J.-Y. Choe

Regulatory T-cells in systemic lupus erythematosus-associated thrombocytopenia: a comparison with idiopathic thrombocytopenic purpura.

Sir, Thrombocytopenia is a common hematologic manifestation of systemic lupus erythematosus (SLE) that occurs in 15–20% of patients, but the exact pathophysiologic mechanism necessary to differentiate it from idiopathic thrombocytopenic purpura (ITP) remains unknown. ITP is an acquired immune disorder diagnosed by excluding other possible causes of thrombocytopenia. Although many complex mechanisms are associated with the development of thrombocytopenia, accelerated platelet destruction due to the presence of autoantibodies against platelet glycoproteins is an important pathophysiological mechanism. The most-studied subset of suppressor CD4 T-cells are the CD4CD25 regulatory T-cells (Treg). The role of Treg in maintaining the balance between self-tolerance by immunosuppression and host defense has been a focus of research since the 1970s. The results of several previous investigations were conflicting in that active SLE patients typically demonstrate lower levels of Treg or functionally defective Treg, as well as a lower number of FoxP3 than inactive SLE patients or normal controls, yet another group reported no difference, and some other groups have even described a significant increase of Treg in the SLE group. Recently, Kuhn et al. reviewed aberrant proportions of Treg in SLE and highlighted the need for further studies. In this study, to investigate the immunological differences between SLE-associated thrombocytopenia and ITP, we assessed ratios of CD4CD25FoxP3 Treg in peripheral blood mononuclear cells (PBMCs) using flow cytometry in active SLE patients, and compared them with ratios drawn from non-thrombocytopenic SLE patients, ITP patients and healthy controls. The expression of Treg in 12 patients with SLE-associated thrombocytopenia (Group 3) and in 5 patients with chronic ITP (Group 4) were compared with the Treg expression of 12 patients with SLE (Group 2) who had never been thrombocytopenic and 10 healthy controls (Group 1). We performed intercellular staining of PBMCs to detect FoxP3 according to the manufacturer’s recommendations (eBioscience, San Jose, CA, USA). PBMCs (1 10 cells) were incubated in a 20ml cocktail of FITC-conjugated anti-human CD4 antibody and PE-conjugated anti-human CD25 antibody (eBioscience). The stained cells were analyzed by flow cytometry (Beckman Coulter). Serum levels of IL-2, IL-10, and TGF-b1 were measured by enzyme-linked immunosorbent assay (ELISA) using commercial assays (R&D Systems, Minneapolis, MN, USA). The Mann–Whitney Utest and Spearman’s correlation test were used to compare Treg cell populations and cytokine levels in each group of patients. The percentage of Treg in the PBMCs of Group 2 patients was 0.05% (SD1⁄4 0.33), which was significantly lower than in healthy controls (0.39%, SD1⁄4 0.20, Group 1). The percentage of Treg in Group 3 patients (0.17%) was significantly lower than in ITP patients (Group 4, 0.27%). The TGF-b1 level of Group 3 patients was 90.07 pg/ml (SD1⁄4 18.88 pg/ml), which was significantly higher than the TGF-b1 level of Group 4 patients (54.14 pg/ml, SD1⁄4 20.60 pg/ml). The serum levels of IL-2 were below the detection limits of the assay in all groups, and there was no significant difference between Group 3 and Group 4 in IL-10 levels. There were no significant correlations detected between Treg percentages and IL-10 or TGFb1 levels in Group 3 and Group 4 (Table1). In terms of the effect of immunosuppressive treatment on population of Treg, all Group 2 and 3 patients were on low doses of steroid and antimalarial agent. One patient in Group 2 was prescribed mycophenolate for nephritis and three patients in Group 3 were taking azathioprine. There were no significant correlations detected between Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores and Treg percentages across groups. The results of non-parametric analyses demonstrated lower levels of Treg in patients with SLE-associated thrombocytopenia than in patients with ITP. Considering the role of Treg in autoimmune function, further large-scale investigations are warranted. Correspondence to: Jung-Yoon Choe, Namgu Daemyung 4dong 3056-6, Daegu 705-718, Korea. Email: jychoe@cu.ac.kr Received 19 August 2009; accepted 13 November 2009

Urine β2-microglobulin is associated with clinical disease activity and renal involvement in female patients with systemic lupus erythematosus

Objective We investigated the association of serum and urine β2-microglobulin (β2MG) with renal involvement and clinical disease activity in systemic lupus erythematosus (SLE). Methods Sixty-four female patients with SLE were enrolled. We assessed SLE disease activity (SLEDAI)-2K and measured serum and urine β2MG levels, as well as complement (C3 and C4) and anti-dsDNA levels. According to the SLEDAI scores, two groups were categorized: low (0–5 of SLEDAI) and high (6–19 of SLEDAI) disease activity groups. The presence of renal involvement was determined by renal SLEDAI score. Statistical analysis was performed using Spearman’s correlation analysis, Mann-Whitney U test, multivariate regression analysis, and logistic regression analysis. Results Urine β2MG levels were significantly different between low and high SLEDAI groups (p = 0.001), but not for serum β2MG levels (p = 0.579). Patients with renal involvement showed higher urine β2MG levels compared to those without renal involvement (p < 0.001), but again there was not a difference in serum β2MG levels (p = 0.228). Urine β2MG was closely associated with SLEDAI (r = 0.363, p = 0.003), renal SLEDAI (r = 0.479, p < 0.001), urine protein/Cr (r = 0.416, p = 0.001), and ESR (r = 0.347, p = 0.006), but not serum β2MG (r = 0.245, p = 0.051). Urine β2MG level was identified as a surrogate for renal involvement (p = 0.009, OR = 1.017, 95% CI 1.004–1.030) and overall disease activity (p = 0.009, OR = 1.020, 95% CI 1.005–1.036). Conclusions We demonstrated that urine β2MG levels are associated with renal involvement and overall clinical disease activity in SLE.

Pain, xerostomia, and younger age are major determinants of fatigue in Korean patients with primary Sjögren’s syndrome: a cohort study

Objectives: Fatigue is a common clinical manifestation in patients with primary Sjögren’s syndrome (pSS). The aims of this study were to investigate the association between fatigue severity and other clinical characteristics in pSS patients and to determine the factors contributing to fatigue. Method: We analysed 257 participants from the Korean Initiative of pSS (KISS), a prospective pSS cohort. Fatigue was assessed according to the fatigue domain of the European League Against Rheumatism (EULAR) Sjögren’s Syndrome Patient-Reported Index (ESSPRI). Health-related quality of life (HRQoL) was evaluated using the EuroQol-5 dimensions (EQ-5D) questionnaire. Multiple linear regression analysis was used to estimate the effect of each variable on fatigue severity. Results: The median total ESSPRI score was 5 [interquartile range (IQR) 4–6]. Thirty-four per cent of patients reported a fatigue score > 5. Younger and premenopausal patients presented with more fatigue (p = 0.013 and p < 0.001, respectively). Higher Xerostomia Inventory (XI) scale (p < 0.001) and Ocular Surface Dryness Index (OSDI) (p < 0.001) scores were observed in patients with a fatigue score > 5. Pain, xerostomia, and age were determined to be significantly associated with fatigue severity after adjusting for depression/anxiety, OSDI score, and the presence of fibromyalgia using a multivariate general linear model. The ESSPRI fatigue score was correlated with the EQ-5D by time trade-off (TTO) values and visual analogue scale (VAS) scores. Conclusions: In Korean patients with pSS, younger age, xerostomia, and pain were correlated significantly with fatigue, and fatigue was associated with HRQoL.

OP0011 A Randomized, Double-Blind, Phase 3 Equivalence TRIAL Comparing the Etanercept Biosimilar, Hd203, with Enbrel®, in Combination with Methotrexate (MTX) in Patients with Rheumatoid Arthritis (RA)

Background Etanercept is a recombinant fusion protein that blocks TNF activity. HD203 is a biosimilar of etanercept. In a double-blind, randomized study in healthy volunteers, HD203 and Enbrel® were comparable with regards to pharmacokinetics, safety and tolerability. Objectives To evaluate the equivalence in efficacy and to compare the safety of HD203 (biosimilar etanercept) and Enbrel® (reference etanercept), in combination with MTX in patients with RA. (ClinicalTrials.gov identifier NCT01270997). Methods Patients (male or female aged ≥20 years) with active RA were randomly assigned (1:1) to 25 mg HD203 or Enbrel®, administered subcutaneously twice weekly with MTX for 48 weeks. The primary endpoint was the proportion of patients achieving ACR20 at week 24. Secondary endpoints included ACRn, change in DAS28, and EULAR response at week 24 and 48, safety and immunogenicity. Efficacy and safety were evaluated at screening, week 0, 2, 4, 8, 12, 16, 20, and 24. Immunogenicity, efficacy and safety were also evaluated at week 36 and 48. Results In total, 294 patients were randomized: 147 to HD203 and 147 to Enbrel®. The proportion of patients achieving ACR20 at week 24 (primary endpoint) was not significantly different for HD203 and Enbrel® (Table) and equivalence in efficacy was demonstrated within predefined margins. In addition, there were no statistically significant differences between proportions achieving ACR20 at weeks 12 and 48. Similar trends were seen for ACR50 and ACR70, however the proportion of patients achieving ACR50 at week 24 and 48 was higher with HD203 than with Enbrel®. There were no statistically significant differences between the groups for ACRn, change in DAS28, and EULAR response at week 24 and 48. Table 1. Proportion of patients achieving ACR20 at week 24 and week 48 HD203 Enbrel® Difference (95% CI) P-value 24-week PPS 83.48% (96/115) 81.36% (96/118) 2.12 (−7.65, 11.89) 0.6706† FAS 79.10% (106/134) 75.56% (102/135) 3.55 (−6.45, 13.55) 0.4870† 48-week PPS 86.27% (88/102) 81.90% (86/105) 4.37 (−5.57, 14.31) 0.3905† CI, confidence interval; PPS, per-protocol set; FAS, full analysis set; †Pearsons chi-square test. Analysis of the safety set (HD203, n=147; Enbrel®, n=146) revealed no statistically significant difference in the number of treatment-emergent (all-causality) adverse events (AEs): HD203 76.87% vs. Enbrel® 78.08% (p=0.8040). Furthermore, no statistically significant differences between HD203 and Enbrel® were observed with regards to adverse drug reactions, serious AEs, or discontinuations due to AEs. No unexpected AEs were observed, and few patients tested positive for anti-drug antibodies. Conclusions The study met the primary endpoint of demonstrating equivalence in efficacy of HD203 compared with Enbrel®. HD203 was well tolerated, with a safety profile comparable to that of Enbrel® in this population of Korean patients with RA. Disclosure of Interest S.-C. Bae: None declared, J.-S. Kim Grant/research support: Hanwha Chemical, J.-Y. Choe: None declared, W. Park: None declared, S.-R. Lee Employee of: Hanwha Chemical, Y. Ahn Employee of: Hanwha Chemical, Y. Seo Consultant for: Hanwha Chemical DOI 10.1136/annrheumdis-2014-eular.3558

Salivary gland ultrasonography findings are associated with clinical, histological, and serologic features of Sjögren's syndrome.

Objective: Salivary gland ultrasonography (SGUS) has been applied in the diagnosis of Sjögren’s syndrome (SS). The aim of this study is to investigate the association of SGUS findings with clinical, histological, and serologic features of SS. Methods: A total of 104 patients with suspected SS underwent SGUS for evaluation of salivary gland involvement. Patients with primary SS were determined according to the classification criteria for SS. The parenchymal inhomogeneity of bilateral parotid and submandibular glands was graded from 0 (homogeneity) to 4 (gross inhomogeneity). Receiver operating characteristic curve analysis was performed to compare the diagnostic performance of different SGUS scoring methods. Clinical and serologic features were compared between groups classified by SGUS score. The association between SGUS and these features of SS was explored by multivariable linear regression analysis. Results: Study participants were predominantly women (96.2%) and had a mean age of 54.1 years. Eighty-seven patients and 88 patients with primary SS were identified based on AECG criteria and ACR/EULAR classification criteria for SS, respectively. Among the different scoring methods, the sum of the grades of four salivary glands (range 0–16) had the best diagnostic performance, with sensitivity of 77.3% and specificity of 87.5% (cutoff value, 7) for distinguishing primary SS from sicca non-SS. SGUS score was associated with focus score in labial salivary gland biopsy (β = 0.240, p = 0.033) and anti-Ro/SSA serology (β = 0.283, p = 0.016) and inversely associated with unstimulated whole salivary flow (β = −0.298, p = 0.011). Conclusion: Ultrasonography of major salivary glands is associated with histopathology of minor salivary glands, serology of SS, and salivary gland function.

AB0627 Physical activity by self-reported physical activity and lupus nephritis in patients with systemic lupus erythematosus

Background Physical activity is found to be associated with clinical status such as disease activity, organ damage, disability, fatigue, and quality of life in systemic lupus erythematosus (SLE). Objectives The aim of this study was to identify the association between physical activity and disease activity and organ damage in patients with SLE. Methods A total of 415 patients with SLE were consecutively enrolled from KORean lupus Network (KORNET) registry. This registry assessed clinical features, disease activity Systemic Lupus Erythematosus Disease Activity Index 2000 [SLEDAI-2K]), and disease damage (Systemic Lupus International Collaborating Clinics/American College of Rheumatology [SLICC/ACR] damage index [SDI]) at the enrollment of study. Self-reported physical activity was measured by International Physical Activity Questionnaire (IPAQ). Statistical analyses were used by Mann-Whitney U test and multivariate logistic regression analysis. Results There is significant difference of vigour activity between patients with lupus nephritis (n=93) and without lupus nephritis (n=322) (p=0.012), but not moderate and walking activities. In contrast, the differences of each physical activity, walking, moderate, and vigorous intensity, according to SLEDAI-2K and SDI were not found. In addition to high PCS of SF-36 (p=0.006) and SLEDAI-2K (p=0.038), less vigorous physical activity were related with lupus nephritis (p=0.033). However, the risk of CVD was not associated with physical activity of SLE. Conclusions This study showed that patients with lupus nephritis had less vigorous physical activity. It implicates that SLE-related organ damage might be associated with levels of physical activity. References [1] Costenbader KH, Wright E, Liang MH, Karlson EW. Cardiac risk factor awareness and management in patients with systemic lupus erythematosus. Arthritis Rheum2004;51:983–8. [2] Katz P, Julian L, Tonner MC, Yazdany J, Trupin L, Yelin E, Criswell LA. Physical activity, obesity, and cognitive impairment among women with systemic lupus erythematosus. Arthritis Care Res (Hoboken)2012;64:502–10. Disclosure of Interest None declared

AB0077 High-mobility group box 1 mediated monosodium urate crystal-induced nlrp3 inflammasome activation in human macrophages

Background High-mobility group box 1 (HMGB1) was identified originally as a highly conserved non-histone DNA-binding factor and recently noted as a potent inflammatory mediator under inflammatory conditions. Objectives This study is to investigate the inflammatory cascade between HMGB1 protein and activation of NLRP3 inflammasome in human macrophage under uric acid-induced inflammation. Methods The study used human U937 macrophage cell line under stimulation with monosodium urate (MSU) crystal or HMGB1. Total reactive oxygen species (ROS) were measured by flow cytometry. Interleukin-1b (IL-1b), NLRP3, TXNIP, HMGB1, NF-kB, IkBa, and caspase-1 protein expression was detected using western blotting. IL-1b, IL-18, caspase-1, and HMGB1 gene expression were assessed by quantitative real-time polymerase chain reaction. Intracellular HMGB1 expression was assessed by immunofluorescent staining with MitoTracker Red. Results MSU crystals induced HMGB1 and ROS production by activation of NF-kB signal pathway in human macrophages. HMGB1 mRNA expression was markedly attenuated under stimulation using TXNIP siRNA. Enhanced release of IL-1 was noted through increased HMGB1 expression and TXNIP-mediated NLRP3 infammasome activation under stimulation of MSU. Combination of MSU and HMGB1 augmented NLRP3 inflammasome, compared to either MSU or HMGB1 stimulation. Conclusions This study demonstrated that HMGB1 is a crucial molecule for ROS-mediated TXNIP and NLRP3 inflammasome activation in uric acid-induced inflammation. Disclosure of Interest None declared

Effect of alcohol consumption and smoking on disease damage in systemic lupus erythematosus: data from the Korean Lupus Network (KORNET) registry:

Background We assessed correlations of smoking habits and alcohol consumption with disease activity or damage in patients with systemic lupus erythematosus (SLE). Methods A total of 505 patients with SLE were enrolled in the Korean Lupus Network (KORNET) SLE registry from January 2014 to January 2016. Disease activity and organ damage were measured by the SLE Disease Activity Index 2000 (SLEDAI-2K) and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index, respectively. Multivariate logistic regression analysis was used to analyze associations with cutaneous lesions. Results There were no differences in SLEDAI-2K and SLICC/ACR damage indices according to either smoking status or alcohol consumption. More frequent cutaneous damage was observed in current alcohol drinkers compared with non-current alcohol drinkers (p = 0.020). Cutaneous damage was significantly associated with alcohol consumption (odds ratio (OR) 4.048, 95% confidence interval (CI) 1.251–12.102, p = 0.020). Both low (1–5 glasses/week) and high (≥6 glasses/week) amounts of alcohol consumption had a significant impact on cutaneous damage compared with the absence of current alcohol consumption (p = 0.033 and p = 0.027, respectively). Pairwise comparison of alcohol consumption and smoking status with cutaneous damage showed that only alcohol consumption was significantly associated with the presence of cutaneous damage, compared with non-current alcohol consumption and non-current smoking (OR 3.513, 95% CI 1.130–10.920, p =0.030). Conclusions Current alcohol consumption, but not smoking, might influence the development of cutaneous damage in patients with SLE.

THU0257 Enhanced activation of NLRP3 inflammasomes in patients with SJÖGREN's syndrome

Background There has been data about pathogenic role of NLRP3 inflammasome in Sjögrens syndrome. However, linkage between their clinical features and NLRP3 inflammasome has not been clearly defined. Objectives The aim of this study is to identify the association of NLRP3 inflammasome with clinical features in patients with primary Sjögrens syndrome. Methods A total 25 female patients with Sjögrens syndrome and gender-matched 25 healthy controls were consecutively enrolled. The mRNA expression for target genes including NLRP3, ASC, caspase-1, IL-1b, and IL-18 in peripheral blood mononuclear cells (PBMCs) were measured using real-time polymerase chain reaction. Serum IL-1b and IL-18 expression were also measured by ELISA method. Clinical information and disease activity and damage for Sjogrens syndrome such as EULAR Sjögrens Syndrome Disease Activity Index (ESSDAI) and Sjögrens Syndrome Disease Damage Index (SSDDI) were collected at the time of enrollment. Statistical analysis were applied including Spearmans correlation coefficient and Mann-Whitney t-test. Results Patients with Sjögrens syndrome was found to be highly expressed in mRNA IL-1b and its protein, compared to controls (p<0.001 and p=0.001, respectively). The mRNA levels of caspase-1 and ASC were significantly higher than those in controls (p=0.021 and p=0.008, respectively), but not mRNA level of NLRP3. The mRNA level of IL-1b is closely related with mRNA level of NLRP3 and ESR (r =0.549, p<0.001 and r =0.577, p=0.003, respectively). Serum IL-1b protein expression in Sjögrens syndrome was found to be associated with mRNA level of caspase-1. Based on SSDDI, patients with SSDDI ≥1 was older and higher IL-1b and NLRP3 mRNA expression, compared to those with SSDDI =0 (p=0.035, p=0.005, and p=0.016, respectively). Conclusions This study confirmed that activation of NLRP3 inflammasome might implicated the pathogenesis of Sjögrens syndrome. Disclosure of Interest None declared

AB0326 Influence of nonsteroidal anti-inflammatory drugs on arterial stiffness in patients with rheumatic diseases

Background The association with adverse cardiovascular (CV) events and NSAIDs has been the topic of much debate. Objectives The aim of the present study was to investigate the effects of continuing NSAIDs therapy on predictable parameters for CV events. Methods We enrolled 155 patients with variable rheumatic diseases (95 rheumatoid arthritis, 49 systemic lupus erythematosus, 3 behçets disease, 3 gout, 5others.) who were free from established CV diseases and had taken cardiovascular function tests from June 2015 to June 2016. They were divided into two groups depending on whether or not to have taken NSAIDs therapy for at least 5 years: NSAIDs taking group (91 patients) vs. non NSAIDs taking group (64 patients). For evaluating heart function, transthoracic echocardiography was used. Arterial stiffness was assessed using brachial-ankle pulse wave analysis. Results There were no significant differences in blood pressure, serum creatinine, serum hemoglobin, total cholesterol, erythrocyte sediment rate, C-reactive protein, disease duration, age, and smoking history between the groups. The NSAIDs taking group had a higher median (95% Cl) baPWV (brachial-ankle pulse wave velocity) and median (95% Cl) mean pulmonary artery pressure (mPAP) than non-NSAIDs taking group: baPWV 13.72 (12.77–15.62) vs. 15.29 (13.93–17.63) m/s, p=0.005; mPAP 26.5 (22.8–30.5) vs. 30.5 (27.3–32.3) mmHg, p=0.011. But baPWV and mPAP were not significantly different between selective cyclooxygenase-2 inhibitor (22 patients) and nonselective NSAIDs (69patients): baPWV 15.33 (13.98–17.63) vs. 14.83 (13.82–17.39) m/s, p=0.191; mPAP 29.0 (24.5–34.5) vs. 30.0 (26.0–33.0) mmHg, p=0.960. Conclusions Our study suggests that continuing NSAIDs therapy is associated with increased arterial stiffness in patients with rheumatic diseases, independently noted to increase the incidence of cardiovascular disease. Disclosure of Interest None declared