the cardiac form. Megaesophagus and megacolon, the most common manifestations of the digestive form, are characterized by achalasia and colon partial /total obstruction, respectively. Histopathologic findings demonstrate the involvement of enteric nervous system in these visceromegalies pathogenesis, but few studies have addressed the limit of what is necessary for denervation changes in motility. In this study, Beagle dogs were infected with strains Y or Berenice-78 of T. cruzi and necropsied during the acute phase (30 days post infection-DPI) or chronic (730DPI) of the experimental disease for esophagus and colon post-mortem histopathological evaluation. Both strains were able to infect the esophagus and colon and cause an inflammatory response during the acute phase. In the chronic phase, the conventional PCR detected only tissue parasitism in the group infected with Be-78 strain that is associated with the inflammatory process maintenance in this group. Only in animals infected with the Y strain was observed fibrosis in chronic phase. Denervation of myenteric ganglia was observed in infection with both strains during the acute phase, but in chronic phase, denervation was persistent only in animals infected with Be-78 strain. The glial cells involvement was earlier in animals infected with Y strain while animals infected with Be-78 strain showed a reduction in enteric glial cells GFAP-IR only in the chronic phase. These results suggest that although the two strains are capable of causing injury in the digestive tract, the Y strain control these changes earlier, while the Be-78 strain sustain alterations throughout the experimental infection, suggesting the development of chronic digestive lesions

Influência da infecção por uma cepa policlonal do Trypanosoma cruzi sobre a resposta imune no coração e cólon durante a fase aguda da infecção experimental de camundongos.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa de Pós Graduação, Universidade Federal de Ouro Preto.Fatores intrínsecos ao Trypanosoma cruzi e relacionados ao hospedeiro agem sobre a patogênese da doença de Chagas. Neste contexto, o polimorfismo genético do parasito parece ter um papel crítico no prognóstico da doença, e tem sido demonstrada uma associação entre o desenvolvimento das distintas formas clínicas e a distribuição geográfica de cepas do T. cruzi. Diante disso, esse projeto propôs avaliar a influência do polimorfismo biológico e molecular de subpopulações do T. cruzi sobre a resposta inflamatória no coração e cólon durante a fase aguda da infecção experimental. A primeira etapa consistiu na caracterização biológica (meio de cultura acelular e celular) e do perfil gênico (Low-Stringency Single Specific Primer- Polymerase Chain Reaction/LSSP-PCR) de subpopulações do parasito obtidas a partir de hemoculturas periódicas (3, 6 e 12 meses após a infecção-mai) em 24 camundongos experimentalmente infectados com 5000 formas tripomastigotas sanguíneas da cepa Berenice- 78 (Be-78 parental) do T. cruzi. Foi possível identificar isolados com comportamento in vitro, assim como perfis de assinatura gênica, distintos do observado para a cepa Be-78 parental. Em alguns casos, os isolados mostraram certo grau de semelhança com a cepa Berenice-62 (Be-62), isolada da paciente Berenice, em fase crônica tardia, 16 anos antes do xenodiagnóstico que originou a cepa Be-78. Esses dados sugerem a presença de distintas subpopulações na cepa Be-78 e reafirmam a plasticidade do parasito frente à pressão seletiva exercida pela interação parasito-hospedeiro. Na segunda etapa, a infecção com um isolado selecionado na primeira etapa, que apresentou perfil distinto da cepa parental, foi comparada com a infecção com as cepas Be-78 parental e Be-62 em modelo murino com o objetivo de investigar a participação de diferentes subpopulações do parasito sobre o desenvolvimento do processo inflamatório durante os estágios iniciais da doença experimental. Nossos dados demonstraram um perfil cardiomiotrópico e retardo no controle da parasitemia na infecção pela cepa Be-78 parental, seguido por processo inflamatório intenso ao final da fase aguda, ao contrário da cepa Be-62 e do isolado Be-78is5-3mai, que por sua vez, apresentou-se preferencialmente parasitando o cólon, com parasitemia significativamente superior, porém, controlada precocemente, e processo inflamatório intenso ao longo da fase aguda. No coração, apesar de já observada no grupo Be-62 aos 14dai, no 28º, a densidade de macrófagos, linfócitos T CD4+ e CD8+ foi semelhante entre as cepas Be-62 e Be-78 parental, com sobreposição de células T CD8+ em relação às células T CD4+. O isolado Be-78is5-3mai apresentou miocardite, embora de menor intensidade, também apenas no 28ºdai, composta essencialmente por macrófagos e linfócitos T CD8+. De forma diferente ao observado no coração, o perfil de resposta imunológica no cólon foi cepa-dependente. Apenas o grupo Be- 78 parental apresentou aumento precoce de células T CD4+, que foram substituídas por células T CD8+ e linfócitos B, no 14ºdai. Todos esses resultados são condizentes com o comportamento da parasitemia e do parasitismo tecidual nesse grupo. Embora com menor intensidade, o perfil imunofenotípico detectado nos animais infectados com o isolado Be- 78is5-3mai foi próximo àquele demonstrado pelo grupo Be-62, e sobretudo, distinto da cepa Be-78 parental. Nas primeiras duas semanas de infecção, o processo inflamatório foi constituído principalmente por macrófagos e células T CD8+, com aumento expressivo de linfócitos T CD4+ apenas ao final da fase aguda. Estes resultados indicam que o isolado Be- 78is5-3mai, consistente com os dados da avaliação in vitro, demonstrou durante a fase aguda da infecção de camundongos BALB/c, um comportamento intermediário entre a cepas de referência utilizadas, apresentando virulência (parasitemia e mortalidade) compatível com a cepa Be-78 parental, no entanto, perfil histotrópico compartilhado com a cepa Be-62, reforçando a hipótese de que a infecção a longo prazo com a cepa Be-78 foi capaz de selecionar uma subpopulação que compartilha características com a cepa Be-62, ambas originada da paciente Berenice, com 16 anos de intervalo entre as coletas.Factors intrinsic to Trypanosoma cruzi and other host-related act in Chagas disease pathogenesis. In this context, the parasite genetic polymorphism seems to have a critical role in disease prognosis, and have been demonstrated an association between the distincts clinical forms development and geographic distribution of T. cruzi strains. Polyclonal strains are common in the natural infections, and may involve specific interactions parasite-organ. Therefore, this project proposed to evaluate the influence of T. cruzi subpopulations polymorphism on the inflammatory response in the heart and colon during the acute phase of the experimental infection. The first step consisted in biological (cellular and acellular growth media culture) and molecular (Low-Stringency Single-Specific Primer Polymerase Chain Reaction/LSSP-PCR) profile characterization of parasite subpopulations obtained from periodic blood cultures (3, 6, 12 months after the infection-mai) in 24 mice experimentally infected with 5000 blood trypomastigotes forms of Berenice-78 (Be-78 parental strain) T. cruzi strain. It was possible to identify isolates with in vitro behavior, as well as gene signature profile distincts from that observed for the Be- 78 parental strain. In some cases, the isolates showed some degree of resemblance with the Berenice-62 (Be- 62) strain, isolated from patient Berenice in Chagas disease chronic phase, 16 years before the xenodiagnostic that originated the Be- 78 strain. These data suggest the presence of distinct subpopulations in Be-78 strain and underline the plasticity of the parasite against the selective pressure exerted by the parasite-host interaction. In the second stage, infection with one isolate selected in the first step, presenting distinctive profile of the to parental strain, will be compared to infection with Be-78 parental and Be-62 strains in isogenic murine model, in order to investigate the participation of the different subpopulations on the inflammatory process during the early stages of experimental disease. Our data demonstrated a cardiomyotropic profile and a delay in parasitemia controlling in Be-78 parental infected mice, followed by severe inflammation at the end of the acute phase, unlike the Be-62 strain and Be-78is5-3mai isolate, which in turn is preferably parasitizing the colon, with significantly higher parasitaemia, however, early controlled, and intense inflammation during the acute phase. At heart, despite already observed in the Be-62 group to 14dai, on the 28th, the density of macrophages, CD4+ and CD8+ T cells were similar between the Be-62 and Be-78 parental strains, with T-cell overlap CD8+ over CD4+ T cells. The isolated Be-78is5-3mai presented myocarditis, although of lower intensity, also in 28ºdai essentially composed of macrophages and CD8+ T lymphocytes. Differently to that observed in the heart, the immune response profile in the colon was strain-dependent. As Be-78 parental group showed an early increase of CD4+ T cells, which were replaced by CD8+ T cells and B lymphocytes in 14ºdai. All these results are consistent with the parasitaemia and tissue parasitism behavior in that group. Although to a lesser extent, the immunophenotypic profile detected in the animals infected with the isolated Be-78is5-3mai was close to that shown by Be-62 group. In the first two weeks of infection, the inflammation was composed primarily of macrophages and CD8+ T cells, with significant increase of CD4+ T cells only at the end of the acute phase. These results indicate that the isolate Be-78is5-3mai, consistent with the in vitro evaluation, shown during the acute phase of infection of BALB/c mice, an intermediate behavior between the both reference strains used, with virulence (parasitemia and mortality) compatible with the Be-78 parental strain, however, shared histotropic profile with the Be-62 strain, reinforcing the hypothesis that longterm infection with the Be-78 strain was able to select a subpopulation that shares characteristics with the Be-62 strain, both originated from Berenice patient, 16-year interval between collections

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

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

A High-Fat Diet Exacerbates the Course of Experimental Trypanosoma cruzi Infection That Can Be Mitigated by Treatment with Simvastatin

The protozoan Trypanosoma cruzi is responsible for triggering a damage immune response in the host cardiovascular system. This parasite has a high affinity for host lipoproteins and uses the low-density lipoprotein (LDL) receptor for its invasion. Assuming that the presence of LDL cholesterol in tissues could facilitate T. cruzi proliferation, dietary composition may affect the parasite-host relationship. Therefore, the aim of this study was to evaluate myocarditis in T. cruzi-infected C57BL/6 mice—acute phase—fed a high-fat diet and treated with simvastatin, a lipid-lowering medication. Animals (n=10) were infected with 5×103 cells of the VL-10 strain of T. cruzi and treated or untreated daily with 20 mg/kg simvastatin, starting 24 h after infection and fed with a normolipidic or high-fat diet. Also, uninfected mice, treated or not with simvastatin and fed with normolipidic or high-fat diet, were evaluated as control groups. Analyses to measure the production of chemokine (C-C motif) ligand 2 (CCL2), interferon- (IFN-) γ, interleukin- (IL-) 10, and tumor necrosis factor (TNF); total hepatic lipid dosage; cholesterol; and fractions, as well as histopathological analysis, were performed on day 30 using cardiac and fat tissues. Our results showed that the high-fat diet increased (i) parasite replication, (ii) fat accumulation in the liver, (iii) total cholesterol and LDL levels, and (iv) the host inflammatory state through the production of the cytokine TNF. However, simvastatin only reduced the production of CCL2 but not that of other inflammatory mediators or biochemical parameters. Together, our data suggest that the high-fat diet may have worsened the biochemical parameters of the uninfected and T. cruzi-infected animals, as well as favored the survival of circulating parasites

Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single <i>Trypanosoma cruzi</i> Strain Identified <i>In Vitro via</i> LSSP-PCR

<div><p>The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of <i>Trypanosoma cruzi</i> is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of <i>Trypanosoma cruzi</i> Berenice-78 and characterized <i>via</i> LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and <i>in vitro</i> profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct <i>Trypanosoma cruzi</i> isolates may be obtained upon experimental murine infection with a single polyclonal <i>Trypanosoma cruzi</i> strain.</p></div