Makoto Sueishi

Prediction of Relapse After Discontinuation of Biologic Agents by Ultrasonographic Assessment in Patients With Rheumatoid Arthritis in Clinical Remission: High Predictive Values of Total Gray‐Scale and Power Doppler Scores That Represent Residual Synovial Inflammation Before Discontinuation

This prospective study aimed to determine whether the comprehensive ultrasonographic assessment of synovial inflammation predicts relapse after discontinuation of treatment with a biologic agent in patients with rheumatoid arthritis (RA) in clinical remission.

Prediction of Therapeutic Responses to Tocilizumab in Patients With Rheumatoid Arthritis: Biomarkers Identified by Analysis of Gene Expression in Peripheral Blood Mononuclear Cells Using Genome‐Wide DNA Microarray

The aim of this prospective multicenter study was to identify biomarkers that can be used to predict therapeutic responses to tocilizumab in patients with rheumatoid arthritis (RA).

Age dependence of early symptomatic vertebral fracture with high-dose glucocorticoid treatment for collagen vascular diseases.

OBJECTIVES Collagen vascular diseases requiring treatment with high-dose glucocorticoids are frequently complicated by vertebral fracture. We investigated the incidence of symptomatic vertebral fractures for 20 yr among patients who were treated with high-dose glucocorticoids in the Chiba-Shimoshizu Rheumatic Cohort. METHODS A total of 2631 patients with collagen vascular diseases (aged >or=18 yr) was registered between 1986 and 2006. The prevalence of symptomatic vertebral fracture was compared between the high-dose glucocorticoid group newly treated with high-dose glucocorticoids (>or=20 mg/d prednisolone equivalent) (n = 700), and the non-glucocorticoid controls not treated with glucocorticoids (n = 194). RESULTS During the 20-yr study period, symptomatic vertebral fractures occurred more frequently in the high-dose glucocorticoid group (23.9%) than in the non-glucocorticoid controls (2.6%). According to a Kaplan-Meier analysis, the cumulative incidence of symptomatic vertebral fracture was significantly higher in the high-dose glucocorticoid group than in the non-glucocorticoid controls (P < 0.001). Stratified into age quartiles of the high-dose glucocorticoid group (age 18-31, 32-47, 48-59, and 60-88 yr), the patients had a markedly increased incidence of symptomatic vertebral fracture with aging. The hazard ratios were also significantly higher in the older age quartile of 60-68 than in the younger age quartile of 32-47 (P < 0.001 for trend). The hazard ratio was 26-fold higher in patients aged 60-88 than in patients aged 18-31 (P < 0.01). In the group with fractures, the treatment duration before fracture was negatively associated with the initial age (r = -0.6587; P < 0.001). CONCLUSIONS The prevalence of symptomatic vertebral fractures was higher in the patients treated with high-dose glucocorticoids than the untreated controls. Vertebral fractures were age dependent in patients treated with high-dose glucocorticoids. Treatment duration before fracture incidence was significantly shorter in the older patients.

Sonoporation: Gene transfer using ultrasound

Genes can be transferred using viral or non-viral vectors. Non-viral methods that use plasmid DNA and short interference RNA (siRNA) have advantages, such as low immunogenicity and low likelihood of genomic integration in the host, when compared to viral methods. Non-viral methods have potential merit, but their gene transfer efficiency is not satisfactory. Therefore, new methods should be developed. Low-frequency ultrasound irradiation causes mechanical perturbation of the cell membrane, allowing the uptake of large molecules in the vicinity of the cavitation bubbles. The collapse of these bubbles generates small transient holes in the cell membrane and induces transient membrane permeabilization. This formation of small pores in the cell membrane using ultrasound allows the transfer of DNA/RNA into the cell. This phenomenon is known as sonoporation and is a gene delivery method that shows great promise as a potential new approach in gene therapy. Microbubbles lower the threshold of cavity formation. Complexes of therapeutic genes and microbubbles improve the transfer efficiency of genes. Diagnostic ultrasound is potentially a suitable sonoporator because it allows the real-time monitoring of irradiated fields.

Methylprednisolone Pulse Therapy in Dermatomyositis

A high dose of methylprednisolone (pulse therapy) was given to 3 patients with uncontrolled dermatomyositis (DM) accompanied by severe muscle weakness. The effects of pulse therapy were compared with findings in 5 patients with severe DM who were treated with the usual dose of prednisolone (controls). The pulse therapy led to an improvement in the clinical symptoms in the 3 patients with uncontrolled DM, who were in danger of aspiration pneumonia. A rapid reduction in muscle enzyme levels in this group was also evident.

Survival of Primary Human Hepatocytes and Death of Induced Pluripotent Stem Cells in Media Lacking Glucose and Arginine

Background Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. Materials and Methods Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. Results The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. Conclusion Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.

Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

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.

Incidence of symptomatic vertebral fractures in women of childbearing age newly treated with high-dose glucocorticoid

BACKGROUND The treatment and prevention of glucocorticoid (GC)-induced osteoporosis have been controversial in premenopausal women during their childbearing years. OBJECTIVE This study assessed the incidence and risk factors for symptomatic vertebral fracture in women of childbearing age newly treated with high-dose GC. METHODS An observational cohort study was conducted at the rheumatic center of Shimoshizu National Hospital in Chiba, Japan, from 1986 to 2006. The prevalence of symptomatic vertebral fractures, as determined by x-rays, was assessed in premenopausal (aged <50 years) women with collagen vascular disease newly treated with high-dose GC (> or =20 mg/d prednisolone equivalent) compared with their counterparts who did not receive GC. Differences in the incidences of vertebral fractures were compared between groups by the Kaplan-Meier method and evaluated by the log-rank test. Hazard ratios (HRs) with 95% CIs were estimated using the Cox proportional hazards regression model. RESULTS A total of 373 women were assessed: 292 patients in the high-dose GC treatment group (mean [SD] initial age, 32.4 [8.2] years; initial dose, 43.8 [14.9] mg/d; follow-up time, 124.2 [75.4] months) and 81 patients in the non-GC control group (initial age, 39.3 [7.8] years; follow-up time, 106.5 [79.7] months). Symptomatic vertebral fractures occurred more frequently in the high-dose GC group (11.3%) than in the non-GC group (1.2%). Using the Cox model, the adjusted HR for the high-dose GC group was 13.96 (95% CI, 1.87-104.22) relative to the non-GC group. In the high-dose GC group, Kaplan-Meier analyses revealed that the incidence of fractures in women in their forties was significantly higher in comparison with those in their twenties (P < 0.001) and thirties (P < 0.05), and that the incidence of fractures in those who consumed alcohol (>80 g/wk of pure alcohol) was significantly higher than in those who did not (P < 0.05). The Cox model also revealed that the risk was independently higher with every 10-year increment of initial age (HR = 2.27; 95% CI, 1.46-3.53), with every GC dose increase (HR = 2.28; 95% CI, 1.58-3.31), and with each 1-gram decrease of cumulative GC dose (HR = 0.95; 95% CI, 0.93-0.98). CONCLUSIONS In this study, high-dose GC use was associated with a significantly high prevalence of symptomatic vertebral fractures in premenopausal women with collagen vascular disease during their childbearing years. However, the fracture risk was relatively low in women of childbearing age, especially those in their twenties and thirties during the early years of treatment.

Reduced hemoglobin and increased C-reactive protein are associated with upper gastrointestinal bleeding

AIM To investigate the early upper gastrointestinal endoscopy (endoscopy) significantly reduces mortality resulting from upper gastrointestinal (GI) bleeding. METHODS Upper GI bleeding was defined as 1a, 1b, 2a, and 2b according to the Forrest classification. The hemoglobin (Hb), and C-reactive protein (CRP) were examined at around the day of endoscopy and 3 mo prior to endoscopy. The rate of change was calculated as follows: (the result of blood examination on the day of endoscopy - the results of blood examination 3 mo prior to endoscopy)/(results of blood examination 3 mo prior to endoscopy). Receiver operating characteristic curves were created to determine threshold values. RESULTS Seventy-nine men and 77 women were enrolled. There were 17 patients with upper GI bleeding: 12 with a gastric ulcer, 3 with a duodenal ulcer, 1 with an acute gastric mucosal lesion, and 1 with gastric cancer. The area under the curve (AUC), threshold, sensitivity, and specificity of Hb around the day of endoscopy were 0.902, 11.7 g/dL, 94.1%, and 77.1%, respectively, while those of CRP were 0.722, 0.5 mg/dL, 70.5%, and 73%, respectively. The AUC, threshold, sensitivity, and specificity of the rate of change of Hb were 0.851, -21.3%, 76.4%, and 82.6%, respectively, while those of CRP were 0.901, 100%, 100%, and 82.5%, respectively. CONCLUSION Predictors for upper GI bleeding were Hb < 11.7 g/dL, reduction rate in the Hb > 21.3% and an increase in the CRP > 100%, 3 mo before endoscopy.

Elevated levels of alanine transaminase and triglycerides within normal limits are associated with fatty liver.

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.