Takashi Kagaya

Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

ABSTRACTPurposeSolute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.MethodsWe first constructed octn1 gene knockout (octn1−/−) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.ResultsThe metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1−/− mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1−/− mice. The octn1−/− mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn’s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.ConclusionsThese results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.

Comparative analysis of various tumor-associated antigen-specific t-cell responses in patients with hepatocellular carcinoma.

Many tumor‐associated antigens (TAAs) recognized by cytotoxic T cells (CTLs) have been identified during the last two decades and some of them have been used in clinical trials. However, there are very few in the field of immunotherapy for hepatocellular carcinoma (HCC) because there have not been comparative data regarding CTL responses to various TAAs. In the present study, using 27 peptides derived from 14 different TAAs, we performed comparative analysis of various TAA‐specific T‐cell responses in 31 HCC patients to select useful antigens for immunotherapy and examined the factors that affect the immune responses to determine a strategy for more effective therapy. Twenty‐four of 31 (77.4%) HCC patients showed positive responses to at least one TAA‐derived peptide in enzyme‐linked immunospot assay. The TAAs consisting of cyclophilin B, squamous cell carcinoma antigen recognized by T cells (SART) 2, SART3, p53, multidrug resistance‐associated protein (MRP) 3, alpha‐fetoprotein (AFP) and human telomerase reverse transcriptase (hTERT) were frequently recognized by T cells and these TAA‐derived peptides were capable of generating peptide‐specific CTLs in HCC patients, which suggested that these TAAs are immunogenic. HCC treatments enhanced TAA‐specific immune responses with an increased number of memory T cells and induced de novo T‐cell responses to lymphocyte‐specific protein tyrosine kinase, human epidermal growth factor receptor type 2, p53, and hTERT. Blocking cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) resulted in unmasking of TAA‐specific immune responses by changing cytokine and chemokine profiles of peripheral blood mononuclear cells stimulated by TAA‐derived peptides. Conclusion: Cyclophilin B, SART2, SART3, p53, MRP3, AFP, and hTERT were immunogenic targets for HCC immunotherapy. TAA‐specific immunotherapy combined with HCC treatments and anti‐CTLA‐4 antibody has the possibility to produce stronger tumor‐specific immune responses. (HEPATOLOGY 2011;)

Enhancement of tumor‐associated antigen‐specific T cell responses by radiofrequency ablation of hepatocellular carcinoma

Radiofrequency ablation (RFA) is one of the treatments for hepatocellular carcinoma (HCC) and is known to enhance host immune response. However, the epitopes to which enhanced immune responses occur, the impact on patient prognosis, and the functions and phenotype of T cells induced are still unclear. To address these issues, we analyzed immune responses before and after RFA in 69 HCC patients using 11 tumor‐associated antigen (TAA)‐derived peptides that we identified to be appropriate to analyze HCC‐specific immune responses. The immune responses were analyzed using enzyme‐linked immunospot (ELISPOT) assay and tetramer assays using peripheral blood mononuclear cells. An increase in the number of TAA‐specific T cells detected by interferon‐γ ELISPOT assays occurred in 62.3% of patients after RFA. The antigens and their epitope to which enhanced T cell responses occur were diverse, and some of them were newly induced. The number of TAA‐specific T cells after RFA was associated with the prevention of HCC recurrence, and it was clarified to be predictive of HCC recurrence after RFA by univariate and multivariate analyses. The number of TAA‐specific T cells after RFA was inversely correlated with the frequency of CD14+HLA‐DR−/low myeloid‐derived suppressor cells (MDSCs). The modification of T cell phenotype was observed after RFA. The number of TAA‐specific T cells at 24 weeks after RFA was decreased. Conclusion: Although RFA can enhance various TAA‐specific T cell responses and the T cells induced contribute to the HCC recurrence‐free survival of patients, besides immunosuppression by MDSCs, the memory phenotype and lifetime of TAA‐specific T cells are not sufficient to prevent HCC recurrence completely. Additional treatments by vaccine or immunomodulatory drugs might be useful to improve the immunological effect of RFA. (Hepatology 2013;1448–1457)

Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.

Prevention of intrahepatic metastasis of liver cancer by suicide gene therapy and chemokine ligand 2/monocyte chemoattractant protein-1 delivery in mice

The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, largely as a result of intrahepatic metastasis. Using a mouse model of intrahepatic metastasis, we investigated whether chemokine ligand 2/monocyte chemoattractant protein‐1 (CCL2/MCP‐1) could potentiate the antitumor effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV‐tk/GCV) system.

Enhanced anti-tumor effects of herpes simplex virus thymidine kinase/ganciclovir system by codelivering monocyte chemoattractant protein-1 in hepatocellular carcinoma

The therapeutic efficacy of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system in many types of tumors is unsatisfactory due to the insufficient spread of gene transfer and insufficient cell killing. In the current study, we investigated whether adenovirally delivered monocyte chemoattractant protein (MCP)-1 potentiates the antitumor effects of the HSV-tk/GCV system in hepatocellular carcinoma (HCC) cells. Subcutaneous tumor foci of the human HCC cell line, HuH7, established in athymic mice were directly transduced with a recombinant adenovirus (rAd) harboring an HSV-tk gene driven by a human α-fetoprotein promoter, followed by GCV administration. Subsequently, another rAd expressing MCP-1 under the universal CAG promoter was injected. The growth of tumors was markedly suppressed by codelivering HSV-tk and MCP-1 genes compared to that by either HSV-tk/GCV or MCP-1 delivery. In the tumor tissues, monocyte/macrophage infiltration was detected immunohistochemically. The antitumor effects of the rAd expressing MCP-1 were markedly reduced by the administration of carrageenan, a compound known to inactivate macrophage. These results indicate that adenovirally delivered MCP-1 enhanced the antitumor effects of the HSV-tk/GCV system synergistically by recruitment/activation of macrophages in tumor tissues, suggesting an effective immunotherapy for HCC and other lineages of tumors when used adjuvantly with a suicide gene. Cancer Gene Therapy (2001) 8, 695–704

Feasibility and efficacy of hepatic arterial infusion chemotherapy for advanced hepatocellular carcinoma after sorafenib

Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, although there is no proven therapeutic procedure following the termination of sorafenib, hepatic arterial infusion chemotherapy (HAIC) may be a treatment option in advanced HCC. The aim of this study was to evaluate feasibility and efficacy of HAIC for patients with advanced HCC as subsequent therapy.

Blood neutrophil to lymphocyte ratio as a predictor in patients with advanced hepatocellular carcinoma treated with hepatic arterial infusion chemotherapy

Inflammation plays a critical role in cancer. The aim of the present study was to investigate the impact of neutrophil to lymphocyte ratio (NLR) on patients with advanced hepatocellular carcinoma (HCC) treated with hepatic arterial infusion chemotherapy (HAIC).

The development and clinical features of splenic aneurysm associated with liver cirrhosis.

Abstract: Objectives: Splenic artery aneurysm (SAA) is usually asymptomatic, but can be fatal if it ruptures. Portal hypertensive patients with varix or splenomegaly are sometimes complicated by SAA. However, there have been no large‐scale clinical studies regarding whether liver cirrhosis itself is associated with splenic aneurysm regardless of varix or splenomegaly.