Made available in DSpace on 2014-12-17T14:13:23Z (GMT). No. of bitstreams: 1 AlexandreFSQ.pdf: 1970606 bytes, checksum: 5b6e9381595f936a1ed9bffd4f4fef7a (MD5) Previous issue date: 2008-05-12 / A galactose and sucrose specific lectin from the marine sponge Cliona varians named CvL was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. Models of leukocyte migration in vivo were used to study the inflammatory activity of CvL through of mouse paw oedema and peritonitis. Effect of CvL on peritoneal macrophage activation was analyzed. Effects of corticoids and NSAIDS drugs were also evaluated on peritonitis stimulated by CvL. Results showed that mouse hind-paw oedema induced by sub plantar injections of CvL was dependent dose until 50?g/paw. This CvL dose when administered into mouse peritoneal cavities induced maxima cell migration (9283 cells/?L) at 24 hours after injection. This effect was preferentially inhibited by incubation of CvL with the carbohydrates D-galactose followed by sucrose. Pre-treatment of mice with 3% thioglycolate increases the peritoneal macrophage population 2.3 times, and enhanced the neutrophil migration after 24h CvL injection (75.8%, p<0.001) and no significant effect was observed in presence of fMLP. Finally, Pre-treatment of mice with dexamethason (cytokine antagonist) decreased 65.6%, (p<0.001), with diclofenac (non-selective NSAID) decreased 34.5%, (p<0.001) and Celecoxib (selective NSAID) had no effect on leukocyte migration after submission at peritonitis stimulated by CvL, respectively. Summarizing, data suggest that CvL shows pro-inflammatory activity, inducing neutrophil migration probably by pathway on resident macrophage activation and on chemotaxis mediated by cytokines / A lectina denominada CvL, com especificidade para D-galactose e sacarose foi purificada da esponja marinha Cliona varians atrav?s de fracionamento com acetona seguido por cromatografia de afinidade em Sepharose CL 4B. No presente trabalho, foi estudada a atividade inflamat?ria de CvL in vivo atrav?s dos modelos de edema de pata e migra??o de leuc?citos em peritonite induzida em camundongos. Foram analisados os efeitos de carboidratos na inibi??o da peritonite, o efeito da lectina na ativa??o de macr?fagos e o efeito de antiinflamat?rios no modelo de peritonite provocada pela CvL. Os resultados mostraram que o edema de pata induzido pela CvL foi dose dependente at? 50 ?g/pata. Essa mesma dose quando administrada na cavidade peritoneal, foi capaz de induzir migra??o celular (9.283 c?lulas/?L) com pico em 24 horas ap?s a administra??o. Este efeito foi inibido quando a CvL foi previamente incubada com os carboidratos D-galactose e sacarose. O pr?-tratamento dos camundongos com 3% de tioglicolato aumentou a popula??o de macr?fagos em 2,3 vezes o que provocou o aumento da migra??o de neutr?filos em 75,58% (p<0,0001) ap?s 24 horas da inje??o da CvL. No entanto, com a inje??o de fMLP n?o foi observado nenhum efeito significativo. O pr?-tratamento dos camundongos com dexametasona (cortic?ide antagonista de citocinas) reduziu a migra??o de neutr?filos em 65,6% (p<0,001), com diclofenaco ( antiinflamat?rio n?o ester?ide n?o seletivo) reduziu em 34,5% (p<0,001) e com Celecoxibe (antiinflamat?rio n?o ester?ide seletivo) n?o apresentou efeito. Os resultados do presente trabalho s?o indicativos de que a CvL possui atividade pr?-inflamat?ria, induzindo a migra??o de neutr?filos, provavelmente atrav?s da indu??o da libera??o de citocinas quimiot?cticas na ativa??o de macr?fagos residentes no perit?nio

Made available in DSpace on 2014-12-17T14:03:25Z (GMT). No. of bitstreams: 1 GiocondaEDDM.pdf: 827312 bytes, checksum: dd690ec78d2921013aa0e24b334bac19 (MD5) Previous issue date: 2007-12-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NF&#61547;B) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway / Neste trabalho, a linhagem K562 de c?lulas de leucemia miel?ide cr?nica, expressando a prote?na oncog?nica BCR-ABL, foi usada como modelo para investigar a atividade antitumoral da lectina CvL purificada da esponja marinha Cliona varians. CvL inibiu o crescimento de c?lulas K562 com um IC50 de 70 g/mL, mas n?o afetou a viabilidade celular de linf?citos normais de sangue perif?rico humano no mesmo intervalo de concentra??es testadas (1 80 g/mL). A morte celular ocorreu ap?s 72 horas de exposi??o ? lectina e com altera??es nucleares t?picas de apoptose como analisado pela fluoresc?ncia de DAPI. Investiga??o dos poss?veis efetores deste processo mostrou que a morte celular ocorreu sem ativa??o de caspases e na presen?a de express?es aumentadas de Bcl-2 e Bax. O fato de CvL desencadear a libera??o de catepsina B, como evidenciado pela microscopia de fluoresc?ncia, e do inibidor E-64 bloquear completamente a morte celular induzida por CvL, sugerem papel central dessa protease lisossomal na ativa??o de uma via alternativa de morte celular. CvL tamb?m induziu o aumento de express?o do receptor de morte TNFR-1 e a diminui??o dos n?veis de NF&#954;B. Estes efeitos foram acompanhados pelo aumento significativo na express?o de p21 e pela modula??o negativa de pRb, mostrando que CvL foi capaz de bloquear a progress?o do ciclo celular. Juntos, estes dados sugerem que catepsina B age como mediador da citotoxicidade induzida por CvL possivelmente atrav?s de uma conex?o ainda n?o caracterizada com a via dos receptores de morte