ATIVIDADE LEISHMANICIDA in vitro DE FRAÇÕES DO EXTRATO HIDROALCOÓLICO DAS FOLHAS DE Chenopodium ambrosioides L.

A leishmaniose é uma doença infecciosa causada por protozoários do gênero Leishmania e representa sério problema de saúde pública em paises da África, Ásia e América Latina. Chenopodium ambrosioides L.,  popularmente conhecido como mastruz, tem sido utilizado no Maranhão para o tratamento tópico de úlceras leishmanióticas. O trabalho avaliou a eficácia das frações do extrato hidroalcoólico das folhas de C. ambrosioides contra formas promastigotas da espécie Leishmania amazonensis. O fracionamento foi realizado pela partição sequencial do extrato hidroalcoólico de folhas de C. ambrosioides com solventes de polaridade crescente (hexano, clorofórmio e acetato de etila). As formas promastigotas foram cultivadas em meio RPMI 1640 com e sem as frações. Após 24h de incubação a 26ºC, o número de promastigotas viáveis foi contado pelo método direto em câmara de Neubauer. As concentrações que inibem o crescimento de 50% (CI50 ) das promastigotas de L. amazonensis foram calculadas a partir da avaliação da mortalidade das promastigotas in vitro. De acordo com escores padrões nas frações de acetato de etila e hidroalcoólica remanescente não foi observada atividade leishmanicida significativa, mas as frações hexânica e clorofórmica apresentaram-se ativas. Os resultados obtidos demonstram uma ação leishmanicida promissora das frações. Estudos futuros são necessários para investigar a eficácia destas frações no tratamento da leishmaniose em modelos experimentais in vivo.Descritores: Chenopodium ambrosioides L.; Mastruz; Leishmaniose; in vitro. Abstract:  In vitro leishmanicidal activity of fractions obtained from hydroalcoholic extracts of  Chenopodium ambrosioides’  leaves. Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania and represents serious public health problem in Africa, Asia and Latin America. Chenopodium ambrosioides, popularly known as “mastruz”, has been used in Maranhão for the topical treatment of leishmanial ulcers. The study evaluated the effectiveness of the fractions from hydroalcoholic extract of C. ambrosioides’  leaves against the promastigotes of Leishmania amazonensis. Fractionation as accomplished by partition sequential extract of leaves of C. ambrosioides with solvents of increasing polarity (hexane, chloroform and ethyl acetate). The promastigotes forms were cultured in supplemented RPMI 1640 with or without fractions. After 24h incubation at 26°C, the number of viable promastigotes was counted by the direct method in a Neubauer chamber. The concentrations that inhibit growth of 50% (IC50 ) of L. amazonensis’ promastigotes were calculated from the assessment of the mortality of promastigotes in vitro. According to standard scores, in the ethyl acetate and hydroalcoholic remaining fractions there was no leishmanicidal activity, while the hexane and chloroform fractions were actives. The results showed a promissor leishmanicidal activity of fraction. Future studies are necessary to investigate the effectiveness of these fractions in the treatment of these fractions in experimental models in vivo.Descriptors: Chenopodium ambrosioides L.; Mastruz; Leishmaniasis; in vitro

INIBIÇÃO DA INFECÇÃO in vitro DE MACRÓFAGOS POR Leishmania amazonensis POR EXTRATO E FRAÇÕES DE Chenopodium ambrosioides L.

A utilização de espécies vegetais, como Chenopodium ambrosioides L., para o tratamento da leishmaniose na terapêutica tradicional tem despertado interesse na busca de novos compostos mais eficazes e menos tóxicos. Nosso grupo demonstrou as atividades imunoestimuladora e anti-Leishmania in vivo do extrato bruto hidroalcoólico (EBH) de C. ambrosioides e efeito anti-promastigota in vitro do EBH e das suas frações. Neste trabalho, avaliou-se a atividade anti-Leishmania do EBH e suas frações acetato de etila (FAc) e clorofórmica (FCHCl3) em macrófagos infectados in vitro por Leishmania amazonensis. Foram realizados dois modelos: “proflático” e “terapêutico”. No primeiro, macrófagos peritoneais de camundongos Swiss  foram tratados com EBH, FAc ou FCHCl3 nas concentrações de 62,5µg/mL, 125µg/mL e 250µg/mL e, após 4 horas, infectados com formas promastigotas do parasito na razão de 1:10 por 24 horas. No segundo, os macrófagos foram infectados com promastigotas (1:10) e, após 4 horas, tratados com EBH, FAc ou FCHCl3 por 24 horas. Foram então realizados a quantifcação das amastigotas fagocitadas e o cálculo das taxas de infecção. No modelo “proflático”, apenas os macrófagos expostos ao EBH nas maiores concentrações apresentaram  taxas de  infecção  inferiores ao controle negativo. Entretanto, no modelo “terapêutico”, as  três concentrações de EBH e também da FAc reduziram a infecção de macrófagos em relação ao controle negativo, sendo a maior concentração do EBH mais efetiva inclusive que o controle positivo.  Em conclusão, o EBH de folhas de C. ambrosioides e a sua FAc possuem efeito terapêutico anti-Leishmania na infecção in vitro de macrófagos.Descritores: Leishmaniose; Leishmania amazonensis; Chenopodium ambrosioides.AbstractInhibit  of  in  vitro  macrophage  infection  by  Leishmania  amazonensis  by  extract  and  fractions  from Chenopodium ambrosioides L. The use of plant species such as Chenopodium ambrosioides L. for the treatment of leishmaniasis in traditional medicine has aroused interest in fnding new, more effective and less toxic compounds. Our group demonstrated the immunostimulatory and in vivo anti-Leishmania activities of the crude hydroalcoholic extract (HCE) from C. ambrosioides L. and the in vitro anti-promastigote effect of the HCE and its fractions. In this study, we evaluated the anti-Leishmania activity of the HCE and its fractions ethyl acetate (FAc) and chloroform (FCHCl3) in macrophages infected in vitro with Leishmania amazonensis. Two models, “prophylactic” and “therapeutic”, were performed. In the frst, Swiss mice peritoneal macrophages were treated with CHE, FAc or FCHCl3 in concentrations of 62,5μg/mL, 125μg/mL and 250μg/mL and, after 4 hours, infected with promastigote forms in the ratio of 1:10 for 24 hours. In the second model, the macrophages were infected with promastigotes (1:10)  and,  after 4 hours,  treated with HCE, FAc or FCHCl3 for 24 hours. Quantifcation of phagocytosed amastigotes and calculation  of  infection  rates were  then  perfomed.  In  the  “prophylactic” model,  only macrophages  exposed  to  the  highest concentrations of HCE presented  infection  rates  lower  than  the negative  control. However,  in  the  “therapeutic” model,  the three concentrations of both the HCE and FAc reduced the infection of macrophages compared to the negative control, with the highest concentration of HCE being even more effective than the positive control. In conclusion, the HCE from leaves of Chenopodium ambrosioides and its FAc have an anti-Leishmania therapeutic effect on the in vitro macrophages infection.Descriptors: Leishmaniasis. Leishmania amazonensis. Chenopodium ambrosioides

Antimicrobial activity against oral pathogens and immunomodulatory effects and toxicity of geopropolis produced by the stingless bee Melipona fasciculata Smith

<p>Abstract</p> <p>Background</p> <p>Native bees of the tribe Meliponini produce a distinct kind of propolis called geopropolis. Although many pharmacological activities of propolis have already been demonstrated, little is known about geopropolis, particularly regarding its antimicrobial activity against oral pathogens. The present study aimed at investigating the antimicrobial activity of <it>M. fasciculata </it>geopropolis against oral pathogens, its effects on <it>S. mutans </it>biofilms, and the chemical contents of the extracts. A gel prepared with a geopropolis extract was also analyzed for its activity on <it>S. mutans </it>and its immunotoxicological potential.</p> <p>Methods</p> <p>Antimicrobial activities of three hydroalcoholic extracts (HAEs) of geopropolis, and hexane and chloroform fractions of one extract, were evaluated using the agar diffusion method and the broth dilution technique. Ethanol (70%, v/v) and chlorhexidine (0.12%, w/w) were used as negative and positive controls, respectively. Total phenol and flavonoid concentrations were assayed by spectrophotometry. Immunotoxicity was evaluated in mice by topical application in the oral cavity followed by quantification of biochemical and immunological parameters, and macro-microscopic analysis of animal organs.</p> <p>Results</p> <p>Two extracts, HAE-2 and HAE-3, showed inhibition zones ranging from 9 to 13 mm in diameter for <it>S. mutans </it>and <it>C. albicans</it>, but presented no activity against <it>L</it>. <it>acidophilus</it>. The MBCs for HAE-2 and HAE-3 against <it>S. mutans </it>were 6.25 mg/mL and 12.5 mg/mL, respectively. HAE-2 was fractionated, and its chloroform fraction had an MBC of 14.57 mg/mL. HAE-2 also exhibited bactericidal effects on <it>S. mutans </it>biofilms after 3 h of treatment. Significant differences (p < 0.05) in total phenol and flavonoid concentrations were observed among the samples. Signs toxic effects were not observed after application of the geopropolis-based gel, but an increase in the production of IL-4 and IL-10, anti-inflammatory cytokines, was detected.</p> <p>Conclusions</p> <p>In summary, geopropolis produced by <it>M. fasciculata </it>can exert antimicrobial action against <it>S. mutans </it>and <it>C. albicans</it>, with significant inhibitory activity against <it>S. mutans </it>biofilms. The extract with the highest flavonoid concentration, HAE-2, presented the highest antimicrobial activity. In addition, a geopropolis-based gel is not toxic in an animal model and displays anti-inflammatory effect.</p

TRPV1 Antagonism by Capsazepine Modulates Innate Immune Response in Mice Infected with Plasmodium berghei ANKA

Thousands of people suffer from severe malaria every year. the innate immune response plays a determinant role in host's defence to malaria. Transient receptor potential vanilloid 1 (TRPV1) modulates macrophage-mediated responses in sepsis, but its role in other pathogenic diseases has never been addressed. We investigated the effects of capsazepine, a TRPV1 antagonist, in malaria. C57BL/6 mice received 10(5) red blood cells infected with Plasmodium berghei ANKA intraperitoneally. Noninfected mice were used as controls. Capsazepine or vehicle was given intraperitoneally for 6 days. Mice were culled on day 7 after infection and blood and spleen cell phenotype and activation were evaluated. Capsazepine decreased circulating but not spleen F4/80(+)Ly6G(+) cell numbers as well as activation of both F4/80(+) and F4/80(+)Ly6G(+) cells in infected animals. in addition, capsazepine increased circulating but not spleen GR1(+) and natural killer (NK) population, without interfering with natural killer T (NKT) cell numbers and blood NK and NKT activation. However, capsazepine diminished CD69 expression in spleen NKT but not NK cells. Infection increased lipid peroxidation and the release of TNF alpha and IFN gamma, although capsazepine-treated group exhibited lower levels of lipid peroxidation and TNF alpha. Capsazepine treatment did not affect parasitaemia. Overall, TRPV1 antagonism modulates the innate immune response to malaria.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Amparo a Pesquisa e Desenvolvimento Cientifico do Maranhao (FAPEMA; Brazil)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ CEUMA, BR-65075120 Sao Luis, MA, BrazilKings Coll London, Div Cardiovasc, London WC2R 2LS, EnglandUniv São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Diadema, BrazilUniv Fed Maranhao, Sao Luis, BrazilInst Florence Ensino Super, Sao Luis, BrazilUniversidade Federal de São Paulo, Diadema, BrazilWeb of Scienc

<i>In vitro</i> suppression capacity of liver Tregs after infection with iRBCs.

<p>Foxp3-GFP mice were intraperitoneally infected (or not) with Pc-iRBCs. All animals were divided into two groups: Control (non-infected) or infected, on day 7 of infection (INF d7). (A) Conventional CD4+Foxp3- T cells (Tconv) were isolated from INF d7 liver (Liver 7d) or spleen (Spleen 7d) samples or from non-infected spleens (Spleen 0d) by flow cytometry. Next, these cells were labeled with VioletCell tracer to measure proliferation by tracer dilution among cells. Note that Tconv from infected animals did not proliferate after <i>in vitro</i> anti-CD3 stimulation; proliferation was observed in non-infected Tconv only. (B) CD4+Foxp3+ T cells (Tregs) isolated from the same animals described above were co-cultured with APCs and naïve Tconv, with anti-CD3 stimulation. The histograms show the VioletCell tracer dilution after culture. (C) Percentages of naïve Tconv cells that proliferated with different concentrations of Tregs in culture. These results are representative of 3 independent experiments.</p

<i>In vivo</i> accumulation of iRBCs and leukocyte sequestration within the liver.

<p>B10.PL mice were intraperitoneally infected or not with Pc-AS-mCherry. All animals were divided into three groups: (A) infected, on day 7 of infection (INF d7); infected, on day 21 of infection (INF d21); and (B) Control (non-infected) mice that were freshly injected with iRBCs and immediately imaged. Each square area represents 20×20 µm. The red arrows indicate red-iRBC accumulation points within the liver blood vessels. Red circles represent foci of intense iRBC accumulation. To allow for the observation of blood vessels, FITC-Dextran was injected just after image acquisition. (C) Livers from Control, INF d7, and INF d21 mice were frozen, cut, and analyzed by IF. Green, red, blue, and DAPI colors correspond to CD11c, CD4, CD8, and DAPI staining, respectively. Red bar  = 20 µm. These results are representative of 3 repetitions.</p