Nóra Bodnár

Assessment of Subclinical Vascular Disease Associated with Ankylosing Spondylitis

Objective. Studies indicate that ankylosing spondylitis (AS), as well as rheumatoid arthritis, may be associated with accelerated atherosclerosis and vascular disease. We assessed endothelial dysfunction, carotid atherosclerosis, and aortic stiffness in AS in context with clinical and laboratory measurements. Methods. Forty-three patients with AS and 40 matched healthy controls were studied. We assessed common carotid intima-media thickness (ccIMT), flow-mediated vasodilation (FMD), and pulse-wave velocity (PWV) in association with age, disease duration, smoking habits, body mass index, patient’s assessment of pain and disease activity, Bath AS Disease Activity Index, Bath AS Functional Index (BASFI), metric measurements, erythrocyte sedimentation rate, C-reactive protein, and HLA-B27 status. Results. We found impaired FMD (6.85 ± 2.98% vs 8.30 ± 3.96%; p = 0.005), increased ccIMT (0.65 ± 0.15 vs 0.54 ± 0.15 mm; p = 0.01), and higher PWV (8.64 ± 2.44 vs 8.00 ± 1.46 m/s; p = 0.03) in patients with AS compared to controls, respectively. We also found that ccIMT negatively correlated with FMD (r = −0.563; p = 0.0001) and positively correlated with PWV (r = 0.374; p = 0.018). Both ccIMT and PWV correlated with disease duration (r = 0.559; p = 0.013 and r = 0.520; p = 0.022, respectively), BASFI (r = 0.691; p = 0.003 and r = 0.654; p = 0.006), decreased lumbar spine mobility (r = −0.656; p = 0.006 and r = −0.604; p = 0.013), chest expansion (r = −0.502; p = 0.047 and r = −0.613; p = 0.012), and increased wall-occiput distance (r = 0.509; p = 0.044 and r = 0.614; p = 0.011). Conclusion. In this well characterized AS population, impaired FMD and increased ccIMT and PWV indicate abnormal endothelial function and increased atherosclerosis and aortic stiffness, respectively. The value of noninvasive diagnostic tools needs to be further characterized.

Biologics — beyond the joints

Biologics including tumor necrosis factor α (TNF-α), interleukin-6 receptor (IL-6R), T and B cell inhibitors are very effective therapeutic agents for the treatment of arthritides. These compounds effectively improve articular symptoms and inhibit joint damage. In this respect, there are no major differences in the efficacy of the available biologics. However, many arthritis patients also exert extra-articular features, systemic manifestations of the disease. These associated conditions include uveitis, inflammatory bowel disease, psoriasis, secondary bone loss and cardiovascular disease. There have been data suggesting that there may be differences in the effects of various TNF inhibitors, rituximab and tocilizumab on the systemic manifestations described above. At present, we do not always have sufficient evidence to confirm these differences, therefore, more information should be obtained from large trials and long-term observational studies.

Increased production of asymmetric dimethylarginine (ADMA) in ankylosing spondylitis: Association with other clinical and laboratory parameters

OBJECTIVE Asymmetric dimethylarginine (ADMA) has been associated with atherosclerosis, vascular diseases and, recently, also with arthritis including rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS Serum ADMA, arginine and symmetric dimethylarginine (SDMA) levels were assessed by liquid chromatography in 61 AS and 26 osteoarthritis (OA) patients with no known cardiovascular disease. RESULTS Serum ADMA levels were significantly increased in AS compared to OA patients (0.95 ± 0.17 μM versus 0.70 ± 0.25 μM; p < 0.001). There were no differences in serum arginine and SDMA levels. Serum ADMA levels also positively correlated with age (R = 0.258; p = 0.043), body mass index (R = 0.368; p = 0.003), erythrocyte sedimentation rate (R = 0.329; p = 0.009) and ADMA levels negative correlated with chest expansion (R = -0.251; p = 0.04). No correlations were found between ADMA levels and disease duration, pain intensity, BASDAI, BASFI, BASMI, quality of life, CRP, HLA-B27 positivity, endothelial dysfunction or carotid atherosclerosis. CONCLUSION ADMA may serve as a marker of systemic inflammation and may reflect functional immobility in AS. Further studies are needed to assess the possible role of ADMA in AS and AS-related vascular disease.

Longterm effects of rituximab on B cell counts and autoantibody production in rheumatoid arthritis: Use of high-sensitivity flow cytometry for more sensitive assessment of B cell depletion

Objective. To assess the efficacy and safety of longterm rituximab (RTX) therapy for rheumatoid arthritis (RA) and study correlations among B cell depletion, clinical response, and autoantibody production. Methods. Seventy-seven patients with moderate or high RA activity received RTX and were re-treated every 6 months regardless of clinical response. All patients received at least 5 cycles. We assessed 28-joint Disease Activity Score (DAS28), IgM rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) levels at baseline, after 15 days, and then every 6 months for 24 months. Absolute CD19+ B lymphocyte counts were determined in 50 patients using high-sensitivity flow cytometry (hsFACS) by reading 100,000 events. Results. After 6, 12, 18, and 24 months, 51.6%, 51.9%, 73.3%, and 83.8% of patients, respectively, showed good European League Against Rheumatism responses. Significant and sustained decreases in IgM RF and ACPA levels were observed as early as 6 months and 12 months, respectively. The baseline mean absolute B cell number was 0.234 g/l. B cell numbers diminished significantly after the very first infusion by Day 15 (0.104 g/l; p = 0.007); they further decreased until 24 months (0.0013 g/l; p < 0.001). One RTX infusion resulted in incomplete depletion in 76.7% of patients. Upon RTX treatment, changes in CD19+ B cell numbers positively correlated with changes in DAS28 (r = 0.963, p = 0.008) and IgM RF (r = 0.859, p = 0.028), but not with changes in ACPA production (r = 0.726, p = 0.102). The correlations between B cell numbers and DAS28 were observed in both ACPA-seropositive (r = 0.999, p < 0.0001) and ACPA-negative patient subpopulations (r = 0.962, p = 0.009). The correlation between CD19+ cell numbers and IgM RF was observed only in the ACPA-positive population (r = 0.944, p = 0.005) but not in seronegative patients (r = 0.398, p = 0.435). No safety issues arose. Conclusion. In RA, clinical response to RTX is associated with the extent of B cell depletion and with autoantibody production. Changes in CD19+ B cell numbers correlate with those in disease activity and, in seropositive patients, also with IgM RF, but not with ACPA production. We found that hsFACS may be a useful method to more accurately assess incomplete B cell depletion.

Anti-mutated citrullinated vimentin (anti-MCV) and anti-65 kDa heat shock protein (anti-hsp65): New biomarkers in ankylosing spondylitis

INTRODUCTION Citrullination as well as anti-citrullinated protein/peptide antibodies (ACPA) have been implicated in the pathogenesis of rheumatoid arthritis (RA). While ACPAs are specific and sensitive markers for RA, there have been hardly any reports regarding ACPAs in ankylosing spondylitis (AS). The possible role of antibodies to Mycobacterial 65 kDa heat shock protein (hsp65) has not been characterized in AS. As new laboratory biomarkers of AS are needed, we investigated the prevalence of anti-mutated citrullinated vimentin (MCV) and anti-hsp65 antibodies in AS. METHODS Altogether 43 AS and 44 healthy controls were included in the study. Anti-MCV and anti-hsp65 were determined in sera by commercial and in-house ELISA, respectively. Serum autoantibody levels were correlated with ESR, CRP, HLA-B27 status, smoking habits, pain intensity, BASDAI, BASFI and BASMI indices. RESULTS Patients with AS had significantly higher serum anti-MCV levels (17.3 U/mL, range: 8.3-31.5 U/mL) in comparison to healthy subjects (8.9 U/mL, range: 5.4-13.3 U/mL) (p<0.01). Sixteen of the 43 AS patients (37%) and none of the 44 healthy controls (0%) were anti-MCV positive using the cut-off value recommended by the manufacturer (>20 U/mL). The mean anti-hsp65 concentration in AS sera was 124.8 AU/mL (range: 27.2-1000 AU/mL), while controls exerted significantly lower anti-hsp65 levels (mean: 51.8 AU/mL; range: 22.5-88.5 AU/mL) (p<0.001). Correlation analysis revealed that both anti-MCV positivity (r=0.613; p=0.012) and absolute serum anti-MCV levels (r=0.553; p=0.021) correlated with anti-hsp65 levels. Anti-MCV positivity also correlated with ESR (r=0.437; p=0.03). CONCLUSIONS Anti-MCV and anti-hsp65 may be novel biomarkers in AS.

A4.3 Effects of anti-TNF therapy on markers of bone homeostasis in rheumatoid arthritis and ankylosing spondylitis

Background and objectives Uncoupling of bone resorption and formation has been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Generalised bone loss and erosions are characteristic for RA, while in AS, bone formation overrides resorption. The RANKL/OPG and the Wnt/DKK-1/sclerostin systems have been implicated in disturben bone homeostasis in arthritides. Anti-TNF biologics may beneficially influence erosions and bone loss in RA, however, they have little effect on bone formation in AS. In the present study, we assessed the effects of 1-year anti-TNF treatment on various bone biomarkers. Patients and methods Altogether 43 arthritis patients including 30 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 13 AS patients treated with ETN were included in a 12-month follow-up study. Disease activity (DAS28 or BASDAI), CRP, IgM rheumatoid factor, anti-CCP, calcium (Ca), phosphate (P), osteocalcin (OC), P1NP, CTX, BNP, sclerostin (SOST), DKK-1, soluble RANKL (sRANKL), cathepsin K (cathK) and vitamin D3 (vitD) levels were assessed at baseline, as well as 6 and 12 months after treatment initiation. Results Anti-TNF treatment was highly effective in both diseases, as the mean DAS28 decreased from 6.32 to 3.16 (p = 0.02) in RA, mean BASDAI decreased from 5.87 to 1.84 (p < 0.001) in AS. In RA, anti-TNF treatment significantly decreased DKK-1 (60.5 ± 28.9 pM and 54.7 ± 20.8 pM, p = 0.036) and CathK (28.7 ± 6.2 pm and 26.8 ± 4.0 pm, p = 0.014) but increased SOST (107.0 ± 47.5 pM and 131.2 ± 85.2 pM, p = 0.04) levels from baseline to 12 months, respectively. In RA, ETN and CZP treatment also increased OPG/sRANKL ratio after 6 months (51.9) vs. baseline (43.9) (p = 0.01). In AS, ETN therapy significantly increased the bone formation marker P1NP (49.4 ± 19.0 pM and 56.9 ± 28.7 pM, p = 0.03) and SOST (70.6 ± 29.0 pM and 82.4 ± 48.3 pM, p = 0.022) levels from baseline to 12 months, respectively. ETN therapy also increased OPG/sRANKL ratio after 6 months (44.5) and 12 months (46.9) compared to baseline (34.5) in AS (p < 0.01). Both baseline and 12-month SOST levels were significantly lower in AS compared to RA (p < 0.001). When RA and AS data were pooled, TNF inhibition resulted in significantly decreased DKK-1 (p = 0.035) and cathK (p = 0.008) but increased P1NP (p = 0.04) and SOST (p = 0.04) after 12 months. In this study, biologics did not affect Ca, P, OC, CTX, BNP, vitD levels. Conclusions In a mixed cohort of RA and AS patients, anti-TNF therapy resulted in a restoration of bone homeostasis by decreasing DKK-1 and CathK, increasing P1NP, SOST and OPG/sRANKL ratio. Lower SOST levels in AS compared to RA, as well as the induction of bone formation over resorption may account for the inefficacy of TNF inhibitors on syndesmophyte formation in AS.

A1.83 Long-term effects of etanercept and certolizumab pegol treatment on vascular function and lipid parameters in rheumatoid arthritis and ankylosing spondylitis

Background and Objectives Cardiovascular (CV) morbidity and mortality are increased in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Biologics may influence vascular function and lipids in arthritides, however, most studies have been short-term and less information has become available on etanercept (ETN) and certolizumab pegol (CZP). We wished determine the effects of these TNF blockers on common carotid intima-media thickness (ccIMT), brachial artery flow-mediated, endothelium-dependent vasodilatation (FMD) and the arterial stiffness marker pulse wave velocity (PWV) in context with laboratory assessments in RA and AS patients after 12 months of biological therapy. Materials and Methods Twenty-seven RA patients (23 female, 4 male) and 17 AS patients (14 males, 3 females) were included. RA patients received either ETN or CZP and AS patients were treated with ETN for 12 months. Brachial and carotid ultrasonography was performed to determine FMD, ccIMT and PWV, respectively. We also assessed immunological, inflammatory and metabolic laboratory markers. Results In RA, at baseline, mean ccIMT was 0.56 mm (normal range: 0.4-0.9 mm), mean FMD was 6,5% (normal: >10%) and the mean PWV was 8.4 m/s (normal range: 4-20m/s). At baseline, ccIMT correlated with disease duration (R = 0.446, p = 0.015), while FMD and PWV did not. ccIMT (R = 0.393, p = 0.023) and PWV (R = 0.511, p = 0.005) also correlated with age at RA onset. PWV correlated with serum triglyceride levels. In AS, at baseline, mean ccIMT was 0.47 mm (normal range: 0.4-0.9 mm), mean FMD was 6,9% (normal: >10%) and the mean PWV was 6.4 m/s (normal range: 4-20m/s). At baseline, a significant inverse correlation was observed between CRP and HDL-C levels (R = -0.518, p = 0.033). ccIMT strongly correlated with BASDAI R = 0.881, p = 0.001). After 12 months of anti-TNF treatment in RA, DAS28 (p < 0.001), CRP (p = 0.004). FMD (p = 0.04) and PWV (p = 0.035) significantly improved. In AS, 12 months of ETN treatment resulted in significant improvement in BASDAI (from 6.14 to 1.25; p < 0.001), CRP (from 13.4 to 2.3 mg/l; p = 0.002). FMD (from 6.9% to 9.3%; p = 0.01) and PWV (from 6.4 to 5.4 m/sec, p = 0.045), while ccIMT showed no change. There have been no significant changes in lipid levels in AS. Conclusions In patients with RA and AS, FMD, a marker of endothel dysfunction and PWV, a marker of arterial stiffness significantly improved after 12 months of anti-TNF treatment. ccIMT may require more time to improve. These beneficial vascular changes were associated with improved disease activity.

Safety and efficacy of etanercept therapy in ankylosing spondylitis patients undergoing phacoemulsification surgery

Safety and efficacy of etanercept therapy in ankylosing spondylitis patients undergoing phacoemulsification surgery SIR, AS is a chronic, progressive inflammatory disease that generally begins in the third decade of life. Ocular manifestations, namely acute anterior uveitis, or iridocyclitis are its most common extra-articular manifestations; 25– 30% of the patients develop these symptoms in the course of the disease [1]. Besides traditional immunosuppressive agents, biological therapy, primarily TNF-a inhibitors, resulted in significantly better outcome in patients with AS. Among TNF-a blockers, etanercept is a 75-kDa dimeric soluble form of the p75 TNF-a receptor linked to human IgG1 [2]. Although the number of AS patients treated with biologics is increasing, unfortunately there are scant data on the safety of etanercept therapy in surgically treated AS patients [3], in particular in cataract surgery. On the basis of the available data, it is proved that during the use of etanercept therapy there is a slightly increased incidence of bacterial infection; nevertheless, the risk of serious infections does not seem to be increased [4]. However, discontinuing TNF-a inhibitor therapy may result in exacerbation of AS [5]. AS patients present a challenge to the cataract surgeon because of chronic uveitis and sometimes locomotor deformities. Phacoemulsification is a routinely performed operation in cataract surgery, whereby the opacified crystalline lens is extracted through a 1.8to 3.2-mm clear corneal, or scleral incision and an artificial lens is implanted. In the present study, we aimed to assess the possible effects of etanercept therapy in AS patients undergoing 1-day phacoemulsification surgery. In our current study, 10 patients were on biological therapy (etanercept) (Group I), while 14 received nonbiological therapy (Group II). The diagnosis of AS was established based on the New York classification criteria [6]. All the patients developed secondary cataract after anterior uveitis and required cataract extraction. The study was approved by the institutional ethics committee (Regional and Institutional Ethics Committee, Medical and Health Science Centre, University of Debrecen). Written informed consent was obtained from all patients. Simultaneously, 14 patients also with definitive, advanced AS without receiving biological therapy were operated for cataract with phacoemulsification (Group II). Seven patients of Group II were administered 4 mg methylprednisolone daily for 3 weeks pre-operatively as steroid therapy (Group II/a); the remaining seven received no pre-operative steroid therapy (Group II/b). Disease activity of AS patients was monitored by the standard BASDAI, BASFIs and BASMI, respectively. In addition, ESR and CRP levels, as biomarkers of systemic inflammation, were also determined pre-operatively. None of the patients had any episode of uveitis in the last 3 months before phacoemulsification, i.e. the uveitis was inactive in the last 3 months. The follow-up time was a minimum of 24 months. Neither in Group I nor in Group II were there any intra-operative complications. Regarding the aqueous flare and cells, the Tyndall effect was greater on the first post-operative day and then declined rapidly in the first week and more gradually thereafter in a normal population without AS [7]. Out of 10 patients in Group I, 8 had an uneventful post-operative period with a regular healing rate. Only two patients developed mild post-operative anterior uveitis, i.e. Tyndall effect 1þ, and none of them had posterior synechiae (adhesion between the anterior surface of the artificial posterior chamber intraocular lens and the iris). Anterior uveitis is successfully treated with combined local steroid (dexamethasone) and antibiotic (gentamycin) injection administered for 2 days. During the follow-up period, none of the patients developed intraocular infection, or had any other complications. Concerning the 14 AS patients without biological therapy (Group II), 3 patients out of the 7 being administered pre-operative steroid therapy (Group II/a) had Tyndall effect 1þ and 1 had 2þ post-operatively. Two of them had posterior iris synechiae. Two patients of Group II/b had Tyndall effect 1þ post-operatively, two also had Tyndall phenomenon 2þ, one had Tyndall effect 3þ and three of them had posterior synechiae. We did not discontinue the otherwise highly effective etanercept therapy in patients, and indeed, we did not observe any complications in 8 out of these 10 patients. Only two patients had easily manageable, mild postoperative anterior uveitis. AS patients without biological therapy had a more severe form of post-operative anterior uveitis, whereas patients given etanercept had milder forms of uveitis and none had synechiae post-operatively. Although some data have shown that TNF blockers can induce new uveitis flares [8], AS patients seem to have greater improvement in the frequency of uveitis flares after treatment with infliximab than etanercept [9]. Infliximab resulted in better clinical responses than etanercept in patients with JRA [10]. In summary, anti-TNF-a therapy, such as etanercept, may control the musculoskeletal disease, as well as uveitis simultaneously in patients with AS. Furthermore, if these patients need elective cataract surgery, the discontinuation of anti-TNF-a treatment is unnecessary.

A6.15 Genetic signatures may be associated with vascular pathology in rheumatoid arthritis

Background and objectives Accelerated atherosclerosis, increased cardiovascular (CV) morbidity and mortality have been associated with rheumatoid arthritis (RA). In single SNP studies, CD40, HLADRB1, MTHFR, SMAD3 and possibly other alleles have been associated with cardiovascular disease (CVD) or vascular pathophysiology in RA. Endothelial dysfunction, carotid atherosclerosis and arterial stiffness that may predict the development of CVD are assessed by bracial artery flow-mediated vasodilation (FMD), common carotid intima-media thickness (ccIMT) and arterial pulse-wave velocity (PWV), respectively. In this study, we wished to determine expression profiles of multiple genes that may differentiate between physiological and pathological vascular function in RA. Patients and methods Altogether 16 RA patients were recruited. FMD, ccIMT and PWV were assessed in all patients using standard B-mode ultrasound techniques. FMD < 6%, ccIMT > 0.6mm and PWV > 9 m/sec were considered abnormal. Peripheral blood mononuclear cell samples were obtained and used in microarray. The signature of those genes were determined by principal component analysis (PCA) and hierarchic clustering (GeneSpring software), which significantly differentiated patient subsets with normal vs. abnormal FMD, ccIMT and PWV. Results Among RA patients, 11 had low (impaired) and 5 had normal FMD, 11 had high (increased) and 5 had normal ccIMT and 9 had high (increased) and 7 had normal PWV. Altogether 20 genes differentiated patients with low vs. normal FMD. Altogether 33 genes separated high vs. normal PWV. Finally, 240 genes differentiated increased vs. normal ccIMT. Conclusions Using microarray, genetic signatures may differentiate RA patients with and without vascular pathology.

AB0082 Effects of Anti-Tnf Therapy on Circulating Oxldl-Beta2Gpi Complex Levels in Arthritis

Background and objectives Accelerated atherosclerosis, increased cardiovascular (CV) morbidity and mortality have been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Oxidised LDL (oxLDL) and beta 2 glycoprotein I (beta2gpI) antigens have been implicated in atherosclerosis, as well as antiphospholipid syndrome. High circulating oxLDL/beta2gpI levels may reflect vascular damage in acute coronary, syndrome, SLE, and other autoimmune diseases. However, the role of these complexes in RA and AS has not yet been evaluated in relation to therapy. Therefore, circulating complex levels, as well as the effects of anti-TNF therapy on these complexes were assessed in RA and AS patients. Complex levels were also correlated with various autoimmune-innflammatory and metabolic markers. Patients and methods Altogether 43 arthritis patients including 30 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 13 AS patients treated with ETN were included in a 12-month follow-up study. Circulating oxLDL/beta2gpI complexes were assessed by an AtherOx® ELISA system (Corgenix). In addition, disease activity (DAS28 or BASDAI), CRP, IgM rheumatoid factor, anti-CCP, and lipid levels (total cholesterol, TC; LDL-C, HDL-C and triglyceride) were also assessed. Assessments were performed at baseline, as well as 6 and 12 months after treatment initiation. Results Anti-TNF treatment was highly effective in both diseases, as the mean DAS28 decreased from 6.32 to 3.16 (p = 0.02) in RA, mean BASDAI decreased from 5.87 to 1.84 (p < 0.001) in AS. In RA, AS and the mixed arthritis population (n = 43) baseline oxLDL/beta2gpI levels were 0.235 ± 0.1, 0.245 ± 0.1 and 0.238 ± 0.1, respectively. There were no significant differences between RA and AS patients. In RA, ETN/CZP treatment resulted in non-significant decreases in complex levels after 6 months (0.214 ± 0.1) and 12 months (0.206 ± 0.1). In AS, oxLDL/beta2gpI complex levels did not change after 6 months of ETN therapy, but significantly decreased after one year (0.195 ± 0.1; p = 0.01). In the RA+AS population, anti-TNF treatment significantly decreased oxLDL/beta2gpI levelés after 12 months (0.203 ± 0.1, p = 0.02). In addition, baseline oxLDL/beta2gpI complex levels positively correlated with TC (RA: r = 0.563, p = 0.002; AS: r = 0.542, p = 0.049; RA+AS: r = 0.532, p < 0.001), and LDL-C (RA: r = 0.630, p < 0.001; AS: r = 0.756, p = 0.004; RA+AS: r = 0.648, p < 0.001) in both diseases. Circulating oxLDL/beta2gpI levels did not correlate with DAS28, BASDAI or CRP. Conclusions In a mixed cohort of RA and AS patients, anti-TNF therapy suppressed the circulating levels of oxLDL/beta2gpI complexes, markers of atherosclerosis and vascular disease in SLE or APS. Moreover, oxLDL/beta2gpI levels correlated with TC and LDL-C in arthritides. oxLDL/beta2gpI complexes do not seem to be markers of disease activity in RA or AS.