Karina Roxana Gheorghe

Autoantibodies to cytosolic 5′-nucleotidase 1A in inclusion body myositis

Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy characterized by both degenerative and autoimmune features. In contrast to other inflammatory myopathies, myositis‐specific autoantibodies had not been found in sIBM patients until recently. We used human skeletal muscle extracts as a source of antigens to detect autoantibodies in sIBM and to characterize the corresponding antigen.

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH2 to PGE2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids

IntroductionIt was previously shown that lipoxygenase (LO) pathways are important in the rheumatoid arthritis (RA) inflammatory process and that synovial fluid from RA patients contains high amounts of leukotrienes. We therefore aimed to investigate the 5-LO and 15-LO-1 expression pattern in RA and ostheoarthritis (OA) synovial tissue and to study the effect of intraarticular glucocorticoid (GC) therapy on enzyme expression.MethodsExpression of LOs was evaluated by immunohistochemistry in RA and OA synovial biopsies. Cellular localization of these enzymes was analyzed by double immunofluorescence. In synovial biopsies from 11 RA patients, 5-LO and 15-LO-1 expression was evaluated before and after triamcinolone hexacetonide knee injection and assessed by image analysis to quantify their expression. We also investigated the presence of 15-LO-1 by immunohistochemistry in synovial fluid (SF) cells as well as their ability to form 15-hydroxyeicosatetraenoic acid (15-HETE) following treatment with arachidonic acid (AA).Results5-LO and 15-LO-1 are present in RA and OA synovium, with 5-LO being mostly expressed in lining and sublining macrophages, neutrophils and mast cells and 15-LO-1 mainly in lining macrophages, fibroblasts and sublining endothelial cells. Intraarticular GC treatment resulted in a significant suppression of 5-LO expression, but did not influence the 15-LO-1 enzyme significantly. Also, SF cells express a functional 15-LO-1 and produce 15-HETE when challenged with AA.ConclusionsThese data demonstrate that local therapy with GC decreases 5-LO expression in RA synovium and offer an additional possible mechanism for the efficiency of intraarticular adjuvant therapy in RA.

Cytosolic 5′-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis

Objectives Autoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. Materials and methods Data from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Results Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. Interpretation Differences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

Prostaglandin E2 Synthesizing Enzymes in Rheumatoid Arthritis B Cells and the Effects of B Cell Depleting Therapy on Enzyme Expression

Introduction B cells may play an important role in promoting immune activation in the rheumatoid synovium and can produce prostaglandin E2 (PGE2) when activated. In its turn, PGE2 formed by cyclooxygenase (COX) and microsomal prostaglandin E2 synthase 1 (MPGES1) contributes to the rheumatoid arthritis (RA) pathological process. Therapeutic depletion of B cells results in important improvement in controlling disease activity in rheumatoid patients. Therefore we investigated the expression of PGE2 pathway enzymes in RA B cells and evaluated the effects of B cell depleting therapy on their expression in RA tissue. Methods B cells expressing MPGES1 and COX-2 were identified by flow cytometry in in vitro stimulated and control mononuclear cells isolated from synovial fluid and peripheral blood of RA patients. Synovial biopsies were obtained from 24 RA patients before and at two consecutive time points after rituximab therapy. Expression of MPGES1, COX-1 and COX-2, as well as interleukin (IL)-1β and IL-6, known inducers of MPGES1, was quantified in immunostained biopsy sections using computerized image analysis. Results Expression of MPGES1 or COX-2 was significantly upregulated upon stimulation of B cells from blood and synovial fluid while control cells displayed no detectable enzymes. In synovial biopsy sections, the expression of MPGES1, COX-1 or COX-2 was resistant to rituximab therapy at 8 or 16 weeks after start of treatment. Furthermore expression of IL-1β in the synovial tissue remained unchanged, while IL-6 tended to decrease after therapy. Conclusions Therapy with B cell depleting agents, although efficient in achieving good clinical and radiographic response in RA patients, leaves important inflammatory pathways in the rheumatoid synovium essentially unaffected.

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue.

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E2 (PGE2) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE2. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.MethodsSynovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.Results15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE2 pathway unaltered both in biopsies ex vivo and in cultured FLS.ConclusionsEarly methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE2 synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE2 synthesis may find a rationale in additionally reducing local inflammatory mediators.

THU0253 ANTI-PM-SCL autoantibodies in polymyositis and dermatomyositis

Background Idiopathic inflammatory myopathies (IIM) are frequently accompanied by presence of autoantibodies. A correlation of anti-Jo-1 and anti-SRP antibody levels with disease activity and creatine kinase levels has been described. Recently a new anti-PM1α quantitative ELISA test detecting antibodies to the main epitope of PM-Scl-100 has become available. Objectives To investigate the prevalence of anti-PM1α autoantibodies and association with clinical symptoms, clinical disease activity and serum CK in a multicenter myositis cohort. Methods Two hundred and thirty eight consecutive patients with established IIM (103 DM, 99 PM, 8 JDM, 3 IBM and 25 IIM/CTD-overlap syndrome) were tested for the presence of anti PM-Scl autoantibodies by immunoblot and by immunoprecipitation. Eight additional anti-PM-Scl positive sera were also obtained. Serum levels of anti-PM1α autoantibodies were detected by ELISA kit (Dr. FOOKE Laboratorien GmbH, Neuss, DE). Clinical data were derived from the Euromyositis database (www.euromyositis.eu). Global and organ clinical disease activities were assessed by Myositis Activity Assessment Tool (MYOACT) 10 cm visual analogue scales. Results 19/238 (8%) patients (11 DM, 2 PM and 6 IIM/CTD-overlap syndrome (3 DM + SSc, 2 PM + SSc, 1 PM + SjS)) were positive for anti-PM-Scl in immunoblot, immunoprecipitation and anti-PM1α ELISA. 19 PM-Scl+ were 16/181 women and 3/57 men, average age 50.5 years (22.0 – 74.2) and average disease duration 3.0 years (0 – 11.6 years). Good agreement between detection tests was found. The most common clinical features among anti-PM-Scl positive patients were muscle weakness (96%), interstitial lung disease (77%), Raynaud’s phenomenon (69%) and mechanic’s hands (65%). One patient had a cancer. Anti-PM1α levels correlated with serum CK activity in the DM group (p=0.041, r=0.533) and with constitutional disease activity and seriousness of dysphagia in the PM group (p=0.048, r=0.768 and p=0.007, r=0.927). Furthermore longitudinal change in anti-PM1α levels correlated with HAQ scores in the whole group (p=0.016, r=0.730) and with global disease activity score in DM group (p=0.023, r=0.685), but not with other MYOACT parameters. Conclusions Anti-PM-Scl autoantibodies are detected in 8% of patients with myositis. They are more frequent in those with DM and overlap syndromes. Presence of these autoantibodies is associated with muscle weakness, ILD, Raynaud’s phenomenon and mechanic’s hands. Anti-PM1α levels correlate with serum CK levels in DM and their change is correlated with change of HAQ scores in the whole group and with MYOACT scores in DM group. This suggests that the quantitative measurement of anti-PM-1α may be a useful tool in the assessment of patients with this syndrome. Disclosure of Interest None Declared

THU0232 Pregnancy outcome in idiopathic inflammatory myopathy patients in a multicenter study

Background Immunological changes during pregnancy are in the centre of scientific researches. We already know that immunological pathways may go wrong during pregnancy in autoimmune diseases with some general complications like fetal loss and low birth weight but we have less informations about the outcome of pregnancy in idiopathic inflammatory myopathies, polymyositis (PM) and dermatomyositis (DM). Objectives Our aim was to assess the clinical features, organ complications, functional course, therapeutic response in patients with PM/DM according to the pregnancy status. Methods Medical records were retrospectively revised using a standardised protocol. 23 PM/DM patients with pregnancy were identified in four countries: Hungary (n=9), Czech Republic (n=8); Sweden (n=5) and Poland (n=1). Immunological and serological characteristics were measured by ELISA, immunoblot and immuneprecipitation. Autoantibodies to Jo-1, PL-7, PL-12, EJ, OJ, SRP, PM-Scl, Ku, Mi-2, U1-, SSA, SSB were detected by LIA. We used the Fisher’s exact test for statistical analysis. Results These 23 patients had 33 pregnancies after or at the time of disease onset. 10 of them suffered from PM and 13 had DM or JDM. The most frequent symptoms were ILD, mechanic’s hand, Raynaud phenomenon and arthritis. 19 pregnancies ended without complications giving life to healthy babies. In 13 cases complications, like intra-uterine growth retardation, intra-uterine death, abortion, extra-uterine gravidity occurred. These complications were most frequently seen in patients with PM than DM (p=0,0729). The relative risk of complicated pregnancy in PM was 1,923 (CI 95% 1,041-3,553). This “almost significant” finding could be caused by PM and by anti-Jo1 and anti-phospholipid autoantibodies as well, because we found these antibodies frequently associated to PM in these cases. 13 pregnancies started during the active phase of myositis and the majority of these patients didn’t reach remission after the delivery. The mean weight (2599g in active disease vs. 3342g in remission) and height (48 cm vs. 51 cm) of babies was lower in the active cases and the pregnancy ended earlier than in patients with remission. Most of the affected patients used only glucocorticoid treatment during pregnancy. Conclusions Complications during pregnancy were frequently reported in PM/DM although no myositis specific complications could be found. In patients with PM there was a tendency for a higher risk of fetal problems than in DM. Disclosure of Interest None Declared

Limited effects of methotrexate on enzymes of the PGE2 pathway in rheumatoid arthritis synovial tissue

Objectives Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E2 (PGE2) displays important pathogenic role in the affected joints. Whereas the enzymes involved in its synthesis, microsomal PGE2 synthase-1 and cyclooxygenases (COX1 and COX2), are highly expressed in the inflamed synovium, little is known about 15-prostaglandin dihydrogenase (15-PGDH) that metabolises PGE2. In this study the authors aimed to investigate the localisation of 15-PGDH in the synovial tissue and the influence of common RA therapy on its expression. Materials and methods Synovial tissue specimens were obtained from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients and immunohistochemical analysis and double immunfluorescence staining was used to describe the expression pattern of 15-PGDH. In addition, synovial biopsies were obtained by arthroscopy from 10 RA patients with active knee arthritis undergoing intra-articular glucocorticoid therapy as well as from 13 newly diagnosed RA patients starting methotrexate therapy, before and after start of treatment. After immunohistochemical staining, 15-PGDH was analysed in patients receiving glucocorticoid injection and the expression of enzymes involved in the PGE2 cascade was evaluated in the methotrexate cohort. Prostaglandins deriving from the COX pathway were determined by liquid-chromatography coupled with mass spectrometry in supernatants from in vitro cultured fibroblast-like synoviocytes (FLS) activated with IL-1β and treated with methotrexate. Results 15-PGDH is present in synovial tissue of RA, ostheoarthritis and psoriatic arthritis patients but also in healthy individuals, mainly in lining macrophages, fibroblasts and vessels. Intra-articular show a trend towards reduced 15-PGDH in RA synovium while methotrexate treatment to leaves unaltered the PGE2 pathway both in biopsies ex vivo and in cultured FLS. Conclusion 15-PGDH, the enzyme implicated in degradation and removal of pro-inflammatory PGE2 in the rheumatoid joint is expressed in the synovial tissue. While local glucocorticoids may decrease its expression, methotrexate has little influence on any of the PGE2 pathway enzymes 8 weeks after therapy start. Thus therapeutical strategies involving blocking PGE2 synthesis may find a rationale in additionally reducing local inflammatory mediators.

Structure of microsomal prostaglandin E synthase 1 as determined by electron crystallography

Structure of Microsomal Prostaglandin E Synthase 1 as Determined by Electron Crystallography