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[Abstract] Introduction: Non-infectious uveitis include a heterogeneous group of sight-threatening and incapacitating conditions. Their correct management sometimes requires the use of immunosuppressive drugs (ISDs), prescribed in monotherapy or in combination. Several observational studies showed that the use of ISDs in combination could be more effective than and as safe as their use in monotherapy. However, a direct comparison between these two treatment strategies has not been carried out yet. Methods and analysis: The Combination THerapy with mEthotrexate and adalImumAb for uveitis (CoTHEIA) study is a phase III, multicentre, prospective, randomised, single-blinded with masked outcome assessment, parallel three arms with 1:1:1 allocation, active-controlled, superiority study design, comparing the efficacy, safety and cost-effectiveness of methotrexate, adalimumab or their combination in non-infectious non-anterior uveitis. We aim to recruit 192 subjects. The duration of the treatment and follow-up will last up to 52 weeks, plus 70 days follow-up with no treatment. The complete and maintained resolution of the ocular inflammation will be assessed by masked evaluators (primary outcome). In addition to other secondary measurements of efficacy (quality of life, visual acuity and costs) and safety, we will identify subjects’ subgroups with different treatment responses by developing prediction models based on machine learning techniques using genetic and proteomic biomarkers. Ethics and dissemination: The protocol, annexes and informed consent forms were approved by the Reference Clinical Research Ethic Committee at the Hospital Clínico San Carlos (Madrid, Spain) and the Spanish Agency for Medicines and Health Products. We will elaborate a dissemination plan including production of materials adapted to several formats to communicate the clinical trial progress and findings to a broad group of stakeholders. The promoter will be the only access to the participant-level data, although it can be shared within the legal situation. Trial registration number: 2020-000130-18; NCT04798755. This work was supported by the Instituto de Salud Carlos III, grant number [ICI19/00020]. Sponsor: Fundación para la Investigacion Biomédica del Hospital Clínico San Carlos. Executive Committee: Administrative and executive arm of the clinical trial, providing overall oversight for the study and making decisions on day-to-day operational issues (Study Coordinator (Luis Rodriguez-Rodriguez), a representative from the Spanish Clinical Trial Network (Amanda López Picado), and 5 Site Directors (these seats will be rotatory, with changes every 6 months months)); Data Coordinating and Analysis Committee: Supervising data collection,management and quality control, designing the statistical analysis plan, performing unmasked data analysis and preparing interim and final reports for the Data Security Monitoring Board and the Executy Committee (Study Coordinator (Luis Rodriguez-Rodriguez), a representative from the Spanish Clinical Trial Network (Amanda López Picado) and Ester Carreño); Biobank and Biomarker Identification Committee (Maintaining an up-to-date manual of operations for blood extraction, processing and storage, and monitoring procedures adherence, supervising biological sample collection, sample shipment coordination, coordinating the phamacogenetic and proteomic analysis (Study Coordinator (Luis Rodriguez-Rodriguez), a representative from the Instituto de Salud Carlos III Biobank Platform (Elena Molino), a representative the Instituto de Investigación Biomédica de A Coruña, a representative from, the Data Coordinating and Analysis Committee); Data Security Monitoring Committee (PierGiogio Neri, Andrew Dick, Loreto Carmona)

ObjectivesTo identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids.MethodsThree prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing.ResultsmtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes.ConclusionsThe presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

Osteoarthritis (OA) is a disease primarily characterized by the loss of cartilage extracellular matrix. We have followed a peptidomic strategy to identify endogenous peptides (neopeptides) released from healthy and OA human knee and hip articular cartilage, which may serve as disease markers. This study provides a comprehensive neopeptidomic profile of healthy and diseased tissues, and the identification and validation of a panel of eight endogenous peptides that are differentially released from the extracellular matrix because of the pathogenic process., Graphical Abstract Highlights Shotgun identification of neopeptides released from osteoarthritic cartilage. Specific endogenous peptides from the cartilage ECM are measured by MRM. Identification of neopeptides differentially generated from diseased tissue. The peptide DSNKIETIPN shows the best metrics as biomarker of OA cartilage., Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

[Abstract] Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment. This research was funded by the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, grant numbers PI17/00027, PI20/00078, RD16/0012/0008 and RD16/0012/0002, co-financed by Fondo Europeo de Desarrollo Regional (FEDER)

The Osteoarthritis Initiative (OAI) is a public-private partnership comprised of five contracts (N01-AR-2-2258; N01-AR-2-2259; N01-AR-2-2260; N01-AR-2-2261; N01-AR-2-2262) funded by the National Institutes of Health, a branch of the Department of Health and Human Services, and conducted by the OAI Study Investigators. Private funding partners include Pfizer, Inc.; Novartis Pharmaceuticals Corporation; Merck Research Laboratories; and GlaxoSmithKline. Private sector funding for the OAI is managed by the Foundation for the National Institutes of Health. This work has been supported by grants from Fondo Investigación Sanitaria (PI16/02124, PI17/00404, PI19/01206, DTS17/00200, CIBER-CB06/01/0040 and RETIC-RIER-RD16/0012/0002), integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”. This study has been also supported by grants IN607A2017/11, IN607D2020/10 and AE CICA-INIBIC (ED431E 2018/03) from Xunta de Galicia. The Proteomics Unit of GIR belongs to ProteoRed, PRB3- ISCIII (PT17/0019/0014). L.L. is supported by Xunta de Galicia (IN606B-2016/2005) and Contrato Sara Borrell (CD19/00229) Fondo de Investigación Sanitaria, ISCIII. I.R.P. is supported by Contrato Miguel Servet-II Fondo de Investigación Sanitaria (CPII17/00026). This work was also supported by the KTH Center for Applied Precision Medicine funded by the Ehrling Persson foundation, as well as the Human Protein Atlas project funded by Knut and Alice Wallenberg Foundation Xunta de Galicia; IN607A2017/11 Xunta de Galicia; IN607D2020/10 Xunta de Galicia; ED431E 2018/03 Xunta de Galicia; IN606B-2016/2005

Data availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/archive/projects/PXD012879) with the dataset identifier PXD012879 and the project name of MSCs OA SILAC Mass spectrometry imaging [Abstract] In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis. This work has been funded by Fondo Investigación Sanitaria-Spain (grant numbers PI14/01707, PI16/02124, PI17/00404, DTS17/00200, CIBER-CB06/01/0040 and RETIC-RIER-RD12/0009/0018), a part of the National Plan for Scientific Program Development and Technological Innovation 2013–2016, funded by the ISCIII-General Subdirection of Assessment and Promotion of Research - European Regional Development Fund (FEDER) “A way of making Europe.” The Proteomics Unit of GIR belongs to ProteoRed, PRB2- ISCIII (grant number PT17/0019/0014). B.R. is supported by Xunta de Galicia (IN606B-2016/004). The research was partially performed within the M4I research program, financially supported by the Dutch Province of Limburg as part of the “LINK” program Xunta de Galicia; IN606B-2016/004 https://www.ebi.ac.uk/pride/archive/projects/PXD012879

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.

[Abstract] Background: In the present study, we explored potential protein biomarkers useful to predict the therapeutic response of knee osteoarthritis (KOA) patients treated with pharmaceutical grade Chondroitin sulfate/Glucosamine hydrochloride (CS+GH; Droglican, Bioiberica), in order to optimize therapeutic outcomes. Methods: A shotgun proteomic analysis by iTRAQ labelling and liquid chromatography–mass spectrometry (LC-MS/MS) was performed using sera from 40 patients enrolled in the Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES). The panel of proteins potentially useful to predict KOA patient’s response was clinically validated in the whole MOVES cohort at baseline (n = 506) using commercially available enzyme-linked immunosorbent assays kits. Logistic regression models and receiver-operating-characteristics (ROC) curves were used to analyze the contribution of these proteins to our prediction models of symptomatic drug response in KOA. Results: In the discovery phase of the study, a panel of six putative predictive biomarkers of response to CS+GH (APOA2, APOA4, APOH, ITIH1, C4BPa and ORM2) were identified by shotgun proteomics. Data are available via ProteomeXchange with identifier PXD012444. In the verification phase, the panel was verified in a larger set of KOA patients (n = 262). Finally, ITIH1 and ORM2 were qualified by a blind test in the whole MOVES cohort at baseline. The combination of these biomarkers with clinical variables predict the patients’ response to CS+GH with a specificity of 79.5% and a sensitivity of 77.1%. Conclusions: Combining clinical and analytical parameters, we identified one biomarker that could accurately predict KOA patients’ response to CS+GH treatment. Its use would allow an increase in response rates and safety for the patients suffering KOA. Insituto de Salud Carlos III; PI14/01707 Instituto de Salud Carlos III; PI16/02124 Insituto de Salud Carlos III; PI17/00404 Instituto de Salud Carlos III; DTS17/00200 Instituto de Salud Carlos III; CIBER-CB06/01/0040 Insituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002

Background Immune-Mediated Inflammatory Diseases (IMIDs) are a group of conditions/diseases characterized by common pathways leading to inflammation, which may result from a dysregulation of the normal immune response. The activity of these diseases is usually measured by the assessment of clinical symptoms. Objectives To apply a three-stage proteomic strategy to find plasma biomarkers useful to monitor disease activity in patients three different IMIDs: rheumatoid arthritis (RA), psoriatic arthritis (PsA) and systemic lupus erythematosus (SLE). Methods A three-stage proteomic strategy was conducted: 1) Discovery stage: a random selection of 80 plasma samples from patients with different RA activities according to the DAS28, which were classified as low activity (LA, n=40) and high activity (HA, n=40). The analysis was conducted by shotgun mass spectrometry (MS) applying an iTRAQ 8-plex strategy, by nanoLC-MS/MS using three different MS instruments. 2) Verification stage: a targeted proteomics strategy was used to verify the candidate biomarkers from the screening, using the same 80 samples of this first stage. This analysis was performed by Multiple Reaction Monitoring (MRM) with the aid of synthetic heavy-labelled peptides, by nanoLC-MS/MS in an ESI-QTRAP 5500. 3) Validation stage: ELISA experiments were performed to confirm the previous results on an independent set of plasma samples from patients with RA (n=170), PsA (n=80) and SLE (n=80). Results 1) Discovery stage: The MS analysis led to the identification of 186 proteins. A panel of 9 proteins was identified as candidate biomarkers for disease activity monitoring in RA. Most of these proteins are related with the pathogenic process and the effects caused by this type of disease (inflammation and immune disorder in joints). 2) Verification stage: four from the 9 proteins (SAA1, AACT, HPT, A1AG1) were found to be quantitatively altered between patients with extreme disease activities (p Conclusion Using a three-stage proteomic strategy, we have identified HPT, SAA1 and AACT as protein biomarker candidates for monitoring PsA activity. Disclosure of Interests Lucia Gonzalez Rodriguez: None declared, Valentina Calamia: None declared, Rocio Paz Gonzalez: None declared, Patricia Fernandez Puente: None declared, Cristina Ruiz-Romero: None declared, Juan D. Canete: None declared, Antonio Julia: None declared, Antonio Fernandez Nebro: None declared, Jesus Tornero: None declared, Sara Marsal: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB

Background Osteoarhthritis (OA) is a chronic, degenerative and multifactorial disease, and the main cause of pain and dysfunction among elder people. It is characterized by a progressive loss of function of synovial joints (1). The role of chondrocytes in this pathology has been widely studied (2), but other joint cells are also involved, including the synovial fibroblast (SF) (3-5). During joint destruction, inflammatory and degradative mediators are released by joint cells and from the extracellular matrix (ECM), including fibronectin degradation fragments (Fn-fs) (3, 4, 6). On the other hand, vasoactive intestinal peptide (VIP) exerts anti-inflammatory and immunomodulatory actions in several autoimmune and inflammatory disorders, including OA (3, 5). The study of the mediators released by joint cells and their modulation by pro- and anti-inflammatory mediators would be useful for the design of novel therapies for OA treatment. Objectives To analyse the mediators released from co-cultures of OA chondrocytes and SF, and to elucidate the effect of Fn-fs and VIP on these cells. Methods Human articular chondrocytes (HAC) and SF from 4 OA patients were provided by the Rheumatology Service at Complejo Hospitalario Universitario A Coruna. Isolated cells were recovered and plated in SILAC DMEM-Flex lacking Arginine and Lysine. In the case of medium and heavy media, isotope-labeled L-lysine and L-arginine were used. When 100% of labeling was reached, cells were put in co-culture and incubated in serum-free medium with or without Fn-fs (10-8M) or Fn-fs + VIP (10-8M) for 48h. Cell secretomes were separated on a 10% SDS PAGE gel. Gels were stained with Coomassie blue and the resulting lanes were cut into slices and subjected to in-gel digestion. Extracted peptide mixtures were desalted and concentrated via NuTip, subjected to liquid chromatography, using a Tempo nano LC equipped with a Sun Collect MALDI Spotter, and analyzed by MALDI-TOF/TOF. Identification of peptides and proteins and relative quantification were performed using Protein Pilot software (Sciex). Results Cell secretomes were analysed in 4 OA patients in duplicate. Database search (UniprotKB/Swissprot) and Venn’s diagrams drawing tool (Venny 2.1.0) allowed us to identify 79 common proteins in the HACs-SF co-cultures. Among them, VIP was able to modulate 33 different proteins, significantly reducing 9 of them: CH3L1, PTX3, PGS2, MMP2, Complement C1R, Complement C3, TBA1, QSOX1, and CATB. These proteins include inflammatory and ECM proteins, proteases and complement system proteins among others, which play a main role in the OA pathogenesis. Conclusion VIP decreases inflammatory and degradative mediators in HAC-SF co-cultures, potentially slowing the progression of the disease and supporting its therapeutic role in osteoarthritis. References [1] Loeser RF, et al. Arthritis Rheum. 2012;64(6):1697-707. [2] Calamia V, et al. J Proteome Res. 2011;10(8):3701-11. [3] Perez-Garcia S, et al. Journal of molecular neuroscience : MN. 2014;52(1):18-27. [4] Perez-Garcia S, et al. Am J Pathol. 2016;186(9):2449-61. [5] Perez-Garcia S, et al. J Cell Mol Med. 2016;20(4):678-87. [6] Zack MD, et al. Arthritis Rheum. 2006;54(9):2912-22. Acknowledgement This work has been supported by Instituto de Salud Carlos III, Spain, cofinanced by FEDER, European Union: RETICS program, RIER (RD16/0012/0002 and RD16/0012/0008), and the projects PI17/00027 and PI16/2124. Disclosure of Interests Selene Perez Garcia: None declared, Valentina Calamia: None declared, Tamara Hermida Gomez: None declared, Mar Carrion: None declared, Raul Villanueva Romero: None declared, Yasmina Juarranz: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, Rosa P. Gomariz: None declared

The methods currently available for the diagnosis and monitoring of osteoarthritis (OA) are very limited and lack sensitivity. Being the most prevalent rheumatic disease, one of the most disabling pathologies worldwide and currently untreatable, there is a considerable interest pointed in the verification of specific biological markers for improving its diagnosis and disease progression studies. Considering the remarkable development of targeted proteomics methodologies in the frame of the Human Proteome Project, the aim of this work was to develop and apply a MRM-based method for the multiplexed analysis of a panel of 6 biomarker candidates for OA encoded by the Chromosome 16, and another 8 proteins identified in previous shotgun studies as related with this pathology, in specimens derived from the human joint and serum. The method, targeting 35 different peptides, was applied to samples from human articular chondrocytes, healthy and osteoarthritic cartilage, synovial fluid and serum. Subsequently, a verification analysis of the biomarker value of these proteins was performed by single point measurements on a set of 116 serum samples, leading to the identification of increased amounts of Haptoglobin and von Willebrand Factor in OA patients. Altogether, the present work provides a tool for the multiplexed monitoring of 14 biomarker candidates for OA, and verifies for the first time the increased amount of two of these circulating markers in patients diagnosed with this disease. Significance We have developed an MRM method for the identification and relative quantification of a panel of 14 protein biomarker candidates for osteoarthritis. This method has been applied to analyze human articular chondrocytes, articular cartilage, synovial fluid, and finally a collection of 116 serum samples from healthy controls and patients suffering different degrees of osteoarthritis, in order to verify the biomarker usefulness of the candidates. HPT and VWF were validated as increased in OA patients.

Background:The improvement of the existing diagnostic methods to detect pre-radiographic knee OA (KOA) may facilitate the development of preventive strategies. It has been postulated that combining biochemical with clinical markers, may increase the prognostic power to detect who is at high risk for developing KOA.Objectives:To validate and qualify the ability of 6 proteins with biomarker potential to generate a prognostic model of knee OA prediction through the combination of validated OA biomarkers and clinical markers.Methods:In thevalidation phase(Figure 1), 749 sera at the baseline visit belonging to participants from the Osteoarthritis Initiative (OAI) Cohort were randomly selected to blindly quantify 6 biomarkers using in-house custom sandwich microarrays built using the xMAP technology. Among these, only 540 participants have a Kellgren and Lawrence (KL) grade = 0-1 at the beginning of the OAI study in at least one knee. After a follow-up period of 96 months, 209 participants developed KOA in at least one knee (KL ≥ 2) and were classified as incident group, whereas 331 did not developed the disease (KL = 0-1) and were classified as not-incident group. Statistical differences between the outcome groups were assessed by non-parametric Mann-Whitney U tests. In thequalification phase(n=540), univariate regression analyses were carried out to investigate whether the individual biomarkers were associated with the risk of KOA development. A clinical prognostic model was defined by stepwise regression analysis using clinical non-radiographic variables significantly associated with the OA incidence. The utility of the potential biomarkers, alone or in combination, was evaluated by comparing the Area Under the Curve (AUC) of the clinical prognostic model with the biomarkers plus clinical prognostic models. In addition, senFigure 1.Study desingResults:The incident group showed significant higher serum concentrations at the baseline visit (p < 0.05) for all the potential biomarkers analyzed in this study. Moreover, 5 of them were also significantly associated with the future appearance of radiographic KOA, yielding Odds Ratios (OR) ≥ 10 per 10 µg/ml increase. Among all the possible combinations, the inclusion of 2 biomarkers to the clinical prognostic model showed a significant improvement of the predictive capacity (AUCs = 0.78 vs 0.82, p= 0,044) with 65% (95% Confidence Interval (95%CI): 60-70%) specificity and 88% (95%CI: 81-91%) sensitivity. Variables included in the regression model and all metrics comparing the biomarkers plus clinical prognostic model with the clinical prognostic model are shown in Figure 2A. The ROC curves of the biomarkers-only model, clinical prognostic model and biomarkers plus clinical prognostic model are represented in Figure 2B.Figure 2.(A) Multivariate regression analysis (B) ROC curves of the modelsConclusion:We have generated a prognostic model for the prediction of KOA by combining biomarkers and clinical variables, which showed a putative utility in the clinical setting by improving the predictive capacity of a clinical prognostic model to identify patients at a higher risk to develop radiographic KOA.Disclosure of Interests:Maria Camacho Encina: None declared, Vanesa Balboa-Barreiro: None declared, Ignacio Rego-Perez: None declared, Rocío Paz González: None declared, Valentina Calamia: None declared, Lucía Lourido: None declared, Cristina Ruiz-Romero: None declared, Francisco J. Blanco Grant/research support from: Sanofi-Aventis, Lilly, Bristol MS, Amgen, Pfizer, Abbvie, TRB Chemedica International, Glaxo SmithKline, Archigen Biotech Limited, Novartis, Nichi-iko pharmaceutical Co, Genentech, Jannsen Research & Development, UCB Biopharma, Centrexion Theurapeutics, Celgene, Roche, Regeneron Pharmaceuticals Inc, Biohope, Corbus Pharmaceutical, Tedec Meiji Pharma, Kiniksa Pharmaceuticals, Ltd, Gilead Sciences Inc, Consultant of: Lilly, Bristol MS, Pfizer

Endometrial cancer (EC) is the most frequent gynecological cancer. Tumor dissemination affecting ∼20% of EC patients is characterized at the primary carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the microenvironment is crucial for metastatic colonization, with a participation of the extracellular vesicles (EVs). We comprehensively approached these primary and secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the primary carcinoma, whereas CTCs are characterized by an EMT phenotype. We modeled this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized the epithelial-like EVs in circulation by SILAC proteome analysis. The identification of proteins involved in cell-cell and cell-matrix interaction and binding, together with in vitro evidence of an improved adhesion of CTC to a functionalized endothelium, suggests a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites. Accordingly, adhesion protein LGALS3BP was found to be significantly enriched in circulating EVs from a cohort of EC patients with a high risk of recurrence by targeted proteomics (multiple reaction monitoring), highlighting its potential in liquid biopsy in EC.

[Abstract] Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent traditional non-steroidal anti-inflammatory drugs (tNSAIDs) gastro-intestinal adverse effects. VA692, a recently disclosed selective COX-2 inhibitor, structurally related to well-known marketed coxibs, showed anti-inflammatory, and anti-nociceptive properties. The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. At this purpose we evaluated the pro-inflammatory cytokines and anti-oxidant enzymes gene expression, apoptosis and ROS production, and PGE2 release in chondrocytes (both primary cultures and immortalized T/C-28a2 cell line) treated with the two drugs. Furthermore, a proteomic analysis has been performed in T/C-28a2 cell line to evaluate modifications in their proteomic profile following drug treatment in presence of IL-1β. Our results demonstrated the anti-inflammatory effect of the novel synthesized VA692, and confirmed those of celecoxib, in counteracting the stimulus of IL-1β in both osteoarthritic (OA) chondrocytes and T/C-28a2 cell line. Furthermore, the data underlined the possible anti-apoptotic and anti-oxidant role of VA692, implying its regulation in superoxide anion production as indicated by the modulation of anti-oxidant enzymes. The proteomic analysis provides new information about the effect of VA692 on human T/C-28a2 intracellular proteome, demonstrating the usefulness of this approach in the identification and quantifications of several proteins. Modulation of some proteins such as Hsp90 and SOD by VA692 could explain its role in the therapeutic approach of OA. Based on our results, we can affirm that VA692 has more beneficial effect compared with celecoxib particularly regarding the modulation of oxidant/anti-oxidant system and proteome profile of human articular chondrocytes.

Background The treatment of rheumatoid arthritis (RA) aims to control a patient‘s signs and symptoms, prevent joint damage, and maintain his/her quality of life. Among the best known disease-modifying antirheumatic drugs, Methotrexate (MTX) is one of the most effective and widely used medications. It is used as a general first-choice drug, although some patients will not respond to this treatment and it is not free from side effects. Objectives To identify circulating proteins that could be useful as predictors of the patient‘s response to MTX. Methods Serum samples from patients enrolled in the Pathobiology of Early Arthritis Cohort (PEAC) were collected before treatment with MTX. Response to therapy was determined after 6 months by calculating the initial and final DAS28 of the patients. Their classification was performed following the EULAR response criteria. Sixty samples at baseline from this cohort (30 good responders and 30 non-responders) were depleted from the 14 most abundant proteins by affinity chromatography to remove background. Then, they were analysed by reversed-phase nanoliquid chromatography coupled to mass spectrometry using a SWATH strategy in a tripleTOF MS (Sciex). The quantitative data obtained in this proteomic analysis were processed using the ProteinPilot 5.0.1 and PeakView 2.1 software (Sciex). Machine learning analyses were performed on a train set of 30 samples (15 responders and 15 non-responders) via support vector machine (SVM) using the Classyfire, e1071 and caret R packages. Results were verified in an independent set of 24 samples by a two-stage support vector machine (TSSVM) with RBF kernel and 10 cross-fold validation for each meta-model. Results The proteomic analysis led to the identification and quantification of 229 proteins that were common between the screening and validation sets. Independent screening and validation data sets were preprocessed by PCA for dimension reduction and analysed by machine learning tools, leading to the definition of a panel of 8 proteins (one of them involved in MTX metabolism) differentiating the groups of responders and non-responders to MTX with strong agreement (Kappa >0.80), very high accuracy and good relevant metrics (table 1). Conclusions We have defined a panel of circulating proteins useful to predict the response to MTX therapy in rheumatoid arthritis patients. Disclosure of Interest None declared

Background The immune system can detect the changes involved in the osteoarthitic (OA) joint, triggering the production of immunoglobulins against self-proteins (Autoantibodies or AAbs). As AAbs might be generated in a stage prior to the disease, they can be potentially used to identify an increased risk for the disorder, allowing the diagnosis of asymptomatic OA. Objectives The discovery of an AAb signature useful for the early diagnosis of knee OA. Methods Nucleic Acid-Programmable Protein Arrays (NAPPA) were used to screen the presence of AAbs against 2200 human proteins in sera belonging to two different subcohorts from the Osteoarthritis Initiative (OAI): Incidence and Non-exposed subcohorts. Participants in the incidence subcohort had not developed any symptomatic knee OA at baseline, but they did present an increased risk of developing the disease. Non-exposed subcohort incorporate participants which have no radiographic findings or symptoms of knee OA, nor any reported risk factors. A hundred sera from both subcohorts at baseline were used. In order to know if the profile of AAbs was specific of asymptomatic OA, samples belonging to the same patients selected from the incidence subcohort were analysed after 72 months of follow up, when all of them have developed OA. Data were normalised following the Biodesing Institute criteria. A 1.1 cutoff was used to determine reactivity and all proteins over the cutoff were analysed by Wilcoxon test. The Partial Area Under the Curve (pAUC) at 95% specificity was analysed with a p value Results We detect AAbs against six proteins showing different reactivity (see table 1) in a stage prior to the disease –samples from the incidence subcohort at baseline compared with the non-exposed subcohort–. These proteins were implicated in the colesterol biosynthesis (Diphosphomevalonate decarboxylase, MVD), and the elimination of pottentially toxic xenobiotic or endogenous compounds (ASB7 and UGT1A7, repectively). We also found a GTPase from the Rho family (RAC3), the Vacuolar protein sorting-associated protein 4B (VPS4B), which has been recently reported to facilitate chondrocyte apoptosis in a OA rat model, and Methionine adenosyltransferase 2 subunit beta (MAT2B). The latter is the regulatory subunit of the enzyme responsible of the catalysis of S-adenosylmethionine, a dietary supplement widely used in the management of OA symptoms. None of the six AAbs were found after the disease has been established (samples from the incidence subcohort at 72 months). Conclusions This work is the first to screen a large number of human proteins for the discovery of OA-associated AAbs. We define a panel of six AAbs, which are increased prior to the development of symptomatic knee OA. These results suggest that a serum AAbs signature can facilitate the discovery of early OA biomarkers useful for clinical diagnosis. Acknowledgements This work has received financial support from the Xunta de Galicia and the European Union (European Social Fund – ESF). Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is a long-lasting inflammatory autoimmune disorder that ultimately leads to the destruction of joint architecture. The activity of this disease is measured by the assessment of clinical symptoms. Objectives To apply a proteomic strategy to find serum biomarkers able to discriminate patients with different RA activities. Methods To facilitate the complex measurement of serum by proteomics, a simple, fast and reproducible albumin-specific depletion method using ethanol was optimised and applied to the samples employed in this study. 80 samples from the IMID consortium, classified according to the DAS28 score into low activity40 and high activity40 were analysed by mass spectrometry. Four independent pools of the high RA activity samples (10 samples per pool) and 4 pools of the low RA activity samples were firstly albumin-depleted, and then the remnant serum proteins were digested and differentially labelled with iTraq 8-plex reagents. Subsequently, the 8 labelled pools were combined and cleaned using StageTips-C18. Finally, the pool mixture was fractionated by HPLC (Zorbax-C18) and the resulting fractions were analysed by nanoLC-MS/MS using MALDI-TOF/TOF, TripleTOF and LTQ-Orbitrap. Results The mass spectrometry analysis led to the identification of 186 proteins. In this screening step, the abundance of 9 proteins was found to be significantly different between patients with high (H) and low (L) RA activity. Orthogonal experiments, using Western Blot, protein bead arrays and Multiple Reaction Monitoring (MRM) with synthetic heavy-labelled peptides, were conducted in order to verify some of these these biomarker candidates of RA activity. The results obtained from the verification phase, conducted by MRM on 50 samples from the same cohort, show a decrease of Apolipoprotein B (ratio H/L=0.84, p=0.00), Histidine Rich Glycoprotein (ratio H/L=0.86, p=0.01) and Plasma protease C1 inhibitor (ratio H/L=0.84, p=0.02). Furthermore, this verification phase shows also an increase of Haptoglobin (ratio H/L=1.34, p=0.01) and Serum Amyloid A1 (ratio H/L=1.64, p=0.05), in accordance with that observed in the screening. These proteins are related with the RA process and the effects caused by this type of disease (inflammation and immune disorder in joints). Conclusions In this proteomic study, 5 proteins were found to be quantitatively altered between patients with high and low RA activity using shotgun and targeted mass spectrometry. Disclosure of Interest None declared

Background Two different phenotypes of hand osteoarthritis (HOA) have been defined: nodal hand osteoarthritis (NHOA) and erosive hand osteoarthritis (EHOA). NHOA involve bone enlargement of the underlying interphalangeal joints, which may typically give rise to Heberden’s nodes, synovitis and swelling. EHOA is a particularly aggressive form characterised by an abrupt onset, as well as signs of inflammation and subchondral erosions. In the absence of efficient diagnostic methods, searching for specific biomarkers for each subtype may help to characterise them. Objectives To define a panel of specific protein markers for the characterisation of EHOA and NHOA and its potential use in clinic. Methods A proteomic approach based on peptide labelling with Isobaric tags for relative and absolute quantitation (iTRAQ) was performed using two different sets of sera (n=55). Samples were classified in 4 groups of study (EHOA, n=10; non-EHOA, n=10; NHOA n=10; non-NHOA, n=5) and 2 control groups (rheumatoid arthritis (RA), n=10 and psoriatic arthritis (PSA), n=10). Serum proteins were digested and peptides from each condition to be compared were differentially labelled with iTRAQ reagents (Sciex). Then, samples were combined and analysed by two-dimensional liquid chromatography coupled to mass spectrometry in a TripleTOF 5600 Mass Spectrometer System (Sciex). Protein identification and quantitation was carried out using ProteinPilot software v.5.0.1. Results A total of 257 different proteins were identify with more than two peptides and a total score ≥2 at 95% confidence. In order to identify specific biomarkers for the characterisation of NHOA and EHOA phenotypes, each group was compared with the non-NHOA or non-EHOA respectively, and also with the control groups. After all the comparisons were made, 26 unique different proteins were found specific of the nodular phenotype. Vasorin (VAS) showed elevated levels in patients diagnosed with NHOA when compared to non-NHOA, RA and PSA groups. On the other hand, 36 unique proteins were identified in those patients with EHOA. Extracellular matrix protein 1 (ECM1) was found with higher concentrations in EHOA than in non-EHOA, RA and PSA patients. In addition, both HOA phenotypes were compared to the control groups and a panel of 30 different proteins were defined. Among these proteins, vascular cell adhesion molecule-1 (VCAM1) was found increased in HOA compared to RA and PSA groups. Conclusions A specific protein profile for the characterisation of EHOA and NHOA disorders has been established. VAS showed elevated levels in patients with NHOA, whereas ECM1 was increased in patients diagnosed with the erosive form of the disease. As none of them were identify in the other phenotype, they might be phenotype-specific biomarkers. In addition, VCAM1 was found with higher levels in both phenotypes of HOA when compared with RA and PSA and might be used to differentiate hand osteoarthritis from other rheumatic diseases. Acknowledgements Financial support (IN606A-2016/012) from the Xunta de Galicia and the European Union (European Social Fund – ESF), is gratefully acknowledged. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is a disease characterised by high clinical variability and an underlying cellular and molecular heterogeneity. Efforts to find tools for the classification of the different disease phenotypes and patient stratification are essential to develop tailored therapies and improve its management. According to this, specific pathological phenotypes of synovial tissue (pathotypes) have emerged as associated with diverse clinical evolution and response to therapy.1 Objectives To identify signatures of circulating proteins associated with specific synovial pathotypes in RA patients. Methods A proteomic analysis was carried out on samples from patients enrolled in the Pathobiology of Early Arthritis Cohort. Ultrasound-guided synovial biopsies from these patients allowed their classification into three groups: lymphoid (L), myeloid (M) or fibroid (F), according to the pathotype. The study was performed using 54 serum samples at baseline. Sera were analysed by nanoliquid chromatography coupled to mass spectrometry using a SWATH strategy on a tripleTOF (Sciex). The proteomic data were processed using ProteinPilot and PeakView. A two-stage support vector machine (TSSVM) with RBF kernel and 10 cross-fold validation for each meta-model was applied using the Classyfire, e1071 and caret R packages. Results The proteomic analysis led to the identification and quantification of 229 proteins in all samples. A screening was performed on a group of 30 samples (Train set: 10 L, 10 M and 10 F). Data were pre-processed by PCA for dimension reduction. Then, application of machine learning tools led to the identification of a panel of 11 proteins whose different abundance is associated with a specific synovial phenotype (either L, M or F) in RA patients. As shown in the table 1, a very high accuracy and Kappa coefficient were achieved with this classification tool. The results were confirmed on an independent validation set of 24 samples (12 L, 8 M and 4 F) with also good performance. This protein signature allowed the correct classification of the samples into the three pathotypes with very high sensitivity and specificity (see table 1). Conclusions A signature of 11 circulating proteins has been identified as associated with synovial pathotypes in RA patients. The putative correlation of this signature with the clinical evolution and/or response to therapy of the patients remains to be elucidated. Reference [1] New learnings on the pathophysiology of RA from synovial biopsies. Pitzalis C, Kelly S, Humby F. Curr Opin Rheumatol. 2013May;25(3):334–44. Disclosure of Interest None declared

Background The prediction of drug responses based on the analysis of multiple clinical variables and omics data is mandatory for accomplishing the promise of precision medicine in rheumatology. Objectives Integrating clinical-radiological-analytical variables and proteomics data for predicting patient‘s response to different treatments, in order to optimise therapeutic outcomes in OA. Methods A panel of 10 serum proteins potentially useful to predict OA patient‘s response was qualified using ELISA Kits in the whole Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES) cohort. Patients were classified as responders (R) and non-responders (NR), either to Chondroitin sulfate/Glucosamine hydrochloride (CS+GH; Droglican, Bioiberica, Spain) or Celecoxib, after 6 months of treatment. Logistic regression analyses, adjusted by confounder variables previously reported as significant in bivariate approaches, were used to analyse the contribution of the measured proteins to our prediction models of drug response in knee OA. Appropriate receiver-operating-characteristics (ROC) curves were also calculated. Results In the discovery phase of the study, two different panels of putative predictive biomarkers useful to stratify OA patients according to their unique protein profiles was identified by shotgun proteomics (n=80). In the verification phase , the panel of 6 proteins specific for Droglican treatment (APOA2, APOA4, APOH, ITIH1, C4BPa and ORM2), and the panel of 4 proteins specific for Celecoxib treatment (α2HS, SHBG, CD5L and TSP1) were verified in a larger cohort of OA patients (n=262 for CS+GH group and n=244 for Celecoxib group) by ELISA assays. In the qualification phase , the sensitivity and specificity of a panel of 4 validated proteins (ORM2, APOA2, ITIH1, and TSP1) were tested in blind in the whole MOVES cohort at baseline (n=506). In CS+GH group, only ORM2 levels were significantly lower in responders compared to non-responders (R: 192,8 ug/mL vs NR: 261,6 ug/mL; p=0,042), while no statistically significant differences were found in the Celecoxib group. Five clinical and two analytical parameters recorded at baseline significantly influence patients‘ response regardless of treatment. Notably, if we include ORM2 as covariate, we found a specific interaction between response to CS+GH and baseline protein levels (p=0,007) thus increasing the power of our prediction model (ROC up to 0,843). In the Celecoxib group, non parametric analysis showed increased levels only of TSP1 at baseline in responders compared to non-responders (R: 363,03±104,83 ng/mL vs NR: 331,95±92,59 ng/mL; p=0,041), while no statistically significant differences were found for this protein in the CS+GH group. When we include in the regression model 4 predictive variables (2 clinical and 2 analytical) and TSP1 as covariate, we found a specific interaction between response to Celecoxib and baseline protein levels (p=0,045) thus increasing the predictive power of this model (ROC up to 0,749). Conclusions Combining clinical and analytical parameters, we clinically validated (qualified) 2 panels of biomarkers that could efficiently predict OA patients‘ response to CS+GH with an accuracy of 84,3% or to Celecoxib with an accuracy of 74,9%. Disclosure of Interest V. Calamia: None declared, F. Picchi: None declared, I. Rego: None declared, M. Camacho: None declared, L. Gonzalez: None declared, P. Fernandez-Puente: None declared, M. Herrero Employee of: Bioiberica S.A.U., H. Martinez Employee of: Bioiberica S.A.U., J. Verges: None declared, C. Ruiz-Romero: None declared, F. J. Blanco: None declared

Background Aging-related Osteoarthritis (OA) is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation. Autophagy is essential to maintain chondrocyte homeostasis by regulating the intracellular macromolecule and organelle turnover (1). Previous findings indicated that autophagy is defective in Aging and OA articular cartilage (2,3). However, the specific target/-s that regulates this homeostatic mechanism and affect cartilage integrity are still unknown. Objectives The objective of study is to identify targets regulating autophagy in human chondrocytes. Methods We performed quantitative proteomic analysis of Atg5 knockdown primary uman chondrocytes using iTRAQ (isobaric tags for relative and absolute quantitation) labeling coupled with on-line 2D LC/MS/MS. Protein identification and quantification were performed using Protein Pilot Software v 4.0. Each MS/MS spectrum was searched in the Uniprot/Swissprot database for Homo sapiens. Human chondrocytes and human cartilage from healthy, aged and OA patients were employed to confirm the role of the identified target by Western Blot (WB), Inmunofluorescence (IF) and Inmunohistochemistry (IHC). Importanly, CRISPR/Cas9 genome editing technology was used for mechanism of action studies. Results 24 out of 487 proteins were significantly altered (p Conclusions Lamin A/C, a nuclear protein contributing to structural integrity to the nucleus and matrix was identified as candidate target for regulating cartilage function under defective autophagy, such as aging and OA. These results support the hypothesis that autophagy is decreased with aging. Therefore, targeting Lamin A/C might be a promising strategy to find novel therapeutics for cartilage aging and OA. References Lotz MK and Carames B. Autophagy and cartilage homeostasis mechanisms in joint health, aging and OA. Nat Rev Rheumatol 2011;7:579–587. Carames B, Olmer M, Kiosses WB, Lotz MK. The relationship of autophagy defects to cartilage damage during joint aging in a mouse model. Arthritis Rheumatol 2015;67:1568–76. Acknowledgements This study was supported by Instituto de Salud Carlos III- Ministerio de Economia y Competitividad, Spain-CP11/00095 and Fondo Europeo de Desarrollo Regional (FEDER). Disclosure of Interest None declared

Background Peptidomics can be defined as the comprehensive, qualitative and quantitative study of all native peptides in a sample in a defined space at a defined time under defined biological conditions. LC-MS/MS targeted proteomics have been used to identify and quantify candidate molecular biomarkers in diverse range of samples, including cells, tissues, serum/plasma, and other types of body fluids. Objectives In the present work, we have developed a targeted method to detect a panel of endogenous peptides that were identified as released from human healthy (N) and/or osteoarthritic (OA) hip and knee cartilage. Secretomes were obtained separately from the wounded (WZ) and unwounded (UZ) zones of OA cartilage. The enrichment of endogenous peptides was achieved by ultrafiltration and solid phase extraction (SPE). After peptide extraction, the different peptide profiles of these secretomes were relative quantified by target proteomics. Methods Proteins secreted from human articular cartilage (secretomes) were obtained by culture of tissue explants [1]. The enrichment of endogenous peptides was standardized using ultrafiltration procedures and solid phase extraction with reversed phase (C18) resins. A method for the targeted identification and relative quantitation of endogenous peptides by Multiple Reaction Monitoring (MRM)-mass spectrometry was developed employing cartilage secretome samples, and then applied on synovial fluid and serum. The peptides were separated by nano-LC coupled to a 5500 QTRAP mass spectrometer using stable isotope-labeled (SI) peptides as an internal control. Peptide identifications were searched using the ProteinPilot program. Data analysis was performed using the Skyline software. Results 23 endogenous peptides belonging to 9 proteins related with OA, as Cartilage Oligemeric Matrix Protein (COMP), Cartilage Intermediate Layer Protein (CILP), Prolargin (PRELP), Dermcidin (DCD), Fibronectin (FINC) and Glia Derived Nexin (GDN) among others, were differentially detected and relatively quantified in the cartilage secretomes. Some of the endogenous peptides belonging to COMP, FINC and GDN were found with a significant high release in the UZ of hip cartilage, however in knee cartilage the release is higher in the WZ. On the other hand, certain peptides belonging to proteins like CILP or PRELP were found to be mostly increased in the UZ of both hip and knee OA cartilages when compared to healthy tissue. Conclusions A panel of endogenous peptides has been identified in articular cartilage, which are differentially released between OA and healthy patients, as well as between knee and hip OA patients. These endogenous peptides may be potential biomarkers to differentiate osteoarthritis from hip and knee. Our targeted proteomics approach would be widely applicable to quantify low abundant peptides of interest in complex biological samples as synovial fluid and serum in order to unravel their putative biomarker value for osteoarthritis. References Lucia Lourido et al. “Quantitative Proteomic Profiling of Human Articular Cartilage Degradation in Osteoarthritis”. J Proteome Res. 2014 Dec 5;13(12):6096–106. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is a long-lasting inflammatory autoimmune disorder that ultimately leads to the destruction of joint architecture. Objectives Using the DAS28 activity index, 80 RA serum samples (40 with low activity and 40 with high activity) were selected in order to be analyzed by mass spectrometry. The aim of this study was to find possible protein biomarkers that could discriminate patients with different RA activity in the daily clinical routine. Methods In order to facilitate the complex measurement of these serum samples, a simple, fast and reproducible albumin-specific depletion method using ethanol was optimized and applied to this study. Four independent pools of the 40 high RA activity samples (10 samples per pool) and 4 pools of the 40 low RA activity samples were firstly albumin-depleted, and then the remnant serum proteins were digested and differentially labelled with iTRAQ 8-plex reagents. Subsequently, the 8 labelled pools were combined and cleaned using StageTips-C18. Finally, the pool mixture was fractionated by HPLC (Zorbax-C18) and the resulting fractions were analyzed by nanoLC-MS/MS using two different equipments for validation (MALDI-TOF/TOF and TripleTOF). Results The mass spectrometry analysis led to the identification of 186 proteins. Among these, Haptoglobin, Kininogen-1, Alpha-2-HS-glycoprotein, Serum Amyloid A, Afamin and Histidine-rich-glycoprotein, exhibited a differential relative abundance depending on the RA activity of the patients (p Conclusions In this proteomic study, 9 proteins were found to be modulated between patients with high and low RA activity. Most of these proteins are related with the RA process and the effects caused by this type of disease (inflammation and immune disorder in joints). Therefore, these proteins are possible biomarker candidates for improving RA monitoring. Future validation experiments and prospective studies are needed to facilitate their implementation in the clinical routine. Disclosure of Interest None declared

Background Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent NSAIDs gastro-intestinal adverse effects. VA692, a new hydroxyetyl selective COX-2 inhibitor, showed anti-inflammatory, anti-nociceptive and chondroprotective properties. Proteomics is being applied for the study of drug mode of action, toxicity and to identify new drugs targets. Objectives The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. By iTRAQ methodology, we quantitatively analyzed the different expressed profiles in T/C-28a2 cell line treated with the studied drugs in presence of IL-1β. Methods Human T/C-28a2 chondrocytes cell line were generated by Goldring group. Human articular cartilage was obtained from femoral heads of five OA patients. Cells were incubated with VA692 and celecoxib (1, 0.5 and 0.2μM) in presence of Interleukin (IL)-1β (5ng/ml) for 48h. The expression of inflammatory cytokines and anti-oxidant enzymes was evaluated by quantitative qRT-PCR, PGE2 release by ELISA, and apoptosis and ROS production by flow cytometry. T/C-28a2 cell line was also processed to carry out western blot tests and finally employed for the iTRAQ analysis. Statistical analysis was performed by ANOVA and Bonferroni multiple comparison tests. Results IL-1β-stimulated chondrocytes showed a significant increase (p Conclusions Our data demonstrated the anti-inflammatory effect of VA692, suggesting also its anti-apoptotic and anti-oxidant role. The proteomic profile showed that VA692 induced not only an anti-inflammatory effect in chondrocytes but, interestingly, this compound also seemed to regulate their anabolic response. References Hochberg MC et al.Arthritis Care Res 2012. McAlindon TE et al. Osteoarthritis Cartilage 2014. Bingham CO III. Cleve Clin J Med 2002. Anzini M et al. J Med Chem 2013. Calamia V et al.Arthritis Res Ther 2010. Disclosure of Interest None declared

Background A shotgun proteomic analysis performed on sera from patients enrolled in the Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES) led to the discovery of a panel of putative predictive protein biomarkers useful to stratify osteoarthritis (OA) patients into responders and non-responders, either to Chondroitin sulfate/Glucosamine hydrochloride (Droglican®, Bioiberica S.A.,Barcelona,Spain) or Celecoxib. Objectives To validate the sensitivity and specificity of a panel of six serum proteins useful to predict the patient response to Droglican treatment, in order to optimize therapeutic outcomes in OA. Methods We analyzed the serum levels of a panel of six putative predictive protein biomarkers by enzyme-linked immunosorbent assays (ELISAs): APOA2, APOA4, APOH, C4BPa, ITIH1 and ORM2. All the subjects studied belonged to the MOVES cohort at baseline (Droglican sub-cohort, n=260). Non-parametric and multivariate analysis were performed to test the effects of the clinical variables, including gender, age, BMI, radiologic Kellgren/Lawrence (K/L) grade and WOMAC score at baseline, as well as the serum levels of each of the six mentioned proteins, on the response to Droglican treatment according to the OMERACT-OARSI criteria and the WOMAC pain score (20%, 30%, 50% and 70% reduction) recorded at the end of the trial (after 6 months of treatment). Results Non parametric analysis showed decreased serum levels of ORM2 at baseline in responders to Droglican according to the OMERACT-OARSI criteria compared to non-responders (76,11±53,25 vs 104,25±84,93; n=171 vs 46; p=0,047), meanwhile the values for APOA2 appeared statistically increased in responders with a 50% reduction in WOMAC pain score compared to non-responders (79,95±58,53 vs 66,05±46,49; n=129 vs 112; p=0,028). Patients with lower levels of ORM2 (median concentration=69,8 ug/mL) and/or higher level of APOA2 (median concentration=63,8 ug/mL) showed a markedly better response to pharmacotherapy. Statistical interactions between ORM2 and APOA2 levels and radiologic K/L grade were also detected (p=0,048 and p=0,002 respectively). No statistically significant differences were found for the other four proteins. Conclusions Our results show that ORM2 and APOA2 levels significantly correlates with patients response to Droglican suggesting the possibility of their use in predictive assays in order to optimize therapeutic outcomes in OA. Validation studies in different cohorts are needed to identify and validate a cut-off point for these biomarkers. Disclosure of Interest V. Calamia: None declared, M. Camacho-Encina: None declared, L. Gonzalez-Rodriguez: None declared, P. Fernandez-Puente: None declared, I. Rego-Perez: None declared, M. Herrero Employee of: Bioiberica S.A., H. Martinez Employee of: Bioiberica S.A., C. Ruiz-Romero: None declared, F. J. Blanco: None declared

Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.

Due to the complex structure of the articular joint, it requires great effort to fully understand joint disease pathogenesis. The proteomic analysis of articular joint tissues could contribute greatly to our insight into the endogenous control mechanisms of matrix turnover and the unravelling of the molecular and cellular mechanisms involved in the progression of the arthritides. To date, most proteome analysis strategies use the two-dimensional gel electrophoresis (2-DE) technique to separate proteins according to their isoelectric point, molecular mass, solubility and relative abundance. In this work, we describe optimization of human joint sample preparation techniques to obtain high quality 2-DE maps of human joint tissues (cartilage and synovium), cells (chondrocytes and synoviocytes) and synovial fluid. These techniques improve the performance of gel-based differential proteomic analysis, and facilitate the application of proteomics to rheumatology studies.

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-alpha receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis.

Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.

The aim of this pilot study was to analyze the eVects of glucosamine (GlcN) and its N-acetyl-phenylalanine derivative (NAPA) in Vitamin A model of osteoarthritis (OA) in rabbits. GlcN or NAPA or saline solution was intra-articularly administered in rabbit OA knees. Histological analysis revealed that treatment with GlcN or NAPA was associated with more homogeneous chondrocyte cellularity, absence of Wssures and fragmentation and more intense staining of the matrix with Alcian Blue compared to the articular surfaces of the knees treated with saline solution. Comparative in vitro study performed on rabbit primary chondrocytes revealed that GlcN and NAPA were also able to counteract the IL-1-upregulation of genes coding for metalloproteases and inXammatory cytokines. Our preliminary in vivo and in vitro studies suggest that GlcN and NAPA could play a disease-modifying protective role in OA by an anti-catabolic eVect and an anti-inXammatory activity on chondrocytes.

The Spanish Chromosome 16 consortium is integrated in the global initiative Human Proteome Project, which aims to develop an entire map of the proteins encoded following a gene-centric strategy (C-HPP) in order to make progress in the understanding of human biology in health and disease (B/D-HPP). Chromosome 16 contains many genes encoding proteins involved in the development of a broad range of diseases, which have a significant impact on the health care system. The Spanish HPP consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. Proteomics strategies have enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. BIOLOGICAL SIGNIFICANCE: In this manuscript we describe how the Spanish HPP-16 consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. We show how the Proteomic strategy has enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. This article is part of a Special Issue entitled: HUPO 2014.

Osteoarthritis is the most common rheumatic pathology and one of the leading causes of disability worldwide. It is a very complex disease whose etiopathogenesis is not fully understood. Furthermore, there are serious limitations for its management, since it lacks specific and sensitive biomarkers for early diagnosis, prognosis and therapeutic monitoring. Proteomic approaches performed in the last few decades have contributed to the knowledge on the molecular mechanisms that participate in this pathology and they have also led to interesting panels of putative biomarker candidates. In the next few years, further efforts should be made for translating these findings into the clinical routines. It is expected that targeted proteomics strategies will be highly valuable for the verification and qualification of biomarkers of osteoarthritis.

Purpose Osteoarthritis (OA) is a degenerative joint pathology characterized by articular cartilage degradation that lacks from efficient therapy. Since previous epidemiological data show a high controversy regarding the role of smoking in OA, we aimed to evaluate the effects of nicotine (the most physiologically active compound of tobacco) on the joint. Experimental design Secretome analyses, based on metabolic labeling followed by LC-MALDI-TOF/TOF analysis, were carried out using an in vitro model of articular inflammation (primary human articular chondrocytes treated with interleukin-1β), and also on osteoarthritic cells. ELISA and Western blot assays were performed to verify some of the results. Results Nineteen proteins were altered by nicotine in the model of articular inflammation, including several cytokines and proteases. We confirmed the increased secretion by nicotine of matrix metalloproteinase 1 and two proposed markers of OA, fibronectin, and chitinase 3-like protein 1. Finally, four components of the extracellular matrix of cartilage were decreased by nicotine in OA chondrocytes. Conclusions and clinical relevance Our data contribute to a better understanding of the molecular mechanisms that are modulated by nicotine in cartilage cells, suggesting a negative effect of this drug on the joint.