Predicting Flare in Patients With Rheumatoid Arthritis in Biologic Induced Remission, on Tapering, and on Stable Therapy.

OBJECTIVE: The tapering of biologic disease-modifying antirheumatic drug (b-DMARD) therapy for patients with rheumatoid arthritis (RA) in stable remission is frequently undertaken, but specific guidance on how to successfully taper is lacking. The objective of this study is to identify predictors of flare in patients in stable b-DMARD-induced clinical remission, who did or did not follow structured b-DMARD tapering. METHODS: Patients with RA receiving b-DMARD treatment who had achieved sustained remission according to a Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) <2.6 for ≥6 months were offered tapering. Clinical, ultrasound (US) (total power Doppler [PD]/grayscale abnormalities), CD4+ T cell subsets, and patient-reported outcomes (PROs) were collected at inclusion. The primary endpoint was the occurrence of flare (loss of DAS28-CRP remission) over 12 months. Logistic regression analyses identified predictors of flare. Dichotomization into high/low-risk groups was based on 80% specificity using the area under the receiving operator curve (AUROC). RESULTS: Of 63 patients choosing tapering, 23 (37%) flared compared with 12 of 60 (20%) on stable treatment (P = 0.043). All patients who flared regained remission upon reinstating treatment. In the tapering group, flare was associated with lower regulatory T cell (Treg) (P < 0.0001) and higher CRP levels (P < 0.0001), erythrocyte sedimentation rate (P < 0.035), and inflammation-related cells (IRCs) (P = 0.054); stepwise modeling selected Tregs (odds ratio [OR] = 0.350, P = 0.004), IRCs (OR = 1.871, P = 0.007), and CRP level (OR = 1.577, P = 0.004) with 81.7% accuracy and AUROC = 0.890. In the continued therapy group, modeling retained the tender joint count, total PD, and visual analog scale pain score, with 82.1% accuracy and AUROC = 0.899. Most patients in the study were considered low risk of flare (80 of 123 patients [65%]). Only 5 of 37 (13.5%) of the low-risk patients who tapered flared, which was notable compared with the continued therapy group (20% flare). CONCLUSION: Flare on tapering b-DMARDs was predicted by lower Tregs and elevated inflammation biomarkers (IRCs/CRP level); flare on continued b-DMARDs was associated with raised pain parameters and US inflammation. Knowledge of these biomarkers should improve outcomes by targeted selection for tapering, and by increased monitoring of those on continued therapy predicted to flare.

DNA Hypomethylation in the TNF-Alpha Gene Predicts Rheumatoid Arthritis Classification in Patients with Early Inflammatory Symptoms.

Biomarkers for the classification of rheumatoid arthritis (RA), and particularly for anti-citrullinated peptide antibody (ACPA)-negative patients, remain an important hurdle for the early initiation of treatment. Taking advantage of DNA-methylation patterns specific to early RA, quantitative methylation-specific qPCR (qMSP) offers a robust technology for the development of biomarkers. We developed assays and established their value as RA classification biomarkers. METHODS: DNA-methylation data were screened to select candidate CpGs to design qMSP assays. Eight assays were developed and tested on two early inflammatory arthritis cohorts. Logistic regression and bootstrapping were used to demonstrate the added value of the qMSP assays. RESULT: Differentially methylated CpG data were screened for candidate CpG, thereby meeting the qMSP assay requirements. The top CpG candidate was in the TNF gene, for which we successfully developed a qMSP assay. Significantly lower DNA-methylation levels were observed in RA (p < 4 × 10-9), with a high predictive value (OR < 0.54/AUC < 0.198) in both cohorts (n = 127/n = 157). Regression using both datasets showed improved accuracy = 87.7% and AUC = 0.944 over the model using only clinical variables (accuracy = 85.2%, AUC = 0.917). Similar data were obtained in ACPA-negative patients (n = 167, accuracy = 82.6%, AUC = 0.930) compared to the clinical variable model (accuracy = 79.5%, AUC = 0.892). Bootstrapping using 2000 datasets confirmed that the AUCs for the clinical+TNF-qMSP model had significant added value in both analyses. CONCLUSION: The qMSP technology is robust and can successfully be developed with a high specificity of the TNF qMSP assay for RA in patients with early inflammatory arthritis. It should assist classification in ACPA-negative patients, providing a means of reducing time to diagnosis and treatment.

Lymphocyte subset phenotyping for the prediction of progression to inflammatory arthritis in anti-citrullinated-peptide antibody-positive at-risk individuals.

OBJECTIVES: Inflammatory arthritis (IA) is considered the last stage of a disease continuum, where features of systemic autoimmunity can appear years before clinical synovitis. Time to progression to IA varies considerably between at-risk individuals, therefore the identification of biomarkers predictive of progression is of major importance. We previously reported on the value of three CD4+T-cell subsets as biomarkers of progression. Here, we aim to establish the value of 18 lymphocyte subsets (LS) for predicting progression to IA. METHODS: Participants were recruited based on a new musculoskeletal complaint and being positive for anti-citrullinated-peptide Antibody. Progression (over 10 years) was defined as the development of clinical synovitis. LS analysis was performed for lymphocyte lineages, naïve/memory subsets, inflammation-related cells (IRC), and regulatory cells (Treg/B-reg). Modelling used Logistic/Cox regressions. RESULTS: Of 210 patients included, 93 (44%) progressed to IA, 41/93 (44%) within 12 months (rapid progressors). 5/18 LS were associated with progression (Treg/CD4-naïve/IRC (adjusted p < 0.0001), CD8 (p = 0.021), B-reg (p = 0.015)) and 3 trends (NK-cells/memory-B-cells/plasmablasts).Unsupervised hierarchical clustering using these 8 subsets segregated 3 clusters of patients, one cluster being enriched (63/109(58%)) and one poor (10/45(22%)) in progressors.Combining all clinical and LS variables, forward logistic regression predicted progression with accuracy=85.7% and AUC=0.911, selecting smoking/Rheumatoid-Factor/HLA-Shared-Epitope/Tender-Joint-Count-78 and Treg/CD4-naive/CD8/NK-cells/B-reg/plasmablasts.To predict rapid progression, a Cox regression was performed resulting in a model combining smoking/rheumatoid factor and IRC/CD4-naïve/Treg/NK-cells/CD8+T-cells (AUC=0.794). CONCLUSION: Overall, progression was predicted by specific LS, suggesting potential triggers for events leading to the development of IA, while rapid progression was associated with a different set of subsets.

Predicting Inflammatory Arthritis in At-Risk Persons: Development of Scores for Risk Stratification.

BACKGROUND: Inflammatory arthritis (IA) is an immune-related condition defined by the presence of clinical synovitis. Its most common form is rheumatoid arthritis. OBJECTIVE: To develop scores for predicting IA in at-risk persons using multidimensional biomarkers. DESIGN: Prospective observational cohort study. SETTING: Single-center, Leeds, United Kingdom. PARTICIPANTS: Persons with new musculoskeletal symptoms, a positive test result for anticitrullinated protein antibodies, and no clinical synovitis and followed for 48 weeks or more or until IA occurred. MEASUREMENTS: A simple score was developed using logistic regression, and a comprehensive score was developed using the least absolute shrinkage and selection operator Cox proportional hazards regression. Internal validation with bootstrapping was estimated, and a decision curve analysis was done. RESULTS: Of 455 participants, 32.5% (148 of 455) developed IA, and 15.4% (70 of 455) developed it within 1 year. The simple score identified 249 low-risk participants with a false negative rate of 5% (and 206 high-risk participants with a false-positive rate of 72%). The comprehensive score identified 119 high-risk participants with a false-positive rate of 29% (and 336 low-risk participants with a false-negative rate of 19%); 40% of high-risk participants developed IA within 1 year and 71% within 5 years. LIMITATIONS: External validation is required. Recruitment occurred over 13 years, with lower rates of IA in later years. There was geographic variation in laboratory testing and recruitment availability. CONCLUSION: The simple score identified persons at low risk for IA who were less likely to need secondary care. The comprehensive score identified high-risk persons who could benefit from risk stratification and preventive measures. Both scores may be useful in clinical care and should also be useful in clinical trials. PRIMARY FUNDING SOURCE: National Institute for Health and Care Research Leeds Biomedical Research Centre.

B-cell capacity for expansion and differentiation into plasma cells are altered in osteoarthritis.

OBJECTIVE: Autoantibody (autoAbs) production in osteoarthritis (OA), coupled with evidence of disturbed B-cell homoeostasis, suggest a potential role for B-cells in OA. B-cells can differentiate with T-cell help (T-dep) or using alternative Toll like recptor (TLR) co-stimulation (TLR-dep). We analysed the capacity for differentiation of B-cells in OA versus age-matched healthy controls (HCs) and compared the capacity of OA synovitis-derived stromal cells to provide support for plasma cell (PC) maturation. METHODS: B-cells were isolated from OA and HC. Standardised in vitro models of B-cell differentiation were used comparing T-dep (CD40 (cluster of differentiation-40/BCR (B-cell receptor)-ligation) versus TLR-dep (TLR7/BCR-activation). Differentiation marker expression was analysed by flow-cytometry; antibody secretion (immunnoglobulins IgM/IgA/IgG) by ELISA (enzyme-linked immunosorbent assay), gene expression by qPCR (quantitative polymerase chain reaction). RESULTS: Compared to HC, circulating OA B-cells showed an overall more mature phenotype. The gene expression profile of synovial OA B-cells resembled that of PCs. Circulating B-cells differentiated under both TLR-dep and T-dep, however OA B-cells executed differentiation faster in terms of change in surface marker and secreted more antibody at Day 6, while resulting in similar PC numbers at Day 13, with an altered phenotype at Day 13 in OA. The main difference was reduced early B-cells expansion in OA (notably in TLR-dep) and reduced cell death. Stromal cells support from OA-synovitis allowed better PC survival compared to bone marrow, with an additional population of cells and higher Ig-secretion. CONCLUSION: Our findings suggest that OA B-cells present an altered capacity for proliferation and differentiation while remaining able to produce antibodies, notably in synovium. These findings may partly contribute to autoAbs development as recently observed in OA synovial fluids.

Can biomarkers predict successful tapering of conventional disease-modifying therapy in rheumatoid arthritis patients in stable remission?

OBJECTIVES: Specific guidelines for managing RA patients in clinical remission for ≥6 months on cs-DMARDs are lacking. Tapering of treatment is encouraged, however, without validated biomarkers for success. We aimed to assess the rate of sustained remission after 12 months in patients who either (i) followed structured cs-DMARD tapering or (ii) continued therapy, focusing on the added value of biomarkers as predictors of outcome. METHODS: RA patients fulfilling 3v-DAS28CRP<2.6 for ≥6 months on stable cs-DMARD therapy were included. Patients were offered structured tapering, with 117 accepting tapering and 83 continuing therapy. Clinical, ultrasound, immunological (T-cell subsets) and patient-reported outcome (PRO) data were collected. The primary endpoint was the proportion of patients in sustained remission without relapse after 12 months. Regression analyses were used to identify predictors of sustained remission. RESULTS: Of those who tapered, 64% remained in clinical remission after 12 months compared with 80% (p=0.018) of patients on stable treatment. In the tapering group, higher levels of CRP, TJC, % inflammation-related T-cell (IRC) and PROs were associated with flare (all p<0.05), with a trend for total PD (p=0.066). A model predicting sustained remission retained RAQoL, total PD and IRC (85% accuracy, AUROC=0.893, p<0.0001). In the non-tapering group, higher CRP, ESR, SJC and shorter disease duration (all p<0.05) were associated with flare, with no parameter able to predict sustained remission. CONCLUSIONS: In the tapering group, the combination of clinical, PRO, US and T-cell parameters demonstrated added value for predicting sustained remission compared with clinical parameters alone. These data may inform best tapering practice.

Association of circulating lymphocyte subsets with response to IL17i and TNFi in axial spondyloarthritis.

OBJECTIVES: Biologic disease-modifying anti-rheumatic drugs (b-DMARDs) have qualitatively improved the management of axial spondyloarthritis (axSpA), but up to 30-40% of patients do not respond. Although lymphocytes are clearly implicated in the pathology of SpA, circulating lymphocyte subsets (LS) dynamics has been poorly studied. The objective of this pilot study was to comprehensively analyse circulating LS abnormalities in axSpA, and to determine their potential association with response to b-DMARDs. METHODS: Sixty-nine patients with axSpA and 141 control subjects (HC) were included. The clinical features were measured at baseline, and additionally at 6 months in a subgroup of patients who received TNFi (n=36) or IL17i (n=26). Clinical response was defined as a 50% reduction of BASDAI or decrease in ASDAS of 1.1 point. CD4/CD8 T-cells, B-cells and NK-cells and their subsets were analysed by flow cytometry at inclusion. RESULTS: At baseline, alterations in LS were observed in axSpA with reduced/increased frequencies of 10/27 subsets (p<0.003 after correction) and trends for another 5. There was no association of response to bDMARDs with clinical data. Response to IL17i (61% cases) was associated with a higher frequency of NK-cells (p=0.003), trends for change in naïve/memory-CD8+T-cells (p<0.055) and increased expression of KIR3DL2 on Th17-cells (p=0.052). No LS was associated with response to TNFi (69% cases) although trends were observed (CD4+T-cells subsets, higher IL-6R on CD4+/CD8+T-cells). CONCLUSIONS: This pilot work demonstrated a dysregulation of LS in axSpA. The association observed between several LS and clinical response to IL17i (NK/CD8 subsets/Th17-KIR3DL2) was very different to that observed for TNFi (CD4/IL-6R).

Added value of multiple autoantibody testing for predicting progression to inflammatory arthritis in at-risk individuals.

BACKGROUND: Predicting progression to clinical arthritis in individuals at-risk of developing rheumatoid arthritis is a prerequisite to developing stratification groups for prevention strategies. Selecting accurate predictive criteria is the critical step to define the population at-risk. While positivity for anti-citrullinated protein antibodies (ACPA) remains the main recruitment biomarker, positivity for other autoantibodies (AutoAbs) identified before the onset of symptoms, may provide additional predictive accuracy for stratification. OBJECTIVE: To perform a multiple AutoAbs analysis for both the prediction and the time of progression to inflammatory arthritis (IA). METHODS: 392 individuals were recruited based on a new musculoskeletal complaint and positivity for ACPA or rheumatoid factor (RF). ELISAs were performed for ACPA, RF, anti-nuclear Ab, anti-carbamylated protein (anti-CarP) and anti-collagen AutoAbs. Logistic and COX regression were used for analysis. RESULTS: Progression to IA was observed in 125/392 (32%) of cases, of which 78 progressed within 12 months. The AutoAbs ACPA, RF, anti-CarP were individually associated with progression (p<0.0001) and improved prediction when combined with demographic/clinical data (Accuracy >77%; area under the curve (AUC) >0.789), compared with prediction using only demographic/clinical data (72.9%, AUC=0.760). Multiple AutoAbs testing provided added value, with +6.4% accuracy for number of positive AutoAbs (AUC=0.852); +5.4% accuracy for AutoAbs levels (ACPA/anti-CarP, AUC=0.832); and +6.2% accuracy for risk-groups based on high/low levels (ACPA/RF/anti-CarP, AUC=0.837). Time to imminent progression was best predicted using ACPA/anti-CarP levels (AUC=0.779), while the number of positive AutoAbs was/status/risk were as good (AUC=0.778). CONCLUSION: We confirm added value of multiple AutoAbs testing for identifying progressors to clinical disease, allowing more specific stratification for intervention studies.

Poor Clearance of Free Hemoglobin Due to Lower Active Haptoglobin Availability is Associated with Osteoarthritis Inflammation.

INTRODUCTION: Circulating plasma proteins play an important role in various diseases, and analysis of the plasma proteome has led to the discovery of various disease biomarkers. Osteoarthritis (OA) is the most common chronic joint disease, mostly affecting people of older age. OA typically starts as a focal disease (in a single compartment, typically treated with unicompartmental knee replacement), and then progresses to the other compartments (if not treated in time, typically treated with total knee replacement). For this, identification of differential proteins was carried out in plasma samples of OA cases and compared with healthy controls. The aim of this study was to identify circulatory differentially expressed proteins (DEPs) in knee-OA patients undergoing total knee replacement or unicompartmental knee replacement compared to healthy controls and assess their role, in order to have better understanding of the etiology behind OA pathophysiology. METHODS: DEPs were identified with two-dimensional gel electrophoresis (2DE) and isobaric tags for relative and absolute quantification (iTRAQ), followed by liquid chromatography with tandem mass spectrometry. Validation of DEPs was carried out using Western blot and ELISA. Posttranslational modifications were checked after running native gel using purified protein from patients, followed by detection of autoantibodies. RESULTS: In total, 52 DEPs were identified, among which 45 were distinct DEPs. Haptoglobin (Hp) was identified as one of the most significantly upregulated proteins in OA (P=0.005) identified by both 2DE and iTRAQ. Decreased levels of Hp tetramers and increased levels of autoantibodies against Hpß were observed in OA plasma. CONCLUSION: Our data suggest that poor clearance of free hemoglobin and low levels of Hp tetramers may be associated with OA pathogenesis and inflammation.

Biomarkers of Joint Damage in Osteoarthritis: Current Status and Future Directions.

Osteoarthritis (OA) is a disease of the whole joint organ, characterized by the loss of cartilage, and structural changes in bone including the formation of osteophytes, causing disability and loss of function. It is also associated with systemic mediators and low-grade inflammation. Currently, there is negligible/no availability of specific biomarkers that can be used to facilitate the diagnosis and treatment of OA. The most unmet clinical need is, however, related to the monitoring of disease progression over a short period that can be used in clinical trials. In this review, the value of biomarkers identified over the past decade has been highlighted. These biomarkers are associated with the synthesis and breakdown of cartilage, including collagenous and noncollagenous biomarkers, inflammatory and anti-inflammatory biomarkers, expressed in the biological fluid such as serum, synovial fluid, and urine. Broad validation of novel and clinically applicable biomarkers and their involvement in the pathways are particularly needed for early-stage diagnosis, monitoring disease progression, and severity and examining new drugs to mitigate the effects of this highly prevalent and debilitating condition.

Interleukin-7: a potential factor supporting B-cell maturation in the rheumatoid arthritis synovium.

OBJECTIVES: The exact function of interleukin-7 (IL-7) in autoimmune diseases remains unclear although it is a recognised therapeutic target for cytokine blockade. Our objective was to investigate the regulation and downstream effect of IL-7 in diseased tissue from rheumatoid arthritis (RA) patients notably with respect to its function as bone turnover regulator and tissue architecture (TA) organiser. METHODS: Synovial tissues (fresh, frozen or xed) were obtained from our tissue bank and distributed between experiments for live cell cultures, histology, immunohistochemistry or gene expression array by qPCR. RESULTS: IL-7 expression in synoviocyte cultures was up-regulated by pro-in ammatory cytokines, notably IL-6. Gene expression pro ling segregated synovial biopsies based on the presence of B/plasma cells and ectopic TA. IL-7 gene expression was associated with that of several genes whose function was to support B-cell maturation in tissue with distinct B-cell aggregates (despite the lack of IL-7-Receptor expression on B-cells) as well as with ectopic germinal-like centres. IL-7 was associated with bone turnover regulation in biopsies with diffuse in ltration. A novel relationship between the IL-7 and IL-6 axis was also highlighted in human tissue. CONCLUSIONS: Overall, IL-7 may contribute to the maintenance of the pro-in ammatory cycle perpetuating in ammation in RA synovium. We therefore propose a novel role for IL-7 as an orchestrator of TA with an impact on B-cell maturation in relation with IL-6.

Development of a long-term, IL7 dependent cell death rescue assay in CD4+ T-cells.

Interfering with signalling pathways by targeting cell surface proteins has become an important strategy in the development of novel therapeutic agents. Notably, interfering with cytokine signalling revolutionised the treatment of chronic diseases. Cytokines can induce a range of effects that are not always accounted for in assays detecting cytokine binding to cell surface receptors and/or proximal signalling interference. Hence, robust assays are needed to characterise the activity of potential drug candidates targeting such effects. We chose interleukin-7 (IL7) as a cytokine model due to its long-term effect on T-cells. In this report we describe the development and refinement of an in vitro assay for measuring the long-term effect of IL7, more specifically on CD4+ T-cells, while the assay could be adapted to look at CD8+ T-cells. PBMCs and/or purified CD4+ T-cells stained with VPD450 (cell cycle dye) were expanded for 5 days using the mitogen Phytohemagglutinin and/or CD3/CD28 agonists. This resulted in cell proliferation (VPD450 dilution) and activation-induced cell death (7-AAD uptake) which was rescued by the addition of IL7, resulting in cell survival over a further 5 days. JAK-inhibitor (Tofactinib) or a blocking anti-IL7Rα antibody (clone R34.34) abolished cell survival suggesting antagonism, while another antibody (clone A019D5) displayed an agonist effect. These results were confirmed at the proximal signalling level using an IL7/STAT5-luciferase reporter assay. This novel assay for a biological long term effect may be useful for the characterisation of potential therapeutic drugs targeting the IL7/IL7R in CD4+ T-cells.

B-cell capacity for differentiation changes with age.

BACKGROUND: Age-related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin. AIMS: To compare the intrinsic B-cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T-cell help or activation via TLR engagement. METHODS: B cells were isolated from healthy individuals, in younger (30-38 years) and older (60-64 years) donors. An in vitro model system of B-cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T-dependent (TD: CD40/BCR stimulation) or T-independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR. RESULTS: TI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B-cell-executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age-related differences in B-cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient. CONCLUSION: Altogether, B-cell differentiation into PC appeared similar between age groups when provided with T-cell help, in contrast to TI differentiation, where multiple age-related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.

Interferon-related gene expression in response to TNF inhibitor treatment in ankylosing spondylitis patients: a pilot study.

OBJECTIVE: Ankylosing spondylitis (AS) is a chronic inflammatory arthritis primarily affecting the spine and sacroiliac joints. TNF inhibitor (TNFi) drugs are recommended for patients not responding to NSAIDs; however, there is a significant need for biomarkers of response. IFN-regulated genes (IRGs) and other cytokines/chemokines are linked to autoimmune diseases and have been associated with treatment response. Our objective was to explore whether IRGs and cytokines/chemokines can be associated with response to TNFiagents in AS. METHODS: Peripheral blood mononuclear cells were obtained from 26 AS patients who were to receive a TNFi (I, n = 15) or placebo (P, n = 11) at week 0 and week 22. Response (R)/non-response (NR) was defined as reduction in ASDAS ≥ 1.2 points or reduction in sacroiliac/vertebral MRI lesions. The expression of 96 genes was quantified using TaqMan assays. Finally, ELISA was used to measure IL-6 in serum samples from another 38 AS patients. RESULTS: Analysis of gene expression in 26 baseline samples segregated patients into four groups defined by a signature of 15 genes (mainly IRGs). ASDAS response was associated with one group independently of treatment received. We then analysed response to the TNFi (n = 15) and identified a 12-gene signature associated with MRI response. A third IRG signature was also associated with a reduction in IRGs expression post-TNFi samples (n = 10 pairs). Finally, decreased circulating IL-6 was associated with BASDAI-R. CONCLUSION: This pilot study suggests an association between IRG expression and response to TNFi in AS. These findings require validation in a larger cohort in order to construct predictive algorithms for patient stratification.

Receptor activator of nuclear factor kappa-Β ligand (RANKL) serum levels are associated with progression to seropositive/negative rheumatoid arthritis.

OBJECTIVES: The aim of this study was to establish whether serum RANKL levels in early inflammatory arthritis (IA) were associated with rheumatoid arthritis (RA) diagnosis at follow-up, and to evaluate the added value of RANKL for RA diagnosis. METHODS: Serum from 298 patients was collected. Demographic and clinical (swollen/tender joint counts, CRP, DAS28-CRP, RF, ACPA and shared-epitope data were recorded. Baseline ultrasound of 26 joints was performed, including total power Doppler (PD). An ELISA was used to measure RANKL. Predictors of progression were identified using multivariable logistic regression analysis. Area under the receiver operating characteristics (AUROC) was used to assess the performance of the prediction models and quantify the added value of RANKL in RA diagnosis. RESULTS: 151 patients developed RA and 147 were non-RA (undifferentiated IA, other inflammatory diagnoses or non-persistent inflammation). RANKL levels were significantly higher in RA (median [IQR]: 474.1 [270.8-1430.6]) than in non-RA (median [IQR]: 301.0 [174.1-477.5]. Three clinical factors (age, SJC and PD) were identified by multivariable logistic regression with model performance AUROC of 77.9% (95% CI 72.1-83.8%). Adding RANKL resulted in a relative increase of 6.5% in the model classification performance of an AUROC of 83.0% (95% CI 77.9-88.1%). In ACPA-negative patients, the model performance increased from 77.6% (95% CI 69.5-85.7%) with clinical data only to 81.9% (95% CI 73.7-89.8%) with added value of RANKL and imaging. CONCLUSIONS: RANKL levels can predict RA diagnosis over clinical biomarkers alone, both seropositive and particularly in seronegative IA patients.

Anti-carbamylated protein antibodies: are they useful for the diagnosis of rheumatoid arthritis?

OBJECTIVES: ACR/EULAR-2010 classification criteria for rheumatoid arthritis (RA) rely heavily on the presence of anti-citrullinated peptide antibody (ACPA). The role of anti-carbamylated protein antibodies (anti-CarP) in this context is uncertain. We aimed to investigate the value of anti-CarP for RA classification in patients with early inflammatory arthritis. METHODS: Patients (n=402) were recruited from an early arthritis clinic and followed for 24 months. Healthy controls (n=95) were included. An anti-CarP ELISA was performed (aU/mL). Statistical analysis used regression and AUC analysis. RESULTS: The criteria for RA were met by 195/402 patients at inclusion; 28 developed RA during follow-up and 179 had other diagnosis (non-RA). 97/195 (49%) RA patients were anti-CarP+ (median 250 uA/mL [IQR 25-762]). In the group that progressed to RA, 7/28 (25%) were positive (82 uA/mL [13-235]) compared to non-RA (p=0.001) with 13/179 (7%) positive (26 uA/mL [5-80]). Being anti-CarP+ alone was observed in 17 patients of whom 7 (41%) were RA. Levels/positivity were not associated with other parameters. Anti-CarP+ had an odds ratio (OR) 6.5 for predicting RA (OR=17.1 for ACPA+ and OR=2.5 for RF+). In ACPA- patients, anti-CarP+ was also predictive of RA (OR=2.39). Being ACPA+/anti-CarP+/RF+ had a high predictive value for RA (OR=29.9 sensitivity/specificity (sen/spe) 33%/99%, positive/negative predictive values (ppv/npv) 97%/54%), however, being ACPA+/anti-CarP+ was superior (OR=36.1 sen/spe=41%/99%, ppv/npv=98%/57%) while being ACPA+/RF+ was inferior (OR=11.9, sen/spe=54%/95%, ppv/npv=94%/62%). CONCLUSIONS: For RA classification, anti-CarP+ was less sensitive than ACPA, but more specific than RF. Anti-CarP+ may prove useful, classifying early arthritis patients, notably ACPA- patients.

Identification of a Human SOCS1 Polymorphism That Predicts Rheumatoid Arthritis Severity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by an autoimmune response in the joints and an exacerbation of cytokine responses. A minority of patients with RA experience spontaneous remission, but most will show moderate/high disease activity, with aggressive joint damage and multiple systemic manifestations. There is thus is a great need to identify prognostic biomarkers for disease risk to improve diagnosis and prognosis, and to inform on the most appropriate therapy. Here we focused on suppressor of cytokine signaling 1 (SOCS1), a physiological negative regulator of cytokines that modulates cell activation. Using four independent cohorts of patients with arthritis, we characterized the correlation between SOCS1 mRNA levels and clinical outcome. We found a significant inverse correlation between SOCS1 mRNA expression and disease activity throughout the follow-up of patients with RA. Lower baseline SOCS1 levels were associated with poorer disease control in response to methotrexate and other conventional synthetic disease-modifying anti-rheumatic drugs in early arthritis, and to rituximab in established (active) RA. Moreover, we identified several single nucleotide polymorphisms in the SOCS1 gene that correlated with SOCS1 mRNA expression, and that might identify those patients with early arthritis that fulfill RA classification criteria. One of them, rs4780355, is in linkage disequilibrium with a microsatellite (TTTTC)3-5, mapped 0.9 kb downstream of the SNP, and correlated with reduced SOCS1 expression in vitro. Overall, our data support the association between SOCS1 expression and disease progression, disease severity and response to treatment in RA. These observations underlie the relevance of SOCS1 mRNA levels for stratifying patients prognostically and guiding therapeutic decisions.

T-cell subset abnormalities predict progression along the Inflammatory Arthritis disease continuum: implications for management.

The presence of a disease continuum in inflammatory arthritis (IA) is a recognised concept, with distinct stages from at-risk stage (presence of anti citrullinated-peptide autoantibody) to diagnosis of rheumatoid arthritis (RA), including therapy-induced remission. Despite T-cell dysregulation being a key feature of RA, there are few reports of T-cell phenotyping along the IA-continuum. We investigated the disturbances of naïve, regulatory and inflammation related cell (IRC) CD4+ T-cell subsets in 705 individuals across the IA-continuum, developing a simple risk-score (summing presence/absence of a risk-associated with a subset) to predict progression from one stage to the next. In 158 at-risk individuals, the 3 subsets had individual association with progression to IA and the risk-score was highly predictive (p < 0.0001). In evolving IA patients, 219/294 developed RA; the risk-score included naïve and/or Treg and predicted progression (p < 0.0001). In 120 untreated RA patients, the risk-score for predicting treatment-induced remission using naïve T-cells had an odds ratio of 15.4 (p < 0.0001). In RA patients in treatment-induced remission, a score using naïve T-cells predicted disease flare (p < 0.0001). Evaluating the risk of progression using naïve CD4+ T-cells was predictive of progression along the whole IA-continuum. This should allow identification of individuals at high-risk of progression, permitting targeted therapy for improved outcomes.

Dynamics of Early Signalling Events during Fracture Healing and Potential Serum Biomarkers of Fracture Non-Union in Humans.

To characterise the dynamic of events during the early phases of fracture repair in humans, we investigated molecular events using gene expression profiling of bone fragments from the fracture site at different time points after trauma and immune/stromal cells recruitment at the fracture site using flow cytometry. Bone and inflammatory markers were expressed at low levels at homeostasis, while transcripts for bone constituent proteins were consistently detected at higher levels. Early after fracture (range 2-4 days), increased expression of CXCL12, suggested recruitment of immune cells associated with a change in the balance of degradation enzymes and their inhibitors. At intermediate time after fracture (4-8 days), we observed high expression of inflammatory cytokines (IL1-beta, IL6), CCL2, the T-cell activation marker CD69. Late after fracture (8-14 days), high expression of factors co-operating towards the regulation of bone turnover was detected. We identified potential soluble factors and explored circulating levels in patients for whom a union/non-union (U/NU) outcome was known. This showed a clear difference for PlGF (p = 0.003) at day 1. These findings can inform future studies further investigating the cascade of molecular events following fractures and for the prediction of fracture non-union.

Defining remission in rheumatoid arthritis: does it matter to the patient? A comparison of multi-dimensional remission criteria and patient reported outcomes.

OBJECTIVES: In a cross-sectional study, we evaluated the prevalence of 'multi-dimensional remission' (MDR) and its component parameters, assessed using objective measures in a cohort of RA patients in treatment-induced DAS28-remission, and their relationship with patient-reported outcome measures. We sought to confirm the feasibility and face validity of the MDR construct, providing a platform for future longitudinal studies in which its clinical utility might be further established. METHODS: 605 patients were selected from an inflammatory arthritis register using DAS28(CRP)<2.6. Demographic, clinical and patients reported outcomes (PRO) data were collected. Ultrasound power doppler synovitis (n = 364) and T-cell subsets (n = 297) were also measured. Remission using clinical parameters was defined as: tender and swollen joint count (TJC/SJC) and CRP all ⩽1; ultrasound remission: total power doppler = 0 and T cell remission: positive normalized naïve T-cell frequency. MDR was defined as the achievement of all three dimensions. RESULTS: Overall, only 53% (321/605) of the patients achieved clinical parameters, failures being mainly due to raised CRP (52%), TJC (28)>1 (37%) or SJC (28)>1 (16%). 211/364 (58%) of patients achieved ultrasound remission and 193/297 (65%) patients showed T-cell remission. Complete data were available for 231 patients. MDR was observed in only 35% and was associated with the best (lower) PRO scores (all P ⩽ 0.05 vs non-MDR) when compared with the other definitions of remission assessed. The MDR rate was similar in early and established RA patients on b-DMARDs; however, it was lower in established RA patients who received multiple cs-DMARDs (P = 0.011). CONCLUSIONS: In this study, MDR, which may represent a state closer to normality, was found to occur in about a third of DAS28-remission patients and was associated with better patient-reported outcome measures. MDR could be a novel optimal treatment target, notably from a patient's perspective. The relevance of these findings needs further assessment.