A GC polymorphism associated with serum 25-hydroxyvitamin D level is a risk factor for hip fracture in Japanese patients with rheumatoid arthritis: 10-year follow-up of the Institute of Rheumatology, Rheumatoid Arthritis cohort study

INTRODUCTION: Vitamin D deficiency has been reported to be common in patients with rheumatoid arthritis (RA) who have a higher prevalence of osteoporosis and hip fracture than healthy individuals. Genetic variants affecting serum 25-hydroxyvitamin D (25(OH)D) concentration, an indicator of vitamin D status, were recently identified by genome-wide association studies of Caucasian populations. The purpose of this study was to validate the association and to test whether the serum 25(OH)D-linked genetic variants were associated with the occurrence of hip fracture in Japanese RA patients. METHODS: DNA samples of 1,957 Japanese RA patients were obtained from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort DNA collection. First, five single nucleotide polymorphisms (SNPs) that were reported to be associated with serum 25(OH)D concentration by genome-wide association studies were genotyped. The SNPs that showed a significant association with serum 25(OH)D level in the cross-sectional study were used in the longitudinal analysis of hip fracture risk. The genetic risk for hip fracture was determined by a multivariate Cox proportional hazards model in 1,957 patients with a maximum follow-up of 10 years (median, 8 years). RESULTS: Multivariate linear regression analyses showed that rs2282679 in GC (the gene encoding group-specific component (vitamin D binding protein)) locus was significantly associated with lower serum 25(OH)D concentration (P = 8.1 × 10(-5)). A Cox proportional hazards model indicated that rs2282679 in GC was significantly associated with the occurrence of hip fracture in a recessive model (hazard ratio (95% confidence interval) = 2.52 (1.05-6.05), P = 0.039). CONCLUSIONS: A two-staged analysis demonstrated that rs2282679 in GC was associated with serum 25(OH)D concentration and could be a risk factor for hip fracture in Japanese RA patients

Studies of the efficacy and safety of methotrexate at dosages over 8 mg/week using the IORRA cohort database

The maximum dosage of methotrexate (MTX) for treatment of rheumatoid arthritis (RA) formally approved in Japan is 8 mg/week. We intended to examine the efficacy and safety of MTX at dosages over 8 mg/week in Japanese rheumatoid arthritis patients using the large Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort database. Among 9,122 patients registered in the IORRA database from the October 2000 survey to the October 2007 survey, 5,201 patients who had been treated with MTX were selected. We attempted to overcome the drawbacks innate to nonrandomized studies by using longitudinal analyses and multifactorial logistic regression analyses. Cross-sectional analysis of data obtained from the October 2007 survey indicated that dosages of MTX higher than 8 mg/week were used in 27.5% of patients treated with MTX. Longitudinal analyses based on data from three consecutive phases showed that final Disease Activity Score-28 (DAS28) values were significantly lower [n = 260, mean difference 0.563, 95% confidence interval (CI) 0.438–0.688, P < 2.2 × 10−22, two-sided paired t test] than initial values when MTX was increased from 8 mg/week or lower to over 8 mg/week. In addition, longitudinal analyses based on data from two consecutive phases indicated decreases in DAS28 values of 0.26 ± 1.04 (n = 690, P = 6.78 × 10−11, two-sided paired t test) when MTX dosages were increased from 8 mg/week or lower to over 8 mg/week, compared with decreases of 0.07 ± 0.89 (n = 2,125, P = 0.000307) when the dosage was maintained at 8 mg/week. The decreases in DAS28 values were significantly larger in the former than the latter (P = 2.27 × 10−6, two-sided unpaired t test). Concerning safety of MTX at dosages over 8 mg/week, we performed logistic regression analysis in which the objective variable was the existence or nonexistence of self-reported side-effects and the explanatory variable was the MTX dosage in the former phase, with adjustments made for age, sex, body mass index (BMI), steroid administration, folic acid administration, concomitant pulmonary diseases, and renal dysfunction. The results indicated that MTX dosages over 8 mg/week did not have any association with either severe or severe + moderate side-effects. These data regarding both efficacy and safety of MTX at dosages over 8 mg/week in Japanese RA patients would provide the basis for use of the drug at dosages currently not formally approved by the Japanese government

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury

AbstractImpairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified.In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions

Evaluation of oral immunotherapy efficacy and safety by maintenance dose dependency: A multicenter randomized study

Background Generally, oral immunotherapy (OIT) aims for daily administration. Recently, the efficacy of treatment with OIT at a low dose has been reported. However, the optimal dose and the evaluation of dose-dependent OIT outcome have not been described. Methods A multicenter, parallel, open-labeled, prospective, non-placebo controlled, randomized study enrolled 101 Japanese patients for treatment with OIT. We hypothesized that target dose OIT would induce short-term unresponsiveness (StU) earlier than reduced dose OIT. StU was defined as no response to 6200 mg whole egg, 3400 mg milk, and 2600 mg wheat protein, as evaluated by oral food challenge after 2-week ingestion cessation. To compare the two doses of OIT efficacy, the maximum ingestion doses during the maintenance phase of OIT were divided into 100%-dose or 25%-dose groups against their target StU dose, respectively. A total of 51 patients were assigned to the 100%-dose group [hen's egg (HE) = 26, cow's milk (CM) = 13, wheat = 12] and 50 to the 25%-dose group (HE = 25, CM = 13, wheat = 12). Primary outcome was established by comparing StU at 1 year. Secondary outcome was StU at 2 years and established by comparing allergic symptoms and immunological changes. Results The year 1 StU rates (%) for the 100%- and 25%-dose groups were 26.9 vs. 20.0 (HE), 7.7 vs. 15.4 (CM), and 50.0 vs. 16.7 (wheat), respectively. The year 2 StU rates were 30.8 vs. 36.0 (HE), 7.7 vs. 23.1 (CM), and 58.3 vs. 58.3 (wheat), respectively. There were no statistically significant differences in StU between years 1 and 2. The total allergic symptom rate in the 25%-dose group was lower than that in the 100%-dose group for egg, milk, and wheat. Antigen-specific IgE levels for egg-white, milk, and wheat decreased at 12 months. Conclusions Reduced maintenance dose of egg OIT showed similar therapeutic efficacy to the target dose. However, we were not able to clearly demonstrate the efficacy, particularly for milk and wheat. Reducing the maintenance dose for eggs, milk, and wheat may effectively lower the symptoms associated with their consumption compared to the target OIT dose. Furthermore, aggressive reduction of the maintenance dose might be important for milk and wheat, compared to the 25%-dose OIT

Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells

Genome-Wide Association Study and Gene Expression Analysis Identifies CD84 as a Predictor of Response to Etanercept Therapy in Rheumatoid Arthritis

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (ΔDAS) in the etanercept subset of patients (P = 8×10-8), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3′ UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1×10-11 in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better ΔDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry. © 2013 Cui et al

Genetics of rheumatoid arthritis contributes to biology and drug discovery

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological datasets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA)1. Here, we performed a genome-wide association study (GWAS) meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ~10 million single nucleotide polymorphisms (SNPs). We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 1012–4. We devised an in-silico pipeline using established bioinformatics methods based on functional annotation5, cis-acting expression quantitative trait loci (cis-eQTL)6, and pathway analyses7–9 – as well as novel methods based on genetic overlap with human primary immunodeficiency (PID), hematological cancer somatic mutations and knock-out mouse phenotypes – to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery