Cotransfection of Poly(I: C) and siRNA of IL-10 into fusions of dendritic and glioma cells enhances antitumor T helper type 1 induction in patients with glioma.

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-ß 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-ß, 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.

Clinical effectiveness of probiotics therapy (BIO-THREE) in patients with ulcerative colitis refractory to conventional therapy.

OBJECTIVE: Intestinal microflora has been implicated in the etiology of ulcerative colitis (UC). Over the past few years, the use of probiotics in UC has gained attention. The aim of this study was to evaluate the efficacy of probiotics therapy for mild to moderate distal UC refractory to conventional therapies. MATERIAL AND METHODS: Twenty patients with mild to moderate distal UC took 9 BIO-THREE tablets per day for 4 weeks. Clinical symptoms and endoscopic findings were evaluated as ulcerative colitis disease activity index (UCDAI) scores before and after administration of BIO-THREE. Fecal samples were collected from all patients before and after probiotics administration, and fecal microflora was analyzed by the terminal restriction fragment length polymorphism (T-RFLP) method. RESULTS: Remission (UCDAI score < or =2) was observed in 45% (9/20) of the patients; response (decrease in UCDAI > or = 3, but final score > or = 3) in 10% (2/20); no response in 40% (8/20); and worsening (UCDAI > 3) in 5% (1/20). T-RFLP analysis indicated that the principal alteration in microflora was an increase in bifidobacteria. CONCLUSIONS: 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 leukocyte adsorption apheresis in patients with active ulcerative colitis: clinical efficacy, effects on plasma IL-8, and expression of Toll-like receptor 2 on granulocytes.

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.

Fusion of dendritic cells with multiple myeloma cells results in maturation and enhanced antigen presentation.

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 gamma-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.

Inhibition of spontaneous development of liver tumors by inoculation with dendritic cells loaded with hepatocellular carcinoma cells in C3H/HeNCRJ mice.

We attempted to prevent spontaneous development of liver tumors by s.c. inoculation with DCs loaded with syngeneic HCC cells in C3H/HeNCrj mice. A new cell line, MIH-2, was established from an HCC that had developed spontaneously in a C3H/HeNCrj mouse. Bone marrow-derived DCs were loaded with irradiated MIH-2 cells by treatment with PEG. Fluorescence microscopy and flow-cytometric analysis showed that about 45% of PEG-treated DCs and MIH-2 cells (DC/MIH-2) were DCs loaded with MIH-2 cells. Thirteen-month-old mice received inoculations of DC/MIH-2 (9 x 10(5)/mouse) 4 times at 6-day intervals and were killed at 16 months of age to assess liver tumors. The incidence of liver tumors in these mice was significantly lower than that in mice not receiving inoculations (p < 0.05) but similar to that in 13-month-old mice (the age at which inoculation started), indicating that inoculation inhibited the development of new tumors. Splenocytes from inoculated mice, but not those from uninoculated mice, showed cytotoxic activity against MIH-2 cells. Cytotoxic activity was not elicited by CD4(+) T cells, CD8(+) T cells, or DX5(+) cells isolated from splenocytes but was elicited by adherent cells, identified as CD11b(+) macrophages. CD4(+) T cells, but not CD8(+) T cells, from inoculated mice produced IFN-gamma by incubation with DC/MIH-2. Cytotoxicity by splenocytes was attenuated by anti-IFN-gamma antibody. Immunization with DCs loaded with syngeneic HCC cells induces CD4(+) T cells that produce IFN-gamma by response to antigen of HCC, which would lead to macrophage activation to kill liver tumor cells at an early stage.

Immunotherapy using fusions of autologous dendritic cells and tumor cells showed effective clinical response in a patient with advanced gastric carcinoma.

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.