Avaliação da eficácia antitumoral e toxicidade de lipossomas pH-sensíveis de circulação prolongada contendo cisplatina no tratamento de camundongos portadores de tumor ascítico de Ehrlich

Submitted by Nuzia Santos ([email protected]) on 2012-08-22T13:27:09Z No. of bitstreams: 1 Tese_Lais Maroni Portugal.pdf: 2267253 bytes, checksum: 795ea3bb95d9876df036c514007c1d1c (MD5)Made available in DSpace on 2012-08-22T13:27:09Z (GMT). No. of bitstreams: 1 Tese_Lais Maroni Portugal.pdf: 2267253 bytes, checksum: 795ea3bb95d9876df036c514007c1d1c (MD5)FAPEMIG: Fundação de Amparo à Pesquisa do Estado de Minas GeraisCPqRR/FIOCRUZ: Centro de Pesquisas René Rachou/Fundação Oswaldo CruzCNPq: Conselho Nacional de Desenvolvimento Científico e TecnológicoFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Cisplatina (CDDP) é um dos agentes ativos citotóxicos mais comumente usados no tratamento da carcinomatose peritoneal. A inconveniência de seu uso clínico são os efeitos colaterais sistêmicos, como nefrotoxicidade e mielotoxicidade. Lipossomas pH-sensíveis de circulação prolongada contendo CDDP (SpHL-CDDP) foram desenvolvidos por nosso grupo de pesquisa com o objetivo de promover a liberação de CDDP mais próximo do tumor, bem como diminuir a toxicidade. O objetivo deste estudo foi avaliar a eficácia antitumoral e toxicidade de SpHL-CDDP, após a administração intraperitoneal em camundongos portadores de tumor nas fases inicial ou avançada, com uma dose de 12 mg/Kg. A sobrevida foi monitorada e amostras de sangue foram coletadas para análises bioquímicas e hematológicas. Rins, fígado e baço foram removidos para exame histopatológico. As células tumorais foram avaliadas, segundo sua viabilidade e ciclo celular. Os resultados demonstraram que a sobrevida de animais tratados com SpHL-CDDP foi maior do que aqueles tratados com CDDP livre. A morte celular causada pelo tratamento com SpHL-CDDP ocorreu através da indução de apoptose com a parada do ciclo celular na fase G0/G1. O tratamento de camundongos que apresentam câncer inicial com ambas as formulações provocou a supressão de granulócitos. Camundongos tratados com CDDP livre apresentaram também diminuição da contagem de plaquetas, o que sugere alta mielotoxicidade. No modelo de câncer avançado, o tratamento com SpHL-CDDP permitiu melhoria da resposta imune. Camundongos portadores de câncer em estágio inicial e tratados com CDDP livre ou SpHL-CDDP apresentaram menor índice de uréia/creatinina, em comparação ao grupo controle salina. Esses achados indicam que ambos os tratamentos foram capazes de reduzir o dano renal causado por carcinomatose peritoneal. A análise microscópica dos rins de camundongos tratados com SpHL-CDDP mostrou alteração morfológica discreta, enquanto necrose tubular foi observada para animais tratados com CDDP livre. Em relação à hepatotoxicidade, nenhuma alteração nos parâmetros de química clínica foi observada. Estes achados revelam que SpHL-CDDP pode melhorar a eficácia antitumoral e diminuir a toxicidade renal e da medula óssea. A dosagem de VEGF no líquido ascítico mostrou que ambas as formulações contendo CDDP, em ambos os estágios de tratamento, diminuíram a capacidade angiogênica do tumor de Ehrlich, apresentando efeito antitumoral. O estudo imunológico mostrou que o tratamento com CDDP livre ou SpHLCDDP apresenta um perfil modulado de resposta imune, com diminuição das citocinas próinflamatórias e de citocinas reguladoras. Portanto, estes resultados abrem a possibilidade de uso futuro de SpHL-CDDP para o tratamento da carcinomatose peritonealCisplatin (CDDP) is one of the most active cytotoxic agents commonly used on treatment of peritoneal carcinomatosis. The inconvenience of its clinical use is systemic side effects, such as nephrotoxicity and myelotoxicity. Long-circulating and pH-sensitive liposomes containing CDDP (SpHL-CDDP) were developed by our research group in order to promote the release of CDDP near the tumor as well decrease of its toxicity. The aim of this study was to evaluate the antitumor efficacy and toxicity of SpHL-CDDP after intraperitoneal administration in initial or disseminated tumor bearing mice, at a dose of 12 mg/Kg. The survival was monitored and blood samples were collected for biochemical and hematological analysis. Kidney, liver and spleen were removed to histopathological examination. Tumor cells were evaluated according to their viability and cell cycle. The survival of animals treated with SpHCDDP was higher than those treated with free CDDP. The cell death caused by treatment with SpHL-CDDP occurred through induction of apoptosis with a cell cycle arrest at G0/G1 phase. The treatment of mice presenting initial cancer with both formulations provoked a suppression of granulocytes. Mice treated with free CDDP showed also a decrease of platelets count, which suggests a high myelotoxicity. Mice affected by cancer at an early stage and treated with free CDDP or SpHL-CDDP showed lower urea/creatinine index as compared to the saline control group. These findings indicate that both treatments were able to reduce the renal damage caused by peritoneal carcinomatosis. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed a discrete morphological alteration, while tubular necrosis was observed for free CDDP treated mice. Concerning hepatotoxicity, no alteration in clinical chemistry parameters was observed. These findings reveal that SpHL-CDDP can improve the antitumor efficacy and decrease renal and bone marrow toxicity. VEGF levels in ascitic fluid showed that both formulations containing CDDP administered in both development stages, decreased angiogenic capacity of Ehrlich tumor, showing their antitumor effect. The immunological study showed that treatment with free CDDP or SpHL-CDDP presents an immune response modulated profile, with reduction of pro-inflammatory and regulatory cytokines. Overall, the results presented in this thesis indicate a promising future application of SpHL-CDDP to peritoneal carcinomatosis treatment

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of <it>Schistosomiasis mansoni</it> infection

Abstract Background The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. Methods The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. Results The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4+ T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8+ T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. Conclusions The data indicate that SEA-stimulated CD4+ T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.</p

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection

Submitted by Nuzia Santos ([email protected]) on 2014-06-16T12:44:46Z No. of bitstreams: 1 Cytokine profile.pdf: 3149759 bytes, checksum: cf43916157d8279f1d5361396fd1143b (MD5)Made available in DSpace on 2014-06-16T12:44:46Z (GMT). No. of bitstreams: 1 Cytokine profile.pdf: 3149759 bytes, checksum: cf43916157d8279f1d5361396fd1143b (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Escola de Enfermagem. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais.Escola de Enfermagem. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Brasilia, DF, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrazilUniversidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Clínica Médica, Cirurgia. Belo Horizonte, MG, Brazil/Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Belo Horizonte, BrazilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil/Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais. Belo Horizonte, BrazilUniversidade Federal de Minas Gerais. Faculdade de Medicina. Departamento de Cirurgia. Belo Horizonte, MG, BrazilBackground :The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. Methods : The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. Results : The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4+ T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8+ T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. Conclusions : The data indicate that SEA-stimulated CD4+ T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group