Skip to main content
Top
Published in: Rheumatology International 5/2018

01-05-2018 | Biomarkers

Cartilage oligomeric protein, matrix metalloproteinase-3, and Coll2-1 as serum biomarkers in knee osteoarthritis: a cross-sectional study

Authors: Tsvetoslav Georgiev, Mariana Ivanova, Aleksandar Kopchev, Tsvetelina Velikova, Asen Miloshov, Ekaterina Kurteva, Kalina Yuzeir, Marin Penkov, Plamena Kabakchieva, Rasho Rashkov, Rumen Stoilov

Published in: Rheumatology International | Issue 5/2018

Login to get access

Abstract

Biochemical markers reflecting joint remodeling in osteoarthritis (OA) are a promising diagnostic tool. The aim of this study was to investigate serum levels of candidate biomarkers in subjects with and without knee OA and assess their correlation with clinical parameters and knee structural damage. 56 patients with primary knee OA and 31 healthy controls participated in this study. Patients were separated into two groups: isolated knee OA and generalized OA. Clinical parameters were obtained by validated self-reported questionnaires and a visual analogue scale. Serum levels of cartilage oligomeric protein (COMP), matrix metalloproteinase-3 (MMP-3), and Coll2-1 were quantified by enzyme-linked immunosorbent assay. Knee structural damage was determined by plain X-ray and 1.5 T magnetic resonance imaging (MRI), using Kellgren–Lawrence (KL) grading scale and Whole-Organ Magnetic Resonance Imaging Score (WORMS), respectively. Compared to controls, patients had significantly higher median serum COMP (985 vs. 625 ng/ml; p < 0.001) and MMP-3 (36.85 vs. 22.10 ng/ml; p = 0.003) levels. Patients with radiographic evidence of KLII/III knee OA had greater median COMP levels than KLI patients (1095 vs. 720 ng/ml; p = 0.001). In the generalized OA group, mean MMP-3 levels were higher than in the isolated knee OA group (30.40 vs. 55.13 ng/ml; p < 0.001). COMP correlated positively with WORMS (r s = 0.454, p < 0.001) and MMP-3 (r s = 0.337, p = 0.003). Cut-off values for serum COMP and MMP-3 were determined. We observed higher serum COMP and MMP-3 levels in knee OA patients compared to controls. COMP may reflect knee structural damage, while MMP-3—OA “generalization”.
Literature
1.
go back to reference Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R, Hochberg M, Howell D, Kaplan D, Koopman W, Longley S III, Mankin H, McShane DJ, Medsger T Jr, Meenan R, Mikkelsen W, Moskowitz R, Murphy W, Rothschild B, Segal M, Sokoloff L, Wolfe F (1986) Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheumatol 29(8):1039–1049CrossRef Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R, Hochberg M, Howell D, Kaplan D, Koopman W, Longley S III, Mankin H, McShane DJ, Medsger T Jr, Meenan R, Mikkelsen W, Moskowitz R, Murphy W, Rothschild B, Segal M, Sokoloff L, Wolfe F (1986) Development of criteria for the classification and reporting of osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheumatol 29(8):1039–1049CrossRef
2.
go back to reference Hart DJ, Spector TD, Brown P, Wilson P, Doyle DV, Silman AJ (1991) Clinical signs of early osteoarthritis: reproducibility and relation to X-ray changes in 541 women in the general population. Ann Rheum Dis 50(7):467–470CrossRefPubMedPubMedCentral Hart DJ, Spector TD, Brown P, Wilson P, Doyle DV, Silman AJ (1991) Clinical signs of early osteoarthritis: reproducibility and relation to X-ray changes in 541 women in the general population. Ann Rheum Dis 50(7):467–470CrossRefPubMedPubMedCentral
3.
go back to reference Biomarkers Definitions Working Group (2001) Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. Clin Pharmacol Ther 69: 89–95CrossRef Biomarkers Definitions Working Group (2001) Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. Clin Pharmacol Ther 69: 89–95CrossRef
4.
go back to reference Eckstein F, Burstein D, Link TM (2006) Quantitative MRI of cartilage and bone: degenerative changes in osteoarthritis. NMR Biomed 19(7):822–854CrossRefPubMed Eckstein F, Burstein D, Link TM (2006) Quantitative MRI of cartilage and bone: degenerative changes in osteoarthritis. NMR Biomed 19(7):822–854CrossRefPubMed
5.
go back to reference Pelletier JP, Cooper C, Peterfy C, Reginster JY, Brandi ML, Bruyère O, Chapurlat R et al (2013) What is the predictive value of MRI for the occurrence of knee replacement surgery in knee osteoarthritis? Ann Rheum Dis 72(10):1594–1604CrossRefPubMed Pelletier JP, Cooper C, Peterfy C, Reginster JY, Brandi ML, Bruyère O, Chapurlat R et al (2013) What is the predictive value of MRI for the occurrence of knee replacement surgery in knee osteoarthritis? Ann Rheum Dis 72(10):1594–1604CrossRefPubMed
6.
go back to reference Kraus VB, Burnett B, Coindreau J et al (2011) Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis. Osteoarthr Cartil 19(5):515–542CrossRefPubMedPubMedCentral Kraus VB, Burnett B, Coindreau J et al (2011) Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis. Osteoarthr Cartil 19(5):515–542CrossRefPubMedPubMedCentral
7.
go back to reference Lotz M, Martel-Pelletier J, Christiansen C, Brandi ML, Bruyère O, Chapurlat R, Collette J et al (2013) Value of biomarkers in osteoarthritis: current status and perspectives. Ann Rheum Dis 72(11):1756–1763CrossRefPubMedPubMedCentral Lotz M, Martel-Pelletier J, Christiansen C, Brandi ML, Bruyère O, Chapurlat R, Collette J et al (2013) Value of biomarkers in osteoarthritis: current status and perspectives. Ann Rheum Dis 72(11):1756–1763CrossRefPubMedPubMedCentral
8.
go back to reference Georgiev TA, Stoilov R, Ivanova M (2013) Biomarkers in osteoarthritis. Revmatologiia 21(4):21–25 Georgiev TA, Stoilov R, Ivanova M (2013) Biomarkers in osteoarthritis. Revmatologiia 21(4):21–25
9.
go back to reference Smith RKW, Heinegård D (2000) Cartilage oligomeric matrix protein (COMP) levels in digital sheath synovial fluid and serum with tendon injury. Equine Vet J 32(1):52–58CrossRefPubMed Smith RKW, Heinegård D (2000) Cartilage oligomeric matrix protein (COMP) levels in digital sheath synovial fluid and serum with tendon injury. Equine Vet J 32(1):52–58CrossRefPubMed
10.
go back to reference Bauer DC, Hunter DJ, Abramson SB et al (2006) Classification of osteoarthritis biomarkers: a proposed approach. Osteoarthr Cartil 14(8):723–727CrossRefPubMed Bauer DC, Hunter DJ, Abramson SB et al (2006) Classification of osteoarthritis biomarkers: a proposed approach. Osteoarthr Cartil 14(8):723–727CrossRefPubMed
11.
12.
go back to reference Bruyere O, Collette J, Kothari M et al (2006) Osteoarthritis, magnetic resonance imaging, and biochemical markers: a one year prospective study. Ann Rheum Dis 65:1050–1054CrossRefPubMedPubMedCentral Bruyere O, Collette J, Kothari M et al (2006) Osteoarthritis, magnetic resonance imaging, and biochemical markers: a one year prospective study. Ann Rheum Dis 65:1050–1054CrossRefPubMedPubMedCentral
13.
go back to reference Vilım V, Olejarova M, Macháček S, Gatterova J, Kraus VB, Pavelka K (2002) Serum levels of cartilage oligomeric matrix protein (COMP) correlate with radiographic progression of knee osteoarthritis. Osteoarthr Cartil 10(9):707–713CrossRefPubMed Vilım V, Olejarova M, Macháček S, Gatterova J, Kraus VB, Pavelka K (2002) Serum levels of cartilage oligomeric matrix protein (COMP) correlate with radiographic progression of knee osteoarthritis. Osteoarthr Cartil 10(9):707–713CrossRefPubMed
14.
go back to reference Singh S, Shahi U, Kumar D, Shahi NT (2014) Serum Cartilage Oligomeric Matrix Protein: Tool for early diagnosis and grading of severity of primary knee osteoarthritis. Int J Osteol Orthop 1:1–7 Singh S, Shahi U, Kumar D, Shahi NT (2014) Serum Cartilage Oligomeric Matrix Protein: Tool for early diagnosis and grading of severity of primary knee osteoarthritis. Int J Osteol Orthop 1:1–7
15.
go back to reference Awadallah AM, Gehan HS, Tarek MK (2010) Serum level of cartilage oligomeric matrix protein as a screening modality for osteoarthritis among knee joint pain patients. J Am Sci 6(12) Awadallah AM, Gehan HS, Tarek MK (2010) Serum level of cartilage oligomeric matrix protein as a screening modality for osteoarthritis among knee joint pain patients. J Am Sci 6(12)
16.
go back to reference Clark AG, Jordan JM, Vilim V, Renner JB, Dragomir AD, Luta G, Kraus VB (1999) Serum cartilage oligomeric matrix protein reflects osteoarthritis presence and severity: the Johnston County Osteoarthritis Project. Arthritis Rheumatol 42(11):2356–2364CrossRef Clark AG, Jordan JM, Vilim V, Renner JB, Dragomir AD, Luta G, Kraus VB (1999) Serum cartilage oligomeric matrix protein reflects osteoarthritis presence and severity: the Johnston County Osteoarthritis Project. Arthritis Rheumatol 42(11):2356–2364CrossRef
17.
go back to reference Ribbens CY, Porras MM, Franchimont N et al (2002) Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis 61(2):161–166CrossRefPubMedPubMedCentral Ribbens CY, Porras MM, Franchimont N et al (2002) Increased matrix metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis 61(2):161–166CrossRefPubMedPubMedCentral
18.
go back to reference Kopchev A, Monov S, Kyurkchiev D, Ivanova I, Georgiev T (2017) Vascular endothelial growth factor (VEGF), cartilage oligomeric protein (COMP) and matrix metalloproteinase 3 (MMP-3) as serum biomarkers in psoriatic arthritis. Int J Pharm Sci Inven 6(7):08–12 Kopchev A, Monov S, Kyurkchiev D, Ivanova I, Georgiev T (2017) Vascular endothelial growth factor (VEGF), cartilage oligomeric protein (COMP) and matrix metalloproteinase 3 (MMP-3) as serum biomarkers in psoriatic arthritis. Int J Pharm Sci Inven 6(7):08–12
19.
go back to reference Connelly AE, Tucker AJ, Kott LS, Duncan AM, Wright AJ (2014) Serum biochemical markers of joint metabolism and inflammation in relation to clinical symptoms and physical function in adults with symptomatic knee osteoarthritis. Osteoarthr Cartil 22:S66CrossRef Connelly AE, Tucker AJ, Kott LS, Duncan AM, Wright AJ (2014) Serum biochemical markers of joint metabolism and inflammation in relation to clinical symptoms and physical function in adults with symptomatic knee osteoarthritis. Osteoarthr Cartil 22:S66CrossRef
20.
go back to reference Li W, Du C, Wang H, Zhang C (2014) Increased serum ADAMTS-4 in knee osteoarthritis: a potential indicator for the diagnosis of osteoarthritis in early stages. Genet Mol Res 13(4):9642–9649CrossRefPubMed Li W, Du C, Wang H, Zhang C (2014) Increased serum ADAMTS-4 in knee osteoarthritis: a potential indicator for the diagnosis of osteoarthritis in early stages. Genet Mol Res 13(4):9642–9649CrossRefPubMed
21.
go back to reference Pelletier J, Raynauld J, Caron J, Mineau F, Abram F, Dorais M et al (2010) Decrease in serum level of matrix metalloproteinases is predictive of the disease-modifying effect of osteoarthritis drugs assessed by quantitative MRI in patients with knee osteoarthritis. Ann Rheum Dis 69(12):2095–2101CrossRefPubMed Pelletier J, Raynauld J, Caron J, Mineau F, Abram F, Dorais M et al (2010) Decrease in serum level of matrix metalloproteinases is predictive of the disease-modifying effect of osteoarthritis drugs assessed by quantitative MRI in patients with knee osteoarthritis. Ann Rheum Dis 69(12):2095–2101CrossRefPubMed
22.
go back to reference Deberg M, Labasse A, Christgau S, Cloos P et al (2005) New serum biochemical markers (Coll 2-1 and Coll 2-1 NO 2) for studying oxidative-related type II collagen network degradation in patients with osteoarthritis and rheumatoid arthritis. Osteoarthr Cartil 13(3):258–265CrossRefPubMed Deberg M, Labasse A, Christgau S, Cloos P et al (2005) New serum biochemical markers (Coll 2-1 and Coll 2-1 NO 2) for studying oxidative-related type II collagen network degradation in patients with osteoarthritis and rheumatoid arthritis. Osteoarthr Cartil 13(3):258–265CrossRefPubMed
23.
go back to reference Henrotin Y, Conrozier T, Deberg M, Walliser-Lohse A, Richette P, Mulleman D et al (2013) Early decrease of serum biomarkers of type II collagen degradation (Coll2-1) and joint inflammation (Coll2-1 NO2) by hyaluronic acid intra-articular injections in patients with knee osteoarthritis: A research study part of the Biovisco study. J Orthop Res 31(6):901–907CrossRefPubMed Henrotin Y, Conrozier T, Deberg M, Walliser-Lohse A, Richette P, Mulleman D et al (2013) Early decrease of serum biomarkers of type II collagen degradation (Coll2-1) and joint inflammation (Coll2-1 NO2) by hyaluronic acid intra-articular injections in patients with knee osteoarthritis: A research study part of the Biovisco study. J Orthop Res 31(6):901–907CrossRefPubMed
24.
go back to reference Deberg MA, Labasse AH, Collette J, Seidel L, Reginster JY, Henrotin YE (2005) One-year increase of Coll 2-1, a new marker of type II collagen degradation, in urine is highly predictive of radiological OA progression. Osteoarthr Cartil 13:1059–1065CrossRefPubMed Deberg MA, Labasse AH, Collette J, Seidel L, Reginster JY, Henrotin YE (2005) One-year increase of Coll 2-1, a new marker of type II collagen degradation, in urine is highly predictive of radiological OA progression. Osteoarthr Cartil 13:1059–1065CrossRefPubMed
25.
go back to reference Lequesne MG, Mery C, Samson M, Gerard P (1987) Indexes of severity for osteoarthritis of the hip and knee. Validation–value in comparison with other assessment tests. Scand J Rheumatol Suppl 65:85–89CrossRefPubMed Lequesne MG, Mery C, Samson M, Gerard P (1987) Indexes of severity for osteoarthritis of the hip and knee. Validation–value in comparison with other assessment tests. Scand J Rheumatol Suppl 65:85–89CrossRefPubMed
26.
go back to reference Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW (1988) Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. J Rheumatol 15:1833–1840PubMed Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW (1988) Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. J Rheumatol 15:1833–1840PubMed
28.
go back to reference Peterfy CG, Guermazi A, Zaim S et al (2004) Whole-organ magnetic resonance imaging score (WORMS) of the knee in osteoarthritis. Osteoarthr Cartil 12(3):177–190CrossRefPubMed Peterfy CG, Guermazi A, Zaim S et al (2004) Whole-organ magnetic resonance imaging score (WORMS) of the knee in osteoarthritis. Osteoarthr Cartil 12(3):177–190CrossRefPubMed
29.
go back to reference Schiphof D, Boers M, Bierma-Zeinstra SM (2008) Differences in descriptions of Kellgren and Lawrence grades of knee osteoarthritis. Ann Rheum Dis 67(7):1034–1036CrossRefPubMed Schiphof D, Boers M, Bierma-Zeinstra SM (2008) Differences in descriptions of Kellgren and Lawrence grades of knee osteoarthritis. Ann Rheum Dis 67(7):1034–1036CrossRefPubMed
30.
go back to reference Verma P, Dalal K (2013) Serum cartilage oligomeric matrix protein (COMP) in knee osteoarthritis: a novel diagnostic and prognostic biomarker. J Orthop Res 31(7):999–1006CrossRefPubMed Verma P, Dalal K (2013) Serum cartilage oligomeric matrix protein (COMP) in knee osteoarthritis: a novel diagnostic and prognostic biomarker. J Orthop Res 31(7):999–1006CrossRefPubMed
31.
go back to reference Naito К, Takahashi М, Kushida К, Suzuki М, Ohishi Т, Miura М, Inoue Т, Nagano А (1999) Measurement of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in patients with knee osteoarthritis: comparison with generalized osteoarthritis. Rheumatology 38(6):510–515CrossRefPubMed Naito К, Takahashi М, Kushida К, Suzuki М, Ohishi Т, Miura М, Inoue Т, Nagano А (1999) Measurement of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in patients with knee osteoarthritis: comparison with generalized osteoarthritis. Rheumatology 38(6):510–515CrossRefPubMed
32.
go back to reference Recht MP, Kramer J, Marcelis S, Pathria MN et al (1993) Abnormalities of articular cartilage in the knee: analysis of available MR techniques. Radiology 187:473–478CrossRefPubMed Recht MP, Kramer J, Marcelis S, Pathria MN et al (1993) Abnormalities of articular cartilage in the knee: analysis of available MR techniques. Radiology 187:473–478CrossRefPubMed
33.
go back to reference Rifai N, Paul MR (2001) High-sensitivity C-reactive protein: a novel and promising marker of coronary heart disease. Clin Chem 47(3):403–411PubMed Rifai N, Paul MR (2001) High-sensitivity C-reactive protein: a novel and promising marker of coronary heart disease. Clin Chem 47(3):403–411PubMed
34.
go back to reference Danesh J, Wheeler JG, Hirschfield GM, Eda S et al (2004) C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med 350(14):1387–1397CrossRefPubMed Danesh J, Wheeler JG, Hirschfield GM, Eda S et al (2004) C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med 350(14):1387–1397CrossRefPubMed
35.
go back to reference Pearson TA, Mensah GA, Alexander RW et al (2003) Markers of inflammation and cardiovascular disease. Circulation 107(3):499–511CrossRefPubMed Pearson TA, Mensah GA, Alexander RW et al (2003) Markers of inflammation and cardiovascular disease. Circulation 107(3):499–511CrossRefPubMed
36.
go back to reference Pearle AD, Scanzello CR, George S et al (2007) Elevated high-sensitivity C-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis. Osteoarthr Cartil 15(5):516–523CrossRefPubMed Pearle AD, Scanzello CR, George S et al (2007) Elevated high-sensitivity C-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis. Osteoarthr Cartil 15(5):516–523CrossRefPubMed
37.
go back to reference Miller ME, Rejeski WJ, Messier SP et al (2001) Modifiers of change in physical functioning in older adults with knee pain: the Observational Arthritis Study in Seniors (OASIS). Arthritis Rheum 45:331–339CrossRefPubMed Miller ME, Rejeski WJ, Messier SP et al (2001) Modifiers of change in physical functioning in older adults with knee pain: the Observational Arthritis Study in Seniors (OASIS). Arthritis Rheum 45:331–339CrossRefPubMed
38.
go back to reference Bedson J, Croft PR (2008) The discordance between clinical and radiographic knee osteoarthritis: a systematic search and summary of the literature. BMC Musculoskelet Disord 9:116CrossRefPubMedPubMedCentral Bedson J, Croft PR (2008) The discordance between clinical and radiographic knee osteoarthritis: a systematic search and summary of the literature. BMC Musculoskelet Disord 9:116CrossRefPubMedPubMedCentral
39.
go back to reference Stoilov R, Ivanova M, Garbeva K, Marinova N, Manolova I, Kalitchin Z (2011) Evaluation of lubrication and friction in knee joint attacked by osteoarthritis treated with ostenil, ostenil plus, yaral forte and durolane. J Balk Tribol Assoc 17(3):456–461 Stoilov R, Ivanova M, Garbeva K, Marinova N, Manolova I, Kalitchin Z (2011) Evaluation of lubrication and friction in knee joint attacked by osteoarthritis treated with ostenil, ostenil plus, yaral forte and durolane. J Balk Tribol Assoc 17(3):456–461
40.
go back to reference Felson DT, Naimark A, Anderson J, Kazis L, Castelli W, Meenan RF (1987) The prevalence of knee osteoarthritis in the elderly. Fram Osteoarthr Study Arthritis Rheumatol 30(8):914–918CrossRef Felson DT, Naimark A, Anderson J, Kazis L, Castelli W, Meenan RF (1987) The prevalence of knee osteoarthritis in the elderly. Fram Osteoarthr Study Arthritis Rheumatol 30(8):914–918CrossRef
41.
Metadata
Title
Cartilage oligomeric protein, matrix metalloproteinase-3, and Coll2-1 as serum biomarkers in knee osteoarthritis: a cross-sectional study
Authors
Tsvetoslav Georgiev
Mariana Ivanova
Aleksandar Kopchev
Tsvetelina Velikova
Asen Miloshov
Ekaterina Kurteva
Kalina Yuzeir
Marin Penkov
Plamena Kabakchieva
Rasho Rashkov
Rumen Stoilov
Publication date
01-05-2018
Publisher
Springer Berlin Heidelberg
Published in
Rheumatology International / Issue 5/2018
Print ISSN: 0172-8172
Electronic ISSN: 1437-160X
DOI
https://doi.org/10.1007/s00296-017-3887-y

Other articles of this Issue 5/2018

Rheumatology International 5/2018 Go to the issue
Live Webinar | 27-06-2024 | 18:00 (CEST)

Keynote webinar | Spotlight on medication adherence

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

WHO estimates that half of all patients worldwide are non-adherent to their prescribed medication. The consequences of poor adherence can be catastrophic, on both the individual and population level.

Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.

Prof. Kevin Dolgin
Prof. Florian Limbourg
Prof. Anoop Chauhan
Developed by: Springer Medicine
Obesity Clinical Trial Summary

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.

Developed by: Springer Medicine

Highlights from the ACC 2024 Congress

Year in Review: Pediatric cardiology

Watch Dr. Anne Marie Valente present the last year's highlights in pediatric and congenital heart disease in the official ACC.24 Year in Review session.

Year in Review: Pulmonary vascular disease

The last year's highlights in pulmonary vascular disease are presented by Dr. Jane Leopold in this official video from ACC.24.

Year in Review: Valvular heart disease

Watch Prof. William Zoghbi present the last year's highlights in valvular heart disease from the official ACC.24 Year in Review session.

Year in Review: Heart failure and cardiomyopathies

Watch this official video from ACC.24. Dr. Biykem Bozkurt discusses last year's major advances in heart failure and cardiomyopathies.