Several reports of clinical trials of immunotherapy using dendritic cells have been published to date. In this study, we investigated the safety and clinical response of immunotherapy with fusions of dendritic and glioma cells for the treatment of patients with malignant glioma. Eight patients with malignant glioma, ranging in age from 4 to 63 years old, participated in this study. Dendritic cells were generated from peripheral blood. Cultured autologous glioma cells were established from surgical specimens in each case. Fusion cells of dendritic and glioma cells were prepared with polyethylene glycol, and the fusion efficiency ranged from 9.2 to 35.3% (mean, 21.9%). All patients received the fusion cells every three weeks for a minimum of 3, and a maximum of 7, immunizations. Fusion cells were injected intradermally, close to a cervical lymph node. The percentage of CD16- and CD56-positive cells in peripheral blood lymphocytes slightly increased after immunization in 4 out of 5 cases investigated. Peripheral blood mononuclear cells were incubated with irradiated autologous glioma or U87MG cells and supernatants were harvested. In 6 cases analyzed, the concentration of interferon-gamma in the supernatant increased after immunization. Clinical results showed that there were no serious adverse effects and two partial responses. Although the results of the phase I clinical trial of fusion cells indicated that this treatment safely induced immune responses. we were unable to establish a statistically significant treatment-associated response rate, due to the limited sample population. Therefore, further evaluation of the role of adjuvant cytokines is necessary.

Summary Dendritic cells (DCs) are potent antigen‐presenting cells that are uniquely capable of inducing primary immune responses. Although tumour cells may directly inhibit DC maturation, exposure to tumour products may also result in their activation. Fusions of cancer cells and DCs are being explored as cancer vaccines. The effect of tumour cell fusion on DC maturation and their functional characteristics has not been defined. In the present study, immature and mature DC generated from human CD34+ and peripheral blood precursors were fused to multiple myeloma cells in the presence of polyethylene glycol. Fusion of both immature and mature DCs with tumour cells resulted in an activated phenotype. In this regard, fusion cells expressed interleukin‐12, a cytokine essential for the induction of T‐helper cell type 1 immunity. In contrast to immature DCs, fusion cells also strongly expressed CC‐chemokine receptor R7, which is responsible for DC migration to draining lymph nodes. Fusions generated with both immature and mature DCs also potently stimulated T‐cell expression of γ‐interferon and cytotoxic T lymphocyte killing of tumour targets. These findings demonstrate that tumour cell fusion induces DC maturation and the development of an activated phenotype necessary for their effectiveness as cancer vaccines.

This study showed that administration of BIO-THREE improved the clinical symptoms and endoscopic findings in patients with UC, indicating that administration of BIO-THREE is safe and efficacious for the treatment of UC.

Adacolumn selective granulocyte and monocyte apheresis (GMA) depletes activated leukocytes in patients with ulcerative colitis (UC). However, this per se cannot fully explain the efficacy of GMA. We have investigated the effects of GMA on the expression of toll-like receptors (TLRs) and plasma interleukin-8 (IL-8). Twenty-two patients with clinical activity index (CAI) of 5-17, 15 with total colitis and 7 with left-sided colitis, were included. Each patient could receive up to 10 GMA sessions, at 1 or 2 sessions per week. GMA was added to the patients' ongoing medication following a relapse or worsening UC, but no additional medication was given. Further, at entry and pre-GMA, blood samples were taken for full blood cell count, expression of TLRs on leukocytes, and plasma IL-8. Seventy-five percent of patients achieved remission after the 10th session (CAI, < or =4; P < 0.005) and there was a marked fall in C-reactive protein (P < 0.01), plasma IL-8 (P < 0.001), and granulocytes (P < 0.05) but an increase in lymphocytes (P < 0.05). The expression of TLR2 on granulocytes was down-modulated (P < 0.05) together with suppression of inflammatory cytokines produced by peripheral blood leukocytes. In conclusion, GMA appears to be an effective adjunct therapy to induce remission in the majority of patients, who are then spared from excess drug therapy. The procedure is associated with sustained immunomodulation. Control studies should strengthen these findings.

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Immunotherapy using tumor antigen-loaded dendritic cells is a new approach for the treatment of various types of malignant tumors. Here, we describe a patient with advanced gastric carcinoma who received immunotherapy using fused autologous dendritic cells and carcinoma cells (fusions) and showed effective clinical responses to the treatment. A 74-year-old man showed massive ascitic effusion due to peritonitis carcinomatosa after surgical operation for gastric carcinoma. A gastric carcinoma cell line was established from the patient's tumor tissue. Dendritic cells were obtained by cultivation of the adherent cell fraction of the patient's peripheral blood mononuclear cells (PBMCs) with granulocyte macrophage-colony stimulating factor, interleukin-4, and tumor necrosis factor-alpha. The cells were mixed with irradiated tumor cells and treated with 50% polyethyleneglycol (PEG) for the generation of fusions, as described previously. The PEG-treated cells were injected subcutaneously every 2 weeks. Low-grade fever was observed after the first and second treatments. After the third treatment, ascitic effusion and leg edema decreased markedly, without any other treatments. Serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 decreased to levels lower than those at the initiation of treatment. PBMCs collected after the fifth treatment elicited cytotoxic activity against autologous tumor cells. Although treatment was continued in the same way, recurrence of the disease was observed about 5 months after the start of the treatment. This is the first report of immunotherapy utilizing fusions of autologous dendritic cells and tumor cells resulting in effective clinical responses in advanced gastric carcinoma, without severe adverse effects.

Apart from generating T-helper (Th) effector responses, dendritic cells (DCs) are capable of initiating tolerance against the inciting antigens. Therefore, successful DC-based immunotherapy against malignant tumors requires an additional strategy to activate antigen-processing DCs. We studied the antitumor immune responses conferred by fusions of DCs and glioma cells in vitro. Fusion cells (FCs) were stimulated with polyriboinosinic polyribocytidylic acid [Poly(I:C)] and/or small interference RNA (siRNA) of IL-10 (IL-10-siRNA). Increased IFN-β expression induced by Poly(I:C) transfection was accompanied by enhanced production of IL-10 and IL-12p70 in the FCs. We also found that the ability of Poly(I:C)-transfected FCs to produce IL-12p70, but not IFN-β, was preserved when endogenous IL-10 was suppressed by IL-10-siRNA. To analyze the antigen-presenting function further, DCs, glioma cells, and peripheral lymphocytes were established from patients newly diagnosed with glioma. In this experiment, peripheral lymphocytes were stimulated with autologous FCs and restimulated with autologous glioma cells. CD4T cells isolated from the stimulated lymphocytes were subjected to the ELISPOT and WST-1 assays, which revealed that the IL-10-siRNA/Poly(I:C)-cotransfected FCs elicit an efficient tumor-specific Th1 response. These findings support the relevance of using Poly(I:C) and IL-10-siRNA in clinical immunotherapy protocols with an FC-based vaccine for patients with malignant glioma as a means of promoting Th1-induced tumor antigen presentation.

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