P. Fardellone

Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using citrullinated and non-citrullinated recombinant proteins as antigens are highly diagnostic for rheumatoid arthritis

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti‐filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non‐RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow‐up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti‐keratin autoAb (AKA) and anti‐perinuclear factor (APF) were tested; for group 2, anti‐CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non‐RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58·4% and 30·7%, and specificity for RA was 99·5% and 98·4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92·1%), APF (95·2%), AKA (96·8%) and anti‐CCP (95·2%). dACRF positive predictive value was high (98·2) and close to that given by the concomitant positivity of RF and anti‐CCP autoAb. Despite a high positive correlation between AKA, APF, anti‐CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti‐citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serums reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.

Diagnostic and prognostic values of anti glucose-6-phosphate isomerase antibodies in community-recruited patients with very early arthritis.

The objective of this study was to determine the diagnostic and prognostic values of antiglucose‐6‐phosphate isomerase (GPI) antibodies in patients with very early arthritis. Anti‐GPI antibodies were measured by ELISA using purified GPI from rabbit muscle in: (i) 383 sera from healthy blood donors (n = 120), well‐established rheumatoid arthritis (RA) (n = 99) and non‐RA differentiated arthritis (NRADA) (n = 164) patients; (ii) 195 sera obtained from community‐recruited patients with very early inflammatory arthritis (VErA cohort) that were studied for 1 year and classified as having RA (n = 116), NRADA (n = 41), and undifferentiated arthritis (UA) (n = 38) after the follow‐up period. The criterion for severity was the progression of radiographic damage. Prevalence of anti‐GPI antibodies was significantly higher in well‐established RA patients (45·4%) compared to healthy subjects (2·5%). Anti‐GPI antibodies were also present in sera from NRADA: systemic lupus erythematosus 53%, polymyositis 45·4%, adult‐onset Stills disease 44%, systemic sclerosis 42·8%, spondylarthropathies 25% and primary Sjögren’s syndrome 5·8%. No significant association was found between the presence of anti‐GPI antibodies and the 3 diagnostic groups from the VErA cohort. No correlation was observed between anti‐GPI and autoantibodies usually associated with RA. Anti‐GPI antibodies were not predictive of radiological progression in patients with very early arthritis. Thus, anti‐GPI antibodies are not useful for discriminating RA from non‐RA rheumatic diseases and do not constitute a predictive factor of structural damage.

Epistatic Interaction between BANK1 and BLK in Rheumatoid Arthritis: Results from a Large Trans-Ethnic Meta-Analysis

Background BANK1 and BLK belong to the pleiotropic autoimmune genes; recently, epistasis between BANK1 and BLK was detected in systemic lupus erythematosus. Although BLK has been reproducibly identified as a risk factor in rheumatoid arthritis (RA), reports are conflicting about the contribution of BANK1 to RA susceptibility. To ascertain the real impact of BANK1 on RA genetic susceptibility, we performed a large meta-analysis including our original data and tested for an epistatic interaction between BANK1 and BLK in RA susceptibility. Patients and Methods We investigated data for 1,915 RA patients and 1,915 ethnically matched healthy controls genotyped for BANK1 rs10516487 and rs3733197 and BLK rs13277113. The association of each SNP and RA was tested by logistic regression. Multivariate analysis was then used with an interaction term to test for an epistatic interaction between the SNPs in the 2 genes. Results None of the SNPs tested individually was significantly associated with RA in the genotyped samples. However, we detected an epistatic interaction between BANK1 rs3733197 and BLK rs13277113 (Pinteraction = 0.037). In individuals carrying the BLK rs13277113 GG genotype, presence of the BANK1 rs3733197 G allele increased the risk of RA (odds ratio 1.21 [95% confidence interval 1.04–1.41], P = 0.015. Combining our results with those of all other studies in a large trans-ethnic meta-analysis revealed an association of the BANK1 rs3733197 G allele and RA (1.11 [1.02–1.21], P = 0.012). Conclusion This study confirms BANK1 as an RA susceptibility gene and for the first time provides evidence for epistasis between BANK1 and BLK in RA. Our results illustrate the concept of pleiotropic epistatic interaction, suggesting that BANK1 and BLK might play a role in RA pathogenesis.

Determination and Modulation of Total and Surface Calcium-Sensing Receptor Expression in Monocytes In Vivo and In Vitro

Expression of the calcium-sensing receptor (CaSR) has previously been demonstrated in human circulating monocytes (HCM). The present study was designed to measure CaSR expression in HCM and to examine its potential modulation by pro-inflammatory cytokines, Ca2+, vitamin D sterols in U937 cell line. Twenty healthy volunteers underwent blood sampling with subsequent isolation of peripheral blood mononuclear cells (PBMC) at 3 visits. Flow cytometry analysis (FACS) was performed initially (V1) and 19 days later (V2) to examine intra- and intersubject fluctuations of total and surface CaSR expression in HCM and 15 weeks later (V3) to study the effect of vitamin D supplementation. In vitro experiments were conducted to assess the effects of pro-inflammatory cytokines, calcidiol, calcitriol and Ca2+ on CaSR expression in U937 cell line. By FACS analysis, more than 95% of HCM exhibited cell surface CaSR staining. In contrast, CaSR staining failed to detect surface CaSR expression in other PBMC. After cell permeabilization, total CaSR expression was observed in more than 95% of all types of PBMC. Both total and surface CaSR expression in HCM showed a high degree of intra-assay reproducibility (<3%) and a moderate intersubject fluctuation. In response to vitamin D supplementation, there was no significant change for both total and surface CaSR expression. In the in vitro study, U937 cells showed strong total and surface CaSR expression, and both were moderately increased in response to calcitriol exposure. Neither total nor surface CaSR expression was modified by increasing Ca2+ concentrations. Total CaSR expression was concentration dependently decreased by TNFα exposure. In conclusion, CaSR expression can be easily measured by flow cytometry in human circulating monocytes. In the in vitro study, total and surface CaSR expression in the U937 cell line were increased by calcitriol but total CaSR expression was decreased by TNFα stimulation.

AB0128 Total Calcium-Sensing Receptor Expression in Circulating Monocytes is Increased in Rheumatoid Arthritis Patients with Severe Coronary Artery Calcification

Background Human circulating monocytes express the calcium-sensing receptor (CaSR) and are involved in atherosclerosis. Vascular calcification is commonly used as a subclinical marker of atherosclerosis and has been linked to increased all-cause mortality, cardiovascular mortality and coronary events. Moreover, patients with RA are known to develop early-onset, widespread calcification in various vascular beds. We have previously assessed CaSR expression by flow cytometry in human circulating monocytes and have provided evidence that this type of approach could be useful in certain clinical situations, in which changes in CaSR expression can be expected. Objectives This study investigated the potential association between vascular calcification in rheumatoid arthritis (RA) and CaSR expression in circulating monocytes. Methods In this cross-sectional study, 50 RA patients were compared to 25 control subjects matched for age and gender. Isolation of peripheral blood mononuclear cells and flow cytometry analysis were performed to study the surface and total CaSR expression in circulating monocytes. Coronary artery calcium (CAC) and abdominal aortic calcification (AAC) scores were evaluated by computed tomography and an association between these scores and the surface and/or total CaSR expression in circulating monocytes in RA patients was investigated. Results The two groups were similar in terms of age (RA: 60.9±8.3 years, versus controls: 59.6±5.3 years) and gender (RA: 74.0% females versus 72.0% females). RA patients did not present a higher prevalence or a greater burden of CAC or AAC compared to age- and gender-matched controls. When compared with control subjects, RA patients did not exhibit greater total CaSR (101.6% ±28.8 vs. 99.9% ±22.0) or surface CaSR (104.6% ±20.4 vs. 99.9% ±13.7) expression, but total CaSR expression in circulating monocytes was significantly higher in RA patients with severe CAC (Agatston score ≥200, n=11) than in patients with mild-to-moderate CAC (1-199, n=21) (p=0.01). Conclusions This study demonstrates, for the first time, that total CaSR expression in human circulating monocytes is increased in RA patients with severe coronary artery calcification. References Paccou J, Brazier M, Mentaverri R, et al. Vascular calcification in rheumatoid arthritis: prevalence, pathophysiological aspects and potential targets. Atherosclerosis 2012;224:1418-23. Paccou J, Boudot C, Mary A, et al. Determination and modulation of total and surface calcium-sensing receptor expression in monocytes in vivo and in vitro. PLoS one 2013;8:e74800. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2377

Biological Effects of Supplementation with Vitamin D and Calcium in Postmenopausal Women with Low Bone Mass Receiving Alendronate

ObjectiveTo investigate the biological effects of supplementation with vitamin D and calcium versus supplementation with calcium alone during the first 3 months of treatment with alendronate in postmenopausal women with a risk of fracture and with vitamin D and calcium insufficiency.DesignRandomised, double-blind trial.SubjectsThe study randomised 48 osteopenic and osteoporotic women, mean age 70 ± 6 years and at least 5 years after menopause, who were living at home. Inclusion criteria were low bone mineral density (more than 1 SD below reference value), serum 25-hydroxy-vitamin D3 (25-OHD, calcifediol) <12 μg/L and dietary calcium intake <1 g/day.MethodsThe women were divided into two groups. The first group (n = 23) received alendronate 10mg once daily supplemented with calcium and vitamin D (elemental calcium 500mg, colecalciferol [vitamin D3] 400IU) twice daily for 3 months. The second group (n = 25) received the same dosage of alendronate and a placebo with calcium alone (500 mg/day). Blood, serum and urine samples were obtained for measurement of calcaemia, intact parathyroid hormone (i-PTH) and markers of bone remodelling such as the N- and C-terminal telopeptides of type I collagen (NTX and CTX).ResultsSupplementation with calcium and vitamin D caused a rapid increase of 25-OHD levels without changes in calcaemia or i-PTH levels. In the two groups, serum and urinary CTX and urinary NTX were dramatically and significantly decreased after as little as 15 days of treatment and remained decreased throughout the course of treatment. No significant difference between the two treatments was observed, but the combined treatment resulted in a more pronounced effect as assessed by the Hodge-Lehman test, particularly after 1 month for the bone resorption markers serum CTX (p = 0.064) and urinary NTX (p = 0.076).ConclusionSupplementation with calcium and vitamin D could be appropriate in elderly women with calcium and vitamin D insufficiencies being treated with alendronate in order to achieve rapid reduction of bone remodelling.

Prevalence of treatment of hyperuricemic in patients admitted to the rheumatology ward and evaluation of compliance with the 2012 ACR Guidelines.

Joint Bone Spine - In Press.Proof corrected by the author Available online since mercredi 2 avril 2014

Soluble vascular endothelial (VE) cadherin and autoantibodies to VE-cadherin in rheumatoid arthritis patients treated with etanercept or adalimumab

OBJECTIVES The aim of this study was to investigate the clinical value of sVE and anti-vascular endothelial-cadherin antibodies (AAVE) in RA treated with etanercept or adalimumab combined with methotrexate. METHODS This was an 18-month prospective multicenter study in which patients had active RA, requiring TNF antagonist. sVE rates and AAVE titers were measured respectively by dot blot and ELISA. The relationship of these biomarkers with parameters reflecting articular or systemic disease activity, progression of structural damage, and response or remission to treatment was analyzed. RESULTS Forty-eight patients received TNF blocking agents. Variation of sVE rates were significantly correlated with that of C-reactive protein (CRP) levels at weeks 6, 12, 26 and 52. A significant decrease in sVE levels was observed in the group of patients exhibiting a decrease in CRP levels as compared to the patient group with unmodified CRP. AAVE at baseline were correlated with rheumatoid factor. Kinetics analysis of sVE levels and AAVE titers showed that their level were not associated with disease activity score and to methotrexate/adalimumab or etanercept response. CONCLUSIONS sVE is a biomarker associated with systemic RA activity under anti-TNF. AAVE are related to autoantibodies usually associated to RA.

Intraosseous venous drainage anomaly at the tibia

Joint Bone Spine - In Press.Proof corrected by the author Available online since vendredi 2 juin 2017

AB0025 Evolution of Regulatory B Cells under Treatment by Biologics in Rheumatoid Arthritis

Background In clinical daily practice, the response to biologics is unpredictable in patients with rheumatoid arthritis (RA) and there is a crucial need for biomarkers of response. Objectives The main objective of our study is to describe the evolution of regulatory B cells (Bregs) in patients with RA treated with biologics and to investigate whether there is a correlation between the rate of Bregs and the clinical response. Methods This was a propective single-center pilot study, descriptive and not randomized. Patients were included with RA fulfilling the ACR/EULAR 2010 criteria, with an active disease according to the DAS28 score despite treatment, and in whom initiation or switch of a biologic except rituximab was required. Acontrol group of patients without any autoimmune disease or immune dysfunction was also assessed. B and T lymphocytes whole blood phenotyping, as well as an analysis of the production of IL-10 were performed by multicolor flow cytometry (FCM) in both patients and controls at baseline (M0) and in patients after 1 (M1), 3 (M3) and 6 (M6) months of treatment. CD24hiCD38hi CD24hiCD27+ subpopulations were selected for the phenotypic study of Bregs (24–38 and 24–27 Breg). The primary endpoint was the rate and absolute value of Bregs as a percentage and absolute value measured at each time of the study and compared with disease activity. Results Thirty patients and 15 controls were included in this study. Nowadays, phenotypic analysis was performed at baseline in all patients, but only in 15 patients at M1 and 13 patients at M3. The complete data will be available for the EULAR congress. There was an increase in circulating 24–38 Bregs between M1 and M3 (p=0.06) whatever the biologic prescribed. At baseline, the 24–38 Bregs were significantly lowered in patients compared to controls in percentage and absolute value (p=0.001 and p=0.013 respectively). The DAS28 remission and EULAR response at M3 were associated with a quantitative increase of 24–38 and 24–27 Breg at baseline compared to the values of the non-responders patients. Thus, a high initial rate of 24–27 and 24–38 Breg seemed associated with a good clinical response and an EULAR remission at 3 months. The rate of CD19 + B producing IL-10 obtained by PBMC culturing seemed lower in controls as compared to the patients. Conclusions We observed an increase of Bregs under treatment by biologics and a quantitative decrease in Bregs in patients with active RA compared to healthy subjects and patients with inactive disease. Thus, Bregs could be a marker of activity of the disease and their level may represent a potential predictor of the therapeutic response. Full data are required to confirmed these preliminary results. Disclosure of Interest None declared