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ObjectivesSpontaneous pneumomediastinum (SPNM) historically has been considered a poor prognostic factor in dermatomyositis/polymyositis patients complicated with interstitial lung disease (ILD). However, there is a lack of actual data regarding the association between SPNM occurrence and mortality in dermatomyositis/polymyositis patients. This study aimed to assess the association between SPNM occurrence and mortality in myositis patients with ILD according to antimelanoma differentiation-associated gene 5 (MDA5) antibody status.MethodsDermatomyositis/polymyositis patients with ILD who were hospitalised at five Japanese hospitals from 2016 to 2020 were included in this retrospective observational study. We collected data about baseline characteristics including myositis-specific autoantibodies, treatments, SPNM and death within 1 year from therapy initiation or strengthening. Baseline characteristics and outcomes were compared between patients with and without SPNM (the SPNM group and the non-SPNM group, respectively).ResultsA total of 119 patients were analysed. SPNM occurred in 23 patients, and 15 patients died. Fifteen patients with SPNM were anti-MDA5 antibody positive. The mortality rate was significantly higher in the SPNM group (34.8%) than in the non-SPNM group (7.3%) (p=0.001). All deaths in the SPNM group occurred in anti-MDA5 antibody-positive patients (8/15), whereas none of the anti-MDA5 antibody-negative patients in the SPNM group died (0/8). In anti-MDA5 antibody-positive patients, the mortality rate was significantly higher in patients with SPNM occurrence (53.3%) than in those without SPNM occurrence (4.0%) (p=0.001).ConclusionSPNM occurred more frequently in anti-MDA5 antibody-positive than in anti-MDA5 antibody-negative myositis patients. SPNM occurrence was associated with higher mortality risk, especially in anti-MDA5 antibody-positive patients.

Importance The current standard induction therapy for antineutrophil cytoplasm antibody (ANCA)-associated vasculitis is the combination of high-dose glucocorticoids and cyclophosphamide or rituximab. Although these regimens have high remission rates, they are associated with considerable adverse events presumably due to high-dose glucocorticoids. Objective To compare efficacy and adverse events between a reduced-dose glucocorticoid plus rituximab regimen and the standard high-dose glucocorticoid plus rituximab regimen in remission induction of ANCA-associated vasculitis. Design, setting, and participants This was a phase 4, multicenter, open-label, randomized, noninferiority trial. A total of 140 patients with newly diagnosed ANCA-associated vasculitis without severe glomerulonephritis or alveolar hemorrhage were enrolled between November 2014 and June 2019 at 21 hospitals in Japan. Follow-up ended in December 2019. Interventions Patients were randomized to receive reduced-dose prednisolone (0.5 mg/kg/d) plus rituximab (375 mg/m2/wk, 4 doses) (n = 70) or high-dose prednisolone (1 mg/kg/d) plus rituximab (n = 70). Main outcomes and measures The primary end point was the remission rate at 6 months, and the prespecified noninferiority margin was -20 percentage points. There were 8 secondary efficacy outcomes and 6 secondary safety outcomes, including serious adverse events and infections. Results Among 140 patients who were randomized (median age, 73 years; 81 women [57.8%]), 134 (95.7%) completed the trial. At 6 months, 49 of 69 patients (71.0%) in the reduced-dose group and 45 of 65 patients (69.2%) in the high-dose group achieved remission with the protocolized treatments. The treatment difference of 1.8 percentage points (1-sided 97.5% CI, -13.7 to ∞) between the groups met the noninferiority criterion (P = .003 for noninferiority). Twenty-one serious adverse events occurred in 13 patients in the reduced-dose group (18.8%), while 41 occurred in 24 patients in the high-dose group (36.9%) (difference, -18.1% [95% CI, -33.0% to -3.2%]; P = .02). Seven serious infections occurred in 5 patients in the reduced-dose group (7.2%), while 20 occurred in 13 patients in the high-dose group (20.0%) (difference, -12.8% [95% CI, -24.2% to -1.3%]; P = .04). Conclusions and relevance Among patients with newly diagnosed ANCA-associated vasculitis without severe glomerulonephritis or alveolar hemorrhage, a reduced-dose glucocorticoid plus rituximab regimen was noninferior to a high-dose glucocorticoid plus rituximab regimen with regard to induction of disease remission at 6 months. Trial registration ClinicalTrials.gov Identifier: NCT02198248.

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

This study researched the efficacy of rituximab (RTX) in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated hypertrophic pachymeningitis (HP).Eight patients were identified by retrospective chart review from local registries at four hospitals in Japan. All patients met the Chapel Hill 2012 Consensus Conference definitions of ANCA-associated vasculitis and had disease complicated with HP. We assessed the dose of glucocorticoids, C-reactive protein (CRP) levels, Birmingham vasculitis activity score (BVAS) and contrast-enhanced magnetic resonance imaging (MRI) findings of HP before and after RTX administration.Three of eight patients were female. The median age was 68 years. No patients had HP at onset of vasculitis. Two patients had a relapse of HP before RTX administration. RTX was used as the initial treatment for HP in three patient. The daily dose of glucocorticoids, CRP levels and BVAS decreased from baseline to 6 months after RTX treatment in all patients. Evaluation of HP by contrast-enhanced MRI showed improvement in seven of eight cases. All of seven patients achieved sustained remission at 6 months after RTX treatment. No serious adverse events were observed in any patient.Our case series highlights the efficacy of RTX in patients with difficult-to-treat ANCA-associated HP. Future prospective studies are warranted to establish B-cell depletion therapy by RTX as a treatment option for ANCA-associated HP.

Objective: To describe the pre-conception status, pregnancy outcomes, and medication prevalence in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn’s disease (CD), and ulcerativ...

Objective Pneumocystis pneumonia (PCP) is one of the most common opportunistic infections. In systemic autoimmune disease patients receiving immunosuppressive treatments, low lymphocyte count, old age and coexisting lung disease have been known as risk factors for the occurrence of PCP. However, factors relevant to prognosis of PCP have not been fully studied. Methods A total of 95 sequential patients who developed PCP during immunosuppressive treatment for systemic autoimmune diseases was identified from five Japanese centres. We retrospectively assessed baseline characteristics, immunosuppressive treatment prior to the onset of PCP, treatment for PCP and survival. Univariate and multivariate analyses were performed to identify prognostic factors. Results Forty-two deaths (44.2%) were observed in this study. Age at the diagnosis of PCP was higher in non-survivors than in survivors (74 years vs. 64 years, p = 0.008). Non-survivors more frequently had lung involvement than did survivors (47.6% vs. 13.2%, p

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.

Transcription factors and culture media were investigated to determine the condition to initiate the differentiation of human-induced pluripotent stem (iPS) cells most efficiently. The expression of genes in human adult liver was compared with that in 201B7 cells (iPS cells) using cDNA microarray analysis. Episomal plasmids expressing transcription factors were constructed. 201B7 cells were transfected with the episomal plasmids and cultured in ReproFF (feeder-free media maintaining pluripotency), Leibovitz-15 (L15), William's E (WE), or Dulbecco's modified Eagle medium/Nutrient F-12 Ham (DF12) for 7 days. RNA was isolated and subjected to real-time quantitative PCR to analyze the expression of alpha-feto protein (AFP) and albumin. cDNA microarray analysis revealed 16 transcription factors that were upregulated in human adult liver relative to that in 201B7 cells. Episomal plasmids expressing these 16 genes were transfected into 201B7 cells. CCAAT/enhancer-binding protein alpha (CEBPA), CCAAT/enhancer-binding protein beta (CEBPB), forkhead box A1 (FOXA1), and forkhead box A3 (FOXA3) up-regulated AFP and down-regulated Nanog. These four genes were further analyzed. The expression of AFP and albumin was the highest in 201B7 cells transfected with the combination of CEBPA, CEBPB, FOXA1, and FOXA3 and cultured in WE. The combination of CEBPA, CEBPB, FOXA1, and FOXA3 was suitable for 201B7 cells to initiate differentiation to the hepatocyte lineage and WE was the most suitable medium for culture after transfection. J. Cell. Biochem. 117: 2001-2009, 2016. © 2016 Wiley Periodicals, Inc.

Objective.Patients with eosinophilic granulomatosis with polyangiitis (EGPA) frequently experience relapses, which lead to cumulative organ damage. In this retrospective observational study, we aimed to reveal the risk factors for relapse in EGPA.Methods.A total of 188 Japanese patients with EGPA diagnosed between 1996 and 2015 were identified from medical records in 10 hospitals. The diagnosis was based on the American College of Rheumatology 1990 criteria or Lanham’s criteria. Baseline characteristics, treatments, asthma exacerbation, and relapses were evaluated by retrospective chart review.Results.The median followup period was 56 months. The median age at disease onset was 59.7 years. At the disease onset, 95.2% of the patients had a history of bronchial asthma and 44.7% were positive for antineutrophil cytoplasmic antibodies. The cumulative survival and relapse-free survival rates at 5 years were 89.6% and 64.0%, respectively. Multivariate analysis with 2 models, proportional hazards, and competing risk models, was performed to identify the factors associated with relapse. The proportional hazards model identified azathioprine (AZA) maintenance therapy and high eosinophil counts at onset as independent factors with lower relapse risks, and high immunoglobulin E (IgE) levels at onset as a risk factor for relapse. The competing risk model identified no statistically significant factors.Conclusion.Although potential benefit of AZA maintenance therapy in preventing relapse of EGPA was suggested by the proportional hazards model, there was a discrepancy in the results between the models. Eosinophil counts and IgE levels at onset were also identified as candidates of factors associated with relapse in EGPA.

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

Activin A maintains the pluripotency of human induced pluripotent stem (hiPS) cells. A combination of activin A and CHIR99021 (CHIR), a specific inhibitor of glycogen synthase‑3β, is suitable for feeder‑free culture of hiPS cells. In the present study, the specific role of the Wnt signaling pathway in cells cultured under different conditions was investigated. Following transfection with the reporter plasmids, TOPflash and FOPflash, hiPS cells were cultured in medium, containing activin A, CHIR, leukemia inhibitory factor (LIF) or SB431542, a specific inhibitor of activin A. A luciferase reporter assay was performed 48 h later. Western blot analysis was performed to determine the expression levels of β‑catenin and tubulin‑α. The activity of Wnt in hiPS cells was suppressed by culture in the presence of activin A. The activation of the Wnt pathway was most marked when the cells were cultured with a combination of activin A and CHIR. Addition of SB431542 into the culture revealed no significant change in the Wnt pathway. Western blot analysis revealed that β‑catenin accumulated most often in cells cultured with activin A and CHIR. β‑catenin also accumulated in cells cultured with activin A alone. Culture with activin A and CHIR most effectively stimulated the Wnt signaling pathway, as measured by luciferase assays using TOPflash and FOP flash as reporter plasmids. β‑catenin accumulated in the hiPS cells cultured with activin A, via a mechanism, which remains to be elucidated. The Wnt signaling pathway may be important for hiPS cell growth in feeder‑free culture.

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.

Aims/introduction Although high-dose glucocorticoids have been reported to cause new-onset diabetes mellitus (glucocorticoid-induced diabetes mellitus), its risk factors have remained to be determined. We investigated the risk factors related to glucocorticoid-induced diabetes mellitus diagnosed within 2 months after the high-dose treatment (newly treated with an initial high dose of > 20 mg prednisolone (PSL) equivalent per day for at least more than 6 months) in collagen vascular diseases. Methods A total of 2,631 patients with collagen vascular diseases was registered between 1986 and 2006 in the Chiba-Shimoshizu Rheumatic Cohort. We analyzed 681 patients newly treated with high-dose glucocorticoid who did not have diabetes mellitus and/or its previous diagnosis (age: 46.3 ± 16.7 years, PSL dose: 40.0 ± 14.1 mg/day). Glucocorticoid-induced diabetes mellitus was diagnosed by two or more glucose measurements in patients with fasting glycaemia ≥ 7 mmol/L and 120 minutes post-load glycaemia ≥ 11.1 mmol/L. Results Glucocorticoid-induced diabetes mellitus was observed in 26.3% of patients, and the glucocorticoid-induced diabetes mellitus group had higher age, higher BMI, lower rates of females and systemic lupus erythematosus, higher rates of smoking, alcohol use, and microscopic polyangiitis. Multivariate logistic regression analysis demonstrated that the risk of glucocorticoid-induced diabetes mellitus was independently higher in every 10-year increment of initial age with adjusted odds ratio (OR) 1.556 (95% confidence interval: 1.359 - 1.783), in every 1 kg/m2 increment of BMI with OR 1.062 (1.002 - 1.124), in current smoking with OR 1.664 (1.057 - 2.622), and in every 10 mg increment of initial dose of prednisolone with OR 1.250 (1.074 - 1.454). Conclusions High-dose glucocorticoids caused diabetes mellitus with high prevalence within a short period, and current smokers should be considered at higher risk of glucocorticoidinduced diabetes mellitus in addition to age, BMI, and initial dose.

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.

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.