Phylloseptin-1 is leishmanicidal for amastigotes of Leishmania amazonensis inside infected macrophages

Leishmania protozoans are the causal agents of neglected diseases that represent an important public health issue worldwide. The growing occurrence of drug-resistant strains of Leishmania and severe side effects of available treatments represent an important challenge for the leishmaniases treatment. We have previously reported the leishmanicidal activity of phylloseptin-1 (PSN-1), a peptide found in the skin secretion of Phyllomedusa azurea (=Pithecopus azureus), against Leishmania amazonensis promastigotes. However, its impact on the amastigote form of L. amazonensis and its impact on infected macrophages are unknown. In this work, we evaluated the effects of PSN-1 on amastigotes of L. amazonensis inside macrophages infected in vitro. We assessed the production of hydrogen peroxide and nitric oxide, as well as the levels of inflammatory and immunomodulatory markers (TGF-β, TNF-α and IL-12), in infected and non-infected macrophages treated with PSN-1. Treatment with PSN-1 decreased the number of infected cells and the number of ingested amastigotes per cell when compared with the untreated cells. At 32 µM (64 µg/mL), PSN-1 reduced hydrogen peroxide levels in both infected and uninfected macrophages, whereas it had little effect on NO production or TGF-β release. The effect of PSN-1 on IL-12 and TNF-α secretion depended on its concentration, but, in general, their levels tended to increase as PSN-1 concentration increased. Further in vitro and in vivo studies are needed to clarify the mechanisms of action of PSN-1 and its interaction with the immune system aiming to develop pharmacological applications

Influência da pravastatina na evolução da lesão, capacidade fagocitária, produção de radicais de oxigênio, nitrogênio e fator de necrose tumoral-α em camundongos infectados pela Leishmania (L.) amazonensis

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Núcleo de Medicina Tropical, 2011.Introdução: As leishmanioses constituem um grupo de doenças de grande impacto na saúde pública mundial alcançando uma incidência de dois milhões de novos casos por ano. O quadro clínico é complexo com apresentações clínicas que vão desde formas cutâneas, mucocutâneas e viscerais, que dependem da espécie do parasito e do hospedeiro, mas, sobretudo, da resposta imunitária predominante no momento da infecção. O controle da leishmaniose é dificultado pela pouca opção terapêutica, alta toxicidade dos medicamentos e crescente resistência dos parasitos. Diante disso, é importante a busca por alternativas terapêuticas eficazes e de baixa toxicidade. As estatinas constituem um grupo de drogas imunomoduladoras, potencialmente capazes de alterar o equilíbrio leishmânia/resposta imunitária. Objetivo: O presente estudo objetivou verificar a influência da pravastatina sobre a evolução da doença e função dos fagócitos pela análise da sobrevida e desenvolvimento de lesões cutâneas em camundongos BALB/c, C57BL/6 e hamsters sírio, infectados por L. (L.) amazonensis. Verificar os efeitos in vitro de diferentes concentrações da pravastatina sobre formas promastigotas de L. (L.) amazonensis e sobre células peritoneais. E sua influência sobre as funções dos fagócitos, produção de peróxido de hidrogênio, óxido nítrico e FNT- α pelos macrófagos peritoneais de camundongos BALB/c. Métodos: camundongos BALB/c, C57BL/6 e hamster sírio foram infectados com formas promastigotas de L. (L.) amazonensis e tratados ou não com 20 mg/kg/dia de pravastatina ou salina, por via oral, durante 90 dias e então, acompanhados até o óbito com controle semanal do peso e diâmetros das patas. Para avaliar a toxicidade da pravastatina para as leishmânias e para os fagócitos, os efeitos de diferentes concentrações de pravastatina foram avaliados in vitro por meio da redução do MTT sobre formas promastigotas de L. (L.) amazonensis ou sobre macrófagos peritoneais de camundongos BALB/c. As funções dos fagócitos foram avaliadas em camundongos BALB/c tratados ou não com a pravastatina por 30 ou 90 dias. A capacidade fagocitária foi avaliada utilizando S. cerevisiae sensibilizados ou não com soro fresco. A produção de peróxido de hidrogênio foi avaliada pela oxidação do vermelho fenol em presença de peroxidase. A produção de óxido nítrico foi avaliada pela reação de Griess. A produção de FNT-α foi avaliada por ensaio imunoenzimático. Resultados: A pravastatina aumentou a sobrevida dos camundongos BALB/c infectados, mas não a dos camundongos C57BL/6 ou hamster sírio. O tratamento com a pravastatina diminui a perda de peso dos camundongos C57BL/6 e hamster sírio infectados pela leishmânia, mas não influenciou a evolução do peso dos camundongos BALB/c. A pravastatina retardou o surgimento da lesão da pata dos camundongos BALB/c e as lesões foram menores no camundongo C57BL/6, mas não interferiu no diâmetro das patas do hamster. A pravastatina diminuiu o percentual de redução de MTT por formas promastigotas de L. (L.) amazonensis em concentrações ≥ 4 μg/mL, mas não afetou a atividade mitocondrial de células peritoneais até a concentração de 256 μg/mL. A pravastatina aumentou a capacidade fagocitária dos macrófagos peritoneais pelos receptores para padrões moleculares de patógeno aos 30 dias, retornando aos valores do grupo controle aos 90 dias. Ao mesmo tempo aumentou o índice fagocitário e a média de leveduras fagocitadas por macrófagos quando avaliado pelos receptores para opsoninas aos 90 dias de tratamento. Camundongos BALB/c infectados com L. (L.) amazonensis tratados com pravastatina apresentaram diminuição do índice fagocitário bem como da proporção de macrófagos envolvidos na fagocitose e da media de leveduras fagocitadas por macrófagos após 90 dias de tratamento quando a fagocitose foi avaliada pelos receptores para padrões moleculares de patógenos, mas IX não houve alterações na fagocitose pelos receptores para complemento após 30 ou 90 dias de tratamento com a droga. A pravastatina aumentou a produção de peróxido de hidrogênio após 30 dias em animais infectados e em animais não infectados após 90 dias. Também aumentou a produção de óxido nítrico em camundongos infectados tratados com pravastatina comparados a camundongos infectados tratados com salina após 90 dias de tratamento. A pravastatina reduziu a produção de FNT-α em animais infectados tratados com pravastatina por 30 dias, em relação aos animais infectados tratados com salina, mas retornou essa produção aos valores basais aos 90 dias de tratamento. Conclusão: o aumento da sobrevida de camundongos BALB/c, a clínica favorável em animais infectados e os efeitos observados sobre os macrófagos apontam para a potencialidade da pravastatina como co-adjuvante no tratamento da leishmaniose, de forma a contribuir para diminuir a morbidade da doença. _________________________________________________________________________________ ABSTRACTIntroduction: Leishmaniosis is a group of diseases with great impact on the world public health, with as many as 2 millions new cases by year. The clinical profile of the disease is complex, presenting many clinical forms, such as cutaneous, mucocutaneous and visceral; these forms depend not only on the species of the parasite and of the host, but also on the immune response of the host mainly at the time of infection. Controlling leishmaniosis is a difficult task due to the few therapeutical options available, and due to the high toxicity of the available drugs and due to the increasing resistance of the parasites. In that case, it is important to search for new and efficient therapeutical alternatives, presenting lower toxicity. Statins are a group of immunomodulatory drugs with the potential to alter the balance between Leishmania and immune response of the host. Objective: This study aimed to evaluate the influence of pravastatin on the evolution of the disease and to verify the function of phagocytes by analyzing the survival time and the development of cutaneous lesions on BALB/c and C57BL/6 mice and Syrius hamsters infected by L. (L.) amazonensis. The in vitro effects of different concentrations of pravastatin on promastigotes forms of L. (L.) amazonensis and on peritoneal cells, and its influence on phagocytic functions and on the production of hydrogen peroxide, nitric oxide and FNT-α by peritoneal macrophages of BALB/c mice, were assessed. Material and Method: BALB/c and C57BL/6 mice and Syrius hamsters were infected or not with promastigota forms of L. (L.) amazonensis and treated or not with 20 mg/kg/day of pravastatin, per os, during 90 days. They were then followed until their death, with a weekly control of weight and footpad thickness. In order to evaluate the toxicity of pravastatin to leishmania and to the phagocytes, the effects of different concentrations of pravastatin were evaluated in vitro by MTT reduction over promastigotes forms of L. (L.) amazonensis or over peritoneal macrophages of BALB/c mice. The functions of the phagocytes were evaluated in BALB/c mice infected or not with L. (L.) amazonensis and treated or not with pravastatin for 30 or 90 days. The phagocytic capacity was evaluated using S. cerevisiae, sensitized or not with fresh mouse sera. Hydrogen peroxide production was assessed by the oxidation of fenol red in the presence of peroxidase. Nitric oxide production was assessed by the Griess reaction. FNT-α production was assessed by imunoenzimatic assay Results: Pravastatin increased the survival of infected BALB/c mice, but it did not influence the survival of the C57BL/6 mice or Syrius hamsters. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL/6 mice and Syrian hamsters, but not infected BALB/c mice. Low footpad thickness was found on BALB/c pravastatin treated mice, the drug delayed the footpad thickness of the C57BL/6 mice, and pravastatin had no influence on footpad thickness of the Syriam hamsters. Pravastatin decreased the MTT reduction percent by promastigotes forms of L. (L.) amazonensis in concentrations ≥ 4 μg/mL, but did not affect the mitochondrial activity of peritoneal cells until 256 μg/mL. Pravastatin increased the phagocytic capacity through pathogen-associated molecular pattern receptors on the 30th day, but the value returned to the control group level on the 90th day. At the same time it increased the phagocytic index and the number of phagocytosed yeasts by macrophages when we evaluated the phagocytosis through opsonin receptors on the 90th day of treatment. BALB/c mice infected with L. (L.) amazonensis and treated with pravastatin presented a decrease in the phagocytic index and a decrease in the proportion of macrophages involved in phagocytosis and a decrease in the number of phagocytosed yeasts by macrophages by pathogen-associated molecular pattern receptors after 90 days of treatment, but there were no alteration in complement receptors after 30 or 90 days. Pravastatin increased hydrogen peroxide production after 30 days in infected animals and after 90 days in animals not infected. After 90 days, it also increased nitric oxide production in infected mice treated with pravastatin when compared to infected mice treated with saline. Pravastatin reduced FNT-α production in infected animals treated with pravastatin by the 30th day of treatment in relation to infected animals treated with saline, returning to their base levels by the 90th day of treatment. Conclusion: The survival increase for BALB/c mice, the favorable clinical evolution observed in the infected animals and the effects observed on the macrophages point to the potential use of pravastatin as a co-adjuvant drug in the therapy of leishmaniasis, to contribute to lower the morbidity of the disease

Guillain Barré syndrome in a population less than 15 years old in Brazil

To know the impact of the Guillain Barré syndrome (GBS) in the population less than 15 years old, after the eradication of poliomyelitis. Data bank from the program of epidemiological surveillance of acute flaccid palsies (AFP) from the Fundação Nacional de Saúde were analyzed between 1990 -- 1996. From 3619 notifications of AFP there were 1678 GBS. GBS yearly incidence rates is 0.39-0.63 cases/100,000. No consistent seasonal variation existed or relationship to vaccines. Weakness at inclusion were, moderate 52.1%, severe in 47.9%, sixty days after 57.1% normal, 7.4% mild, 15.7% moderate, 10.4% with severe deficits, death in 5.4%. 67 (4.0%) cases unknown. Death rates varies from 2.8% in southeast to 7.9% in the northeast. GBS was the most frequent cause of AFP. In spite of the severity of this disease being similar in the different regions, the outcome varies according to origin of the cases, possibly reflecting the economical conditions in those places