Fibrocartilage in the extensor tendons of the human metacarpophalangeal joints.
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The extensor tendons of the fingers cross both the metacarpophalangeal (MCP) and interphalangeal joints. Previous studies have shown that where the extensor tendons replace the capsule of the proximal interphalangeal (PIP) joint, they contain a sesamoid fibrocartilage that articulates with the proximal phalanx during flexion. The fibrocartilage labels immunohistochemically for a variety of glycosaminoglycans and collagens. In the current study, we investigate the molecular composition of the extensor tendons at the level of the MCP joints. This is of particular interest because the tendon has a greater moment arm at this location (and might thus be subject to greater compression), but is separated from the joint cavity by the capsule and peritendinous tissue. Six hands were removed from elderly cadavers (39-85 years of age) and the MCP joints were fixed in 90% methanol. The extensor tendons were dissected from all fingers, cryosectioned, and immunolabelled with a panel of monoclonal and polyclonal antibodies for types I, II, III, and VI collagens, chondroitin 4 and 6 sulphates, dermatan, and keratan sulphate and aggrecan. Antibody binding was detected with the Vectastain ABC 'Elite' avidin/biotin/peroxidase kit. The extensor tendons in all the fingers had a metachromatic sesamoid fibrocartilage on their deep surface which immunolabelled for types I, III, and VI collagens, and for all glycosaminoglycans and aggrecan. Labelling for type II collagen was also seen in some fibrocartilages and was a constant feature of all index fingers. This probably relates to the greater use of that digit and the higher loads to which its tendons are subject. Chondroitin 6 sulphate and type II collagen are the most consistent markers of the fibrocartilage phenotype and most of the chondroitin 6 sulphate is probably associated with aggrecan. It is concluded that the labelling profile of the tendon fibrocartilage in the different fingers at the MCP joints is broadly similar to that at the PIP joints. Thus, the potentially greater level of compression on the extensor tendons may be counterbalanced by the lack of fusion of the tendon with the joint capsule. It is suggested that the maintenance of a similar level of fibrocartilage differentiation at two different points along the length of the extensor tendon ensures that the tensile strength is the same in the two regions and that no weak link is present. (+info)
The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulfate proteoglycan.
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Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and they are key components of germ cell tumors (GCTs). They express several high molecular weight glycoprotein antigens that are down-regulated upon differentiation. One of these antigens, defined by monoclonal antibody TRA-1-60, can be detected in the serum of GCT patients and provides a useful complement to the established serum markers human chorionic gonadotropin and alpha-fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or alpha-fetoprotein. To examine the relationship of the TRA-1-60-defined antigen to similar antigens defined by other monoclonal antibodies, we have carried out comparative Western blot and immunoprecipitation analyses of human GCT-derived cell lines with monoclonal antibodies TRA-1-60, TRA-1-81, GCTM2, and K21. The TRA-1-60 antigen was detected by Western blot analysis in extracts of all human EC cell lines and in clinical specimens of GCT tested as a diffuse band with a molecular weight of >200,000. A similar but noticeably fainter band was detected in GCT composed of seminoma only. The antigen was not expressed by GCT-derived lines without an EC phenotype. Affinity bead-purified TRA-1-60, TRA-1-81, GCTM2 and K21 antigens reacted in Western blot analysis with each of the other antibodies tested, indicating that the epitopes recognized by each antibody are carried by the same molecular species. This molecule could be metabolically labeled with inorganic [35S]sulfate and was degraded by keratanase. Glycopeptides produced from affinity-purified TRA-1-60 antigen by extensive digestion with Pronase exhibited a molecular weight in excess of 10,000 and were degraded by keratanase. The TRA-1-60 epitope was destroyed by digestion with neuraminidase, but the epitopes defined by TRA-1-81, GCTM2, and K21 were not. Our results indicate that human EC cells generally express a cell surface sialylated keratan sulfate proteoglycan that is subject to modification to yield a variety of epitopes, one of which is recognized by the monoclonal antibody TRA-1-60. Sensitivity to milk alkaline digestion suggests that the oligosaccharides of this proteoglycan are O-linked to a core polypeptide. (+info)
Serum levels of hyaluronan, antigenic keratan sulfate, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 change predictably in rheumatoid arthritis patients who have begun activity after a night of bed rest.
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OBJECTIVE: To evaluate whether and how moderate physical activity following a night of rest influences serum levels of matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), antigenic keratan sulfate (Ag KS), and hyaluronan (HA) in 10 normal subjects and 38 patients with rheumatoid arthritis (RA). METHODS: Blood was obtained from 20 RA patients before they arose from a night's sleep, and again 1 and 4 hours after they had begun to perform moderate physical activity. Another 18 RA patients remained in bed and blood was sampled at the same time periods. Serum levels of MMP-3, TIMP-1, Ag KS, and HA were measured by enzyme-linked immunosorbent assay. Clinical activity was evaluated by the Lansbury index. RESULTS: Both in normal subjects and in RA patients who did not remain in bed throughout the period of blood sampling, levels of HA, Ag KS, and MMP-3 increased significantly during the first hour after the subjects arose: the increase in HA and Ag KS correlated with the Lansbury index in the RA group. Three hours later, levels of Ag KS had dropped to baseline values in both groups of subjects. Levels of HA remained significantly and moderately elevated in the RA group but not in the control group, while levels of MMP-3 did not drop significantly in either group. In contrast, levels of HA, Ag KS, and MMP-3 did not change significantly in RA patients who had remained in bed. Unlike the other markers, the levels of TIMP-1 remained unchanged at the different time periods in all 3 groups studied. CONCLUSION: Significant changes in serum levels of some metabolic markers occur during the first hour after one arises from a night of sleep, especially in patients with RA. Measurement of the magnitude of these changes at different times in individual patients provides very different information about metabolic changes occurring in joint tissue than does measurement of the level of the markers at a single time point, as is usually currently reported. (+info)
Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.
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Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion. (+info)
600 MHz NMR studies of human articular cartilage keratan sulfates.
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The use of high-field two-dimensional 1H-correlation data is described for the detailed comparison of intact keratan sulfate polymer chains derived from human articular cartilage sources as a function of age. For fetal material the nonreducing chain termini are shown to be sparsely capped by sialyl groups which, if present, are exclusively (alpha2-3)-linked to an unsulfated galactose residue. The asialo capping segment has the structure: Gal-GlcNAc6S-Gal-GlcNAc6S-. Examination of keratan sulfate from 10-year-old cartilage shows that capping by sialyl groups is complete, with (alpha2-3)-linkages predominant; for both this and the 38-year-old cartilage the three capping structures: NeuAc(alpha2-3)-Gal-GlcNAc6S-Gal-GlcNAc6S-, NeuAc(alpha2-3)-Gal-GlcNAc6S-Gal6S-GlcNAc6S-, and NeuAc(alpha2-3)-Gal6S-GlcNAc6S-Gal6S-GlcNAc6S- are clearly recognizable. The level of (alpha2-6)-linked chain capping sialyl groups is significant for 38-year-old cartilage keratan sulfate. Structural information concerning the linkage region to protein and the distribution of galactose environments is readily obtained from the spectra. Signal complexities severely limit the usefulness of two-dimensional correlation spectroscopy at 600 MHz for the examination of N-acetylglucosamine residues within the poly(N-acetyllactosamine) repeat sequence and signals representing fucose placements remain undifferentiated. This nondestructive approach complements current degradative methods for the structural examination of keratan sulfates. (+info)
Mosaic characteristics of human endometrial epithelium in vitro: analysis of secretory markers and cell surface ultrastructure.
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Specific terminal carbohydrate structures and mucin-associated glycans increase in expression within the human endometrial epithelium during the secretory phase of the menstrual cycle but exhibit wide intercellular variation. We postulated that variation in glycosylation between cells would produce differences in the glycocalyx and result in complex mixtures of cells bearing different combinations of glycans. MUC-1 mucin, keratan sulphate and fucosylated lactosaminoglycans were examined in epithelial gland fragment cultures with antibodies (HMFG1, 5D4) and a lectin (Dolichos biflorus agglutinin). The glycocalyx was examined by transmission and high resolution scanning electron microscopy. The data were related to patterns of expression seen in vivo. The MUC-1 mucin was expressed relatively uniformly in culture, but heterogeneity was evident in mucin sialylation within the epithelial cell population. Double labelling of gland explant cultures for combinations of fucosylated lactosaminoglycans, keratan sulphate and MUC-1 demonstrated cells expressing all combinations of these markers. Ultrastructural examination confirmed remarkable intercellular variation in the glycocalyx. Though the human endometrial epithelium is relatively morphologically homogeneous, these observations reveal complex variations of cell surface glycosylation between neighbouring cells and suggest that secretory function might vary in a similar fashion. (+info)
Repair of human articular cartilage after implantation of autologous chondrocytes.
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Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage. (+info)
Oncostatin M induces leukocyte infiltration and cartilage proteoglycan degradation in vivo in goat joints.
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OBJECTIVE: To evaluate the effect of intraarticular injections of recombinant human oncostatin M (rHuOSM) in the goat joint. METHODS: One milliliter of endotoxin-free normal saline (vehicle) containing either 40 ng, 200 ng, or 1,000 ng of rHuOSM was injected into the right radiocarpal joints (RCJs) of 12 male angora goats, while the left RCJs were injected with an equivalent volume of vehicle alone. In subsequent studies, the right and left RCJs of 8 male angora goats were injected with 200 ng of rHuOSM, and 1 hour later, the right RCJs were injected with either 5 microg of recombinant murine leukemia inhibitory factor binding protein (rMuLBP) or 1 mg of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) in 1 ml of vehicle, while the left RCJs received 1 ml of vehicle alone. Goat joints were examined for clinical features of inflammation, and synovial fluid (SF) was aspirated on day 0 (before injection) and at days 2 and 6 postinjection. RESULTS: Injections of rHuOSM stimulated dose-dependent increases in the carpal:metacarpal ratio, SF volume, and SF leukocyte numbers, and stimulated dose-dependent decreases in the cartilage proteoglycan (PG) content ex vivo and PG synthesis. No significant changes were observed in the control joints that received saline alone, or between RCJs that were injected with 200 ng rHuOSM followed by 5 microg rMuLBP and RCJs that were injected with 200 ng of rHuOSM alone, except in respect to synovial fluid keratan sulfate concentrations, where a modest statistically significant reduction was observed in the joints injected with the combination of rHuOSM and rMuLPB. In contrast, RCJs injected with 200 ng rHuOSM followed by 1 mg of rHuIL-1Ra had significantly lower SF volumes (P<0.0001) and a significantly higher rate of ex vivo PG synthesis (P<0.0001). CONCLUSION: These results indicate that rHuOSM stimulates inflammation and modulates cartilage PG metabolism in vivo. Some of the effects of rHuOSM in vivo appear to be due, in part, to elaboration of IL-1. Even at very high doses, however, the rHuIL-1Ra did not attenuate OSM-mediated cartilage PG resorption. Thus, OSM has the potential to contribute to synovitis in vivo and can stimulate cartilage PG resorption in vivo, independent of IL-1. (+info)