Virchows Archiv B

, 64:45 | Cite as

Inhibition of the effects of rheumatoid synovial fluid cells on chondrogenesis and cartilage breakdown in vitro: possible therapeutical conclusions

A morphological—biochemical study
  • Hassan Mohamed-Ali
  • Peter Scholz
  • Hans-Joachim Merker


Short-term co-cultivation of blastemal cells from 12-day-old mouse limb buds and human rheumatoid synovial fluid cells in high density cultures (Trowell culture system) resulted, depending on when co-cultivation started, either in (1) an inhibition of chondrogenesis (co-cultivation right from the start) or in (2) an extensive breakdown of cartilaginous matrix (co-cultivation after formation of embryonic cartilage). These synovial effects were markedly impeded if Avarol (a dioxygenase inhibitor) was applied singly or in combination with PAI-2 (a u-PA-inhibitor). PAI-2 alone, however, had no effect on the synovial-induced inhibition of chondrogenesis, but produced a pronounced inhibitory effect on matrix breakdown. The effects of both inhibitors were studied electron microscopically and biochemically (determination of sulfated-glycosaminoglycans in the high density cultures by Alcian Blue binding assay). The results of this study are consistent with the presumption that rheumatoid synovial cells are capable of inhibiting chondrogenesis and enhancing the breakdown of the cartilaginous matrix. Amongst others, the possible mediators involved are prostaglandins and plasminogen activators. The response to the inhibitors Avarol and PAI-2 is compatible with their mode of action. The chondroprotective action of these substances may be useful in developing potential antirheumatic drugs.

Key words

Synovial fluid cells Cartilage synthesis Cartilage breakdown Enzyme activator inhibition 


  1. Arner EC, Pratta MA (1989) Independent effects of interleukin-1 on protoeglycan synthesis, and prostaglandin E2 release from cartilage in organ culture. Arthritis Rheum 32:288–297PubMedGoogle Scholar
  2. Astrup T (1966) Tissue activators of plasminogens. Fed Proc 25:42–51PubMedGoogle Scholar
  3. Barrett AJ (1975) The enzyme degradation of cartilage matrix. In: Burleigh PMC, Poole AR (eds) Dynamics of connective tissue macromolecules. North Holland, Amsterdam, pp 189–215Google Scholar
  4. Bergström S, Danielsson H, Samuelsson B (1964) The enzymatic formation of prostaglandin E2 from arachidonic acid prostaglandins and related factors 32. Biochim Biophys Acta 90:207–210Google Scholar
  5. Biddulph DM, Sawyer LM, Smales WP (1984) Chondrogenesis of chick limb mesenchyme in vitro: effects of prostaglandin on cyclic AMP. Exp Cell Res 153:270–274PubMedGoogle Scholar
  6. Blackwell GJ, Flower RJ (1983) Inhibition of phospholipase. Br Med Bull 39:260–264PubMedGoogle Scholar
  7. Campbell IK, Roughley PJ, Mort JS (1986) The action of human articular cartilage metalloproteinase on proteoglycan and link protein. Biochem J 237:117–122PubMedGoogle Scholar
  8. Carmichael DJ, Gillard GC, Lowther DA, Handley CJ, Santer VB (1977) Carrageenin-induced arthritis. IV. Rate changes in cartilage matrix proteoglycan synthesis. Arthritis Rheum 20:834–840PubMedGoogle Scholar
  9. Carty TJ, Eskra JD, Lombardino JG (1978) Piroxicam, a novel anti-inflammatory compound. Effect of modification of chemical structure on prostaglandin (PG) biosynthesis inhibition and anti-inflammatory activity. Fed Proc 37:2359Google Scholar
  10. Chang W-C, Abe M, Murota SI (1977) Stimulation by prostaglandin F2 α of acidic glycosaminoglycan production in cultured fibroblasts. Prostaglandins 13:15–63Google Scholar
  11. Chapman HA, Stone OL (1985) Characterization of a macrophage-derived plasminogen activator inhibitor. Similarities with placental urokinase inhibitor. Biochem J 230:109–116PubMedGoogle Scholar
  12. Chepenik KP, Ho WC, Waite BM, Parker CL (1984) Arachidonate metabolism during chondrogenesis in vitro. Calcif Tissue Int 36:175–181PubMedGoogle Scholar
  13. Christman JK, Silverstein SC, Acs G (1977) Plasminogen activators. In: Barrett AJ (ed) Research monographs in cells and tissues. North Holland Publishing Co, Amsterdam, pp 90–149Google Scholar
  14. Cushman DW, Cheung HS (1976) Effect of sustrate concentration on inhibition of prostaglandin synthetase of bull seminal vesicles by anti-inflammatory drugs and fenamic acid analogs. Biochim Biophys Acta 424:449–459PubMedGoogle Scholar
  15. Dayer JM, de Rockemonteix B, Burrus B, Demczuk S, Dinarello CA (1986) Human recombinant interleukin-1 stimulates collagenase and prostaglandin E2 production by human synovial cells. J Clin Invest 77:645–648PubMedGoogle Scholar
  16. Dingle JT (1984) The effects of synovial catabolin on cartilage synthetic activity. Connect Tissue Res 12:277–286PubMedGoogle Scholar
  17. Dingle JT, Horsefield P, Fell HB et al. (1975) Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture. Ann Rheum Diss 34:303–311Google Scholar
  18. D'Onofrio C, Paradisi F (1983) In-vitro differentiation of human monocytes into mature macrophages during long-term cultures. Immunobiol 164:13–22Google Scholar
  19. Eastgate JA, Symons JA, Wood NC, Grinlinton FM, di Giovine FS, Duff GW (1988) Correlation of plasma interleukin-1 levels with disease, activity in rheumatoid arthritis. Lancet II:706–709Google Scholar
  20. Evequoz V, Bettens F, Kristensen F, Trechsel U, Stadler BM, Dayer JM, de Weck AL, Fleisch H (1984) Interleukin 2-independent stimulation of rabbit chondrocyte collagenase and prostaglandin E2 production by an interleukin 1-like factor. Eur J Immunol 14:490–495PubMedGoogle Scholar
  21. Fell HB, Jubb RW (1977) The effect of synovial tissue on the breakdown of articular cartilage in organ culture. Arthritis Rheum 20:1359–1371PubMedGoogle Scholar
  22. Ferreira SH, Moncada S, Vane JR (1971) Indomethacin, and aspirin abolish prostaglandin release from the spleen. Nature 231:237–239Google Scholar
  23. Fulkerson JP, Ladenbauer-Bellis IM, Chrisman D (1979) In vitro hexosamine depletion of intact articular cartilage by E-prostaglandins. Arthritis Rheum 22:1117–1121PubMedGoogle Scholar
  24. Genton C, Kruithof EKO, Schleuning W-D (1987) Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinasetype plasminogen activator in human U-937 lymphoma cells. J Cell Biol 104:705–712PubMedGoogle Scholar
  25. Gillard GC, Lowther DA (1976) Carrageenin-induced arthritis. II. Effect of intra-articular injection of carrageenin on the synthesis of proteoglycan in articular cartilage. Arthritis Rheum 19:918–922PubMedGoogle Scholar
  26. Golder JP, Stephens RW (1983) Minactivin: a human monocyte product which specifically inactivates urokinase-type plasminogen activator. Eur J Biochem 136:517–522PubMedGoogle Scholar
  27. Grossley MJ, Hunneyball IM (1984) Studies on the release of proteolytic enzymes during synovium-induced cartilage breakdown in vitro and the actions of anti-inflammatory drugs. Biochem Pharmacol 33:1263–1271Google Scholar
  28. Hamberg M, Samuelsson B (1973) Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc Natl Acad Sci USA 70:899–903PubMedGoogle Scholar
  29. Hamberg M, Svensson J, Wokabayashi T, Samuelsson B (1974) Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Natl Acad Sci USA 71:345–349PubMedGoogle Scholar
  30. Harris ED Jr, Vater CA (1980) Methodology of collagenase research: Substrate preparation, enzyme activation and purification. In: Woolley DE, Evanson JM (eds) Collagenase in normal and pathological connective tissues. Wiley & Sons, New York, pp 51–53Google Scholar
  31. Henderson B, Edwards JCW (1987) The synovial lining in health and disease. Chapman, and Hall, London, pp 289–341Google Scholar
  32. Higgs GA, Vane JR, Hart FD, Wojtulewski JG (1974) Effect of anti-inflammatory drugs on prostaglandins in rheumatoid arthritis. In: Robinson HJ, Vane JR (eds) Prostaglandin synthetase inhibitors. Raven Press, New York, pp 165–173Google Scholar
  33. Hollander AP, Atkins RM, Eastwood DM, Dieppe PA, Elson CJ (1991) Degradation of human cartilage by synovial fluid but not cytokines in vitro. Ann Rheum Dis 50:57–58.PubMedGoogle Scholar
  34. Horodniak JW, Jilius M, Zarembo JE, Bender AD (1974) Inhibitory effects of aspirin and indomethacin on the biosynthesis of PGE2 and PGF2α. Biochem Biophys Res Commun 57:539–545Google Scholar
  35. Jubb RW, Fell HB (1980) The effect of synovial tissue on the synthesis of proteoglycan by the articular cartilage of young pig. Arthritis Rheum 23:545–555PubMedGoogle Scholar
  36. Kawano T, Morimoto K, Uemura Y (1968) Urokinase inhibitor in human placenta. Nature 217:253–254PubMedGoogle Scholar
  37. Keller K, Shortkroff S, Sledge CB, Thornhill TS (1990) Effects of isolated rheumatoid synovial cells on cartilage degradation in vitro. J Orthop Res 8:345–352PubMedGoogle Scholar
  38. Kirchheimer JC, Renold HG (1989) Functional characteristics of receptor-bound urokinase on human monocytes: Catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors. Blood 74:1396–1402PubMedGoogle Scholar
  39. Kopitar M, Rozman B, Babnik J, Turk V, Mullins DE, Wun TC (1985) Human leukocyte urokinase inhibitor-purification, characterization and comparative studies against different plasminogen activators. Thromb Haemostasis 54:750–755Google Scholar
  40. Kosher RA, Walker KH (1983) The effect of prostaglandins on in vitro limb cartilage differentiation. Exp Cell Res 145:145–153PubMedGoogle Scholar
  41. Kruithof EKO, Vassalli JD, Schleuning W-D, Mattaliano RJ, Bachmann F (1986) Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell-line U-937. J Biol Chem 261:11207–11213PubMedGoogle Scholar
  42. Lack CH, Rogers HJ (1958) Actions of plasmin on cartilage. Nature 182:948–949PubMedGoogle Scholar
  43. Lackner H, Javid JP (1973) The clinical significance of the plasminogen level. Am J Clin Pathol 60:175–181PubMedGoogle Scholar
  44. Lands WEM (1979) The biosynthesis and metabolism of prostaglandins. Ann Rev Physiol 41: 633–652Google Scholar
  45. Lands WEM (1978) Somite chondrogenesis in vitro; stimulation by exogenous extracellular matrix components. Devl Biol 66:151–171Google Scholar
  46. Laug WE, Aebersold R, Jong A, Rideout W, Bergman BL, Baker J (1989) Isolation of multiple types of plasminogen activator inhibitors from vascular smooth muscle cells. Thromb Haemostasis 61:517–521Google Scholar
  47. Lecander I, Astedt B (1986) Isolation of a new specific plasminogen activator inhibitor from pregnancy plasma. Br J Haematol 62:221–228PubMedGoogle Scholar
  48. Lippiello L, Yamamoto K, Robinson D, Mankin HJ (1978) Involvement of prostaglandins from rheumatoid synovium in inhibition of articular cartilage metabolism. Arthritis Rheum 21:909–917PubMedGoogle Scholar
  49. Loskutoff DJ, Van Mourik JA, Erickson LA, Lawrence D (1983) Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc Natl Acad Sci USA 80:2956–2960PubMedGoogle Scholar
  50. Lowther DA, Sandy JD, Santer VB, Brown HLG (1978) Antigen-induced arthritis: decreased proteoglycan content and inhibition of proteoglycan synthesis in articular cartilage. Arthritis Rheum 21:675–680PubMedGoogle Scholar
  51. Malemud CJ, Sokoloff L (1977) The effect of prostaglandins, on cultured lapine articular chondrocytes. Prostaglandins 13:845–860PubMedGoogle Scholar
  52. Malemud CJ, Moskowitz RW, Papay RS (1982) Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes. Biochim Biophys Acta 715:70–79PubMedGoogle Scholar
  53. Matsuo O, Tanaka S, Kikuchi H (1988) Effect of urinary trypsin inhibitor on osteoarthritis. Thromb Res 52:237–245PubMedGoogle Scholar
  54. Merker H-J, Zimmermann B, Barrach HJ, Grundmann K, Ebel H (1980a) Simulation of steps of limb bud skeletogenesis in vitro. In: Merker H-J, Nau H, Neubert D (eds) Teratology of the limbs. Walter de Gruyter Verlag. Berlin New York, pp 137–151Google Scholar
  55. Merker H-J, Zimmermann B, Grundmann K (1980b) Differentiation of isolated blastemal cells from limb buds into chondroblasts. In: Richards RJ, Rajan KT (eds) Tissue culture in medical research (II). Pergamon Press, Oxford New York, pp 31–39Google Scholar
  56. Merker H-J, Krüger U, Zimmermann B (1981) Simulation of limb bud skeletogenesis in vitro. In: Neubert D, Merker H-J (eds) Culture Techniques. Walter de Gruyter Verlag, Berlin New York, pp 119–133Google Scholar
  57. Minale L, Riccio R, Sodano G (1974) Avarol, a novel sesquiterpenoid hydroquinone with a rearranged drimane skeleton from the sponge Dysidea avara. Tetrahedron Lett 38:3401–3404Google Scholar
  58. Miyasaka N, Sato K, Goto M, et al. (1988) Augmented interleukin-1 production and HLA-DR expression in the synovium of rheumatoid arthritis patients. Arthritis Rheum 31:480–486PubMedGoogle Scholar
  59. Mochan E, Keler T (1984) Plasmin degradation of cartilage proteoglycan. Biochim Biophys Acta 800:312–315PubMedGoogle Scholar
  60. Mohamed-Ali H (1991a) Proteolytische Enzyme und die Destruktion des Gelenkknorpels bei der Arthrose und der chronischen Polyarthritis. Wien Med Wochenschr 4:77–85Google Scholar
  61. Mohamed-Ali H, Hauer RW, Soerensen H (1991b) Morphologie und Wachstumsverhalten von Synoviazellen in der Monolayer-Kultur. Z Rheumatol 50:74–81PubMedGoogle Scholar
  62. Mohamed-Ali H (1992) Influence of synovial cells on cartilage in vitro. Induction of breakdown and inhibition of synthesis. Virchow's Arch [B] 62:227–236Google Scholar
  63. Müller WEG, Maidhof A, Zahn RK, Schröder HC, Gasic MJ, Heidemann D, Bernd A, Kurelec B, Eich E, Seibert G (1985) Potent antileukemic activity of the novel cytostatic agent avarone and its analogues in vitro and in vivo. Cancer Res 45:4822–4826PubMedGoogle Scholar
  64. Myhre-Jensen O, Larsen SB, Astrup T (1969) Fibrinolytic activity in serosal and synovial membranes. Rats, guinea pigs and rabbits. Arch Pathol 88:623–630Google Scholar
  65. Nietfeld JJ, Wilbrink B, Hele M, van Roy JLAM, Den Otter W, Swaak AJG, Huber-Bruning O (1990) Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage. Arthritis Rheum 33:1695–1701PubMedGoogle Scholar
  66. Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff D (1986) Cloning and sequence of a cDNA coding for the human betamigrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 83:6776–6781PubMedGoogle Scholar
  67. Okada Y, Nakanishi I (1989) Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (gelatinase) by human neutrophil elastase and kathepsin G. FFB 249:353–356Google Scholar
  68. Oronsky AL, Perper RJ, Schroder HC (1973) Phagocytic release and activation of human leukocyte procollagenase. Nature 246:417–419PubMedGoogle Scholar
  69. Pandolfi M, Ahlberg A, Traldi A et al. (1972) Fibrinolytic activity of human synovial membranes in health and in haemophilia. Scand J Haematol 9:572–576PubMedGoogle Scholar
  70. Robbins JC, Summaria L, Hsieh B, Shah RJ (1967) The peptide chains of human plasmin. Mechanism of activation of human plasminogen to plasmin. J Biol Chem 242:2333–2342PubMedGoogle Scholar
  71. Rome LH, Lands WEM (1975) Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. Proc Natl Acad Sci 72:4863–4865PubMedGoogle Scholar
  72. Roughley PJ (1977) The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation. Biochem J 167: 639–646PubMedGoogle Scholar
  73. Sajdera SW, Hascall VC (1969) Protein polysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J Biol Chem 244: 77–87.PubMedGoogle Scholar
  74. Saklatvala J (1986) Tumor necrosis factor-α stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature 322:547–549PubMedGoogle Scholar
  75. Sandy JD, Lowther DA, Brown HLG (1980) Antigen-induced arthritis. Studies on the inhibition of proteoglycan synthesis observed in articular cartilage during short-term joint inflammation. Arthritis Rheum 23:433–447PubMedGoogle Scholar
  76. Sapolsky AT, Keiser H, Howell DS et al. (1976) Metalloproteinases of human articular cartilage that digest proteoglycan at neutral and acid pH. J Clin Invest 58:1030–1041PubMedGoogle Scholar
  77. Saxne T, Heinegard D, Wollheim FA (1988) Human arthritic synovial fluid influences proteoglycan biosynthesis and degradation in organ culture of bovine nasal cartilage. Coll Rel Res 8:233–247Google Scholar
  78. Schröder HC, Begin ME, Klöcking R, Matthes E, Sarma AS, Gasic M, Müller WEG (1991) Avarol restores the altered prostaglandin and leukotriene metabolism in monocytes infected with human immunodeficiency virus type 1. Virus Res 21:213–223PubMedGoogle Scholar
  79. Scott JE, Quintarelli G, Dellovo MC (1964) The chemical and histochemical properties of Alcian Blue: I. The mechanism of Alcian Blue staining. II. Dye binding of tissue polyanions. Histochemie 4:73–98PubMedGoogle Scholar
  80. Seibert G, Raether W, Dogovic N, Gasic MJ, Zahn RK, Müller WEG (1985) Antibacterial and antifungal activity of avarone and avarol. Zbl Bakt [Hyg A] 260:379–386Google Scholar
  81. Smith JB, Willis AL (1971) Aspirin selectively inhibits prostaglandin production in human platelets. Nature 231:235–237Google Scholar
  82. Stanfield AB, Stephens CAL Jr (1963) Studies of cells cultured from 188 rheumatoid and non-rheumatoid, synovial tissues. Texas Reports Biol Med 21:400–411Google Scholar
  83. Steinberg JJ, Tsukamoto S, Sledge CB (1983) Breakdown of cartilage proteoglycan in a tissue culture model of rheumatoid arthritis. Biochim Biophys Acta 757:47–58PubMedGoogle Scholar
  84. Stone KJ, Mathes SJ, Gibson PP (1975) Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone. Prostaglandins 10:241–251PubMedGoogle Scholar
  85. Storti E, Magrini U, Castello A, Pandolfi M, Ascari E (1973) The histochemistry of fibrinolysis in haemophylic synovial membranes. Acta Haematol 49:142–153PubMedGoogle Scholar
  86. Thorsen S, Philips M, Selmer J, Lecander I, Astedt B (1988) Kinetics of inhibition of tissue-type and urokinase-type plasminogen activators by plasminogen-activator inhibitor type 1 and type 2. Eur J Biochem 175:33–39PubMedGoogle Scholar
  87. Tolman EL, Birnbaum JE, Chiccarelli FS, Panagides J, Sloboda AE (1976) Inhibition of prostaglandin activity and synthesis by fenbufen (a new non-steroidal anti-inflammatory agent) and one of its metabolites. In: Samuelsson B, Paoletti R (eds) Advances in prostaglandin and thromboxane research, vol I. Raven Press, New York, pp 133–138Google Scholar
  88. Travis J, Salvesen GS (1983) Human plasma proteinase inhibitors. Ann Rev Biochem 52:655–709PubMedGoogle Scholar
  89. Trowell OA (1959) The culture of mature organs in a synthetic medium. Exp Cell Res 16:118–147PubMedGoogle Scholar
  90. Tyler JA (1985) Articular cartilage cultures with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules. Biochem J 227:869–878PubMedGoogle Scholar
  91. Unkeles JC, Gordon S, Reich E (1974) Secretion of plasminogen activator by stimulated macrophages. J Exp Med 139:834–850Google Scholar
  92. Van Beuningen HM, Arntz OJ, van den Berg WB (1991) In vivo effects of interleukin-1 on articular cartilage: prolongation of proteoglycan metabolism disturbance in old mice. Arthritis Rheum 34:606–615PubMedGoogle Scholar
  93. Van de Loo AAJ, Arntz OJ, Otternes IG, van den Berg WB (1992) Protection against cartilage proteoglycan synthesis inhibition by antiinterleukin 1 antibodies in experimental arthritis. J Rheumatol 19:348–356PubMedGoogle Scholar
  94. Van Dorp DA, Beerthuis RK, Nugteren DH, Vonkeman H (1964) The biosynthesis of prostaglandins. Biochim Biophys Acta 90:204–207Google Scholar
  95. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like, drugs. Nature 231:232–235Google Scholar
  96. Vaubel E (1933) The form and function of synovial cells in tissue cutures. J Exp Med 58:63–83Google Scholar
  97. Werb Z, Aggeler J (1978) Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. Proc Natl Acad Sci USA 75:1839–1843PubMedGoogle Scholar
  98. Werb Z, Mainardi CL, Vater CA, Harris ED Jr (1977) Endogenous activation of latent collagenase by rheumatoid synovial cells. New Engl J Med 296:1017–1023PubMedGoogle Scholar
  99. Whiteman P (1973) The quantitative measurement of Alcian Blueglycosaminoglycan complexes. Biochem J 131:343–351PubMedGoogle Scholar
  100. Wiseman EH (1980) Piroxicam (Felden): Theorie und Wirkungsweise. Akt Rheumatol 5:1–8CrossRefGoogle Scholar
  101. Wohlwend A, Belin D, Vassalli JD (1987) Plasminogen activatorspecific inhibitors produced by human monocytes/macrophages. J Exp Med 165:320–339PubMedGoogle Scholar
  102. Ye RD, Wun TC, Sadler JE (1987) cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta. J Biol Chem 262:3718–3725PubMedGoogle Scholar
  103. Zimmermann B (1987) Lung organoid culture. Differentiation 36:86–109PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Hassan Mohamed-Ali
    • 1
  • Peter Scholz
    • 2
  • Hans-Joachim Merker
    • 1
  1. 1.Institute of AnatomyFree University of BerlinBerlin 33Germany
  2. 2.Department of Protein ChemistrySchering AG BerlinBerlinGermany

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