Kuniaki Arai

Nucleolin Interacts with Telomerase

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex.

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;)

Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma

Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT‐derived, HLA‐A*2402–restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT‐specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA‐A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT‐specific CTLs in both hTERT cDNA‐immunized HLA‐A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA‐A*2402–restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope‐specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 × 105 PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT‐specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T‐cell–based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC. (HEPATOLOGY 2006;43: 1284–1294.)

Impact of diabetes on recurrence of hepatocellular carcinoma after surgical treatment in patients with viral hepatitis.

OBJECTIVES: Consensus has been reached that diabetes is a risk factor for development of HCC, but the impact on postoperative recurrence is still controversial. To clarify this point, we analyzed the relationship of postoperative recurrence rate of HCC and coexistence of diabetes in the patients with viral hepatitis.METHODS: A total of 90 patients who had undergone curative resection for HCC were analyzed. They were divided into two groups with and without diabetes, and the recurrence-free survival rates after surgical treatment and the factors contributing to recurrence were examined.RESULTS: Kaplan-Meier survival analysis showed the recurrence-free survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005) and overall survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005). These results were emphasized in the analysis of patients infected with hepatitis C virus. Univariate and multivariate analyses showed diabetes was a significant factor contributing to HCC recurrence after treatment. Furthermore, multivariate analysis in HCC patients with diabetes showed Child-Pugh classification B (P = 0.001) and insulin therapy (P = 0.049) were significant factors contributing to HCC recurrence after treatment.CONCLUSIONS: The results of the present study suggest that diabetes is a risk factor for the recurrence of HCV-related HCC and decreases the overall survival rates after surgical treatment. HCV-related HCC patients with diabetes should be closely followed for postoperative recurrence.

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes.

Aims/hypothesisMitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown.Materials and methodsWe analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT.ResultsBoth gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-α or peroxisome proliferator-activated receptor-γ, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p = 0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-γ coactivator-1α or nuclear respiratory factor 1.Conclusions/interpretationOur results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.

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)

Histological course of nonalcoholic fatty liver disease in Japanese patients: Tight glycemic control, rather than weight reduction, ameliorates liver fibrosis

OBJECTIVE The goal of this study was to examine whether metabolic abnormalities are responsible for the histological changes observed in Japanese patients with nonalcoholic fatty liver disease (NAFLD) who have undergone serial liver biopsies. RESEARCH DESIGN AND METHODS In total, 39 patients had undergone consecutive liver biopsies. Changes in their clinical data were analyzed, and biopsy specimens were scored histologically for stage. RESULTS The median follow-up time was 2.4 years (range 1.0–8.5). Liver fibrosis had improved in 12 patients (30.7%), progressed in 11 patients (28.2%), and remained unchanged in 16 patients (41%). In a Cox proportional hazard model, decrease in A1C and use of insulin were associated with improvement of liver fibrosis independent of age, sex, and BMI. However, ΔA1C was more strongly associated with the improvement of liver fibrosis than use of insulin after adjustment for each other (χ2; 7.97 vs. 4.58, respectively). CONCLUSIONS Tight glycemic control may prevent histological progression in Japanese patients with NAFLD.

Randomized, Phase II Study Comparing Interferon Combined with Hepatic Arterial Infusion of Fluorouracil plus Cisplatin and Fluorouracil Alone in Patients with Advanced Hepatocellular Carcinoma

Objective: This randomized phase II trial compared the response rates to treatment with interferon (IFN) combined with hepatic arterial infusion of fluorouracil (FU) plus cisplatin (CDDP) or FU alone in patients with advanced hepatocellular carcinoma (HCC). Methods: A total of 114 patients with measurable advanced HCC were enrolled and randomized into 2 groups. FU (300 mg/m2, days 1–5, days 8–12) with or without CDDP (20 mg/m2, days 1 and 8) was administered via the hepatic artery. IFNα-2b was administered 3 times per week for 4 weeks. Results: The response rates were 45.6% for the IFN/FU + CDDP group and 24.6% for the IFN/FU group. The response rate was significantly higher in the IFN/FU + CDDP group (p = 0.030). The median overall survival period was 17.6 months in the IFN/FU + CDDP group versus 10.5 months in the IFN/FU group (p = 0.522). The median progression-free survival period was 6.5 months in the IFN/FU + CDDP group versus 3.3 months in the IFN/FU group (p = 0.0048). Hematological toxicity was common, but no toxicity-related deaths were observed. Conclusion: These results show the clinical efficacy of adding CDDP to the hepatic arterial infusion of FU in combined chemotherapy regimens with IFN.

Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and lipid metabolism. Recent studies have suggested that the quality of fat accumulated in the liver is associated with the development of nonalcoholic steatohepatitis (NASH). In this study, we investigated the fatty acid composition in liver tissue and its association with the pathology in NAFLD patients.

Prolonged recurrence-free survival following OK432-stimulated dendritic cell transfer into hepatocellular carcinoma during transarterial embolization

Despite curative locoregional treatments for hepatocellular carcinoma (HCC), tumour recurrence rates remain high. The current study was designed to assess the safety and bioactivity of infusion of dendritic cells (DCs) stimulated with OK432, a streptococcus‐derived anti‐cancer immunotherapeutic agent, into tumour tissues following transcatheter hepatic arterial embolization (TAE) treatment in patients with HCC. DCs were derived from peripheral blood monocytes of patients with hepatitis C virus‐related cirrhosis and HCC in the presence of interleukin (IL)‐4 and granulocyte‐macrophage colony‐stimulating factor and stimulated with 0·1 KE/ml OK432 for 2 days. Thirteen patients were administered with 5 × 106 of DCs through arterial catheter during the procedures of TAE treatment on day 7. The immunomodulatory effects and clinical responses were evaluated in comparison with a group of 22 historical controls treated with TAE but without DC transfer. OK432 stimulation of immature DCs promoted their maturation towards cells with activated phenotypes, high expression of a homing receptor, fairly well‐preserved phagocytic capacity, greatly enhanced cytokine production and effective tumoricidal activity. Administration of OK432‐stimulated DCs to patients was found to be feasible and safe. Kaplan–Meier analysis revealed prolonged recurrence‐free survival of patients treated in this manner compared with the historical controls (P = 0·046, log‐rank test). The bioactivity of the transferred DCs was reflected in higher serum concentrations of the cytokines IL‐9, IL‐15 and tumour necrosis factor‐α and the chemokines CCL4 and CCL11. Collectively, this study suggests that a DC‐based, active immunotherapeutic strategy in combination with locoregional treatments exerts beneficial anti‐tumour effects against liver cancer.