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Title: 活性化リンパ球を用いた犬の腫瘍免疫療法の臨床応用に関する基礎的研究
Other Titles: A basic study on clinical application of canine antitumor immunotherapy using activated lymphocytes
Authors: 星野, 有希1 Browse this author
Authors(alt): Hoshino, Yuki1
Issue Date: 30-Jun-2008
Abstract: Conventional tumor therapy, including surgery, radiotherapy, and chemotherapy, effectively prolongs the survival time of tumor-bearing patients. However, critical adverse effects frequently occur during conventional tumor therapy, disturbing the quality of life (QOL) of these patients. Immunotherapy is therefore expected to be an alternative therapy for accelerating antitumor immunity, while inducing less adverse effects. In tumor-bearing animals, the activation of spontaneous cellular immunity to tumorous tissue is relatively limited, and this suppression of immune function is not easily compensated even after the removal of the tumor mass. The induction of both specific and nonspecific immunity, especially T cell function, is imperative for the activation of antitumor immunity, which is suppressed in tumor-bearing animals. Passive immunotherapy by administration of artificially proliferated effector cells that are activated ex vivo is considered to be applicable for these immunosuppressed animals. Activated lymphocyte therapy is a type of passive immunotherapy; it involves sequential administration of autologous lymphocytes stimulated and cultured with anti-cluster of differentiation (CD)-3 antibody and human recombinant (hr) interleukin (IL)-2. The majority of these cultured cells are speculated to be CD3+ T cells and natural killer (NK) cells, which can induce both specific and nonspecific immunity against the tumor mass. It is essential to determine the optimum conditions for lymphocyte culture and cell administration and to evaluate this method, which is not yet well established. The purpose of the present study was to establish the methodology for the administration of activated lymphocyte therapy in tumor-bearing dogs and to evaluate this therapy. In the first part of this study, the dynamics of canine CD56+ cells in peripheral blood collected from healthy beagles were investigated. CD56 is considered to be one of the markers for NK cells in human beings; thus, canine CD56 is expected to be a candidate marker for canine NK cells. Quantitative polymerase chain reaction (PCR) method was used to determine canine CD56 messenger ribonucleic acid (mRNA) expression in normal tissues, peripheral blood mononuclear cells (PBMCs), and stimulated lymphocytes. The highest CD56 mRNA expression was observed in the brain. The CD56 mRNAexpression in stimulated lymphocytes was higher than that in PBMCs. To localize CD56 mRNA, in situ hybridization was performed. The number of CD56+ cells of stimulated lymphocytes was extremely higher than that of PBMCs. CD56 mRNAexpression in canine lymphocytes was considerably lower; most lymphocytes were CD56-. However, in the lymphocytes activated by stimulated PBMCs, both the CD56 mRNAexpression and the number of CD56+ cells increased. In the second part of the study, PBMCs collected from clinically healthy dogs were activated using anti-CD3 antibody and hr IL-2 and reactivated using hr interferon (IFN)-α and IL-2. The properties of the cells thus obtained were analyzed in order to investigate the adaptation of canine activated lymphocyte therapy. In the obtained cells, the proportion of CD4+CD8- cells and CD4-CD8+ cells was significantly increased; the cytotoxicity of the cultured cells was reinforced; and CD56 mRNA levels tended to be increased. These cells were confirmed to be activated lymphocytes. Furthermore, we investigated the effects of sequential administration of these cells in healthy dogs. This sequential administration was shown to increase the cell proliferative activity of PBMCs, the proportions of CD4+CD8- cells and CD4-CD8+ cells, and the serum IFN-γ concentration; no severe adverse effects were observed. We concluded that activated lymphocytes could be administered to healthy dogs, and sequential administration of these cells reinforced the recipient’s immunity. In the third part of the study, autogenous activated lymphocytes, which were activated in the second part of the study, were sequentially administered as an adjuvant antitumor therapy to 10 dogs with spontaneous malignant tumors, in Veterinary Teaching Hospital of Hokkaido University. Peripheral blood lymphocytes from tumor-bearing dogs were proliferated and activated for 14 days in culture. After sequential administration of these activated lymphocytes, the proliferation activities of PBMCs, proportions of CD4+CD8- cells and CD4-CD8+ cells, and serum IFN-γ concentration increased in some cases. The general conditions of all 10 patients remained stable, and the patients were comfortable during the entire period of lymphocyte administration. The owners of these dogs considered this new concept of antitumor therapy acceptable. This shows that the QOL of these tumor-bearing dogs was maintained in a relatively favorable state, with no evidence of any adverse effects. Activated lymphocyte therapy was therefore thought to be applicable and effective as an adjuvant antitumor therapy; moreover, it maintained the QOL in immunosuppressed tumor-bearing dogs. In conclusion, CD56+ cells existed in canine peripheral blood, and these cells proliferated during the process of activation to lymphocytes. Expression of canine CD56 mRNApossibly indicates the activation of lymphocytes. The conditions of activation of lymphocytes were then optimized, using anti-canine CD3 antibody, hr IL-2, and hr IFN-α in healthy and tumor-bearing dogs. Sequential administration of these activated lymphocytes was an applicable and effective therapy for tumor-bearing small animals with immunosuppression induced by malignant tumors.
Conffering University: 北海道大学
Degree Report Number: 甲第8786号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 星野 有希

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