RESUMO
Uncaria tomentosa is a plant native to the Amazon that has immunomodulatory and antitumor properties due to the alkaloids found in the plant, being able to modify the immune response by potentiating or suspending the action of cytokines secreted by macrophages that induce the immune response, either by the classical route (M1) or through the alternative route (M2). Macrophages activated by M1 convert L-arginine into L-citrulline and nitric oxide (NO), whereas macrophages activated by the M2 pathway use the enzymatic activity of arginase to convert the same substrate into L-ornithine and urea. The aim of this work was to evaluate the immunostimulating activity of the crude hydroalcoholic extract from the bark of the U. tomentosa stem in RAW 264.7 macrophages. Concentrations of 0.2, 0.1 and 0.05 mg/mL of U. tomentosa extract associated with LPS, INF-γ and IL-4 inducers were tested by determining NO production and arginase enzyme activity. Nitric oxide production was enhanced by the extract when associated with LPS and LPS + INF-γ inducers. In the activity of the arginase enzyme, the extract decreased the stimulation of IL-4 on the enzyme, mainly at 0.2 mg/mL concentration. Therefore, it is concluded that the crude hydroalcoholic extract of the stem bark of U. tomentosa in RAW 264.7 cells, at a concentration of 0.2 mg/mL, showed considerable pro-inflammatory activity.
Assuntos
Unha-de-Gato , Arginase , Interleucina-4 , Lipopolissacarídeos/farmacologia , Óxido Nítrico , Macrófagos , Extratos Vegetais/farmacologiaRESUMO
Abstract Uncaria tomentosa is a plant native to the Amazon that has immunomodulatory and antitumor properties due to the alkaloids found in the plant, being able to modify the immune response by potentiating or suspending the action of cytokines secreted by macrophages that induce the immune response, either by the classical route (M1) or through the alternative route (M2). Macrophages activated by M1 convert L-arginine into L-citrulline and nitric oxide (NO), whereas macrophages activated by the M2 pathway use the enzymatic activity of arginase to convert the same substrate into L-ornithine and urea. The aim of this work was to evaluate the immunostimulating activity of the crude hydroalcoholic extract from the bark of the U. tomentosa stem in RAW 264.7 macrophages. Concentrations of 0.2, 0.1 and 0.05 mg/mL of U. tomentosa extract associated with LPS, INF-γ and IL-4 inducers were tested by determining NO production and arginase enzyme activity. Nitric oxide production was enhanced by the extract when associated with LPS and LPS + INF-γ inducers. In the activity of the arginase enzyme, the extract decreased the stimulation of IL-4 on the enzyme, mainly at 0.2 mg/mL concentration. Therefore, it is concluded that the crude hydroalcoholic extract of the stem bark of U. tomentosa in RAW 264.7 cells, at a concentration of 0.2 mg/mL, showed considerable pro-inflammatory activity.
Resumo A Uncaria tomentosa é uma planta nativa da Amazônia que possui propriedades imunomoduladoras e antitumorais devido aos alcaloides encontrados no vegetal, sendo capaz de modificar a resposta imune ao potencializar ou suspender a ação de citocinas secretadas por macrófagos que induzem a resposta imune, seja pela via clássica (M1) ou pela via alternativa (M2). Macrófagos ativados pela M1 converte L-arginina em L-citrulina e óxido nítrico (NO), já os macrófagos ativados pela via M2 utilizam a atividade enzimática da arginase para converter o mesmo substrato em L-ornitina e ureia. O trabalho teve como objetivo avaliar a atividade imunoestimuladora do extrato hidroalcoólico bruto da casca do caule da U. tomentosa em macrófagos RAW 264.7. Foram testadas as concentrações 0.2, 0.1 e 0.05 mg/mL do extrato de U. tomentosa associadas aos indutores LPS, INF-γ e IL-4, por meio da determinação da produção de NO e atividade da enzima arginase. A produção de óxido nítrico foi potencializada pelo extrato quando associado aos indutores LPS e LPS + INF-γ. Na atividade da enzima arginase, o extrato diminuiu a estimulação da IL-4 sobre a enzima, principalmente na concentração 0.2 mg/mL. Portanto, conclui-se que o extrato hidroalcoólico bruto da casca do caule de U. tomentosa em células RAW 264.7, na concentração 0.2 mg/mL, demostrou atividade pró-inflamatória considerável.
RESUMO
Sepsis induces a severe systemic inflammatory response that may result in multiple organ dysfunction and death. Studies using a protein derived from natural Hevea brasiliensis (rubber tree) latex, denominated Hev b 13, have demonstrated important anti-inflammatory effects, but no data have been published regarding its effects on sepsis. The aim of this study was to investigate the effects of Hev b 13 on the inflammatory response and lung lesions of septal rats. Male Wistar rats were submitted to cecal ligation and puncture (CLP), randomized into groups and treated with subcutaneously administered doses of 0.5/2.0/3.0 mg/Kg of Hev b 13. Next, animals were subdivided into three different points in time (1, 6 and 24 hours after treatments) for collection of blood samples and euthanasia accompanied by organ removal. Total and differential leukocyte counts, cytokine dosage and histological assessment were analyzed. Treatment with Hev b 13 resulted in a significant decline in total and differential leukocytes as well as suppression of TNF-α and IL-6 production, associated with the increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it reduced morphological and pathological changes found in the lungs, including neutrophil infiltration, edema and alveolar thickening. The present study concluded that Hev b 13 exerts anti-inflammatory effects and attenuates lung lesions in septal rats, showing potential for clinical application.(AU)
Sepse induz uma resposta inflamatória sistêmica grave podendo resultar em disfunção de múltiplos órgãos e morte. Pesquisas utilizando uma proteína derivada do látex natural de Hevea brasiliensis (seringueira), denominada Hev b 13 tem demonstrado importantes efeitos anti-inflamatórios, mas nenhum dado foi publicado dos seus efeitos na sepse. O objetivo deste estudo foi investigar os efeitos da Hev b 13 na resposta inflamatória e na lesão pulmonar de ratos com sepse. Ratos machos da linhagem Wistar foram submetidos a ligação e perfuração do ceco (LPC), randomizados em grupos e tratados com as doses 0,5/2,0/3,0 mg/Kg de Hev b 13 subcutâneo. Após subdividiu-se os animais em três pontos diferentes de tempo (1, 6 e 24 horas após os tratamentos) para coleta de amostras sanguíneas e eutanásia com remoção dos órgãos. Contagem total e diferencial de leucócitos, dosagem de citocinas e avaliação histológica foram analisadas. O tratamento com a Hev b 13 resultou em diminuição significativa de leucócitos totais e diferenciais bem como suprimiu a produção de TNF-α e IL-6, associado ao aumento de IL-10 e IL-4 no plasma e tecido pulmonar. Além disso, reduziu as alterações morfológicas e patológicas encontradas nos pulmões, incluindo infiltrado de neutrófilos, edema e espessamento alveolar. Este estudo concluiu que a Hev b 13 tem efeitos anti-inflamatórios e atenua lesões pulmonares em ratos com sepse, apresentando potencialidades para aplicabilidade clínica.(AU)
Assuntos
Animais , Ratos , Hevea , Látex/uso terapêutico , Sepse/terapia , Proteínas Recombinantes/uso terapêutico , Anti-Inflamatórios/farmacologiaRESUMO
Abstract Sepsis induces a severe systemic inflammatory response that may result in multiple organ dysfunction and death. Studies using a protein derived from natural Hevea brasiliensis (rubber tree) latex, denominated Hev b 13, have demonstrated important anti-inflammatory effects, but no data have been published regarding its effects on sepsis. The aim of this study was to investigate the effects of Hev b 13 on the inflammatory response and lung lesions of septal rats. Male Wistar rats were submitted to cecal ligation and puncture (CLP), randomized into groups and treated with subcutaneously administered doses of 0.5/2.0/3.0 mg/Kg of Hev b 13. Next, animals were subdivided into three different points in time (1, 6 and 24 hours after treatments) for collection of blood samples and euthanasia accompanied by organ removal. Total and differential leukocyte counts, cytokine dosage and histological assessment were analyzed. Treatment with Hev b 13 resulted in a significant decline in total and differential leukocytes as well as suppression of TNF-α and IL-6 production, associated with the increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it reduced morphological and pathological changes found in the lungs, including neutrophil infiltration, edema and alveolar thickening. The present study concluded that Hev b 13 exerts anti-inflammatory effects and attenuates lung lesions in septal rats, showing potential for clinical application.
Resumo Sepse induz uma resposta inflamatória sistêmica grave podendo resultar em disfunção de múltiplos órgãos e morte. Pesquisas utilizando uma proteína derivada do látex natural de Hevea brasiliensis (seringueira), denominada Hev b 13 tem demonstrado importantes efeitos anti-inflamatórios, mas nenhum dado foi publicado dos seus efeitos na sepse. O objetivo deste estudo foi investigar os efeitos da Hev b 13 na resposta inflamatória e na lesão pulmonar de ratos com sepse. Ratos machos da linhagem Wistar foram submetidos a ligação e perfuração do ceco (LPC), randomizados em grupos e tratados com as doses 0,5/2,0/3,0 mg/Kg de Hev b 13 subcutâneo. Após subdividiu-se os animais em três pontos diferentes de tempo (1, 6 e 24 horas após os tratamentos) para coleta de amostras sanguíneas e eutanásia com remoção dos órgãos. Contagem total e diferencial de leucócitos, dosagem de citocinas e avaliação histológica foram analisadas. O tratamento com a Hev b 13 resultou em diminuição significativa de leucócitos totais e diferenciais bem como suprimiu a produção de TNF-α e IL-6, associado ao aumento de IL-10 e IL-4 no plasma e tecido pulmonar. Além disso, reduziu as alterações morfológicas e patológicas encontradas nos pulmões, incluindo infiltrado de neutrófilos, edema e espessamento alveolar. Este estudo concluiu que a Hev b 13 tem efeitos anti-inflamatórios e atenua lesões pulmonares em ratos com sepse, apresentando potencialidades para aplicabilidade clínica.
Assuntos
Animais , Masculino , Ratos , Proteínas de Plantas/farmacologia , Antígenos de Plantas/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pneumopatias/metabolismo , Proteínas de Plantas/administração & dosagem , Distribuição Aleatória , Citocinas/imunologia , Citocinas/metabolismo , Citocinas/sangue , Ratos Wistar , Sepse/metabolismo , Modelos Animais de Doenças , Antígenos de Plantas/administração & dosagem , Pneumopatias/imunologiaRESUMO
Sepsis induces a severe systemic inflammatory response that may result in multiple organ dysfunction and death. Studies using a protein derived from natural Hevea brasiliensis (rubber tree) latex, denominated Hev b 13, have demonstrated important anti-inflammatory effects, but no data have been published regarding its effects on sepsis. The aim of this study was to investigate the effects of Hev b 13 on the inflammatory response and lung lesions of septal rats. Male Wistar rats were submitted to cecal ligation and puncture (CLP), randomized into groups and treated with subcutaneously administered doses of 0.5/2.0/3.0 mg/Kg of Hev b 13. Next, animals were subdivided into three different points in time (1, 6 and 24 hours after treatments) for collection of blood samples and euthanasia accompanied by organ removal. Total and differential leukocyte counts, cytokine dosage and histological assessment were analyzed. Treatment with Hev b 13 resulted in a significant decline in total and differential leukocytes as well as suppression of TNF-α and IL-6 production, associated with the increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it reduced morphological and pathological changes found in the lungs, including neutrophil infiltration, edema and alveolar thickening. The present study concluded that Hev b 13 exerts anti-inflammatory effects and attenuates lung lesions in septal rats, showing potential for clinical application.
Assuntos
Antígenos de Plantas/farmacologia , Pneumopatias/metabolismo , Pulmão , Proteínas de Plantas/farmacologia , Sepse/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Animais , Antígenos de Plantas/administração & dosagem , Citocinas/sangue , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pneumopatias/imunologia , Masculino , Proteínas de Plantas/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Wistar , Sepse/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologiaRESUMO
Visceral leishmaniasis (VL) is a tropical neglected disease endemic in 98 countries and affects more than 58 000 individuals per year. Several serological tests are available for VL diagnosis, including an immunochromatographic (IC) test with the rK39 antigen and finger prick-collected blood, a rapid and low-invasive test. Here, we investigate the possibility to use saliva as a non-invasive source of biological material for the rK39 IC test. Blood samples from 84 patients with suspected VL were screened by the rK39 IC test, and 29 were confirmed as being infected by a positive rK39 IC test and the presence of amastigotes on smears slides or parasite DNA (detected using PCR-RFLP) from bone marrow aspirate. The rK39 IC test using saliva samples was positive for 17 of the 29 confirmed VL cases (58.6%). The amount of Leishmania-specific IgG or total IgG, as evaluated by an immunoenzymatic assay, was higher in the saliva of patients who had rK39 IC test positivity using saliva, whereas the amount of Leishmania-specific IgA or total IgA was similar to the healthy donors. These results suggest that saliva is not an appropriated material for diagnosing VL with this test.
Assuntos
Anticorpos Antiprotozoários/análise , Antígenos de Protozoários/imunologia , Cromatografia de Afinidade/métodos , Leishmania/imunologia , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/imunologia , Saliva/imunologia , Adolescente , Adulto , Idoso , Sangue/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1ß, IL-6, TGF-ß and IL-23 production, whereas IL-10 and TGF-ß are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1ß, IL-6 and TGF-ß. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.
Assuntos
Citocinas/imunologia , Leishmania braziliensis/imunologia , Leishmaniose/imunologia , Leucócitos Mononucleares/imunologia , Animais , Citocinas/genética , Feminino , Humanos , Leishmania braziliensis/isolamento & purificação , Masculino , Camundongos , Camundongos Knockout , Células Th17/imunologiaRESUMO
Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post-infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6-8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti-L. amazonensis IgG and increased production of IL-10 and IL-17. Because IL-10 is a mediator of susceptibility to Leishmania infection, we blocked IL-10 signalling in neutrophil-depleted mice using anti-IL-10R. Interestingly, inhibition of IL-10 signalling abrogated the increase in parasite loads observed in neutrophil-depleted mice, suggesting that parasite proliferation is at least partially mediated by IL-10. Additionally, we tested the effect of IL-17 in inflammatory macrophages and observed that IL-17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.
Assuntos
Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Neutrófilos/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Arginase/metabolismo , Feminino , Imunoglobulina G/sangue , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Cinética , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Carga Parasitária , Linfócitos T Reguladores/imunologiaRESUMO
The larval stage of Taenia crassiceps has been used to study human cysticercosis as these larvae have antigenic similarity to the cysticerci of Taenia solium. The aim of this study was to evaluate the histopathological and immunological changes that followed the inoculation of T. crassiceps cysticerci into the subcutaneous tissue of C57BL/6 mice. Microscopically, granulomas formed of neutrophils and macrophages developed at the sites of inoculation. The serum concentration of the cytokine interferon (IFN)-γ increased throughout the course of infection, while the serum concentration of interleukin-4 increased during the period of transition from the initial phase (7-30 days postinoculation [dpi]) to the late phase (60-90 dpi) of infection. Destruction of the parasite therefore appears to be associated with an increase in IFN-γ, suggesting that a type 1 immune response is important in the control of the parasite.
Assuntos
Cisticercose/patologia , Granuloma/patologia , Doenças Parasitárias em Animais/patologia , Tela Subcutânea/patologia , Animais , Cisticercose/sangue , Cisticercose/imunologia , Modelos Animais de Doenças , Granuloma/sangue , Granuloma/imunologia , Interações Hospedeiro-Parasita/fisiologia , Interferon gama/sangue , Interleucina-4/sangue , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/patologia , Doenças Parasitárias em Animais/sangue , Doenças Parasitárias em Animais/imunologia , Tela Subcutânea/imunologia , Tela Subcutânea/parasitologia , Fatores de TempoRESUMO
ABSTRACT This study was aimed to morphologically and quantitatively analyze the types of hemocytes in Diatraea saccharalis F. thirdinstar caterpillar immunologicaly challenged by Metarhizium anisopliae (Metsch.) Sorok. and Beauveria bassiana (Bals.) Vuill. in concentrations of 103, 105 and 107 conidia mL-1, compared to nontreated specimens. A 10µL-sample of hemolymphs from five caterpillars for each treatment, in the intervals of 24, 36, 48 and 60h; 300 cells were counted in random fields of the lamina, using the immersion objective (100X). The most frequent hemocytes were the spherulocytes (34.8%), plasmatocytes (29.7%) and granulocytes (22.2%), while the least frequent were the prohemocytes (8.3%), the adipohemocytes (2.6%) and the oenocytoids (2.4%). B. bassiana promoted effective interference in the population of granulocytes at the concentration of 107 conidia mL-1 (24 and 36h) and of plasmatocytes at the concentrations of 103 conidia mL-1 (60h) and 107 conidia mL-1 (24 and 36h); there occurred an increase of the number of the granulocytes and a decrease of the number of plasmotocytes at the highest concentration, and an increase in the number of plasmatocytes at the concentration of 103 conidia mL-1. M. anisopliae do not interfere quantitatively in the granulocytes and plasmatocytes. Regarding the spherulocytes, there was observed a higher tendency of reduction in the number of these cells in the interval of 24 and 36h for both fungi, but a meaningful difference only occurred at the concentration of 105 conidia mL-1 in the 60h interval, presenting the smallest average. It was possible to conclude that B. bassiana has a more effective action over the populational dynamics of the granulocytes and plasmatocytes of D. saccharalis caterpillars in association with M. anisopliae.
RESUMO A pesquisa objetivou analisar, morfológica e quantitativamente, os tipos de hemócitos em lagartas do terceiro ínstar de Diatraea saccharalis F., desafiadas imunologicamente por Metarhizium anisopliae (Metsch.) Sorok. e Beauveria bassiana (Bals.) Vuill., nas concentrações 103, 105 e 107 conídios mL-1, em comparação a lagartas não tratadas. Foram coletados 10 µL de hemolinfa de cinco lagartas por tratamento, nos intervalos de 24, 36, 48, e 60 horas pós-inoculação, sendo contadas 300 células em campos aleatórios da lâmina, utilizando-se a objetiva de imersão (100X). Os hemócitos mais freqüentes foram os esferulócitos (34,8%), plasmatócitos (29,7%) e granulócitos (22,2%), enquanto os menos freqüentes foram os prohemócitos (8,3%), adipohemócitos (2,6%) e oenocitóides (2,4%). B. bassiana promoveu interferência efetiva na população de granulócitos na concentração de 107 conídios mL-1 (24 e 36h) e plasmatócitos nas concentrações 103 conídios mL-1 (60h) e 107 conídios mL-1 (24 e 36h), onde ocorreu aumento do número de granulócitos e redução do número de plasmatócitos na maior concentração e, ainda, o aumento do número de plasmatócitos na concentração 103 conídios mL-1. M. anisopliae não interferiu quantitativamente nos granulócitos e plasmatócitos. No que se refere aos esferulócitos, verificou-se maior tendência de redução do número dessas células no intervalo de 36h para ambos os fungos, porém, diferença significativa só ocorreu na concentração de 105 conídios mL-1 no intervalo de 60h, apresentando a menor média. Conclui-se que B. bassiana tem ação mais efetiva sobre a dinâmica populacional dos granulócitos e plasmatócitos em lagartas de D. saccharalis em relação ao M. anisopliae.