ASPECTOS IMUNOPATOGÊNICOS, CLÍNICOS E FARMACOLÓGICOS DA HANSENÍASE: UMA REVISÃO DE LITERATURA

Hanseníase é uma infecção crônica causada pelo bacilo Mycobacterium lepraelevando a lesões de pele e nervos periféricos. O Brasil trata-se do único país que permanece acima do limite de prevalência de 1 caso/10.000 habitantes. O objetivo do presente artigo é abordar de forma interdisciplinar os principais aspectos imunopatogênicos, clínicos, laboratoriais e farmacológico desta doença, de modo a contribuir para a compreensão da doença. A pesquisa foi realizada através das bases de dado PubMed, Scielo, Lilacs. O diagnóstico da hanseníase, classicamente, é baseado na presença de anestesia em lesões cutâneas, espessamento de nervos periféricos e demonstração do agente etiológico na linfa ou cortes histológicos. Além da baciloscopia e histopatologia, o diagnóstico também pode ser feito pelos teste de Mitsuda e ML-Flow. A doença apresenta amplo espectro clínico, estando intimamente associado à resposta imunológica do hospedeiro. O diagnóstico precoce da hanseníase com o tratamento oportuno, ainda, é o principal ponto para cessar a transmissão e incapacidades. Assim, o reconhecimento precoce da doença é a parte que apresenta, ainda, maior deficiência dentro dos serviços de saúde

Liver accumulation of Plasmodium chabaudi-infected red blood cells and modulation of regulatory T Cell and dendritic cell responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (PciRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these\ud molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.CAPES-FCT grant 258/2010CAPES-IGC grant 04/2012Fundação de Apoio à Pesquisa do Estado de São Paulo – FAPESP grant 2009/53.889-0CAPES-FCT grant 258/2010FCT grant PTDC/EBB-BIO/115514/200

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

Fetal-Derived MyD88 Signaling Contributes to Poor Pregnancy Outcomes During Gestational Malaria

Placental malaria (PM) remains a severe public health problem in areas of high malaria transmission. Despite the efforts to prevent infection poor outcomes in Plasmodium endemic areas, there is still a considerable number of preterm births and newborns with low birth weight resulting from PM. Although local inflammation triggered in response to malaria is considered crucial in inducing placental damage, little is known about the differential influence of maternal and fetal immune responses to the disease progression. Therefore, using a PM mouse model, we sought to determine the contribution of maternal and fetal innate immune responses to PM development. For this, we conducted a series of cross-breeding experiments between mice that had differential expression of the MyD88 adaptor protein to obtain mother and correspondent fetuses with distinct genetic backgrounds. By evaluating fetal weight and placental vascular spaces, we have shown that the expression of MyD88 in fetal tissue has a significant impact on PM outcomes. Our results highlighted the existence of a distinct contribution of maternal and fetal immune responses to PM onset. Thus, contributing to the understanding of how inflammatory processes lead to the dysregulation of placental homeostasis ultimately impairing fetal development

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

ESTUDOS PRÉ-CLÍNICOS DE ATIVIDADE GIARDICIDA DE Chenopodium ambrosioides L. E A PADRONIZAÇÃO DOS EXTRATOS NA PESQUISA E DESENVOLVIMENTO DE FITOTERÁPICOS

Chenopodium ambrosioides L. (Chenopodiaceae), conhecida como erva-de-Santa-Maria ou mastruz, representa espécie exótica, de grande ocorrência no estado do Maranhão, muito utilizada na prática popular para diversos fins terapêuticos. Estudos farmacológicos têm comprovado o potencial da espécie para desenvolvimento de bioprodutos como alternativa terapêutica. Assim, o objetivo deste trabalho foi desenvolver metodologia analítica para padronização dos extratos de C. ambrosioides empregando ensaios químicos e biológicos de atividade anti-Giardia, visando o desenvolvimento de tecnologia farmacêutica na obtenção de novos fármacos no tratamento da giardíase. Foram obtidos extratos das folhas de C. ambrosioides por planejamento fatorial dos parâmetros: natureza do solvente (etanol 70% e água), operação de extração (maceração, percolação e extração por Soxhlet) e hidromódulo; submetidos a análise química e avaliação da atividade giardicida in vitro. Os resultados permitem concluir que a obtenção dos extratos hidroalcoólicos das folhas de C. ambrosioides pelo procedimento extrativo de percolação e maceração representam os extratos com melhor perfil químico e/ou atividade giardicida; evidenciando que a natureza do solvente, o procedimento extrativo e o hidromódulo são variáveis que  infuenciam na obtenção dos extratos da espécie em estudo.Descritores: Chenopodium ambrosioides; Padronização de Extratos; Atividade Giardicida; in vitro.Abstract: Chenopodium ambrosioides L., Chenopodiaceae, (herb-of-Santa-Maria or mastruz), represents high occurrence alien species, in the State of Maranhão, widely used in popular practice for many therapeutic purposes. Pharmacological studies have demonstrated the potential of species to develop therapeutic bioproducts such as new alternatives. Thus, the goal of this work was to developanalytical methodology for standardized extracts of Chenopodium. ambrosioides employing chemical and biological tests of giardicidal activity, aiming at the development of pharmaceutical technology in getting new drugs for Giardia treatment. Were obtained extracts of leaves of C. ambrosioides by factorial planning factors: nature of solvent (ethanol 70 and water), operation of extraction (maceration, percolation and Soxhlet extraction) and hydromodule (plant:solvent); subjected to chemical analysis and evaluation of giardicidal activity  in vitro. The results suggest that the obtaining of hydroalcoholic extracts from the leaves of C. ambrosioides extractive of percolation and maceration represents the extract with better chemical profle and/or giardicidal activity; evidencing that the nature of the solvent, extractive procedure and hydromodule are variables that infuence in obtaining extracts of the species under study.Descriptors: Chenopodium ambrosioides; Standardization of Extracts; Giardicidal Activity; in vitro

TLR4-Mediated Placental Pathology and Pregnancy Outcome in Experimental Malaria

Malaria-associate pregnancy has a significant impact on infant morbidity and mortality. The detrimental effects of malaria infection during pregnancy have been shown to correlate with immune activation in the placental tissue. Herein we sought to evaluate the effect of Toll-like receptors (TLRs) activation on placental malaria (PM) development by using the Plasmodium berghei NK65(GFP) infection model. We observed that activation of the innate immune system by parasites leads to PM due to local inflammation. We identified TLR4 activation as the main pathway involved in the inflammatory process in the placental tissue since the absence of functional TLR4 in mice leads to a decrease in the pro-inflammatory responses, which resulted in an improved pregnancy outcome. Additionally, a similar result was obtained when infected pregnant mice were treated with IAXO-101, a TLR4/CD14 blocker. Together, this study illustrates the importance of TLR4 signalling for the generation of the severe inflammatory response involved in PM pathogenesis. Therefore, our results implicate that TLR4 blockage could be a potential candidate for therapeutic interventions to reduce malaria-induced pathology both in the mother and the fetus.Fundação de Amparo a Pesquisa do Estado de São Paulo - FAPESPCoordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESConselho Nacional de Desenvolvimento Científico e Tecnológico - CNPqUniv Fed São Paulo, Dept Ciencias Biol, Diadema, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Parasitol, São Paulo, BrazilHosp Israelita Albert Einstein, São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, São Paulo, BrazilUniv Estadual Campinas, Dept Genet Evolucao & Bioagentes, Inst Biol, Campinas, SP, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Imunol, São Paulo, BrazilUniv São Paulo, Dept Analises Clin & Toxicol, Fac Ciencias Farmaceut, São Paulo, BrazilUniv Fed São Paulo, Dept Ciencias Biol, Diadema, BrazilFAPESP: 2009/53889-0FAPESP: 2014/09964-5FAPESP: 2014/20451-0FAPESP: 2012/16525-2FAPESP: 2011/17880-8FAPESP: 2013/16417-8FAPESP: 2011/19048-8FAPESP: 2013/00981-1FAPESP: 2015/06106-0]CAPES: AUX-PE-PNPD 2751/2010CNPq: 475771/2009-5Web of Scienc

Brazilian Green Propolis: Anti-Inflammatory Property by an Immunomodulatory Activity

The immunomodulatory and anti-inflammatory activities of green propolis extracts from Apis mellifera were investigated using acute and chronic inflammation models. Swiss mice were anesthetized and a cotton pellet granuloma was implanted in subcutaneous tissue. Then the mice were divided into six groups and received apyrogenic water or different propolis extracts by oral route (5 mg/kg). According to the treatment the groups were designated as E1A, E1B, E10, E11, and E12. The control group received apyrogenic water. The treatment was performed by six days when the mice were killed. The blood and the bronchoalveolar lavage (BAL) were collected to measure the leukocyte recruitment. In acute pulmonary inflammation, Balb/c mice received lipopolysaccharide (LPS) of Escherichia coli by intranasal route for three days. Concomitantly the mice received by oral route apyrogenic water (control) or E10 and E11 propolis extracts. BAL was performed to assess the inflammatory infiltrate and cytokine quantification. The results showed that the E11 extract has anti-inflammatory property in both models by the inhibition of proinflammatory cytokines and increase of anti-inflammatory cytokines suggesting an immunomodulatory activity