Cartilage proteoglycan-induced arthritis in BALB/c mice. Antibodies that recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis.

Human fetal cartilage proteoglycan (PG) induces the development of an erosive polyarthritis and spondylitis in BALB/c mice. We have examined the properties of 3 monoclonal antibodies (MAb) to human fetal cartilage PG isolated from immunized mice that cross-react with mouse cartilage PG. Compared with sera from arthritic mice, which contain antibodies reactive with keratan sulfate, MAb 202 (IgG1) reacted only with a protein-related epitope that is distributed on both hyaluronic acid-binding and chondroitin sulfate-attachment regions. MAb 813 (IgG1) reacted with the same fragments and recognized an epitope with the immunologic characteristics of keratan sulfate. MAb 945 (IgM) remains to be further characterized. Introduction of hybridomas secreting MAb 202 and MAb 945 into irradiated mice resulted in the loss of PG from articular cartilage and from growth plate cartilage (with MAb 202 only), as revealed by a loss of staining with toluidine blue. There was no synovial hyperplasia with MAb 202, but some hyperplasia and mononuclear cell infiltration was seen with MAb 945. This was accompanied by the binding of immunoglobulins to articular cartilage, as demonstrated by immunofluorescence. The hybridoma secreting MAb 813 produced no cartilage changes or synovitis, and there was no immunoglobulin binding to cartilage. Polymorphonuclear leukocyte infiltration was never observed with these antibodies. These studies indicate that MAb reactive with mouse cartilage PG can cause the depletion of PG from hyaline cartilage by mechanisms that may be both complement dependent and complement independent. Antibodies may serve to release and expose PG antigen to immune cells, as well as causing a loss of the mechanical properties of cartilage that are PG dependent.

Performance, carcass, cartilage calcium, sensory and collagen traits of longissimus muscles of open versus 30-month-old heifers that produced one calf.

One hundred eleven Simmental x Hereford (3/8 to 5/8 Simmental) heifers were used to determine the effects of age, parturition and implantation on performance, carcass and meat-sensory traits, muscle-collagen characteristics and thoracic-button calcification. Eighty-five heifers that calved at about 2 yr of age, designated as single-calf heifers (SCH), were either implanted (I-SCH) with Synovex-H or not implanted (NI-SCH). The remaining 26, 2-yr-old non-pregnant heifers (2-OH) served as controls. Additionally, 24, 1-yr-old open heifers (1-OH) from the same genetic source were utilized as the standard heifer-production system. The 1-OH and 2-OH were slaughtered after being fed a high-grain diet for 137 and 112 d, respectively. The SCH were fed the same high-grain diet beginning about 1 mo after calving and were fed 137 d before slaughter. The 33 I-SCH were implanted when started on the high-grain diet. Calves were weaned about 5 wk before the SCH were slaughtered. The 2-OH had the highest (P less than .05) feedlot ADG, whereas no differences (P greater than .05) occurred among other treatments. Dressing percentages were higher (P less than .01) for I-SCH than for NI-SCH. Carcass weights were lowest (P less than .05) and percentage kidney, pelvic and heart fat was highest (P less than .01) for 1-OH. Fat thickness, yield grades, marbling scores and quality grades were similar (P greater than .05) and desirable for all treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

Micromechanical spectroscopy of cartilage proteoglycans: hydration.

Proteoglycan subunits extracted from calf cartilage have been studied with a high resolving power mechanical spectroscopy: the Thermostimulated Creep (TSC). The influence of hydration on TSC spectra shows the existence of two types of bound water: the weakly bound water increases the inertia of proteoglycan and stiffens their structure; the strongly bound water is responsible to a compensation law indicating the existence of a resonance phenomenon at the physiological temperature. Because of the looseness of bonds in weakly bound water, an increase of the local pressure may induce, in vivo, a release of water in tissues. This hypothesis explains perfectly the role of a water pump of proteoglycans in cartilage.

An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein.

A glycosylated protein (small, cartilage-derived glycoprotein, SCGP) of approximately 18 kDa with unknown function has been isolated from dissociative extracts of bovine nasal cartilage and its primary structure determined. The protein has 121 amino acids, giving a calculated protein molecular weight of 13,878, four disulfide bonds, two N-linked oligosaccharides and one O-linked oligosaccharide. In nasal cartilage, this glycoprotein is in molar concentrations equivalent to 1/5-1/2 that of the link protein of cartilage proteoglycan aggregates, and it has also been isolated from bovine articular cartilage and from bovine fetal epiphysis. The N-terminal, glycosylated region of the molecule is relatively rich in arginine, proline, glycine, and threonine. The C-terminal 82 amino acids (which contains all four of the disulfide bonds and none of the carbohydrate) can be found as a discrete entity in cartilage extracts, indicating that the N-terminal domain is readily removed by extracellular proteolytic attack.

Identification of an inhibitor of neovascularization from cartilage.

Certain tissues such as cartilage are resistant to vascular invasion, yet no single tissue-derived molecule that can inhibit angiogenesis has been reported. A protein derived from cartilage was purified that inhibits angiogenesis in vivo and capillary endothelial cell proliferation and migration in vitro in three separate bioassays. This protein is also an inhibitor of mammalian collagenase. These findings may help elucidate the mechanisms by which neovascularization is controlled in both normal and pathological states.

Concentration of ciprofloxacin in bone tissue after single parenteral administration to patients older than 70 years.

The concentrations of ciprofloxacin produced in bone, cartilage and menisci after a single administration of 200 mg were determined at different intervals in a group of patients with an average age of 80 years. Concentrations of 0.11 to 0.94 mg/kg bone tissue were measured after 0.5 to 5 hours. In the cartilage a concentration of active substance was measureable only once (4.18 mg/kg). In the presence of marked circulatory disorders the active substance concentrations reached in the bone were above those found in the seriously damaged muscle. Although the concentrations reached in the bone are effective, no risk should be taken in osteomyelitis. Ciprofloxacin should therefore be used at high dosage and possibly be combined with another substance. Given for therapeutic purposes, a single dose of ciprofloxacin is naturally not effective enough, and given for prophylactic purposes, not safe enough to prevent a post-traumatic osteitis.

Glial fibrillary acidic protein immunoreactivity of chondrocytes in immature and mature teratomas.

The immunoreactivity of chondrocytes for glial fibrillary acidic protein (GFAP), other intermediate filament proteins and S-100 protein was studied in formalin-fixed paraffin-embedded sections. A total of 95 cartilage specimens were examined from five immature teratomas, 12 mature teratomas, and a teratocarcinoma. GFAP-immunoreactive chondrocytes were abundant in immature cartilages, and as the cartilages maturated, these chondrocytes decreased and became distributed peripherally. Elastic cartilage had more GFAP-immunoreactive chondrocytes than non-elastic cartilage. GFAP-immunoreactive cartilage was often located close to central nervous tissue. Immunostaining for vimentin and S-100 protein revealed extensive distribution of immunoreactive chondrocytes in immature and mature cartilages, but in mature cartilage, chondrocytes at the center had less vimentin immunoreactivity. GFAP-immunoreactive chondrocytes also showed apparent immunostaining for vimentin. There was no difference in immunohistochemical staining for the alpha and beta subunits of S-100 protein. The immunoreactivities of teratoma cartilage specimens were quite similar to those of respiratory tract cartilage.

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules.

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.

Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan with an extraordinarily large chondroitin sulfate chain and short alpha 1 polypeptide.

We have prepared a high buoyant density proteoglycan fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an alpha 1(IX) chain Mr approximately 23,000 shorter than the known alpha 1 in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the alpha 2(IX) polypeptide; its Mr is approximately 350,000 indicating that it is approximately 10 times larger in vitreous humor than in cartilage. Examination of vitreous bodies at different developmental stages indicates that a transition occurs in the size of alpha 1(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type alpha 1(IX) of Mr 84,000 is the predominant species, whereas at stage 36 and thereafter, a Mr 61,000 species appears with a concomitant disappearance of the Mr 84,000 species. Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fibrils, thus contributing to structural as well as functional properties of this unique matrix.

In situ cross-linking of cartilage proteoglycans.

Although the in vitro interactions between purified cartilage matrix components have been studied extensively, little is known about these interactions in situ. In this study, cartilage was treated with a cross-linking reagent with a span of 1.2 nm between its reactive terminal groups in order to preserve the native relationships between closely associated matrix components throughout extraction, purification, and preparation for electron microscopy. After in situ cross-linking, electron microscopy and gel chromatography revealed that about one-half of the guanidine hydrochloride extractable proteoglycans were polymeric, usually with two to five proteoglycan subunits in each polymer. Cross-linking consistently involved the thin segments of the proteoglycan subunits. Some of the proteoglycan polymers were capable of binding hyaluronic acid and were parts of aggregates under associative conditions. SDS-polyacrylamide gel electrophoresis revealed that link proteins were present within the polymers, and studies in which purified proteoglycans were cross-linked in vitro confirmed that the link proteins increased the proportion of polymeric proteoglycans. These findings suggest that individual proteoglycans within cartilage have intimate associations with other proteoglycans that are mediated by link proteins.

Metabolic fate of exogenous chondroitin sulfate in the experimental animal.

After the administration of tritiated chondroitin sulfate (CS) by oral and intramuscular route, the distribution of radioactivity was investigated in two opportunist omnivorous animals, namely the rat and the dog. More than 70% of the orally administered radioactivity was absorbed. Independently of the administration route, radioactivity was mainly excreted through the urine. Plasma levels showed a rapid increase after oral administration, followed by a large plateau with a maximum at the 14th and 28th h in the rat and in the dog, respectively. A tropism of the radioactivity was observed towards glycosaminoglycan-rich tissues, such as joint cartilage. The analysis of the molecular weight of the radioactive material showed that compounds with a molecular weight corresponding to those of CS, poly-, oligo- and monosaccharides as well as of tritiated water, were present in the plasma, urine, synovial fluid and cartilage. The level of radioactive low molecular weight material, derived from the metabolism of CS and from the exchange reaction, increased with the time after administration. The high molecular weight fraction represented at least 10% of the orally administered CS.

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes.

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.

Inhibition of sulphate incorporation by chondrocytes in intact cartilage by hyaluronate from foetal cartilage.

A number of regulators are available in cartilage to effect the local control of matrix production by chondrocytes. A cartilage slice assay has been used in this study to investigate the influence of such regulators (extracted from foetal cartilage) on intact cartilage. A net inhibition of sulphation was found, rather than stimulation as reported for extracts rich in the somatomedin-like, cartilage derived factor (CDF). Inhibition was due, to a high molecular weight component identified as hyaluronic acid (based on enzyme sensitivity, chromatographic behaviour and temperature stability). Its inhibition of sulphation in intact cartilage was more profound than that produced by commercially available umbilical cord hyaluronate. We conclude that foetal cartilage hyaluronate is a far more potent inhibitor of sulphation than hyaluronate from other sources, suppressing sulphation even in the presence of a somatomedin-like activator and in intact cartilage, which responds poorly to commercial hyaluronate.

[Structural characteristics of collagens from the skin and rib cartilage of patients with Ehlers-Danlos syndrome type II]. / Strukturnye kharakteristiki kollagenov kozhi i rebernogo khriashcha bol'nykh s sindromom Elersa-Danlo II tipa]

Collagens were analyzed in skin and rib cartilage of 9 patients with Ehlers-Danlos syndrome of the II type. Electrophoresis and CNBr-peptide mapping showed that extended inserts and deletions as well as rough impairments of post-translation processing were not detected in collagens of the I, II and III types from these patients. In the patients with Ehlers-Danlos syndrome of the II type distinct increase was observed both in the total ratio of collagens III/I (P = 0.95) and in the ratio of intact collagens III/I free of cross-links. A decrease in content of dimers beta 11 and beta 12 was found in two patients. The data obtained suggest that the Ehlers-Danlos syndrome of the II type involved deteriorations in the structure of collagens I responsible for decrease in stability and sometimes for impairments in cross-link formation. Increase in content of collagen II fraction, predisposed to proteolytic hydrolysis of terminal sites, as well as elevated sensitivity of collagen II to pepsin hydrolysis were found in collagens of rib cartilage from patients with the syndrome and with funnel chest deformation. This suggests the lowered stability of collagen II from rib cartilage in funnel chest deformation.

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate.

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Isolation and analysis of ribonucleic acids from skeletal tissues.

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.