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1.	Andersson, Elin, et al. (författare) Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study 2023 Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3, s. 1-11 Tidskriftsartikel (refereegranskat)abstract ObjectiveTo validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples.DesignSynovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry.ResultsUnsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low.ConclusionsThe HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.
2.	Andersson, Elin, et al. (författare) Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study 2023 Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3 Tidskriftsartikel (refereegranskat)abstract Objective: To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. Design: Synovial fluid from osteoarthritis (OA, n = 25) and knee-injury (n = 13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. Results: Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. Conclusions: The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.
3.	Andersson, Elin, et al. (författare) Quantification of chondroitin sulfates and hyaluronan in synovial fluid using high performance liquid chromatography 2022 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 30:Suppl 1, s. 106-106 Konferensbidrag (refereegranskat)abstract Purpose: Extracellular proteins such as aggrecan may be primed with specific glycan-patterns which result in their degradation, and hence may play a role in the pathogenesis of osteoarthritis (OA). To be able to use glycans as molecular biomarkers, the method of analysis of these molecules needs to be validated. The primary aim of this study was to validate quantitative high performance liquid chromatography (HPLC) of chondroitin sulfate (CS) and hyaluronan (HA) in synovial fluid samples. The secondary aim was to examine the glycan-pattern in different subject groups, and the correlation between age and the concentration of specific glycans.Methods: OA (n=25, age=36-86 years, 40% women) and recent knee injury patients (0-5 days from injury; n=13, age=36-64 years, 46% women) were selected from a cross-sectional convenience cohort. Individual synovial fluid samples, a synovial fluid pool (SF-control; n=7) and a CS quality control sample (CS-QC; Sigma #C2905) were digested with chondroitinase ABC overnight. Samples and glycan standards (CS [n=8] and HA [n=1] standards from Iduron) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative HPLC assay. Aggrecan from synovial fluid samples (n=6) was purified using density centrifugation (D1 mini-prep). Sulfated glycosaminoglycans (sGAG) were quantified using Alcian blue precipitation. Since the CS, HA and sGAG data were not normally distributed, non-parametric analyses for group comparisons were done. P-values less than 0.05 were considered statistically significant.Results: Synovial fluid samples were digested with varying concentrations of chondroitinase ABC and analyzed with the HPLC assay; 5 mU chondroitinase ABC per μg sGAG gave the highest CS- and HA-signals and were chosen for the rest of the study (data not shown).The CS profiles in synovial fluid and on aggrecan purified from corresponding synovial fluids were assessed from six knee injury patients. Of the six CS-markers that were detected, uronic acid (UA)-N-acetylgalactosamine (GalNAc) was only present in aggrecan samples, while UA2S-GalNAc and UA2S-GalNAc6S were found only in the synovial fluids. Similar proportions of UA-GalNAc4S and UA-GalNAc6S were found in synovial fluids and aggrecan samples (Figure 1), and these CS-glycans accounted for 95% of all glycans in the SF-control sample (Table 1).The technical performance of CS- and HA-markers using HPLC-assay were evaluated (Table 1). Of the nine markers, five were present in the majority of the synovial fluid samples (N=20-38) and included in the investigation of the technical performance. The mean intra coefficient of variation (CV) for the synovial fluid samples was between 1.2 and 12.9%. For the SF-control sample, the mean intra CV was 3.3-12.1% and the inter CV was 11.0-18.5%. For the CS-QC sample, the mean intra CV was 2.5-9.5% and the mean inter CV was 3.3-31.7%. For the glycan standards, the mean intra CV was 0.2-7.0%. With dilution of the SF-control sample up to 1:10, the dilution recovery rate for the five CS- and HA-markers was mainly between 75 and 125%.The synovial fluid concentration of biomarkers UA-GalNAc6S and UA2s-GalNAc6s and sGAG were approximately 2 to 3 times higher for the recent injury group compared to the age-matched OA group, while the HA levels were 3.7 times lower for the recent injury group (data not shown). No difference in biomarker concentrations were found between the sexes in any of the patient groups (data not shown). For correlation assessments, the two patient groups were merged (total N=38, assessment N = between 20 and 38). Synovial fluid concentrations of HA and UA-GalNAc4S,6S correlated positively with age (rS=0.420 and 0.532, respectively) while UA-GalNAc6S and sGAG correlated negatively with age (rS=-0.333 and -0.528, respectively). HA correlated negatively with UA-GalNAc6S (rS=-0.462) and sGAG (rS=-0.472) and positively with UA-GalNAc4S,6S (rS=0.868).Conclusions: The technical performance of the HPLC-assay indicates that the method is suitable for analyzing CS and HA markers in synovial fluid samples. Our results suggest that: the vast majority of CS in synovial fluid derives from aggrecan, the glycan pattern differs between OA and knee injured subjects and that the concentrations of some of the CS-markers seem to be associated with HA and age.
4.	Andersson, Elin, et al. (författare) Quantification Of Glycosaminoglycans In Knee Synovial Fluid From Different Patient Groups And Knee-Healthy Subjects Using High Performance Liquid Chromatography 2023 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 31:Suppl 1, s. 109-109 Konferensbidrag (refereegranskat)abstract Purpose: The glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are important for the normal function of articular cartilage, and changes of their sulfation and concentration may play a role in the pathogenesis of osteoarthritis (OA). Therefore, these glycans may be clinically useful as biomarkers in OA management. The purpose of this study was to analyze the CS and HA pattern in knee synovial fluid from different subject groups.Methods: OA patients (n=20, age=34-75 years, 45% women), recently knee-injured patients (0-77 days from injury, n=46, age 15-64 years, 15% women), previously knee-injured patients (88 days-21 years from injury, n=30, age 25-65 years, 17% women) and knee-healthy subjects (n=22, age 17-48 years, 23% women) were selected from a cross-sectional convenience cohort. CS and HA in individual synovial fluid samples, a synovial fluid quality control sample (SF-QC; a pool of synovial fluids) and a CS quality control sample (CS-QC) were digested with chondroitinase ABC and glucose oxidase overnight. Samples and glycan standards (CS [n=6] and HA [n=1] standards) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative high performance liquid chromatography (HPLC) assay. In total, 118 synovial fluid samples were run, whereof 34 in duplicates. SF-QC, CS-QC and standards were run in duplicates.Since the CS and HA data were not normally distributed, non-parametric analyses for group comparisons were done (Student’s T-test for age analysis, Chi-square test for sex analysis and Mann-Whitney U test for CS and HA analysis). The significance level was set at pResults: HPLC assay validation: The intra experiment coefficient of variation (CV) for the synovial fluid samples (n=36; including SF-QC) was 0.03-36.9% (median 5.4%) for CS and 0.4-44.9% (median 7.6%) for HA; intra CV for the CS-QC sample was 0.01-3.7%, and for the standards it was 0.2-6.7% for CS and 1.9-7.1% for HA. The inter experiment CV for SF-QC (n=5 experiments) was 9.8-17.5% for CS and 15.1% for HA; the inter CV for CS-QC (n=4 experiments) was 3.4-6.1%, and for the standards (n=5 experiments) it was 0.01-0.07% for CS and 0.1% for HA. Glucose oxidase was added to remove glucose that otherwise co-elutes with non-sulfated CS; it did not affect the CS and HA standards (data not shown). Group comparisons: Comparisons were made between the knee-healthy control group and the OA, recent injury and previous injury groups respectively, as well as between the recent injury and previous injury groups. There was no difference in sex between either of the groups (p=0.074-0.866). There was no difference in age between the knee-healthy group and the recent injury group (p=0.330), but the knee-healthy group was younger than the previous injury group and the OA group (pConclusions: Our data suggests that the groups with knee pathologies have higher concentrations of some CS variants and HA than the knee-healthy group. We also see a trend of higher levels of CS variants in the recent knee injury group compared to the previous knee injury group. This indicates that there is both an acute and chronic increase in the concentrations of CS variants and HA in synovial fluid following knee injury and/or cartilage damage.
5.	Angelini, Federico, et al. (författare) Osteoarthritis endotype discovery via clustering of biochemical marker data 2022 Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 81:5, s. 666-675 Tidskriftsartikel (refereegranskat)abstract Objectives Osteoarthritis (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. Method Data 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. Results Three 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. Conclusions This 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 number NCT03883568.
6.	Björklund, Maria, et al. (författare) Proteolysis Of Human Aggrecan With Mmp-3 2010 Ingår i: Osteoarthritis and Cartilage. - 1063-4584. ; 18, s. 61-62 Konferensbidrag (refereegranskat)
7.	Black, Rebecca Mae, et al. (författare) Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model 2020 Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 2:4 Tidskriftsartikel (refereegranskat)abstract 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.
8.	Black, Rebecca Mae, et al. (författare) Proteomic clustering reveals the kinetics of disease biomarkers in bovine and human models of post-traumatic osteoarthritis 2021 Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 3:4 Tidskriftsartikel (refereegranskat)abstract Objectives: In this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome. Design: Mass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein. Results: Our proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins. Conclusions: We demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis.
9.	Bricca, Alessio, et al. (författare) Impact of exercise therapy on molecular biomarkers related to cartilage and inflammation in people at risk of, or with established, knee osteoarthritis : a systematic review and meta-analysis of randomized controlled trials 2019 Ingår i: Arthritis care and research : the official journal of the Arthritis Health Professions Association. - : Wiley. - 2151-4658. ; 71:11, s. 1504-1515 Tidskriftsartikel (refereegranskat)abstract 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.
10.	Cronström, Anna, et al. (författare) Is good muscle function a protective factor for early signs of knee osteoarthritis after anterior cruciate ligament reconstruction? The SHIELD cohort study protocol 2020 Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier. - 2665-9131. ; 2:4 Tidskriftsartikel (refereegranskat)abstract 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 Skåne 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.
11.	Dwivedi, Garima, et al. (författare) Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system 2022 Ingår i: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6354 .- 1478-6362. ; 24:1 Tidskriftsartikel (refereegranskat)abstract Background: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. Methods: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin’s grade 0–2, 23–83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. Results: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. Conclusion: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.
12.	Einarsson, Emma, et al. (författare) The role of cartilage glycosaminoglycan structure in gagCEST 2020 Ingår i: NMR in Biomedicine. - : Wiley. - 0952-3480 .- 1099-1492. ; 33:5 Tidskriftsartikel (refereegranskat)abstract 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.
13.	Fredlund, Kenneth M., et al. (författare) Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes 1994 Ingår i: Plant Physiology. - 0032-0889 .- 1532-2548. ; 106:3, s. 1103-1106 Tidskriftsartikel (refereegranskat)abstract The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.
14.	Hagemans, Frans J.A., et al. (författare) An Anterior Cruciate Ligament Rupture Increases Levels of Urine N-terminal Cross-linked Telopeptide of Type I Collagen, Urine C-terminal Cross-linked Telopeptide of Type II Collagen, Serum Aggrecan ARGS Neoepitope, and Serum Tumor Necrosis Factor–α 2021 Ingår i: American Journal of Sports Medicine. - : SAGE Publications. - 0363-5465 .- 1552-3365. ; 49:13, s. 3534-3543 Tidskriftsartikel (refereegranskat)abstract 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.
15.	Hannani, Monica T., et al. (författare) From biochemical markers to molecular endotypes of osteoarthritis : a review on validated biomarkers 2024 Ingår i: Expert Review of Molecular Diagnostics. - 1473-7159. ; 24:1-2, s. 23-38 Forskningsöversikt (refereegranskat)abstract Introduction: Osteoarthritis (OA) affects over 500 million people worldwide. OA patients are symptomatically treated, and current therapies exhibit marginal efficacy and frequently carry safety-risks associated with chronic use. No disease-modifying therapies have been approved to date leaving surgical joint replacement as a last resort. To enable effective patient care and successful drug development there is an urgent need to uncover the pathobiological drivers of OA and how these translate into disease endotypes. Endotypes provide a more precise and mechanistic definition of disease subgroups than observable phenotypes, and a panel of tissue- and pathology-specific biochemical markers may uncover treatable endotypes of OA. Areas covered: We have searched PubMed for full-text articles written in English to provide an in-depth narrative review of a panel of validated biochemical markers utilized for endotyping of OA and their association to key OA pathologies. Expert opinion: As utilized in IMI-APPROACH and validated in OAI-FNIH, a panel of biochemical markers may uncover disease subgroups and facilitate the enrichment of treatable molecular endotypes for recruitment in therapeutic clinical trials. Understanding the link between biochemical markers and patient-reported outcomes and treatable endotypes that may respond to given therapies will pave the way for new drug development in OA.
16.	Holm, Patur M., et al. (författare) The Effects of Different Management Strategies or Rehabilitation Approaches on Knee Joint Structural and Molecular Biomarkers Following Traumatic Knee Injury : A Systematic Review of Randomized Controlled Trials for the OPTIKNEE Consensus 2023 Ingår i: Journal of Orthopaedic and Sports Physical Therapy. - : Journal of Orthopaedic & Sports Physical Therapy (JOSPT). - 0190-6011 .- 1938-1344. ; 53:4, s. 172-193 Forskningsöversikt (refereegranskat)abstract OBJECTIVE: To summarize the effectiveness of management strategies and rehabilitation approaches for knee joint structural and molecular biomarker outcomes following anterior cruciate ligament (ACL) and/or meniscal tear. DESIGN: Intervention systematic review. LITERATURE SEARCH: We searched the MEDLINE, Embase, CINAHL, CENTRAL, and SPORTDiscus databases from their inception up to November 3, 2021. STUDY SELECTION CRITERIA: We included randomized controlled trials (RCTs) investigating the effectiveness of management strategies or rehabilitation approaches for structural/molecular biomarkers of knee joint health following ACL and/or meniscal tear. DATA SYNTHESIS: We included 5 RCTs (9 papers) with primary ACL tear (n = 365). Two RCTs compared initial management strategies (rehabilitation plus early vs optional delayed ACL surgery), reporting on structural biomarkers (radiographic osteoarthritis, cartilage thickness, meniscal damage) in 5 papers and molecular biomarkers (inflammation, cartilage turnover) in 1 paper. Three RCTs compared different post-ACL reconstruction (ACLR) rehabilitation approaches (high vs low intensity plyometric exercises, accelerated vs nonaccelerated rehabilitation, continuous passive vs active motion), reporting on structural biomarkers (joint space narrowing) in 1 paper and molecular biomarkers (inflammation, cartilage turnover) in 2 papers. RESULTS: There were no differences in structural or molecular biomarkers between post-ACLR rehabilitation approaches. One RCT comparing initial management strategies demonstrated that rehabilitation plus early ACLR was associated with greater patellofemoral cartilage thinning, elevated inflammatory cytokine response, and reduced incidence of medial meniscal damage over 5 years compared to rehabilitation with no/delayed ACLR. CONCLUSION: Very low-certainty evidence suggests that different initial management strategies (rehabilitation plus early vs optional delayed ACL surgery) but not postoperative rehabilitation approaches may influence the incidence of meniscal damage, patellofemoral cartilage loss and cytokine concentrations over 5 years post-ACL tear.
17.	Huang, Shan, et al. (författare) Cathepsin g Degrades Both Glycosylated and Unglycosylated Regions of Lubricin, a Synovial Mucin 2020 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1 Tidskriftsartikel (refereegranskat)abstract 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. © 2020, The Author(s).
18.	Huang, Shan, et al. (författare) Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1α : Interplay between O-linked glycosylation and inflammatory cytokines 2022 Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9 Tidskriftsartikel (refereegranskat)abstract The primary aim of the study was to identify inflammatory markers relevant for osteoarthritis (OA)-related systemic (plasma) and local (synovial fluid, SF) inflammation. From this, we looked for inflammatory markers that coincided with the increased amount of O-linked Tn antigen (GalNAcα1-Ser/Thr) glycan on SF lubricin. Inflammatory markers in plasma and SF in OA patients and controls were measured using a 44-multiplex immunoassay. We found consistently 29 markers detected in both plasma and SF. The difference in their concentration and the low correlation when comparing SF and plasma suggests an independent inflammatory environment in the two biofluids. Only plasma MCP-4 and TARC increased in our patient cohort compared to control plasma. To address the second task, we concluded that plasma markers were irrelevant for a direct connection with SF glycosylation. Hence, we correlated the SF-inflammatory marker concentrations with the level of altered glycosylation of SF-lubricin. We found that the level of SF-IL-8 and SF-MIP-1α and SF-VEGFA in OA patients displayed a positive correlation with the altered lubricin glycosylation. Furthermore, when exposing fibroblast-like synoviocytes from both controls and OA patients to glycovariants of recombinant lubricin, the secretion of IL-8 and MIP-1α and VEGFA were elevated using lubricin with Tn antigens, while lubricin with sialylated and nonsialylated T antigens had less or no measurable effect. These data suggest that truncated glycans of lubricin, as found in OA, promote synovial proinflammatory cytokine production and exacerbate local synovial inflammation.
19.	Kumahashi, Nobuyuki, et al. (författare) Type II collagen C2C epitope in human synovial fluid and serum after knee injury - associations with molecular and structural markers of injury. 2015 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 23:9, s. 1506-1512 Tidskriftsartikel (refereegranskat)abstract Investigate in a cross-sectional study time-dependent changes of synovial fluid type II collagen epitope C2C concentrations after knee injury and correlate to other joint injury biomarkers.
20.	Larsson, Staffan, et al. (författare) An ARGS-aggrecan assay for analysis in blood and synovial fluid. 2014 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 22:2, s. 242-249 Tidskriftsartikel (refereegranskat)abstract To validate a modified ligand-binding assay for the detection of aggrecanase generated aggrecan fragments with the ARGS neoepitope in synovial fluid (SF) and blood, and to verify the identity of aggrecan fragments found in blood.
21.	Larsson, Staffan, et al. (författare) Association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression in knee osteoarthritis. 2010 Ingår i: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 12:6 Tidskriftsartikel (refereegranskat)abstract ABSTRACT: INTRODUCTION: Aggrecanase cleavage at the 392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal 393ARGS fragments, is an early key event in arthritis and joint injuries. We determined whether synovial fluid (SF) levels of ARGS-aggrecan distinguish subjects with progressive radiographic knee osteoarthritis (ROA) from those with stable or no ROA. METHODS: We studied 141 subjects who, at examination A, had been given meniscectomies an average of 18 years earlier (range, 15 to 22 years). Seventeen individuals without surgery, and without known injury to the menisci or cruciate ligaments, were used as references. At examinations A and B, with a mean follow-up time of 7.5 years, we obtained SF and standing tibiofemoral and skyline patellofemoral radiographs. SF ARGS-aggrecan was measured with an electrochemiluminescence immunoassay, and we graded radiographs according to the OARSI atlas. The association between SF ARGS levels at examination A and progression of radiographic features of knee OA between examinations A and B was assessed by using logistic regression adjusted for age, gender, body mass index, and time between examinations, and stratified by ROA status at examination A. RESULTS: We found a weak negative association between SF ARGS concentrations and loss of joint space: the likelihood of progression of radiographic joint space narrowing decreased 0.9 times per picomole per milliliter increase in ARGS (odds ratio (OR) 0.89; 95% confidence interval (CI), 0.79 to 0.996). In subjects with and without preexisting ROA at examination A, the association was OR, 0.96; 0.81 to 1.13; and 0.77; 0.62 to 0.95, respectively. Average levels of SF ARGS 18 years after meniscectomy were no different from those of reference subjects and were not correlated to radiographic status at examination A. CONCLUSIONS: In subjects with previous knee meniscectomy but without ROA, levels of SF ARGS-aggrecan were weakly and inversely associated with increased loss of joint space over a period of 7.5 years.
22.	Larsson, Staffan, et al. (författare) Biological variation of human aggrecan ARGS neoepitope in synovial fluid and serum in early-stage knee osteoarthritis and after knee injury 2022 Ingår i: Osteoarthritis and Cartilage Open. - : Elsevier BV. - 2665-9131. ; 4:4, s. 1-7 Tidskriftsartikel (refereegranskat)abstract OBJECTIVE: To determine biological variation of the aggrecanase-generated aggrecan ARGS neoepitope in serum (sARGS) and synovial fluid (sfARGS) within and between patients with osteoarthritis (OA) or anterior cruciate ligament (ACL) injury.DESIGN: Matched samples of serum and synovial fluid were available, as parts of clinical trials, from i) 16 subjects with early-stage OA on 8 occasions over 1 year, and ii) 120 subjects with acute ACL injury with samples available from at least 2 of 6 visits over 5 years. We used an in-house immunoassay to quantify ARGS and one-way ANOVA for statistical analyses.RESULTS: Variability in ARGS was higher in synovial fluid than in serum in both patient groups. Subjects with OA had the lowest variability both within and between patients and showed no variation over time in the degree of variability or in the cross-sectional mean, neither in serum nor in synovial fluid. After ACL injury, the concentration and the variability of ARGS was highest immediately after injury, with a subsequent decline both in concentration and in variability with time. In both patient groups there was a positive correlation between sfARGS and sARGS both within and between individuals (correlation coefficients between 0.16 and 0.20).CONCLUSIONS: The biological variation of ARGS is lower in serum than in synovial fluid, and lower in OA than after ACL injury. Serum ARGS is a measure of the total release of ARGS aggrecan from the whole body and a poor reflection of the release of ARGS aggrecan within the affected joint.
23.	Larsson, Staffan, et al. (författare) Interleukin-6 and tumor necrosis factor alpha in synovial fluid are associated with progression of radiographic knee osteoarthritis in subjects with previous meniscectomy. 2015 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 23:11, s. 1906-1914 Tidskriftsartikel (refereegranskat)abstract To explore potential associations between proinflammatory cytokines in synovial fluid and progression of osteoarthritis (OA) in meniscectomized subjects.
24.	Larsson, Staffan, et al. (författare) Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study 2009 Ingår i: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 11:3 Tidskriftsartikel (refereegranskat)abstract Introduction Aggrecanase cleavage at the (392)Glu-(393)Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal (393)ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays. Methods Synovial fluid (SF) from 26 people with healthy knees (reference) and 269 patients were analyzed in a cross-sectional study. Patient groups were acute inflammatory arthritis, acute knee injury, chronic knee injury and knee osteoarthritis (OA). Aggrecan ARGS fragments were assayed by ELISA using the monoclonal antibody OA-1. Total aggrecan content was analyzed by an ELISA using the monoclonal antibody 1-F21, and sulfated glycosaminoglycan by Alcian blue precipitation. Results Aggrecan ARGS fragment concentrations in all groups differed from the reference group (P < 0.001). The acute inflammatory arthritis group had the highest median level, 177-fold greater than that of the reference group. Median levels (in pmol ARGS/ml SF) were: reference 0.5, acute inflammatory arthritis 88.5, acute knee injury 53.9, chronic knee injury 0.5 and OA 4.6. In contrast, aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference. Conclusions Levels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the (392)Glu-(393)Ala bond in the IGD by aggrecanase. An assay that specifically quantified these fragments better distinguished samples from joints with pathology than assays monitoring aggrecan or glycosaminoglycan concentrations. The newly developed ARGS fragment assay can be used to monitor aggrecanase activity in human joint disease and experimental models.
25.	Larsson, Staffan, et al. (författare) The association between changes in synovial fluid levels of ARGS-aggrecan fragments, progression of radiographic osteoarthritis and self-reported outcomes: a cohort study. 2012 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 20:5, s. 388-395 Tidskriftsartikel (refereegranskat)abstract OBJECTIVE: To investigate whether change in concentrations over time of aggrecanase generated ARGS-aggrecan in synovial fluid (SF ARGS) associates with progression of radiographic knee Osteoarthritis (OA) and patient-reported outcome in subjects with previous meniscectomy. METHODS: We studied 141 subjects at two time points after meniscectomy. Time point A was on average 18years after meniscectomy, time point B was on average 7.5years later; 74 subjects had SF available from both examinations. We measured SF ARGS by an electrochemiluminescence immunoassay, graded radiographic features of tibiofemoral or patellofemoral OA according to the Osteoarthritis Research Society International (OARSI) atlas, and scored patient-reported outcomes using the Knee Injury and Osteoarthritis Outcome Score (KOOS). Using logistic regression (adjusted for age, gender, body mass index, time between examinations, and SF ARGS at first examination) we assessed associations between change in SF ARGS between first and second examinations and progression of radiographic OA and KOOS. RESULTS: In subjects with decreasing SF ARGS between examinations, the likelihood of loss of joint space and worsening of KOOS pain between examinations was increased 6- and 4-fold respectively compared to those increasing in SF ARGS (OR 5.72; 95% CI 1.53-21.4 and 3.66; 1.01-13.2, respectively). No significant associations were seen between decreasing SF ARGS and progression of osteophytes (OR 0.88; 0.28-2.78), or for patient-reported outcomes other than KOOS pain. CONCLUSION: Having decreasing levels of SF ARGS over time was associated with an increased risk of loss of joint space and pain worsening, but showed no association with other patient-reported outcomes or osteophyte progression.
26.	Larsson, Staffan, et al. (författare) The association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression of knee osteoarthritis 2010 Ingår i: Osteoarthritis and Cartilage. - 1063-4584. ; 18:Supplement 2, s. 59-60 Konferensbidrag (övrigt vetenskapligt/konstnärligt)
27.	Larsson, Staffan, et al. (författare) α1-microglobulin protects against bleeding-induced oxidative damage in knee arthropathies 2018 Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 9:NOV Tidskriftsartikel (refereegranskat)abstract 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.
28.	Liu, Yang, et al. (författare) Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing 2023 Ingår i: Acta Biomaterialia. - : Elsevier BV. - 1878-7568 .- 1742-7061. ; 162, s. 164-181 Tidskriftsartikel (refereegranskat)abstract Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.
29.	Lohmander, Stefan, et al. (författare) Use of the plasma stromelysin (matrix metalloproteinase 3) concentration to predict joint space narrowing in knee osteoarthritis. 2005 Ingår i: Arthritis and Rheumatism. - : Wiley. - 1529-0131 .- 0004-3591. ; 52:10, s. 3160-3167 Tidskriftsartikel (refereegranskat)
30.	Neuman, Paul, et al. (författare) Higher aggrecan 1-F21 epitope concentration in synovial fluid early after anterior cruciate ligament injury is associated with worse knee cartilage quality assessed by gadolinium enhanced magnetic resonance imaging 20 years later 2020 Ingår i: BMC Musculoskeletal Disorders. - : Springer Science and Business Media LLC. - 1471-2474. ; 21:1 Tidskriftsartikel (refereegranskat)abstract Background: To investigate if cartilage related biomarkers in synovial fluid are associated with knee cartilage status 20 years after an anterior cruciate ligament (ACL) injury. Methods: We studied 25 patients with a complete ACL rupture without subsequent ACL reconstruction or radiographic knee OA. All had a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) 20 years after the ACL injury, using the T1 transverse relaxation time in the presence of gadolinium (T1Gd) which estimates the concentration of glycosaminoglycans in hyaline cartilage. Synovial fluid samples were aspirated acutely (between 0 and 18 days) and during 1 to 5 follow up visits between 0.5 and 7.5 years after injury. We quantified synovial fluid concentrations of aggrecan (epitopes 1-F21 and ARGS), cartilage oligomeric matrix protein, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by immunoassays, and sulfated glycosaminoglycans by Alcian blue precipitation. Western blot was used for qualitative analyses of aggrecan fragments in synovial fluid and cartilage samples. Results: Western blot indicated that the 1-F21 epitope was located within the chondroitin sulfate 2 region of aggrecan. Linear regression analyses (adjusted for age, sex, body mass index and time between injury and sampling) showed that acute higher synovial fluid 1-F21-aggrecan concentrations were associated with shorter T1Gd values 20 years after injury, i.e. inferior cartilage quality (standardized effects between − 0.67 and − 1.0). No other statistically significant association was found between molecular biomarkers and T1Gd values. Conclusion: Higher acute synovial fluid 1-F21-aggrecan concentrations in ACL injured patients, who managed to cope without ACL reconstruction and were without radiographic knee OA, were associated with inferior knee cartilage quality assessed by dGEMRIC 20 years after injury.
31.	Pratta, M. A., et al. (författare) Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments 2006 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:7, s. 702-713 Tidskriftsartikel (refereegranskat)abstract Objective: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the (373)Glu-(374)Ala bond within the aggrecan interglobular domain. Methods: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. Results: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. Conclusion: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
32.	Roemer, Frank W., et al. (författare) Molecular and Structural Biomarkers of Inflammation at Two Years After Acute Anterior Cruciate Ligament Injury Do Not Predict Structural Knee Osteoarthritis at Five Years 2019 Ingår i: Arthritis and Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 71:2, s. 238-243 Tidskriftsartikel (refereegranskat)abstract 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.
33.	Roemer, Frank W, et al. (författare) Reply to letter by Deng et al. "Could the levels of inflammation biomarkers predict OA? : Comment on the article by Roemer FW et al." 2019 Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 71:9, s. 1588-1588 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract 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.
34.	Stattin, Eva-Lena, et al. (författare) A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans 2010 Ingår i: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297 .- 1537-6605. ; 86:2, s. 126-137 Tidskriftsartikel (refereegranskat)abstract Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.
35.	Stattin, Eva-Lena, 1960-, et al. (författare) A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans with short stature and early osteoarthritis Annan publikation (övrigt vetenskapligt/konstnärligt)
36.	Stattin, Eva-Lena, et al. (författare) Novel missense ACAN gene variants linked to familial osteochondritis dissecans cluster in the C-terminal globular domain of aggrecan 2022 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12, s. 1-13 Tidskriftsartikel (refereegranskat)abstract The cartilage aggrecan proteoglycan is crucial for both skeletal growth and articular cartilage function. A number of aggrecan (ACAN) gene variants have been linked to skeletal disorders, ranging from short stature to severe chondrodyplasias. Osteochondritis dissecans is a disorder where articular cartilage and subchondral bone fragments come loose from the articular surface. We previously reported a missense ACAN variant linked to familial osteochondritis dissecans, with short stature and early onset osteoarthritis, and now describe three novel ACAN gene variants from additional families with this disorder. Like the previously described variant, these are autosomal dominant missense variants, resulting in single amino acid residue substitutions in the C-type lectin repeat of the aggrecan G3 domain. Functional studies showed that neither recombinant variant proteins, nor full-length variant aggrecan proteoglycan from heterozygous patient cartilage, were secreted to the same level as wild-type aggrecan. The variant proteins also showed decreased binding to known cartilage extracellular matrix ligands. Mapping these and other ACAN variants linked to hereditary skeletal disorders showed a clustering of osteochondritis dissecans-linked variants to the G3 domain. Taken together, this supports a link between missense ACAN variants affecting the aggrecan G3 domain and hereditary osteochondritis dissecans.
37.	Struglics, André, et al. (författare) A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid. 2010 Ingår i: Matrix Biology. - : Elsevier BV. - 1569-1802 .- 0945-053X. ; 29, s. 74-83 Tidskriftsartikel (refereegranskat)abstract In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented. Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative-dissociative cesium chloride density gradient centrifugation (A1D1-D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions. The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800-1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample. This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.
38.	Struglics, André, et al. (författare) Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C-terminus. 2012 Ingår i: Arthritis and Rheumatism. - : Wiley. - 1529-0131 .- 0004-3591. Tidskriftsartikel (refereegranskat)abstract OBJECTIVE: To understand aggrecan degradation in juvenile idiopathic arthritis (JIA), the pattern and abundance of aggrecan fragments in synovial fluid aspirates from JIA patients were analysed and compared with aggrecan fragments in synovial fluids from patients with other arthritides, juvenile knee injury and a knee-healthy reference group. METHODS: The concentration of sulphated glycosaminoglycans in synovial fluid was measured by the Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density gradient centrifugation, deglycosylated and analysed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE and KEEE neoepitopes, or the aggrecan G3-domain. RESULTS: The concentration of sulphated glycosaminoglycans in JIA synovial fluids was significantly lower compared with the levels in fluids from OA (P<0.001), juvenile knee injury (P=0.006) and knee-healthy reference (P=0.022) groups. Western blot analysis detected KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulphate-rich region of JIA aggrecan. The pattern of JIA aggrecan fragments was not identical to that in synovial fluids pooled from OA patients, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in the JIA synovial fluids, in marked contrast to the levels of ARGS fragments in OA synovial fluids. CONCLUSIONS: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in aggrecan's chondroitin-sulphate rich region. The results suggest that unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event in JIA. © 2012 American College of Rheumatology.
39.	Struglics, André (författare) Biomarkers have to make sense 2024 Ingår i: Osteoarthritis and Cartilage. - 1063-4584. ; 32:3, s. 232-233 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
40.	Struglics, André, et al. (författare) Calpain is involved in C-terminal truncation of human aggrecan 2010 Ingår i: Biochemical Journal. - 0264-6021. ; 18, s. 10-10 Tidskriftsartikel (refereegranskat)abstract Mature aggrecan is generally C-terminally truncated at several sites in the CS (chondroitin sulfate) region. Aggrecanases and MMPs (matrix metalloproteinases) have been suggested to be responsible for this digestion. To identify whether calpain, a common intracellular protease, has a specific role in the proteolysis of aggrecan we developed neoepitope antibodies (anti-PGVA, anti-GDLS and anti-EDLS) against calpain cleavage sites and used Western blot analysis to identify alpain-generated fragments in normal and OA (osteoarthritis) knee cartilage and SF (synovial fluid) samples. Our results showed that human aggrecan contains six calpain cleavage sites: one in the IGD (interglobular domain), one in the KS (keratan sulfate) region, two in the CS1 and two in the CS2 region. Kinetic studies of calpain proteolysis against aggrecan showed that the aggrecan molecule was cleaved in a specific order where cuts in CS1 was the most preferred and cuts in KS region was the second most preferred cleavage. OA and normal cartilage contained low amounts of a calpain-generated G1-PGVA fragment (0.5-2%) compared with aggrecanase-generated G1-TEGE (71-76%) and MMP-generated G1-IPEN (23-29%) fragments. Significant amounts of calpain-generated GDLS and EDLS fragments were found in OA and normal cartilage, and a ARGS-EDLS fragment was detected in arthritic SF samples. The results of the present study indicate that calpains are involved in the C-terminal truncation of aggrecan and might have a minor role in arthritic diseases.
41.	Struglics, André, et al. (författare) Changes in synovial fluid and serum cytokines and ARGS-aggrecan, and urine CTX-II and NTX-I over five years after anterior cruciate ligament rupture: An exploratory analysis in the KANON trial. 2015 Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5205 .- 2326-5191. ; 67:7, s. 1816-1825 Tidskriftsartikel (refereegranskat)abstract Prospectively monitor levels of synovial fluid and serum pro-inflammatory cytokines and aggrecan ARGS neoepitope, and urine C-terminal type II (CTX-II) and N-terminal type I (NTX-I) collagen cross-linked telopeptides after acute anterior cruciate ligament (ACL) rupture.
42.	Struglics, André, et al. (författare) Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot. 2006 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:9, s. 898-905 Tidskriftsartikel (refereegranskat)abstract Objective: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Method. Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified. Results: The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a + g), or as K = 1.085 m/(a + g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels. Conclusions: With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
43.	Struglics, André, et al. (författare) Human aggrecanase generated synovial fluid fragment levels are elevated directly after knee injuries due to proteolysis both in the inter globular and chondroitin sulfate domains. 2011 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 19, s. 1047-1057 Tidskriftsartikel (refereegranskat)abstract OBJECTIVE: To examine different aggrecanase generated fragments in synovial fluid (SF) from patients with acute and chronic knee injuries and from knee healthy subjects. METHODS: We prepared SF-D1 samples from acute (n=35) and chronic (n=35) knee injury patients and knee healthy subjects (n=10). Aggrecan fragments were analyzed in the SF-D1 samples by quantitative (G1, ARGS, KEEE and G3 antibodies) and non-quantitative (GRGT and AGEG antibodies) Western blot. RESULTS: ARGS-SELE, ARGS-chondroitin sulfate (CS)1, GRGT-, GLGS- and AGEG-G3 fragments were the main ARGS and G3 fragments in injured and reference samples. In the acute injury samples the concentrations of these fragments were increased compared to the reference, and the level of the ARGS-SELE remained elevated for at least 2 years after the joint injury. Both SF ARGS fragments and aggrecanase generated G3 fragments had high sensitivity and specificity as biomarkers in distinguishing injured from healthy knee joints, although the ARGS fragments had higher area under the receiver operating characteristic curve (AUC) values for injuries (74-86%) than the G3 fragments (AUC values 63-68%). CONCLUSION: Our results suggest that during the acute phase after knee injury there is an increased aggrecanase activity against both the interglobular domain (IGD) and the CS2 cleavage sites of joint cartilage aggrecan. This increase in SF aggrecanolytic fragments is present for several years after the injury. SF ARGS fragments are better biomarkers than the aggrecanase generated G3-fragments in distinguishing injured from healthy knee joints.
44.	Struglics, André, et al. (författare) Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments. 2006 Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:2, s. 101-113 Tidskriftsartikel (refereegranskat)abstract Objective: To identify the major aggrecanase-and matrix metalloproteinase (MMP)-generated aggrecan fragments in human osteoarthritis (OA) synovial fluid and in human OA joint cartilage. Method Aggrecan fragments were prepared by CsCl gradient centrifugation. Fragment distributions were compared with aggrecanase-1 (ADAMTS-4) and MMP-3 digested human aggrecan by analysis with neoepitope antibodies and an anti-G1 domain antibody, using Western immuno-blots. Results: The overall fragment pattern of CA synovial fluid aggrecan was similar to the fragment pattern of cartilage aggrecan cleaved in vitro by ADAMTS-4. However, multiple glycosaminoglycan (GAG) containing aggrecanase and MMP-generated aggrecan fragments were identified in OA synovial fluid and some of these fragments were produced by the action of both types of proteinases. The synovial fluid content of large size aggrecan fragments with (374)ARGS- and (342)FFGV-N-terminals was about 107 and 40 pmoles per ml, respectively, out of a total concentration of aggrecan fragments of about 185 pmoles per ml. CA synovial fluid contained insignificant amounts of the G1-IPEN341 fragment as compared to the G1-TEGE(373) fragment, while CA cartilage contained significant amounts of both fragments. OA cartilage contained several GAG-containing aggrecan fragments with N-terminals of G1- or (342)FFGV- but no fragments with an N-terminal of (374)ARGS-. Conclusions: The overall pattern of aggrecan fragments in human CA synovial fluid and cartilage supports an important role for aggrecanase in aggrecan degradation. However, the fragment patterns and their differential distribution between cartilage and synovial fluid are consistent with the existence of at least two proteolytic pathways for aggrecan degradation in human OA, generating both (342)FFGV- and (374)ARGS-fragments. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
45.	Struglics, André, et al. (författare) Juvenile idiopathic arthritis patients have a distinct cartilage and bone biomarker profile that differs from healthy and knee-injured children 2020 Ingår i: Clinical and Experimental Rheumatology. - : Clinical and Experimental Rheumatology. - 0392-856X .- 1593-098X. ; 38:2, s. 355-365 Tidskriftsartikel (refereegranskat)abstract OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
46.	Struglics, André, et al. (författare) MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover 2012 Ingår i: Biochemical Journal. - 0264-6021. ; 446, s. 213-223 Tidskriftsartikel (refereegranskat)abstract Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS I region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.
47.	Struglics, André, et al. (författare) Phosphoproteins and protein kinase activities intrinsic to inner membranes of potato tuber mitochondria 1999 Ingår i: Plant and Cell Physiology. - 0032-0781. ; 40:12, s. 1271-1279 Tidskriftsartikel (refereegranskat)abstract Inside-out submitochondrial particles (IO-SMP) were isolated and purified from potato (Solanum tuberosum L. cv.) tubers. When these IO-SMP were incubated with [γ32P]ATP more then 20 proteins became labelled as a result of phosphorylation. The 32P incorporation was stimulated by the oxidizing reagent ferricyanide. Except for a 17 kDa protein which was phosphorylated only in the absence of divalent cations, the protein phosphorylation required Mg2+. The time for half-maximum 32P incorporation was 4 min for the 22 kDa phospho-F1 δ-subunit and 2 min for the 28 kDa phospho-F0 b-subunit of the proton-ATPase. The K(m) for ATP for the detected phosphoproteins was between 65 μM and 110 μM. The pH optimum for protein phosphorylation in inner membranes was between pH 6 and 8, and for the F1 δ-subunit and the F0 b-subunit the pH optima were 6.5-8 and pH 8, respectively. A 37 kDa phosphoprotein was phosphorylated on a histidine residue while the remainder of the inner membrane proteins were phosphorylated on serine or threonine residues. Two autophosphorylated putative kinases were identified: one at 16.5 kDa required divalent cations for autophosphorylation, while another at 30 kDa did not. A 110 kDa protein was labelled only with [α-32P]ATP, suggesting adenylylation.
48.	Struglics, André, et al. (författare) Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria 2000 Ingår i: FEBS Letters. - 0014-5793. ; 475:3, s. 213-217 Tidskriftsartikel (refereegranskat)abstract Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with [γ-32P]ATP. Proteins were separated by SDS-PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of [γ- 32P]ATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins. (C) 2000 Federation of European Biochemical Societies.
49.	Struglics, André (författare) Protein phosphorylation in plant mitochondria 1998 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Protein phosphorylation in the subcompartments of plant mitochondria was investigated by labelling with [gamma-32P]ATP and by SDS-PAGE/autoradiography. About 20 proteins in inside-out inner mitochondrial membranes from potato tubers were phosphorylated by endogenous protein kinases when incubated with [gamma-32P]ATP. None of the phosphorylated proteins were labelled when using [alpha-32P]ATP as a phosphate donor. The reversible protein phosphorylation in these membranes is mainly a result of the action of inner membrane serine/threonine protein kinases/phosphatases, although the dephosphorylation of inner membrane proteins appears to be catalysed by protein phosphatases located in the matrix. In addition, a 37 kDa protein contained acid-labile phosphate groups, indicating modification of histidine residues. The phosphorylation of inner membrane proteins requires divalent cations (i.e. Mg2+ or Mn2+) and is stimulated under oxidising conditions. Two autophosphorylated inner membrane proteins with molecular mass of 16.5 and 30 kDa are putative protein kinases. Two inner membrane phosphoproteins with molecular mass of 22 and 28 kDa were identified by N-terminal sequencing as the delta?-subunit and b-subunit, respectively, of the FoF1-ATPase. A 17 kDa phosphoprotein has some characteristics of a nucleoside diphosphate kinase. All other plant inner mitochondrial membrane phosphoproteins remain to be identified. A 39.5 and 41 kDa protein are the most heavily labelled phosphoproteins in the matrix of potato tuber mitochondria. The 41 kDa protein is probably the alpha-subunit of pyruvate dehydrogenase, and the 39.5 kDa protein, which was also labelled with 32Pi, may be the alpha-subunit of succinyl-CoA synthetase. In pea leaf mitochondria, a 17.4 kDa protein, which is probably located in the intermembrane space, was purified and identified as a nucleoside diphosphate kinase. This pea protein is autophosphorylated on both the catalytic histidine and on serine residues. The labelling on serine residues was higher then for the histidine, and kinetic studies demonstrated a faster rate of phosphorylation of serine compared to histidine.
50.	Struglics, André, et al. (författare) Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum 1999 Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 262:3, s. 765-773 Tidskriftsartikel (refereegranskat)abstract For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [γ-32]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino- acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali- stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.

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Lärosäte: Lunds universitet (65); Göteborgs universitet (4); Karolinska Institutet (4); Umeå universitet (3); Uppsala universitet (2); Luleå tekniska universitet (1); visa fler...; Jönköping University (1); visa färre...

Språk: Engelska (67)

Forskningsämne (UKÄ/SCB): Medicin och hälsovetenskap (56); Naturvetenskap (8)

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