Luca Petricca

MicroRNA-155 influences B-cell function through PU.1 in rheumatoid arthritis

MicroRNA-155 (miR-155) is an important regulator of B cells in mice. B cells have a critical role in the pathogenesis of rheumatoid arthritis (RA). Here we show that miR-155 is highly expressed in peripheral blood B cells from RA patients compared with healthy individuals, particularly in the IgD-CD27- memory B-cell population in ACPA+ RA. MiR-155 is highly expressed in RA B cells from patients with synovial tissue containing ectopic germinal centres compared with diffuse synovial tissue. MiR-155 expression is associated reciprocally with lower expression of PU.1 at B-cell level in the synovial compartment. Stimulation of healthy donor B cells with CD40L, anti-IgM, IL-21, CpG, IFN-α, IL-6 or BAFF induces miR-155 and decreases PU.1 expression. Finally, inhibition of endogenous miR-155 in B cells of RA patients restores PU.1 and reduces production of antibodies. Our data suggest that miR-155 is an important regulator of B-cell activation in RA.

B-cell subsets in the joint compartments of seropositive and seronegative rheumatoid arthritis (RA) and No-RA arthritides express memory markers and ZAP70 and characterize the aggregate pattern irrespectively of the autoantibody status.

The aim of the present study was to determine whether different subsets of B cells characterize synovial fluid (SF) or synovial tissue (ST) of seropositive or seronegative rheumatoid arthritis (RA) with respect to the peripheral blood (PB). PB, SF and ST of 14 autoantibody (AB)-positive (rheumatoid factor (RF)-IgM, RF-IgA, anti-citrullinated peptide (CCP)), 13 negative RA and 13 no-RA chronic arthritides were examined for B-cell subsets (Bm1-Bm5 and IgD-CD27 classifications), zeta-associated protein kinase-70 (ZAP70) expression on B cells and cytokine levels (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-8 and monocyte chemotactic protein (MCP)-1). Synovial tissues were classified as aggregate and diffuse patterns. No differences were found in B-cell percentages or in subsets in PB and SF between AB+ and AB− RA and no-RA. In both AB+ and AB− RA (and no-RA), the percentage of CD19+/ZAP70+ was higher in SF than in PB (AB+: P= 0.03; AB−: P = 0.01; no-RA: P = 0.01). Moreover, SF of both AB+ and AB− RA (and no-RA) patients was characterized by a higher percentage of IgD-CD27+ and IgD-CD27− B cells and lower percentage of IgD+CD27− (P < 0.05) B cells compared to PB. In SF, ZAP70 positivity is more represented in B cell CD27+/IgD−/CD38−. The aggregate synovitis pattern was characterized by higher percentages of Bm5 cells in SF compared with the diffuse pattern (P = 0.05). These data suggest that no difference exists between AB+ and AB− in B-cell subset compartmentalization. CD27+/IgD−/ZAP70+ memory B cells accumulate preferentially in the joints of RA, suggesting a dynamic maturation of the B cells in this compartment.

Polymorphisms of the IgH enhancer HS1.2 and risk of systemic lupus erythematosus

Objective To determine whether the allelic frequency variation of the HS1.2 enhancer of the immunoglobulin heavy chain (IgH) 3′ regulatory region (3′RR-1) locus represents a risk factor for systemic lupus erythematosus (SLE) and to identify a possible functional difference in the two most frequent alleles (*1 and *2) in binding nuclear factor- κB (NF-κB) and Sp1. Methods The frequency of the enhancer HS1.2 alleles was determined in two cohorts of patients with SLE (n=293) and in 1185 controls. Electrophoretic mobility shift assays (EMSA) were carried out with B cell nuclear extracts with different probes of HS1.2 alleles *1 and *2 to map the consensus binding sites of the nuclear factors. A confirmatory cohort of 121 patients with SLE was also included. Results The frequency of allele *2 of the HS1.2 enhancer was significantly increased in patients with SLE compared with controls (OR 1.60, 95% CI 1.33 to 1.92, p<0.001). EMSA experiments showed the presence of the Sp1 binding site in both alleles whereas only allele *2 carried the consensus for the NF-κB factor. The presence versus absence of allele *2 in patients with SLE correlated with a higher concentration of IgM levels and with the expression of B cell activating factor receptor (BAFF-R). Conclusions The increased frequency of allele *2 in patients with SLE identifies a new genetic risk factor for SLE. A possible biological effect of the polymorphism could be the difference observed in the localisation of an NF-κB binding site which is specific for allele *2 and absent in allele *1. These observations suggest a functional effect of the HS1.2 enhancer in this disease.

MicroRNA-34a dependent regulation of AXL controls the activation of dendritic cells in inflammatory arthritis

Current treatments for rheumatoid arthritis (RA) do not reverse underlying aberrant immune function. A genetic predisposition to RA, such as HLA-DR4 positivity, indicates that dendritic cells (DC) are of crucial importance to pathogenesis by activating auto-reactive lymphocytes. Here we show that microRNA-34a provides homoeostatic control of CD1c+ DC activation via regulation of tyrosine kinase receptor AXL, an important inhibitory DC auto-regulator. This pathway is aberrant in CD1c+ DCs from patients with RA, with upregulation of miR-34a and lower levels of AXL compared to DC from healthy donors. Production of pro-inflammatory cytokines is reduced by ex vivo gene-silencing of miR-34a. miR-34a-deficient mice are resistant to collagen-induced arthritis and interaction of DCs and T cells from these mice are reduced and do not support the development of Th17 cells in vivo. Our findings therefore show that miR-34a is an epigenetic regulator of DC function that may contribute to RA.

Diagnostic performance of anti-citrullinated peptide antibodies for the diagnosis of rheumatoid arthritis: the relevance of likelihood ratios.

Abstract Background: The goal of our study was to evaluate the diagnostic performance of the anti-cyclic citrullinated peptide 2 (anti-CCP2) assay in patients with autoimmune and inflammatory disorders. Methods: We tested the specificity and sensitivity of anti-CCP2 antibodies measured by ELISA in 787 patients with rheumatoid arthritis (RA), 1024 patients with other autoimmune/inflammatory rheumatic disease and 401 subjects without autoimmune rheumatic disease. The optimal cut-off value was defined as the value with the highest diagnostic accuracy (receiver operating characteristic curve analysis). Interval-specific likelihood ratios (LRs) were calculated for each range bounded by defined anti-CCP2 values. Results: To distinguish between patients with RA and controls, the cut-off value with the highest diagnostic accuracy for anti-CCP2 was 2.8 U/mL. Comparing the optimal cut-off value for anti-CCP2 to that recommended by the manufacturer (5.0 U/mL), an increase in prevalence between the proportions of test-positive patients was found for RA, undifferentiated connective tissue disease and undifferentiated arthritis. Evaluating interval-specific LRs for the selected ranges bound by two anti-CCP2 values, in RA and diseased controls, the LRs were 0.40 for values <5.0 U/mL, 6.66 for 5.0–15.0 U/mL, 27.01 for 15.1–30.0 U/mL and 28.89 for >30.0 U/mL. Conclusions: The cut-off value of 2.8 U/mL for anti-CCP2 has the highest diagnostic accuracy. A value of anti-CCP2 >15 U/mL is associated with an increase in the likelihood of RA disease. Clin Chem Lab Med 2010;48:829–34.

Synovial features of patients with rheumatoid arthritis and psoriatic arthritis in clinical and ultrasound remission differ under anti-TNF therapy: a clue to interpret different chances of relapse after clinical remission?

Objective To define the synovial characteristics of patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA) in clinical and ultrasound remission achieved by combination therapy with methotrexate (MTX) and tumour necrosis factor (TNF) blockers. Methods Patients with RA in remission (n=25) (disease activity score (DAS)<1.6 for at least 6 months), patients with RA in low disease activity (LDA) (n=10) (1.6<DAS<2.4 for at least 6 months) and patients with PsA in remission (n=18) (DAS<1.6 and Psoriasis Area Severity Index (PASI)=0 for at least 6 months) achieved by MTX+anti-TNF (adalimumab 40 mg or etanercept 50 mg) with power Doppler (PDUS)-negative synovial hypertrophy underwent synovial tissue biopsy. Patients with RA with high/moderate disease naïve to treatment (n=50) were included as a comparison group. Immunostaining for cluster designation (CD)68, CD21, CD20, CD3, CD31 and collagen was performed. Results PDUS-negative patients with RA in remission showed lower histological scores for synovial CD68+, CD20+, CD3+ cells and CD31+ vessels and collagen deposition (p<0.05 for both lining and sublining) compared with PDUS-positive patients with RA with high/moderate disease. In addition, there was no significant difference in terms of lining and sublining CD68+, CD20+, CD3+, CD31+ cells and collagen comparing PDUS-negative patients with RA in remission and in LDA, respectively. On the contrary, PDUS-negative patients with PsA in remission showed higher histological scores for sublining CD68+ (p=0.02) and CD3+ cells (p=0.04) as well as CD31+ vessels (p<0.001) than PDUS-negative patients with RA in remission. Conclusions PDUS-negative patients with RA in remission have comparable synovial histological features than PDUS-negative patients with RA in LDA. However, patients with PsA in remission are characterised by a higher degree of residual synovial inflammation than patients with RA in remission, despite PDUS negativity under TNF inhibition.

SAT0066 Histological and ultrasound synovial predictors of clinical differentiation to defined arthritis in patients with seronegative undifferentiated peripheral inflammatory arthritis

Background Undifferentiated Peripheral Inflammatory Arthritis (UPIA) is a common diagnosis at the first clinical evaluation in rheumatological settings. However, the likelihood of developing a well-defined rheumatic disease in UPIA patients is still matter of debate. Objectives To examine the role of ultrasound (US) and histological parameters in the disease outcome of patients with seronegative UPIA. Methods Fourty-two patients with IgA/IgM-Rheumatoid Factor and anti-citrullinated peptide antibodies negative UPIA, naïve to any Disease-Modifying Anti-Rheumatic Drugs, underwent Gray Scale (GSUS) and power Doppler (PDUS) evaluation and US guided synovial tissue biopsy. Synovial expression of CD68, CD3, CD21, CD20 and CD31 was evaluated by immunohistochemistry. IL-6, VEGF-A and VEGF-D peripheral blood (PB) and synovial fluid (SF) levels were measured by ELISA. To exclude Reactive Arthritis, each patient underwent genital and throat swabs. Afterwards, each UPIA patient was treated with chloroquine 250 mg/daily and followed every 3 months for 1 year and classified as having UPIA, Rheumatoid Arthritis (RA), Spondyloarthritis (SpA) or Psoriatic Arthritis (PsA), respectively. Results During the follow-up 6 (14.3%) UPIA reached a defined diagnosis (2 RA, 2 SpA and 2 PsA, respectively). At baseline, UPIA who differentiated had higher GSUS (p=0.01) and PDUS scores (p=0.02) compared to patients who remained as UPIA within 1 year. At baseline, UPIA who differentiated towards defined arthritis had higher histological scores for lining and sublining CD68+ (p=0.005 and p=0.04 for lining and sublining, respectively), sublining CD3+ cells (p=0.002) and CD31+ vessels count (p<0.001) than patients who remained as UPIA. In addition, there were direct correlations between baseline GSUS and PDUS scores with lining CD68+ cells scores (p<0.001 for GSUS and p=0.02 for PDUS scores respectively), sublining CD68+ cells scores (p=0.02 for GSUS and p=0.03 for PDUS scores respectively), sublining CD3+ cells score (p=0.002 for GSUS and p=0.002 for PDUS scores respectively) and CD31+ vessels count (p<0.001 for GSUS and p=0.01 for PDUS scores respectively) in UPIA. Finally, the areas under the receiver operating characteristic (ROC) curves CD31+ vessels count (cut-off value: 24.3), GS score (cut-off value: 1.5) and PDUS score (cut-off value: 1.5) were calculated to assess the best cut-off points to identify the differentiation likelihood during the follow-up in UPIA patients. The logistic regression analysis, demonstrated that having baseline GSUS and PDUS scores ≥1.5 [OR:13.64 (95% CI: 0.98–242.59); p=0.05] and CD31+ vessels count ≥24.3 [OR:51.13 (95% CI: 3.15–829.16); p=0.01] were independent factors associated with the achievement of defined arthritis. Conclusions Histological and US assessment may help in the identification of patients with seronegative UPIA with high likelihood of clinical differentiation towards defined arthritis. Disclosure of Interest None declared

Immunosuppressive Therapy (Methotrexate or Cyclophosphamide) in Combination with Corticosteroids in the Treatment of Giant Cell Arteritis: Comparison with Corticosteroids Alone

tion group were less likely to be prefrail or frail (P = .048). Frail persons were more likely to use anticholinergic medications (83%, n = 56) than prefrail (64%, n = 195) and robust (42%, n = 103) persons (p < .001). Mean anticholinergic scores were 0.73 1.14 for robust individuals, 1.4 1.75 for prefrail individuals, and 2.62 2.52 for frail individuals (P < .001). Factors associated with anticholinergic medication use (ADS score >0) were being prefrail or frail, having a FCI score of 2 or greater, and being aged 85 and older (Table 1). In longitudinal analysis, 84 of those who were robust in 2005 were classified as prefrail or frail in 2007. In the control group but not the intervention group, robust persons were more likely to become prefrail or frail if they used anticholinergic medications in 2005 (unadjusted P = .04).

THU0089 Biomarkers of Erosive Disease in a Cohort of Early-Rheumatoid Arthritis Patients Treated According to the Treat to Target Strategy

Background Seropositive and seronegative rheumatoid arthritides (RA) have been considered as two different clinical entities, and traditionally autoantibodies have been associated to worse prognosis. Objectives To assess differences in disease characteristics, presence of erosions and outcome between anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF)-IgM and -IgA seropositive and seronegative early rheumatoid arthritis (ERA) patients. Methods A total of 386 ERA patients with a disease duration of less than 12 months were enrolled in the study. Seronegative ERA patients were defined as those negative for ACPA, RF-IgM and -IgA [113 (29.3%)]; all the other subjects were defined as seropositive [273 (70.7%)]. ERA patients fulfilled the 2010 ACR criteria for RA and were followed according to the treat-to-target strategy. Subjects with symptom duration less than 3 months were defined as having “very early RA” (VERA). At baseline, and every three months, the ACR/EULAR core data set [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), swollen joint count (SJC), tender joint count (TJC), physician and patient global assessment, pain, health assessment questionnaire (HAQ)] were recorded. At each visit, clinical improvement and remission were evaluated according to EULAR criteria. Results At diagnosis, seronegative patients were older (p=0.002) and had shorter disease duration, comprising a greater number of VERA (p=0.04), compared to seropositive ones, and at baseline had higher average of SJC (p<0.0001), TJC (p=0.004) and consequently DAS28 (p=0.02), CDAI (p=0.001) and SDAI (p=0.001). There were no differences in gender and presence of erosion between the two analyzed cohorts. Similar percentages of good-EULAR response and DAS-remission were seen over time between seropositive and seronegative patients. Dividing patients according to the positive autoantibody, between ACPA seropositive and seronegative subjects we observed the same previously reported differences. RF-IgM and RF-IgA seropositive patients had higher ESR value (p=0.03) and were more smokers (p=0.03), compared to seronegative ones. Moreover, a higher percentage of RF-IgA seropositive patients had erosions at baseline (p=0.03), a longer disease duration (p=0.03) and BMI>30 (p=0.02), compared to seronegative subjects. At 12th month of follow-up, patients that developed new erosions or had a worsening of Sharp erosion score (13.1%, of which 6.9% were seronegative and 20.0% were seropositive, p=0.02), differed from non erosive subjects only for ACPA seropositivity (p=0.04). At the multivariate analysis, RF-IgA seropositivity [OR (95%CI): 2.25 (1.32-3.82)] and age at diagnosis [OR (95%CI): 1.02 (1.01-1.04)] arose as significant predictors of erosive disease at baseline. At 12th month of follow-up, ACPA seropositivity [OR (95%CI): 3.61 (1.38-9.45)] and age at diagnosis [OR (95%CI): 1.03 (1.01-1.06)] were the variables significantly associated to development of new erosions and to worsening of Sharp erosion score. Conclusions Even though seronegative early RA had higher disease activity scores at diagnosis, the presence of RF-IgA and ACPA represent the strongest risk factors for being erosive and developing new erosions. Disclosure of Interest None declared

FRI0021 Weight Loss in Obese Rheumatoid Arthritis Patients Improves Disease Activity Without Modifying RA Treatment

Background Obesity is one of the potentially preventable risk factors for RA being associated with RA onset, disease severity and poor response to therapy. New evidence suggests that weight loss may lead to outcome improvement in obese osteoarthritis and psoriatic arthritis patients. Objectives To evaluate whether, in obese RA patients with a low-moderate disease activity, a weight loss obtained with a controlled nutritional intervention may lead to an improvement of disease activity and to a reduced need of increasing therapeutic regimens, without modifying RA treatment during the study period. Methods 64 consecutive obese RA patients (BMI>30 kg/m2) with DAS>1.6, treated according to a treat-to-target strategy and in a stable therapy with conventional DMARDs (cDMARDs) and/or biological DMARDs (bDMARDs) for at least 12 weeks, were enrolled. All patients underwent a scheduled diet under a Nutritionist guide, aimed at a weight loss >5% at 6 months (T6), maintaining unchanged the RA therapy. Patients were evaluated by rheumatologist and nutritionist every 2 months and at each visit clinical and laboratory data and the ACR/EULAR core data set was registered. Disease activity was evaluated by Disease Activity Score on 44 joints (DAS) and Simplified Disease Activity Index (SDAI). Results Of the 64 RA patients (82.8% female, age 56.5±12.5 years, disease duration 8.1±8.1 years, 66.9% seropositive, baseline (T0) DAS 2.8±0.7, baseline BMI 35.3±4.3), 36 (56.3%) were under cDMARDs-only therapy and 28 (43.8%) under biologic bDMARDs therapy ± cDMARDs. At now, 56 patients reached the 6 months follow-up. At T6, the mean reduction in percentage of body weight was 6.6±5.5% and of DAS was 25.4±28.5% (DAS T6: 2.0±0.7, p<0.01 vs DAS T0). Moreover, at T6 patients showed a significant improvement of SDAI with respect to baseline (p<0.01), of tender (p<0.01) and swollen (p<0.01) joint count, of systemic inflammatory parameters (ESR p=0.001, CRP p=0.003), GH (p<0.01), VAS pain (p<0.01) and HAQ (p<0.01), without any change of RA therapy nor corticosteroids need. Dividing patients according to the percentage of weight loss at T6, the 34 (60.1%) RA patients reaching a weight reduction >5% of the baseline body weight obtained higher rates of DAS remission than patients with a weight reduction <5% (DAS remission at T6: 35.3% vs 13.6%, respectively, p=0.07), as well as of SDAI remission (33.3% vs 4.5%, p=0.02). The differences were even more significant when considering a weight reduction >10% (DAS remission at T6: 57.1% in patients reaching 10% weight reduction vs 16.7% in not reaching patients, p=0.01; SDAI remission at T6: 57.1% vs 9.8%, p=0.001). Results were similar between patients under cDMARDs-only and bDMARDs treatment. Conclusions A weight loss obtained with a controlled diet in obese still active RA patients can allow to obtain a better disease control without changing the treatment of RA, and in particular a weight loss >10% permits to reach disease remission in a significant percentage of patients, and therefore reduces the need of an increase in therapy. The effects of weight loss based on a nutritional intervention, and so applicable at the population level, on the RA disease course appears to be crucial in terms of potential clinical and pharmacoeconomics perspectives. Disclosure of Interest None declared