Dong-Hyuk Sheen

Genome-Wide Copy Number Variation Analysis Identifies Deletion Variants Associated With Ankylosing Spondylitis

To identify ankylosing spondylitis (AS)–associated copy number variations (CNVs) in Korean subjects and their synergistic roles in the development of AS.

Association of FOXJ1 polymorphisms with systemic lupus erythematosus and rheumatoid arthritis in Korean population

The forkhead-box J1 (FOXJ1) transcription factor could suppress a spontaneous activation of T cells and B cells through an induction of IκBβ that results in repression of NF-κB activity. In Foxj1 deficiency mice, systemic autoimmune inflammation is quite common symptom. Therefore, deregulated Foxj1 is supposed to be associated with autoimmune diseases and/or other inflammatory diseases. Previously, we identified that polymorphisms of human FOXJ1 gene (g.-460C>T, g.1805G>T and g.3375G>C) are associated with allergic rhinitis in a Korean population. In present study, we compared the genotype and allele frequencies of these SNPs between healthy controls and systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) patients. We also investigated the relationships between each genotype and the expression levels of anti-nuclear antibodies in SLE patients, and rheumatoid factor and anti-cyclic citrullinated peptide in RA patients. The frequencies of haplotypes constructed by these FOXJ1 SNPs were compared between controls and SLE (or RA) patients. The results of genotype and allele analysis showed that the prevalence of polymorphism g.3375G>C was associated with the susceptibility of SLE (P = 0.0072 and 0.0042, respectively). But no significant association was found with RA. In the haplotype analysis, however, the main CGG showed a weak association between controls and RA patients (P = 0.048).

Associations of Vitamin D Binding Protein Gene Polymorphisms with the Development of Peripheral Arthritis and Uveitis in Ankylosing Spondylitis

Objective. Genetic factors account for more than 90% of overall susceptibility to ankylosing spondylitis (AS), and recent studies have focused on non-major histocompatibility complex genes. Vitamin D binding protein (DBP) is a highly polymorphic protein that transports vitamin D and its metabolites. In addition to its sterol binding capacity, DBP has many other roles in the inflammatory and immune systems, and has been reported to be associated with autoimmune diseases. We investigated the association between DBP polymorphisms and susceptibility to AS. Methods. This case-control study was conducted in 223 patients with AS and 239 ethnically matched controls who were genotyped for 8 single-nucleotide polymorphisms (SNP) in the DBP and its promoter. Genomic DNA was isolated from peripheral blood leukocytes using the standard phenolchloroform method, and the GoldenGate assay was used for genotyping. Results. No significant association was found between the susceptibility to AS and DBP polymorphisms. In a subgroup analysis of patients with AS, G alleles at rs222016 and rs222020 (OR 0.63, 95% CI 0.42–0.95, p = 0.03; OR 0.63, 95% CI 0.42–0.95, p = 0.03, respectively) and A allele at rs3733359 (OR 0.59, 95% CI 0.39–0.90, p = 0.01) showed the decreased risk of peripheral arthritis. G allele at rs4752 showed increased risk of uveitis (OR 2.04, 95% CI 1.12–3.72, p = 0.02). On the haplotype analyses, haplotype 2 (AGGA) protected against the development of peripheral arthritis (p = 0.01) and haplotype 3 (GAAG) was associated with an increased likelihood of uveitis (p = 0.02). Conclusion. DBP gene polymorphisms are associated with the development of peripheral arthritis and uveitis in Korean patients with AS. Given the influence of different DBP variants on the immune system, larger-scale studies are warranted to elucidate the role of DBP in the pathogenesis of AS.

Anti-Cyclic Citrullinated Peptide Antibodies Distinguish Hepatitis B Virus (HBV)-associated Arthropathy from Concomitant Rheumatoid Arthritis in Patients with Chronic HBV Infection

Objective. To determine whether anti-cyclic citrullinated peptide (anti-CCP) antibodies, which are a highly specific test for rheumatoid arthritis (RA), could differentiate between hepatitis B virus (HBV)-associated arthropathy and concomitant RA in Korean patients with chronic HBV infection. Methods. We investigated 240 patients with HBV infection. Anti-CCP antibodies were measured by ELISA and rheumatoid factor (RF) by the latex fixation test. Patient records were reviewed, and a standard form was used to record all demographic, clinical, and laboratory characteristics. Patients were divided into 4 groups according to joint symptoms: asymptomatic, arthralgia, oligoarthritis, and RA. We categorized liver disease into 3 groups: carrier, chronic hepatitis, and cirrhosis. Results. Anti-CCP antibodies and RF were detected in 11 and 28 of 240 patients, respectively. Anti-CCP antibodies were detected in 9 of 10 RA (90%) and 2 of 230 non-RA patients (0.86%). The positive rate for RF was 90% in RA and 8.3% in non-RA. Eight of 10 RA patients were positive for both RF and anti-CCP antibodies. RF was detected in 11 patients without joint symptoms, 4 with arthralgia, and 4 with oligoarthritis, whereas anti-CCP antibodies were found in 1 patient without joint symptoms and 1 with oligoarthritis. Specificity of anti-CCP antibody for RA was 99.1%, whereas RF showed 91.7% specificity (p < 0.0002). We compared the titers and positive detection rates of anti-CCP antibodies and RF among liver disease subgroups. There was no significant between–subgroup difference. Conclusion. Measurement of anti-CCP antibodies is better than RF detection to discriminate HBV-associated arthropathy from concomitant RA in patients with chronic HBV infection.

Anti-cyclic citrulline peptide antibody in non-tuberculous mycobacteria sera: a negative association

Dear Editor, We read with great interest the article by Guedes-Barbosa [1] titled “Anticitrulline peptide antibodies (anti-CCP) in leprosy sera: a negative association.” They emphasized high specificity of anti-CCP for the diagnosis of rheumatoid arthritis (RA); however, anti-CCP has been less well studied in many other diseases [2]. Recent studies showed 0–32% anti-CCP positive in patients with active pulmonary tuberculosis [3–5]. Guedes-Barbosa [1] reported two patients positive, among 64 patients, with leprosy [1]. Various musculoskeletal manifestations by non-tuberculous mycobacteria (NTM), such as tenosynovitis, arthritis, spondylitis, and so on, are difficult to distinguish between infections associated arthropathy and concomitant RA. To date, anti-CCP in NTM has not been studied. The aim of the present study was to determine the prevalence anti-CCP in patients with NTM. The study population consisted of 35 patients with NTM pulmonary diseases satisfying the clinical, radiographic, and microbiological diagnostic criteria published by the American Thoracic Society [6]. Blood samples were collected before starting treatment. All the subjects were recruited from Eulji University Hospital and Asan Medical Center. Ethics approval for study was obtained. Acid-fast bacilli staining was done using Ziehl–Neelson staining and culture using egg-based Ogawa medium. The cultured colonies were identified as Mycobacterium tuberculosis or NTM using the AccuProbe test (GenProbe Inc., San Diego, USA) or duplex PCR kit (M&D, Wonju, Korea). The species of NTM was identified using a polymerase chain reaction–restriction fragment length polymorphism method, based on the rpoB gene [7]. AntiCCP was determined by enzyme-linked immunosorbent assay using DIASTAT anti-CCP kit (MBL Co., Nagoya, Japan) and read by automated EIA analyzer, CODA (BioRAD Co., Japan). According to the manufacturer’s instructions, anti-CCP was considered positive when the absorbance was higher than the cutoff value (5 U/ml). RF was measured by the latex fixation test using Hitachi 7170 S (Hitachi Co., Tokyo, Japan). The cutoff value for positivity was 18 IU/ml. All patients had pulmonary manifestations, but no joint symptoms. Serological HIV tests were performed in all subjects and all with negative results. We could find no patients with anti-CCP positive, whereas, five patients (14%) were positive for RF (Table 1). The average titers of anti-CCP and RF were 1.06±0.54 and 14±5.22 IU/ml, respectively. Among five patients with RF+ (one female and four males), two patients were infected by MycobacM.-K. Lim :D.-H. Sheen : S.-C. Shim (*) Division of Rheumatology, Department of Internal Medicine, School of Medicine and Medical Sciences Research Institute, Eulji University, Dunsandong 1306, 302-799 Daejeon, South Korea e-mail: ssc@eulji.ac.kr

Exosomal amyloid A and lymphatic vessel endothelial hyaluronic acid receptor-1 proteins are associated with disease activity in rheumatoid arthritis

BackgroundExosomes are thought to play an important role in exchanging information between cells. The proteins and lipids in exosomes play roles in mediating inflammatory and autoimmune diseases. The aim of this study was to identify exosomal candidate proteins that are related to other inflammatory parameters in rheumatoid arthritis (RA).MethodsThe study population consisted of 60 patients with RA: 30 in the clinical remission (CR) group with a Disease Activity Score in 28 joints based on erythrocyte sedimentation rate (DAS28-ESR) ≤2.6 and 30 in the non-clinical remission (non-CR) group with a DAS28-ESR >2.6. Preparation of exosomes from patient serum samples was performed with the ExoQuick kit, and protein identification/quantification was performed using tandem mass tag labeling/mass spectrometry and an enzyme-linked immunosorbent assay. Comparisons between groups were made using Student’s t test or the Mann-Whitney U test, as appropriate. Spearman’s correlation coefficients (ρ) were calculated.ResultsWe identified six candidate proteins. Exosomal levels of amyloid A (AA) and lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) differed between the CR and non-CR groups. Both serum and exosomal AA levels were higher in the non-CR group than in the CR group (p = 0.001). Significant positive correlations were found between exosomal AA and C-reactive protein (CRP) as well as between serum AA and CRP (ρ = 0.614, p = 0.001, and ρ = 0.624, p = 0.001, respectively). Although serum levels of LYVE-1 did not differ between the non-CR and CR groups, exosomal levels of LYVE-1 were lower in the non-CR group than in the CR group (p = 0.01). We identified positive correlations between serum/exosomal LYVE-1 and CRP only in the non-CR group (serum ρ = 0.376, p = 0.04; exosome ρ = 0.545, p = 0.002).ConclusionsExosomal LYVE-1 shows potential for use as an additional marker of disease activity in patients with RA, and exosomes may carry other useful markers for RA.

Proteomics Analysis for Verification of Rheumatoid Arthritis Biomarker Candidates Using Multiple Reaction Monitoring

Rheumatoid arthritis (RA) is an autoimmune disease in which autoantibodies attack the synovial membrane, causing joint inflammation. Blood tests would offer a powerful, minimally invasive method for early diagnosis of RA. However, no reliable biomarkers for RA are presently available. The aim is to develop biomarkers for RA by multiple reaction monitoring (MRM)‐based quantification of candidate biomarkers.

Long-term Treatment with Anti-platelet Agents for Collagen-induced Arthritis Improves Radiological Findings

Objectives The objectives of this study were to evaluate the long-term effect of anti-platelet treatment on the radiological progression of collagen-induced arthritis in rats. Methods Female Lewis rats with collagen-induced arthritis were divided into three experimental groups: saline, aspirin monotherapy (n = 12), and aspirin–clopidogrel dual therapy (n = 12). Drugs were administered daily and continued up to 70 days after the induction of arthritis. The clinical arthritis index (weight, morphology score, and paw thickness) and radiological scores were evaluated. Results The clinical arthritis index peaked on day 20, while the radiological scores peaked on day 35. No intergroup difference was observed in the clinical arthritis index throughout the experiment. The aspirin–clopidogrel dual therapy group had a significantly higher mean radiological score than the other groups (p = 0.045) on day 35. Further treatments resulted in significantly improved radiological findings in the aspirin monotherapy and aspirin–clopidogrel dual therapy groups on day 70 but no significant improvement in the saline group. Conclusion Anti-platelet agent treatment improved radiological findings on day 70. These observations emphasize the importance of a future long-term study of the effects of anti-platelet agent treatment on arthritis.

THU0438 Peptidylarginine Deiminase Polymorphisms Associated with Susceptibility to Tuberculosis

Background Anti–cyclic citrullinated peptide antibodies (as a serologic marker of RA) bind to citrulline residue which is now considered to play a pivotal role in various human diseases such as multiple sclerosis, various tumors, and many other infectious diseases. The hypothesis of the association between PADI4 gene and TB susceptibility was introduced, but didn’t identified. Therefore, we hypothesized that the diversity in PADI4 genes could affect susceptibility to tuberculosis in RA patients and preliminarily designed the association study of PADI4 polymorphisms in the patients with mycobacteria infection. Objectives To examine the possibility for single nucleotide polymorphisms of peptidylarginine deiminase 4 to discriminate who’s susceptible to active tuberculosis disease. Methods The study population consisted of 47 patients with bacteriological-confirmed tuberculosis, 35 patients with non-tuberculous mycobacterial disease, 50 rheumatoid arthritis patients and 83 healthy controls. Blood samples were collected before starting treatment for TB and NTM. All subjects were genotyped for three SNPs in PADI4, namely PADI489, PADI490, and PADI4104. In addition, antCCP antibodies were determined by enzyme-linked immunosorbent assay and rheumatoid factor was measured by the latex fixation test. Results In TB patients, the AG and CT genotypes for PADI4_89 and PADI490, respectively were significantly lower than those of control subjects. Adjusted odd ratiosfor PADI489 and PADI490 were 0.34 (p = 0.005). The frequencies of haplotypes and minor allele were not significantly different among groups. IgM RF-positive was observed in 42 patients with RA (84%), in 7 patients with TB (15.0%), in 5 patients with NTM (14%), and in 5 healthy controls (6%) (p < 0.0001). There were significant differences in the frequencies of the presence of antiCCP antibodies among groups: RA (88%), TB (8%), NTM (0%), and healthy controls (1%) (p < 0.0001) Conclusions This study showed that polymorphisms of PADI were associated with susceptibility to TB in a Korean population References Baronnet L, Barnetche T, Kahn V, Lacoin C, Richez C, Schaeverbeke T. Incidence of tuberculosis in patients with rheumatoid arthritis. A systematic literature review. Joint, bone, spine : revue du rhumatisme. 2011 May;78(3):279-84. PubMed PMID: 21273108. Epub 2011/01/29. eng. Dixon WG, Hyrich KL, Watson KD, Lunt M, Galloway J, Ustianowski A, et al. Drug-specific risk of tuberculosis in patients with rheumatoid arthritis treated with anti-TNF therapy: results from the British Society for Rheumatology Biologics Register (BSRBR). Annals of the rheumatic diseases. 2010 Mar;69(3):522-8. PubMed PMID: 19854715. Pubmed Central PMCID: 2927681. Epub 2009/10/27. eng. Seong SS, Choi CB, Woo JH, Bae KW, Joung CL, Uhm WS, et al. Incidence of tuberculosis in Korean patients with rheumatoid arthritis (RA): effects of RA itself and of tumor necrosis factor blockers. The Journal of rheumatology. 2007 Apr;34(4):706-11. PubMed PMID: 17309133. Epub 2007/02/20. eng. Disclosure of Interest None Declared