BIOMARKERS IN AXIAL SPONDYLOARTHRITIS

Axial spondyloarthritis (axSpA) is a common chronic inflammatory rheumatic disease affecting predominantly axial skeleton. Long-term duration of the disease causes bone erosions, new bone formation and can gradually lead to ankylosis of the joints. Despite new possibilities for detection of early disease, the delay from the occurrence of the first symptom(s) to the diagnosis is still striking. Recent studies have focused on biomarkers that would help to diagnose the disease earlier, to determine disease activity and to select patients with potential rapid progression. To this date the only widely used biomarker with some diagnostic value is HLA-B27 antigen. Other potential biomarkers can be found among acute-phase reactants. Some of them have been already well studied (calprotec tin, IL-27), however some are newly discovered (defensin-2, lipocalin-2). Recently even relations between biomarkers of fat metabolism (triglycerides, glycerol) and fatty MRI lesions were studied for diagnostic utility. Second presented group could be entitled as disease activity biomarkers, where C-reactive protein (CRP) together with active magnetic resonance imaging (MRI) lesions are used as common indicators of disease activity. Other inflammatory biomarkers are serum amyloid A, some interleukins and tissue turnover biomarkers (metalloproteinases and their products of degradation). The last part of the presentation will be related to prognostic biomarkers. Except for already mentioned biomarkers (e.g. CRP or MMPs), vasoactive endothelial growth factor (VEGF) together with vimentin fragments, biomarkers of bone remodelling (DKK-1 and sclerostin) and some adipokines have been found to predict radiographic progression. Recently altered microRNAs (miRNAs) expression and target gene dysregulation have been shown to potentially predict progression of the disease. Several biomarkers have been identified as potential diagnostic candidates, disease activity reflectors or markers of disease prognosis. The problem of inadequate sensitivity and specificity of these biomarkers however still remains and therefore future studies are needed for further validation. References: 1. Walter P. Maksymowych (2017) An update on biomarker discovery and use in axial spondyloarthritis,Expert Review of Molecular Diagnostics, 17:11, 965–974 2. Prajzlerova K, Grobelna K, Pavelka K, et al. An update on biomarkers in axial spondyloarthritis. Autoimmunity reviews 2016 doi: 10.1016/j.autrev.2016.02.002 3. Mattey DL, Packham JC, Nixon NB, et al. Association of cytokine and matrix metalloproteinase profi les with disease activity and function in ankylosing spondylitis. Arthritis Res Th er 2012; 14(3): R127. 4. Taylan A, Sari I, Akinci B, et al. Biomarkers and cytokines of bone turnover: extensive evaluation in a cohort of patients with ankylosing spondylitis. BMC Musculoskelet Disord 2012;13:191. doi: 10.1186/1471-2474-13-191 5. Baraliakos X, Landewe R, Heijde DVD, et al. Th e Relationship of Biomarkers and Radiographic Progression in Patients with Ankylosing Spondylitis. Arthritis and rheumatism 2010;62(10):105

BIOMARKERS IN AXIAL SPONDYLOARTHRITIS

Axial spondyloarthritis (axSpA) is a common chronic inflammatory rheumatic disease affecting predominantly axial skeleton. Long-term duration of the disease causes bone erosions, new bone formation and can gradually lead to ankylosis of the joints. Despite new possibilities for detection of early disease, the delay from the occurrence of the first symptom(s) to the diagnosis is still striking. Recent studies have focused on biomarkers that would help to diagnose the disease earlier, to determine disease activity and to select patients with potential rapid progression. To this date the only widely used biomarker with some diagnostic value is HLA-B27 antigen. Other potential biomarkers can be found among acute-phase reactants. Some of them have been already well studied (calprotec tin, IL-27), however some are newly discovered (defensin-2, lipocalin-2). Recently even relations between biomarkers of fat metabolism (triglycerides, glycerol) and fatty MRI lesions were studied for diagnostic utility. Second presented group could be entitled as disease activity biomarkers, where C-reactive protein (CRP) together with active magnetic resonance imaging (MRI) lesions are used as common indicators of disease activity. Other inflammatory biomarkers are serum amyloid A, some interleukins and tissue turnover biomarkers (metalloproteinases and their products of degradation). The last part of the presentation will be related to prognostic biomarkers. Except for already mentioned biomarkers (e.g. CRP or MMPs), vasoactive endothelial growth factor (VEGF) together with vimentin fragments, biomarkers of bone remodelling (DKK-1 and sclerostin) and some adipokines have been found to predict radiographic progression. Recently altered microRNAs (miRNAs) expression and target gene dysregulation have been shown to potentially predict progression of the disease. Several biomarkers have been identified as potential diagnostic candidates, disease activity reflectors or markers of disease prognosis. The problem of inadequate sensitivity and specificity of these biomarkers however still remains and therefore future studies are needed for further validation. References: 1. Walter P. Maksymowych (2017) An update on biomarker discovery and use in axial spondyloarthritis,Expert Review of Molecular Diagnostics, 17:11, 965–974 2. Prajzlerova K, Grobelna K, Pavelka K, et al. An update on biomarkers in axial spondyloarthritis. Autoimmunity reviews 2016 doi: 10.1016/j.autrev.2016.02.002 3. Mattey DL, Packham JC, Nixon NB, et al. Association of cytokine and matrix metalloproteinase profi les with disease activity and function in ankylosing spondylitis. Arthritis Res Th er 2012; 14(3): R127. 4. Taylan A, Sari I, Akinci B, et al. Biomarkers and cytokines of bone turnover: extensive evaluation in a cohort of patients with ankylosing spondylitis. BMC Musculoskelet Disord 2012;13:191. doi: 10.1186/1471-2474-13-191 5. Baraliakos X, Landewe R, Heijde DVD, et al. Th e Relationship of Biomarkers and Radiographic Progression in Patients with Ankylosing Spondylitis. Arthritis and rheumatism 2010;62(10):105

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur. CONCLUSIONS: We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

Abstract The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients

Identification of Novel Native Autoantigens in Rheumatoid Arthritis

The majority of patients diagnosed with rheumatoid arthritis (RA) have developed autoantibodies against neoepitopes in proteins that have undergone post-translational modification, e.g., citrullination or carbamylation. There is growing evidence of their molecular relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Autoantibodies reacting to native proteins may also have a role in RA pathogenesis, however, their reactivity patterns remain much less studied. We hypothesized that a high-density protein array technology could shed light onto the normal and disease-related autoantibodies produced in healthy and RA patient subgroups. In an exploratory study, we investigated the global reactivity of autoantibodies in plasma pools from 15 anti-cyclic citrullinated peptide (CCP)-positive and 10 anti-CCP-negative RA patients and 10 healthy donors against more than 1600 native and unmodified human proteins using a high-density protein array. A total of 102 proteins recognized by IgG autoantibodies were identified, hereof 86 were recognized by antibodies from CCP-positive RA patients and 76 from anti-CCP-negative RA patients, but not by antibodies from healthy donors. Twenty-four of the identified autoantigens have previously been identified in synovial fluid. Multiple human proteins in their native conformation are recognized by autoantibodies from anti-CCP-positive as well as anti-CCP-negative RA patients

Metastasis-inducing S100A4 protein is associated with the disease activity of rheumatoid arthritis

Objectives. To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients. Methods. Plasma levels of the S100A4 protein were analysed in 40 anti-TNF-alpha naive patients with active RA. Of the 40 patients, 25 were treated with adalimumab and monitored over time. The conformational form of S100A4 was analysed using size-exclusion gel chromatography. TNF-alpha mRNA expression and protein synthesis were analysed by RT-PCR and ELISA, respectively. Results. Baseline levels of S100A4 were significantly correlated with disease activity in RA patients (r = 0.41; P < 0.01). After 12 weeks of treatment with adalimumab, there was an obvious shift in the conformations of S100A4 from the multimeric to the dimeric forms, whereas the total levels of the S100A4 protein remained unchanged. This suggests that the bioactive (multimer) S100A4 may decline in response to successful treatment with adalimumab. In addition, we showed significant up-regulation of TNF-alpha mRNA (P < 0.01), and protein release to the cell culture medium of monocytes stimulated with the S100A4 multimer compared with those treated with the dimer and to the unstimulated monocytes (P < 0.001). Conclusions. This is the first study to show that the levels of the S100A4 protein are correlated with RA disease activity. Furthermore, only the bioactive form, but not the total amount of S100A4, decreases after successful TNF-alpha blocking therapy in patients with RA. These data support an important role for the S100A4 multimer in the pathogenesis of R