| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
[Background] Dendritic cells (DCs) are specialized to present antigen for initiation of immune response, and have shed light on tumor therapy. However, the development and characteristics of DCs in the dog has not been studied thoroughly. Therefore, we conducted a series of experiments to understand in-vitro differentiation, characterization of canine DCs and regulatory factors for optimizing their activity in the tumor therapy.
[Results] 1) Generation of canine DCs from peripheral blood monocytes without using purified cytokines. To seek the appropriate condition for in vitro DC differentiation, we have developed a conditioned media by activated T cells (TCCM), and attempted to induce differentiation of DCs from peripheral blood monocytes (PBMOs). When purified PBMOs were cultured with TCCM, the PBMOs had extended dendritic processes, and significantly increased the expression of MHC class II and CD1a. Also, they increased stimulatory activity for T cells. Moreover, the cells significa
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ntly increased IL-18 and IFN-gamma expression with stimulation with Poly (I:C), and reduced phagocytic activity. It follows from these results that TCCM provide optimal condition for the in vitro differentiation of DCs from PBMOs. 2) Treatment of cancers in dogs using in vitro-generated DC and interferon gamma. It is reported that the in vitro-generated DCs are acutely inactivated when they are put back into life body, and therefore, can not exert to develop immune responses against cancers. To tackle this problem, we injected interferon gamma (IFN_Y), a typical Th1 and DC activator, together with in vitro-generated DCs into patient dogs with tumor including mammalian carcinoma, fibrosarcoma, lung-epithelial cell carcinoma, sebacerous epithelioma. In all of five cases of the dog tumor, the treatment with DC plus IFN_Y exerted complete remission or significant decrease of tumor lesion. In contrast, treatment with IFN_Y alone did not result any decrease of tumors. These results suggest that maintenance of DC activation in vivo is necessary for success of immune cancer therapy. 3) Cloning and production of canine cytokine genes for in vivo activation and recruitment of DCs. To make in vivo activation and recruitment of DCs, we cloned cDNA of canine IL-12, IL-18, GM-.CSF and IL-4 gene, and made the improved IL-18 gene by fusing signal sequence to mature cDNA for facilitated secretion of bioactive protein. Expressed protein by the improved IL-18 gene significantly enhanced IFN-gamma production of T cells. Expressed proteins by the cloned GM-CSF and IL-4 gene mediated the induction of DCs from PBMOs, and the protein by cloned IL-12 gene activated the induced DCs. These results suggest that the cytokine genes can be utilized for in vivo initiation and activation of immune response against tumors.
[Conclusion] These studies make advances for DC based tumor therapy in the dog. Less
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