Martin Andersen

Ultrasound colour Doppler is associated with synovial pathology in biopsies from hand joints in rheumatoid arthritis patients: a cross-sectional study

Objectives Little is known regarding the association between ultrasound-determined pathological synovial blood flow and synovial pathology in rheumatoid arthritis (RA). We therefore examined the association between colour Doppler ultrasound imaging and synovitis assessed by histopathology and specific cell markers by immunohistochemistry in patients with RA. Methods 81 synovial sites from wrist and finger joints from 29 RA patients were evaluated by ultrasound colour Doppler and subsequently biopsied by needle arthroscopy. The association between ultrasound colour fraction and an overall synovitis score and immunohistochemical staining for CD3, CD68, Ki67 and von Willebrand factor was investigated, including repeated samples from the same patients. The overall synovitis score (total 0–9) assessed synovial lining hyperplasia (0–3), stromal activation (0–3) and inflammatory infiltration (0–3). Data were clustered within patients, thus a linear mixed model was applied for the statistical tests. Parsimony in the statistical models was achieved omitting covariates from the model in the case of what was judged no statistical significance (p>0.1). Results Doppler colour fraction showed an association with the overall synovitis score (approximated Spearman, approximately r=0.43, p=0.003). The density of all immunohistochemical stainings showed a significant association with Doppler colour fraction: von Willebrand factor (approximately r=0.44, p=0.01), CD68 (approximately r=0.53, p=0.02), Ki67 (approximately r=0.57, p=0.05) and CD3 (approximately r=0.57, p=0.0003). Conclusions Colour Doppler activity is associated with the extent of inflammation present in the synovial biopsies from RA patients. However, synovial pathology was also seen in biopsies taken from Doppler negative sites.

Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

IntroductionDespite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.MethodsRA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.ResultsTissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.ConclusionsThe association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.

Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

Association between IL-6 production in synovial explants from rheumatoid arthritis patients and clinical and imaging response to biologic treatment: A pilot study

Introduction The need for biomarkers which can predict disease course and treatment response in rheumatoid arthritis (RA) is evident. We explored whether clinical and imaging responses to biologic disease modifying anti-rheumatic drug treatment (bDMARD) were associated with the individual’s mediator production in explants obtained at baseline. Methods RA Patients were evaluated by disease activity score 28 joint C-reactive protein (DAS 28-)), colour Doppler ultrasound (CDUS) and 3 Tesla RA magnetic resonance imaging scores (RAMRIS). Explants were established from synovectomies from a needle arthroscopic procedure prior to initiation of bDMARD. Explants were incubated with the bDMARD in question, and the productions of interleukin-6 (IL-6), monocyte chemo-attractive protein-1 (MCP-1) and macrophage inflammatory protein-1-beta (MIP-1b) were measured by multiplex immunoassays. The changes in clinical and imaging variables following a minimum of 3 months bDMARD treatment were compared to the baseline explant results. Mixed models and Spearman’s rank correlations were performed. P-values below 0.05 were considered statistically significant. Results 16 patients were included. IL-6 production in bDMARD-treated explants was significantly higher among clinical non-responders compared to responders (P = 0.04), and a lack of suppression of IL-6 by the bDMARDS correlated to a high DAS-28 (ρ = 0.57, P = 0.03), CDUS (ρ = 0.53, P = 0.04) and bone marrow oedema (ρ = 0.56, P = 0.03) at follow-up. No clinical association was found with explant MCP-1 production. MIP-1b could not be assessed due to a large number of samples below the detection limit. Conclusions Synovial explants appear to deliver a disease-relevant output testing which when carried out in advance of bDMARD treatment can potentially pave the road for a more patient tailored treatment approach with better treatment effects.

BLOCKING THE INHIBITORY CD94/NKG2A NK CELL RECEPTOR WITH A NOVEL ANTI-NKG2A MAB ENHANCES THE SUSCEPTIBILITY OF RHEUMATOID ARTHRITIS FIBROBLAST-LIKE SYNOVIOCYTES (FLS) TO NK CELL-MEDIATED CYTOTOXICITY

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive bone erosion and cartilage degradation [1]. Fibroblast-like synoviocytes (FLS) present in the inflamed joints of RA patients are central components of the aggressive, tumor-like structure named pannus that erodes the joints [2]. CD94/NKG2A+ natural killer (NK) cells are found in the inflamed joints of RA patients [3], and their role in the disease process is emerging. Objectives The aims of the present study were to characterize the expression of potential ligands for NK cell receptors on rheumatoid arthritis synovial tissue-derived FLS, investigate the susceptibility of FLS to NK cell-mediated lysis in vitro, as well as characterize, phenotype and localize NK cells and FLS in inflamed RA joints. Methods FLS were derived from human synovial tissue obtained from the small joints of RA patients undergoing arthroscopic synovectomy. NK cells from the blood of healthy donors, or the synovial fluid of RA patients, were co-cultured with FLS, and the susceptibility of FLS to NK cell-mediated cytotoxicity was assessed by degranulation of NK cells and subsequent expression of CD107a/b by flow cytometry. Flow cytometry was performed on FLS and NK cells to characterize the expression of NK cell ligands and receptors, respectively. Results RA synovial NK cells express several activating receptors of which primarily NKG2D, DNAM-1 and NKp44 are involved in killing of FLS upon co-culture in vitro. Furthermore, FLS express numerous ligands for such activating NK cell receptors, and also express HLA-E, a molecule responsible for restraining NK cell-mediated cytotoxicity through interactions with CD94/NKG2A inhibitory receptors expressed on most synovial NK cells. We found that blocking the interaction between HLA-E and CD94/NKG2A with a novel humanized anti-NKG2A mAb (NNC141-0100) enhanced NK cell degranulation towards FLS. Conclusions This study is the first to suggest that NK cells may play a role in the elimination of FLS, a process that is enhanced upon blocking the ability of HLA-E to engage the inhibitory CD94/NKG2A NK cell receptor. Exploitation of the cytotoxic potential of NK cells by blocking CD94/NKG2A with the novel humanized anti-NKG2A mAb NNC141-0100 may thus yield an opportunity for therapeutic treatment of chronic inflammation. NNC141-0100 is currently being tested in a phase I clinical trial for rheumatoid arthritis. References Gerlag DM et al. Novel approaches for the treatment of rheumatoid arthritis: lessons from the evaluation of synovial biomarkers in clinical trials. Best Pract Res Clin Rheumatol. 2008 Apr;22(2):311-23. Neumann E et al. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends Mol Med. 2010 Oct;16(10):458-68. Teixeira de Matos C et al. Activating and inhibitory receptors on synovial fluid natural killer cells of arthritis patients: role of CD94/NKG2A in control of cytokine secretion. Immunology. 2007 Oct;122(2):291-301. Disclosure of Interest N. Nielsen Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, E. Galsgaard Shareholder of: Novo Nordisk, Employee of: Novo Nordisk, D. Ahern: None Declared, M. Andersen Employee of: Novo Nordisk, P. Spee Shareholder of: Novo Nordisk, M. Feldmann: None Declared, F. Brennan: None Declared, K. Söderström Employee of: Novo Nordisk

OP0016 The Effects of Biologics on Imaging Pathology Reflects Changes in Ex Vivo Mediator Release from Rheumatoid Arthritis Synovial Explants: A Cohort Study

Background It remains to be clarified whether disease changes detected by color Doppler ultrasound (CDUS) and magnetic resonance imaging (MRI) during biological treatment reflect alterations of synovial pathology in rheumatoid arthritis (RA). Objectives Compare the changes CDUS and MRI scores during biological (bDMARD) treatment of RA patients with the effects of the same bDMARD on inflammatory mediator release from synovial explants obtained at baseline. Methods RA patients opted for bDMARD treatment with synovial hypertrophy in hand joints were eligible. CDUS and MRI and subsequent synovectomy by a needle athroscopic procedure was performed on the hand joint with most pronounced ultrasound pathology. Patients were re-evaluated with imaging after a minimum three months bDMARD treatment. Synovectomies were performed in ≤6 positions. Explant cultures, were established from the synovectomy specimens at baseline and incubated for 2 weeks with the bDMARD prescribed for the patient and isotype control (10μg/mL). CDUS activity was defined as the systolic color pixel/gray scale ratio (CFmax). MRI outcomes were the RA MRI score (RAMRIS) components for synovitis (0-3), bone marrow edema (BMI, 0-3) and erosion (0-10). Synovial mediator release of interleukin 6 and 8 (IL-6 and IL-8), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 beta (MIP-1b) were detected by multiplex immuno assays (Myriad RBM™). Heterophilic antibody interference was blocked. Mixed linear models were applied for the statistical analyses for the associations of changes in imaging and the fold change (72 hours/two weeks culture) in mediator concentration. Approximative correlation coefficients (approx.rho) were calculated using Spearman rank correlation. P-values <0.05 were considered significant. Results 16 RA patients initiated bDMARD treatment. Median follow up time was 7 months (inter quartile range 7-11 months). Fifteen patients had matching CDUS and explant data while 11 patients had matching MRI and explant data, respectively. The release of IL-6 from bDMARD treated explant samples were statistically significantly associated to changes in CFmax (approx. rho =0.52, P=0.04) and RAMRIS BME score (approx.rho=0.56, P=0.03), while MCP-1 production was only significantly associated to RAMRIS BME (approx.rho=0.49, P=0.01). There were no significant associations to change in RAMRIS synovitis or erosion score. Analysis of IL-8 and MIP-1b was discarded due to a large number of samples outside assay detection limits. Conclusions In RA, changes in CDUS and MRI pathology during bDMARD treatment reflected the changes in cytokine production induced by the same biologic in synovial biopsy cultures. This substantiates the use of imaging parameters as markers of local disease activity. The lack of associations with explant activity and changes in MRI synovitis and erosions may be due to the synovectomy procedure and relative short follow up, respectively. Acknowledgements The OAK Foundation, Novo Nordisk A/S and The Danish Agency for Science, Technology and Innovation for unrestricted grants. Disclosure of Interest M. Andersen Employee of: Novo Nordisk A/S, M. Boesen: None declared, K. Ellegaard: None declared, K. Söderström Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, N. Søe: None declared, P. Spee Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, U. Mørch Employee of: Data collected during employment at Novo Nordisk A/S, S. Torp-Pedersen: None declared, E. Bartels: None declared, B. Danneskiold-Samsøe: None declared, L. Karlsson Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, H. Bliddal: None declared

SYNOVIAL EXPLANT INFLAMMATORY MEDIATOR PRODUCTION IS CORRELATED WITH DISEASE ACTIVITY IN RHEUMATOID ARTHRITIS: A CROSS SECTIONAL STUDY

Background In vitro assays are widely used for characterisation of rheumatoid arthritis (RA) pathology and for testing of potential disease modifying anti-rheumatic drugs (DMARDs). However, little is known to what extent in vitro disease activity measures are correlated with in vivo disease activity. Objectives The aim of this study was to investigate whether the release of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 beta (MIP-1b) correlated with serum c-reactive protein (CRP), tender joint count (TJC, 28 joints), swollen joint count (SJC, 28 joints) and disease activity score of 28 joints (DAS-28-CRP) of RA patients. Methods Forty synovial sites from the hand joints of sixteen RA patients scheduled for biologic DMARD treatment were synovectomised by a needle-arthroscopic procedure. Synovial tissue was cultured for 72h, and the concentrations of IL-6, IL-8, MCP-1 and MIP-1b in the culture supernatants were measured by Multiplex immunoassays using heterophilic antibody blocking agents. Spearman rank correlations (r) were calculated for outcome measures using averaged values from the various explant positions. A p-value<0.05 was considered significant. Results All four mediators were statistically significantly (least significant p-value =0.01) correlated with SJC (r=0.64, r=0.71, r=0.62 and r=0.65 for IL-6, IL-8, MCP-1 and MIP-1b, respectively). IL-6, IL-8 and MCP-1 were highly significantly correlated with TJC (r=0.78, 0.74 and r=0.77, respectively, p=0.001 for IL-8 and p=0.0004 for IL-6 and MCP-1, respectively), whereas MIP-1b had a lower correlation of r=0.58 (p=0.02) for TJC. IL-6 (r=0.58, p=0.02), IL-8 (r=0.56, p=0.03) and MCP-1 (r=0.70, p=0.003) were significantly correlated with DAS-28-CRP. In contrast no statistically significant correlation was found with DAS-28-CRP and MIP-1b (r=0.33, p=0.27) or any of the three mediators with CRP-levels. Conclusions The production of pro-inflammatory mediators, IL-6, IL-8, MCP-1 and MIP-1b, by synovial explants from RA patients with active arthritis shows a high degree of correlation with clinical disease activity measures. This is indicative of a disease model that can facilitate translation of in vitro findings to in vivo effects. Acknowledgements Supported by unrestricted grants from Novo Nordisk, The Danish Agency for Science Technology and Innovation and the Oak Foundation. Disclosure of Interest M. Andersen: None declared, M. Boesen: None declared, K. Ellegaard: None declared, R. Christensen: None declared, K. Söderström Employee of: Nono Nordisk, N. Søe: None declared, P. Spee Employee of: Nono Nordisk, U. Mørch Shareholder of: Nono Nordisk, Employee of: Nono Nordisk, S. Torp-Pedersen: None declared, E. Bartels: None declared, B. Danneskiold-Samsøe: None declared, N. Vendel: None declared, L. Karlsson Employee of: Nono Nordisk, H. Bliddal Grant/research support: Nono Nordisk DOI 10.1136/annrheumdis-2014-eular.4690

OP0234 Association between ultrasound colour doppler activity and synovial pathology in biopsies from small hand joints in patients with rheumatoid arthritis: A cross sectional study

Background Little is known regarding the association between ultrasound determined pathological synovial blood flow and synovial pathology in rheumatoid arthritis (RA). Objectives To examine the association between colour Doppler ultrasound imaging and synovitis according to histopathology in patients with RA. Methods 51 synovial sites from the small hand joints from 19 RA patients were evaluated by ultrasound colour Doppler and subsequently biopsied by needle arthroscopy. Association between ultrasound colour fraction and an overall synovitis score (1) and immunohistochemical stainings for CD3, CD68, Ki67 and von Willebrandt Factor were investigated (2;3) using repeated samples from the same patients. The overall synovitis score (total 0-9) assessed synovial hypertrophy (0-3), stromal proliferation (0-3) and cellular infiltration (0-3). Data was clustered within patients, thus a linear mixed model was applied for the statistical analysis. Parsimony in the statistical models was achieved omitting covariates from the model in the case of, what was judged, no statistical significance (P>0.1). Results For the RA patients Doppler colour fraction showed an association with the overall synovitis score (Approximated Spearman, Appr.rho =0.43, P=0.002). The density of all immunohistochemical staining showed a significant association with Doppler colour fraction: von Willebrandt Factor (Appr.rho =0.49, P=0.01), CD68 (Appr.rho=0.49, P=0.0002,), Ki67 (Appr.rho =0.47, P=0.002) and CD3 (Appr.rho=0.66, P<0.0001). Conclusions Colour Doppler activity significantly correlates with the extent of inflammation present in the synovium of RA patients. Absence of a detectable colour fraction does not rule out synovial pathology. Acknowledgement Supported by unrestricted grants from Novo Nordisk and the Oak Foundation. B. Jørgensen for preparation of the synovial biopsies. References Krenn V et al.Grading of chronic synovitis–a histopathological grading system for molecular and diagnostic pathology. Pathol Res Pract 2002;198(5):317-25. Ogdie A et al. Identification of broadly discriminatory tissue biomarkers of synovitis with binary and multicategory receiver operating characteristic analysis. Biomarkers 2010 March;15(2):183-90 Pessler F et al. The synovitis of “non-inflammatory” orthopaedic arthropathies: a quantitative histological and immunohistochemical analysis. Ann Rheum Dis 2008 August;67(8):1184-7. Disclosure of Interest M. Andersen Employee of: Novo Nordisk A/S, K. Ellegaard: None Declared, J. Hebsgaard Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, R. Christensen: None Declared, P. Kvist Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, N. Vendel: None Declared, J. Rømer Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, N. Søe: None Declared, H. Bliddal: None Declared

THU0348 Localisation of IL-20 in Synovium From Patients with Psoriatic Arthritis

Background Interleukin-20 (IL-20) is a pro-inflammatory cytokine that belongs to the IL-10 family of cytokines. Increased expression of IL-20 is found in autoimmune diseased tissue, such as lesional psoriatic skin and synovium from patients with rheumatoid arthritis (RA). A neutralising anti-IL-20 monoclonal antibody (NNC0109-0012) has shown efficacy in phase 2A clinical testing in patients with RA. Objectives The aim of the present study is to investigate the expression of IL-20 in synovial samples from patients with psoriatic arthritis (PsA). Methods Three synovial biopsies were obtained during synovectomy from the joints of the hands from patients with PsA. Five ‘normal’ synovial biopsies were obtained during surgical treatment of patients with non-inflamed shoulder disorders, such as subacromial impingement. Samples from eight synovial biopsies from patients with PsA and seven ‘normal’ synovial biopsies were obtained during joint surgery and represented as 2-mm spots on tissue micro array sections. All synovial samples were formalin-fixed and paraffin-embedded. The localisation of IL-20 was analysed in synovial sections by immunohistochemistry using a rabbit polyclonal anti-IL-20 antibody (2313b), and immunoreactivity was visualised with tyramide signal amplification and immunoperoxidase. The specificity of the antibody was verified with IL-20-transfected cells and using antigen pre-absorption and pre-immune IgG control. Results IL-20 was detected in 6 out of 11 synovial samples from patients with PsA. In 4 of these samples, IL-20 was present in both synovial lining and sublining. In 2 samples, IL-20 was present in the synovial lining, but not in the sublining. The three synovial samples obtained by synovectomy were all positive for IL-20, while IL-20 positive cells were observed in only 3 out of the 8 synovial samples obtained during joint replacement surgery. A few IL-20 positive cells were detected in the lining in one ‘normal’ synovial sample, while 11 out of the 12 ‘normal’ samples were negative for IL-20. The localisation pattern of IL-20 in synovial tissue from patients with PsA was similar to what we have previously observed in synovium from patients with RA. The IL-20 immunoreactivity was completely eliminated following antigen pre-absorption of the antibody. Conclusions The results demonstrate that IL-20 is differentially expressed in synovial samples from patients with PsA compared with ‘normal’ synovial tissue, and that the distribution of IL-20 in synovium from patients with PsA and RA appears to be similar. Disclosure of Interest M. Jackerott Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, J. Mandelbaum Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, L.-L. Kruse Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, N. Vendel: None Declared, E. Bartels: None Declared, M. Andersen Employee of: Novo Nordisk A/S, C. Jensen: None Declared, N. Søe: None Declared, H. Bliddahl: None Declared, J. Rømer Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S