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Efficient stent implantation among others depends on avoiding the aggregation of platelets in the blood vessels and appropriate proliferation of endothelial cells and controlled proliferation of smooth muscle cells, which reduces the development of pathology, such as neointimal hyperplasia, thrombosis, and restenosis. The current article provides an elegant solution for prevention of platelet and smooth muscle cell adhesion and activation on stent surfaces while obtaining surface conditions to support the growth of human coronary artery endothelial cells. This was achieved by surface nanostructuring and chemical activation of the surface. Specific nanotopographies of titanium were obtained by electrochemical anodization, while appropriate chemical properties were attained by treatment of titanium oxide nanotubes by highly reactive oxygen plasma. Surface properties were studied by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Wettability was evaluated by measuring the water contact angle. The influence of nanostructured morphology and plasma modification on in vitro biological response with human coronary artery endothelia and smooth muscle cells as well as whole blood was studied. Our results show that a combination of nanostructuring and plasma modification of the surfaces is an effective way to achieve desired biological responses necessary for implantable materials such as stents.

In this study, we explored expression of microRNA (miR), miR-target genes and matrix remodelling molecules in temporal artery biopsies (TABs) from treatment-naïve patients with giant cell arteritis (GCA, n = 41) and integrated these analyses with clinical, laboratory, ultrasound and histological manifestations of GCA. NonGCA patients (n = 4) served as controls. GCA TABs exhibited deregulated expression of several miRs (miR-21-5p, -145-5p, -146a-5p, -146b-5p, -155-5p, 424-3p, -424-5p, -503-5p), putative miR-target genes (YAP1, PELI1, FGF2, VEGFA, KLF4) and matrix remodelling factors (MMP2, MMP9, TIMP1, TIPM2) with key roles in Toll-like receptor signaling, mechanotransduction and extracellular matrix biology. MiR-424-3p, -503-5p, KLF4, PELI1 and YAP1 were identified as new deregulated molecular factors in GCA TABs. Quantities of miR-146a-5p, YAP1, PELI1, FGF2, TIMP2 and MMP9 were particularly high in histologically positive GCA TABs with occluded temporal artery lumen. MiR-424-5p expression in TABs and the presence of facial or carotid arteritis on ultrasound were associated with vision disturbances in GCA patients. Correlative analysis of miR-mRNA quantities demonstrated a highly interrelated expression network of deregulated miRs and mRNAs in temporal arteries and identified KLF4 as a candidate target gene of deregulated miR-21-5p, -146a-5p and -155-5p network in GCA TABs. Meanwhile, arterial miR and mRNA expression did not correlate with constitutive symptoms and signs of GCA, elevated markers of systemic inflammation nor sonographic characteristics of GCA. Our study provides new insights into GCA pathophysiology and uncovers new candidate biomarkers of vision impairment in GCA.

The research presented herein follows an urgent global need for the development of novel surface engineering techniques that would allow the fabrication of next-generation cardiovascular stents, which would drastically reduce cardiovascular diseases (CVD). The combination of hydrothermal treatment (HT) and treatment with highly reactive oxygen plasma (P) allowed for the formation of an oxygen-rich nanostructured surface. The morphology, surface roughness, chemical composition and wettability of the newly prepared oxide layer on the Ti substrate were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) analysis. The alteration of surface characteristics influenced the material’s bio-performance, platelet aggregation and activation was reduced on surfaces treated by hydrothermal treatment, as well as after plasma treatment. Moreover, it was shown that surfaces treated by both treatment procedures (HT and P) promoted the adhesion and proliferation of vascular endothelial cells, while at the same time inhibiting the adhesion and proliferation of vascular smooth muscle cells. The combination of both techniques presents a novel approach for the fabrication of vascular implants, with superior characteristics.

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.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL) that cause endothelial injury and thrombophilia. Extracellular vesicles are involved in endothelial and thrombotic pathologies and may therefore have an influence on the prothrombotic status of APS patients. Intercellular communication and connectivity are important mechanisms of interaction between healthy and pathologically altered cells. Despite well-characterized in vitro and in vivo models of APS pathology, the field of extracellular vesicles is still largely unexplored and could therefore provide an insight into the APS mechanism and possibly serve as a biomarker to identify patients at increased risk. The analysis of EVs poses a challenge due to the lack of standardized technology for their isolation and characterization. Recent findings in the field of EVs offer promising aspects that may explain their role in the pathogenesis of various diseases, including APS.

BackgroundImmunoglobulin A vasculitis (IgAV) is a small vessel leukocytoclastic vasculitis, characterized by vascular IgA deposits, and clinically by skin, join, kidney and bowel involvement. Renal involvement, present in up to 84% of adult patients, is associated with an increased risk of progression to chronic kidney failure. A skin or renal biopsy is still the golden standard for diagnosis. Omics data from NCBI-GEO and Array Express databanks are lacking from IgAV samples of adult patients.ObjectivesOur aim was to perform RNA sequencing and to identify differentially expressed genes and dysregulated molecular pathways in the affected skin of adult IgAV patients with renal involvement, patients with skin-limited IgAV and healthy controls (HC).MethodsSkin biopsy samples were collected from treatment-naive adult IgAV patients at time of diagnostic procedure: 1) skin-limited disease (n=3), 2), IgAV with renal involvement (n=3), and age-/sex-matched HC (n=3). RNA was isolated and 100 bp paired-end sequenced using the Illumina HiSeq 4000 platform (25.9 to 30.8 million reads were obtained per sample). Reads were mapped with Salmon tool against the human transcriptome (Ensemble Release 104). Differentially expressed genes (log2 fold change ≥ │1│, padj < 0.05) were computed using the R package DESeq2. Data were clustered using principal component analysis (PCA), and KEGG pathway over-representation analysis (ORA) was performed with R package clusterProfiler.ResultsWe identified 72 differentially expressed genes (34 up- and 38 down-regulated) between skin-limited IgAV and HC, while 686 genes were differentially expressed (421 up- and 265 down-regulated) in the affected skin between adult IgAV patients with renal involvement and healthy controls (HC). PCA distinguished between IgAV (renal and skin-limited) and healthy controls, suggesting specific transcriptome signatures in IgAV. 21 overlapping genes were differentially expressed in both groups of patients in comparison to HC. 196 genes were differentially expressed between IgAV patients with renal involvement compared to skin-limited IgAV.Genes, differentially expressed in patients with skin-limited disease were enriched in the oxidative phosphorylation (padj = 3.08 x 10-5) and cell adhesion molecules (padj = 6.32 x 10-4) KEGG Pathways. Differentially expressed genes in patients with renal involvement exhibited enriched KEGG Pathways involved in the ribosome (padj = 0 x 10-7), the regulation of lipolysis in adipocytes (padj 9,066 x 10-4), cell adhesion molecules (padj = 8.0008 x 10-3), renin secretion (padj = 4.2882 x 10-3), complement and coagulation cascades (padj = 3.58547 x 10-2), vascular smooth muscle contraction (padj = 3.76132 x 10-2) and aldosterone synthesis and secretion (padj = 3.76132 x 10-2).ConclusionDeregulated genes in skin of adult IgAV patients with renal manifestation could serve as biomarkers for organ-specific involvement. Further studies are needed that would support deciphering the involvement of perturbed molecular pathways and the contribution of individual cell types to skin manifestations of adult IgAV patients.AcknowledgementsThe authors also wish to thank all the patients who volunteered to participate into this study and the funding.Disclosure of InterestsNone declared

BackgroundActivated fibroblasts are the main drivers of skin fibrosis in SSc. We have recently identified dimethyl alpha-ketoglutarate (dm-aKG) as a potential repressor of myofibroblast differentiation and profibrotic activity in cultured skin fibroblasts.ObjectivesTo further analyse the clinical translation of our findings by investigating the antifibrotic capacity of dm-aKG on explanted skin biopsies from SSc patients.MethodsWe cultured forearm punch skin biopsies from SSc patients (n=10) for 24h ex vivo in the presence/absence of 6 mM dm-aKG. Thereafter, skin biopsies (n=4) were dissociated into single cells using a combined mechanical-enzymatic dissociation protocol, followed by single cell (sc)RNA-seq library preparation (10x Genomics) and sequencing (Illumina, NovaSeq6000, 50,000 reads/cell). We mapped the scRNA-seq reads to the reference genome GRCh38.p13 and analysed the data with R/Bioconductor tools. We deconvoluted cell types in bulk skin transcriptomes from SSc cohorts (GSE: 45485, 59785, 9285, 32413) using human skin scRNA-seq data1. The secretion of IL-6, procollagen-1, PRO-C1 (N-terminal type I collagen pro-peptide), C1M (MMP-degradation fragment of type I collagen), and fibronectin (FBN-C) from cultured skin (n=10) was measured in supernatants by ELISA. We analysed gene and protein expression in TGFβ-activated healthy and SSc dermal fibroblasts (DF, n=10) treated or not with dm-aKG using qPCR, Western blot and ELISA. Contractile properties of DF were assessed by gel contraction assay. Traction forces generated by DF were determined by reference-free traction force microscopy.ResultsDissociated cultured SSc skin exhibited comparable cell yield and viability in the presence (20,203; 89%) and absence (25,280; 93%) of dm-aKG, respectively. scRNA-seq skin analysis included 20,869 high quality single cell profiles segregating into 10 distinct skin cell populations (Figure 1A). This analysis demonstrated decreased proportion of fibroblasts and increased proportion of keratinocytes in dm-aKG treated skin (p0.5, FDRIL6, STAT1) and extracellular matrix (ECM) organization (hub genes MMP1, ITGB3) as top downregulated biological processes in fibroblasts in dm-aKG treated SSc skin (Figure 1C), coinciding with a decreased abundance of proinflammatory skin fibroblast subpopulation. Specifically, these cells were identified as the main source of IL6 (Figure 1D) and were enriched in SSc skin as revealed by deconvolution analysis of skin transcriptomes. Furthermore, dm-aKG reduced the secretion of IL-6, procollagen-1 and C1M, but not pro-C1 and FBN-C, from cultured skin explants. In cultured DF, dm-aKG blocked the inflammatory (IL-6, pSTAT3), profibrotic (aSMA, Fibronectin, Procollagen-1, Pro-C1) and contractile activities, and significantly diminished traction forces exerted by DF on the matrix substrate.Figure 1.scRNA-seq – comparison of untreated and dm-aKG treated paired skin biopsies. (A) UMAP plot with annotated skin cells, (B) differential abundance of main skin cell types, (C) volcano plot of DE genes with top downregulated gene ontology (GO) pathways in dm-aKG treated skin fibroblasts, (D) IL6 expression in untreated (blue) and treated (pink) skin fibroblasts.ConclusionDm-aKG broadly interferes with inflammatory and ECM organizational activities of skin fibroblasts in culture and in explanted skin from SSc patients. These results confirm that dm-aKG might represent a potential new therapeutic approach for efficient targeting of skin inflammation and fibrosis in SSc.References[1]He H et al. J Allergy Clin Immunol 2020AcknowledgementsThis work was supported by a research grant from FOREUM Foundation for Research in Rheumatology and University Medical Centre Ljubljana.Disclosure of InterestsBlaž Burja: None declared, Dominique Paul: None declared, Reto Gerber: None declared, Sam G. Edalat: None declared, Muriel Elhai Speakers bureau: BMS, Elena Pachera: None declared, Rahel S. Zingg: None declared, Francesca Michela Pramotton: None declared, Sofie Falkenløve Madsen: None declared, Kristina Buerki: None declared, Giampietro Costanza: None declared, Michael Whitfield: None declared, Anne-Christine Bay-Jensen: None declared, Snežna Sodin-Šemrl: None declared, Matija Tomsic: None declared, Gabriela Kania: None declared, Hubert Rehrauer: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Ziga Rotar: None declared, Mark Robinson: None declared, Katja Lakota: None declared, Mojca Frank Bertoncelj: None declared.

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

Influenza may cause severe complications in patients with autoimmune inflammatory rheumatic disease (AIRD), to whom vaccinations are especially recommended. However, AIRD patients require cautious scrutiny of immunogenicity as they might exhibit poor antibody response to vaccination, especially when taking immunomodulatory medications.The aim was to determine immunogenicity of seasonal and pandemic influenza vaccine in AIRD patients, its timeline/persistence, and influence of medications on immune response.One hundred and thirty-seven AIRD and 54 healthy controls were vaccinated with trivalent seasonal influenza. After 3-5 weeks, 15 healthy controls and 93 AIRD were vaccinated with pandemic influenza vaccine, and 63 of patients were vaccinated a second time after 3-5 weeks. Sera were collected before vaccination, 18-90 days after each vaccination, and more than 180 days after the last vaccination. The immune response was measured using hemagglutination inhibition (HI) assay and IgG/IgA antibodies against influenza A/B with ELISA.Our findings indicate that following vaccination with seasonal influenza vaccine, seroprotection, seroresponse, and change in geometric mean titers (GMT) in AIRD patients was not compromised compared to healthy. Similarly, we report for pandemic influenza vaccination little added benefit of the second dose. We confirm lowest increase in HI titer in rituximab-treated AIRD compared to other medications. Vaccination largely tilts the balance from negative ELISA A IgG and IgA titers to positive titers in seasonal H1N1 seroresponsive AIRD patients and controls. A significant decrease in HI GMT and seroprotection was observed only in AIRD at 180 days after vaccination highlighting an absent persistence of immunogenic response in AIRD patients. Due to high initial HI titers for influenza vaccine, we foresee their benefit in personalized medicine in the future.Influenza vaccination is immunologically active for AIRD, with little value of the second dose of the pandemic vaccine and further scrutiny on persistence of immune response to vaccine in AIRD is needed.

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.

It was the aim of this work to determine whether the plasma concentration of extracellular vesicles (EVs) in active diabetic Charcot neuroarthropathy (CN) is connected to the inflammatory markers, temperature elevation in the affected foot and concentration of soluble receptor for advanced glycation end products (RAGE). EVs were isolated from peripheral blood of 35 patients with active CN. EVs were counted after repetitive centrifugation and washing of samples, by flow cytometry. Foot temperature was measured by infrared thermometer. Concentration of soluble receptor for advanced glycation end products (RAGE) was determined by enzyme-linked immunosorbent assay (ELISA). We found statistically significant correlations of EV concentration (but not soluble RAGE concentration) with C-Reactive Protein (CRP) and with temperature difference between the affected and the contralateral foot (r=0.40, p=0.032; r=0.89, p

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

Heparins represent an efficient treatment of acute thrombosis and obstetric complications in antiphospholipid syndrome (APS). Enhanced microvesiculation of cell membranes, as detected by reduced membrane adhesion, can contribute to hypercoagulability in APS. Healthy donor IgG antibodies significantly increased beta2-glycoprotein I (beta2-GPI)-induced membrane adhesion, indicating that IgG antibodies might supplement the role of beta2-GPI in the regulation of membrane microvesiculation in healthy individuals. Anti-beta2-GPI IgG antibodies significantly reduced beta2-GPI-induced membrane adhesion, suggesting a direct role of anti-beta2-GPI antibodies in enhancing membrane microvesiculation in APS. Therapeutic concentration of nadroparin completely restored beta2-GPI-induced membrane adhesion in the presence of anti-beta2-GPI IgG antibodies. A novel anticoagulant mechanism of nadroparin in APS is suggested that supplements its direct effect on the coagulation cascade. Restoration of adhesion between negatively charged membranes in the presence of nadroparin might decrease shedding of microvesicles into the surrounding solution and could thus contribute to the efficacy of heparin treatment in APS.

Lipoxin A4 (LXA4) and scrum amyloid A (SAA) are endogenous negative and positive modulators of inflammation, respectively. Both molecules bind the shared lipoxin A4 receptor (ALX) and elicit opposing effects on the production of inflammatory cytokines and matrix metalloproteinases. The aim of these studies is to examine the divergence of the intracellular signaling pathways triggered by lipid LXA4 (1 nM) and protein SAA (200 nM) ligands of ALX. Phospholipase D (PLD) is a phosphohydrolase enzyme that catalyzes the generation of phosphatidic acid (PA) from membrane phospholipids. Our results showed that in fibroblast-like synoviocytes, activation of PLD occurred only in response to LXA4, and not SAA. PA (30 μM) mimicked LXA4 and demonstrated inhibition of IL-8 production induced by SAA or interleukin-1β. In sharp contrast to LXA4, SAA confirmed the stimulation of IL-8 release as determined previously. Taken together, these findings suggest that two physiologic ligands sharing the common ALX receptor, LXA4 and SAA, differentially regulate the level of PLD activation and differentially modulate IL-8. These results may have important implications for understanding the regulation of inflammatory responses under physiologic and pathological conditions.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

In order to elucidate beta2-GPI at the DNA level and characterize its polymorphisms, mRNA expression, protein levels and clinical significance at each of these steps, a molecular review of beta2-GPI literature was performed. The human beta2-GPI complete nucleotide sequence has been reported and it consists of 8 exons separated by large introns. The beta2-GPI gene is polymorphic with four alleles. The distribution of point mutations can be significantly different between various racial populations. DNA variation studies of the beta2-GPI gene identified a total of 151 single-nucleotide polymorphisms, 26 of which are within regions with potential clinical significance. Southern blot analysis indicated the presence of one gene product only. An atypical TATA box and a hepatic nuclear factor-1 element are both essential for beta2-GPI promoter activity. Transcription factor binding sites for STAT, CREB, C/EBPbeta, NF-1, AP-1, NFAT, HNF-3beta and HNF-1 have been identified in the promoter region of the beta2-GPI gene by computer analysis. The beta2-GPI transcriptional signal of 1.5 kb was detected in Northern blot analysis and its 326-amino-acid sequence was found to be one of the most proline-rich eukaryotic proteins. Amino acid substitutions have been shown to be associated with loss of phospholipid binding, development and recognition of antiphospholipid antibodies.

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.

Aim To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. Methods Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunoflourescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. Results FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (

The aim of this report was an overview of beta2-glycoprotein I (beta2-GPI)- and annexin A5 (AnxA5)-phospholipid interactions including candidate beta2-GPI receptors, and their relevance to the investigation of physiological/pathological processes. Both beta2-GPI and AnxA5 have thrombomodulatory functions in vivo and their antigenicity is particularly important for thrombotic manifestations and pregnancy complications in antiphospholipid syndrome. Specific elements of beta2-GPI- and AnxA5-phospholipid interactions are different. The crucial elements for beta2-GPI are conformational change and dimerization, both of which are also required and necessary for receptor signaling, leading to the prothombotic state. AnxA5 differs in its ability to crystallize into a protective shield, the disruption of which seems to be the major prothrombotic mechanism. These differences may explain some of the functional consequences of both molecules seen in the pathological conditions. Future alternative therapies of antiphospholipid syndrome are proposed to be based on the expanding knowledge of beta2-GPI- and AnxA5-phospholipid interactions, specifically antagonizing beta2-GPI receptors, as well as inhibiting their signaling pathways.

Aims Oxidation reactions can modify protein activity or specificity. Recently, a novel redox-reactive family of autoantibodies was described, which indicated involvement of altered antibodies (beside altered antigens) into autoimmune reactions. The aim of our study was to determine the binding capacity alterations of electro-oxidized blood donors' IgGs, and to evaluate their effects on released proinflammatory interleukin 6 in HUVEC. Results We found out that 1.) Isolated blood donor IgGs bound after electro-oxidation to β2-glycoprotein I, cardiolipin, citrullinated cyclic peptide and protein 3 by enzyme-linked immunosorbent assay, extractable nuclear antigens by counterimmuno-electrophoresis, and cell antigens by indirect immunofluorescence; 2.) Alterations in immunoreactivity of IgGs due to oxidation highly depend on electric current, time of exposure and the presence of antioxidants, 3.) Treatment of HUVEC with oxidized IgGs resulted in changed cell morphology, accompanied by an increase in released interleukin-6. Conclusions Our data suggest repeatable transformation of antibodies present in the blood of healthy persons and patients. Inter-individual differences in chemical stability of antibodies, patient's antioxidant status, and the microenvironmental changes at the cellular level may influence the range of antibody alterations and their involvement in pathophysiological autoimmune processes.

The way proteins associate with the lipid bilayer defines not only their function, but in autoimmune diseases, such as antiphospholipid syndrome (APS), also represents the conformational change of an antigen necessary for the exposure of critical epitopes recognized by potential autoantibodies. These interactions between the antigen (bound to phospholipid bilayers) and its antibodies could lead to clinical implications. Our aim was to review and characterize the requirements of the plasma protein β2‐glycoprotein I (β2‐GPI), one of the major proteins implicated in autoantibody development in APS for interactions with phospholipids, to provide primary sequence and secondary structure information of its Vth phospholipid binding domain, and to perform a NCBI search for comparison between homology of the Vth β2‐GPI domain with other known proteins. The second part addresses the effects of phospholipid membrane composition, charge, and specifically, the mechanism of interaction with artificial negatively charged membranes as in vitro model systems (vesicle and other studies). The third part focuses on the influence of environmental factors on β2‐GPI–phospholipid binding, and the fourth part addresses antibody binding to membrane‐bound β2‐GPI, exposure of its epitopes, recognition by its autoantibodies, and the functional (clinical) implications (specifically in APS). The development of antigenicity is addressed, with an emphasis on human studies and pathogenicity of antibodies. In the future, we plan to concentrate also on the numerous, important and critical reports centered on cell‐specific and receptor‐mediated studies, which we have omitted in the present compilation, since they are beyond the scope of this chapter.

The loss of membrane asymmetry is an important feature in mechanisms involved in maintaining immunity. Negatively charged phospholipids are present only in small amounts in the outer leaflet of the membranes of viable cells. In some pathological conditions and in apoptosis, negatively charged phospholipids become exposed on the outer membrane surface facing among other surroundings, the blood plasma. There, they can contribute to the catalytic potential for chemical reactions leading to blood clot formation. Also, they serve, in connection with protein cofactors, as antigens in the immune system leading to autoantibody formation and autoimmune diseases, among them of special emphasis, antiphospholipid syndrome (APS). APS is manifested clinically by venous and arterial thromboses and fetal loss. Laboratory analysis reveals a presence of antiphospholipid antibodies (aPL) in the patients' blood and research analysis indicates an increased amount of cell-derived microvesicles (MVs). MVs often have an impaired membrane asymmetry and can be procoagulant. Elevation of MV quantity in patient sera from different cells has been shown to be associated with thromboses. It is indicated that MVs can also be antigenic, depending on the properties and content of the blood plasma, that is, the presence in plasma of certain proteins called protein cofactors. Although numerous methods have been used in attempt to understand the antigen—phospholipid—antibody interactions and their role in APS, knowledge of the underlying mechanisms remains fragmentary. In this chapter, we study complex interactions between protein cofactors, phospholipid membranes, and aPL in giant phospholipid vesicles and in erythrocytes. We consider the process of budding and vesiculation in giant phospholipid vesicles and in erythrocytes, plasma protein-mediated attraction of negatively charged phospholipid membranes, and effects of annexin A5 (ANXA5) on interaction of protein cofactors and aPL with the phospholipid membranes. Our results indicate that phospholipid vesicles and erythrocytes can be useful not only in elucidating basic mechanisms involved in APS but can serve as models of antigen—phospholipid—antibody interactions with potential for improving clinical diagnosis and/or prognosis of this debilitating and sometimes lethal autoimmune disorder. Further studies in this direction should be undertaken.

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