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GLPG1972/S201086 is a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5) inhibitor in development as an osteoarthritis disease-modifying therapy. We report the safety, tolerability, pharmacokinetics, and pharmacodynamics (turnover of plasma/serum ARGS-aggrecan neoepitope fragments [ARGS]) of GLPG1972 in 3 randomized, double-blind, placebo-controlled phase 1 trials. Study A, a first-in-human trial of single (≤2100 mg [fasted] and 300 mg [fed]) and multiple (≤1050 mg once daily [fed]; 14 days) ascending oral (solution) doses, investigated GLPG1972 in healthy men (N = 41; NCT02612246). Study B investigated multiple ascending oral (tablet) doses of GLPG1972 (≤300 mg once daily [fed]; 4 weeks) in male and female participants with osteoarthritis (N = 30; NCT03311009). Study C investigated single (Japanese: ≤1500 mg; White: 300 mg [fasted]) and multiple (Japanese, ≤1050 mg once daily; White, 300 mg once daily [fed]; 14 days) ascending oral (tablet) doses of GLPG1972 in healthy Japanese and White men (N = 88). The pharmacokinetic profile of GLPG1972 was similar between healthy participants and participants with osteoarthritis, with low to moderate interindividual variability. GLPG1972 was rapidly absorbed (median time to maximum concentration, 4 hours), and eliminated with a mean apparent terminal elimination half-life of ≈10 hours. Steady state was achieved within 2 days of dosing, with minimal accumulation. Steady-state plasma exposure after 300 mg of GLPG1972 showed no or minor differences between populations. Area under the plasma concentration-time curve (56.8-67.6 μg · h/mL) and time to maximum concentration (4 hours) were similar between studies. Urinary excretion of GLPG1972 (24 hours) was low (

Background:An anterior cruciate ligament (ACL) rupture results in an increased risk of developing knee osteoarthritis (OA) at an early age. Before clinical signs become apparent, the OA process has already been initiated. Therefore, it is important to look at the cascade of changes, such as the activity of cytokines and proteases, which might be associated with the later development of OA.Purpose:To compare biomarker levels in patients with a recent ACL rupture with those in controls with a healthy knee and to monitor biomarker levels over 2 years after an ACL rupture.Study Design:Descriptive laboratory study.Methods:Patients were enrolled after an ACL tear was identified. Serum and urine samples were collected at the time of enrollment in the study (3-25 weeks after the injury) and then at 14 and 27 months after the injury between January 2009 and November 2010. Reference samples were obtained from participants with healthy knees. The following biomarkers were measured with immunological assays: aggrecan ARGS neoepitope (ARGS-aggrecan), tumor necrosis factor–α (TNF-α), interferon-γ, interleukin (IL)–8, IL-10, IL-13, N-terminal cross-linked telopeptide of type I collagen (NTX-I), and C-terminal cross-linked telopeptide of type II collagen (CTX-II).Results:Samples were collected from 152 patients with an acute ACL rupture, who had a median age of 25 years (interquartile range [IQR], 21-32 years). There were 62 urine reference samples (median age, 25 years [IQR, 22-36 years]) and 26 serum reference samples (median age, 35 years [IQR, 24-39 years]). At a median of 11 weeks (IQR, 7-17 weeks) after trauma, serum levels of both ARGS-aggrecan and TNF-α were elevated 1.5-fold ( P < .001) compared with reference samples and showed a time-dependent decrease during follow-up. Urine NTX-I and CTX-II concentrations were elevated in an early phase after trauma (1.3-fold [ P < .001] and 3.7-fold [ P < .001], respectively) compared with reference samples, and CTX-II levels remained elevated compared with reference samples at 2-year follow-up. Strong correlations were found between serum ARGS-aggrecan, urinary NTX-I, and urinary CTX-II ( rs= 0.57-0.68).Conclusion:In the first few months after an ACL injury, there was a measurable increase in serum levels of ARGS-aggrecan and TNF-α as well as urine levels of NTX-I and CTX-II. These markers remained high compared with those of controls with healthy knees at 2-year follow-up.

Previous research has suggested an association between physical activity (PA), joint function, and molecular biomarkers, but more studies are needed. The aim of this study was to explore the associations between PA or self-reported joint function and molecular biomarkers of cartilage and inflammation in individuals with hip and/or knee osteoarthritis (OA). Specific objectives were to explore the correlations between (1) the change over 3 months in self-reported PA/joint function and the change in molecular biomarkers (2) objectively measured PA and molecular biomarkers measured at 3-month follow-up.Working age participants (n = 91) were recruited from a cluster randomized controlled trial. Self-reported PA, joint function, and serum samples were collected at baseline and after 3 months. Serum concentrations of the inflammatory marker C-reactive protein (CRP) and the cartilage markers Alanine-Arginine-Glycine-Serine (ARGS)-aggrecan, cartilage oligomeric matrix protein (COMP), and type II collagen C2C were analyzed by immunoassays. Objectively measured PA (steps/day) was collected during 12 weeks from activity trackers used by 53 participants. Associations were analyzed with Spearman's rank correlation.There was a weak negative correlation between the change in self-reported PA and the change in COMP (In general, no or only weak associations were found between PA/joint function and molecular biomarkers. Future research recommends including participants with lower PA, extend the follow-up, and use a design that allows comparisons.

ObjectivesOsteoarthritis (OA) patient stratification is an important challenge to design tailored treatments and drive drug development. Biochemical markers reflecting joint tissue turnover were measured in the IMI-APPROACH cohort at baseline and analysed using a machine learning approach in order to study OA-dominant phenotypes driven by the endotype-related clusters and discover the driving features and their disease-context meaning.MethodData quality assessment was performed to design appropriate data preprocessing techniques. The k-means clustering algorithm was used to find dominant subgroups of patients based on the biochemical markers data. Classification models were trained to predict cluster membership, and Explainable AI techniques were used to interpret these to reveal the driving factors behind each cluster and identify phenotypes. Statistical analysis was performed to compare differences between clusters with respect to other markers in the IMI-APPROACH cohort and the longitudinal disease progression.ResultsThree dominant endotypes were found, associated with three phenotypes: C1) low tissue turnover (low repair and articular cartilage/subchondral bone turnover), C2) structural damage (high bone formation/resorption, cartilage degradation) and C3) systemic inflammation (joint tissue degradation, inflammation, cartilage degradation). The method achieved consistent results in the FNIH/OAI cohort. C1 had the highest proportion of non-progressors. C2 was mostly linked to longitudinal structural progression, and C3 was linked to sustained or progressive pain.ConclusionsThis work supports the existence of differential phenotypes in OA. The biomarker approach could potentially drive stratification for OA clinical trials and contribute to precision medicine strategies for OA progression in the future.Trial registration numberNCT03883568.

Objective: To determine the role of inflammatory biomarkers at 2 years post–anterior cruciate ligament (ACL) injury to predict radiographic knee osteoarthritis (OA) and magnetic resonance imaging (MRI)–defined knee OA at 5 years postinjury, with a secondary aim of estimating the concordance of inflammatory biomarkers assessed by MRI and synovial fluid (SF) analysis. Methods: We studied 113 patients with acute ACL injury. Knee scans using 1.5T MRIs were read for Hoffa- and effusion-synovitis. Biomarkers of inflammation that we assessed included interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor, and interferon-ɣ in serum and SF, and IL-12p70 in serum. We defined the outcome as radiographic knee OA (ROA) or MRI-defined OA (MROA) at 5 years. The area under the receiver operating characteristic curve (AUC), sensitivity, and specificity were evaluated in models that included MRI features only (model 1), inflammation biomarkers only (serum [model 2a] or SF [model 2b]), both MRI features and serum biomarkers (model 3a), or both MRI features and SF (model 3b) biomarkers. Linear regression analysis was used to evaluate the association between MRI features and SF biomarkers. Results: At 5 years postinjury, ROA was present in 26% of the injured knees, and MROA was present in 32%. The AUCs for ROA in each model were 0.44 (95% confidence interval [95% CI] 0.42, 0.47) for model 1, 0.62 (95% CI 0.59, 0.65) for model 2a, 0.53 (95% CI 0.50, 0.56) for model 2b, 0.58 (95% CI 0.55, 0.61) for model 3a, and 0.50 (95% CI 0.46, 0.53) for model 3b. The AUCs for MROA in each model were 0.67 (95% CI 0.64, 0.70) for model 1, 0.49 (95% CI 0.47, 0.52) for model 2a, 0.56 (95% CI 0.52, 0.59) for model 2b, 0.65 (95% CI 0.61, 0.68) for model 3a, and 0.69 (95% CI 0.66, 0.72) for model 3b. The concordance between MRI and SF biomarkers was statistically significant only for effusion-synovitis and IL-8. Conclusion: Neither MRI-detected inflammation nor selected SF/serum inflammation biomarkers at 2 years postinjury predicted ROA or MROA at 5 years postinjury. Concordance between MRI and SF inflammatory biomarkers was weak. (Less)

Osteoarthrithis (OA) is an endemic disease due to the increase of the world’s elderly population. Previously thought to be a consequence of an imbalance between cartilage degradation and biosynthesis, it is now recognized as a disease also involving inflammation, hence influencing the level of inflammatory cytokines, growth factors and chemokines. Lubricin is a mucin type molecule where its OA induced glycosylation truncation propels a deteriorating lubrication of the articular cartilage. The objective of this study was to explore the OA driven truncation of O-linked glycosylation of synovial lubricin and its cross talk with systemic and local (synovial fluid, SF) inflammation. We compared the systemic level of cytokines/chemokine in OA patients’ and controls’ plasma with their local level in SF using a 44 plex screen. The level of 27 cytokines and chemokines was consistently measured in both plasma and SF. The data showed that the levels of cytokines and chemokines in OA plasma display limited correlation to their counterpart in SF. The level of synovial IL-8 and MIP-1α and VEGFA in OA patients, but not their plasma level, where the only cytokines that displayed a significant correlation to the observed lubricin O-linked glycosylation truncation. These cytokines were also shown to be upregulated exposing fibroblast like synoviocytes from healthy and OA patients to recombinant lubricin with truncated glycans mainly consisting of Tn-antigens, while lubricin with sialylated and non-sialylated T anigens did not have any effect. The data suggest that truncated glycans of lubricin, as found in OA, promotes the synovial cytokine production and exerebate the local synovial inflammation.

Summary Objectives In this exploratory study, we used discovery proteomics to follow the release of proteins from bovine knee articular cartilage in response to mechanical injury and cytokine treatment. We also studied the effect of the glucocorticoid dexamethasone (Dex) on these responses. Design Bovine cartilage explants were treated with either cytokines alone (10 ng/ml TNFα, 20 ng/ml IL-6, 100 ng/ml sIL-6R), a single compressive mechanical injury, cytokines and injury, or no treatment, and cultured in serum-free DMEM supplemented with 1% ITS for 22 days. All samples were incubated with or without addition of 100 nM Dex. Mass spectrometry and Western blot analyses were performed on medium samples for the identification and quantification of released proteins. Results We identified 500 unique proteins present in all three biological replicates. Many proteins involved in the catabolic response of cartilage degradation had increased release after inflammatory stress. Dex rescued many of these catabolic effects. The release of some proteins involved in anabolic and chondroprotective processes was inconsistent, indicating differential effects on processes that may protect cartilage from injury. Dex restored only a small fraction of these to the control state, while others had their effects exacerbated by Dex exposure. Conclusions We identified proteins that were released upon cytokine treatment which could be potential biomarkers of the inflammatory contribution to cartilage degradation. We also demonstrated the imperfect rescue of Dex on the effects of cartilage degradation, with many catabolic factors being reduced, while other anabolic or chondroprotective processes were not.

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff base in gels, and with proteomics. Full length lubricin (∼300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain (∼250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in all domains of lubricin, including the mucin domain. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the pathological decrease of the lubrication in degenerative joint disease.

To monitor longitudinal changes of cartilage oligomeric matrix protein (COMP) in synovial fluid (sf) and serum (s) over 5 years after acute anterior cruciate ligament (ACL) rupture, and to compare results from two commercial COMP immunoassays.Bio-fluids were collected from 121 patients on six occasions over 5 years after acute ACL injury, and from 25 knee healthy reference subjects. Concentrations of sf- and sCOMP were measured by AnaMar (sCOMP-Ana) and by BioVendor (sf- and sCOMP-Bio) immunoassays; other biomarkers were previously assessed. We used ANCOVA for group comparisons and linear mixed models for associations between biomarkers over 5-years with P 0.05 considered a statistically significant difference or association.Compared to the reference group, sfCOMP-Bio concentrations were 2-fold elevated within 6 weeks after ACL injury and remained elevated 5 years thereafter, whereas sCOMP-Bio and sCOMP-Ana concentrations were no different from reference levels at any time point. Over the 5-year period, there was an association between sCOMP-Bio and sCOMP-Ana concentrations, although neither sCOMP-Bio nor sCOMP-Ana associated with sfCOMP-Bio. sfCOMP-Bio associated with SF ARGS-aggrecan, urine type I and II collagens (uNTX-I and uCTX-II) and SF cytokines, while sCOMP-Bio associated inversely with uCTX-II, uNTX-I and SF cytokines.The local process after an acute ACL injury generates increased SF COMP concentrations in the injured knee up to 5 years after injury. This response is not detected in serum. Discrepancies in associations between sCOMP measured by BioVendor and AnaMar immunoassays with other biomarkers indicate differences in detected COMP fragments.

Summary Objective To explore the involvement of the wingless-type MMTV integration site (WNT) and bone morphogenetic protein (BMP) antagonists dickkopf-related protein 1 (DKK1), frizzled-related protein (FRZB) and gremlin 1 (GREM1) in knee injury and osteoarthritis (OA). Design The antagonists were immunoassayed in synovial fluid from a cross-sectional cohort of nine knee healthy reference subjects, patients with recent (0–77 days, n = 158) or old (1–37 years, n = 50) knee injuries, and OA ( n = 22). Cartilage (ARGS-aggrecan, cartilage oligomeric matrix protein and C2C type II collagen) and other biomarkers were assessed in synovial fluid in a subset of samples. Statistical analysis was by Kendall's tau ( τ ) correlation, Mann–Whitney U test, and linear regression analysis. Results Compared to references, median concentration of GREM1 (but not DKK1 and FRZB) was elevated 1.5-fold immediately after injury, and FRZB was reduced 1000-folds in OA. All three antagonists decreased with increasing time after injury as well as with increasing age, but the temporal change after injury was less accentuated for FRZB (peaked 8–22 days after injury) compared to that of DKK1 and GREM1 (peaked immediately after injury). In the recent injury group, there was a correlation between GREM1 and DKK1 ( τ = 0.172); FRZB concentrations correlated with concentrations of cartilage biomarkers ( τ between 0.257 and 0.369), while DKK1 and GREM1 were inversely correlated ( τ between −0.177 and −0.217) with these markers. Conclusions Our results indicate separate roles for the antagonists, where DKK1 and GREM1 had similarities in response to injury and in OA, with a different response for FRZB.

The aim of this study was to identify molecules that trigger complement activation in rheumatic joints. C4d, the final cleavage product of C4 activation, is found in the diseased joint and can bind covalently to complement-activating molecules. By using a highly specific Ab against a cleavage neoepitope in C4d, several molecules that were specifically bound to C4d were identified from pooled synovial fluid (SF) from four rheumatoid arthritis (RA) patients. One of these molecules, pigment epithelium–derived factor (PEDF), is a broadly expressed multifunctional member of the serine proteinase inhibitor family. Using ELISA, we confirmed the presence of various amounts of complexes between PEDF and C4d in the SF from 30 RA patients, whereas none were detected in SF from control subjects. Correlation analyses suggested that, in arthritis patients, C4d–PEDF complexes found in sera arise from the joints, as well as from other tissues, and levels of the complexes did not differ in sera of RA patients and healthy controls. When immobilized, recombinant PEDF expressed in eukaryotic cells activated the classical complement pathway but not the alternative or lectin pathways. C1q protein was demonstrated to bind immobilized PEDF, and PEDF was shown to bind to immobilized C1q, in particular its head regions, which are known to interact with other activators of the classical pathway. Our results call for further investigation into the role of PEDF in inflammatory processes in the joint, which, in combination with classical complement activation, appears to be part of a (patho-)physiologic response.

Background:The disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) is a key enzyme in OA (Verma P,et al.J Cell Biochem 2011;112:3507-14). In preclinical models of OA, S201086, a potent and selective ADAMTS-5 inhibitor, protects from cartilage degradation (Clement-Lacroix,et al.Osteoarthritis Cartilage 2017;25:S58). Therefore, S201086 is co-developed by Servier and Galapagos (GLPG1972) as a potential new treatment in OA.Objectives:This phase I study was to assess and compare the safety, tolerability, pharmacokinetics and pharmacodynamics of S201086 in healthy male Japanese and Caucasian subjects.Methods:Six ascending single doses of S201086 (50 to 1500 mg) were administered orally as tablets in fasting conditions in 6 groups of 8 Japanese subjects (aged 20-45 years) and 3 ascending multiple doses (300, 600 or 1050 mg/day for 14 days) were administered orally as tablets in fed conditions in 3 groups of 8 Japanese subjects. Additionally, 2 cohorts of 8 Caucasian subjects received either a single oral dose of 300 mg or repeated oral dose of 300 mg once daily for 14 days. In each group, 6 subjects received S201086 and 2 placebo. Plasma was collected at several time points for the quantification of S201086 by LC-MS/MS. Plasma pharmacokinetic parameters were calculated via non-compartmental analysis using Phoenix® WinNonlin® software. Pharmacodynamics was assessed by measurement of the aggrecan ARGS neoepitope levels in serum by ELISA.Results:No serious adverse drug reactions were observed after administration of single and multiple ascending doses of S201086 in either Japanese or Caucasian subjects. Most adverse events were of mild to moderate intensity and were resolved by the end of the study. Following single administration to Japanese subjects, Cmax and AUC increased proportionally with doses from 50 to 600 mg and slightly less than dose-proportionally from 600 to 1500 mg, consistent with what was previously observed in the first in-human study in healthy Caucasian subjects (van der Aar E,et al.Osteoarthritis Cartilage 2018;26:S310). In Japanese subjects, after 14-days administration, a limited 1.2- to 1.4-fold accumulation was observed on both Cmax and AUC at Day14 as compared to Day1, and the steady-state exposure increased dose-proportionally within the 300-1050 mg dose range. Taking the inter-subject variability into account, no difference was observed in Cmax and AUC between Caucasian and Japanese subjects after single administrations of 300 mg and repeated administrations of 300 mg once daily for 14 days. The serum ARGS neoepitope levels decreased between baseline and last day of treatment for S201086-treated subjects but not for subjects receiving placebo. This decrease was similar for the 3 tested doses in Japanese subjects and was significant as compared to the placebo-treated subjects.Table.Comparison of ARGS concentration relative change from baseline to last day of treatment according to dose in Japanese participantsS 201086 300 mg (N = 6)S 201086 600 mg (N = 6)S 201086 1050 mg (N = 6)Placebo (N = 6)Descriptive Statistics(D14 pre-dose – Baseline) / Baselinen6646Mean ± SD-42.67 ± 8.81-53.70 ± 6.50-54.11 ± 5.2013.35 ± 16.53Statistical analysisE (SE)56.01 (6.156)67.04 (6.156)67.45 (6.883)95% CI43.08; 68.9554.11; 79.9752.99; 81.91p-value< 0.0001< 0.0001< 0.0001Conclusion:S201086 was shown to be safe and well-tolerated in Japanese and Caucasian healthy male subjects with a suitable PK profile and a significant decrease of the ARGS neoepitope, indicating target engagement. A global Phase 2 study to evaluate the efficacy of S201086 in patients with knee OA is ongoing (NCT03595618).Disclosure of Interests:Agnès Lalande Employee of: Institut de Recherches Internationales Servier, Nadya KUZNIATSOVA-MOUCHETTE Employee of: Institut de Recherches Internationales Servier, Florian CHASSEREAU Employee of: Institut de Recherches Internationales Servier, Julia GERONIMI Employee of: Institut de Recherches Internationales Servier, Katy Bernard Employee of: Institut de Recherches Internationales Servier, Staffan Larsson: None declared, André Struglics: None declared, Stefan Lohmander: None declared, Maria Pueyo Employee of: Institut de Recherches Internationales Servier

Lubricin (PRG4) is a mucin type protein that plays an important role in maintaining normal joint function by providing lubrication and chondroprotection. Improper lubricin modification and degradation has been observed in idiopathic osteoarthritis (OA), while the detailed mechanism still remains unknown. We hypothesized that the protease cathepsin G (CG) may participate in degrading lubricin in synovial fluid (SF). The presence of endogenous CG in SF was confirmed in 16 patients with knee OA. Recombinant human lubricin (rhPRG4) and native lubricin purified from the SF of patients were incubated with exogenous CG and lubricin degradation was monitored using western blot, staining by Coomassie or Periodic Acid-Schiff in gels, and with proteomics. Full length lubricin (∼300 kDa), was efficiently digested with CG generating a 25-kDa protein fragment, originating from the densely glycosylated mucin domain (∼250 kDa). The 25-kDa fragment was present in the SF from OA patients, and the amount was increased after incubation with CG. A CG digest of rhPRG4 revealed 135 peptides and 72 glycopeptides, and confirmed that the protease could cleave in different domains of lubricin. Our results suggest that synovial CG may take part in the degradation of lubricin, which could affect the lubrication of OA joints.

Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (-A and -C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTRasym ) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTRasym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS-A and CS-C compared with phantoms containing mainly CS-A. The difference in response corresponds well to the difference in CS-A concentration. GAG bound in aggrecan from calf articular cartilage, where CS-A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS-A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS-A while for CS-C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo.

Summary Objective To estimate the association between molecular or imaging inflammatory biomarkers at 2 years after anterior cruciate ligament (ACL) injury and patient-reported outcomes at 5 years. Methods For 116 ACL-injured patients, molecular biomarkers of inflammation (synovial fluid and serum cytokines) and Hoffa- and effusion-synovitis as visualized on magnetic resonance imaging (MRI) were assessed 2 years post-injury. Knee injury and Osteoarthritis Outcome Score (KOOS) and SF-36 were assessed at 2 and 5 years. We used multiple imputation to handle biomarker values that were below the level of detection or missing, and linear regression for statistical analyses. Results None of the synovial fluid cytokines or imaging biomarkers of inflammation at 2 years were associated with any of the patient-reported outcomes at 5 years. With each log10 unit higher of serum tumor necrosis factor concentration the knee-related quality of life of KOOS was increased (i.e., better outcome) by 35 (95% confidence interval 7 to 63) points. No other serum biomarker measured at 2 years was associated with patient-reported outcome at 5 years. Conclusion Local joint inflammation assessed by biomarkers in synovial fluid and Hoffa- and effusion-synovitis on MRI at 2 years after an ACL injury did not associate with patient-reported outcomes at 5 years. Thus, chronic inflammation in the ACL-injured knee, as reflected by the biomarkers studied here, seems not to be a key determinant for the long-term patient-reported outcomes.

As explained in the methods-section, we chose to analyze the 2-year data in regard to inflammatory activity based on MRI and synovial biomarkers (and not earlier time points) as our aim was to capture patients that experienced "prolonged" or chronic inflammation, which may be regarded as a potential risk factor for osteoarthritis (OA) development. Virtually all patients of an acute injury cohort (like the KANON patients) will exhibit acute post-traumatic inflammation in their injured knee to a various extent as manifested by marked changes on imaging (i.e. effusion, traumatic synovitis and haemarthrosis) or in biochemical parameters. This article is protected by copyright. All rights reserved.

Joint destruction is a hallmark of juvenile idiopathic arthritis (JIA). Clinical evaluation and radiographic imaging are current methods to identify destruction. Biomarkers could aid an earlier and more sensitive diagnosis. Our aim was to investigate levels of bone and cartilage degradation biomarkers in JIA patients, compared to healthy children or juveniles with knee injuries.Triple-paired synovial fluid, plasma and urine samples from 29 JIA patients were compared to 61 plasma samples from healthy children and synovial fluid from 41 knee-injured juveniles. Cartilage biomarkers ARGS neoepitope of aggrecan (ARGS), cartilage oligomeric matrix protein (COMP), type II collagen epitope (C2C), bone biomarkers N-terminal type I collagen cross-linked telopeptide (NTX-I) and tartrate-resistant acid phosphatase 5b (TRAP5b) were analysed by immunoassays.Plasma levels of ARGS, C2C, COMP and TRAP5b were increased in JIA compared to healthy children. Compared to knee-injured juveniles, synovial fluid C2C and TRAP5b were increased in JIA, while ARGS and COMP were decreased. For JIA patients, local (synovial fluid) and systemic (plasma/urine) levels of bone biomarkers correlated positively; age correlated negatively to plasma levels of C2C and TRAP5b; no correlation was found between biomarkers and gender, affected joint count, disease duration or medication.Elevated levels of destruction biomarkers in JIA compared to healthy children indicate a potential to serve as clinical tools for destructive joint disease. High levels of TRAP5b, NTX-I and collagen II in JIA in contrast to more pronounced aggrecan and COMP degradation in juvenile knee injuries, suggests that JIA patients have a unique biomarker pattern, different from healthy and knee-injured children.

Knee injury increases the risk of developing knee osteoarthritis (OA). Recent evidence suggests involvement of oxidative stress induced by inflammation and bleeding in the joint. This study investigates the role in this process of α1-microglobulin (A1M), a plasma and tissue antioxidant protein with reducing function, and heme- and radical-binding properties. We studied matched knee synovial fluid (sf) and serum (s) samples from 122 subjects (mean age 40 years, 31% females): 10 were knee healthy references, 13 had acute inflammatory arthritis (AIA), 79 knee injury 0–10 years prior to sampling, and 20 knee OA. Using immunoassays, we measured sf-A1M and s-A1M, sf-hemoglobin (sf-Hb), sf-total free heme (sf-Heme), and sf-carbonyl groups (sf-Carbonyl). We explored associations by partial correlation, or linear regression models with adjustments for age, sex and diagnosis, and evaluated diagnostic capacity by area under the receiver operator characteristics curve (AUC). The AIA group had 1.2- to 1.7-fold higher sf-A1M and s-A1M concentrations compared to the other diagnostic groups; other biomarkers showed no between-group differences. sf-A1M and s-A1M were with AUC of 0.76 and 0.78, respectively, diagnostic for AIA. In the injury group, the amount of bleeding in the joint was inversely correlated to time after injury when measured as sf-Heme (r = -0.41, p < 0.001), but not when measured as sf-Hb (r = -0.19, p = 0.098). A similar inverse association with time after injury was noted for sf-A1M (r = -0.30, p = 0.007), but not for s-A1M and sf-Carbonyl. Linear regression models showed that sf-Heme was more strongly associated with sf-A1M and sf-Carbonyl than sf-Hb. Independent of diagnosis, sf-Heme explained 5.7% of the variability in sf-A1M and 3.0% in the variability in sf-Carbonyl, but appeared unrelated to s-A1M. High sf-A1M and low sf-Heme or sf-Hb were independently associated with low sf-Carbonyl. In conclusion, our results demonstrate that independent of disease, Hb and heme within a knee joint correlates with an increased sf-A1M concentration that appears to be protective of oxidative damage, i.e., a reduction in carbonyl groups. High concentrations of A1M in synovial fluid and serum was further diagnostic for AIA.

Summary Introduction Knee injury history and increased joint load, respectively, are major risk factors for the development of knee osteoarthritis (OA). Lower extremity muscle function, such as knee muscle strength, influence joint load and may be important for the onset of knee OA. However, the role of muscle function as a possible modifiable protective mechanism for the development of OA after anterior cruciate ligament reconstruction (ACLR) is not clear. Methods and analysis In this prospective cohort study, 100 patients (50% women, 18–35 years) with ACLR will be recruited from Skane University Hospital, Sweden and Oslo University Hospital, Norway. They will be assessed with a comprehensive test battery of muscle function including muscle strength, muscle activation, hop performance, and postural orientation as well as patient-reported outcomes, one year (baseline) and three years (follow-up) after ACLR. Primary predictor will be knee extension strength, primary outcome will be patient-reported knee pain (Knee injury and Osteoarthritis Outcome Score, subscale pain) and secondary outcomes include compositional MRI (T2 mapping) and turnover of cartilage and bone biomarkers. Separate linear regression model will be used to elucidate the influence of each baseline muscle function variable on the outcomes at follow-up, adjusted for baseline values. Twenty non-injured individuals will also be assessed with MRI. This study is approved by The Regional Ethical Review Board in Lund (Sweden) and Oslo (Norway). Discussion This study may have important clinical implications for using muscle function to screen for risk of early-onset knee OA and for optimizing exercise therapy after knee injury.

Anterior cruciate ligament (ACL) trauma often occurs in combination with injury to the articular cartilage of the knee, this can result in earlier radiographic evidence of post traumatic osteoarthritis (OA) of the knee compared to the contralateral, ACL intact knee; however, the biomechanical and biological mechanisms associated with the onset and progression of this disease are not understood. We sought to gain insight into the mechanisms by determining the relationship between articular cartilage injury associated with ACL trauma and the expression of synovial fluid biomarkers of articular cartilage metabolism, and to evaluate the relationship between these biomarkers and the patient's perspective of the outcomes. Synovial fluid samples were acquired from 39 ACL injured subjects at an average of 10 weeks after injury, and 32 control subjects with normal knees (documented with clinical exam and MRI assessment). Subjects in the ACL-injured group were classified as low-risk for future OA if they displayed an International Cartilage Repair Society (ICRS) Grade 2 articular cartilage lesion or less and high-risk for future OA if they had an ICRS Grade 3A articular cartilage lesion. The patient's perspective of the injury was evaluated with the Knee Injury and Osteoarthritis Outcomes Score (KOOS). There were no significant differences in mean concentrations of the markers of type II collagen metabolism (CPII, C2C, and C1,2C) or the aggrecan breakdown Alanine-Arginine-Glycine-Serine (ARGS) -fragment between control subjects and the subjects in the low- and high-risk groups (p-value range: 0.80-0.43). Associations between ARGS-aggrecan concentration and KOOS subscales of symptoms and pain were significantly different between the low- and high-risk groups (p = 0.03 and p = 0.01, respectively). Likewise, there was strong evidence in support of an association between the markers of type II collagen metabolism (C1,2C and CPII concentrations) and the KOOS subscale of pain between the low- and high-risk groups (p = 0.051 and 0.077, correspondingly). In ACL injured subjects with concomitant Grade 3A articular cartilage injuries, concentrations of synovial fluid ARGS-aggrecan were directly associated with improvements in KOOS symptoms and pain. These findings suggest the possible involvement of ARGS-aggrecan in a localized tissue repair response involving an increase in aggrecan turnover following severe knee trauma. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:820-827, 2016.

OBJECTIVE: To explore potential associations between proinflammatory cytokines in synovial fluid and progression of osteoarthritis (OA) in meniscectomized subjects.DESIGN: We studied 132 subjects on average 18 years after meniscectomy, with a second examination 4-10 years later. We measured concentrations of interleukin (IL)-6, -8 and tumor necrosis factor (TNF)-α by multiplex immunoassay, graded radiographic features of tibiofemoral and patellofemoral OA according to the Osteoarthritis Research Society International (OARSI) atlas, scored patient-reported outcomes using the Knee Injury and Osteoarthritis Outcome Score (KOOS), and used logistic regression (adjusted for age, gender, body mass index, and time between examinations) for assessment of associations.RESULTS: Higher first examination concentrations of IL-6 and TNF-α were associated with increased risk for subsequent osteophyte progression (odds ratios (OR); 95% confidence intervals 1.05; 1.00-1.09 and 1.35; 1.03-1.75). Higher second examination concentrations of TNF-α were associated with having progressed in loss of joint space (OR 1.70; 1.15-2.52) or having worsened in the activity of daily living subscale of KOOS (OR 1.50; 1.07-2.09) in the preceding years. Subjects with increasing concentrations of IL-6 or TNF-α between examinations were five times more likely to have progressed in joint space narrowing between the same examinations, as compared to those with stable or decreasing concentrations (OR 5.17; 1.54-17.32 and 5.01; 1.32-18.92).CONCLUSIONS: In subjects with previous meniscectomy, higher or over time increasing synovial fluid levels of IL-6 and TNF-α seems to be associated with increased risk for progression of radiographic OA.

Background Trauma-induced cytokine response and local inflammation after knee injury may be important in the development of posttraumatic osteoarthritis (OA). It has been reported that synovial fluid levels of inflammatory markers remain increased up to 5 years after anterior cruciate ligament (ACL) injury, indicative of extended local inflammation in the injured joint.1 Inflammation potentially represents a valid target for treatment in the early and subacute phase after joint trauma in order to prevent or delay onset of post-traumatic knee OA. Objectives To determine the role of inflammatory biomarkers at 2 years after anterior cruciate ligament (ACL) injury for predicting radiographic and magnetic resonance imaging (MRI)-defined knee OA 5 years post injury. Secondary aim was to estimate the concordance of inflammatory biomarkers assessed by MRI and in synovial fluid. Methods We studied 113 patients with acute ACL injury. 1.5 Tesla knee MRIs were read for Hoffa- and effusion-synovitis. Biomarkers of inflammation included IL-6, IL-8, IL-10, TNF-α and IFN-γ in serum and synovial fluid, and IL-12p70 in serum. The outcome was radiographic knee OA (ROA) or MRI-defined OA (MROA) at 5 years. Area under receiver operating characteristic curve (AUC), sensitivity and specificity were evaluated in models including MRI features only (M1), inflammation biomarkers only (serum [M2a] – synovial [M2b]) or both MRI and serum [M3a] or synovial [M3b] markers. Linear regression was used for evaluating association between MRI and synovial biomarkers. Results At 5 years, ROA was present in 26% and MROA was present in 32% of patients’ injured knee. The AUCs (95% CI) for ROA were 0.44 (0.42–0.47;M1), 0.62 (0.59–0.65;M2a), 0.58 (0.55–0.61;M2b), 0.53 (0.50–0.56;M3a) and 0.50 (0.46–0.53;M3b) for each model. The corresponding AUCs for MROA were 0.67 (0.64–0.70), 0.49 (0.47–0.52), 0.65 (0.61–0.68), 0.56 (0.52–0.59) and 0.69 (0.66–0.72) (table 1). The associations between MRI and synovial biomarkers were weak and not statistically significant, apart from effusion–synovitis and IL-8 (log 10 IL8 levels were 0.23 and 0.43 higher in persons with grade 1 or 2/3, respectively). Conclusions Neither MRI-defined inflammation, nor synovial/serum inflammation biomarkers at 2 years as analysed here predicted ROA or MROA at 5 years. The concordance between MRI and synovial inflammatory biomarkers was weak. Additional studies with longer follow-up will be needed to confirm or refute our findings and to more firmly define the role of inflammation for OA development following acute ACL injury. Reference [1] Struglics A, et al. Changes in cytokines and aggrecan ARGS neoepitope in synovial fluid and serum and in C-terminal crosslinking telopeptide of type II collagen and N-terminal crosslinking telopeptide of type I collagen in urine over five years after anterior cruciate ligament rupture: An exploratory analysis in the knee anterior cruciate ligament, nonsurgical versus surgical treatment trial.Arthritis Rheumatol2015;67:1816–25. Acknowledgements We thank the participants of the KANON study without this work would not have been possible. Disclosure of Interest F. Roemer Shareholder of: Boston Imaging Core Lab (BICL), LLC., M. Englund: None declared, A. Turkiewicz: None declared, A. Struglics: None declared, A. Guermazi Shareholder of: Boston Imaging Core Lab (BICL), LLC., Consultant for: Sanofi-Aventis, Merck Serono, OrthoTrophix, AstraZeneca, Pfizer, GE, and TissuGene, L. S. Lohmander: None declared, S. Larsson: None declared, R. Frobell: None declared

OBJECTIVE: To investigate the impact of exercise therapy on molecular biomarkers related to cartilage and inflammation in people at risk of, or with established, knee osteoarthritis by conducting a systematic review of randomized controlled trials (RCTs).METHODS: Literature search up to September 2017 in five major databases with no restriction on publication year or language. Data were extracted from the first available follow-up time point and we performed a narrative synthesis for the effect of exercise therapy on molecular biomarkers related to cartilage and inflammation. A subset of studies reporting sufficient data was combined in a meta-analysis, using an adjusted random effects model.RESULTS: Twelve RCTs, involving 57 study comparisons at 4 to 24 weeks following an exercise therapy intervention were included. Exercise therapy decreased molecular biomarkers in 17 (30%) study comparisons, had no effect in 36 (63%), and increased molecular biomarkers in four (7%) study comparisons. Meta-analyses of nine biomarkers showed that exercise therapy was associated with non-significant reductions of C-reactive protein, C-terminal crosslinking telopeptide of type II collagen, tumor necrosis factor alpha (TNF-α), soluble TNF-α receptor-1 and -2, C2C neoepitope of type II collagen and cartilage oligomeric matrix protein compared to non-exercising control groups and had no effect on interleukin-6 and soluble interleukin 6 receptor.CONCLUSIONS: Exercise therapy is not harmful, as it does not increase the concentration of molecular biomarkers related to cartilage turnover and inflammation, implicated in osteoarthritis progression. The overall quality of evidence was downgraded to low because of the limited number of RCTs available. This article is protected by copyright. All rights reserved. (Less)

Severe knee trauma, such as an ACL disruption, produces aggrecan degradation as evidenced by elevated synovial fluid (SF) N-terminal (393) Alanine-Arginine-Glycine-Serine (ARGS) neoepitope (or ARGS-aggrecan) and is associated with inflammatory activity soon after injury. However, it is not known if this process persists for a substantial time interval following the initial trauma. The purpose of this study was to evaluate relationships between SF ARGS concentrations and an array of cytokines, matrix metalloproteases (MMPs), and tissue inhibitor of metalloproteases (TIMPs) during the initial 6 months following ACL rupture. SF samples from 67 ACL-injured subjects (29 women) were analyzed within 6 months of injury (18-155 days), immediately prior to surgical ACL reconstruction. Relationships between ARGS and individual analyte concentrations, as well as MMP/TIMP ratios were evaluated. Statistically significant relationships were found between ARGS and basic fibroblast growth factor (FGF2) (p=0.03) and TIMP-3 (p=0.01). Our findings suggest that FGF2, considered to be primarily catabolic in articular cartilage, is not downregulated as ARGS concentration declines over time since injury. In addition, these results support the hypothesis that an upregulation of TIMP-3, the primary aggrecanase inhibitor, is elicited in response to increased aggrecan degradation, which may inhibit further cleavage.

Risk stratification of patients presenting with acute chest pain is crucial for immediate and long-term management. Traditional predictors are suboptimal; therefore inflammatory biomarkers are studied for clinical assessment of patients at risk. Recently, we reported the association of IgM-uria with worse cardiovascular outcome in patients with acute chest pain. In this study, in the same cohort of patients with chest pain, we compared the value of IgM-uria to pro-inflammatory cytokines in predicting the occurrence of subsequent cardiovascular events.A total of 178 consecutive patients presenting with acute chest pain to the emergency department at the University Hospital of Lund, were recruited. Twenty-seven of 57 patients with acute coronary syndrome (ACS), and 18 of 118 patients with non-specific chest pain at baseline developed a subsequent major cardiovascular event during the 18 months follow-up. Urinary proteins (IgM-uria and Microalbuminuria) and plasma inflammatory markers (IL-6, Il-8, IL-10, IFN-γ and TNF-α) were measured at time of admission.Using the receiver operating characteristic curves, the area under the curve for predicting cardiovascular events was 0.71 (95%CI 0.61-0.81) for IgM-uria, 0.61 (95%CI 0.51-0.71) for IL-6, 0.63 (95%CI 0.53-0.72) for IL-8, 0.65 (95%CI 0.56-0.74) for IL-10, and 0.64 (95% CI 0.54-0.74) for TNF-α. In multivariate Cox-regression analysis adjusted for age, microalbuminuria, IgM-uria, IL-10, TNF-α, troponin T, hsCRP and ACS at baseline; IgM-uria was the only biomarker that remained an independent predictor of outcome (HR = 4.2, 95%CI 2.2-7.8, p0.001).In patients with chest pain with or without acute coronary syndrome, IgM-uria could better predict the occurrence of cardiovascular events than plasma pro-inflammatory cytokines.

OBJECTIVE: To prospectively monitor levels of proinflammatory cytokines and aggrecan ARGS neoepitope in synovial fluid and serum as well as levels of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and N-terminal crosslinking telopeptide of type I collagen (NTX-I) in urine after acute anterior cruciate ligament (ACL) rupture.METHODS: Synovial fluid, serum, and urine were collected from 121 adults on 6 occasions over 5 years after acute ACL injury. Reference samples were obtained from subjects without knee injury. Concentrations of interleukin-6 (IL-6), IL-8, IL-10, interferon-γ (IFNγ), tumor necrosis factor (TNF), aggrecan ARGS neoepitope, CTX-II, and NTX-I were measured by enzyme-linked immunosorbent assay.RESULTS: Shortly after ACL injury, cytokine concentrations in synovial fluid were elevated 6-fold (TNF) to 1,050-fold (IL-6) compared to reference levels, while concentrations of aggrecan ARGS neoepitope in synovial fluid and serum and CTX-II in urine were elevated 1.4-fold to 8-fold. Thereafter, concentrations of cytokines and aggrecan ARGS neoepitope in synovial fluid decreased with different half-lives (in years: IL-6 0.9, IL-8 2.2, IL-10 2.3, IFNγ 3.1, TNF 3.6, aggrecan ARGS neoepitope 4.0). After 5 years, the TNF concentration in synovial fluid remained higher than the reference level. There was a correlation between the concentrations of aggrecan ARGS neoepitope in synovial fluid and serum (rs = 0.36). Concentrations of aggrecan ARGS neoepitope in synovial fluid and of CTX-II and NTX-I in urine were correlated with concentrations of cytokines in synovial fluid (rs = 0.41-0.49 and rs = 0.21-0.31, respectively).CONCLUSION: Acute ACL injury induced highly increased levels of inflammatory cytokines in the joint, and these were associated with proteolysis of aggrecan and type II collagen. Cytokine levels remained increased up to 5 years after injury, indicative of extended local inflammation in the joint.