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Validated methods for assessment of subclinical atherosclerosis in rheumatology.

Rheumatoid arthritis, as well as other types of arthritides and connective tissue diseases, is associated with accelerated atherosclerosis, and increased cardiovascular morbidity and mortality. The early signs of cardiovascular disease therefore need to be recognized in patients with these conditions so that effective cardiovascular protection can be introduced. This Review provides an overview of validated techniques that are currently available to determine subclinical atherosclerosis in patients with rheumatic conditions. Techniques for early assessment of endothelial dysfunction include brachial artery flow-mediated vasodilation and laser Doppler flowmetry. Coronary circulation can be assessed by measuring coronary flow reserve using CT, MRI or PET based techniques. The standard indicators of arterial stiffness are pulse-wave velocity and the augmentation index. Carotid atherosclerosis is determined by the common carotid intima–media thickness (ccIMT) measurement or by the assessment of plaques and plaque areas. The combination of ccIMT with plaque assessment is likely to increase the predictive value of this approach. The potential use of a multimarker approach to increase the diagnostic and prognostic value of these clinical assessments is also discussed.

Changes in the expression of Na+/K+-ATPase isoenzymes in the left ventricle of diabetic rat hearts: effect of insulin treatment

SummaryNa+/K+-ATPase related strophanthidin sensitive 3-O-methylfluorescein-phosphatase activity, [3H]ouabain binding and expression of Na+/K+-ATPase subunit isoforms were measured in the left ventricle of the heart of normal and streptozotocin-diabetic rats with and without insulin treatment. Compared to control animals, the enzyme activity was 0.75 ± 0.09 and 0.62 ± 0.06 times lower in rats diabetic for 2 and for 4 weeks, respectively. This was associated with a proportional decrease of the [3H]ouabain binding sites. Immunoblots indicated a 0.76 ± 0.08 and 0.61 ± 0.08-fold decrease of alpha1, a 0.68 ±0.09 and 0.41 ± 0.04-fold decrease of alpha2 subunit in 2- and 4-week diabetic rats, respectively relative to controls. Beta1 subunit decreased proportionally 0.71 ± 0.07 and 0.38 ± 0.06-fold, and beta2 decreased 0.75 ± 0.08 and 0.31 ± 0.06-fold, respectively. Northern blot analysis revealed a significant reduction in mRNA level of Na+/K+-ATPase subunit isoforms after 2 and 4 weeks of diabetes (for alpha1 66.2 ± 8.2 and 55.9 ± 7.8% of controls for alpha2 91.7 ± 12.1 and 41.1 ± 7.1 % of controls and for beta subunit 93.4 ±11.1 and 49.8 ±6.8% of controls, respectively). Although, mRNA levels of isoform reverted to even higher levels than the control values after insulin treatment, insulin caused only a partial recovery of enzyme activity, [3H]ouabain binding capacity and protein expression. We have obtained evidence that in cardiac left ventricle there are more than one type of Na+/K+-ATPase alpha and beta subunit isoforms which are affected in diabetes and by insulin treatment. The time course of diabetes induced changes and the degree of involvement suggest that the Na+/K+-ATPase isoforms are altered individually.

Na+,K+‐ATPase is modulated by angiotensin II in diabetic rat kidney – another reason for diabetic nephropathy?

Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes in man and in streptozotocin‐induced (STZ) diabetic rats. This study investigates the effect of untreated STZ‐diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na+,K+‐ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ‐diabetes (i.v. 65 mg kg−1) a subgroup of control (C) and diabetic (D7) Wistar rats were treated with ANGII (s.c. minipump 33 μg kg−1 h−1 for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA α‐1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA α‐1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A, CA versus D7A). NKA α‐1 Ser23 phosphorylation was higher both in D7 and ANGII‐treated rats in the non‐cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA α‐1 immunopositivity on the basolateral membrane of proximal tubular cells, while both D7 and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na+ pump is no longer functional. ANGII also caused translocation from the basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.

Pharmacogenetics and pharmacogenomics in rheumatology

Pharmacogenetics and pharmacogenomics deal with possible associations of a single genetic polymorphism or those of multiple gene profiles with responses to drugs. In rheumatology, genes and gene signatures may be associated with altered efficacy and/or safety of anti-inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs) and biologics. In brief, genes of cytochrome P450, other enzymes involved in drug metabolism, transporters and some cytokines have been associated with responses to and toxicity of non-steroidal anti-inflammatory drugs, corticosteroids and DMARDs. The efficacy of biologics may be related to alterations in cytokine, chemokine and FcγR genes. Numerous studies reported multiple genetic signatures in association with responses to biologics; however, data are inconclusive. More, focused studies carried out in larger patient cohorts, using pre-selected genes, may be needed in order to determine the future of pharmacogenetics and pharmacogenomics as tools for personalized medicine in rheumatology.

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.

Autoimmune atherosclerosis in 3D: How it develops, how to diagnose and what to do

Autoimmune-inflammatory rheumatic diseases, such as rheumatoid arthritis (RA) have been associated with autoimmune atherosclerosis leading to increased cardiovascular risk. Traditional risk factors, genetics, as well as the role of systemic inflammation including inflammatory cells, cytokines, chemokines, proteases, autoantibodies, adhesion receptors and others have been implicated in the development of these vascular pathologies. Cardiovascular risk may be determined by the use of currently available tools. In addition, non-invasive assessment of vascular pathophysiology by imaging, as well as laboratory biomarkers can help to refine risk assessment. With respect to prevention and therapy, traditional vasculoprotection using statins, ACE inhibitors, aspirin should be applied to patients at risk. Non-steroidal antiinflammatory drugs and corticosteroids may be pro-atherogenic, on the other hand, they may also be beneficial due to their anti-inflammatory nation. Traditional and biologic DMARDs may have significant vascular and metabolic effects. Decreasing inflammatory activity by any of these agents may lead to better CV outcome. The official EULAR recommendations on the assessment and management of cardiovascular disease in arthritides may guide the rheumatologist during the process of CV screening, prevention and treatment.

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.

THU0119 Rheumatoid Arthritis and Periodontal Disease: Association between Salivary Citrulline, ACPA Levels and Clinical Presentation

Background Periodontitis and other dental conditions have been associated with the pathogenesis of rheumatoid arthritis (RA). In addition, these dental pathologies are also important comorbidities for RA. Objectives We wished to evaluate the periodontal involvement of rheumatoid arthritis (RA) patients, and we correlated with various laboratory biomarkers including lipids, autoantibodies, serum vitamin D levels, markers of bone metabolism in relation with the periodontal condition and cariological indices; and also correlated them with salivary citrulline and anti citrullinated protein autoantibody levels with the above mentioned clinical and blood test findings. Methods Twenty-three RA patients were recruited for the study. Saliva samples were taken following whole scale periodontal and cariological examination. Protein concentration, peptidyl-citrulline and anti-cyclic citrullinated protein (anti-CCP) levels were measured from saliva samples. Blood test results were provided by rheumatologists. Citrullinated enolase protein-1 (CEP-1) level from serum was also measured. Results Periodontal diagnoses (scores) seem to have a positive dependency on LDL (R=0.722, p=0.008), PTH1 (R=0.586, p=0.022), D3 vitamin level (R=0.586, 0.022), the sum of D3/D2 (R=0.634, p=0.011) respectively, in these patients. Anti-CEP-1 positive patients had significantly higher periodontal scores (2.71±0.11 vs 2.50±0.09, p<0.05) compared to anti-CEP-1 negative subjects. Interestingly, anti-CEP-1 positive patients had significantly higher triglyceride levels compared to seronegative ones (1.81±0.17 vs 1.42±0.05 mmol/l; p<0.05). Salivary citrulline and salivary anti-CCP, (p=0.007, R: 0.583) level has a correlation with the maximum of clinical probing depth. Conclusions Our results may add further pieces to the mosaic of RA-periodontitis connection. The possible role of antimicrobial immunity, as well as the possible role of lipids and bone metabolism have also been delineated. Future therapies should aim the disruption of this framework. Disclosure of Interest None declared

AB0012 Genetic Signatures in Rheumatoid Arthritis: Changes upon Anti-TNF Therapy and Association with Response To Biological Treatment

Background Genetic signatures may be involved in the pathogenesis of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). In addition, such genetic patterns may change overtime upon treatment with anti-TNF biologics. With respect to pharmacogenomics, pre-treatment genomics may predict response or non-response to biological therapy. Objectives In the present study, we wished to determine gene expression changes due to anti-TNF therapy. Furthermore, we wished to study associations between baseline genetic signature and response to TNF blockade. Methods Altogether 23 RA and 17 AS patients were recruited for the study. Among RA patients, 10 received certolizumab pegol (CZP), and 13 etanercept (ETN). All AS patients were treated with ETN. Gene expression analysis using Affymetrix microarray and PrimeView array was performed at baseline and after 2 weeks of treatment. EULAR response criteria were used to differentiate responders (R) from non-responders (NR) after 12 weeks of treatment using GeneSpring software. Principal Component Analysis (PCA) was also performed. Changes in gene expression patterns were also determined between baseline and 2 weeks. Results In the CZP-treated RA group, 4 patients were R and 6 were NR. Altogether 453 genes showed significantly differential expression between N and NR. In the RA-ETN group, 10 patients were R, and 3 were NR. Here, 836 genes exerted differential expression. When a CZP- and ETN-treated patients, were pooled, 165 genes separated R from NR. In the AS population, 14 patients were R and 3 were NR. 177 genes differentiated between R and NR. When changes in gene expression patterns from baseline to week 2 were determined, in the RA-CZP, RA-ETN, RA-all and AS groups 370, 79, 24 and 76 genes showed significant changes in expression. Conclusions Using microarray, genetic signatures may differentiate RA patients responding or not responding to anti-TNF therapy. Furthermore, a large set of genes show differential expression before versus 2 weeks after biologic treatment. Disclosure of Interest None declared