| 1. |
- Larsson, Staffan, et al.
(author)
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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
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In: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 11:3
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Journal article (peer-reviewed)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.
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| 2. |
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| 3. |
- Pratta, M. A., et al.
(author)
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Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments
- 2006
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:7, s. 702-713
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Journal article (peer-reviewed)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.
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| 4. |
- Struglics, André, et al.
(author)
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Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.
- 2006
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:9, s. 898-905
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Journal article (peer-reviewed)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.
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| 5. |
- Struglics, André, et al.
(author)
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Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments.
- 2006
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 14:2, s. 101-113
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Journal article (peer-reviewed)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.
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| 6. |
- Struglics, André, et al.
(author)
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Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases.
- 2009
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 17, s. 497-506
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Journal article (peer-reviewed)abstract
- OBJECTIVE: To develop a Western blot method for quantification of multiple aggrecan fragments in human synovial fluids (SFs). METHOD: SF aggrecan fragments were prepared from knee healthy (reference), knee injury and arthritis subjects by CsCl gradient centrifugations collecting D1 fractions. Samples were analyzed by Western blot, using antibodies against the N-terminal epitope ARGS and the G3 domain, and fragments were quantified using a digital luminescence image analyzer. RESULTS: The method had a coefficients of variation of 10-30%, and a high correlation (r(S)=0.86) with a corresponding enzyme-linked immunosorbent assay (ELISA). The SFs from reference, knee injured and arthritic subjects contained two major ARGS fragments, ARGS-SELE and ARGS-CS1, and three major G3 fragments (GRGT-G3, GLGS-G3 and AGEG-G3). Compared to the reference, the acute arthritis and acute joint injury groups had a 30-fold elevated concentration of ARGS fragments, and both groups had a higher proportion of the aggrecan in joint fluid as ARGS fragments compared to the other groups. The reference and chronic injury groups had an excess of ARGS-CS1 fragments over ARGS-SELE fragments, while subjects with acute arthritis or osteoarthritis had a more even distribution between these fragments. CONCLUSIONS: We have developed a novel Western blot quantification method for quantification of SF aggrecan fragments which can differentiate fragments of different sizes sharing the same epitope. The anti-ARGS and anti-G3 quantitative Western blots provided information important for a better understanding of the proteolytic pathways in aggrecan breakdown, information that discriminates between different joint diseases, and may aid in identification of new biomarkers.
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