Mirjana Sefik-Bukilica

Serum DNase I activity in systemic lupus erythematosus: correlation with immunoserological markers, the disease activity and organ involvement.

Abstract Background: Decreased activity of serum desoxyribonuclease I (DNase I) in systemic lupus erythematosus (SLE) has been reported, but its role as a biomarker in SLE is still unelucidated. Methods: Seventy-seven SLE patients (aged 39.6±13.1 years) were studied for serum DNase I activity, levels of antinuclear (ANA), anti-dsDNA [high-avidity ELISA, conventional ELISA and indirect immunofluorescence (IIF)], anti-nucleosome, anti-histone antibodies, complement components C3 and C4. SLE disease activity was evaluated by disease activity index (SLEDAI-2K). Thirty-five patients were serologically and clinically followed for 3–12 months (mean 5.6±2.8). Thirty-seven healthy blood donors were the control group. Results: DNase I activity in SLE patients was lower than in healthy controls (p<0.01). DNase I activity was in positive correlation with SLEDAI-2K (p<0.01), levels of ANA, anti-dsDNA, anti-nucleosome and anti-histone antibodies (p<0.01) and in negative correlation with C3 concentration (p<0.05). The highest correlation was found between DNase I activity and anti-dsDNA concentrations determined by high-avidity ELISA (r=0.624), followed by IIF (r=0.541) and conventional ELISA (r=0.405). In the follow-up study, DNase I activity also correlated with SLEDAI-2K (p<0.01). SLE patients with low DNase I activity more frequently had SLE-specific cutaneous lesions (p<0.05). Conclusions: Monitoring of DNase I activity simultaneously with SLEDAI-2K might be a useful tool in the follow-up of SLE. An increase of DNase I activity characterized relapse in most SLE patients, although it did not reach the levels of healthy individuals. A decrease of DNase I activity in SLE flare-ups might be a functional biomarker of a subset of patients with specific dysfunction of apoptotic chromatin degradation.

Tumor Necrosis Factor Blockade Differentially Affects Innate Inflammatory and Th17 Cytokines in Rheumatoid Arthritis

Objective. To evaluate the effect of a tumor necrosis factor-α (TNF-α) inhibitor (etanercept) on innate inflammatory and Th17 cytokines in patients with rheumatoid arthritis (RA). Methods. Serum samples were collected from 40 patients with active RA refractory to conventional disease-modifying antirheumatic drugs who initiated therapy with etanercept plus methotrexate (MTX). Treatment response was assessed at Week 24 according to the European League Against Rheumatism response criteria. Serum levels of interleukin 6 (IL-6), TNF-α, IL-32, IL-23, IL-17A, IL-21, and IL-22 were measured in patients with RA and 25 healthy controls. Results. Patients with RA had increased levels of IL-6 (p < 0.001), IL-32 (p < 0.001), IL-23 (p < 0.001), and a trend toward increased IL-21 in the sera compared to controls. At 24 weeks’ posttreatment, followup serum samples of etanercept responders had decreased levels of IL-6 (p < 0.001) and increased IL-21 (p < 0.05) and IL-32 (p < 0.001), while there were no differences in cytokine levels in non-responders. Serum IL-6 levels were positively correlated with levels of erythrocyte sedimentation rate (r = 0.458, p < 0.01), C-reactive protein (r = 0.593, p < 0.01), and 28-joint Disease Activity Score (r = 0.432, p < 0.01) at baseline. Serum IL-21 levels were positively correlated with levels of rheumatoid factor (r = 0.513, r = 0.633, both p < 0.01) and antimutated citrullinated vimentin antibodies (r = 0.515, p < 0.01; r = 0.428, p < 0.05) at baseline and after 24 weeks of treatment with etanercept. Conclusion. Multiple inflammatory pathways contribute to persistent chronic inflammation in RA. In contrast to nonresponders, etanercept therapy modulated serum cytokine levels and caused a marked decrease of IL-6 levels in responders. IL-21 might be involved in the regulation of autoantibody production in RA.

Immunoserological parameters in SLE: high-avidity anti-dsDNA detected by ELISA are the most closely associated with the disease activity.

We assessed the relationship between the serum levels of antibodies against double-stranded DNA (dsDNA), C1q, nucleosomes, histones, C3 and C4 complement components with one another, with organ involvement and overall disease activity in patients with systemic lupus erythematosus (SLE). One hundred seventy-five sera from 99 patients with SLE, 31 sera of patients with other connective tissue diseases, and 20 sera from healthy blood donors were tested. SLE disease activity was assessed by modified SLEDAI-2K (M-SLEDAI-2K), not including complement and anti-dsDNA descriptors. Anti-dsDNA antibodies were measured by indirect immunofluorescence on Crithidia luciliae (CLIFT), standard enzyme-linked immunosorbent assay (ELISA) and ELISA for high-avidity antibodies. The most significant risk factor for renal involvement were anti-C1q antibodies (OR = 3.88, p < 0.05), high-avidity anti-dsDNA antibodies for polyserositis (OR = 7.99, p < 0.01), anti-histone antibodies for joint involvement (OR = 2.75, p < 0.05), and low C3 for cytopenia (OR = 11.96, p < 0.001) and mucocutaneous lesions (OR = 3.32, p < 0.01). Multiple linear regression analysis showed that disease activity in SLE could be predicted by the levels of antibodies against dsDNA determined by standard (p < 0.05) and high-avidity (p < 0.001) ELISA, and inversely associated with concentration of C3 (p < 0.001). Using stepwise method, high-avidity anti-dsDNA antibodies were found to be in the closest association to M-SLEDAI-2K. Moreover, positive test for high-avidity anti-dsDNA antibodies appeared as an independent risk factor for moderately to severely active disease (M-SLEDAI-2K>5) (OR = 5.5, p < 0.01). The presence of high-avidity anti-dsDNA antibodies represented a risk for renal, joint, and most importantly for serosal involvement. Our results suggest that simple and reliable ELISA for high-avidity anti-dsDNA antibodies is the test of good clinical utility for the assessment of global SLE activity.

Influence of Promoter Polymorphisms of the TNF-α (-308G/A) And IL-6 (-174G/C) Genes On Therapeutic Response To Etanercept In Rheumatoid Arthritis

Summary Background: The study was undertaken to assess the influence of functional -308G/A TNF-α (rs 1800629) and -174G/C IL-6 (rs1800795) promoter polymorphisms on the therapeutic response to etanercept, a TNF-α blocker, in patients with rheumatoid arthritis (RA). Methods: Seventy-three patients suffering from active RA were studied, at baseline and 6 and 12 months after therapy. The therapeutic response was estimated according to the European League Against Rheumatism response criteria. Patients were genotyped for -308G/A TNF-α and -174G/C IL-6 polymorphisms by the PCR-RFLP method, and the influence of genotype on etanercept response was assessed. Results: No difference in the percentage of responders (patients who had DAS28 improvement > 1.2) between patients with the TNF-α-308GG and GA and AA genotype was detected after 6 and 12 months of treatment. After 12 months of treatment the percentage of responders was significantly increased in patients with the IL-6 -174GG genotype compared with those with the GC or CC genotype (p=0.006 by Chi-square test). Evaluation of the patients according to their combined IL-6/TNF-α genotypes showed that patients with the IL-6 -174GG / TNF-α -308GG genotype were more frequent among the responders compared to those with other combined genotypes (p=0.022 by Chisquare test). More precisely, all patients with the combined IL-6 -174GG / TNF-α -308GG genotype were responders after 12 months of etanercept treatment. Conclusions: The study suggests that patients who are genetically low TNF-α and IL-6 producers are the best responders to etanercept therapy.

Matrix Metalloproteinases-3 Baseline Serum Levels in Early Rheumatoid Arthritis Patients Without Initial Radiographic Changes: A Two Year Ultrasonographic Study

Objective: To investigate the association of high baseline serum levels of metalloproteinases-3 (MMP-3) with structural damage to hand and feet joints, assessed by ultrasonography (US), in patients with early, treatment-naïve rheumatoid arthritis (RA), without initial X-ray-visible erosions, during 24 months follow-up. Methods: Sixty-three early RA (European League Against Rheumatism/American College of Rheumatology 2010), disease-modifying anti-rheumatic drugs/glucocorticoid naïve patients (mean age 53.4 ± 14.1) with symptom duration ≤12 months, had baseline serum levels of MMP-3 tested. OMERACT US group definition was used to detect the presence, as well as longitudinal diameter of erosions by US at study entry and after 24 months, at the level of wrists, metacarpophalangeal (MCP2/MCP5) joints of both hands, and fifth metatarsophalangeal joints. Results: Complete data were collected from 52 out of 63 patients. High baseline serum levels of MMP-3 (MMP-3-positive) were found in 46/63 patients. 122 bone erosions in total (1.9 bone erosions/patients) were detected by US at baseline visit and 213 erosions (4.3/patients) after 24 months. MMP-3 positive patients had significantly higher total number of erosions than MMP-3-negative (p = 0.039) and higher increase in size of bone erosions in the feet but not in the hand joints after follow-up (OR 4.82 [1.23–18.9], p = 0.024; OR 1.17 [0.320–4.26], p = 0.816 respectively). Conclusion: After 2 years of follow-up, US assessment showed a higher number of new bone erosions in MMP-3-positive compared to MMP-3-negative patients with early RA and no visible initial radiographic changes. High baseline levels of MMP-3 predict significantly higher structural damage progression at the level of feet, but not at the level of hand joints.

AB0317 Do Cigarette Smoking Patients Have More Severe Early Rheumatoid Arthritis

Background Smoking in association with genetic factor can facilitate production of auto antibodies against citrullinated peptide and lead rheumatoid arthritis (RA) development (1). Objectives To investigate the influence of cigarette smoking on severity of early rheumatoid arthritis. Methods The 63 (85.7% female) early RA patients, mean age of 53.79±14.1 yrs. were enrolled in prospective observational study. All pts fulfilled EULAR/ACR 2010 criteria for diagnosis of early RA, had disease duration ≤12 months (in average 3.6 months) and have been without structural changes on radiography of hands and foot. Patients were treatment naïve for disease modifying anti-rheumatic drugs (DMARDs) and steroids. RA activity was assessed by the 28-joint disease activity score (DAS28-ESR) and bone erosions of hands and feet estimated by echosonography at the baseline, when early RA diagnosis was established and after 6 months of MTX (15 mg per week) therapy. Ultrasound examination was done by ESAOTE My Lab70 machine using 8-18 MHz linear probe according to OMERACT recommendations. The severity of smoking was estimated by pack/year: light (smoking ≤5 pack/yrs); moderate (5-20 pack/yrs) and heavy (≥20 pack/yrs), Anova statistical method was performed in data processing. Results The total 33 (52.4%) early RA pts had never smoked, 11 (17.4%) pts. had stopped smoking at list 2 years before early RA onset. Nineteen (30.2%) pts. were current smokers. Three (4.8%) pts were a light-smokers, thirteen (20.6%) pts. were a moderate-smokers and three (4.8%) pts were a heavy smokers. Initially, there was no statistical difference between group of RA non-smokers, light-smokers, moderate or heavy-smokers pts regarding value of DAS28 score (mean value: 5.7; 6.5; 5.2; 6.1 respectively, p=0.387) and number of echo erosions (mean value: 2.4; 1.7; 2.3; 3.3 respectively, p=0.814). The group of moderate and heavy-smokers had statistically significant less decrease in DAS28 score than non-smokers and light-smokers (mean value: 0.9; 0.9; 1.7; 3.2 respectively, p<0.05) after 6 months of treatment. However, there was no statistically significant difference between heavy-smokers and moderate-smokers in comparison to non-smokers and light-smokers in increasing number of new echo erosions (mean value: 1.7; 0.2; 0.3; 1.0 respectively, p>0.05) after six months of therapy with MTX. Conclusions After 6 months of MTX 15 mg/week treatment of sixty-three patients with early rheumatoid arthritis, non-smokers and light-smokers had significantly higher decrease of DAS28 score in comparison with group of moderate and heavy smokers. Number of new erosions observed by ultrasound was not significantly different between these groups. References Klareskog L, Stolt P, Lundberg K et al. Smoking May Trigger HLA–DR (Shared Epitope)–Restricted Immune Reactionsto Autoantigens Modified by Citrullination, Arthritis & Rheumatism 2006; 54 (1): 38–46. Disclosure of Interest None declared

SAT0201 Relationship of Matrix Metalloproteinase-3 With Pulmonary and Joint Involvement in Scl-70 Positive Systemic Sclerosis Patients

Background Extracellular matrix degradation is regulated mainly by matrixmetalloproteinases (MMPs). MMP-3 can degrade many components of the extracellular matrix and an increase in serum MMP-3 concentration has been proposed as a synovial derived marker of inflammation. Objectives To determine matrix metalloproteinase-3 (MMP-3) serum levels in anti Scl 70 positive patients with systemic sclerosis (SSc) and to study clinical significance and the relationship between MMP-3 and pulmonary and joint involvement Methods Forty-seven anti-Scl 70 antibodies positive patients with systemic sclerosis (43 female, 4 male) and 50 healthy controls were included in the study. All SSc patients underwent standard assessment, including laboratory tests, physical examination with joint assessment, chest X-ray and pulmonary functional tests, including diffusion capacity for carbon monoxide (DLco) and forced vital capacity (FVC). Levels of MMP-3 were measured with specific ELISA kit (Aeskulisa DF) with normal range between 18-60 ng/ml for female and 24-120ng/ml for male. Results SSc patients had significantly higher MMP-3 concentration compared to healthy donors 122.64±188.40 vs. 57.4±46.79, p<0.001. Twenty-two (46.8%) SSs pts had increased MMP-3 levels above normal range and those had more frequently pulmonary fibrosis (p<0.001) and arthritis (p<0.001). Patients with increased levels of MMP-3 had significantly more frequently reduction in DLco <75% predicted (p<0.001). Conclusions In 47 patient with systemic sclerosis and anti-Scl 70 positive antibodies, high levels of MMP-3 significantly positively correlate with pulmonary fibrosis, DLCo < 75% predicted and joint involvement (arthritis). Disclosure of Interest None Declared