Tetsu Tomonari

Novel des-γ-carboxy prothrombin in serum for the diagnosis of hepatocellular carcinoma.

Serum des‐γ‐carboxy prothrombin (DCP) levels using a newly developed electrochemiluminescence immunoassay (ECLIA, novel DCP [NX‐DCP]) were measured, and the utility of NX‐DCP and DCP/NX‐DCP ratio for the diagnosis of hepatocellular carcinoma (HCC) was investigated. Antigenic differences in DCP between HCC and non‐HCC patients were elucidated.

MRP3 as a novel resistance factor for sorafenib in hepatocellular carcinoma

The mechanism of resistance of hepatocellular carcinoma (HCC) to sorafenib is unknown and no useful predictive biomarker for sorafenib treatment has been reported. Accordingly, we established sorafenib-resistant HCC cells and investigated the underlying mechanism of resistance to sorafenib. Sorafenib-resistant cell lines were established from the HCC cell line PLC/PRF5 by cultivation under continuous exposure to increasing concentration of sorafenib. The IC50 values of the 2 resistant clones PLC/PRF5-R1 and PLC-PRF5-R2 were 9.2±0.47 μM (1.8-fold) and 25±5.1 μM (4.6-fold) respectively, which were significantly higher than that of parental PLC/PRF5 cells (5.4±0.17 μM) (p < 0.01 respectively), as determined by MTT assay. Western blot analysis of signal transduction-related proteins showed no significant differences in expression of AKT/pAKT, mTOR/pmTOR, or ERK/pERK between the 2 resistant clones versus parent cells, suggesting no activation of an alternative signal transduction pathway. Likewise, when expression of membrane transporter proteins was determined, there were no significant differences in expression levels of BSEP, MDR1, MRP2, BCRP, MRP4 and OCT1 between resistant clones and parent cells. However, the expression levels of MRP3 in the 2 resistant clones were significantly higher than that of parent cells. When MRP3 gene was knocked down by siRNA in PLC-PRF5-R2 cells, the sensitivity of the cells to sorafenib was restored. In the analysis of gene mutation, there was no mutation in the activation segment of Raf1 kinase in the resistant clones. Our data clearly demonstrate that the efflux transporter MRP3 plays an important role in resistance to sorafenib in HCC cells.

Light alcohol consumption plays a protective role against non-alcoholic fatty liver disease in Japanese men with metabolic syndrome

Although excess alcohol consumption has been believed to cause liver injury, light alcohol consumption (LAC) has been reported to play a protective role against fatty liver in recent studies. However, the association between non‐alcoholic fatty liver disease (NAFLD) and LAC in men with metabolic syndrome (MS) is unclear. The aim of this study was to examine the association between NAFLD and LAC in men with MS.

Serum diamine oxidase activity as a predictor of gastrointestinal toxicity and malnutrition due to anticancer drugs.

Objective evaluation of intestinal mucosal damage due to anticancer drugs is generally difficult. Serum diamine oxidase (DAO) activity is reported to reflect the integrity and maturity of the small intestinal mucosa. Therefore, we investigated whether serum DAO activity is an indicator of gastrointestinal toxicity or nutritional status in patients receiving chemotherapy.

APOB codon 4311 polymorphism is associated with hepatitis C virus infection through altered lipid metabolism

BackgroundIt has been reported that some single-nucleotide polymorphisms (SNPs) in lipid regulators such as apolipoproteins and cell surface molecules for hepatitis C virus (HCV) entry into hepatocytes are associated with HCV infection. However, it is unknown how HCV infection is affected by altered lipid metabolism resulting from the SNPs. We investigated the relationship between these SNPs and HCV infection status, and also analyzed the mechanism by which these SNPs mediate HCV infection via lipid metabolism alterations.MethodsSerum lipid and apolipoprotein profiles were tested in 158 HCV-positive and 220 HCV-negative subjects. We selected 22 SNPs in five lipid regulator genes which were related to HCV entry into hepatocytes and to lipid metabolism (APOA1, APOB, SR-B1, LDLR, and APOE), and their polymorphisms were analyzed using the PCR-sequence-specific oligonucleotide probe-Luminex method.ResultsAn APOB N4311S (g.41553a > g) SNP, rs1042034, was significantly associated with HCV positivity; the HCV positivity rate for the minor allele AA genotype was significantly higher than for genotype AG + GG (P = 0.016). Other SNPs except for APOB P2712L SNP rs676210, which is in linkage disequilibrium with rs1042034, showed no significant difference in genotype distribution. The serum level of low density lipoprotein-cholesterol (LDL-C) in the genotype AA group was significantly lower than in the genotype non-AA group (P = 0.032), whereas the triglyceride (TG) level was significantly higher (P = 0.007).ConclusionAn APOB SNP, rs1042034, is closely associated with HCV infection through lipid metabolism alteration. The minor allele AA genotype might contribute to facilitating serum LDL uptake into hepatocytes via LDLR by modifying their affinity and interaction and may have an influence on HCV infection by their entry to the liver through the LDLR.

The differing influence of several factors on the development of fatty liver with elevation of liver enzymes between genders with metabolic syndrome: A cross-sectional study

Background Nonalcoholic fatty liver disease (NAFLD) is known to be strongly associated with obesity, visceral fat, metabolic syndrome (MS), lifestyle, and lifestyle-related diseases in both males and females. However, the prevalence of NAFLD, MS, and clinical backgrounds is different between males and females. Objective We conducted a cross-sectional study to examine the differing influence of lifestyle-related factors and visceral fat on fatty liver (FL) with elevation of liver enzymes between males and females with MS. Methods We enrolled 42,134 persons who underwent a regular health check-up, and after excluding subjects who fulfilled excluding criteria, the remaining subjects were 2,110 persons with MS. We examined the differing influence of lifestyle-related factors and visceral fat on FL with elevation of alanine aminotransferase (ALT) (ALT elevation was defined as ALT level of ≥31 IU/l in the present study). Results The odds rations for FL with ALT elevation were as follows: WC, 1.83 (95% confidence interval (CI) 1.36–2.46); dyslipidemia, 1.89 (95% CI 1.34–2.68); hemoglobin A1c, 1.36 (95% CI 1.00–1.85); visceral fat type MS (V-type MS), 5.78 (95% CI 4.29–7.80); and light drinker, 0.56 (95% CI 0.41–0.78) in males with MS and BMI, 2.18 (95% CI 1.43–3.33); WC, 1.85 (95% CI 1.27–2.70); diastolic blood pressure, 1.69 (95% CI 1.16–2.45); triglyceride, 2.22 (95% CI 1.56–3.17); impaired glucose tolerance, 1.66 (95% CI 1.11–2.47); and V-type MS, 3.83 (95% CI 2.57–5.70) in females with MS. The prevalence of FL with ALT elevation and ALT was significantly higher in V-type MS than in the subcutaneous fat type MS in both males and females with MS (P < 0.001). Conclusion Although V-type MS and WC is a common significant predictor of an increased prevalence of FL with ALT elevation in both males and females with MS, gender, lifestyle-related factors, and MS type in individuals with MS should be considered for the development of FL with ALT elevation.

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

Background and Aim: Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Methods: Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. Results: The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Conclusions: Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.

Tu1725 Angiogenesis-Related Factors At the Residual Inflammation in Patients With Ulcerative Colitis in Clinical Remission Stage

Angiogenesis-Related Factors At the Residual Inflammation in Patients With Ulcerative Colitis in Clinical Remission Stage Miwako Kagawa, Toshiya Okahisa, Yoshifumi Takaoka, Yasuteru Fujino, Jinsei Miyoshi, Toshi Takaoka, Tetsu Tomonari, Shinji Kitamura, Yasuyuki Okada, Kaizo Kagemoto, Masanori Takehara, Kumiko Tanaka, Sayo Matsumoto, Tomofumi Teramae, Hiroshi Miyamoto, Naoki Muguruma, Tetsuji Takayama

Radiofrequency Ablation Using a Balloon Catheter for Hepatocellular Carcinoma Adjacent to the Gastrointestinal Tract: Experimental and Clinical Study

Purpose: Radiofrequency ablation (RFA) is a safe and effective technique for hepatocellular carcinoma and it has minimal morbidity and mortality. One of the most important major complications of RFA is perforation of the gastrointestinal tract, which occurs when the tumor is adjacent to the digestive tract. In particular, the risk is much higher in patients with lesions located within 1 cm from the liver surface in proximity to the digestive tract. In such cases, an artificial ascites technique has been employed. However, the separation of adjacent digestive organ from the liver is insufficient in this method. Therefore, in this study, to overcome this problem, we devised a novel RFA technique using a double-balloon catheter. In the first step, an animal experiment was performed to evaluate the safety and feasibility of the balloon RFA method. In the second step, the human pilot trial was carried out to evaluate the safety, feasibility and effectiveness of this method. Materials and Methods: We produced an 8 Fr silicone catheter equipped with 2 balloons of 2.5 cm diameter. In experiments using pigs, we first inserted this balloon catheter percutaneously into the peritoneal space between the liver and gastrointestinal tracts, filled it with cooled water, and performed RFA for in normal liver 1 cm from the liver surface. Then, heat damage to the excised liver and gastrointestinal tract was evaluated macroscopically andmicroscopically. In a human pilot study, balloon catheter RFA was performed in 4 patients with HCC (1.5 ± 0.7 cm) abutting the gastrointestinal tract. Results: In pigs, we performed each 6 RFA sessions with or without balloon catheter. It was technically easy to place the balloon catheter between liver and gastrointestinal tracts to separate them. Heat damage reached the liver surface in all lesions. In the groupwith balloon catheter, no heat damage of the gastrointestinal tracts was observed (0%, 0/6). In contrast, in the group without balloon catheter, heat damage was observed in 5/6 (83.3%): stomach (2/6), small intestine (2/6), and omentum (1/6). The coolant temperature in the balloon was significantly increased after RFA, suggesting that the heat generated by RFA was absorbed by the coolant. In the human pilot study, balloon catheter RFA was easily performed in all patients without associated complications. CT confirmed complete ablation with an appreciable safety margin in all patients, without recurrence for 20.3 ± 4.5 months. Conclusions: RFA with our balloon catheter is safe and effective for the treatment of HCC abutting the gastrointestinal tract, suggesting an expanded indication of these lesions for RFA.

S1323 Usefulness of Electrocolonography for Evaluation of Colonic Motility

Purpose: Mobility disorders of the alimentary tract including irritable bowel syndrome (IBS) is now increasing worldwide. Measurement of intracolonic pressure has been conventionally employed for assessment of colonic motility. However, it is very laborious and burdensome for a routine examination. Therefore, in this study, we performed electrocolonography (ECoG), an easy and simple methodology, and investigated its usefulness for evaluation of colonic motility in comparison with the conventional methodology. Method: Twenty-five well-informed healthy volunteers were enrolled. ECoG was performed using a portable electrogastrograph (NIPRO EGG, A&D, Tokyo, Japan). After detecting a sigmoid colon using external ultrasonography, 4 electrodes of ECoG were attached to the abdomen. The ECoG was recorded by a bipolar lead between the central electrode and 3 surface probe electrodes (Ch1-3) at 1-second interval with frequencies of 1.5-6.0 cpm. Mosapride (10mg) or butylscopolamine (10mg) was orally administered during the examinations. For the analysis of ECoG data, the dominant frequencies and peak powers in 3 channels were calculated using a Fast Fourier Transform. In the 3 subjects, intracolonic pressure was measured using mobility visualization system (ManoScan 360, Sierra Scientific Instruments, CA) concurrently with ECoG. A catheter with 36 pressure sensors was introduced through the anus to the sigmoid colon, and the change of the pressure at each point was recorded. Results: Colonic peristalsis at about 2 cpm (1.95 0.41 cpm) was observed by mobility measuring system. The pressure was significantly increased by administration of mosapride, and was decreased by butylscopolamine, consistent with the previous reports. While, a dominant frequency at 2 cpm, which represented action potential of colonic peristalsis, was observed in all channels of ECoG. The peak power of the dominant frequency was significantly increased by mosapride (pre 15.0 ± 7.38uV, after 52.0 ± 50.3uV) and was significantly decreased by butylscopolamine (pre 15.2 ± 6.27uV, after 0.35 ± 0.24uV), corresponding to the change of intracolonic pressure. The dominant frequency did not change after administration of mosapride or butylscopolamine in ECoG. Conclusion:We could detect action potential of colonic peristalsis by ECoG, an easy and simple methodology, and showed the usefulness of ECoG for assessing colonic motility.