Thesis (M.S.)--Marshall University, 2003. / Title from document title page. Document formatted into pages; contains vi, 74 p. including illustrations. Includes bibliographical references (p. 71-74).

This thesis evaluated the role of rIL-2 in patients with advanced colorectal cancer and also, as a perioperative regime, in patients with localized colorectal cancer undergoing surgical resection. Twenty patients with advanced colorectal cancer received up to six cycles of chemoimmunotherapy, each consisting of 5-fluorouracil, levamisole and rIL-2 at 18x10<SUP>6</SUP>IU/m<SUP>2</SUP>/24 for 120 hours. Responding patients were found to have significantly lower pre-treatment serum IL-6 and soluble IL-2 receptor levels, compared with non-responders. Differential patterns of host cytokine release were also identified. Haemodynamic monitoring found that indices of rIL-2-mediated toxicity, e.g. weight gain correlated with alterations in serum cytokine concentrations. In a separate study, eighteen patients, undergoing curative surgery for localized colorectal cancer, were randomized to receive placebo or bolus low-dose subcutaneous rIL-2 for three days preoperatively. rIL-2 was found to significantly enhance host antitumour natural cytotoxicity, monocyte activity and immune cell surface activation marker expression (e.g. CD25). Circulating levels of key host cytokines (e.g. interleukin-6, soluble interleukin-2 receptor) were elevated in the immediate postoperative period in these patients. Mesenteric release of key cytokines was determined in patients undergoing resection for benign and malignant colorectal disease through portal sampling at surgery. Higher patterns of release were found in patients with malignancy suggesting local modulation of immune activity. rIL-2 has been found to beneficially enhance host immune reactivity in patients with localized and advanced colorectal cancer. The nephrotoxic potential of rIL-2 therapy was determined through urinary enzyme release, plasma renin and standard blood biochemistry measurements. RIL-2, when administered carefully, may form the basis of further adjuvant immunotherapy in both the peri-operative period and in patients with advanced colorectal cancer.

Activation of immunity to self-antigens is the goal in cancer immunotherapy, whereas blocking such responses is the goal in autoimmune disease. Thus, it is not surprising that investigation into cancer immunotherapy might also produce insights for the treatment of autoimmune disease. Heat shock protein, gp96, based therapies lead to the robust activation of CD8+ cytotoxic T cells that can slow tumor growth in 60-70% of mice, but only lead to the elimination of tumors in 30-40% of animals. The primary goal of the current studies was to understand why vaccination with a secreted gp96 vaccine was not efficacious in a larger proportion of animals, and identify combination therapies that enhanced the anti-tumor activity of gp96-Ig vaccination. It was found that in mice bearing established tumors, some mice responded well to vaccination with gp96-Ig, and that the induction of CD8+ T cells was found to correlate with tumor rejection; indicating that the proportion of mice that failed to reject tumors had established mechanisms of tumor-mediated suppression of anti-tumor immunity. The mechanism of this suppression was found to differ between various tumor models, so combination therapy sought to amplify CD8+ T cell responses directly, rather than by indirectly inhibiting suppressive factors induced by established tumors. It was found that antibody-based therapies leading to the stimulation of TNFRSF25, a powerful T cell co-stimulatory receptor, caused synergistic expansion of tumor-specific T cells when given in combination with gp96-Ig vaccination and led to enhanced rejection of multiple tumor types. Interestingly, TNFRSF25 agonistic antibodies were also found to directly stimulate the proliferation of natural CD4+FoxP3+ regulatory T cells. This activity was found to be beneficial in the prevention of allergic lung inflammation when administered prior to antigen challenge. These studies have therefore identified the conditions required for successful tumor elimination following gp96-Ig vaccination, and discovered that a TNFRSF25 agonistic antibody may be used to enhance anti-tumor immunity induced by gp96-Ig. These studies have also identified TNFRSF25 stimulation as the most powerful, and physiologically relevant, method to selectively induce Treg proliferation in vivo ever discovered, with important consequences for the treatment of autoimmune inflammation.

Transduction of melanoma cells with the aim to induce avidine expression on tumor cell surface was studied. Subsequently the method enabling quantification of binding of ligands to the cell surface was developed.

Orientadores: Lucimara Gaziola de la Torre, Patrícia Cruz Bergami-Santos / O texto da introdução e conclusão estão na lingua portuguesa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T15:41:58Z (GMT). No. of bitstreams: 1 Vitor_MicaelaTamara_M.pdf: 6469643 bytes, checksum: c14b8646a57fec71cc1e0eac8a82cddb (MD5) Previous issue date: 2013 / Resumo: Esta pesquisa teve como objetivo o desenvolvimento tecnológico de uma vacina lipossomal contendo RNA tumoral destinado à imunoterapia do câncer. Nesta estratégia, RNA total codificando o antígeno tumoral Her-2/neu extraído de linhagem de células de adenocarcinoma de mama humano SK-BR-3 foram incorporados em lipossomas catiônicos introduzidos in vitro em células dendríticas (DCs). A vacina de DCs tem a função de auxiliar o sistema imunológico a identificar antígenos tumorais para que as células cancerígenas sejam eliminadas. Porém uma das etapas críticas é a introdução (transfecção) de RNA nas DCs. Lipossomas catiônicos é uma alternativa promissora, pois além de ativarem as DCs, é capaz mediar a transfecção de ácidos nucléicos para células. A experiência prévia do grupo de pesquisa na área de lipossomas catiônicos mostrou a possibilidade da obtenção de lipossomas em larga escala para o desenvolvimento de vacina de DNA contra a tuberculose. Neste contexto, este trabalho avaliou os lipossomas catiônicos com a composição lipídica de fosfatidilcolina natural de ovo (EPC), 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamina (DOTAP) e 1,2-dioleoil-3-trimetilamônio-propano (DOPE), na respectiva proporção molar de 50/25/25%. Metodologicamente, o trabalho foi dividido em quatro partes: na primeira parte foi apresentada uma visão geral do trabalho desenvolvido, demonstrando o potencial dos lipossomas catiônicos complexados com RNA na imunoterapia do câncer. Na segunda parte do trabalho investigaram-se os efeitos dos lipossomas produzidos através do processo laboratorial na diferenciação/maturação das DCs in vitro e as DCs estimuladas por estes lipossomas, na indução da proliferação de linfócitos T, resultando em lipossomas catiônicos incorporados pelas DCs, com a capacidade de ativar as DCs in vitro e de induzir proliferação de linfócitos T. A terceira parte do trabalho teve como finalidade a otimização da produção dos lipossomas catiônicos obtidos através do método de injeção de etanol utilizando a ferramentas estatísticas, obtendo lipossomas com menor polidispersidade e tamanho, que demonstraram in vitro serem incorporadas e ativarem as DCs e induzirem a proliferação de linfócitos T. A última etapa refere-se ao estudo da incorporação do RNA nos lipossomas catiônicos produzidos através do processo escalonado otimizado e comparado com o laboratorial no intuito de serem internalizados pelas DCs, transfectar o RNA e induzir a proliferação de linfócitos T através das DCs. Os resultados demonstraram que os complexos foram internalizados pelas DCs e que estas são capazes de induzir a proliferação de linfócitos T, porém há necessidade de se obter a condição ótima de transfecção. Dessa forma, conclui-se que os lipossomas catiônicos em questão têm potencial para serem usados como ferramenta em futuras estratégias na imunoterapia do câncer / Abstract: This research aimed at the technological development of a liposomal vaccine containing tumor RNA for cancer immunotherapy. In this strategy, total RNA encoding the Her-2/neu tumor antigen extracted from cell line of human breast adenocarcinoma SK-BR-3 were incorporated into cationic liposomes, which were introduced in vitro into dendritic cells (DCs). DCs vaccine has the function of helping the immune system to identify tumor antigens in order to eliminate cancerous cells. However, one of the critical steps is the introduction (transfection) of RNA in DCs. Cationic liposomes are a promising alternative, because besides activating DCs, they are able to mediate transfection of nucleic acids into cells. Previous work of our research group in the cationic liposomes field developed a liposomal nanostructure obtained by a scale up process containing DNA vaccine against tuberculosis. In this context, this work evaluated the cationic liposomes composed by egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE), at 50/25/25% molar proportion respectively. Methodologically, the present work was carried out in four mean steps: in the first step, it was carried out an overview of all work, showing the relevancy of cationic liposomes complexed with RNA for cancer immunotherapy. The second part of this work investigated the effects of liposomes produced via laboratory process upon DCs differentiation/maturation in vitro and induction of T lymphocytes proliferation by DCs stimulated with these liposomes, resulting in cationic liposomes incorporated by DCs, capable to activate DCs in vitro and to induce proliferation of T lymphocytes. The third part of the work aimed at optimizing the production of cationic liposomes obtained via the ethanol injection method using statistical tools, obtaining liposomes with smaller size and polydispersity, which demonstrated to be incorporated and activate DCs in vitro and to induce T lymphocytes proliferation. The last step refers to the study of RNA incorporation in the cationic liposomes produced via optimized scalable process compared to the laboratory process in order to be internalized by DCs, transfected RNA and to induce T lymphocytes proliferation by DCs. The results showed that the complexes were internalized by DCs and they are able to induce T lymphocytes proliferation, however we still have to obtain the optimal transfection condition. In sum, we conclude that the cited cationic liposomes can be used as a potential tool in further strategies in cancer immunotherapy / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.