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The following chapter addresses vascular fibroblasts in a healthy, quiescent state, as well during vascular inflammation, focusing on atherosclerosis. The development of atherosclerosis, an inflammatory disease of medium- and large-sized arteries, has traditionally been viewed as an “inside-out” mechanism, with prominent roles of the innermost layer of the artery, consisting of endothelial cells. However, emerging evidence suggests a new paradigm of “outside-in” mechanism, including an earlier role for fibroblasts, constituents of the outermost adventitial layer of the artery. Phenotypic and functional changes of fibroblasts in adventitia may even occur prior to, or alongside endothelial activation. Activated adventitial fibroblasts, implicated in atherosclerosis progression, begin to transform into myofibroblasts, upregulate production of different proinflammatory cytokines, chemokines, growth factors, proteolytic enzymes, extracellular matrix proteins and reactive oxygen species, leading to extensive matrix remodeling, chemotaxis and recruitment of immune cells. Due to their suitable location for drug delivery systems, preventing fibroblast activation, modulating their activity or inducing myofibroblast dedifferentiation could represent a promising therapeutic approach for atherosclerosis regression.

Background:The heterogeneity of synovial tissues from patients with arthritis could contribute to the interpatient variability in disease course, prognosis and treatment response. Single-cell RNA sequencing (scRNA-seq) permits in-depth analysis of tissue heterogeneity, which could facilitate drug discovery and patient stratification for precision medicine.Objectives:To construct a comprehensive landscape of synovial cell types and molecular pathways in arthritis by integrating our and published scRNA-seq data, generated across different scRNA-seq technologies [Smart-seq2, Drop-seq], cell preparation protocols [dissociated unsorted, sorted cells] and types of arthritis [undifferentiated (UA), rheumatoid arthritis, osteoarthritis].Methods:Synovial tissues were obtained by ultrasound-guided biopsy from patients with UA [not fulfilling the classification criteria for a specific arthritis, n=3]. Biopsies were disintegrated [enzymatic and mechanical disruption] and cell viability assessed with trypan blue. ScRNA-seq libraries [2 per patient] were prepared with 10X Genomics Drop-Seq and sequenced on NovaSeq6000. Bioinformatics analysis of our and published [n=35] datasets1-3was performed using Seurat protocol4with correction for batch effects and filtering low-quality cells. Functional enrichment analysis of marker genes in clusters was done with STRING Protein-Protein networks. Synovitis was assessed with ultrasound and histology.Results:Our tissue disintegration protocol resulted in good cell yield and viability (92%, 72%, 100%). The synovial cellular heterogeneity detected by scRNA-seq reflected the histological findings [Krenn score, pathotype]. These were supported with the ultrasound and clinically assessed disease activity. The integrated analysis of 41 datasets from 38 donors yielded 41845 scRNA-seq cell profiles, 50% contributed by our dataset. An independent analysis of our data and their integration with published data showed that different scRNA-seq methods and protocols can identify all the major synovial cell types and their activation states (Figure 1) with large heterogeneity between donors. We identified a previously undescribed synovial cell population, which was located near the fibroblast cluster, was negative for canonical cell markers, but highly enriched in cell division genes (80% of marker genes). These cells comprised a mixed population of CD34-, podoplanin (PDPN)highor PDPNlowcells that were mostly negative for the sub-lining fibroblast marker THY. Furthermore, they appeared to be highly secretory (extracellular matrix components) and their gene expression profile was inclined towards cell migration, vascular development and insulin growth factor-dependent processes.Figure 1.Heatmap with top 20 cluster gene markers, gene enrichment analysis and UMAP plot of synovial cell clusters.Conclusion:By integrating synovial scRNA-seq data from 41845 cells, we identified a previously undescribed, highly proliferative and secretory synovial cell population in arthritis. We increased the number of known scRNA-seq synovial cell profiles in arthritis by two-fold and demonstrated the robustness of synovial scRNA-seq data outputs across different technologies and protocols. This broadens the current knowledge of synovial tissue heterogeneity and pathology in arthritis.References:[1]Stephenson W. et al. Nat Commun 2017.[2]Mizoguchi F. et al. Nat Commun 2017.[3]Zhang F. et al. Nat Immunol 2018.[4]Stuart T et al. Cell 2019Acknowledgments:This work is supported by Vontobel Foundation and medAlumni University of ZurichDisclosure of Interests:Sam G. Edalat: None declared, Raphael Micheroli: None declared, Tadeja Kuret: None declared, Kristina Buerki: None declared, Chantal Pauli: None declared, Snežna Sodin-Šemrl: None declared, Adrian Ciurea Consultant of: Consulting and/or speaking fees from AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Merck Sharp & Dohme, Novartis and Pfizer., Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Caroline Ospelt Consultant of: Consultancy fees from Gilead Sciences., Gregor Rot: None declared, Mojca Frank-Bertoncelj: None declared

Background:Myofibroblasts are the orchestrators of aberrant extracellular matrix (ECM) remodelling in fibrosis. Actin cytoskeleton is a central hub that integrates mechanical signals to promote myofibroblast differentiation and ECM remodelling. Targeting these pathways could represent a novel antifibrotic strategy. We have recently shown that metabolic intermediate dimethyl-α-ketoglutarate (dm-αKG) blocks TGFβ-driven myofibroblast differentiation in dermal fibroblasts (DF).Objectives:To investigate the mechanisms by which dm-αKG regulates TGFβ-driven myofibroblast differentiation and inflammatory responses in DF.Methods:DF from healthy controls and patients with systemic sclerosis (SSc) were treated with TGFβ (10 ng/ml) and/or dm-αKG (6 mM) for 24h, 48h and 72h. RNA sequencing (Ilumina 2000, n=3 per experimental group) was followed by the analysis of differentially expressed genes (DeSEQ2, log2 fold ≥ |0.5|, padj< 0.01), pathway enrichment analysis (GO terms) and supervised PCA analysis (ClustVis). Protein amounts (fibronectin, αSMA, IL-6), cell contraction and apoptosis were measured with Western blot (n=6), ELISA (n=4), collagen gel contraction assay (n=4) and real time Annexin V assay (n=6). Significance (pResults:TGFβ (24h) altered the expression of 4076 genes in DF as determined by RNA-seq, among which 1864 genes were upregulated. The upregulated genes were enriched in GO biological processes/molecular functions/cellular compartments related to ECM organization (p=1e-07), Wnt signalling (p=5e-06), actin binding (p=3e-07), focal adhesion (p=1e-10), stress fibers (p=3e-07) and actin cytoskeleton (p= 3e-06). Dm-αKG altered the expression of 589 genes in TGFβ-treated DF compared to TGFβ only. The most downregulated pathways in DF treated with dm-αKG + TGFβ compared to TGFβ only included actin binding (p=5e-05), muscle contraction (p=0.001), ECM organization (p=0.008), focal adhesion (p=0.01), Z disk (p=0.01) and stress fibers (p=0.03). Specifically, dm-aKG significantly (p-0.5) decreased the expression of many TGFβ-induced genes involved in actin organization and focal adhesion (NEXN, FRMD5, ANTXR1, ACTC1, LIMCH1, SORBS2, TGM2, CSRP2, CAP2, LMO7, FZD2), muscle contraction (SNTB1, LMOD1, ANKRD1, SULF1, JPH2, CAVIN4, OXTR, DYSF, FBXO32) and ECM organization (COL10A1, COL11A1, HAPLN1, MMP14, MMP3, SPINT2, GREM1, MATN3, ADAMTS4). The PCA analysis revealed that the experimental treatment (PC1, Fig 1A) accounted for 61% variability in the expression of these genes, while 19% was attributed to interdonor variability (PC2). Dm-αKG diminished TGFβ-induced production of αSMA protein (72h, p=0.02, mean O.D. ± SD in TGFβ + dm-αKG vs. TGFβ: 0.34 ± 0.38 vs. 3.1 ± 2.3) and repressed TGFβ-driven secretion of fibronectin protein (72h, p=0.047, 0.5 ± 0.1 vs. 1.2 ± 0.6). Dm-αKG reduced the contractile capacity of TGFβ-stimulated DF in collagen gel contraction assay (p=0.003, 0 vs. 67.1 ± 5.4%). Additionally, dm-αKG decreased TGFβ-driven production of IL-6 transcripts (24h, p=0.05, 2.9 ± 0.6 vs 1.9 ± 0.3) and protein (24h, p=0.0005, 5.9 ± 1.2 vs 3 ± 0.7, Fig 1B), but did not increase the apoptosis of DF (24h, 48h, 72h).Fig 1.A Supervised PCA analysis of RNA-seq data. B. IL-6 secretion (ELISA).Conclusion:Dm-αKG counteracted TGFβ-induced myofibroblast differentiation by regulating the cytoskeleton organization and ECM dynamics in DF and blocked the TGFβ-induced IL-6 production. This closely links metabolism to inflammatory and pro-fibrotic responses in DF. Therefore, regulating intracellular αKG might offer a novel strategy in combating the inflammatory and fibrotic stages of skin fibrosis in SSc.Acknowledgments:This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology.Disclosure of Interests:Blaž Burja: None declared, Gabriela Kania: None declared, Matija Tomsic: None declared, Snežna Sodin-Šemrl: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Katja Lakota: None declared, Mojca Frank-Bertoncelj: None declared

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Background Neutrophils with differential surface protein expression were recently implicated in pathogenesis of Giant Cell Arteritis (GCA)1. However, data are lacking with regard to treatment-naive GCA and their long-term follow-up. Objectives To determine the expression of l-selectin (CD62L) and integrin αM (CD11b) on CD16+ neutrophils in peripheral blood of newly diagnosed, treatment-naive GCA cases, at the time of diagnosis (time 0) and during follow-up - at week 1, 4, 12, 24 and 48. In parallel, we aimed to measure also sera levels of serum amyloid A (SAA) and interleukin-6 (IL-6). Methods Peripheral blood from 33 treatment-naive GCA patients and 16 healthy blood donors (HBD) was stained, lysed, fixed and analysed by flow cytometry (Miltenyi). SAA and IL-6 were measured by nephelometry and ELISA, respectively. 3/33 GCA patients experienced relapse and were analysed separately during follow-up, at the time of relapse and 12 weeks after relapse. At the time of diagnosis, all patients received steroid treatment and therapy tapering started after 4 weeks. At week 12, some of the patients (14/22) received leflunomide, in addition to steroid therapy. Results Expression of CD62L, but not CD11b, was significantly higher on CD16+ neutrophils in treatment-naive GCA patients (median; IQR: 72.5; 56.2–100.7), as compared to HBD (55.9; 44.5–70.6, p=0.017). Longitudinally, the expression of CD62L significantly decreased in GCA patients from day 0 to week 4 (p=0.005). At week 12 there was an elevation in CD62L which escalated also at week 24. Patients receiving steroids only showed a marked increase at week 48, while patients receiving also leflunomide exhibited a decrease (Fig 1). Expression of CD11b declined from day 0 to week 4, but substantially increased throughout weeks 12, 24 and 48, regardless of therapy used. SAA and IL-6 declined sharply from day 0 to week 1 and 4, with gradual elevation of both at week 12. There was a decline in SAA levels observed in all patients at week 24, while IL-6 increased in patients receiving only steroids. These patients exhibited further elevation of both markers at week 48, while patients receiving steroids, in combination with leflunomide, showed a decrease. In 2/3 patients who experienced a relapse, we could observe an increase in the expression of CD62L at the time of relapse, which was found to be decreased again 12 weeks later. A similar trend was observed for IL-6. Conclusions Neutrophil CD62L could represent a good surface marker for detection of relapse in GCA. A distinct dichotomy was found for CD62L, as well as SAA and IL-6 in GCA during long term follow-up, with the combination of steroids with leflunomide showing more optimal results. Reference [1] Nadkarni S, et al. Investigational analysis reveals a potential role for neutrophils in GCA disease progression. Circ. Res. 2014;114:242–48. Acknowledgements The authors would like to thank the Rotary club Zgornji Brnik, Slovenia, as well as Prof. Mauro Peretti and Dr. Suchita Nadkarni from WHRI, Queen Mary, University of London for their support. The research was conducted within the National Research Programme (#P3–0314), financially supported by the Slovenian Research Agency. Disclosure of Interest None declared

Infections by pathogenic microorganisms present a serious concern in health care sector, as such infections kill more people than any other cause. The bacterial infections are commonly treated by antibiotics, but serious problems occur because of biofilm formation, which highly reduce the effectiveness of antibiotic therapy. Moreover, the overuse of antibiotics with excessively broad spectrum has increased the number of antibiotic-resistant bacterial strains, which are life threatening for patients. The bacterial infections due to implantable materials are connected with a high number of postsurgical complications, which cause immense health care costs because of prolonged antimicrobial therapy, multiple surgical intervention, and even implant removal. Thus there is an increasing demand to develop novel superior-quality medical implants that would prevent bacterial adhesion, biofilm formation and at the same time provide appropriate cell support for specific application. The rapid growth of nanotechnology and its immense potential in medicine has offered numerous opportunities for designing surfaces with superior properties, which will be used in new-generation medical devices. In this chapter, some of the antibacterial implantable devices are summarized, with emphasis on vascular implants. Bacterial adhesion and biofilm formation on implantable materials is discussed. Furthermore, the bacterial infections associated with vascular implants and the biological responses on these surfaces are presented in more detail. Research focuses on the major antibacterial mechanisms, reviewing antifouling and bactericidal approaches for the benefit of antibacterial surfaces.

The objectives of this study were (1) to determine how levels of serum amyloid A (SAA), high sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) correlate to autoimmune diseases in patients with or without thrombosis, and (2) to discuss the parameters that influence the relative SAA values. SAA, CRP and IL-6 concentrations were determined by enzyme linked immunosorbent assay (ELISA). 84 patients with secondary antiphospholipid syndrome (SAPS), primary antiphospholipid syndrome (PAPS), systemic lupus erythematosus with antiphospholipid antibodies (SLE+aPL), SLE, venous thrombosis (VT), arterial thrombosis (AT) were compared to healthy donors (n=60). The percentages of patients above cut-off were highest in the SAPS, SLE and SLE+aPL groups. Significant differences were observed between healthy donors and inflammatory groups of patients (SAPS and SLE+aPL) in all three measured parameters. SAA and CRP were shown to be correlated to a greater extent in SAPS patients than SLE+aPL patients. In summary, this cross-sectional, retrospective, small study and accompanying clinical considerations limit the ability to make definite conclusions. SAA would not serve as a useful marker for venous, arterial thrombosis or PAPS (pro-coagulant events). It could however, be a good predictor of progression from a non-inflammatory thrombotic condition to an inflammatory one.

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low ( n = 9), heterogeneous ( n = 31) and high ( n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p

The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy.MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5].Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations.MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.

Vaccines have undoubtedly brought overwhelming benefits to mankind and are considered safe and effective. Nevertheless, they can occasionally stimulate autoantibody production or even a recently defined syndrome known as autoimmune/inflammatory syndrome induced by adjuvants (ASIA). There is scarce data regarding autoimmune response after seasonal/influenza A (H1N1) vaccine in patients with autoimmune inflammatory rheumatic disease (AIRD). The objective of our study was therefore to determine autoimmune response in a large group of AIRD patients vaccinated against seasonal and/or H1N1 influenza. We conducted a prospective cohort study with a 6-month follow-up. Two-hundred and eighteen patients with AIRD (50 vaccinated against seasonal influenza, six against H1N1, 104 against both, 58 non-vaccinated controls) and 41 apparently healthy controls (nine vaccinated against seasonal influenza, three against H1N1, 18 against both, 11 non-vaccinated controls) were included. Blood samples were taken and screened for autoantibodies [antinuclear antibody (ANA), anti-extractable nuclear antigen (anti-ENA), anticardiolipin (aCL) IgG/IgM antibodies, anti-beta 2-glycoprotein I (anti-β2GPI)] at inclusion in the study, before each vaccination, 1 month after the last vaccination and 6 months after inclusion. For non-vaccinated participants (patients and healthy controls) blood samples were taken at the time of inclusion in the study and 6 months later. We report that after the administration of seasonal/H1N1 vaccine there were mostly transient changes in autoantibody production in AIRD patients and in healthy participants. However, a small subset of patients, especially ANA-positive patients, had a tendency towards anti-ENA development. Although no convincing differences between the seasonal and H1N1 vaccines were observed, our results imply that there might be a slight tendency of the H1N1 vaccine towards aCL induction. Although seasonal and H1N1 vaccines are safe and effective, they also have the potential to induce autoantibodies in selected AIRD patients and healthy adults. Follow-up of such individuals is proposed and further research is needed.

Background Chronic inflammation might be involved in the pathogenesis of different types of pulmonary hypertension (PH) [1]. Serum amyloid A (SAA) is an evolutionarily conserved, inflammatory protein expressed in the liver in a manner analogous to that of C-reactive protein (CRP). SAA is also expressed by several extrahepatic cells, including vascular smooth muscle, macrophages and endothelial cells in response to different stimuli, such as interleukin 6 (IL-6) [2]. Data on potential roles of SAA in PH are scarce. We recently reported that increased SAA is associated with pulmonary arterial hypertension (PAH) in patients with systemic sclerosis [3]. Furthermore, children with idiopathic PAH experiencing non-response to specific drugs and a worse prognosis, had significantly elevated SAA [4]. Objectives To check associations of SAA with CRP, IL-6 and noninvasive markers of disease severity in PH referral patients. Methods Seventy five consecutive patients were evaluated in the PH out-patient clinic within one year. Serum SAA and CRP were measured by nephelometry and IL-6 by routine ELISA. Markers of PH severity were evaluated with serum troponin I (TnI), N-terminal pro-B-type natriuretic peptide (NTproBNP), tricuspid annular plane systolic excursion ( TAPSE ), cardiac index (CI) and systolic pulmonary arterial pressure (sPAP), as assessed by routine echocardiography, and the six minute walk test (6MWT) in the whole group of patients, as well as in subgroups of 60 patients with PAH specifically, group I (n=17) or IV (n=43) according to WHO classification. Results Within the whole group of 75 PH patients (64% females, 36% males, mean age 63, range 29-89 years), we found significant correlations of SAA with CRP, diffusing capacity for CO, TnI (r=0.75, r=-0.36, r=0.23, respectively, all p Conclusions We showed for the first time, that SAA is associated with markers of chronic inflammation and disease severity in unselected adult patients with PH. In subgroup PAH type IV, where increased SAA might indicate non-responsiveness to specific drugs and worse prognosis, this association is even stronger. These new findings are pending further evaluation. References Kherbeck N, et al. Clin Rev Allergy Immunol. 2013; 44: 31-8. Eklund KK, et al. Crit Rev Immunol. 2012; 32: 335-48. Lakota K, et al. PLoS One. 2015 Jan 28;10(1):e0110820. Yeager ME, et al. Proteomics Clin Appl. 2012; 6:257-67. Disclosure of Interest None declared

Background Methotrexate (MTX) may reduce the risk of developing type 2 diabetes in rheumatoid (RA) and psoriatic arthritis (PsA). Animal studies suggest that MTX could reduce this risk by inducing intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl-59-monophosphate (ZMP). However, whether MTX therapy leads to chronic increase in ZMP concentration in RA or PsA patients has not been determined. Objectives We explored whether improvements in glucose homeostasis during MTX therapy in RA or PsA are associated with changes in concentration of ZMP or its metabolite 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Methods Newly diagnosed nondiabetic, MTX-naive, RA (2010 ACR/EULAR criteria) and PsA (CASPAR criteria) patients were enrolled between December 2012 and June 2013. Patients were treated according to the treat to target approach. DAS28ESR and the metabolic parameters were followed at baseline and at 1, 2, 3 and 6 months after MTX initiation. Concentration of glycated hemoglobin HbA 1c and HOMA-IR (fasting glucose (mmol/l) x fasting insulin (μU/ml)/22.5) were determined to assess glucose homeostasis. Erythrocyte ZMP concentration and urinary AICAR concentration were measured with liquid chromatography-mass spectrometry at baseline and at 1 and 6 months. Data were analyzed using repeated measures ANOVA or Friedman test, followed by an appropriate post hoc test. Results 16 RA and 10 PsA patients completed the study (16 female, 10 male). Mean age was 53±12, BMI 28.5±4.6 kg/m 2 and the initial DAS28ESR 5.3±1.3. Remission or low disease activity (DAS28ESR 1c concentration was reduced from 5.80%±0.29% (39.0±3.1 mmol/mol) at baseline to 5.52%±0.31% (36.8±3.5 mmol/mol) at 6 months ( P 1c concentration was observed in 19 out of 26 patients. The number of patients with prediabetes (5.7%≤HbA 1c concentration at 6 months were not associated with changes in ZMP or AICAR concentration. Conclusions Here we show that MTX therapy reduces HbA 1c concentration in RA or PsA patients. However, MTX does not produce a sustained increase in erythrocyte ZMP or urinary AICAR concentration, suggesting ZMP did not accumulate during MTX therapy. Collectively, our data do not support the notion that MTX improves glucose homeostasis via chronic accumulation of ZMP. Disclosure of Interest None declared

Background Influenza is a frequent infectious disease affecting more than 20% of the world population yearly. It can cause severe complications in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis (RA). In immunocompromised patients vaccinations are especially recommended. Influenza vaccines are generally safe and effective. However, RA patients require cautiousness in terms of vaccine immunogenicity especially when treated with immunomodulatory drugs. Objectives To determine the immunogenicity of a seasonal influenza vaccine in RA patients, its timeline and the influence of different medications on patients9 immune response. Methods 137 RA patients (38 males, 99 females, median age 33) and 54 healthy controls (HC; 11 males, 43 females, median age 54) were vaccinated with trivalent seasonal influenza vaccine (Sanofi Pasteur) containing purified haemagglutinins A/Brisbane/59/2007 (H1N1), A/Brisbane/10//2007 (H3N2), B/Brisbane/60/2008 (B). Sera were collected just before vaccination (baseline), 18-90 days and >180 days after vaccination. Measurement of antibodies against surface membrane proteins was performed using haemagglutination inhibition (HI) tests, while detection of antibodies against inner viral proteins of influenza A and influenza B viruses was tested by IgG and IgA ELISA. Results Using HI test, at baseline, RA patients were seroprotected (antibody titres ≥40) against seasonal influenza viruses A(H1N1), A(H3N2) and B in 58% (79/137), 97% (133/137) and 90% (123/137) respectively, versus 74% (40/54), 100% (54/54) and 83% (45/54) in HC. At the time of vaccination the IgG and IgA titres against inner proteins of influenza A and B viruses tested with ELISA showed a distinct elevation in the vaccinated (HC and RA) vs. non-vaccinated groups. After vaccination, significantly increased median titres of HI for A (H1N1), A (H3N2) and B were observed in controls and in RA. Compared to adalimumab, etarnercept, tocilizumab, infliximab, methotrexate and leflunomide, rituximab-treated patients had the lowest % of patients with of HI elevation for all three antigens (44%, H1N1; 22% H3N2, 50% B), which confirmed previous seasonal flu vaccination data (1). Seroconversion (postvaccination antibody titers ≥40 in patients whose prevaccination titres were 180 days. Similarly, the seroresponse (seroconversion or fold increase in antibody titers ≥4 in patients whose prevaccination titers were ≥10) in RA patients did not persist >180 days. Conclusions The seroresponse observed in RA compared to controls was higher for A (H1N1) but not for A (H3N2) and B. We found that vaccination largely tilts the balance from negative ELISA IgG and IgA titres to positive titres in those RA patients who respond with H1N1 >4-fold titre elevation. The immune responses (for all antigens tested) of treated RA patients are higher than non-treated patients for all medications used, except rituximab. References Eisenberg R.A. et al. Rituximab-treated patients have a poorer response to influenza vaccination. J Clin Immunol. 2013 Feb; 33(2):388-396. Disclosure of Interest None declared