O Krystufkova

Anti-HMGCR antibodies as a biomarker for immune-mediated necrotizing myopathies: A history of statins and experience from a large international multi-center study

In an effort to find naturally occurring substances that reduce cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), statins were first discovered by Endo in 1972. With the widespread prescription and use of statins to decrease morbidity from myocardial infarction and stroke, it was noted that approximately 5% of all statin users experienced muscle pain and weakness during treatment. In a smaller proportion of patients, the myopathy progressed to severe morbidity marked by proximal weakness and severe muscle wasting. Remarkably, Mammen and colleagues were the first to discover that the molecular target of statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is an autoantibody target in patients that develop an immune-mediated necrotizing myopathy (IMNM). These observations have been confirmed in a number of studies but, until today, a multi-center, international study of IMNM, related idiopathic inflammatory myopathies (IIM), other auto-inflammatory conditions and controls has not been published. Accordingly, an international, multi-center study investigated the utility of anti-HMGCR antibodies in the diagnosis of statin-associated IMNM in comparison to different forms of IIM and controls. This study included samples from patients with different forms of IIM (n=1250) and patients with other diseases (n=656) that were collected from twelve sites and tested for anti-HMGCR antibodies by ELISA. This study confirmed that anti-HMGCR autoantibodies, when found in conjunction with statin use, characterize a subset of IIM who are older and have necrosis on muscle biopsy. Taken together, the data to date indicates that testing for anti-HMGCR antibodies is important in the differential diagnosis of IIM and might be considered for future classification criteria.

Development of autoantibodies against muscle-specific FHL1 in severe inflammatory myopathies

Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed FHL1-related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-FHL1-negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic FHL1-related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM.

A1.48 Enhanced expression of CD11c on non-classical CD16 + peripheral blood monocytes in early rheumatoid arthritis

Background and Objectives Classical (CM), non-classical (NCM) and intermediate populations of human monocytes are characterised by differential expression of CD14 and CD16. NCM and IM are potent antigen presenting cells and producers of pro-inflammatory cytokines. They are enriched in patients with established rheumatoid arthritis (RA) and their numbers correlate with disease activity and response to therapy with methotrexate (MTX) and glucocorticoids (GCS). CD11c plays a role in antigen presentation and production of pro-inflammatory cytokines. Increased expression of CD11c on mononuclear blood cells and a predictive role of higher CD11c mRNA expression for response to TNFα blocking treatment in RA were described. We evaluated the expression of CD11c on monocyte subpopulations in patients with early rheumatoid arthritis (ERA) and its changes after three months of treatment with GCS and non-biological disease modifying drugs. Materials and Methods 29 patients with ERA and 10 gender and age-matched healthy controls (HC) were included in this study. ERA patients had symptom duration for <6 months and fulfilled the 2010 ACR/EULAR classification criteria for RA. Disease activity was assessed using DAS28. Monocyte subpopulations were defined according to IUIS nomenclature (CM:CD14+CD16-, NCM:CD14dimCD16++) using multicolour flow cytometry. Based on isotype and FMO controls for CD14 and CD16; two intermediate (IM) populations were evaluated (CD14+CD16dim and CD14+CD16++). CD11c expression was analysed by median fluorescence intensity. Results Expansion of both intermediate populations (IM-CD16dim, p<0.001; IM-CD16++, p = 0.02) and reduction of CM (p<0.0001) were demonstrated in ERA patients compared to HC. NCM displayed enhanced expression of CD11c compared to CM and to both IM subpopulations (p<0.001). Compared to HC, the expression of CD11c was significantly higher in both NCM (p = 0.04) and CM (p = 0.02) subpopulations. Significantly increased expression of CD11c was associated with shorter symptom duration in both IM populations (rs = -0.38 and -0.44, p<0.05). Trend towards a decrease of intermediate monocytes and increase of classical monocytes during the initial treatment with GCS was demonstrated. Decrease of CD11c expression was associated with the daily GCS dose in all subpopulations (CM, IM-CD16+or++ and NCM: rs = -0.64, -0.55 or -0.50 and -0.40; p = 0.004, 0.08 or 0.02 and 0.09) at month three. No associations with disease activity or with MTX dosage were found. Conclusions The expansion of intermediate monocyte subpopulations in ERA patients and enhancement of CD11c expression with reduction after three months of treatment with GCS suggest their possible role in the pathogenesis of RA at early stages of the disease. Acknowledgement Institutional support MZČR0002372801

The expression regulation of the HSPA1B gene in patients with myositis is not dependent on the presence of HLA-DRB1*03 risk allele

Background and objectives Genes within the major histocompatibility complex (MHC) region has a strong genetic relevance in autoimmunity development. The involvement of the class I and class II genes, as well as class III (non-Class I/II MHC) genes has been proposed. For the myositis development, the HLA-DRB1*03 is a known risk factor in Caucasian population. However, there are another genes appearing to be significant players in the ethiology of myositis located near the HLA-DRB1 locus. In the present study the authors have focused on one of these risk factors – the regulation of MHC-linked inducible HSP70 genes expression and their relation to the known immunogenetic risk factor located within the MHC (HLA-DRB1*03). Materials and methods The authors have investigated the gene specific HSP70 expression in 20 patients with dermatopolymyositis and 15 healthy people matching in age as control samples. Expression levels of the two inducible HSP70 genes (HSPA1A, HSPA1B) were analysed both in patients and controls. Both of the groups were additionally genotyped for HLA-DRB1 locus. Myositis-specific and associated autoantibodies were also identified in patients. Results The expression of both, the HSPA1A and HSPA1B genes was significantly upregulated (p<0.001; p<0.05) in patients suffering from myositis when compared to controls. The expression regulation of the HSPA1A was found to be associated with the presence of the HLA-DRB1*03 risk allele in patients. However, this was not observed for the HSPA1B gene. In contrast, the authors found a positive correlation between the expression regulation of the HSPA1B gene and the presence of disease specific autoantibodies in myositis patients. Additionally, positive correlation between the presence of disease specific autoantibodies and the HLA-DRB1*03 risk allele was found. None of these observations were found in healthy controls. Conclusions The results suggest that the two MHC-linked inducible genes are differentially expressed in dependence on the autoantibody or HLA risk allele presence. The differential gene expression regulation shows that the HSPA1B is an – on HLA-DRB1 – independent molecular marker for myositis development.

08.01 Heat shock protein 90 is increased in muscle tissue and plasma in idiopathic inflammatory myopathies

Background Heat shock proteins (Hsps) are chaperones playing important roles in skeletal muscle physiology, adaptation to exercise or stress, and activation of inflammatory cells. The aim of our study was to assess Hsp90 expression in muscle biopsies and plasma of patients with idiopathic inflammatory myopathies (IIM) and to characterise its association with IIM-related features. Methods Total of 277 patients with IIM (198 females, 79 males; mean age 54.8; disease duration 4.1 years; dermatomyositis (DM, 104)/polymyositis (PM, 104)/cancer associated myositis (CAM, 42)/necrotizing myopathy (IMNM, 27)) and 100 age-/sex-matched healthy individuals were included in plasma analysis and 50 muscle biopsy samples were stained for Hsp90 (in PM, DM, IMNM, myodystrophy, myasthenia gravis, 10 each). Plasma Hsp90 was measured by ELISA (eBioscience, Vienna). CK, LD, ALT, AST and CRP were analysed by routine techniques and IIM-specific autoantibodies by in-line blot/immunoprecipitation. Data are presented as median. Results In muscle biopsies Hsp90 expression was higher in IIM than in myodystrophy (myasthenia gravis used as another control was negative). Increased Hsp90 was detected in perifascicular degenerating and/or regenerating fibres, inflammatory cells (DM, PM), and necrotic and regenerating fibres (IMNM). Plasma Hsp90 levels were increased in IIM patients compared to healthy controls (20.2 vs. 9.2 ng/ml, p<0.0001). Hsp90 levels in all patients positively correlated with LD (r=0.551, p<0.0001) and AST (r=0.372, p<0.0001). Increased Hsp90 was associated with decreased MMT8 values (r=−0.136, p=0.029), in particular in proximal muscles. Hsp90 positively correlated with patient and doctor disease activity (r=0.222, p=0.0004; r=0.217, p=0.0005, respectively), pulmonary (r=0.222, p=0.0004) and muscle disease activity (r=0.146, p=0.018), MITAX (r=0.175, p=0.005) and MYOACT (r=0.159, p=0.012), and with MDI extent/severity (r=0.215, p=0.003; r=0.120, p=0.041, respectively). Higher Hsp90 was found in patients with interstitial lung disease, cardiac involvement and dysphagia (25.4 vs. 18.9, p=0.004; 27.5 vs. 19.3, p=0.004; 25.0 vs. 18.2 ng/ml, p=0.018, respectively). Conclusions We demonstrate increased Hsp90 expression in IIM muscle biopsy samples, specifically in inflammatory cells, degenerating, regenerating and/or necrotic fibres. Increased Hsp90 plasma levels in IIM patients are associated with disease activity and damage, and with the involvement of proximal skeletal muscles, heart and lungs. Acknowledgement Supported by AZV-16–33542A.

AB0599 Interleukin-35 in Idiopathic Inflammatory Myopathies

Background Interleukin-35 (IL-35) is a newly described heterodimeric cytokine that belongs to the IL-12 family and consists of p35 (IL-12a) and EBI3 (IL-27b) subunits. IL-35 exerts immunomodulatory activities in several autoimmune inflammatory diseases. Objectives The aim of this study was to assess IL-35 expression in muscle tissue of patients with idiopathic inflammatory myopathies (IIM) and to compare serum levels of IL-35 in patients with IIM to healthy controls and asses potential association with activity of IIM. Methods The expression of IL-35 was studied in a series of 19 muscle biopsy samples of idiopathic inflammatory myopathies (9 dermatomyositis, 10 polymyositis) and 10 non-inflammatory control muscle biopsies from patients with myasthenia gravis.Serum levels of IL-35 were measured in 23 PM, 28 DM and 15 cancer associated myositis patients as well as in 40 healthy controls.Disease activity was evaluated by the Myositis Disease Activity Assessment Tool (MYOACT) and by serum muscle enzymes. Results Both IL-35 subunits were found in immune cells of the inflammatory infiltrates in IIM muscle biopsies, no immunoreactivity was observed in muscle tissue of control patients.IL-35 serum levels were increased in all IIM patients compared to healthy controls (p<0.001). There were no differences in IL-35 serum levels between myositis subgroups. Serum IL-35 levels correlated with the overall MYOACT score, with extramuscular and muscle domains of MYOACT, with physicians global activity assessment and lactate dehydrogenase levels. Conclusions IL-35 subunits are overexpressed in inflammatory infiltrates in muscle tissue of IIM patients and elevated circulating IL-35 levels correlate with several disease activity parameters. These data suggest potential role of IL-35 in the pathogenesis of inflammatory myopathies. Acknowledgement Supported by MHCR support for conceptual development of a research organization (023728) and BTCure (115142–2). Disclosure of Interest None declared

OP0047 Expression of Heat Shock Protein 90 in Muscle Tissue and Plasma Is Increased in Idiopathic Inflammatory Myopathies and Correlates with Disease Activity, Skeletal Muscle, Heart and Lung Involvement

Background Heat shock proteins (Hsps) are chaperones playing important roles in skeletal muscle physiology, adaptation to exercise/stress, and activation of inflammatory cells. Objectives To assess Hsp90 expression in muscle biopsies and plasma of patients with idiopathic inflammatory myopathies (IIM) and to characterize its association with IIM-related features. Methods Total of 277 patients with IIM (198 females, 79 males; mean age 54.8; disease duration 4.1 years; dermatomyositis (DM, 104)/polymyositis (PM, 104)/cancer associated myositis (CAM, 42)/ necrotizing myopathy (IMNM, 27)) and 100 age-/sex-matched healthy individuals were included in plasma analysis and 50 muscle biopsy samples were stained for Hsp90 (PM-10, DM-10, IMNM-10, myodystrophy-10, myasthenia gravis-10). Patients with PM/DM fulfilled Bohan and Peter criteria and CAM was defined as cancer within 3 years of IIM diagnosis. Plasma Hsp90 was measured by ELISA (eBioscience, Vienna, Austria). Clinical disease circumstances were evaluated by Myositis Disease Activity Assessment (MYOACT), Myositis Intention to Treat Index (MITAX), Myositis Damage Index (MDI), physician and patient global activity using VAS and manual muscle testing (MMT8). CK, LD, ALT, AST and CRP were analyzed by routine techniques and IIM-specific autoantibodies by in-line blot and immunoprecipitation. Data are presented as median. Results In muscle biopsies Hsp90 expression was higher in IIM than in myodystrophy (myasthenia gravis used as another control was negative). Increased Hsp90 was detected in perifascicular degenerating and regenerating fibers, inflammatory cells (DM, PM), and necrotic and regenerating fibers (IMNM). Plasma Hsp90 levels were increased in IIM patients compared to healthy controls (20.2 vs. 9.2 ng/ml, p<0.0001), and in individual subgroups of IIM vs. healthy controls (PM: 19.4, DM: 22.4, CAM: 19.1, IMNM: 19.6 ng/ml, p<0.0001 for all). Hsp90 levels in all patients positively correlated with LD and AST (r=0.551, p<0.0001; r=0.372, p<0.0001, respectively), and there was a trend towards correlation with CK (r=0.111, p=0.068). Increased Hsp90 was associated with decreased MMT8 values (r=-0.136, p=0.029), in particular in proximal muscles. Hsp90 positively correlated with patient and doctor disease activity (r=0.222, p=0.0004; r=0.217, p=0.0005, respectively), pulmonary and muscle disease activity (r=0.201, p=0.001; r=0.146, p=0.018, respectively), MITAX and MYOACT (r=0.175, p=0.005; r=0.159, p=0.012, respectively), and with MDI extent/severity (r=0.215, p=0.003; r=0.120, p=0.041, respectively). Higher Hsp90 was found in patients with interstitial lung disease, cardiac involvement and dysphagia (25.4 vs. 18.9, p=0.004; 27.5 vs. 19.3, p=0.004; 25.0 vs. 18.2, p=0.018, respectively). Conclusions We demonstrate increased Hsp90 expression in IIM muscle biopsy samples, specifically in inflammatory cells, degenerating, regenerating and/or necrotic fibers. Increased Hsp90 plasma levels in IIM patients are associated with disease activity and damage, and with the involvement of proximal skeletal muscles, heart and lungs. Acknowledgement Supported by BT Cure, MHCR-23728. Disclosure of Interest None declared

FRI0520 Association Study of the BAFF Genetic Variations in Two Independent Cohorts with Idiopathic Inflammatory Myopathies

Background B-cell activating factor of the TNF family (BAFF) plays a role in (auto)antibody production. Patients with idiopathic inflammatory myopathies (IIMs or myositis) have elevated levels of BAFF in the serum (sBAFF) which associate with disease activity. Previously an association was shown between rs9514828 (-871 C/T) SNP (single nucleotide polymorphism) in the promoter region of the BAFF gene and idiopathic thrombocytopenic purpura and with phenotypic diversity in autoimmune rheumatic diseases but with inconsistent results. An association between the haplotype from the four SNPs located upstream of the BAFF gene and presence of myositis was found in one cohort of Czech patients (CZE). Objectives To replicate our findings in an independent cohort of myositis patients from Sweden (SWE) and to study correlations between particular SNPs and serum levels of BAFF. Methods Two cohorts of patients with myositis (CZE: n=311, age (years) 54.6, females 74% and SWE: n=307, age 54.1, females 64%) and healthy controls (CZE: n=103, age 42, females 69% and SWE: n=325, age 52.0, females 74%) were included in the study. SNPs (rs9514827, rs3759467, rs1041569 and rs9514828) were analysed by direct DNA sequencing and levels of sBAFF were measured by ELISA. The χ2 or Fisher tests for analysis of allelic, haplotype and genotype associations and nonparametric tests for group comparisons with Bonferronis correction were used. Results The four SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT) in both patients and controls, compatible to results reported from other populations. A significantly higher frequency of the TTTT haplotype was present in the CZE myositis patients compared to healthy controls (16% vs. 10%; OR=1.73, 95%CI=1.05-2.85, p<0.02), but not in the SWE cohort (patients 13% vs. controls 17%, p=0.07). CZE and SWE myositis patients did not differ significantly in allelic, genotype or haplotype frequencies, but a difference between the control groups was observed. SWE controls had higher frequencies of the rs1041569 T-allele and rs3759467 TC-genotype compared to CZE HC (19% vs 12% and 33% vs 19% respectively; p<0.05 for both). No associations were found between alleles or genotypes and serum levels of BAFF for any of the studied SNPs. Conclusions We found different patterns of associations between BAFF genetic polymorphism and myositis in two European populations. Our initial finding in Czech population could not be replicated in an independent cohort from Sweden. The difference between the control groups requires further analyses. Acknowledgements IGA -MZ Czech Republic NT/12438-4, ESF in the framework of the EuMyoNet, Krystufkova O. and Svitalkova T. contributed equally Disclosure of Interest O. Krystufkova Grant/research support: IGA -MZ Czech Republic NT/12438-4, ESF in the framework of the EuMyoNet, T. Svitalkova: None declared, H. Hulejova: None declared, M. Svetla: None declared, L. Plestilova: None declared, O. Pecha: None declared, H. Mann: None declared, A. Notarnicola: None declared, P. Venalis: None declared, L. Padyukov: None declared, P. Novota: None declared, I. Lundberg: None declared, J. Vencovsky: None declared DOI 10.1136/annrheumdis-2014-eular.4065

A7.11 Genetic Variation in Promoter Sequence of B-Cell-Activating Factor of the TNF Family (BAFF) in Patients with Idiopathic Inflammatory Myopathies (IIM)

Background and Objectives B-cell activating factor of the TNF family (BAFF) is important for B cell maturation and plays a role in (auto)antibodies production. Elevated serum levels in relation to autoantibodies were documented in patients with IIM. Promoter region of BAFF gene contains several known sites with single nucleotide polymorphism (SNPs). An association between Rs9514828 (–871 C/T) SNP and susceptibility to idiopathic trombocytopaenic purpura was shown and possible relations to systemic lupus erythematosus, rheumatoid arthritis or Sjögren’s syndrome were suggested but in IIM have not been studied yet. Here, we analysed relation of four BAFF SNPs located in the BAFF gene promoter with the development of IIM. Materials and Methods 146 patients with polymyositis (PM), 150 with dermatomyositis (DM), 11 patients with juvenile DM and 4 patients with inclusion body myositis and 103 healthy individuals were included. Four SNPs located upstream in the BAFF gene (Rs9514827 (–2841 T/C); Rs3759467 (–2704 T/C); Rs1041569 (–2701 T/A); Rs9514828 (–871 C/T)) were analysed by direct DNA sequencing. Serum levels of BAFF (s-BAFF) were evaluated using ELISA. Autoantibodies were detected with immunoprecipitation. The chi square test for analysis of alleles and genotypes associations and SNPStats software for haplotype frequency studies were used. Results Significantly higher frequency of –2701T allele was present in patients (18%) compared to healthy controls (12%) (P = 0.029; OR 1.684 (CI 95% = 1.050 – 2.699)). Additionally, increased –2841T allele (P = 0.086), –2841TT, CT genotype (P = 0.066) and –2701TT, AT genotype (P = 0.079) frequencies were observed in patients. SNPs were in strong linkage disequilibrium and formed four common haplotypes (TTAC, CTAT, TCAC, TTTT), with significantly different frequency (>9%) distributions between patients and controls (global P-value < 0.038). Higher frequency of TTTT haplotype was present in patients (16.2%) compared to controls (9.3%; OR 1.99 (95% CI 1.15–3.47; P < 0.015)) relative to the most frequent haplotype TTAC. Significantly higher s-BAFF levels were detected in patients compared to healthy controls (P < 0.0001) and in patients with anti-Jo-1 or anti-PMScl autoantibodies compared to patients without autoantibodies (P = 0.028, P < 0.037 respectively). Higher s-BAFF levels with –2704T allele within anti-Jo-1+ patients (P < 0.043) were found. Conclusions We describe significant association of SNP (Rs1041569) with myositis and a relation of SNP (Rs3759467) to presence of anti-Jo-1 autoantibodies and s-BAFF levels. These results should be confirmed in an independent cohort of patients and possible relations of s-BAFF levels with disease activity and treatment should be considered. Acknowledgement IGA -Ministry of Health of the Czech Republic NT/12438–4.

SAT0031 Anti-RO52 autoantibody epitope mapping in european cohort of myositis patients

Background The most frequent myositis associated autoautoantibodies are directed to Ro52-kd protein (12-26%). Antibodies to the immunodominant epitope (p200) of Ro52 are associated with SLE and Sjögren’s syndrome (SjS) with suggestion for a pathogenic effect in development of neonatal heart block. The role of anti-Ro52 autoantibodies in myositis is not clear. Presence of anti-Ro52 in combination with anti-Jo-1 autoantibodies is associated with interstitial lung disease (ILD). Objectives The aim was to investigate the associations of reactivity to Ro52 epitopes with disease manifestations, autoantibody profile, smoking status and HLA-DRB1 alleles in myositis. In order to test the hypothesis of epitope spreading the association of disease duration with polyreactivity was evaluated. Methods Serum samples from 468 myositis patients were collected in three countries: Czech (n=133); Hungary (n=202), Sweden (n=133). The reactivity with recombinant full length (FL) Ro52 antigen, its four deletion fragments (Ro52-3,-4,-5 and -6) and 7 overlapping synthetic peptides (p136-200) covering the major immunodominant region (Ro52-3) was measured by ELISA. Autoantibodies to Jo-1, PL-7, PL-12, EJ, OJ, SRP, PM-Scl, Ku, Mi-2β, U1-RNP were detected by LIA. 417 patients were genotyped for HLA-DRB1. Smoking status was known in 434 patients. Results 184 patients with dermatomyositis (DM), 250 polymyositis (PM), 11 inclusion body myositis, 20 juvenile DM/PM and three with mixed connective tissue disease were included in the analysis. Median disease duration was 4.7 (0-41.5) years. ILD was present in 132 (29%) of patients. In total 175 (40%) patients were smokers. Reactivity to Ro52-FL was present in 97 (21%) samples; 87 (90%) of these showed specificity to Ro52-3 and anti-p176 dominated (66; 76%) whereas only 44 (51%) had reactivity to the p200 epitope. Anti-Ro52FL positivity was associated with presence of ILD (OR 3.8; p<0.0001), anti-Jo1 (OR=9.0; p<0.0001) and HLA-DRB1*03 allele (OR=1.9; p=0.009). No significant epitope associations were detected. No association was found between reactivity to Ro52 or polyreactivity to 2 or more fragments or peptides and disease duration or smoking status. Moreover, there was no additional effect of smoking to association with HLA-DRB1*03 allele. Conclusions Our results confirm the association between anti-Ro52 positivity with ILD, anti-Jo-1 autoantibodies and HLA-DRB1*03 allele in myositis. The dominance of reactivity to epitope p176 in myositis patients differs from dominant p200 in SLE and SjS, but its clinical significance is not clear. There was no effect of smoking or evidence for epitope spreading in anti-Ro52 response. Supported by EU 6.FP integrated project AutoCure LSHB CT-2006-018661 and Grant NT 12438 of IGA MZCR Disclosure of Interest O. Krystufkova Grant/Research support from: EU 6.FP integrated project AutoCure LSHB CT-2006-018661 and Grant NT 12438 of IGA MZCR, V. Dzikatite: None Declared, P. Charles: None Declared, H. Mann: None Declared, L. Ekholm: None Declared, M. Vincze: None Declared, I. Putova: None Declared, N. Kasprikova: None Declared, L. Padyukov: None Declared, K. Danko: None Declared, P. Novota: None Declared, J. Vencovsky: None Declared, M. Wahren-Herlenius: None Declared, I. Lundberg: None Declared