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To investigate antitumor mechanisms in interleukin (IL)-4 therapy, we established an IL-4-overexpressing MC38 murine colorectal cancer cell line (MC38-IL4). As a therapy against established tumors, MC38-IL4 cells were inoculated contralaterally 7 days after wild-type (MC38-WT) cells had been injected, significantly reducing growth of wild-type tumors (P=0.030). Immunohistochemical analysis showed numerous granulocytes infiltrating wild-type tumors of MC38-IL4-inoculated mice. Injection of MC38-IL4 cells in leukocyte-depleted mice confirmed that granulocytes were involved in IL-4-related primary antitumor effects. Inoculation of MC38-WT in leukocyte-depleted mice initially injected with MC38-IL4 suggested that T cells contributed to the antitumor effects. To investigate tumor-specific responses, we stimulated splenocytes of MC38-immune mice with MC38-IL4 cells in vitro, resulting in MC38-specific lysis (57.5±7.2%, effector to target ratio=20). Treatment of established wild-type tumors with MC38-IL4 in combination with interferon (IFN)-a-overexpressing MC38 cells (MC38-IFNa) significantly reduced the growth of wild-type tumors (P=0.009). In vitro IFN-?production by splenocytes from mice injected with both MC38-IL4 and -IFNawas greatly enhanced in comparison with MC38-IL4 alone, while IL-10 production was not increased. Thus, granulocytes concern early antitumor effects of IL-4 therapy. Subsequently, IL-4 induces long-lasting, tumor-specific immune responses. IL-4 appears to promote a T-helper 1-type antitumor immune response, which is enhanced in cooperation with IFN-a.Gene Therapy (2005) 12, 733-741. doi:10.1038/sj.gt.3302401 Published online 17 March 2005 [ABSTRACT FROM AUTHOR]

Although chronic constipation is common, colonic functional evaluating tests are uncommon. This study examines whether chronic constipation and gastrointestinal symptoms are correlated with the lateral diameter of the colon measured from MRI images. We included chronic constipation patients in a prospective, cross-sectional study using MRI at three centers. We divided 3D MRI colorectal images into 6 segments using with specified sequences and selected the maximum luminal diameter from each segment. We used the GSRS questionnaire to evaluate gastrointestinal symptoms. We evaluated the correlation between luminal diameters and GSRS scores. We found the following positive correlations: descending colon and unsatisfactory defecation symptoms; sigmoid colon and diarrhea; and rectum and constipation. The sum and ratio of the ascending and sigmoid colon diameters correlated with nausea and diarrhea. The sum of the transvers to the sigmoid colon diameter also correlated with nausea and diarrhea. The sum of all segment diameters correlated with nausea and constipation. In conclusion, we showed cross-sectional study of colonic MRI correlate with gastrointestinal symptoms. MRI might be useful for colonic motility evaluations to determine appropriate constipation treatments (Clinical trial registry number UMIN 000021274)., Competing Interests: No potential conflicts of interest were disclosed.

The number of available studies on the role of human serum albumin (HSA) in the treatment of cirrhotic ascites is currently limited. In this study, we aimed to investigated the parameters associated with diuretic therapy with HSA in patients with advanced cirrhotic ascites. The patient inclusion criteria were cirrhotic ascites and a serum albumin (Alb) concentration of <3.5 g/dl. A total of 49 patients registered and 38 patients were ultimately included in this study. The enrolled patients were mainly treated with oral spironolactone and furosemide, which were not specified; the HSA amount was also not specified, although the administration period was set to a maximum of 7 days. Our results demonstrated that the administration of HSA significantly increased the serum levels of Alb [0.97 g/dl; two-sided 95% confidence interval (CI): 0.83-1.11 g/dl] and decreased body weight (-2.24 kg; 95% CI: -3.06 to -1.43 kg), hematocrit ratio (0.96; 95% CI: 0.94-0.98) and plasma renin concentration (day 4; geometric mean fold change, -0.1528; 95% CI: -0.2510 to -0.0545; log-transformed data) in patients with advanced cirrhotic ascites. The observed weight loss was found to be correlated with the total amount of HSA administered (P=0.0012), as indicated by the results of the multiple linear regression analysis. In conclusion, this study confirmed the efficacy of HSA in patients with advanced cirrhotic ascites.

Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy.

Pre-operative chemoradiotherapy (CRT) is an important neoadjuvant therapy for locally advanced rectal cancer. In the present study, we investigated the factors that influence the efficacy of pre-operative CRT in locally advanced rectal cancer. We divided 50 patients with locally advanced rectal carcinoma treated with pre-operative CRT into two groups according to the grade of tumor response to pre-operative CRT: low-sensitivity group and high-sensitivity group. As candidates for the prediction of sensitivity to pre-operative CRT, clinicopathological factors and 12 biomarkers, including factors related to tumor growth, cell cycle, apoptosis, tumor stroma and cancer stem cells, were examined immunohistochemically in 48 resected specimens. Thirty-one tumors showed high sensitivity and 19 showed low sensitivity to pre-operative CRT. The status of stem cell-related factors, CD133 and CD24, was significantly associated respectively with sensitivity to pre-operative CRT (P=0.003, P=0.029). In 10 tumors positive for both CD133 and CD24, low sensitivity to CRT was found in 9 (90%), whereas in 16 tumors negative for both CD133 and CD24, low sensitivity was found in 3 (19%). Other pathological parameters were not associated with tumor response to pre-operative CRT. In conclusion, overexpression of cancer stem cell-related factors, CD133 and CD24, is associated with the sensitivity of locally advanced rectal cancer to pre-operative CRT.

Immune responses of cytotoxic T lymphocytes (CTLs) are implicated in viral eradication and the pathogenesis of hepatitis C. Weak CTL response against hepatitis C virus (HCV) may lead to a persistent infection. HCV infection impairs the function of HCV-specific CTLs; HCV proteins are thought to actively suppress host immune responses, including CTLs. Induction of a strong HCV-specific CTL response in HCV-infected patients can facilitate complete HCV clearance. Thus, the development of a vaccine that can induce potent CTL response against HCV is strongly expected. We investigated HCV-specific CTL responses by enzyme-linked immuno-spot assay and/or synthetic peptides and identified over 40 novel CTL epitopes in the HCV protein. Our findings may contribute to the development of the HCV vaccine. In this paper, we describe the CTL responses in HCV infection and the attempts at vaccine development based on recent scientific articles.

Dendritic cells (DC) are potent antigen-presenting cells that elicit immune responses to foreign antigens. We have previously demonstrated the synergistic effects of cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODN) and interferon (IFN)-alpha on DC maturation in vitro. In the present study, the antitumor effects of DC preincubated with IFN-alpha gene-overexpressing murine colorectal cancer MC38 cells (MC38-IFN-alpha) and CpG ODN were evaluated in a poorly immunogenic murine cancer system. When we injected DC preincubated with MC38-IFNalpha and CpG ODN subcutaneously to mice bearing MC38 wild-type tumors, the outgrowth of the established parental tumors was suppressed significantly compared with that following administration of DC with MC38-IFN-alpha (P = 0.008). All mice injected with DC preincubated with MC38-IFN-alpha and CpG ODN rejected a subsequent parental tumor challenge. Immunohistochemical and flow cytometric analyses showed that CD4(+), CD8(+), and NK1.1(+) cells markedly infiltrated the established tumors of mice treated with DC preincubated with MC38-IFN-alpha and CpG ODN. From the results in immune cell-depleted mice, CD4(+) and asialo-GM-1(+) cells seemed to contribute to the antitumor effects induced by the combination DC therapy. Furthermore, non-specific cytolysis was detected when splenocytes of mice inoculated with DC preincubated with MC38-IFNalpha and CpG ODN were used as effector cells. Using an interleukin (IL)-12-neutralizing antibody it was suggested that IL-12 stimulates natural killer cells and contributes in part to the antitumor effects induced by DC incubated with CpG ODN and IFN-alpha. As DC-based immunotherapy with CpG ODN and IFN-alpha-expressing tumor cells induces a potent antitumor immune response, it should be considered for clinical application.

Background: Dendritic cells (DCs) play an important role in immune response and cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODN) as well as interferon (IFN)-alpha have been proven to induce DC maturation. In this study, the synergistic effects of CpG-ODN and IFN-alpha on DC maturation were evaluated., Materials and Methods: Surface molecules on DCs and the stimulatory responses of DCs to allogeneic splenocytes were analyzed after cultivation with CpG-ODN and IFN-alpha-overexpressing murine colorectal cancer MC38 cells (MC38-IFNalpha)., Results: Co-incubation with CpG-ODN and MC38-IFNalpha, but not wild-type MC38 cells (MC38-WT), effectively up-regulated co-stimulatory molecules on the DCs. CpG, in combination with IFN-alpha, stimulated IL-1beta and TNF-alpha production by DCs effectively. When DCs preincubated with CpG-ODN and MC38-IFNalpha were co-incubated with allogeneic splenocytes in vitro, the proliferation of these splenocytes was significantly enhanced compared with that of splenocytes incubated with CpG-ODN and MC38-WT cells (p = 0.041)., Conclusion: Since CpG-ODN and IFN-alpha have synergistic effects on DC maturation, they may induce potent antitumor immune responses and combination therapy should be considered for clinical application.

Cytokine gene therapy is applied in clinical studies of tumors, and IFN-alpha and IL-12 are widely used for cancer immunotherapy. Using a poorly immunogenic murine colorectal cancer cell line, MC38, we compared antitumor effects of IFN-alpha and IL-12. Transduced MC38 cell lines expressing IFN-alpha or IL-12 (MC38-IFNalpha or MC38-IL12, respectively) were established using retroviral vectors. Transduction of IFN-alpha or IL-12 gene to MC38 cells significantly reduced tumorigenicity in immunocompetent mice. When tumor-free mice initially injected with MC38-IFNalpha or MC38-IL12 cells were reinjected contralaterally with wild-type MC38 cells (MC38-WT) after 35 days, 7 of 12 or 2 of 12 mice rejected MC38-WT cells, respectively. In therapy-model mice with established tumor derived from MC38-WT cells, inoculation of gene-transduced cells significantly suppressed growth of the tumor in MC38-IFNalpha-inoculated groups, but not in the IL-12-inoculated group. Immunohistologic and flow cytometric analyses showed marked infiltration of CD8(+) cells in wild-type tumors of mice inoculated with IFN-alpha-expressing cells. Leukocyte-depletion experiments implicated CD8(+) T cells in tumor rejection induced by IFN-alpha-transduction; both CD8(+) T cells and natural killer cells were implicated in the more modest antitumor effect from IL-12 expression. To investigate induction of tumor-specific immune responses, we stimulated splenocytes from tumor-free mice twice in vitro with genetically modified MC38 cells. In vitro stimulations with MC38-IFNalpha cells induced definite MC38-specific lysis, but not stimulations with MC38-IL-12 cells. Injecting combination of MC38-IFNalpha and MC38-IL-12 cells caused an additive antitumor effect in the therapy model. These data suggested that IFN-alpha induces cytotoxic T lymphocytes and elicits long-lasting tumor-specific immunity, whereas IL-12 seems to stimulate non-specific killing. With additional refinements, combined IFN-alpha and IL-12 gene therapy might warrant clinical trials.

Dendritic cells (DCs) are very potent antigen-presenting cells and play critical roles in regulating immune responses in cancer. The migrating of DCs from the tumor site to the lymphoid organs is believed to be one of the critical events. To examine this important DC function in tumor situations, bone marrow-derived DCs, cultured for 6 days with granulocyte macrophage colony-stimulating factor and interleukin 4, were inoculated at the tumor site. We have shown (Y. Nishioka et al., Cancer Res., 59: 40354041, 1999) that DCs can migrate from tumor site to the draining lymph nodes within 24 h (approximately 0.1% of administrated DCs). The DCs then form clusters with adjacent lymphoid cells, which produce IFN-gamma (1500-3200pg/10(6) cells/48 h) in response to tumor stimulation. The number of the DCs migrating into lymph nodes were greater when they were inoculated into the tumor rather than the skin. Coculture of DCs and apoptotic tumor cells resulted in decreased expression of CC chemokine receptor (CCR) 1 and increased CCR7 expression at mRNA level without alteration in other phenotypical markers on DCs. Chemotaxis assay showed that CCR7 ligands, macrophage inflammatory protein 3beta and secondary lymphoid-tissue chemokine significantly (P < 0.05) induced the migration of DCs when cocultured with apoptotic tumor cells. To directly examine the involvement of CCR7 expression in DC migration, we investigated the functions of DCs genetically modified to express high levels of CCR7. CCR7 transduction promotes DC migration in response to relevant ligands in vitro and in vivo. These results suggest that the CCR7 expression of DCs is enhanced with direct contact with apoptotic tumor cells and may have a critical role for DC migrating to regional lymph nodes. The means to promote DC delivery to tumor and to nodal sites represent novel targets for the biological therapy of cancer.