Establishment of specific immunotherapy by activation of innate immunity

为了建立一种有效的化疗和免疫治疗的联合治疗，选择那些有效诱导肿瘤细胞凋亡且对全身免疫活性影响较小的化疗药物。本实验观察了抗癌药物parlituel（1XL）和docetaxel（TXT）对人外周血单个核细胞（PBMC）的生长抑制作用、对胃癌细胞的凋亡诱导作用以及对DCs表面Toll样受体（TLR）-4mRNA表达的影响。进一步比较了肿瘤细胞裂解物致敏的DC诱导的细胞毒性T淋巴细胞（CTL）和TXT诱导的凋亡细胞的细胞毒性。采集健康志愿者PBMC，在含有10%PCS抗凝剂的RPM 1640中培养，加入浓度分别为血浆峰浓度（PPC）、0.1xPP和10 xPPC，同时加入PHA，通过测定细胞内Al？程度. 1UNNEL法检测细胞凋亡。获得未成熟DC， ...更多信息 通过在含有白细胞介素-4（IL-4 ; 50 ng/ml）和粒细胞-噬菌体刺激因子（GM-SP 50 ng/ml）的无血清培养基（AIM V）中培养PBMC 5天来获得。然后用从胃癌细胞MKN 45或经TXT诱导的凋亡的MKN 45细胞获得的肿瘤细胞裂解物脉冲DC 12小时，并在加入OK-432（0.1KE/ml）后进一步培养30小时。LDH释放法测定CIL活性。尽管大多数抗癌药物以剂量依赖性方式显示对PBMC增殖的抑制活性，但即使在最高药物浓度下，TXT、去氧氟尿苷和irinobecane也未显示对PBMC的抑制活性。MKN 45细胞在24小时后约有60%发生凋亡。1XL和TXT处理48小时。用肿瘤裂解物致敏的DC诱导的TLR-4 mRNA表达在1×10- 8 M Clis处理后2 h达高峰，凋亡细胞显示与tarwt细胞相似的杀伤活性。这些结果表明，TXT似乎是一个最佳的抗癌药物的联合化疗和肿瘤特异性免疫治疗使用树突状细胞在胃癌。我们然后计划一个前瞻性的临床I/II期试验，使用TXT和5 v-DFUR联合化疗，然后在肿瘤内注射未成熟DC胃癌患者。到目前为止，三名患者被纳入，免疫学和临床病理参数的分析正在进行中。少

In order to establish an efficient combination therapy with chemotherapy and immunotherapy, selection of chemotherapeutic agents those effectively induce apoptosis on tumor cells and have less effect to systemic immunological activity. We investigated the growth inhibitory activity of anticancer agent on human peripheral blood mononudear cells (PBMC), apoptosis inducing activity on gastric cancer cells and expression of toll-like receptor (TLR)-4 mRNA on DCs by parlituel (1XL) and docetaxel (TXT). We further compared the cytotoxidty of cytothdc T lymphocytes (CTLs) induced by DCs pulsed with tumor lysate and apoptotic cells induced by TXT. PBMC were collected from healthy volunteers and cultured in RPM1640 with 10% PCS Anticanoer agents were added at concentrations of peak plasma concentration (PPC), 0.1xPP and 10xPPC concomitant with PHA Proliferating activity of PBMC was determined by measuring intracellular Al? levels. Apoptosis was evaluated by 1UNNEL assay. Immature DCs were obtai … More ned by culturing PBMCs in serum-free medium (AIM V) with interieukin-4 (IL-4 ; 5Ong/ml) and granulocyte-maaophage stimulating factor (GM-SP 5Ong/m1) for 5 days. Then DCs were pulsed with tumor cell lysate obtained from gastric cancer cells MKN45 or apoptotic MKN45 cells induced by TXT for 12hr and further cultured for 30hr following addition of OK-432 (0.1 KE/ml). CIL activity was determined by LDH-releasing assay. Although most of anticancer agents demonstrated the suppression activity on proliferation of PBMC in a dose dependent manner, TXT, doxifluridine and irinobecane did not show the suppressive activity on PBMC even in the highest drug concentration. About 60% of MKN45 cells demonstrated apoptosis after 24hr. and 48hr treatment of both 1XL and TXT. Expression of TLR-4 mRNA in iDCs was up-regulated by TXT, not by TXT, peaked at 2 hr after treatment at a concentration 1×10-8M Clis induced by DCs pulsed with tumor lysate and apoptotic cells showed similar killing activity to tarwt cells. These results suggest that TXT appears to be a optimal anticancer agent for a combination therapy with chemotherapy and tumor specific immunotherapy using dendritic cells in gastric cancer.We then planed a prospective clinical Phase I/II trial using a combination chemotherapy of TXT and 5 v-DFUR followed by intra-tumoral immature DC injection in patients with gastric cancer. So far, three patients were included and the analysis of immunological and clinicopathblogic parameters are now underway. Less