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Proteoglycan-induced arthritis is a mouse model displaying many similarities to human rheumatoid arthritis and ankylosing spondylitis which has been documented by clinical and histopathological studies. The development of the disease in genetically susceptible BALB/c mice is dependent upon the expression of both cell-mediated and humoral immunity to host mouse cartilage proteoglycan. Since both development and regression of acute inflammatory processes in joints correlate directly with the serum antibody level to mouse cartilage proteoglycan, it is believed that these autoreactive antibodies may play a key role in the pathological mechanism of proteoglycan-induced arthritis. The treatment of arthritic animals with an immunomodulating agent (leflunomide) suppressed acute inflammatory events, protected animals from new inflammatory episodes or acute exacerbations in chronically inflamed joints and blocked pathological processes in arthritic joints, which otherwise led to progressive deformities, ankylosis and the loss of articular cartilage. We conclude that the suppressive effect of leflunomide (HWA 486) in proteoglycan-induced arthritis primarily is due to the suppression of autoantibody formation and that the drug may be a potential agent in human therapy as well. Further, we feel that this novel model of murine polyarthritis will extend further the pharmacological repertoire necessary to discover innovative antirheumatic drugs.

Articular cartilage explants from bovine calf and steer were cultured for up to 19 days in medium with or without 20% fetal bovine serum (FBS) or insulin-like growth factor I (IGF-I). Lower concentrations of IGF-I were required for maximal stimulation of PG synthesis in calf than in steer. In calf, but not in steer, IGF-I was as effective as 20% FBS in stimulating PG synthesis. The stimulation by IGF-I or FBS was not accompanied at either age by alterations in the composition of the aggregating PGs nor by changes in the proportions of CS-rich and CS-poor PG subpopulations. In calf, IGF-I and FBS did not markedly alter the rate of turnover of either the 35S-PGs synthesized in vitro or the unlabeled PGs. In steer, explants cultured in the absence of IGF-I or FBS exhibited very fast rates of turnover and depletion of matrix PG with time; IGF-I and FBS were both effective in reducing the turnover rate of 35S-PGs and unlabeled PGs and in preventing PG depletion.

Introduction Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration. Methods We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue. Results The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated protein kinase and NF-κB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects. Conclusion FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.

The interfibrillar spacing of collagen fibrils was measured at twenty different positions across a macular dystrophy cornea using synchrotron X-ray diffraction. Unlike previous work of this type the cornes had not been frozen for storage. The spacings were all significantly lower than the spacings which existed at similar positions across a normal adult human cornea. This close-packing of collagen fibrils seems to be responsible for the reduced thickness of the central cornea in macular dystrophy. Neither the patient's serum or corneal tissue contained appreciable amounts of sulfated keratan sulfate, this classifies the disease as Type I macular corneal dystrophy.

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1 – mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.

Objective To further characterize the role of the carbohydrate sulfotransferase( CHST6 ) gene in macular corneal dystrophy (MCD) through identification of causative mutations in a cohort of affected patients from southern India. Methods Genomic DNA was extracted from buccal epithelium of 75 patients (51 families) with MCD, 33 unaffected relatives, and 48 healthy volunteers. The coding region of the CHST6 gene was evaluated by means of polymerase chain reaction amplification and direct sequencing. Subtyping of MCD into types I and II was performed by measuring serum levels of antigenic keratan sulfate. Results Seventy patients were classified as having type I MCD, and 5 patients as having type II MCD. Analysis of the CHST6 coding region in patients with type I MCD identified 11 homozygous missense mutations(Leu22Arg, His42Tyr, Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Glu274Lys), 2 compound heterozygous missense mutations(Arg93His and Ala206Thr), 5 homozygous deletion mutations (del CG707-708, del C890, del A1237, del1748-1770, and del ORF), and 2 homozygous replacement mutations(ACCTAC 1273 GGT, and GCG 1304 AT). One patient with type II MCD was heterozygous for the C890 deletion mutation, whereas 4 possessed no CHST6 coding region mutations. Conclusion A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I MCD in a cohort of patients in southern India. Clinical Relevance An improved understanding of the genetic basis of MCD allows for earlier, more accurate diagnosis of affected individuals, and may provide the foundation for the development of novel disease treatments.

PURPOSE: Synchrotron x-ray diffraction experiments indicate that corneas with macular corneal dystrophy (MCD) contain unusual 4.6-A periodic repeats thought to reside in proteoglycans or glycosaminoglycans. Recently the 4.6-A x-ray reflection was found to be significantly diminished after incubation of MCD specimens in buffer containing chondroitinase ABC or N-glycanase. We examined the sulfated proteoglycans in these glycosidase-digested MCD corneas. METHODS: Transmission electron microscopy was used in conjunction with cuprolinic blue-staining for sulfated proteoglycans. RESULTS: Incubation of an MCD specimen in enzyme buffer left both small and large proteoglycan filaments in the stromal matrix, whereas incubation in the presence of chondroitinase ABC removed these molecules from the tissue. Incubation in buffer containing N-glycanase, on the other hand, removed the large proteoglycan filaments from the MCD stroma but left unaffected the small collagen-associated proteoglycans. CONCLUSION: These results are consistent with the interpretation that 4.6-A periodic repeats in MCD corneas reside in large sulfated proteoglycan filaments (or aggregates thereof) that may contain chondroitin/dermatan sulfate and keratan sulfate or keratan components.

Injection of chymopapain (CP) into the rabbit knee causes rapid depletion of proteoglycans (PGs) in the articular cartilage. Successful repair of the articular cartilage follows injection of only 0.2 mg CP whereas in animals injected with 0.2 mg CP, the repair phase fails leading to cartilage destruction within 21 days. Previous work by the authors indicated that daily oral diclofenac reduced the severity and incidence of CP-induced cartilage injury after 21 days. The present study was designed to determine if administration of diclofenac promoted articular cartilage repair after CP injury over a longer period of time and if coadministration with misoprostol could reduce the concentration of diclofenac needed to promote repair as well as confer protection to the gastric mucosa. Adolescent male New Zealand white rabbits received CP-induced cartilage injury (2.0 mg) followed by daily oral diclofenac, diclofenac/misoprostol or misoprostol. Control animals received diclofenac, diclofenac/misoprostol, or misoprostol or no drug treatment with no cartilage injury. All animals were sacrificed after 2 months. Examination of the stomachs from animals receiving no drug or misoprostol only were completely normal. Most animals receiving diclofenac only had abnormal gastric mucosa. In contrast, coadministration of diclofenac/misoprostol revealed significantly fewer abnormalities. Analysis of the composite histologic score of patellae and measurements of cartilage PG content revealed no significant differences in groups which received CP injury with and without drug treatment. Histology scores and cartilage PG content from animals in groups which did not receive CP injury, but were treated with diclofenac/misoprostol, diclofenac or misoprostol were within normal limits and thus did not differ significantly from untreated control cartilage. The results of this study indicate that daily administration of diclofenac, diclofenac/misoprostol or misoprostol has no protective effect on CP-induced cartilage injury after 2 months. Administration of diclofenac only results in severe gastric abnormalities which are significantly reduced if diclofenac is coadministered with misoprostol. Administration of diclofenac, diclofenac/misoprostol or misoprostol has no effect on PG content of cartilage from otherwise normal joints.

Continuous passive motion facilitates repair of full-thickness defects in the articular cartilage in rabbits. Studies were conducted to determine whether continuous passive motion would similarly affect the repair process after injection of chymopapain into the rabbit knee. Adolescent rabbits were injected with chymopapain and then given intermittent active motion in the form of free cage activity or continuous passive motion of the injected knee. After injection of either 0.2 or 2 mg chymopapain into the knee, serum keratan sulfate levels rose sharply, indicating proteoglycan loss, and, in all cases, peaked at 24 hours between 200-800% of preinjection levels. Importantly, serum keratan sulfate levels were significantly elevated within 1 hour when joints were submitted to immediate continuous passive motion after the injection. As shown previously, injection of either 0.2 or 2 mg chymopapain into the knee, followed by intermittent active motion, resulted in a pronounced loss of proteoglycans by Day 2, partial restoration of proteoglycans by Day 9, continued proteoglycan synthesis by Day 21 in animals receiving the lower dose, and severe degenerative changes by Day 21 in animals receiving the higher dose. In all animals that received either high or low doses of chymopapain, as well as 2 days of intermittent active motion and then continuous passive motion, the articular cartilage surface was intact by Day 9, and replenishment of proteoglycans had occurred in pericellular and interterritorial areas. By Day 21, the surface of the articular cartilage was still intact, and replenishment of proteoglycans in loaded regions continued in all animals receiving either dose of chymopapain. These results indicate that a period of intermittent active motion followed by continuous passive motion of a chymopapain injected knee may protect and stimulate repair of the articular cartilage matrix after chymopapain-induced injury.

We examined the early changes that follow injection of sodium iodoacetate (IA) into the guinea pig knee joint, using Safranin-O (SO) to stain the proteoglycan (PG)- rich matrix and the incorporation of Na 35 SO4 to deter mine which cells have maintained, lost, or developed the ability to synthesize significant amounts of PGs. Within 24 hours following injection of IA, articular chon drocytes exhibited a marked loss in ability to synthesize PGs; this inhibition was complete after 2 weeks. Focal loss of SO staining of the interterritorial matrix was noted after 24 hours, progressing to a complete loss of staining after 2 weeks. In sharp contrast to the degenerative changes in the habitually loaded articular cartilage, cells at the chondrosynovial junction began incorporating Na 35 SO4 and producing a matrix that stained with SO as early as 72 hours following injection. With time, this resulted in the development of prominent osteophytic cartilage that involved bone remodeling beneath the overlying cartilaginous cap after 3 weeks.

We have developed an enzyme-linked immunosorbent assay (ELISA) with an inhibition step to quantify hen egg white lysozyme (HEWL) on a weight basis. The assay is relatively insensitive to changes in ionic strength or pH. Quantification is not affected by the presence or large amounts of mammalian lysozymes or inhibitors of enzymatic activity such as soluble chitin oligomers. We used this ELISA to show that the HEWL content of chick cartilages increases progressively between days 14 and 18 of embryonic life. The maturation-related increase does not appear to be the result of increased synthesis by the chondrocytes for the latter did not synthesize detectable amounts of lysozyme in vitro. Evidence was obtained to suggest that most, if not all, the lysozyme in cartilage is derived from the surrounding fluids which come in contact with the matrix.

Neutrophil release of elastase is postulated to contribute to the pathogonesis of the adult respiratory distress syndrome (ARDS). This enzyme cleaves basement membrane and connective tissue proteins and is able to activate other inflammatory mediators. Extracellularly released elastase is rapidly complexed with inhibitor proteins, primarily alpha 1 -protease inhibitor. These enzyme-inhibitor complexes diffuse or are washed into the circulation before clearance. We postulated that patients with ARDS would have elevated plasma levels of elastase complex as compared to normals. We utilized an enzyme-linked immunoassay to quantify lavels of elastase bound to alpha 1 -protease inhibitor in plasma samples from patients with well-established ARDS, normal laboratory workers, and hospitalized patients with chronic noninfectious disease processes. The plasma level (mean ± SD) of elastase complex in normais was 98 ± 27 ng/mL and in hospitalized patients was 110 ± 41 ng/mL. Patients with ARDS had markedly elevated plasma lovels of elastase complex, 1,018 ± 901 ng/mL as compared to controls ( P 1 -protease inhibitor in patients with ARDS suggest that neutrophil release of this degradative enzyme may be pathophysiologically related to the pulmonary and multiorgan injury that characterizes this disorder.

The distribution of sulphated proteoglycans within the stromas of three patients (A,B,C) suffering from macular corneal dystrophy was studied using the specific dye Cuprolinic Blue in a ‘critical electrolyte concentration’ method. The corneas were examined using transmission electron microscopy and A and C were further studied by low-angle synchroton X-ray diffraction. Sera from all three patients were analyzed for the presence of keratan sulphate using a monoclonal antibody in an enzyme-linked immunosorbent assay. The serum from Patient A contained keratan sulphate, but the chains were thought to be shorter or less sulphated than normal. Patients B and C, on the other hand, contained no detectable keratan sulphate in their sera. Electron microscopy showed many electron-transparent lacunae randomly distributed through-out the specimens. The average collagen fibril diameter was normal but there were differences in packing between the specimens. Specimen A was closely-packed with most collagen fibrils in contact with their neighbours. Specimens B and C showed fewer regions of close packing; in most of the tissue the interfibrillar spacing appeared normal. Staining with Cuprolinic Blue revealed an unusual distribution of proteoglycans in some parts of the interfibrillar matrix. particularly in A, with ‘small’ proteoglycans running exclusively parallel to the collagen fibrils. Furthermore in A, and to a lesser extent in B and C, some lacunae were filled with clusters of abnormal sulphated proteoglycan filaments (of various sizes) which were chondroitinase ABC susceptible. Clearly defined regions, both within the lacunae and elsewhere, failed to stain with Cuprolinic Blue; this suggests an absence of sulphated proteoglycans within these areas. Equatorial X-ray diffraction of the wet tissues (A and C) gave values for the mean interfibrillar centre-to-centre separation of 43±2 nm in Specimen A and 52±3 nm in Specimen C. The differences observed in the serum keratan sulphate levels, the packing of the collagen fibrils and the distribution of chondroitin/dermatan sulphate proteoglycans confirm the heterogeneity that exists within the macular corneal dystrophies.

Serum levels of keratan sulfate (KS), measured by an enzyme-linked immunosorbent-inhibition assay, were found to be significantly higher in 31 patients with hypertrophic osteoarthritis (OA) than those in 41 adults without joint disease. Seventy-seven percent of patients with OA, but only 12% of control subjects, had serum levels which were more than 1 SD above the mean of the control group. Following replacement of a single osteoarthritic hip joint, serum KS levels decreased, at first, in all patients. Subsequently, the concentration of serum KS progressively increased; 6 months following surgery, KS levels were similar or close to the preoperative levels in virtually all patients. The results suggest that patients with hypertrophic OA may have a generalized imbalance of cartilage proteoglycan metabolism. Measurements of serum KS are likely to prove most useful in studying this particular subset of patients with generalized OA.

Previous studies on chondrosarcoma proteoglycans have suggested that keratan sulfate content and average keratan sulfate chain length may be inversely related to the degree of cartilage tumor malignancy. The present study extended this work to an analysis of serum keratan sulfate in chondrosarcoma patients. Keratan sulfate levels of 24 adult patients bearing cartilage tumors were observed to be higher than that for age- and sex-matched controls. In nine of ten patients, keratan sulfate level exhibited a significant decrease following removal of the tumor. Analysis of five general hospital patients before and after surgery did not detect such a change. The data were suggestive of an inverse relationship between tumor histopathologic grade and presurgical serum keratan sulfate concentration. Finally, despite the wide range of serum values within each histopathologic tumor group, sequential serum keratan sulfate measurements may be useful in monitoring patients for secondary growth of chondrosarcoma tumors after primary excision.

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.

Sensitive measurements of serum keratan sulfate (KS), a glycosaminoglycan found in large quantities in the proteoglycans of human cartilage, can be obtained using an enzyme-linked immunosorbent-inhibition assay. Patients who are undergoing chemonucleolysis (CN) provide a clinical opportunity to monitor the large-scale proteolytic degradation of cartilage. By measuring serum KS levels both pre- and post-CN, we have demonstrated that serum KS levels rise predictably after CN, in a manner that reflects major catabolic events of cartilage.

Objective: To further our understanding of the pathogenesis of chondromalacia of the patella (CM), we have studied the release into knee joint fluid and serum, obtained from patients with CM, of molecules associated with the metabolism of joint cartilage matrix and synovium.Methods: Interleukin-1 α (IL-1α), interleukin-1 β (IL-1β), interleukin-6 (IL-6), stromelysin-1 (MMP-3), interstitial collagenase (MMP-1), tissues inhibitor for metalloproteinases-1 (TIMP-1), phospholipase activity A2(PLA2), hyaluronan (HA), aggrecan fragments (AGN) and antigenic keratan sulfate (KS) were quantified in knee joint lavage fluid from 96 patients with CM; KS and HA also was measured in serum. Chondromalacia was graded on a scale of I to IV according to Outerbridge (1961). The histopathology of the synovial membrane close to the patellofemoral joint was evaluated. Control samples were obtained from nine patients with knee pain presenting with arthroscopically normal knee joints.Results: The concentrations of MMP-3, MMP-1 and TIMP-1 proteins in joint lavage fluid were increased in advanced (grade IV) CM, compared with controls. Levels of MMP-1 in lavage fluid correlated with the severity of CM (r=0.38,P

Summary Objective To examine the effect of β-aminopropionitrile (BAPN), an inhibitor of lysyl oxidase, on growth and remodeling of immature articular cartilage in vitro . Design Immature bovine articular cartilage explants from the superficial and middle layers were cultured for 13 days in serum-containing medium with or without BAPN. Variations in tissue size, accumulation of proteoglycan and collagen (COL), and tensile mechanical properties were assessed. Results The inclusion of serum resulted in expansive tissue growth, stimulation of proteoglycan and COL deposition, and a diminution of tensile integrity. Supplementation of medium with BAPN accentuated this phenotype in terms of a further increase in tissue size in explants from the superficial layer and further diminution of tensile integrity, without affecting the contents of proteoglycan and COL in explants from both the superficial and middle layers. Conclusion COL crosslinking is a major factor in modulating the phenotype of cartilage growth and the associated balance between proteoglycan content and integrity of the COL network.

Summary Objective To determine the effects of exercise and weight loss interventions on serum levels of four biomarkers and to examine if changes in biomarker levels correlate with clinical outcome measures in obese and overweight adults with knee osteoarthritis (OA). Methods Serum was obtained at baseline, 6 and 18 months from 193 participants in Arthritis, Diet and Activity Promotion Trial. This was a single-blind 18-month trial with subjects randomized to four groups: healthy-lifestyle (HL), diet (D), exercise (E) and diet plus exercise (D+E). Serum levels of cartilage oligomeric matrix protein (COMP), hyaluronan (HA), antigenic keratan sulfate (AgKS), and transforming growth factor-β1 (TGF-β1) were measured by enzyme linked immunosorbent assay. Results At baseline there were no significant differences in biomarker levels between intervention groups. When results for all the intervention groups were combined, the levels of HA were found to be negatively correlated with medial joint space width and positively correlated with Kellgren–Lawrence scores (K–L scores) while TGF-β1 levels negatively correlated with K–L scores. When biomarker levels measured at 6 and 18 months were adjusted for baseline values, age, gender, and body mass index, weak but significant differences between intervention groups were present for mean levels of COMP and TGF-β1. Furthermore, AgKS levels averaged over all groups tended to decrease over time. There were no significant associations of baseline biomarkers and the follow-up outcomes. Weak associations were noted between change in the biomarkers at 18 months and change in outcome measures that included change in weight with AgKS and COMP and change in Western Ontario and McMaster Universities Osteoarthritis Index pain with AgKS. Conclusion Overall, the E and D interventions did not show a consistent effect on levels of potential OA biomarkers. The four biomarkers showed differences in correlations with outcome measures suggesting that they may measure different aspects of disease activity in OA. The strongest correlations were between serum HA and radiographic measures of OA at baseline.

A sensitive and highly specific enzyme-linked immunosorbent assay with an inhibition step was used to monitor the concentration of keratan sulfate, a cartilage-related glycosaminoglycan, in the serum of three adult male deer. During the course of one complete annual antler regeneration cycle, keratan sulfate levels were found to fluctuate predictably in relation to the growth and maturation of the antlers: levels are substantially elevated during the growth phase and drop precipitously when growth ceases and the antlers become fully mineralized. In addition, an unanticipated elevation of serum keratan sulfate was observed in early spring prior to casting of the preceding year's antlers and the initiation of regrowth. This suggests that changes in cartilage metabolism occur concomitantly, with this phase of the antlerogenic cycle. These results show predictable and physiologically regulated variation in serum keratan sulfate levels which correlate directly with specific phases of the antler regeneration cycle. Furthermore, the findings provide additional support for the assertion that measurements of keratan sulfate levels in serum can provide useful information about changes in cartilage metabolism in normal as well as diseased states.

Very thin slices of the superficial and deep layers of osteophytic and apparently normal articular cartilage from six human osteoarthritic femoral heads were digested with papain. The digests were analyzed for keratin sulfate content using an enzyme-linked immunosorbent assay (ELISA) with inhibition step, and for chondroitin sulfate and collagen contents using biochemical assays. Although there were marked differences in Safranin-O staining of the superficial and deep layers of osteophytic cartilage, these two layers had identical high ratios of chondroitin sulfate/collagen. Keratan sulfate was present only in small amounts in osteophytic cartilage. However, the deeper layer contained significantly more of this glycosaminoglycan. The deeper layer of articular cartilage contained approximately twice as much chondroitin sulfate and six times more keratan sulfate relative to collagen than the superficial layer. The results of this study showed that this new sensitive approach, which requires as little as 200 micrograms wet cartilage as starting material, provides important qualitative and quantitative information about the major constituents of the matrix.

A monoclonal antibody (ET-4-A-4) was used to identify keratan sulfate (KS) as a constituent of bovine flexor tendon. KS was present in at least 500-fold higher amounts in the fibrocartilaginous region of the tendon that is subjected to compressive forces in vivo (mean = 0.03% of tissue dry weight) than in the more proximal regions subjected only to tensional forces. The KS was associated with proteoglycans of three size categories that could be separated by Sepharose CL-4B chromatography. The largest proteoglycan (Vo) contained approximately 4% KS and was predominant in the surface region of the tendon subjected directly to compressive forces. A population of somewhat smaller molecules (Kav approximately equal to 0.3) contained less than or equal to 20% KS and proportionately less chondroitin sulfate (CS) or dermatan sulfate (DS). The KS chains of this population were not digested by keratanase. This population was dominant in tissue from the middle layer of the fibrocartilaginous region. A third population of small KS proteoglycans or fragments (Kav approximately equal to 0.6) comprised 40% of the KS found in the deepest layer of the compressed tendon region. This smaller component was independent of the small DS proteoglycans.

Articular cartilage is a specialized form of connective tissue which covers the ends of the bones in synovial joints. It supplies elasticity and resistance to compressive forces thereby protecting the more rigid underlying bone and ensuring smooth articulation at the joint surfaces. Cartilage has a very prominent extracellular matrix which is produced and maintained by relatively few cells, the chondrocytes (See recent reviews, [1–3]). The two major matrix constituents synthesized by these cells are collagens and proteoglycans (PGs). The remainder includes other proteins, the functions of which are poorly understood, as well as, lipids, nutrients and inorganic material.

Serum was obtained from 155 children at the time of admission to hospital for elective surgery. The concentration of serum keratan sulphate was determined by an ELISA which uses an antibody specific for keratan sulphate, a molecule found predominantly in cartilage. Concentrations of keratan sulphate rise progressively during the first four years of life (0-2: mean = 357 micrograms/l; 2-4: mean = 422 micrograms/l) and then remain high until 12 years of age (mean = approx. 500 micrograms/l). At this time, concentrations drop markedly (13-year olds: mean = 377 micrograms/l; 14-year olds: mean = 318 micrograms/l). After age 15, concentrations continue to fall toward the concentrations found in normal adults. Serum concentrations did not show significant differences with respect to disease category, sex or race but were found to vary, sometimes markedly, from child to child at any one age. The results suggest human cartilage undergoes significant changes in metabolic activities during maturation. Measurements of keratan sulphate concentration in serum may prove useful in studying the biochemical and physiological bases of these changes and in monitoring growth or endochondral ossification during maturation.

We have developed a new ELISA to quantify hyaluronate. This ELISA takes advantage of an anti-keratan sulfate antibody to differentiate between the coated aggregating rat chondrosarcoma proteoglycan which captures the hyaluronate and the keratan sulfate-bearing aggregating proteoglycan added subsequently. Absorbance in this assay was shown to be linear to the logarithmic concentration of hyaluronate in the range of 15 to 1000 ng/ml. Pre-treatment of hyaluronate with papain or protease did not interfere with its quantification; in contrast, pre-treatment with 0.1N NaOH significantly interferes with the subsequent measurement of the hyaluronate molecules. The size of the hyaluronate molecule was found to be an important factor in quantification: only large size hyaluronate molecules could be quantified accurately. The ELISA was used to show that human lung carcinomas contain 2 to 500 times as much hyaluronate as normal lung tissue from the same patient.

Publisher Summary This chapter describe three procedures which have proved useful in the isolation and characterization of newly synthesized proteoglycan and link protein. The first deals with the isolation of newly synthesized link protein and core protein precursor, as well as completed forms of the proteoglycan by taking advantage of their ability to be incorporated into aggregates recoverable from the bottom of CsCl density gradients. The second presents refinements of immunoprecipitation procedures, while the third describes the separation of two populations of newly synthesized aggregating proteoglycan by agarose gel electrophoresis. Bovine articular cartilage aggregating proteoglycans exhibit age-related differences. Electrophoresis in acrylamide-agarose composite gels has been used successfully to show that the matrix contains two populations of aggregating proteoglycans which are present in different ratios at different ages. Electrophoresis in 1% agarose gels has recently been found to offer a number of advantages over the acrylamideagarose composite gel procedure.

We have developed an enzyme-linked immunosorbent-inhibition assay which makes use of a monoclonal antibody specific for keratan sulfate to quantify keratan sulfate present as single chains in adult human serum. In adults hospitalized with conditions not thought to affect the turnover of keratan sulfate-containing tissues, the serum levels varied from individual to individual (53-1,009 ng/ml) but did not show significant differences with respect to age, sex, or disease category. There were no significant differences between the serum levels of adult hospitalized patients and those of nonhospitalized normal adults. In contrast, the concentration of keratan sulfate in the sera of children aged 5-12 was significantly higher. No keratan sulfate was detected in the sera of 3 adult patients with macular corneal dystrophy, an inherited disorder of the cornea. This may indicate that individuals with macular corneal dystrophy have no keratan sulfate-containing proteoglycans in their cartilage. Adult patients with osteoarthritis have significantly higher concentrations of circulating keratan sulfate. This suggests that the assay could prove valuable in monitoring increased cartilage catabolism in joint diseases.

Following injection of chymopapain into a single knee joint in rabbits, serum keratan sulfate levels rose sharply and remained elevated for at least 48 hours before returning to preinjection levels. These changes were accompanied by depletion of proteoglycans from articular cartilage in the injected joint. We conclude that serum keratan sulfate levels rise predictably following acute loss of proteoglycan from a single joint.

We measured levels of sulfated keratan sulfate in serum using a monoclonal antibody in an enzyme-linked immunosorbent assay. Sulfated keratan sulfate was not detected in the serum of 16 patients with macular corneal dystrophy, but was present at normal levels in 66 patients with other corneal diseases. There were no differences with respect to age, sex, and other ocular findings. This monoclonal antibody recognizes a sulfated carbohydrate epitope present in both corneal and skeletal keratan sulfate. Since most serum keratan sulfate is derived from the cartilages, the defect in keratan sulfate synthesis in macular corneal dystrophy may not be restricted to corneal cells. This assay should prove useful in the diagnosis of macular corneal dystrophy, particularly in children at risk before the appearance of opacification.

Osteoarthritis was induced in 12 normal dogs by severing of the anterior cruciate ligament of the right knees, the left knees serving as sham operated controls. The animals were killed at 7 and 14 weeks postsurgery. The total hexuronate, and thus proteoglycan, content of the articular cartilage of operated knees remained unaltered during the period of study. After pretreatment with a highly purified collagenase and in the presence of selected protease inhibitors, a higher proportion of the tissue hexuronate could be extracted from the different topographical areas of osteoarthritic joints under non dissociative conditions (70-75% versus 55-65% for control knees). The nondissociatively recovered osteoarthritic proteoglycans (a-A1 preparations) displayed progressive and consistent changes in their sedimentation profile. First, the size of the fast sedimenting or more saturated aggregates appeared to be reduced in the different regions of osteoarthritic joints at 7 weeks postoperatively. The disappearance of the faster sedimenting mode as well as a dramatic increase in the proportion of monomers were only detected in the topographical zones exhibiting the most severe surface damage and histologic abnormalities at 14 weeks postsurgery. The proteoglycan molecules present as "free" or "nonaggregated" monomers in a-A1 preparations recovered from normal and osteoarthritic cartilage at different time periods after surgery were separated from their corresponding aggregates by rate zonal centrifugation in isokinetic cesium sulfate gradient. Although they were severely depleted in keratan sulfate, the purified "free" and "aggregated" osteoarthritic monomers appeared to be normal in terms of aggregating capacity and size distribution, and were therefore not degraded. This progressive changes in size distribution of proteoglycan aggregates in the early stages of experimental canine osteoarthritis could contribute significantly to the biochemical and biomechanical alterations of osteoarthritic cartilage.