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Fusion images of diffusion-weighted whole-body imaging with background body signal suppression and T2-weighted image (DWIBS/T2) demonstrate a strong signal for malignancies, with a high contrast against the surrounding tissues, and enable anatomical analysis. In the present study, DWIBS/T2 was compared with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) for diagnosing cancer in the abdomen. Patient records, including imaging results of examination conducted between November 2012 and May 2014, were analyzed retrospectively. In total, 10 men (age, 73.6±9.6 years) and 8 women (age, 68.9±7.1 years) were enrolled into the current study. Of the enrolled patients, 2 were diagnosed with hepatocellular carcinoma, 1 with cholangiocellular carcinoma, 1 with liver metastasis, 2 with pancreatic ductal adenocarcinoma, 1 with renal cell carcinoma and 1 with malignant lymphoma. Benign lesions were also analyzed, including adenomyomatosis of the gallbladder (5 patients), intraductal papillary mucinous neoplasm (4 patients) and right adrenal adenoma (1 case). All the patients with cancer showed positive results on DWIBS/T2 images. However, only 7 out of 8 patients were positive with PET/CT. One patient with right renal cellular carcinoma was positive with DWIBS/T2, but negative with PET/CT. All the patients with benign lesions were negative with DWIBS/T2 and PET/CT. In conclusion, DWIBS/T2 was more sensitive in diagnosing cancer of organs in the abdominal cavity compared with PET/CT. Furthermore, negative results with DWIBS/T2 and PET/CT were useful for the diagnosis of benign lesions, such as adenomyomatosis of the gallbladder and intraductal papillary mucinous neoplasm.

Tissues surrounding hepatocellular carcinomas (HCCs) lack glucose. Hepatocyte selection medium (HSM) is deficient in glucose and is supplemented with galactose. HCC cells were cultured in HSM to investigate the stem cell markers α-fetoprotein (AFP) and cluster of differentiation 44 (CD44). HCC cells (HLF and PLC/PRF/5 cells) were cultured in HSM. Viable cell numbers were determined on days 0 and 7 following culture in HSM. RNA was isolated and subjected to reverse transcription-quantitative PCR (RT-qPCR) to analyze the mRNA expression levels of AFP and CD44. Immunostaining was performed to analyze the protein levels of AFP and CD44. The number of viable cells was significantly decreased on day 7 following culture in HSM. The expression levels of AFP and CD44 increased on day 7 as assessed using RT-qPCR. Immunostaining confirmed the results of RT-qPCR analysis. The number of viable HCC cells was decreased in HSM, whereas the expression levels of AFP and CD44 increased. Therefore, HSM is potentially useful for the enrichment of HCC cells with cancer stem cell characteristics.

Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) images show significant contrast for cancer tissues against non-cancerous tissues. Fusion of a DWIBS and a T2-weighted image (DWIBS/T2) can be used to obtain functional, as well as anatomic, information. In the present study, the performance of DWIBS/T2 in the diagnosis of abdominal solid cancer was evaluated. The records of 14 patients were retrospectively analyzed [5 patients with hepatocellular carcinoma (HCC), 4 with metastatic liver cancer, 3 with pancreatic cancer, 1 with renal cellular carcinoma and 1 with malignant lymphoma of the para-aortic lymph node]. T1WI and T2WI scans did not detect pancreatic cancer in certain cases, whereas DWIs and DWIBS/T2 clearly demonstrated pancreatic cancer in all cases. In addition, metastatic liver cancer and HCC were successfully detected with abdominal US and CECT; however, US did not detect pancreatic cancer in 1 case, while CECT and DWIBS/T2 detected pancreatic cancer in all cases. In conclusion, the diagnostic performance of DWIBS/T2 was the same as that of abdominal US and CECT in detecting primary and metastatic liver cancer. DWIBS/T2 enabled the diagnosis of pancreatic cancer in cases where it was not detected with US, T1WI or T2WI.

To improve the management of patients with acute cholangitis, the present study compared laboratory test variables between acute cholangitis patients with or without common bile duct (CBD) dilatation [CBDdil(+) and CBDdil(-), respectively]. The medical records of patients diagnosed with acute cholangitis and subjected to endoscopic retrograde cholangiopancreatography between February 2008 and May 2015 were retrospectively analyzed. The present study consisted of 40 men (aged 69.4±8.8 years) and 37 women (aged 68.8±11.6 years). It was observed that CBDdil(-) patients were slightly younger than CBDdil(+) patients (P=0.0976), and levels of C-reactive protein (CRP) were significantly higher in CBDdil(-) patients than in CBDdil(+) patients (P=0.0392). In addition, logistic regression analysis indicated that CRP levels were associated with the presence of CBD dilatation (P=0.0392). These data indicate that patients with acute cholangitis without CBD dilatation tend to be younger and have higher levels of CRP. Thus, in acute cholangitis patients without CBD dilatation, diagnosis should be determined using clinical symptoms and laboratory data.

Hepatocyte selection medium (HSM) is deprived of glucose and supplemented with galactose, and is based on Leibovitz's‑15 (L15) medium. HSM may promote the differentiation of human induced pluripotent stem (iPS) cells towards hepatocyte lineage. These culture conditions result in increased expression of galactokinase (GALK)‑1 and GALK2. However, iPS cells do not survive in HSM. Two potential alternatives to glucose deprivation are treatment with 3‑bromopyruvate (3BP), an analogue of pyruvate, and 2‑deoxy‑d‑glucose (2DG), an analogue of glucose. The promoters of GALK1 and GALK2 were subcloned using the pMetLuc2 reporter plasmid to make pMetLuc2/GALK1 and pMetLuc2/GALK2, respectively. 201B7 human iPS cells were transfected with the reporter plasmids, cultured in HSM and analyzed by luciferase assay. Furthermore, 201B7 cells were cultured in L15, William's E (WE) or Dulbecco's modified Eagle's medium/nutrient mixture F‑12 Ham (DF12) supplemented with 3BP, 2DG or a combination of the two, for 15 days, and subjected to reverse transcription‑quantitative polymerase chain reaction to measure the levels of α‑fetoprotein (AFP) mRNA expression. Metridia luciferase activity was significantly higher in cells cultured in HSM compared with those in ReproFF medium (P

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α‑fetoprotein (AFP) and albumin were examined by reverse transcription‑quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all‑trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all‑trans retinoic acid and insulin‑transferrin‑selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post‑stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.

Short hairpin RNA of frizzled-2 (shRNA-Fz2) suppresses the cell proliferation of gastric cancer cells. Endoscopic ultrasound (EUS) is considered a suitable method for the introduction of therapeutic plasmids into cells, since the device enables the access and real-time monitoring of gastric cancer tissues. In the present study, plasmids were introduced into cells by sonoporation, as evidenced by the production of H2O2. The production of H2O2 was measured by absorbance of a potassium-starch solution irradiated with EUS. Luciferase activity was analyzed in the cells irradiated with EUS after the addition of a pMetLuc2-control in the media, and cell proliferation was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay after irradiation with EUS following the addition of shRNA-Fz2. Absorbance levels corresponding to free radical levels were found to be higher in the cells irradiated with EUS. Luciferase activities were found to be significantly higher in the transfected cells (plasmid with Lipofectamine LTX) than in untreated cells and were furthermore found to be higher in MKN45 cells irradiated for 0.5 min than in cells not subjected to irradiation. Luciferase activity was also found to be higher in MKN74 cells irradiated for 2 min than in cells that were not irradiated. Although the cell proliferation of the MKN45 cells tended to be suppressed by irradiation with EUS, this was non-significant suppression, while the cell proliferation of MKN74 cells was found to be suppressed by irradiation with 12 MHz for 2 min (P

Autoimmune hepatitis (AIH) is an uncommon chronic autoimmune liver disease. Several studies reported the association of polymorphisms between CD28, CTLA4 and ICOS gene cluster in 2q33.2 with autoimmune or inflammatory diseases. The previous genome-wide association study on type 1 AIH in a European population has reported a risk G allele of a single nucleotide polymorphism (SNP), rs4325730, in this region. Here, we conducted an association study of this SNP with type 1 AIH in a Japanese population, as a replication study.An association study of rs4325730 was conducted in 343 Japanese AIH patients and 315 controls.We found that rs4325730 is associated with AIH (P=0.0173, odds ratio (OR) 1.30, 95% confidence interval (CI) 1.05-1.62, under the allele model for G allele, P=0.0070, OR 1.62, 95% CI 1.14-2.31, under the dominant model for G allele). This SNP was strongly associated with definite AIH (P=0.0134, OR 1.36, 95% CI 1.07-1.74; under allele model for G, P=0.0035, OR 1.85, 95% CI 1.22-2.81, under dominant model for G).This is the first replication association study of rs4325730 upstream of ICOS with AIH in the Japanese population and rs4325730G is a risk allele.

The present study investigated the potential utility of contrast-enhanced abdominal ultrasonography (CEUS), using Sonazoid™, in colorectal cancer (CRC). Three patients were subjected to CEUS with Sonazoid™. Surgical specimens were immunostained for CD31. Numbers of blood vessels positive for CD31 were analyzed in each of five fields at ×400 magnification and averaged to determine blood vessel density. Blood vessel density was compared between non-tumorous and tumorous areas. Prior to the administration of Sonazoid™, CRC was illustrated as irregular-shaped wall thickening. One minute after the administration of Sonazoid™, the majority of the thickened wall was enhanced, while some parts of the thickened wall remained unenhanced. Blood vessel densities of non-tumorous and tumorous areas in patient two were 25.2±2.5 and 5.2±1.1 (P

The present study aimed to identify novel predictors of upper gastrointestinal (GI) bleeding by assessing change ratios of blood test variables. Records of 1,023 patients (431 men and 592 women) who underwent endoscopy between October 2014 and September 2015 at the National Hospital Organization Shimoshizu Hospital (Yotsukaido, Japan) were retrospectively analyzed. Patients whose blood test variables for the time-point of endoscopy and three months previously were available were enrolled and subsequently categorized into a group with and another one without upper GI bleeding (n=32 and 84, respectively), and the respective change ratios were calculated for each group. One-way analysis of variance revealed that in patients with upper GI bleeding, change ratios of white blood cell count and alkaline phosphatase were significantly higher than those in patients without, while change ratios of hemoglobin (Hb), total protein and albumin were significantly reduced. Logistic regression analysis demonstrated that the change ratio of Hb was significantly correlated with upper GI bleeding. Receiver-operator characteristic analysis revealed that an 18.7% reduction of Hb was the threshold value for the prediction of upper GI bleeding. In conclusion, the present study revealed that a ≥18.7% reduction in Hb over three months has predictive value for upper GI bleeding.

Evaluation of the severity of acute cholecystitis is critical for the management of this condition. Superb microvascular imaging (SMI) enables the assessment of slow blood flow of small vessels without any contrast medium. An 84-year-old man visited our hospital with right upper abdominal pain. Computed tomography and abdominal ultrasonography showed a slight thickening of the gallbladder. White blood cell count and C-reactive protein levels were elevated. He was diagnosed with acute cholecystitis and treated conservatively with antibiotics. Two days later, his condition worsened and percutaneous transhepatic gallbladder drainage (PTGBD) was performed. The patient recovered and was discharged, and his drainage was withdrawn 7 days later. On admission, color-coded SMI (cSMI) showed pulsatory signals on the slightly thickened gallbladder wall. On the day of PTGBD, the intensity of the signal on cSMI had increased. Once the patient was cured, no further signal was observed on the gallbladder wall with either cSMI or mSMI. In conclusion, the strong pulsatory signal correlated with the severity of acute cholecystitis observed with cSMI and mSMI. Illustrating the signal intensity is useful for the evaluation of the severity of acute cholecystitis.

Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) yields positive results for cancer against the surrounding tissues. The combination of DWIBS and T2-weighted images (DWIBS/T2) in the diagnosis of gastrointestinal tract cancers was retrospectively analyzed in the present study. Patients were subjected to magnetic resonance imaging after cancer was diagnosed through specimens obtained via biopsy or endoscopic mucosal resection. Sixteen patients were assessed between July, 2012 and June, 2013 and the correlation between detection with DWIBS/T2 and T staging was analyzed. Regarding patients who underwent surgery, the correlation between detection with DWIBS/T2 and the diameter or depth of invasion was analyzed. All cancers that had advanced to >T2 stage were detectable by DWIBS/T2, whereas all cancers staged as T2) or invading beyond the muscularis propria.

Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is performed to obtain specimens for pathological analysis. For this procedure, 19-gauge (19G), 22-guage (22G), and 25-guage (25G) needles are available. The needles are classified into aspiration type and biopsy type. A 56-year-old woman underwent upper gastrointestinal endoscopy that showed a 38-mm-diameter submucosal tumor. The elevated lesion was diagnosed as a submucosal tumor of the stomach. Contrast-enhanced computed tomography showed a low-density area on the luminal surface of the gastric wall, which was covered with a thin layer of gastric mucosa. EUS showed a hypoechoic lesion in the submucosal layer. Color Doppler image showed a pulsating vascular signal extending into the center of the hypoechoic lesion from the periphery. EUS-FNA was performed with a 25G biopsy needle. The specimen tissue consisted of spindle-shaped cells. The cells were positive for CD117 and CD34. The submucosal tumor was diagnosed as a gastrointestinal stromal tumor.

Lower gastrointestinal (GI) bleeding can be caused by colorectal polyps or cancer. The aim of the present study was to identify blood test variables and medications that can predict lower GI bleeding, which would allow for appropriate colonoscopy. The medical records of patients who underwent colonoscopy from September 2014 to September 2015 were retrospectively analyzed. The selected patients included 278 men (mean age, 67.0±11.5 years) and 249 women (mean age, 69.6±12.0 years). The diagnosis, medications, and blood test variables were compared between patients with and without bleeding. Logistic regression analysis was performed to determine the factors associated with lower GI bleeding. The presence of colorectal polyp and cancer was associated with lower GI bleeding (P=0.0044) with an odds ratio of 6.71 (P=0.0148). No lower GI bleeding was observed in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or anticoagulants. The C-reactive protein (CRP) levels were significantly higher in patients with lower GI bleeding (P=0.0227). The Hb levels were lower in patients with lower GI bleeding, however this finding was not statistically significant (P>0.05). No blood test variable was associated with lower GI bleeding. Elevated CRP was associated with lower GI bleeding, while there was no association between the medications and lower GI bleeding.

Differentiation between adenomyomatosis (ADM) and cancer of the gallbladder is necessary during diagnosis. Diffusion-weighted whole body imaging with background body signal suppression (DWIBS) images are able to indicate cancer and inflammation. The fusion of a DWIBS with a T2 weighted image (DWIBS/T2) facilitates both functional and anatomical investigations. In the present study, patient records and images from patients with surgically confirmed ADM from April 2012 to October 2014 were analyzed retrospectively. The enrolled patients, including 6 men (64.2±13.1 years) and 4 women (57.3±12.4 years) were subjected to DWIBS/T2 during routine clinical practice. The diagnosis of ADM was based on magnetic resonance cholangiopancreatography, transabdominal ultrasonography, and endoscopic ultrasonography; ADM was diagnosed definitively when cystic lesions were observed, indicating the Rokitansky-Aschoff sinus. A single patient was indicated to be positive by DWIBS/T2 imaging. The Rokitansky-Aschoff sinus revealed a relatively high signal intensity; however, it was not as strong as that of the spleen. The signal intensity was also high on an apparent diffusion coefficient map, suggesting T2 shine-through. The thickened wall displayed low signal intensity. The aforementioned results indicate that ADM may be negative upon DWIBS/T2 imaging; one false positive case was determined to be ADM, accompanied by chronic cholecystitis. The majority of patients with ADM displayed negative findings upon DWIBS/T2 imaging, and chronic cholecystitis may cause false positives.

Autoimmune hepatitis (AIH) is an autoimmune liver disease that is characterized by a progressive destruction of the liver parenchyma and the development of liver fibrosis. We aimed to examine the relationship between circulating cytokines/chemokines and the Mac-2 binding protein glycosylation isomer (M2BPGi) levels in Japanese patients with autoimmune hepatitis (AIH). We investigated the relationship between circulating cytokines/chemokines and M2BPGi levels in Japanese patients with AIH. Seventy-seven patients with well-documented AIH were enrolled in the National Hospital Organization (NHO)-AIH-liver-network database. We measured the serum levels of 20 cytokines in 31 selected AIH patients before and after steroid treatment using multisuspension cytokine array. Eleven cytokines and soluble adhesion molecules were increased in untreated AIH patients compared with treated AIH patients. Among these cytokines and soluble adhesion molecules, soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-γ-inducible protein 10 (IP-10) were most downregulated by steroid therapy in AIH patients. We measured serum sICAM-1 and IP-10 by ELISA and found the levels were significantly higher in AIH patients (n = 77) compared with chronic viral hepatitis C patients (n = 32). Furthermore, there was a positive correlation between sICAM-1 or IP-10 and alanine aminotransferase, total bilirubin, and circulating M2BPGi levels. M2BPGi levels were increased in AIH patients with high stages of liver fibrosis. Additionally, M2BPGi levels were correlated with the histological grade of inflammation in AIH. Circulating M2BPGi levels were significantly reduced by steroid treatment in AIH patients. sICAM-1 and IP-10 are useful markers to assess immune-mediated hepatitis activity in AIH and they correlate with circulating M2BPGi. Serum M2BPGi levels increased in untreated AIH patients with active hepatitis and were decreased by steroid therapy. M2BPGi reflects autoimmune-mediated hepatic inflammation as well as liver fibrosis.

Short-hairpin RNA of frizzled-2 (shRNA-Fz2) is known to suppress the proliferation of hepatocellular carcinoma (HCC) cells; however, its effect on HCC cell motility is unknown. In this study, suppression of HCC cell motility by shRNA-Fz2 was analyzed, and introduction of shRNA-Fz2 into HCC cells was facilitated with ultrasound (US) irradiation generated from a diagnostic US device, which was enhanced by the contrast-enhanced US reagent Sonazoid. The HCC cell lines HLF and PLC/PRF/5 that were transfected with shRNA-Fz2 were plated to form monolayers, following which the cell monolayers were scratched with a sterile razor. After 48 h, the cells were stained with hematoxylin and eosin, and the distance between the growing edge of the cell layer and the scratch lines was measured. Total RNA from the cells was isolated and subjected to real-time quantitative PCR to quantify matrix metalloproteinase 9 expression at 48 h after transfection of shRNA-Fz2. Starch-iodide method was applied to analyze the generation of H2O2 following US irradiation with the addition of Sonazoid in the liquid, and cell proliferation was analyzed 72 h later. The distances between the growing edge of the cell layer and the scratch lines and MMP9 expression levels were significantly decreased with transfection of shRNA-Fz2 (P

Metastasis negatively affects the prognosis of hepatocellular carcinoma (HCC). In the present study, niclosamide, which is known to suppress the proliferation of HCC cells, was investigated for possible suppressant effects on the migration of HCC cells. HLF and PLC/PRF/5 HCC cells were cultured in the presence of niclosamide. Cell proliferation was analyzed using the MTS assay. Cell migration was measured by performing a scratch assay. Expression levels of cyclin D1 and matrix metalloproteinase 9 (MMP9) were analyzed by performing revers transcription-quantitative polymerase chain reaction. Compared with the control treatment, treatment with 10 µm niclosamide suppressed the proliferation of the HLF and PRL/PRF/5 cells to 49.9±3.7 and 17.9±11.5% (P

Anti-mitochondrial M2 antibody (AMA-M2) is specific to primary biliary cirrhosis (PBC), but can also be found in certain patients with autoimmune hepatitis (AIH). Effective methods of differentiating between PBC and AIH are required, as their clinical course and management are different. Titers of AMA-M2 were analyzed before and after follow-up in patients with PBC or AIH. Patients who underwent liver biopsy and were diagnosed with either AIH (10 patients) or PBC (3 patients) were enrolled in the study. The AMA-M2 antibody titers of these patients were analyzed upon hospital admission. AMA-M2 reacted with the pyruvate dehydrogenase complex-E2, branched-chain 2-oxo acid dehydrogenase complex and 2-oxoglutaric acid dehydrogenase complex in the assay utilized for this study. The cut-off value for AMA-M2 was 5. Six AIH patients were AMA-M2(-) and 4 were AMA-M2(+). The titer for the AIH patients who were AMA-M2(+) was 24.8±14.8, compared with 324±174 in the patients with PBC (P=0.0138). Three AMA-M2(+) AIH patients were followed-up after liver biopsy. The AMA-M2 levels had decreased in all 3 patients, becoming undetectable in 2 of them. In conclusion, certain patients with AIH in this study were found to be AMA-M2(+), but the titers were significantly lower than those in the patients with PBC. At follow-up, the AIH patients exhibited decreased AMA-M2 titers.

Mesenchymal-epithelial transition factor (c-Met) is associated with the proliferation and motility of cancer cells. c-Met expression has been detected in surgical pancreatic cancer specimens, and its overexpression is associated with a poor prognosis. SU11274 is a specific inhibitor of c-Met. In the present study, the cell proliferation and motility of pancreatic cancer cells treated with SU11274 was investigated. The PANC-1, MIA-Paca2, NOR-P1, PK-45H, PK-1 and PK-59 pancreatic cancer cell lines were used. The expression of c-Met and cyclin D1 was analyzed by quantitative polymerase chain reaction. In addition, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess cell proliferation, and a scratch assay was performed to assess cell motility. c-Met expression was higher in PANC-1, PK-45H, PK-1 and PK-59 cell lines compared with that in normal pancreatic tissue. Following treatment with 30 µM SU11274, the proliferation of MIA-Paca2 and PK-45H cells was suppressed to 19.8±10.7% (P

Human induced pluripotent stem (hiPS) cells are an ideal source for hepatocytes. Glucose and arginine are necessary for cells to survive. Hepatocytes have galactokinase (GALK), which metabolizes galactose for gluconeogenesis, and ornithine transcarbamylase (OTC), which converts ornithine to arginine in the urea cycle. Hepatocyte selection medium (HSM) lacks both glucose and arginine, but contains galactose and ornithine. Although human primary hepatocytes survive in HSM, all the hiPS cells die in 3 days. The aim of this study was to modify HSM so as to initiate hepatocyte differentiation in hiPS cells within 2 days. Hepatocyte differentiation initiating medium (HDI) was prepared by adding oncostatin M (10 ng/ml), hepatocyte functional proliferation inducer (10 nM), 2,2'-methylenebis (1,3-cyclohexanedione) (M50054) (100 μg/ml), 1× non-essential amino acid, 1× sodium pyruvate, nicotinamide (1.2 mg/ml), L-proline (30 ng/ml), and L-glutamine (0.3 mg/ml) to HSM. HiPS cells (201B7 cells) were cultured in HDI for 2 days. RNA was isolated, used as template for cDNA, and subjected to real-time quantitative polymerase chain reaction. Alpha-fetoprotein, γ-glutamyl transpeptidase, and delta-like 1 were upregulated. Expression of albumin was not observed. Expression of transcription factors specific to hepatocytes was upregulated. The expression of GALK2, OTC, and CYP3A4 were increased. In conclusion, differentiation of 201B7 cells to hepatoblast-like cells was initiated in HDI. Limitations were small number of cells were obtained, and the cells with HDI were not mature hepatocytes.

In order to identify novel targets for the molecular therapy of gastric cancer (GC), we investigated the mRNA and protein expression of frizzled-2 (Fz2), a Wnt signaling pathway receptor. Reverse-transcriptase polymerase chain reaction (PCR) amplification was utilized to determine the expression patterns of Fz genes in normal stomach and in the GC cell lines MKN45 and MKN74. Immunostaining was performed on surgical specimens of GC using an antibody against Fz2. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed on MKN45 cells and MKN74 cells transfected with Fz2 short-hairpin (sh) RNA. Cell motility was analyzed by scratch assay following Fz2 shRNA. Real-time quantitative PCR was performed to analyze the expression levels of cyclin D1 and matrix metallopeptidase 9 (MMP-9). Fz1, 3, 6 and 8 were expressed in normal stomach, and in MKN45 and MKN74 cells. Fz2 was expressed in normal stomach and in MKN45, but not in MKN74 cells. Well-differentiated GC tissue was weakly positive for Fz2 in cell membranes. Fz2 was positive in both the cell membrane and cytoplasm of GC tissues of moderately differentiated and poorly differentiated adenocarcinoma. Signet ring cells were positive for cytoplasmic Fz2. Proliferation of MKN45 and MKN74 cells was suppressed by Fz2 shRNA, and a scratch assay demonstrated that Fz2 shRNA suppressed also MKN45 and MKN74 cell motility. Furthermore, Fz2 shRNA application led to downregulated mRNA expression of both cyclin D1 and MMP-9. Fz2, 3, 6 and 8 were expressed in normal stomach, and in MKN45 and MKN74 GC cells. Fz2 shRNA suppressed cell proliferation and motility of MKN45 and MKN74 cells, and downregulated cyclin D1 and MMP-9 expression in these GC cell lines.

Several studies reported that autoimmune diseases share a number of susceptibility genes. Of these genes, a SNP rs7708392 in TNIP1 was reported to be associated with systemic lupus erythematosus (SLE). Autoimmune hepatitis (AIH), a rare chronic progressive liver disease, shares some clinical features with SLE. Therefore, we investigated whether the SNP is associated with Japanese AIH. An association study of rs7708392 was conducted in 343 Japanese AIH patients and 828 controls. We found that rs7708392 is associated with AIH (P = 0.0236, odds ratio (OR) 1.26, 95% confidence interval (CI): 1.03–1.54), under the allele model for C allele. Significant differences of clinical characteristics of the AIH patients with or without G allele of rs7708392 were not detected. Of interest, the association was stronger in AIH without HLA-DRB1*04:05 allele (P = 0.0063, Q = 0.0127, OR 1.48, 95% CI: 1.12–1.96), though the association was not detected in AIH with DRB1*04:05. The C allele of rs7708392 was associated with AIH, especially AIH without DRB1*04:05, an already established risk factor.

In a clinical setting, it is important to diagnose complications of acute cholecystitis accurately. Diffusion-weighted whole body imaging with background body signal suppression/T2-weighted image fusion (DWIBS/T2) provides high signal intensity with a strong contrast against surrounding tissues in anatomical settings. In the present study, patients who were being treated for acute cholecystitis and underwent DWIBS/T2 in the National Hospital Organization Shimoshizu Hospital between December 2012 and August 2015 were enrolled. A total of 10 men and 4 women underwent DWIBS/T2. Records, including DWIBS/T2 and computed tomography (CT) imaging, were retrospectively analyzed for patients with acute cholecystitis. CT images revealed thickened gallbladder walls in patients with acute cholecystitis, and high signal intensity was observed in DWIBS/T2 images for the thickened gallbladder wall. Inflammation of the pericholecystic space and the liver resulted in high intensity signals with DWIBS/T2 imaging, whereas CT imaging revealed a low-density area in the cholecystic space. Plain CT scanning identified a low-density area in the liver, which became more obvious with contrast-enhanced CT. DWIBS/T2 imaging showed the inflammation of the liver and pericholesyctic space as an area of high signal intensity. Detectability of inflammation of the pericholecystic space and the liver was the same for DWIBS/T2 and CT, which suggests that DWIBS/T2 has the same sensitivity as CT scanning for the diagnosis of complicated acute cholecystitis. However, the strong contrast shown by DWIBS/T2 allows for easier evaluation of acute cholecystitis than CT scanning.

Human primary hepatocytes are able to survive in a medium without glucose and arginine, but supplemented with galactose and ornithine (hepatocyte selection medium; HSM). To address the possibility of the application of HSM in cancer therapy, hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells were cultured in HSM. Cell proliferation was analyzed using an MTS assay. Morphological changes were analyzed using hematoxylin and eosin staining. Apoptosis was analyzed using a TUNEL assay and cell motility was assessed with a scratch assay. Cell proliferation was significantly suppressed in cell lines grown in HSM (P

Mesenchymal-epithelial transition factor (c-Met) is a receptor that binds to the hepatocyte growth factor and is upregulated in hepatocellular carcinoma (HCC). The anti-tumor effects of (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4- methyl-piperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-me- thyl-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (SU11274), a c-Met inhibitor, were investigated in the present study. HCC cells (HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2) and human umbilical vein endothelial cells (HUVECs) were used. Quantitative polymerase chain reaction was performed to detect the expression level of c-Met in HCC and HUVECs, and cyclin D1 in HCC. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed to assess the proliferation of the HCC cells and HUVECs cultured with SU11274. Co-culture of HLF or PLC/PRL/5 cells and HUVECs was established as an in vitro model of HCC tissues. The expression levels of c-Met in HLE, HLF, PLC/PRL/5, Hep3B, Huh-6 and HepG2, adult healthy liver and HUVECs were 4.43±0.50, 1.61±0.18, 3.70±0.08, 0.81±0.18, 6.60±1.29, 1.06±0.35, 1.00±0.09 and 88.8±17.3 (mean ± standard deviation), respectively. SU11274 (30 μM) suppressed the proliferation of HLF, PLC/PRL/5 and HUVECs to 11.0±9.4, 46.5±30.7 and 29.4±5.0%, respectively. SU11274 (30 μM) decreased the expression levels of cyclin D1 in HLF and PLC/PRL/5 cells to 45.1±11.6 and 30.1±10.3%, respectively. SU11274, at a concentration of 30 μM damaged the morphology of the co-cultures of HLF or PLC/PRL/5 cells with HUVECs and all the cells died. c-Met is highly expressed in HUVECs and HCC cells, but not in Hep3B. At a 30-μM concentration, SU11274 suppresses the proliferation of HLF, PLC/PRL/5 and HUVECs. SU11274 (30 μM) damages the co-cultures of HLF or PLC/PRL/5 cells with HUVECs.

In the present study, Frizzled-2 (Fz2), a receptor of the Wnt ligand, was investigated as a potential target of molecular therapy for hepatocellular carcinoma (HCC). Quantitative polymerase chain reaction (PCR) was performed to determine the expression levels of Fz2. A surgical specimen of HCC was immunostained with an Fz2 antibody. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay was performed on HCC cell lines, including HLF and Hep3B, 72 h after the transfection of the short hairpin (sh)RNA of Fz2 (shRNA-Fz2). RNA was isolated from the Hep3B and HLF cells 48 h after transfection and subjected to quantitative PCR. All cell lines had elevated levels of Fz2 compared with those in an adult liver. The highest and lowest expression levels of Fz2 were 246.9±15.7 in the HLF cells and 5.8±1.4 in the Hep3B cells, respectively. Fz2 was expressed in the tumorous HCC tissue, but not in the surrounding non-tumorous tissue. Cell proliferation was suppressed to 28.6±6.4% in the HLF cells and to 29.8±4.3% in the Hep3B cells at 100 ng shRNA-Fz2 per well. Levels of cyclin D1 expression decreased to 65.2±5.9% in the HLF cells and to 60.8±14.6% in the Hep3B cells at 2.5 μg per well. In conclusion, Fz2 was upregulated in the HCC cells. shRNA-Fz2 suppressed the proliferation of the Hep3B and HLF cells, decreasing Fz2 expression. As it was not expressed in the surrounding non-tumorous tissue, Fz2 may be an ideal molecular therapeutic target for HCC.

Resistance is one limitation of sorafenib in the treatment of hepatocellular carcinoma (HCC). Insulin-like growth factor-1 receptor (IGF-1R) is involved in cancer cell proliferation. To assess the potential synergistic antitumor effects of picropodophyllin (PPP), an IGF-1R inhibitor, HLF and PLC/PRL/5, HCC cells were treated with PPP alone or PPP in combination with sorafenib, a multikinase inhibitor. Normal human umbilical vein endothelial cells (HUVECs) were also used to analyze the antiangiogenic effects of the drugs. HCC cells and HUVECs were cultured on 96-well plates, and then treated with PPP, with and without the addition of sorafenib. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and hematoxylin and eosin staining were then performed 48 h later. The HCC cells were also analyzed using scratch assays and hematoxylin and eosin staining after 48 h. The proliferation of HLF, PLC/PRF/5 and HUVEC cells was suppressed by the combination of 0.2 μM PPP and 3 μM sorafenib more effectively than by 10 μM sorafenib alone. The motility of HLF and PLC/PRF/5 cells was also suppressed to a greater extent with the combination of PPP at 0.2 μM and sorafenib at 3 μM than with sorafenib at 10 μM alone. The cells that had been treated with 0.2 μM PPP and 3 μM sorafenib also exhibited pyknotic nuclei, which is characteristic of apoptosis. In conclusion, PPP enhanced sorafenib-mediated suppression of proliferation and motility in HCC cells. Therefore, the combination of PPP and sorafenib may exert antitumor and antiangiogenic effects.

In the present study, the threshold values of laboratory data for the diagnosis of non-alcoholic fatty liver disease (NAFLD) were investigated. The study enrolled patients who had undergone abdominal ultrasound (US) between April 2013 and August 2013, and for whom laboratory data were available on the same day. NAFLD was diagnosed following observations of a bright liver or hepatorenal echo contrast on the abdominal US scans. Patients were excluded from the study if they had liver diseases or had been prescribed prednisolone or methotrexate. Receiver operating characteristic curves, the Wilcoxon signed-rank test and Fisher’s exact probability test were used for data analysis. In total, 80 NAFLD and 94 non-NAFLD patients were enrolled in the study. The threshold levels of alanine aminotransferase (ALT) and triglyceride (TG) for the diagnosis of NAFLD were 19.0 IU/l and 101 mg/dl, respectively. Patients were divided into two groups according to the levels of ALT and TG. Those with ALT levels of >19 IU/l and TG levels of >101 mg/dl were defined as the positive group, while the remaining patients were classified as the negative group. The specificity and positive predictive value using the combined threshold levels of ALT >19 IU/l and TG >101 mg/dl were 80.9 and 75.0%, respectively. Therefore, the results indicated that ALT levels of >19 IU/l or TG levels of >101 mg/dl were useful markers for the screening of NAFLD. However, NAFLD was more strongly suspected in patients with ALT levels of >19 IU/l and TG levels of >101 mg/dl.

Minoru Tomizawa,1 Fuminobu Shinozaki,2 Yasufumi Motoyoshi,3 Takao Sugiyama,4 Shigenori Yamamoto,5 Makoto Sueishi,4 Takanobu Yoshida6 1Department of Gastroenterology, 2Department of Radiology, 3Department of Neurology, 4Department of Rheumatology, 5Department of Pediatrics, 6Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, Yotsukaido City, Chiba, Japan Background: The Wnt pathway plays an important role in hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. Methods: We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1. Results: Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. Conclusion: Niclosamide is a potential candidate for the treatment of hepatoma. Keywords: Wnt3a, niclosamide, luciferase assay, cyclin D1, T-cell factor

Human primary hepatocytes are able to survive in a medium without glucose and arginine that is instead supplemented with galactose and ornithine (hepatocyte selection medium; HSM). This is because the cells produce glucose and arginine by the action of galactokinase (GALK) and ornithine carbamoyltransferase (OTC), respectively. It was expected that hepatocellular carcinoma (HCC) cells do not survive in HSM. In the current study, HCC cell lines (namely HLE, HLF, PLC/PRL/5, Hep3B and HepG2) and human umbilical vascular endothelial cells (HUVECs) were cultured in HSM, and the expression levels of GALK1, GALK2 and OTC were analyzed by reverse transcription-quantitative polymerase chain reaction. HLE, HLF and PLC/PRL/5 cells died on day 11, while Hep3B, HepG2 and HUVECs died on day 7. HLF cells were further analyzed as these cells had lower expression levels of GALK1, GALK2 and OTC compared with adult liver cells, and survived until day 11. In these cells, the expression levels of GALK1, GALK2 and OTC did not change on days 3 and 7 as compared to day 0. In addition, a co-culture of HLF cells with HUVECs was established and the medium was changed to HSM. It was observed that HLF cells and HUVECs in co-culture were damaged in HSM. In summary, HCC cells and HUVECs died in a medium without glucose and arginine that was supplemented with galactose and ornithine. HCC cells and HUVECs were damaged in HSM, suggesting a potential application for treatment with the medium.

Upper gastrointestinal (GI) bleeding can be fatal. Blood test variables were reviewed in search of threshold values to detect the presence of occult upper GI bleeding. The records of 1,023 patients who underwent endoscopy at the National Hospital Organization Shimoshizu Hospital from October 2014, to September 2015, were retrospectively reviewed. Of those, 95 had upper GI bleeding. One-way analysis of variance was applied to blood test variables comparing patients with and without upper GI bleeding. Logistic regression analysis was applied to detect the association of blood test parameters with upper GI bleeding, and receiver-operator characteristics were applied to establish threshold values. White blood cell count (WBC), platelet (Plt) count, and blood urea nitrogen (BUN) levels were higher, and hemoglobin (Hb) and albumin (Alb) levels were lower in patients with upper GI bleeding. Logistic regression analysis showed that low Hb was significantly associated with upper GI bleeding and a Hb value of 10.8 g/dl was established as the threshold for the diagnosis. In patients with upper GI bleeding, WBC, Plt count, and BUN levels were higher and Hb and Alb levels were reduced. Hb at 10.8 g/dl was established as a threshold value to detect upper GI bleeding.

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

To initiate hepatocyte differentiation in human induced pluripotent stem (iPS) cells, cells are cultured in a medium lacking glucose but supplemented with galactose (hepatocyte selection medium, HSM) or in medium supplemented with oncostatin M and small molecules (hepatocyte differentiation inducer, HDI). In the present study, 2‑Deoxy‑D‑glucose (2DG), an analogue of glucose, was utilized instead of glucose deprivation and the effect of 2DG supplementation on iPS differentiation was examined. First, 201B7 cells, an iPS cell line, were cultured in HSM or HDI media for 2 days and then subjected to reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in order to analyze expression levels of established hepatocyte markers, including cytosolic aspartate aminotransferase (AST), mitochondrial AST, alanine aminotransferase (ALT), and glycerol kinase. mRNA expression levels of mitochondrial AST, ALT, and glycogen synthase significantly increased following culture in HSM and HDI compared with ReproFF media. Cytosolic AST mRNA expression levels significantly increased following culture in HDI compared with ReproFF media, but not in HSM. To test the effect of 2DG on iPS differentiation, 201B7 cells were cultured in ReproFF, a feeder‑free medium that retains pluripotency, supplemented with 2DG. Following 7 days of culture, the cells were subjected to RT‑qPCR to analyze expression levels of α‑fetoprotein (AFP), a marker of immature hepatocytes. AFP mRNA expression levels significantly increased with the addition of 0.1 µM 2DG in the media, and galactose addition acted synergistically with 2DG to further upregulate AFP expression. In conclusion, the present study demonstrated that hepatocyte differentiation was initiated in iPS cells cultured in HSM and HDI media and that 2DG could be used as a supplement instead of glucose deprivation to initiate hepatocyte differentiation in iPS cells.

Peptic ulcer bleeding can be fatal. Non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and immunosuppressive agents are administered for long-term usage. The present study assessed the association between peptic ulcer bleeding and administration of NSAIDs, corticosteroids and immunosuppressive agents. Furthermore, the efficacy of lowering the risk of peptic ulcer bleeding with proton pump inhibitors (PPI) and histamine 2 receptor antagonists (H2RA) was evaluated. Medical records were retrospectively analyzed for patients subjected to an upper gastrointestinal (GI) endoscopy performed at the National Hospital Organization Shimoshizu Hospital (Yotsukaido, Japan) from October 2014 to September 2015. During this period, a total of 1,023 patients underwent an upper GI endoscopy. A total of 1,023 patients, including 431 males (age, 68.1±12.9 years) and 592 females (age, 66.4±12.3 years), who had been administered NSAIDs, corticosteroids, immunosuppressive agents, PPIs and H2RAs, were respectively enrolled. Endoscopic findings of the patients were reviewed and their data were statistically analyzed. Logistic regression analysis was used to determine the odds ratio of peptic ulcer bleeding for each medication; immunosuppressive agents had an odds ratio of 5.83, which was larger than that for NSAIDs (4.77). The Wald test was applied to confirm the correlation between immunosuppressive agents and peptic ulcer bleeding. Furthermore, χ2 tests were applied to the correlation between peptic ulcer bleeding and administration of PPIs or H2RAs. Immunosuppressive agents had the largest χ2, and the P-value was 0.03. Administration of PPIs was significantly correlated with non-peptic ulcer bleeding (P=0.02); furthermore, a tendency toward non-peptic ulcer bleeding with administration of H2RA was indicated, but it was not statistically significant (P=0.12). In conclusion, immunosuppressive agents were correlated with peptic ulcer bleeding and PPIs were effective at lowering the risk of peptic ulcer bleeding.

Induced pluripotent stem (iPS) cells are ideal sources of hepatocyte for transplantation into patients experiencing hepatic failure. Growth and transcription factors were analyzed to design a single-step protocol for the differentiation of iPS cells into hepatocytes. The expression of transcription factors was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and compared among iPS cells, as well as fetal and adult liver cells. iPS cells were cultured with growth factors and RT-PCR was performed to analyze the expression of transcription factors. iPS cells were introduced with transcription factors, cultured with growth factors and subjected to real-time quantitative PCR. Indocyanine green (ICG) was added to the medium as a hepatocyte marker. Sox17, GATA4, GATA6, FoxA2, HEX, HNF4α and C/EBPα were expressed in fetal and adult liver cells, but not in iPS cells. Sox17, GATA6 and HNF4α were expressed after exposure a combination of oncostatin M, epidermal growth factor, retinoic acid, dexamethasone and ITS (OERDITS). When iPS cells were introduced with FoxA2, GATA4, HEX and C/EBPα and cultured with OERDITS for 8 days, the cells expressed α-fetoprotein, δ-like (Dlk)-1 and γ-glutamyl transpeptidase (GTP), and ICG uptake was observed. Exposure to FoxA2, GATA4, HEX and C/EBPα and culturing with OERDITS supplementation potentially serves as a single-step inducer for the differentiation of iPS cells into hepatic progenitor-like cells within 8 days.

Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)β antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRβ was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRβ may be a potential therapeutic target to inhibit invasion of pancreatic cancer.

To investigate the role of Activin A in the embryoid bodies (EBs) of human induced pluripotent stem (iPS) cells, EBs were transferred onto dishes coated with Matrigel after 4 days of incubation with Activin A and observed under a microscope. Alkaline phosphatase staining and immunostaining were performed to analyze the pluripotency of the cells, and the MTS assay was performed to analyze their proliferative potential. Fourteen days after EB formation, cells cultured with Activin A (100 ng/ml) showed no morphological alterations. Cells cultured with 10–100 ng/ml of Activin A were positive for alkaline phosphatase staining, while cells cultured with 0–3 ng/ml showed negative staining. Cells cultured with 10 ng/ml of Activin A were positive for Oct3/4, Nanog, SSEA-4 and TRA-1-60, while cells cultured with 0 ng/ml Activin A were negative. Cells cultured with 3–30 ng/ml of Activin A maintained their proliferative potential, while loss of proliferative potential was observed in cells cultured with 100 ng/ml Activin A. In conclusion, Activin A maintained pluripotency markers in human iPS cells cultured as EBs with Activin A.

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPα) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPα and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPα upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPα. Actinomycin D was used to analyze half-life of C/EBPα mRNA. IGF-II up-regualted C/EBPα by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPα. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPα while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase. J. Cell. Biochem. 102: 161–170, 2007. © 2007 Wiley-Liss, Inc.

Prompt and accurate diagnosis is critical in the treatment of acute cholecystitis. Diffusion-weighted whole-body magnetic resonance imaging with background body signal suppression/T2 image fusion (DWIBS/T2) identifies areas with high signal intensity, corresponding to inflammation. In the present study, the records and images of patients with acute cholecystitis who underwent DWIBS/T2 between January 2013 and March 2014 were retrospectively analyzed. A total of 11 patients with acute cholecystitis were enrolled. In one patient, DWIBS/T2 identified a thickened wall and high signal intensity, with high signal intensity in the pericholecystic space that suggested localized peritonitis. Positive DWIBS/T2 results indicating acute cholecystitis were obtained in 10/11 patients, with a sensitivity of 90.9%. In addition, wall thickening and high signal intensity were absent in DWIBS/T2 images when wall thickening was not detected by computed tomography. Wall thickening and high signal intensity was attenuated when patients with acute cholecystitis were clinically treated. These data suggest that a thickened gallbladder wall and high signal intensity are indicative of acute cholecystitis and that DWIBS/T2 may be a useful technique in evaluating the severity of acute cholecystitis.

Cancer cells consume more glucose than normal cells, mainly due to their increased rate of glycolysis. 2-Deoxy-d-glucose (2DG) is an analogue of glucose, and sorafenib is a kinase inhibitor and molecular agent used to treat hepatocellular carcinoma (HCC). The present study aimed to demonstrate whether combining 2DG and sorafenib suppresses tumor cell proliferation and motility more effectively than either drug alone. HLF and PLC/PRF/5 HCC cells were incubated with sorafenib with or without 1 µM 2DG, and subjected to a proliferation assay. A scratch assay was then performed to analyze cell motility following the addition of 2DG and sorafenib in combination, and each agent alone. RNA was isolated and subjected to reverse transcription-quantitative polymerase chain reaction to analyze the expression of cyclin D1 and matrix metalloproteinase-9 (MMP9) following the addition of 2DG and sorafenib in combination and each agent alone. Proliferation was markedly suppressed in cells cultured with 1 µM 2DG and 30 µM sorafenib compared with cells cultured with either agent alone (P

Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion (DWIBS/T2) strongly contrasts cancerous tissue against background healthy tissues. Positron emission tomography/computed tomography (PET/CT) applies the uptake of 18-fluorodeoxyglucose in the diagnosis of cancer. Our aim was to compare DWIBS/T2 and PET/CT in patients with upper gastrointestinal cancers. Patient records, including imaging results from July 2012 to March 2015, were analyzed retrospectively. Four men (age, 72.5 ± 5.3 years) and ten women (age, 71.6 ± 4.0 years) were enrolled in this study. The numbers of patients with esophageal cancer, gastric cancer, gastrointestinal stromal tumor, and duodenal cancer were one, eight, three, and two, respectively. Six out of eight patients with gastric cancer had positive results on both DWIBS/T2 and PET/CT. The diameter and depth of invasion of gastric cancer was larger in patients with positive DWIBS/T2 and PET/CT findings than those with negative findings. These results suggested that patients with gastric cancer with larger pixel numbers might tend to show positive results with DWIBS/T2. DWIBS/T2 and PET/CT have similar sensitivity for the diagnosis of upper gastrointestinal cancer. The diameter and depth of invasion affected the detectability of gastric cancer.

Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion (DWIBS/T2) is useful for the diagnosis of cancer as it presents a clear contrast between cancerous and non-cancerous tissue. The present study investigated the limitations and advantages of DWIBS/T2 with regards to the diagnosis of colorectal polyp (CP) or cancer (CRC). The current study included patients diagnosed with CP or CRC following colonoscopy, who were subjected to DWIBS/T2 between July 2012 and March 2015. Patient records were analyzed retrospectively. Patients were subjected to DWIBS/T2 when they presented with abdominal cancers or inflammation. Colonoscopy was performed as part of screening, or if patients had suspected colon cancer or inflammatory bowel disease. A total of 8 male and 7 female patients were enrolled in the present study. All patients, with the exception of one who had been diagnosed with CRC following colonoscopy, had positive results and all patients diagnosed with CP following a colonoscopy, with the exception of one, had negative results on DWIBS/T2. Thus, CRC was detected by DWIBS/T2, while CP was not (P=0.0028). The diameter of CRC lesions was significantly larger than that of CP (P