RESUMO
Chagas disease is caused by the protozoan parasite, Trypanosoma cruzi. This parasite alternates between an insect vector and a mammalian host. T. cruzi epimastigotes reside in the insect vector and coexist with the blood components of the vertebrate host. The metabolic profile of T. cruzi has been extensively studied; however, changes in its metabolism in response to signaling molecules present in the vector are poorly understood. Heme acts as a physiological oxidant that triggers intense epimastigote proliferation and upregulates the expression of genes related to glycolysis and aerobic fermentation in vitro. Here, heme-cultured epimastigotes increased D-glucose consumption. In fact, heme-cultured parasites secreted more succinate (the end product of the so-called succinic fermentation) followed by glucose intake. Increased succinate levels reduced the extracellular pH, leading to acidification of the supernatant. However, the acidification and proliferation stimulated by heme was impaired when glycolysis was inhibited. Otherwise, when glucose amount is enhanced in supernatant, heme-cultured parasites increased its growth whereas the glucose depletion caused a delay in proliferation. Heme supplementation increased epimastigote electron transport system-related O2 consumption rates, while glucose addition reduced both the electron transport system-related O2 consumption rates and spare respiratory capacity, indicating a Crabtree-like effect. These results show that glycolysis predominated in heme-cultured epimastigotes over oxidative phosphorylation for energy supply when glucose is present to sustain its high proliferation in vitro. Furthermore, it provided an insight into the parasite biology in the vector environment that supply glucose and the digestion of blood generates free heme that can lead to the growth of T. cruzi epimastigotes.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Animais , Humanos , Trypanosoma cruzi/genética , Heme/metabolismo , Glucose/metabolismo , Succinatos/metabolismo , Succinatos/farmacologia , MamíferosRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of T. cruzi is unclear. The present work investigated the response of T. cruzi epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of T. cruzi epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.
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AIM: To assess the impact of outpatient dental treatment on oral health-related quality of life (OHRQoL) of children and their families. MATERIALS AND METHODS: This prospective observational study was conducted with children with untreated dental caries, recruited from a public university/the pediatric dentistry clinic of a public university in the northeastern Brazil. Sociodemographic data were collected, a clinical examination was carried out before and after dental treatment, and the Early Childhood Oral Health Impact Scale (ECOHIS) questionnaire was applied to those responsible for the children, before and after dental treatment. Descriptive and bivariate statistics were used, and the Wilcoxon and Student's t tests were applied (p < 0.05). RESULTS: The study included 64 children, of both sexes, aged between 3 and 5 years old. The majority had a dmft index higher than or equal to 6 (60.9%), with a mean value of 7.11 (± 4.11). Sociodemographic conditions such as sex, age, and socioeconomic status had no impact on their OHRQoL (p > 0.05). The mean total ECOHIS questionnaire scores and those of its domains decreased after completion of the treatments (p < 0.05), except for scores in the self-image and distress domains of the parents (p > 0.05). This study reinforces the relevance of adopting oral health policies aimed at the prevention and treatment of ECC. CONCLUSION: Outpatient dental treatment had a positive impact on the OHRQoL of children and their families. CLINICAL RELEVANCE: The possibility of evaluating the services provided by the pediatric dentistry clinic.
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Cárie Dentária , Qualidade de Vida , Criança , Pré-Escolar , Assistência Odontológica , Cárie Dentária/epidemiologia , Cárie Dentária/terapia , Suscetibilidade à Cárie Dentária , Feminino , Humanos , Masculino , Saúde Bucal , Pacientes Ambulatoriais , Pais , Inquéritos e QuestionáriosRESUMO
Purpose: The purpose of this study was to compare the effectiveness of lesion sterilization and tissue repair (LSTR) antibiotic paste comprised of chloramphenicol, tetracycline, and zinc oxide and eugenol (CTZ) versus zinc oxide eugenol (ZOE) pulpectomy in the treatment of primary molars with pulp necrosis. Methods: A total of 70 three- to eight-year-old subjects with 88 primary mandibular molars with pulp necrosis were included. The teeth were randomized to the CTZ group or ZOE group. The time taken to perform both techniques was recorded. The parents of the children and the dentist who performed clinical evaluations were blind to the group assignment, although the radiographic evaluator could see the difference in treatments. Clinical and radiographic assessments were performed at three, six, nine, and 12 months. Results: At the 12-month evaluation, the clinical success was 86.4 percent for CTZ and 90.9 percent for ZOE (P=0.50), the radiographic success was 75.0 percent for CTZ and 72.7 percent for ZOE (P=0.81), and the overall success was 70.5 percent for CTZ and 72.7 percent for ZOE (P=0.81). The mean time taken to perform was 61.4 (±20.5 standard deviation) minutes for CTZ and 145.1 (±53.2) minutes for ZOE (P<0.001). Conclusions: At 12 months, both techniques presented no significant difference in success rates for nonvital pulp therapy in primary molars with necrosis. The lesion sterilization and tissue repair procedure time using chloramphenicol, tetracycline, zinc oxide, and eugenol was significantly shorter than for a zinc oxide eugenol pulpectomy.
Assuntos
Materiais Restauradores do Canal Radicular , Óxido de Zinco , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Necrose da Polpa Dentária/terapia , Eugenol/uso terapêutico , Humanos , Dente Molar/diagnóstico por imagem , Dente Molar/cirurgia , Pulpectomia , Esterilização , Dente Decíduo , Óxido de Zinco/uso terapêutico , Cimento de Óxido de Zinco e Eugenol/uso terapêuticoRESUMO
The scope of this study is to discuss equity and access to mental health care by means of analysis of the distribution of primary care teams (Community Health Agents; Family Health Teams; the Expanded Family Health Nucleus) and the coverage of the Psychosocial Care Network in three Northeastern states (Ceará, Piauí and Rio Grande do Norte) and their respective health regions. It is a descriptive study, supported by exploratory quantitative analysis. For this purpose, the database of the Department of Basic Care/DATASUS was used to collect secondary data in relation to the historical trajectory of training of Primary Care teams. Regarding the points of care of RAPS, the database made available by the National Mental Health Coordination of the Ministry of Health was consulted. The conclusion of the investigation was that prior to 2018 there was a major drive in the country into the interior and expansion of coverage in relation to primary and psychosocial care, impacting the expansion of equity in territories of lesser economic and social development. However, it was also observed that Psychosocial Care Network services are more prevalent in the interior while the other related services are more developed in capitals and large municipalities.
Objetiva-se discutir equidade e acesso em saúde mental por meio da análise da distribuição das equipes da atenção primária (Agentes comunitários de Saúde; Equipes de saúde da Família; Núcleo Ampliado de Saúde da Família) e da cobertura da Rede de Atenção Psicossocial (RAPS) em três estados nordestinos (Ceará, Piauí e Rio Grande do Norte) nas suas respectivas regiões de saúde. Trata-se de um estudo descritivo, apoiado por análise quantitativa exploratória. Para tanto, recorremos à base de dados do Departamento de Atenção Básica/DATASUS para a coleta dos dados secundários em relação às séries históricas de habilitação das equipes da Atenção Primária. Quanto aos pontos de atenção da RAPS, utilizamos a base de dados disponibilizada pela Coordenação Nacional de Saúde Mental do Ministério da Saúde. A investigação concluiu que até 2018 observou-se no país um importante movimento de interiorização e expansão da cobertura em relação à atenção primária e psicossocial, impactando na ampliação da equidade nos territórios de menor desenvolvimento econômico e social. Contudo, verificou-se que os serviços de APS estão mais interiorizados, enquanto os demais dispositivos da RAPS estão mais desenvolvidos nas capitais e grandes municípios.
Assuntos
Serviços de Saúde Mental , Reabilitação Psiquiátrica , Brasil , Cidades , Humanos , Saúde MentalRESUMO
Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 µM] and extracellular amastigotes [IC50/24 h = 3.3 µM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of "proton leak" (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and "ETS" (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration ("ROX") enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.
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Resumo Objetiva-se discutir equidade e acesso em saúde mental por meio da análise da distribuição das equipes da atenção primária (Agentes comunitários de Saúde; Equipes de saúde da Família; Núcleo Ampliado de Saúde da Família) e da cobertura da Rede de Atenção Psicossocial (RAPS) em três estados nordestinos (Ceará, Piauí e Rio Grande do Norte) nas suas respectivas regiões de saúde. Trata-se de um estudo descritivo, apoiado por análise quantitativa exploratória. Para tanto, recorremos à base de dados do Departamento de Atenção Básica/DATASUS para a coleta dos dados secundários em relação às séries históricas de habilitação das equipes da Atenção Primária. Quanto aos pontos de atenção da RAPS, utilizamos a base de dados disponibilizada pela Coordenação Nacional de Saúde Mental do Ministério da Saúde. A investigação concluiu que até 2018 observou-se no país um importante movimento de interiorização e expansão da cobertura em relação à atenção primária e psicossocial, impactando na ampliação da equidade nos territórios de menor desenvolvimento econômico e social. Contudo, verificou-se que os serviços de APS estão mais interiorizados, enquanto os demais dispositivos da RAPS estão mais desenvolvidos nas capitais e grandes municípios.
Abstract The scope of this study is to discuss equity and access to mental health care by means of analysis of the distribution of primary care teams (Community Health Agents; Family Health Teams; the Expanded Family Health Nucleus) and the coverage of the Psychosocial Care Network in three Northeastern states (Ceará, Piauí and Rio Grande do Norte) and their respective health regions. It is a descriptive study, supported by exploratory quantitative analysis. For this purpose, the database of the Department of Basic Care/DATASUS was used to collect secondary data in relation to the historical trajectory of training of Primary Care teams. Regarding the points of care of RAPS, the database made available by the National Mental Health Coordination of the Ministry of Health was consulted. The conclusion of the investigation was that prior to 2018 there was a major drive in the country into the interior and expansion of coverage in relation to primary and psychosocial care, impacting the expansion of equity in territories of lesser economic and social development. However, it was also observed that Psychosocial Care Network services are more prevalent in the interior while the other related services are more developed in capitals and large municipalities.
Assuntos
Humanos , Reabilitação Psiquiátrica , Serviços de Saúde Mental , Brasil , Saúde Mental , CidadesRESUMO
Trypanosoma cruzi has a complex life cycle involving four life stages: the replicative epimastigotes and metacyclic trypomastigotes in the invertebrate host digestive tract, and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. Trypomastigotes can invade any nucleated cell, including macrophages, which produce ROS that enhance intracellular infection. However, how ROS modulate T. cruzi infection in the mammalian cell remains unclear. Therefore, the present work aimed to investigate the role of ROS during the stimulation of amastigogenesis in vitro. Our results showed that H2O2 improves the differentiation process in vitro and that it was impaired by Peg-Catalase. However, the antioxidants GSH and NAC had no influence on induced amastigogenesis, which suggests the specificity of H2O2 to increase intracellular differentiation. Amastigogenesis physiologically occurs in low pH, thus we investigated whether parasites are able to produce ROS during amastigogenesis. Interestingly, after 60 min of differentiation induction in vitro, we observed an increase in H2O2 production, which was inhibited by the mitochondrial-targeted antioxidant, mitoTEMPO and Cyclosporine A (a mitochondrial permeability transition pore -mPTP- inhibitor), suggesting mitochondrion as a H2O2 source. Indeed, quantitative real time (qPCR) showed an increase of the mitochondrial superoxide dismutase (FeSODA) gene expression after 60 min of induced amastigogenesis, reinforcing the hypothesis of mitochondrial ROS induction during intracellular differentiation of T. cruzi. The reduction of cellular respiration and the decreased ΔΨm observed during amastigogenesis can explain the increased mitochondrial ROS through mPTP opening. In conclusion, our results suggest that H2O2 is involved in the amastigogenesis of T. cruzi.
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Peróxido de Hidrogênio/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/citologia , Células VeroRESUMO
Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi, and is transmitted by triatomine insects during its blood meal. Proliferative epimastigotes forms thrive inside the insects in the presence of heme (iron protoporphyrin IX), an abundant product of blood digestion, however little is known about the metabolic outcome of this signaling molecule in the parasite. Trypanosomatids exhibit unusual gene transcription employing a polycistronic transcription mechanism through trans-splicing that regulates its life cycle. Using the Deep Seq transcriptome sequencing we characterized the heme induced transcriptome of epimastigotes and determined that most of the upregulated genes were related to glucose metabolism inside the glycosomes. These results were supported by the upregulation of glycosomal isoforms of PEPCK and fumarate reductase of heme-treated parasites, implying that the fermentation process was favored. Moreover, the downregulation of mitochondrial gene enzymes in the presence of heme also supported the hypothesis that heme shifts the parasite glycosomal glucose metabolism towards aerobic fermentation. These results are examples of the environmental metabolic plasticity inside the vector supporting ATP production, promoting epimastigotes proliferation and survival.
Assuntos
Perfilação da Expressão Gênica , Heme/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/metabolismo , Genes Mitocondriais , Glucose/metabolismo , Insetos Vetores/parasitologia , Microcorpos/metabolismo , Transdução de Sinais , Transcrição Gênica , Triatominae/parasitologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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Recent reports from different world regions suggest ocular syphilis is re-emerging, in parallel with an increasing incidence of the systemic infection globally. We conducted a large observational study of 127 persons consecutively treated for ocular syphilis at public medical centers in Brazil over a 2.5-year period ending July 2015. Of 104 individuals serologically tested for human immunodeficiency virus (HIV), 34.6% were positive. Ophthalmological evaluations included measurement of Snellen visual acuity and intraocular pressure, and assessment of inflammation by slit lamp examination and dilated posterior eye examination. Involvements in 214 eyes were anterior (6.1%), intermediate (8.4%), posterior (76.2%) and pan- (8.4%) uveitis, and scleritis (0.9%). Multiple anterior and posterior eye complications were observed, including cataract in the anterior eye (incidence rate, 0.18/eye-year) and epiretinal membrane in the posterior eye (incidence rate, 0.09/eye-year); incidence rates of reduction in best-corrected visual acuity to ≤20/50 and ≤20/200 were 0.10 and 0.06/eye-year, respectively. Rates of complications and visual acuity loss did not differ significantly between HIV- positive and negative individuals. In an era of re-emergence, syphilis has ocular complications that may compromise vision, despite treatment with appropriate anti-microbial drugs.
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Doenças Transmissíveis Emergentes/complicações , Infecções Oculares Bacterianas/epidemiologia , Sepse/microbiologia , Sífilis/complicações , Transtornos da Visão/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Brasil/epidemiologia , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/microbiologia , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/microbiologia , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Sepse/epidemiologia , Sífilis/tratamento farmacológico , Sífilis/epidemiologia , Sífilis/microbiologia , Resultado do Tratamento , Transtornos da Visão/diagnóstico , Transtornos da Visão/microbiologia , Transtornos da Visão/prevenção & controle , Acuidade Visual , Adulto JovemRESUMO
Resumo Relato de um caso clínico de Xeroderma Pigmentoso com carcinoma espinocelular de conjuntiva bilateral que apresentou regressão importante das dimensões tumorais com o uso de Interferon alfa-2b tópico. Relato de caso: Paciente feminina com Xeroderma Pigmentoso em estágio avançado, com ausência de pele sadia, tendo sido submetida a cerca de 60 exéreses de lesões de pele malignas. A paciente compareceu com tumoração conjuntival em ambos os olhos, correspondendo a carcinoma espinocelular de conjuntiva e neoplasia intraepitelial de conjuntiva em olho esquerdo. Devido as dificuldades cirúrgicas, alta taxa de recidiva e elevada probabilidade de formação de simbléfaro foi-se iniciado terapêutica com Interferon alfa-2beta 1.000.000 unidades tópico, obtendo-se bons resultados com importante regressão do tamanho da lesão e resolução dos sintomas. Conclusão: O uso tópico de interferon alfa-2beta em neoplasia escamosa de conjuntiva, mostrou-se uma boa opção terapêutica em situações de elevado risco cirúrgico e de complicações pós operatórias.
Abstract Report of a case of xeroderma pigmentosum with squamous cell carcinoma of bilateral conjunctiva that showed a significant regression in tumor size with the use of interferon alfa-2b topic. Case report: Female patient with Xeroderma pigmentosum in an advanced stage, with no healthy skin, having been subjected to about 60 excisions of malignant skin lesions. The patient appeared with conjunctival tumors in both eyes, corresponding to squamous cell carcinoma of the conjunctiva. Due to surgical difficulties, high relapse rate and high probability of symblepharon formation, therapy was started with interferon alpha 2beta 1,000,000 topic units, obtaining good results with a significant decrease in lesion size and resolution of symptoms. Conclusion: Topical use of alpha-interferon in 2beta squamous neoplasia of the conjunctiva proved to be a good therapeutic option for high surgical risk and situations of postoperative complications.
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Humanos , Feminino , Adulto , Xeroderma Pigmentoso/complicações , Carcinoma in Situ , Carcinoma de Células Escamosas , Neoplasias da Túnica Conjuntiva/diagnóstico , Neoplasias da Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/tratamento farmacológico , Administração Oftálmica , Interferon alfa-2/uso terapêuticoRESUMO
Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.
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Doença de Chagas/imunologia , Heme/metabolismo , Mitocôndrias/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/metabolismo , Processos de Crescimento Celular , Células Cultivadas , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Humanos , Estágios do Ciclo de Vida , Potencial da Membrana Mitocondrial , Compostos Organofosforados/metabolismo , Consumo de Oxigênio , Piperidinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/metabolismoRESUMO
This paper presents results from a study of albumin from pacu (Piaractus mesopotamicus, Holmberg 1887) and the catfish pintado (Pseudoplatystoma corruscans, Spix & Agassiz, 1829), two neotropical fish species inhabitants of Brazilian rivers, comparing their molecular mass and discussing their secondary structures based on spectropolarimetric (circular dychroism) measurements. Genetic controlled specimens were obtained from two fish hatcheries, located in Mococa (pacu) and in São João da Boa Vista (pintado), both in São Paulo State, Brazil. After a period of adaptation in holding tanks, fish blood samples were taken by punctioning their abdominal aorta. Purified albumin was obtained by gel filtration. SDS-PAGE electrophoresis was performed for the molecular mass estimation. Circular Dichroism spectra were registered for albumins of the two fish species over the range of 190-250 nm (far-UV), which shown two negative bands at 217 and 208 nm, a positive peak at 196 nm and a crossover at 200 nm. This profile is compatible with proteins that content predominantly alpha-helix structure.
Este artigo apresenta os resultados de um estudo sobre as albuminas de pacu (Piaractus mesopotamicus, Holmberg 1887) e pintado (Pseudoplatystoma corruscans, Spix & Agassiz, 1829), duas espécies neotropicais de peixes nativas do Brasil, determinando as suas massas moleculares e discutindo suas estruturas secundárias, com base em medidas de espectropolarimetria (dicroísmo circular). Espécimes controlados geneticamente foram obtidos de duas diferentes pisciculturas, uma localizada na cidade de Mococa (pacu) e a outra, na cidade de São João da Boa Vista (pintado), ambas no Estado de São Paulo, Brasil. Após um período de adaptação em tanques apropriados, amostras de sangue foram coletadas por punção da aorta abdominal dos peixes. Albumina pura foi obtida por gel filtração dessas amostras e as massas moleculares foram determinadas a partir dos dados da eletroforese SDS-PAGE. Espectros de dicroísmo circular das albuminas dos peixes foram registrados na região de 190-250 nm (far-UV), os quais mostraram duas bandas negativas, a 217 e 208 nm, um pico positivo a 196 nm e um crossover a 200 nm; perfil este compatível com proteínas que contem predominantemente estrutura alfa-hélice.
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Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. ß-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.
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Insetos Vetores/parasitologia , Trypanosoma cruzi/citologia , Trypanosoma cruzi/fisiologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Heme/farmacologia , Peróxido de Hidrogênio/farmacologia , Oxirredução/efeitos dos fármacos , Rhodnius/parasitologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Ácido Úrico/farmacologiaRESUMO
BACKGROUND: Sepsis is a prevalent condition in critically ill patients and may be associated with thiamine deficiency (TD). The aim of this study was to evaluate the effect of TD on inflammation, oxidative stress and cellular recruitment in a sepsis model. METHODS: The experimental sepsis model, cecal ligation and puncture (CLP), was utilized on mice in comparison with a sham procedure. The following four groups were compared against each other: SHAM with AIN93G complete chow, SHAM with thiamine deficient (TD) chow, CLP with AIN93G complete chow, and CLP with TD chow. Thiamine pyrophosphate (TPP) blood concentrations were determined, and blood and peritoneal fluid were evaluated for differences in TNF-alpha, IL-1, IL-6, KC and MCP-1/CCL2 levels. In addition, the levels of 4-HNE adducts in liver proteins were evaluated by Western Blot. RESULTS: The mean TPP blood concentration from the mice fed with the complete chow was 303.3 ± 42.6 nmol/L, and TD occurred within 10 days. TNF-α and MCP-1 concentrations in the peritoneal fluid were significantly greater in the CLP with TD chow group when compared with the other groups. The blood IL-1ß level, however, was lower in the CLP with TD chow group. Liver 4-HNE levels were highest in the TD chow groups. Blood mononuclear cell numbers, as well as peritoneal total leukocyte, mononuclear cell and neutrophil numbers were greater in the CLP with TD chow group. Peritoneal bacterial colony forming units (CFU) were significantly lower in the CLP with TD chow group. CONCLUSION: TD was associated with greater bacterial clearance, oxidative stress and inflammatory response changes.
RESUMO
It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.
Assuntos
Antiprotozoários/farmacologia , Produtos Biológicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Anêmonas-do-Mar/química , Serpinas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/isolamento & purificação , Organismos Aquáticos/química , Produtos Biológicos/isolamento & purificação , Doença de Chagas/parasitologia , Relação Dose-Resposta a Droga , Humanos , Leishmania/citologia , Leishmania/efeitos dos fármacos , Leishmania/fisiologia , Microscopia Eletrônica , Organelas/ultraestrutura , Serpinas/isolamento & purificação , Trypanosoma cruzi/citologia , Trypanosoma cruzi/fisiologiaRESUMO
As formas epimastigotas de Trypanosoma cruzi proliferam e se diferenciam no interior de diferentes compartimentos do trato digestivo dos triatomíneos. Esses ambientes antagônicos, no que diz respeito à concentração de nutrientes, pH e status redox, constituem um desafio para o protozoário por conterem moléculas e fatores capazes de deflagrar diferentes sinalizações e respostas no parasito. Por isso, testamos a influência de produtos abundantes do metabolismo do vetor e de status redox distintos, frente aos processos de proliferação e diferenciação in vivo e in vitro. Como exemplo temos o heme e a hemozoína, subprodutos da digestão da hemoglobina, e o urato, rico na urina dos insetos. O heme é uma importante molécula em todos os seres vivos. Nosso grupo mostrou seu papel na proliferação in vitro de T. cruzi e que esse sinal é governado pela enzima redox-sensível CaMKII (Lara et al., 2007; Souza et al., 2009). Esse efeito parece depender de uma sinalização redox, onde o heme e não seus análigos induz a formação de EROs, modulando a atividade da CaMKII (Nogueira et al, 2011). Apesar de gerar espécies reativas de oxigênio (EROs) em formas epimastigotas, o heme não alterou a ultraestrutura desses parasitos mostrando uma adaptação a ambientes oxidantes. Além disso, a adição de FCCP inibiu a formação de EROs mitocondrial, diminuindo a proliferação dos parasitos. Em contrapartida, a AA aumentou drasticamente a produção de EROs mitocondrial levando à morte dos epimastigotas. Estes resultados confirmam a hipótese de regulação redox do crescimento de epimastigotas...
Trypanosoma cruzi epimastigotes proliferate and differentiate inside different compartments of the triatomines gut. These environments are antagonic in terms of nutrient content, pH and redox status. All these factors represent a challenge to the protozoan due to the presence of molecules and factors which are able to induce different signals to the parasite. Thus, we tested the influence of abundant metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis in vitro and in vivo. These molecules are heme and hemozoin, both byproducts of hemoglobin digestion, and urate, present in the urine of insects. Heme is a ubiquitous molecule present in all living organisms. Our group studied its role in T. cruzi growth in vitro, showing that this signal is governed by the redox-sensitive enzyme CaMKII (Lara et al., 2007; Souza et al., 2009). Indeed, it seems to rely on a redox signaling pathway in which heme, but not its analogs, induces ROS formation, thus modulating CaMKII activity (Nogueira et al., 2011). Although it induces ROS in epimastigotes, the heme molecule had no deleterious effect upon the parasites ultrastructure, suggesting an adaptation to oxidative environments. In addition, FCCP inhibited mitochondrial ROS formation, then decreasing the parasite proliferation. On the other hand, AA drastically increased mitochondrial ROS production leading to cell death. These results corroborate the hypothesis of redox regulation of epimastigotes proliferation. Hemozoin (β- hematin) formation is an elegant strategy to minimize the toxic effect of heme in hematophagous insects. However, β-hematin had no influence upon the proliferation or metacyclogenesis in vitro. Also, urate, GSH and NAC impaired epimastigote proliferation. These effects were partially reversed when the antioxidants were incubated along with heme...
Assuntos
Humanos , Doença de Chagas/metabolismo , Oxirredução , Trypanosoma cruzi/crescimento & desenvolvimento , Doença de Chagas/genética , Técnicas In Vitro , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS) which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.
RESUMO
Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.