Kiyotaka Fujise

A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery.

Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

Dendritic Cells Fused with Allogeneic Colorectal Cancer Cell Line Present Multiple Colorectal Cancer–Specific Antigens and Induce Antitumor Immunity against Autologous Tumor Cells

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2−/HLA-24−), carcinoembryonic antigen (CEA)+, and MUC1+ as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2+ and/or HLA-A24+) and allogeneic COLM-6 resulted in MHC class I– and MHC class II–restricted proliferation of CD4+ and CD8+ T cells, high levels of IFN-γ production in both CD4+ and CD8+ T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

A randomized trial of intrahepatic arterial infusion of 4′-epidoxorubicin with Lipiodol versus 4′-epidoxorubicin alone in the treatment of hepatocellular carcinoma

We conducted a prospective randomized trial to evaluate the efficacy of Lipiodol in intrahepatic arterial infusion chemotherapy for patients with hepatocellular carcinoma (HCC). A total of 38 patients with unresectable HCCs and underlying cirrhosis were entered in this trial, and 36 of them were evaluable. Every 4 weeks, 17 patients received 70 mg of 4′-epidoxorubicin (epirubicin) alone (group A), whereas 19 patients received a Lipiodol emulsion containing the same dose of epirubicin (group B) through the hepatic artery. A tumor response (CR+PR) was observed in 12% of group A patients and in 42% of group B patients. The group B patients showed a significantly higher response rate than the group A patients. There was a tendency for an increased duration of survival (P=0.09) in the group B patients. These results suggested that the infusion of the Lipiodol emulsion with epirubicin was more effective than epirubicin alone for the treatment of these patients with HCC.

N‐3 polyunsaturated fatty acid diet therapy for patients with inflammatory bowel disease

Background: N‐3 polyunsaturated fatty acids (PUFA) are considered important pharmaconutrients for modulating mucosal immunity and therapeutic responses in patients with inflammatory bowel disease (IBD). We investigated the influence of diet therapy involving the use of an “n‐3 PUFA food exchange table” (n‐3DP) on the fatty acid composition of the erythrocyte membranes of IBD patients and its remission‐maintaining effects. Methods: We analyzed the fatty acid composition of the erythrocyte membrane before and after n‐3DP intervention in 20 initial‐onset IBD patients who had not undergone any dietary intervention. We then analyzed it again and evaluated disease activity after 12–18 months intervention in 230 IBD patients (168 ulcerative colitis, 62 Crohns disease; follow‐up group) in whom n‐3DP was introduced after remission had been achieved. The follow‐up group was divided into remission and relapse groups. Results: In the 20 initial‐onset patients, the mean n‐3/n‐6 ratio significantly increased after intervention (0.41 ± 0.16 versus 0.70 ± 0.20; P < 0.001). In the follow‐up group the ratio in the remission group (n = 145) was significantly higher than that in the relapse group (n = 85) (0.65 ± 0.28 versus 0.53 ± 0.18; P < 0.001). The ratio significantly decreased in those who suffered a relapse after the beginning of treatment (P < 0.01). Conclusions: N‐3DP significantly increased the erythrocyte membrane n‐3/n‐6 ratio in IBD patients, and this ratio was significantly higher in the remission group, suggesting that n‐3DP alters the fatty acid composition of the cell membrane and influences clinical activity in IBD patients. (Inflamm Bowel Dis 2010)

Peginterferon and ribavirin treatment for hepatitis C virus infection

Pegylated interferon α (IFNα) in combination with ribavirin is currently recommended as a standard-of-care treatment for chronic hepatitis C virus (HCV) infection. This combination therapy has drastically improved the rate of sustained virological response, specifically in difficult-to-treat patients. Recently, individualized treatment, such as response-guided therapy, is being developed based on host-, HCV- and treatment-related factors. Furthermore, modified regimens with currently available medications, novel modified IFNα and ribavirin or combinations with specifically targeted antiviral therapy for HCV agents, are currently being investigated. The purpose of this review is to address some issues and epoch-making topics in the treatment of chronic HCV infection, and to discuss more optimal and highly individualized therapeutic strategies for HCV-infected patients.

Thiopurine S-methyltransferase and inosine triphosphate pyrophosphohydrolase genes in Japanese patients with inflammatory bowel disease in whom adverse drug reactions were induced by azathioprine/6-mercaptopurine treatment

BackgroundThe main cause of azathioprine (AZA)/6-mercaptopurine (6MP)-induced adverse reactions is a reduction in the activities of the metabolizing enzymes thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase (ITPA). Adverse reactions develop at a high frequency in Japanese patients at half the dose required for European and American patients; however, the association with TPMT and ITPA gene polymorphisms in Japanese has not been fully investigated.MethodsGene mutations of TPMT and ITPA, the major AZA/6-MP -metabolizing enzymes, were investigated retrospectively in 16 Japanese patients with inflammatory bowel disease (IBD) in whom AZA/6MP treatment induced adverse reactions.ResultsThe TPMT gene was found to have a wild-type sequence in all patients, but in the ITPA gene a mutation, 94C>A, was detected at a rate of 50% (8/16), with 83.3% (5/6) occurring in patients with acute bone marrow suppression and 75% (3/4) in those with agranulocytosis. The 94C>A allele frequency was 10 of 32 (0.313; 95% CI, 0.180–0.486). Adverse reactions developed earlier in patients with the 94C>A mutation. However, in half the patients, no gene polymorphism was noted.ConclusionsIt is suggested that the ITPA gene mutation is closely related to the adverse reactions of AZA/6-MP in Japanese patients, and screening for the mutant allele is useful for predicting the most serious adverse reactions, agranulocytosis and acute bone marrow suppression.

Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells.

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor‐associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte‐derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA‐derived CEA and MUC1 antigens and DC‐derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4+ and CD8+ T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN‐γ. More importantly, coculture of fusion cells with patient‐derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen‐specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1‐positive and HLA partially matched target cells. Antigen‐specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA‐ and MUC1‐specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen‐specific CTL responses. Our report demonstrates the simultaneous induction of CRCA‐specific CTL responses restricted by HLA‐A2 and ‐A24.

Contribution of ribavirin transporter gene polymorphism to treatment response in peginterferon plus ribavirin therapy for HCV genotype 1b patients.

Standard‐dose ribavirin is crucial for the standard‐of‐care treatment of chronic hepatitis C virus (HCV) infection. Equilibrative nucleoside transporter 1 (ENT1), encoded by SLC29A1 gene, is the main transporter that imports ribavirin into human hepatocytes. Aims:

Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- γ at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+IFN-γ+CD8+ T cells and CD154+ IFN-γ+CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.