Background: The randomized controlled NORD-STAR trial demonstrated remission rates of 40-60% in patients with early rheumatoid arthritis (RA) at 24 and 48 weeks according to the Clinical Disease Activity Index (CDAI) when treated early with methotrexate in combination with prednisolone (active conventional therapy (ACT)), certolizumab pegol (CZP), abatacept (ABA) or tocilizumab (TCZ) (1, 2). However, clinical remission criteria reflect in part subjective measures of pain and well-being and not necessarily inflammatory activity. Ultrasound is an imaging tool that aims to measure joint and tendon inflammation objectively. In the NORD-STAR trial, a subset of patients in Norway and Sweden was assessed by ultrasound of joints and tendons. Objectives: To compare ultrasound remission rates at week 48 in patients receiving active conventional treatment versus each of the three biological treatments in early RA and explore the effect of conventional and biological DMARDs on ultrasound-measured inflammation. Methods: NORD-STAR is an investigator-initiated, randomized, blinded-assessor study. Treatment-naïve early RA patients with moderate-severe disease activity were randomized 1:1:1:1 to methotrexate combined with: 1) oral prednisolone (tapered quickly; discontinued at week 36); 2) CZP; 3) ABA or 4) TCZ. Patients in Norway (all 5 sites) and at Karolinska University Hospital in Sweden were assessed with ultrasound of joints/tendons and ultrasound scores were calculated according to the USRA9 score, including assessment of 8 joints (MCP 1-3, PIP 2-3, radiocarpal, MTP2-3) and 1 tendon (extensor carpi ulnaris) bilaterally with a 0-3 score for grey scale and power Doppler, respectively. Ultrasound remission was defined as Doppler sum score=0. Remission rates (w48) were assessed without imputation of missing values and compared between treatment arms using logistic regression adjusted for multiple testing by the Dunnett's procedure and adjusted for age, gender, center, anti-CCP status at baseline and baseline value for the dependent variable. Results: Of the total of 812 patients in the NORD-STAR trial, 239 patients were included in the ultrasound analyses (Norway n=114, Sweden n=125). Patient characteristics were well balanced across the treatment arms. The patients were representative for the whole NORD-STAR population (1,2). Ultrasound remission rates at week 48 were 74 % for ACT, 94 % for CZP, 90 % for ABA and 87 % for TCZ (Table 1, Figure 1). In the primary analysis (adjusted logistic regression), ultrasound remission rates were higher with borderline statistical significance for CZP vs ACT, OR (95%CI) 5.46 (1.33, 22.52) p=0.05and ABA vs ACT OR 4.51 (1.27, 16.00) p=0.06 and not significant for TCZ vs ACT, OR 2,26 (0.71, 7.19) p=0.40. More patients achieved ultrasound remission compared to clinical remission in all treatment arms at 48 weeks. Patients in ultrasound remission, but not in clinical remission (n=56) had higher tender joint counts (8.5 vs 0.6), patient's global (28.1 vs 4.8) and physician's global (10.8 vs 1.3) scores than patients who were in both ultrasound and clinical remission. Swollen joint count (1.0 vs 0.1), ESR (9.3 vs 10.7) and CRP (2.6 vs 2.5) were similar in both groups. Conclusion: A large proportion of patients with early RA achieved ultrasound remission in the NORD-STAR trial. Higher remission rates were achieved in the biologic treatment arms compared to active conventional treatment which is in line with the clinical results. Substantially more patients achieved ultrasound remission than CDAI remission. REFERENCES: [1] Hetland et al. BMJ 2020;371:m4328. [2] Østergaard M et al. Ann Rheum Dis. 2023;82(10):1286-1295. Table 1. Demographics/baseline (BL) characteristics and 48 weeks results. Values are mean (SD), if not otherwise indicated. Acknowledgements: NIL. Disclosure of Interests: Marte S Heiberg: None declared, Yogan Kisten: None declared, Amirhossein Kazemi: None declared, Hamed Rezaei: None declared, Erik af Klint: None declared, Maud-Kristine A Ljosa: None declared, Eli Brodin: None declared, David Stevens: None declared, Gunnstein Bakland UCB, Lars Fridtjof Karoliussen: None declared, Pernille Bolton-King: None declared, Joakim Lindqvist: None declared, Kristina Lend: None declared, Jon Lampa: None declared, Till Uhlig: None declared, Merete Lund Hetland MLH received research grants from AbbVie, Biogen, BMS, Celtrion, Eli Lily, Janssen Biologics B.V., Lundbeck Foundation, MSD, Pfizer, Roche, Samsung Biopies, Sandoz and Novartis; and institution pay from Pfizer, Medac, AbbVie and Sandoz; chaired the steering committee of the Danish Rheumatology Quality Registry (DANBIO), which receives public funding from the hospital owners and funding from pharmaceutical companies; cochairs EuroSpA, which generates real-world evidence of treatment of psoriatic arthritis and axial spondylorthritis based on secondary data and is partly funded by Novartis., Anna Rudin: None declared, Dan Nordström DCN received consulting fees from AbbVie, BMS, Lilly, MSD, Novartis, Pfizer, Roche and UCB' meeting support from Pfizer; advisory board participation fee from Novartis; and other service fee by BMS., Bjorn Gudbjornsson: None declared, Michael T Nurmohamed MTN received research grants from AbbVie, BMS, Pfizer, Galapagos, Amgen and Eli Lily. BG received consulting fee from Novartis and honorary lecture payment from Novartis and Nordic-Pharma., Mikkel Østergaard MØ received the study drug from BMS and UCB; research grants from Abbvie, BMS, Merck, Novartis and UCB; speaker fees from Abbvie, BMS, Celgene, Eli-Lilly, Galapagos, Gilead, Janssen, MEDAC, Merck, Novartis, Pfizer, Sandoz, and UCB; and consultancy fees from Abbvie, BMS, Celgene, Eli-Lilly, Galapagos, Gilead, Janssen, MEDAC, Merck, Novartis, Pfizer, Sandoz and UCB., Gerdur Grondal: None declared, Tuulikki Sokka-Isler: None declared, Hilde Berner Hammer: None declared, Ronald F. van Vollenhoven RFvV received the study drug from BMS and UCB; research grants from BMS, GSK, UCB and AstraZeneca; consulting fees from AbbVie, AstraZeneca, Biogen, BMS, Galapagos, Janssen, Miltenyi, Pfizer and UCB; expert fees from AbbVie, Galapagos, GSK, Janssen, Pfizer, R-Pharma and UCB; and advisory board fees from AbbVie, AstraZeneca, Biogen, BMS, Galapagos, Janssen, Miltenyi, Pfizer and UCB., Espen Haavardsholm: None declared.

Background Individuals testing positive for anti-cyclic-citrullinated-peptide-antibodies (Anti-CCP) and musculoskeletal (MSK) complaints are at risk for developing rheumatoid arthritis (RA). Objectives We aim to identify factors involved in arthritis progression in a population considered at risk for RA. Methods Anti-CCP-positive individuals with MSK complaints referred to rheumatologist in the Region Stockholm were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥3 years or until arthritis diagnosis was made. Blood samples from inclusion were analyzed for 9 selected anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); as well as a panel of 92 inflammation-associated proteins and HLA-SE alleles. Cox regression was applied to the data and a predictive multivariate model was identified. Results are shown with a confidence interval (CI) of 95 percent. Results 267 individuals were recruited. 101 (38%) developed arthritis in median after 14 months (IQR: 6-27). In the multivariate analysis: ACPA reactivity (HR 8.0, CI 2.9-22, p<0.0001), IL15R-α levels (HR 0.6, CI 0.4-0.9, p 0.006), IL6 levels (HR 1.5, CI 1.2-1.8, p<0.0001) and the presence of tenosynovitis as detected by ultrasound (HR 3.4, CI 2.0-6.0, p<0.0001) were significantly associated with arthritis. Diagnostic accuracy for ACPA reactivity test had a sensitivity of 96% (CI 92-99.8), a specificity of 30% (CI 30-46), a positive predictive value of 51% (CI 43-58) and a negative predictive value of 94% (CI 87-99.7). Diagnostic accuracy for ultrasound assessed tenosynovitis had a sensitivity of 17% (CI 9-24), a specificity of 99% (CI 97-100), a positive predictive value of 89% (CI 74-100) and a negative predictive value of 64% (CI 57-70). Conclusion We propose a high-risk RA phase characterized by the presence of certain ACPA reactivities, IL15-Rα, IL6, and tenosynovitis, parameters that could be used to identify individuals at particular low risk and high risk for arthritis progression. Acknowledgements None. Disclosure of Interests Alexandra Cîrciumaru: None declared, Yogan Kisten: None declared, Monika Hansson: None declared, Linda Mathsson-Alm Employee of: ThermoFisher Scientific, Vijay Joshua: None declared, Heidi Wähämaa: None declared, Malena Loberg Haarhaus: None declared, Joakim Lindqvist: None declared, Fei Guozhong: None declared, Nancy Vivar Pomiano: None declared, Hamed Rezaei: None declared, Erik af Klint: None declared, Aleksandra Antovic: None declared, Bence Réthi: None declared, Anca Catrina: None declared, Aase Hensvold: None declared.

Background:Musculoskeletal ultrasound (MSUS) evaluation of individuals at risk for developing rheumatoid arthritis (RA) having Anti-Citrullinated Protein Antibody (ACPA) positivity and musculoskeletal complaints, may play an important role in the very early detection of RA.Objectives:We aimed to identify which ultrasound markers could predict arthritis development.Methods:Individuals with musculoskeletal complaints with a positive anti-CCP2 test were referred to the rheumatology department for a detailed clinical (68 joint count) and MSUS examination of the hands, feet and any symptomatic joints. Only those without clinical and/or MSUS detected arthritis were included in the RISK RA prospective cohort and followed-up over 3 years/ or until arthritis onset. Using EULAR-OMERACT guidelines1, MSUS markers for synovial hypertrophy (SH) and hyperemia (Doppler activity) were documented for each visit. Finger and wrist tendons were screened for any signs of tenosynovitis (TS), and between metatarsal joints for bursitis. Association of MSUS biomarkers with arthritis development was tested (comparing proportions) using Chi-Squared or Fisher’s exact tests.Results:288 individuals were included from January 2014 to October 2019 (79% female, 35% RF positive, median age 48 years: IQR: 36-58). Within a median of 38 months (IQR: 1-72) since recruitment, 84 individuals (28%) developed an arthritis diagnosis.Prior to obtaining any diagnosis (at inclusion and/or follow-up visit), 95 of the 288 individuals (33%) had at least one type of MSUS anatomical modification present (around the tendons, joint synovium and/or within bursal cavities), and 56% (53/95) of these individuals eventually developed arthritis. Of the remaining 193 that did not present with any obvious MSUS changes, 16% progressed towards arthritis development.The presence of tenosynovitis was detected in 64 of 288 individuals scanned prior to diagnosis and were more frequent in those developing arthritis (44%, 37/84) as compared to those with TS not developing arthritis (13%, 27/204), p<0.0001. The extensor carpi ulnaris wrist tendons were mostly involved. Sonographic changes within the synovium were noted in 11% (32/288) of all individuals, mostly affecting the metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joints. There was a higher incidence of synovial hypertrophy detected in those developing arthritis (22%, 18/24), as compared to those that remained arthritis free (7%, 14/204), p<0.0001. The MCP joints with synovial hypertrophy were more prone to arthritis development as compared to the MTP’s. Furthermore, we observed a higher frequency of bursitis between the MTP joints in individuals developing arthritis, as compared to individuals having a bursitis who did not develop arthritis (13%, 11/84 versus 7%, 14/204, p=0.009).Conclusion:Ultrasound biomarkers such as tenosynovitis of the extensor carpi ulnaris, synovial hypertrophy of the MCP joints and feet bursitis have good potential to predict arthritis development in a population at-risk for rheumatoid arthritis.References:[1]Maria-Antonietta D’Agostino et al. RMD Open 2017;3:e 000428Acknowledgements:All study participants and patients, including researchers that are part of the multidisciplinary laboratory, clinical and academic teams of the RISK RA study group, as well as all assisting this research in one form or the other are greatly acknowledged.Disclosure of Interests:None declared

Anti-citrullinated protein antibodies (ACPA) are characteristic markers for rheumatoid arthritis (RA), developing years before disease onset. Early clinical and biological biomarkers could provide useful information on the onset of RA in predisposed individuals.The aim of the study was to investigate whether ACPA along with inflammatory markers and musculoskeletal ultrasound changes could predict arthritis development in individuals at risk for RA.ACPA-positive individuals with musculoskeletal complaints were referred from primary care to a rheumatology clinic, recruited in the Risk-RA research program and followed-up for up to 3 years, between April 2014 and October 2019. All individuals lacked arthritis both at clinical examination by a trained rheumatologist and ultrasound assessment of hands and feet and any other symptomatic joints (according to EULAR-OMERACT definition). Blood samples were collected at inclusion and were analyzed for 15 ACPA fine specificities (by custom made peptide array), 92 inflammation-associated protein biomarkers (by multiplex immunoassay with Olink extension technology) and HLA-SE (DR low resolution kit). Statistical analysis used univariate and multivariate models with backwards selection and cox regression.268 individuals with a median age of 48 (36-58) were recruited, out of which 212 (79%) were females. 75 (28%) developed arthritis within 11 months of follow-up while the median follow-up for those not developing arthritis was 21 months (14-28). Increased ACPA levels, shorter symptom duration and RF positivity were the main differences between individuals developing arthritis and those who did not. In univariate models, the presence of HLA-SE, specific ACPA reactivities, certain inflammatory markers and ultrasound-detected tenosynovitis were associated with arthritis development. In multivariate analysis the presence of anti-cit-fillagrin (HR 2.1 (95% CI 1.2-3.7, p 0.01), IL6 levels (HR 1.4 (95% CI 1.2-1.7, p 0.0001) and tenosynovitis (HR 2.9 (95% CI 1.7-5.0, p 0.0001) remained significant predictors for arthritis onset.Certain ACPA reactivities together with inflammatory markers and ultrasound-detected tenosynovitis predict arthritis development in predisposed individuals for developing RA.None declared

Background: Rheumatoid arthritis (RA) is a disease characterized by synovial joint inflammation, mainly affecting small joints. Histological findings in synovial biopsies ranges from inflammatory infiltration including ectopic lymphoid structures, to a cell sparse fibroid phenotype. T cells in affected joints are non-naïve and have by flow cytometry approaches been shown to have a wide TCR-beta chain gene usage. New technologies allow for analyses of paired TCR sequences and their antigen-specificities. Objectives: To study the alpha/beta-T cell receptor repertoire in single sorted T cells from synovial biopsies at time of RA-diagnosis. Meth ods: Synovial biopsies were taken, primarily using an ultrasound guided technique, from seventeen patients (12 ACPA+, 5 ACPA-) with rheumatoid arthritis. Fresh biopsies were enzymatically digested, followed by mild mechanical treatment, prior to flow cytometry cell sorting. Single cell index sorting of T cells was made into 384-well plates with PCR-buffer followed by a nested PCR and deep sequencing of the TCR amplicons. TCR-receptor sequences showing clonal expansion from four ACPA+ HLA-DRB1*0401 patients were further cloned into SKW3 cells for studies of their reactivity by in vitro stimulation with peptides of viral and citrullinated origin from the literature. A positive response, as measured by CD69-up regulation or IL-2 production, was used to define specificity. Results: Fourteen of the assessed joints were small (1 MTP, 4 MCP and 8 wrists), whereas the remaining three were large joints (2 knees and 1 ankle), table 1. Individual T cells could be isolated from all of these biopsies, with a variating CD4:CD8 ratio. Based on the flow cytometry phenotyping we could identify CD4 T cells of both Treg and T peripheral helper phenotype already at this early time point. Productive alpha/beta-TCR sequences could be retrieved from 16 out of 17 patients and clonal expansion (>1 copy/TCR) was seen in all but one of these patients, with clone sizes ranging between 2 – 34 copies of each TCR. Table 1. Patient characteristics. Patients Gender (F/M ) HLA-SE alleles Joints Joint swelling prior to biopsy (months ) Stiffness specific joint (median VAS ) Pain specific joint (median VAS ) ACPA+ (n = 12) 9/3 *0401, *0404, *0408, *01 and *10 1 MTP, 4 MCP, 6 wrists, 1 knee 4 (1-12) a 46 (0-84) 45 (22-99) ACPA- (n = 5) 3/2 *0401 1 MCP, 2 wrists, 1 ankle, 1 knee 5 (0.25-7) 59 (15-73) 47 (33-81) SKW3 cell lines (patients n = 4) 4/0 * 0401/0404 n=2 *0401 n=2 1 MTP, 3 wrists 2 (1-6) 50.5 (42-84) 50 (40-99) a Data not available for one patient. One patient with prior RA-diagnosis, but after 9 months of treatment remission lasting for 20 years. Artificial T cell lines were generated from the expanded clones of HLA-DRB1*04:01 RA subjects. Our in vitro stimulation protocol identified virus specific CD4 T cells in all samples. So far, no citrulline reactivity has been found. HCMV, followed by HHV were the most commonly found viral reactivities, whereas others were found only in one donor (e.g. JCV, EBV). The majority of clones are thus “orphans”, to which we are still seeking the driving antigen. Conclusion: Clonally expanded T cells are found in the synovium of early RA patients and include virus-specific CD4+ T cells. Our data show that the local T cell repertoire is broad already at the time of RA diagnosis Disclosure of Interests: Sara Turcinov: None declared, Erik af Klint Paid instructor for: Abbvie (courses and lectures), An De Bondt Employee of: Janssen., Muhammad Sohel Mia: None declared, Anca Catrina: None declared, Frederik Stevenaert Employee of: Janssen, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

Career situation of first and presenting author Post-doctoral fellow Introduction Anti-citrullinated protein antibodies (ACPA) are predictive markers with pathological effects in rheumatoid arthritis (RA). Previous prospective studies have used a clinical definition of arthritis. Thus, we aimed to investigate risk factors of developing arthritis in ACPA-positive subjects with musculoskeletal complaints who did not have any of clinical and ultrasound signs of arthritis. Methods Subjects with positive ACPA-test referred from primary care to rheumatology clinic, lacking arthritis in hands and feet by clinical and ultrasound examination (according to EULAR-OMERACT synovitis definition), were recruited into the Risk-RA research program. Patient included between years 2015–2016 with clinical data up to 2017 were analysed. Blood samples from inclusion were analysed for 13 specific ACPA reactivities using a custom made ImmunoCAP ISAC microarray. Presences of HLA-SE risk gene were analysed using DR low-resolution kit. Results 41% (27 out of 66) of the Risk RA subjects developed arthritis during a median follow up of 8 months. The rest was followed 25 months in median without any signs of arthritis. Subjects developing arthritis tended to have a higher concentration of anti-CCP, more tender joints and rheumatoid factor positivity at inclusion compared to those not developing arthritis. The number of ACPA-reactivities (mean 6 vs 3), the presence of HLA-SE (89% vs 56%) and the occurrence of ultrasound detected tenosynovitis (44% vs 5%) at inclusion were significantly increased in subjects developing arthritis compared to those not developing arthritis. Univariate cox proportional hazards regression showed a hazard ratio (HR) for arthritis development of 1.1 for every increase in number of ACPA reactivities (95% CI 0.99 to 1.2, p 0.07); HR: 4.4 (95% CI 2.0 to 9.5, p 0.0002) for tenosynovitis and for HR: 4.9 (95% CI 1.5 to 16, p 0.01) for HLA-SE carriers. All subjects with tenosynovitis (n=14) prior to arthritis development were carriers of HLA-SE, except for one subject but similar to the majority this HLA-SE no-carrier also progressed to arthritis. Conclusions Subjects with ACPA-positive musculoskeletal complaints lacking any clinical and ultrasound signs of arthritis are at high risk of developing arthritis, especially carriers of HLA-SE with tenosynovitis. The role of inflammatory spreading from tendons (synovial sheath) to synovial tissue within joints need to be further investigated. Disclosure of Interest None declared

Background The pathophysiological processes leading from musculoskeletal (MSK) complaints to clinically manifest rheumatoid arthritis (RA) are not fully understood. The urgency for imaging and serological markers that predict arthritis development in individuals at risk of RA is of clinical importance. Objectives To identify ultrasound (US) markers that can predict arthritis development. Methods Patients presenting with MSK complaints and a positive Anti-Citrullinated Protein Antibody (ACPA) test were referred from primary care units to the Rheumatology Unit. Those lacking clinical signs of arthritis, confirmed by absence of synovial hypertrophy with Doppler activity on US examination, were recruited into the Risk-RA prospective program. A total of 66 patients with complete US records were included between years 2015 up to December 2016. Hands and feet, including symptomatic joints were US-evaluated for synovitis, hyperemia and bone erosions. The presence of wrist (compartments 1–6) and finger (flexor and extensor) tenosynovitis, according to OMERACT guidelines were also recorded in all patients. Serum samples from inclusion were analysed on a multiplex immunoassay Results 66 Risk-RA patients (85% female, median age 50 years, range 22–82) were included and followed up to arthritis onset (median 8 months, range 1–27), or to the end of year 2017 (median 25 months, range 11–43). 27 patients (41%, 86% female, median age 52 years, range 22–74) developed arthritis. Of these 7 had tenosynovitis detected by US at inclusion and 7 more developed tenosynovitis at follow-up visits (in total n=14). At the time of diagnosis, 20 out of 27 patients presented with both tenosynovitis and synovitis. A large majority of patients with tenosynovitis (12 out of 14, 86%) and a minority without tenosynovitis (15 out of 52, 29%) developed arthritis, resulting in an increased relative risk of 3.0 (95% CI 1.8–4.8) to develop arthritis for patients presenting with tenosynovitis at baseline or follow-up visits (p=0.001). Concentrations of the anti-CCP antibodies, anti-CEP antibodies and anti-citrullinated vimentin60–75 antibodies tended to be higher in patients with tenosynovitis developing arthritis (n=12, median of 70 AU/ml, range 2–175 for anti-CCP, median of 68 AU/ml, range 0–673 for anti-CEP, median of 53, range 0–644 for anti-vim) than those without tenosynovitis developing arthritis (n=15, median of 35 AU/ml, range 1–100 for anti CCP, median of 12, range of 0–1179 for anti-CEP, median of 29, range 0–332 for anti-vimentin). Same trend was observed when comparing patients with tenosynovitis developing arthritis to those without-tenosynovitis not-developing arthritis. The 2 patients with tenosynovitis not developing arthritis, had lower levels of the antibodies as compared to those with tenosynovitis developing arthritis. No significant differences in other patient baseline characteristics were seen between those with, and those without tenosynovitis (86 vs 85% female, median (range) 54 years29–71 vs 50 years,22–82 mean visual analogue scale pain 34 vs 31, mean c-reactive protein 2.7 vs 3.2; tender joint count 1.2 vs 0.7). Conclusions Ultrasound detected tenosynovitis in the context of ACPA positivity is a good clinical predictor of rapid arthritis onset in individuals at risk of developing RA. Disclosure of Interest None declared

Introduction Prospective studies of individuals at increased risk of developing rheumatoid arthritis (RA) will further improve the understanding of disease development. Ultrasound emerges clinically useful in detecting subtle inflammatory changes in rheumatic diseases. Objectives To investigate ultrasound (US) detected changes as markers for future arthritis development. Methods Patients presenting with musculoskeletal complaints and a positive Anti-Citrullinated Protein Antibody (ACPA) test at primary care, were referred to Karolinska rheumatology clinic for further rheumatic joint disease assessments. Those lacking arthritis by clinical and US examination (defined as synovial hypertrophy with Doppler activity) were recruited into the Risk-RA clinical research program, and followed-up by our multidisciplinary rheumatology team. A total of 64 patients with complete US records were included between years 2015→2016. Our patient demographics were 84% (n 54/64) females, mean age 49 (range 22–82) years, median relative ACPA (times cut-off) 26 (range 1–174) titer, median visual analogue scale (VAS) pain 30, median VAS patient global 28 and median C-reactive protein was 1 (0–20) at inclusion. Hand (Wrists, MCP’s, PIP’s, DIP’s) and feet joints were US-evaluated for synovial hypertrophy, hyperemia and bone erosions. The presence of wrist (compartments 1–6) and finger (flexor and extensor) tenosynovitis were assessed. Data from inclusion→follow-up visits until September 2017 were analysed. SPSS software version 25 was used (Univariate, Chi-square, T-test and Mann Whitney U-test) for comparisons. Results At inclusion, none of the 64 patients had any signs of active joint inflammation. However, ultrasound changes for tenosynovitis were seen in 7 out of 64 patients, 3 of who also presented with mild hypertrophy without Doppler activity and one patient with mild Doppler hyperemia (without hypertrophy), and none with bone erosions at inclusion. Among all tendons evaluated bilaterally, tenosynovitis of the Extensor Carpi Ulnaris (ECU) wrist tendons (4 of 7 patients) and the 2nd finger flexor-tendons (3 of 7 patients) were most commonly affected. Of the 57 patients without US-tendon changes, one had mild Doppler hyperemia (without hypertrophy). Patients with US-tendon changes were 86% (n 6/7) females, had mean age 56 years, median VAS pain 42, median VAS global health 20, mean 2.7 mg/L CRP, median relative ACPA titer 70 in comparison to patients without US-tendon changes, 75% females, mean age of 48 years, median VAS pain 24, median VAS global health 28, mean 2.7 mg/L CRP and median ACPA titer of 23. The numerical difference in pain and relative ACPA titer were non-significant (p>0.05). After follow up for mean 18 months (range 1–18), 7 out of 7 (100%) with US-tendon changes at inclusion and 18 out of 57 (32%) without US-tendon changes developed arthritis. Patients with US-tendon changes compared to those without tenosynovitis at inclusion, developed arthritis within 12 and 11 mean months follow-up, respectively. Conclusions Our study shows that tenosynovitis is a specific marker for arthritis development in ACPA-positive patients with musculoskeletal symptoms. The role of inflammatory spreading from tendons (synovial sheath) to synovial tissue within joints need to be further investigated. Disclosure of interest None declared

Background There is today a paucity of prospective studies to describe the natural longitudinal history of anti-ccp positive individuals developing RA or not developing RA. Further no study to investigate the detailed ACPA reactivities in such a setting is currently available. Methods Individuals at risk of developing RA were included in a cohort at Karolinska University Hospital, Stockholm. Examinations of peripheral joints was repeated at one year follow-up visit or at any time the patients experienced worsening of their symptoms. Peripheral blood samples were available at inclusion (n=70). Serum was run on a microarray based on the ImmuoCAP ISAC system testing for ACPA reactivities toward 13 different citrullinated peptides (fillagrin, fibrinogen, alpha-enolase, vimentin, histone) (1). Results Individuals referred from primary care with musculoskeletal complaints and positive anti-ccp test were systematically investigated as part of routine care at our rheumatology clinic. Individuals lacking self-reported history of suspect arthritis, clinical arthritis according to rheumatologist and signs of synovitis on ultrasound examination were included in a clinical Risk-RA program with life-style coaching and personalized information on the risk of developing RA. Seventy individuals, with a mean age of 48 years (SD 15) and 86% females, were included in the program. Twenty (29%) individuals developed arthritis during a medium follow up time of 7 months (range 1–25 months). Number of ACPA reactivities at baseline was significant higher among those developing (in mean 6 reactivities) as compared to those not developing arthritis (in mean 4 reactivities). A increased proportion of individuals were showing reactivity towards citrullinated (cit) vimentin (vim) 60–75, fibrinogen (fib) 573 and enolase (eno) (CEP-1) among those developing arthritis (80% for anti-cit-vim 45% for anti-cit-fib and 60% for anti-cit eno) as compared to those not developing arthritis (41% for anti-cit-vim, 30% for anti-cit-fib and 52% for anti-cit-eno). Increased level of anti-cit-vim and anti-cit-eno antibodies was also observed at inclusion for those individuals developing arthritis as compared to those not developing arthritis. Conclusions We describe here the pattern of ACPA reactivities in anti-CCP positive individuals with non-specific musculoskeletal symptoms at risk of developing RA and without clinical and ultrasonograph signs of synovitis and report that 30% of these patients will develop arthritis during a short follow-up. Number, frequency and titers of specific ACPA reactivities appear to be enriched already at inclusion among those patients that developed arthritis during follow-up. References Hansson et al Arthritis Res Ther 2012. Disclosure of Interest None declared

Objectives Fluorescence Optical Imaging (FOI) is an emerging modality that uses an intravenous fluorophore to display altered microcirculation (abnormal perfusion/capillary leakage) in synovial tissues in the hands. FOI can be analyzed visually (FOI-v) or by using automated Disease ACTivity (DACT). Using musculoskeletal ultrasound (MSUS) as a validated reference measure, we previously showed FOI to be highly sensitive and specific in detecting clinically manifest and silent synovitis in patients with various rheumatic diseases. Here, we analyze whether the same is true for early rheumatoid arthritis (eRA). Methods Hands and wrists ineRA patients were assessed by clinical examination, MSUS and FOI-DACT. Active inflammation was defined as having synovial-hypertrophy/effusions and intra-articular Doppler signaling on MSUS, and as increased optical-intensities on FOI-v. Scores on DACT ≥ 1 was considered indicative of disease activity. Results 39 eRA patients were studied [72% females, 56% previous/current smokers, 54% RF(+) and 69% ACCP(+)]. Of the 1326 joints in these patients, 303 were inflamed by clinical assessment, 380 by MSUS, and 400 by FOI-v. The percentages of patients and (mean ± SD) joints by clinical, MSUS and FOI-v were 69%(7.8 ± 8.1), 95%(9.7 ± 7.7), and 95%(10.3 ± 7.2), respectively. Using MSUS as reference, FOI-DACT was 95%(35/37) accurate in identifying patients with active disease, 24%(9/37) of whom had erosive RA. Good correlations noted between MSUS and FOI-v (rho = 0.803; p < 0.001), clinical assessment and FOI-v (rho = 0.732; p < 0.001), and MSUS and clinical (rho = 0.793; p < 0.001). The sensitivity, specificity, NPV and PPV of inflammation by FOI-v was 81%(308/380), 90%(854/946), 61%, and 96% respectively. Of the clinically negative but MSUS positive (145/1023) joints, 68%(98/145) were also FOI-v positive. Remarkably, one patient had 15 joints that were FOI-DACT positive and MSUS negative, but a month later, the same joints became MSUS positive. Although the wrists and MCPs were frequently inflamed, DIP joint inflammation was also seen in 34 and 14 joints in 12 patients by FOI-DACT and MSUS, respectively. Nine of these patients had osteoarthritis by conventional radiography. Conclusions As reported for established rheumatic diseases, here we show high correlations and agreements between clinical examination, MSUS and FOI-v in detecting subtle inflammation in early RA as well. Moreover, DACT-FOI emerges as a useful automated quantitative scoring method for synovial inflammation in eRA. Reference Kisten Y, Györi N, af Klint E, et al. 2015 Detection of clinically manifest and silent synovitis in the hands and wrists by fluorescence optical imaging. RMD Open.1: e000106. doi:10.1136/ rmdopen-2015-000106(http://rmdopen.bmj.com/content/1/1/e000106.full.pdf+html)

Background Altered microcirculation (abnormal perfusion/capillary leakage) of synovial tissue can be detected early using Fluorescence Optical Imaging (FOI). FOI utilizes an intravenous fluorophore1,2 that displays high-resolution hand images that can be analyzed visually (FOI-v) in real-time, or by using digital Disease ACTivity (DACT) scoring methods. We previously reported FOI9s sensitivity and specificity in detecting silent synovitis in various rheumatic diseases1. Objectives Here, we test the diagnostic performance of FOI-DACT in detecting subtle hand joint inflammation in early rheumatoid arthritis (eRA), as compared to clinical evaluation and MusculoSkeletal UltraSound (MSUS). Methods Fingers and wrists of patients with eRA were assessed by clinical examination, MSUS and FOI-DACT imaging. Inflammation was defined as having synovial hypertrophy/effusions and intra-articular Doppler signaling on MSUS, and as increased optical intensities on FOI-v. Scores of DACT≥1 were considered indicative of disease activity. Results 1326 joints of 39 eRA patients [72% females, 56% previous/current smokers, 54% RF(+) and 69% ACPA(+)] were studied. The incidence and mean number ±SD of joints inflamed by clinical, MSUS and FOI-v were 23% (7.8±8.1), 29% (9.7±7.7) and 30% (10.3±7.2), respectively. Using MSUS as a reference, FOI-DACT was 95% (35/37) accurate in identifying patients with active disease, 24% (9/37) of whom had erosive RA. High correlations and agreements emerged between MSUS and FOI-v (r=0.803, p<0.001; kappa±SE:0.70±0.02 [95% CI 0.67–0.75]), clinical and FOI-v (r=0.732, p<0.001; kappa±SE:0.56±0.03 [95% CI 0.51–0.61]) and MSUS and clinical (r=0.793, p<0.001; kappa±SE:0.59±0.03 [95% CI 0.54–0.64]). The sensitivity, specificity, NPV and PPV of inflammation by FOI-v was 81% (308/380), 90% (854/946), 61%, and 96% respectively. Of the clinically negative but MSUS positive (145/1023) joints, 68% (Subclinical: 98/145) were also FOI positive. Remarkably, one patient had 15 joints that were FOI-DACT positive and MSUS negative, but a month later, the same joints became MSUS positive. Although the wrists and MCPs were frequently inflamed, DIP joint inflammation was noted in 12 patients by FOI-DACT and MSUS. Nine of these patients had osteoarthritis by conventional radiography. Conclusions In early RA, Fluorescence Optical Imaging (FOI) coupled with digital Disease ACTivity (DACT) scoring correlates well with MSUS, and has a high positive predictive value. FOI-DACT emerges as a useful automated quantitative scoring method for synovial inflammation, and may be used in monitoring the effects of therapy. References Kisten Y, Györi N, af Klint E, et al. 2015 Detection of clinically manifest and silent synovitis in the hands and wrists by fluorescence optical imaging. RMD Open 2015;1: e000106. doi: 10.1136/rmdopen-2015-000106 (http://rmdopen.bmj.com/content/1/1/e000106.full.pdf+html) Glimm AM, Werner SG, et al. Analysis of distribution and severity of inflammation in patients with osteoarthitis compared to rheumatoid arthritis by ICG-enhanced fluorescence optical imaging and musculoskeletal ultrasound: a pilot study. Annals of the Rheumatic Diseases. Published online Aug. 26, 2015. (http://dx.doi.org/10.1136/annrheumdis-2015-207345). Disclosure of Interest None declared

Background The objective detection and quantification of inflammatory disease activity is critical for achieving optimal therapy results. Fluorescence optical imaging (FOI) is a novel modality designed for imaging the hands and wrists, and the automated quantification of the ensuing scans using DACT (Disease ACTivity)-FOI is a novel algorithm for analyzing these images. Objectives To determine the utility of DACT-FOI in the assessment of hand and wrist inflammation. Methods Bilateral finger and wrist joints (n=1360) of 40 patients with inflammatory arthritis were studied. Synovitis was defined as tender and swollen joints on clinical examination, presence of synovial thickening/effusion and intra-articular Doppler signals on ultrasound (MSUS), and abnormal focal optical signal intensities on FOI, respectively. The DACT score used an automatically generated algorithm of the composite images (of 240 frames per second) to achieve a quantified score for each patient. Using dedicated image parameters and size correction, the enhanced pixels were extracted automatically from the image background, and the high signal intensities calculated. The DACT-FOI formula was based on fluorescence intensity curve thresholds that were used to discriminate intensity variations, and then divided by the 95th centile of intensities in normal individuals as the reference value. DACT-FOI ≤1 was referred to as normal digital activity signals. Subclinical synovitis was defined as being clinically non-inflamed but inflamed on MSUS. Results Out of the 1360 joints evaluated, 215 (16%) were inflamed clinically, 329 (24%) by MSUS, and 347 (26%) by FOI. For overall hand and wrist disease activity (n=40), the number (mean ± SD) of active joints detected by clinical, MSUS and semi-quantitative FOI was 5.4±7.0; 8.2±7.8; and 8.7±7.8, respectively. The automated digital activity (±SD) calculation by DACT-FOI was 3.8 (±2.1). Correlations of high statistical significance was denoted as ** when p<0.01. A strong positive correlation (r =0.458**; p=0.003) between clinical synovitis and DACT-FOI was demonstrated. The mean DACT values also correlated significantly with MSUS (r =0.442**; p=0.004) and semi-quantitative FOI (r =0.439**; p=0.005). There was a highly significant correlation of synovitis detection between clinical examination and MSUS (r =0.730**; p=0.000) and between clinical examination and semi-quantitative FOI (r =0.577**; p=0.000). Agreement between MSUS and FOI in synovitis detection was good, and revealed strong correlations (0.816**; p=0.000). Out of the non-inflamed joints by clinical examination, 142/1145 (12%) were inflamed by MSUS, of which 102/142 (72%) were also inflamed by FOI. Thus, for detecting subclinical synovitis, the sensitivity, specificity, and positive and negative predictive values of FOI were 72% (102/142), 93% (934/1003), 77% and 91%, respectively. Conclusions FOI and the automated analysis DACT-FOI were technically feasible with high reproducibility and strong agreement with clinical scoring. Therefore, this objective digitally quantified measurement of inflammatory disease activity in the hands & wrists may be useful both in diagnosis and in monitoring the effects of clinical therapy. Disclosure of Interest None declared

Background Large clinical studies have demonstrated that TNF blockade delays radiological progression in rheumatoid arthritis (RA) independent of the clinical response. Objectives We aimed to investigate the mechanisms by which adalimumab, a human anti TNF antibody, affects osteoclastogenesis in vivo and in vitro. Methods Expression of OPG, RANK and RANKL was evaluated by immunohistochemistry and semi quantitative double blind analysis of serial synovial biopsies obtained from 12 RA patients treated with adalimumab before and 8 weeks after treatment initiation. The in-vitro effect of adalimumab on RANKL and OPG expression in osteoblast-like cells with or without TNF priming was evaluated by RT-PCR and Western blots. Further investigation of the direct effect of adalimumab on osteoclasts formation (TRAP staining) and function was tested in a bone resorption assay using blood derived CD14 positive monocytes. Resorption area and number of pits were analysed by computer assisted image analysis. Results Treatment with adalimumab significantly decreased the RANKL/OPG ratio and RANK expression in synovial tissue in parallel with a reduction in the number of synovial macrophages, while no changes were seen in the number of synovial lymphocytes. No significant differences were observed between EULAR responders and non-responders. In-vitro adalimumab mimicked the in-vivo effect inducing a decrease in the RANKL/OPG ratio in osteoblast-like cells (SaOS2). In addition adalimumab was able to inhibit in-vitro osteoclast formation and bone resorption even in the presence of RANKL. Conclusions Therapy with adalimumab regulates not only synovial inflammation but also bone metabolism through direct modulation of the RANKL/RANK/OPG pathway and inhibition of osteoclasts formation. These effects might explain the bone sparing effect seen with TNF blockade even in the presence of ongoing inflammation. Disclosure of Interest A. Krishnamurthy: None Declared, S. Revu: None Declared, P. Neregård: None Declared, A. Hensvold: None Declared, M. Engström: None Declared, E. Erik af Klint: None Declared, D. Makrygiannakis: None Declared, A. Catrina Grant/Research support from: AI Catrina has received an unrestricted research grant from Abbott.

Background and objectives The objective detection and quantification of disease activity in its earliest pathophysiological stage is critical for achieving optimal therapy results. Fluorescence optical imaging (FOI) is a novel imaging modality for the hands and wrists, and automated quantification of the ensuing images using DACT (Disease ACTivity)-FOI as a novel algorithm representing activity. This study was designed to determine the utility of FOI as a diagnostic tool, and whether it could be used in lieu of colour/power Doppler ultrasound (US) to quantify and ascertain apparent and non-apparent active synovitis Materials and methods A total of 872 hand/wrist joints in 26 patients (18 female, 8 male, average age 51.5 years) with various rheumatic diseases (RA: 12, JIA, SLE, DM, FM, PsA and polyarthritis 1–2 each) were examined by standard clinical assessment, US and DACT-FOI. Joints swollen and tender or swollen only were considered clinically inflamed. Active synovitis was defined as having synovial thickening and Doppler activity on US. Joints positive by FOI displayed abnormal focal optical intensities by visual inspection. Silent synovitis was defined as showing synovitis by US but not clinically. The DACT value was digitally quantified per patient by an automated computer-based algorithm of the composite image (240 frames). After clinical, US and FOI positive joints for each hand were calculated, the sensitivity, specificity and kappa statistics computed and compared with the mean DACT values for all patients Results Out of 872 joints, 142 (16%) were inflamed clinically, 241 (28%) by US, and 229 (26%) by FOI. There was moderate agreement for synovitis detection between clinical examination and US (kappa 0.524 ± 0.033; 95% CI: 0.459–0.589) and between clinical examination and FOI (kappa 0.450 ± 0.035; 95% CI: 0.381–0.519). Of the 241 inflamed joints by US, 196 (81%) were also inflamed by FOI, while only 119 (49%) were inflamed clinically. Agreement between US and FOI in synovitis detection was good (kappa 0.773 ± 0.024; 95% CI: 0.725–0.821). Depending on the gold standard used to define inflammation, FOI was 73%–83% sensitive and 86%–95% specific for detecting synovitis. Out of 730 non-inflamed joints by clinical examination, 608 (83%) were non-inflamed by US and 605 (83%) were non-inflamed by FOI. Of these clinically non-inflamed joints, 122 (17%) were inflamed by US. For detecting silent synovitis, FOI was 80% (98/122) sensitive and 96% (581/608) specific. The number (mean ± SD) of active joints detected by clinical, US and FOI was 5.4 ± 7.6; 9.4 ± 9.8; and 9.3 ± 9.7 respectively, and the overall automated disease activity DACT-FOI was 4.3 ± 2.1. There was a strong positive correlation (r = 0.556; p = 0.003) between the clinical detection of synovitis and DACT-FOI. The mean DACT values also correlated significantly with US (r = 0.479; p = 0.013) and semi-quantitative FOI (r = 0.515; p = 0.007) Conclusion FOI and the automated analysis DACT-FOI were technically feasible with high reproducibility and agreement with clinical scoring and US. For detecting synovitis semi-quantitatively, FOI had a lower sensitivity but similar specificity compared to US. FOI may be particularly useful in identifying patients with clinically non-apparent hand/wrist inflammation (silent synovitis).

Background The timely identification of synovial inflammation is critical for early diagnosis & treatment of rheumatoid arthritis & other inflammatory arthritides. Fluorescence Optical Imaging (FOI, "Rheumascan"), a novel imaging modality, uses an intravenous fluorescence dye, that enables imaging of the hands & wrists with increased optical intensities in areas of high perfusion &/or capillary leakage. Objectives To determine the sensitivity & specificity of FOI as a diagnostic tool in determining "active" synovitis as compared to the US & clinical findings. Methods A total of 748 joints of both hands & wrists, including 3 wrist joints, 5 MCPs, 5 PIPs, & 4 DIPs in 22 patients (15 female, 7 male) with an age average of 44yrs old, with inflammatory arthritis (RA: 9; JIA, psoriatic arthritis, SLE, polyarthritis & other diagnoses, 1-2 each) were examined clinically, by US & FOI. Only swollen & tender joints were regarded as clinically inflamed. Positive colour Doppler US signals with synovial thickening/fluid were considered as "active" synovitis. FOI was scored visually. Comparisons were done using kappa statistics, and the diagnostic utility (sensitivity & specificity) of FOI was tested. Results 72 out of 748 joints (10%) were considered inflamed by clinical examination, 144 (19%) by US, and 129 (17%) by FOI. Of the clinically inflamed joints, 49 (68%) were identified as "actively" inflamed by ultrasound, and 37 out of 72 (51%) of these joints were inflamed by FOI. Out of 676 joints that were negative by clinical examination, 581 (86%) were negative by ultrasound and 575 (95%) by FOI. The agreement between clinical examination & US was fair (kappa 0.37±0.05) and somewhat stronger than the agreement between clinical examination & FOI (kappa 0.28±0.05). Out of 144 joints that were "actively" inflamed by US, 100 (69%) showed inflammation by FOI, while out of 604 non-inflamed joints by US, 575 (95%) were non-inflamed by FOI. The agreement between US and FOI was good (kappa 0.67±0.04). Out of 49 joints that were inflamed both by clinical examination & by US, 27 (55%) were inflamed by FOI. Conclusions The sensitivity of FOI for inflammation in individual joints of the hands & wrists were 51-76% depending on what "gold standard" was used to define inflammation. The specificity of FOI was 95% (575/604), suggesting that it has lower sensitivity but similar specificity compared to US. These findings, together with good agreement between US & FOI, suggest that the latter may be used as a complementary diagnostic tool in clinical practice, in particular when US is not available, in order to identify synovitis earlier and with greater confidence. Acknowledgements To the patients & all involved in this study in one way or the other. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2484

Background While musculoskeletal ultrasound examination (MSUS) is used increasingly in the work-up of patients with inflammatory joint disease, the exact diagnostic utility has not been established. Objectives To quantify the diagnostic utility of MSUS using a prospective, probabilistic (Bayesian) approach. Methods All patients referred to our clinic for evaluation of arthritis were eligible, unless a prior diagnosis was indicated or a certain diagnosis could be made based on the information in the referral letter. Patients were assessed by history and physical examination including joint examination, laboratory testing including acute-phase reactants, RF, and ACPA, and plain x-ray of hands, wrists and feet if clinically indicated. A diagnostic assessment was then performed by the responsible physician where the probability of a) any inflammatory joint disease and b) rheumatoid arthritis was given on a 5-point scale ranging from unlikely (0-20% probability) to very likely (80-100% probability). Subsequently, an ultrasound examination of the wrist, MCP, PIP 2-5 in both hands, MTP 2-5 in both feet and also symptomatic joints was performed by HR and the results of the examination presented to the responsible physician. The latter then assessed the diagnostic probabilities again, using the same scale. The proportions of patients with maximal and minimal diagnostic certainty pre-test and post-test were compared by Fisher exact test. Results 67 patients were included, 52 were female, average (SD) age 47.9 (16.5) years. Symptom duration 8.9 (4.0) months, 15 patients were positive for RF and 12 for ACPA. The pre-test and post-test probability distributions for (any) inflammatory joint disease and for RA are given in the table. The final diagnoses in these patients were RA (15), other inflammatory joint diseases (21), non-inflammatory joint disease (31). With regard to a diagnosis of (any) inflammatory joint disease, the proportion of patient for whom diagnostic certainty was maximal (<20% OR >80% likelihood) was 20/67 (29.9%) before MSUS and 42/67 (62.7%) after MSUS (p=0.0002). With regard to a diagnosis of RA, the proportions were 21/67 (31.3%) pre-test and 39/67 (58.2%) post-test (p=0.003). Parallel reductions were seen in the proportions of patients with greatest diagnostic uncertainty (40-60% likelihood), from 24/67 (35.8%) to 10/67 (14.9%) (p=0.0093) and from 17/67 (25.3%) to 4/67 (6.0%) (p=0.0035), respectively. Conclusions In this probabilistic (Bayesian) analysis, musculoskeletal ultrasound when added to routine physical and laboratory examination greatly increased the diagnostic certainty in patients referred for the evaluation of arthritis. Disclosure of Interest H. Rezaei: None Declared, E. af Klint: None Declared, R. Van Vollenhoven Grant/research support from: AbbVie, BMS, GSK, MSD, Pfizer, Roche, UCB, Consultant for: AbbVie, BMS, GSK, MSD, Pfizer, Roche, UCB

Objectives: Etanercept is an effective tumour necrosis factor (TNF)-alpha inhibitor drug with the unique ability to block not only TNF-alpha but also lymphotoxin (LT)-alpha, at least in vitro. We aimed to investigate the in vivo effect of etanercept on synovial expression of TNF-alpha and LT-alpha.Method: Synovial biopsies from 12 rheumatoid arthritis (RA) patients started on etanercept and 11 RA patients started on infliximab were obtained at baseline and 8 weeks after treatment initiation. Synovial expression of TNF-alpha and LT-alpha was evaluated by immunohistochemistry followed by computer-assisted image analysis. Differences between paired samples were analysed by the Wilcoxon test and between groups by the Mann Whitney test. A p-value < 0.05 was considered statistically significant.Results: Six out of the 12 of the patients started on etanercept achieved an American College of Rheumatology (ACR)50 response. Macroscopic evaluation of the joints during arthroscopy revealed a significant decrease of local inflammation mainly in good ACR50 responders. Synovial expression of both LT-alpha and TNF-alpha decreased but the differences did not reach statistical significance at a group level. By contrast, a significant decrease in both LT-alpha and TNF-alpha was observed when only good ACR50 responders were analysed. Despite higher levels of baseline synovial TNF-alpha in the good responders, neither baseline LT-alpha nor TNF-alpha could predict clinical response after 8 weeks. A decreasing trend of the synovial levels of LT-alpha was also observed in good responders to infliximab, but the difference did not reach statistical significance.Conclusions: Etanercept treatment modulates the synovial expression of both TNF-alpha and LT-alpha in vivo, a mechanism that might partly explain its clinical efficacy in RA.

Objective Methotrexate (MTX) is one of the most widely used therapies in rheumatoid arthritis (RA) due to its antiinfl ammatory and potential bone protection effect.Bone biology is governed by the RANKL/RANK/OPG system that determines the balance between bone formation by osteoblasts and bone resorption by osteoclasts.We investigated the effects of MTX on the RANKL/RANK/OPG system in vivo and in vitro.Methods 16 patients with newly diagnosed RA (mean disease duration 1 week) were started on MTX 10 mg once a week and increased by 10 mg each week until a stable dose of 20 mg once a week was reached.Patients were naïve for other disease-modifying antirheumatic drugs and allowed to take prednisolone to a maximum dose of 10 mg daily and non-steroidal infl ammatory drugs as clinically indicated.Synovial biopsies were obtained by needle arthroscopy at baseline and 8 weeks after initiation of therapy.x-rays of hands and feet were obtained at baseline and 1 year after diagnosis.Immunohistochemical analysis was performed to detect RANKL, RANK and OPG in the synovial biopsies.We further investigated the in vitro effect of MTX on synovial fl uid-derived mononuclear cells, synovial fl uid mononuclear cells (SFMC) (by immunohistochemistry), osteoblasts (by real-time PCR and western blot analysis) and osteoclast formation (tartrate-resistant acid phosphatase (TRAP) staining).Statistical analysis was performed using the Wilcoxon and Mann-Whitney test when appropriate.Results Nine patients (56%) were responders to therapy according to EULAR criteria.Two patients had erosions at inclusion in the study and fi ve more developed erosions at 1-year follow-up in both the responder and non-responder groups.MTX treatment decreased synovial infl ammation with a signifi cant reduction of synovial cellularity.In parallel, MTX decreased synovial RANK expression and the RANKL/OPG ratio, mainly in the subgroup of RA patients with no radiological progression at 1-year follow-up.We confi rmed the effect on RANK expression in SFMC cultured in vitro with MTX.A decrease in the RANKL/OPG ratio was also observed in cultured osteoblasts at both the mRNA and protein levels.MTX blocks osteoclastogenesis from PBMC despite the presence of macrophage colony stimulating factor and RANKL, indicating that MTX directly inhibits osteoclastogenesis.Conclusions MTX directly affects the RANKL/RANK/OPG system and inhibits osteoclasts formation providing an attractive explanation for the bone-sparing effect of MTX.on July