Lean mass, magnesium, faecal calprotectin and glucocorticoid exposure as risk factors for low bone mineral density in inflammatory bowel disease patients.

BACKGROUND AND AIM: Low bone mineral density (BMD) is a common complication in patients with inflammatory bowel disease (IBD). However, debates are ongoing with regard to the other involved factors, especially in younger patients. This study aimed to evaluate the parameters that contribute to decreased BMD, focusing on premenopausal women and men aged <50 years. METHODS: This study included 81 patients with IBD and 81 age-, sex- and BMI-matched controls. Blood tests were conducted on IBD patients, and a dual-energy X-ray absorptiometry (DXA) scan was performed on both groups. RESULTS: Low BMD and fragility fracture were found to be more prevalent in IBD patients than in healthy subjects (49.3% vs 23.4%, P = 0.001 and 9.8% vs 1.2%, P = 0.01, respectively). Patients with low BMD were older, with a longer disease duration, higher faecal calprotectin (FC) levels and lower magnesium and lean mass (appreciated as appendicular skeletal muscle index (ASMI)). Multiple regression analysis revealed that ASMI, age and use of glucocorticoids were the independent parameters for decreased BMD. Although 91.3% of the patients had a 25-hydroxy vitamin D level of <30 ng/mL, it was not a statistically significant factor for decreased BMD. CONCLUSION: In our study, the levels of vitamin D did not seem to have an important impact on BMD. Conversely, FC, magnesium and lean mass are important factors, suggesting that good control of disease, adequate magnesium intake and increased lean mass can have a good impact on bone metabolism in patients with IBD.

Endoscopic knot tying: In vitro assessment in a porcine stomach model.

AIM: To determine if surgical knotting performed via endoscopy is an effective closure method for natural orifice translumenal endoscopic surgery. METHODS: The proposed method was tested on an in vitro pig stomach model using standard endoscopy suite materials. A single use laparoscopy trocar (Versaport Plus manufactured by Tyco Healthcare) was fixed onto a plastic rectangular box in a horizontal position. A fresh pig stomach was tightly attached via its esophageal end to the trocar opening on the inner side of the box. The stomach cavity was closed at the duodenal end with Kocher forceps. A standard upper gastrointestinal endoscope fitted at its tip with a transparent plastic cap was introduced into the stomach through the outer trocar opening, so that the passage of the surgical trocar would mimic the passage of an esophagus. The stomach was subsequently inflated, followed by irrigation and washing. A neutral electrode of an electrocautery unit was placed inside the plastic box, underneath the pig stomach. The stomach's outer surface was kept moist using normal saline in order to maintain the natural elasticity and to ensure good contact with the electrode. RESULTS: The submucosal space on the anterior face of the stomach was accessed using the technique of endoscopic submucosal dissection. First, a site on the anterior face of the stomach was chosen, near the angle. Then, saline was injected into the submucosa with a standard endoscopic needle, so as to create a 20 mm diameter elevation. A linear 15 mm vertical incision was created at its center using a Dual Knife (KD650U manufactured by Olympus). This incision was used to access the submucosal space, and about 10 mm was dissected on both sides of the incision. The endoscope was then pushed through to the outside of the stomach after dilating a small puncture made by the Dual Knife in the muscularis propria, which simulated the peritoneoscopy procedure. Then, a 0.025" guidewire (Jagwire/450 cm manufactured by Boston Scientific) was inserted into the puncture, followed by a dilating balloon (Quantum TT manufactured by Cook Medical) that was used to enlarge the aperture orifice. After withdrawing the scope back into the stomach, the procedure continued with guidewires being passed from the submucosal space into the gastric lumen through small orifices on the left and right sides of the mucosal opening. These orifices were made with the Dual Knife, and the guidewires were inserted via a guiding catheter (HGC-6 manufactured by Cook Medical). As the guidewires were pulled outside of the stomach, they were replaced with a single surgical suture that had been initially attached to their tip and was now untied. Finally, one loop of this surgical suture was formed on the exterior. One loop end was fixed while the opposite suture end was pulled by biopsy forceps through the endoscope channel as the scope was inserted into the stomach. The loop was advanced until it approached and fixed the two mucosal incision margins. Three alternating loops were made in this manner to create a genuine tight surgical knot. CONCLUSION: Endoscopic knotting of the gastric wall is feasible, but an in vitro survival study is necessary to validate clinical significance.

Genetic biomarkers for neoplastic colorectal cancer in peripheral lymphocytes.

BACKGROUND: Loss of genomic stability appears as a key step in colorectal carcinogenesis. Micronucleus (MN) designates a chromosome fragment or an entire chromosme which lags behind mitosis. MN may be noticed as an additional nucleus within the cytoplasm cell during the intermediate mitosis phases. We tested the hypothesis that MN and its related anomalies may be associated with the presence of neoplastic colorectal lesions. METHOD: Peripheral blood lymphocytes were cultured and microscopically examined. The frequency of micronuclei (FMN) and the presence of nucleoplasmic bridges (NPB) in binucleated cells were compared in patients with of without colorectal neoplastic lesions. RESULTS: We included 45 patients undergoing colonoscopy, 23 males and 22 females, with a median age of 59. 17 patients had polyps, 11 colorectal cancer (CRC) and 17 had a normal colonoscopy. The FMN was significantly higher in women than in men (8.14 vs 4.17, p=0.008); NPB were significantly less frequent in patients with advanced adenomas (>10mm or vilous) or CRC (p=0.044) when compared with patients with normal colonoscopy, hiperplastic polyps or non-advanced adenomas. CONCLUSION: Micronuclei are more frequent in women, but its frequency was not significantly different in patients with advanced adenomas or CRC. Null or low frequency values for nucleoplasmic bridges presence in peripheral lymphocyte may be predictive for advanced adenomas and colorectal cancer.

Nuclear Division Index may Predict Neoplastic Colorectal Lesions.

BACKGROUND: Colorectal cancer (CRC) develops by accumulation of multiple genetic damages leading to genetic instability that can be evaluated by cytogenetic methods. In the current study we used Cytokinesis-Blocked Micronucleus Assay (CBMN) technique to assess the behavior of Nuclear Division Index(NDI) in peripheral lymphocytes of patients with CRC and polyps versus patients with normal colonoscopy. METHODS: Blood samples were collected from patients after informed consent. By CBMN technique we assessed the proportion of mono-nucleated, bi-nucleated, tri-nucleated and tetra-nucleated cells/500 cells, to calculate NDI. Data were statistically analyzed using the SPSS 11.0 package. RESULTS: 45 patients were available for analysis, 23 men and 22 women, with a mean age of 58.7±13.5. 17 had normal colonoscopy, 17 colonic polyps and 11 CRC. The mean NDI values were significantly smaller for patients with CRC or polyps than in patients with normal colonoscopy (1.57 vs 1.73, p=0.013). The difference persisted for patients with neoplastic lesions (adenomas and carcinomas) when compared with patients with normal colonoscopy or non neoplastic (hyperplastic) polyps (1.56 vs.1.71, p=0.018). The NDI cut-off value to predict the presence of adenomas or carcinomas was equal to 1.55 with a 54.2% sensitivity and 81% specificity of lower values (p=0.019). The NDI cut off value to predict the presence of advanced adenomas or cancer was 1.525 for a sensitivity of 56.3% and a specificity of 82.8% (p=0.048). CONCLUSION: NDI may be useful in screening strategies for colorectal cancer as simple, noninvasive, inexpensive cytogenetic biomarker.

Immunoassays for collagenase-mediated cleavage of type I and II collagens.

This chapter describes the production and characterization of antibodies raised against neoepitopes in collagenase-cleaved collagen. It also details the development, validation, and use of immunoassays using such antibodies to measure specifically collagenase-mediated cleavage.

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.

Etanercept exerts beneficial effects on articular cartilage biomarkers of degradation and turnover in patients with ankylosing spondylitis.

OBJECTIVE: Anti-tumor necrosis factor-alpha (TNF-alpha) therapies are not only beneficial for reducing symptoms in rheumatoid arthritis (RA) but also for structural damage visible on plain radiographs and serological biomarkers of articular cartilage damage. It is not known if these therapies also prevent structural damage in ankylosing spondylitis (AS). The low sensitivity to change over time of plain radiographic instruments mandates a search for the effects of these therapies on possible biomarkers of cartilage damage. METHODS: We studied 2 populations of patients with AS: (1) patients recruited to a placebo controlled trial of etanercept in AS for 16 weeks; (2) an observational cohort receiving infliximab for disease refractory to conventional therapy. Clinical (morning stiffness, nocturnal pain, Bath AS Disease Activity Index) and laboratory [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP)] assessments of disease activity were performed at baseline and at either 16 weeks (clinical trial cohort) or at 14 weeks (observational cohort). We measured serum matrix metalloproteinase-1 (MMP-1), MMP-3, human cartilage glycoprotein-39 (YKL-40), and cartilage oligomeric matrix protein by ELISA at the same timepoints. We also measured serum concentrations of 2 novel biomarker epitopes, C2C and 846, by competitive ELISA. The C2C assay detects a neoepitope at the carboxy terminus of the long three-quarter amino-terminal fragment generated following cleavage of type II collagen by collagenases. Aggrecan 846 epitope is a chondroitin sulfate epitope present on intact aggrecan molecules. Both these assays would detect products originating from both hyaline cartilages and intervertebral discs. RESULTS: There was a significant reduction in levels of C2C (p = 0.005) and a significant increase in the 846 epitope (p = 0.01) in patients who received etanercept compared to placebo controls. Changes in C2C correlated significantly with changes in ESR (r = 0.51, p = 0.04) and CRP (r = 0.48, p = 0.048). Significant changes in C2C were not evident in the infliximab observational cohort, although significant reductions were noted in levels of MMP-3 (p = 0.04) and MMP-1 (p = 0.02) at 14 weeks that were not observed in the etanercept group. Analysis of all baseline samples showed a significant correlation between levels of MMP-3 with CRP (r = 0.73, p < 0.0001), and YKL-40 (r = 0.71, p < 0.0001). No correlation was evident at baseline between levels of C2C or 846 epitope and either acute phase reactants or other biomarkers. CONCLUSION: Our data suggest that an anti-TNF-alpha agent, etanercept, may modify cartilage turnover. These include decreased degradation of type II collagen and increased turnover of aggrecan. Additional therapeutic properties of some anti-TNF-alpha agents in AS, such as infliximab, may be related to decreased expression of MMP. Additional studies in larger populations are therefore warranted.

Repeated intraarticular injections of triamcinolone acetonide alter cartilage matrix metabolism measured by biomarkers in synovial fluid.

Although intraarticular (IA) corticosteroids are frequently used to treat joint disease, the effects of their repeated use on articular cartilage remains controversial. The aim of our study was to determine the effects of a clinically recommended dose of IA triamcinolone acetonide (TA), on synovial fluid (SF) biomarkers of cartilage metabolism. Ten adult horses, free of osteoarthritis (OA) in their radiocarpal joints, were studied. One radiocarpal joint of each horse was randomly chosen for treatment and the contralateral anatomically paired joint acted as the control. Aseptic arthrocentesis was performed weekly on both joints for 13 weeks. The initial results from the first 3 weeks of the experimental period established baseline untreated control marker levels for each joint, each being its own control. On weeks 3, 5, and 7, a sterile suspension of 12 mg of TA was injected into the treated joint and an equivalent volume of sterile saline solution (0.9%) was injected into the control joint. SF was immunoassayed for biomarkers of aggrecan turnover (CS 846 & KS), types I and II collagen cleavage (C1,2C) and type II collagen synthesis (CPII). In treated joints, there was a significant increase in CS 846, KS, C1,2C and CPII epitope concentrations following IA TA injections when compared to baseline levels. There was also a significant increase in C1,2C and CPII epitope concentrations in the contralateral control joints following IA TA injections in the treated joint. Significant differences were observed between treated and control joints for all markers except CPII. These findings indicate that TA alters articular cartilage and collagen metabolism in treated and, interestingly, also in control joints, suggesting a systemic effect of the drug. Though intuitively the observed findings would favor the hypothesis that long-term IA TA treatment changes joint metabolism and this may have detrimental effects; further studies would be necessary to confirm this.

Glucosamine sulfate and cartilage type II collagen degradation in patients with knee osteoarthritis: randomized discontinuation trial results employing biomarkers.

OBJECTIVE: To determine whether glucosamine sulfate has an effect on cartilage type II collagen degradation in patients with knee osteoarthritis (OA). METHODS: A randomized, double blind, placebo controlled glucosamine discontinuation trial was conducted in 137 subjects with knee OA, who had had at least moderate relief of knee pain after starting glucosamine. Subjects were randomized to glucosamine at prestudy dose or placebo at an equivalent dose. Treatment was continued to Week 24 or disease flare, whichever occurred first. Serum and urine samples were collected at Weeks 0, 4, 12, and 24 or flare visit. Samples were analyzed in triplicate for 2 type II collagen degradation biomarkers: C2C epitope (COL2-3/4C(long)) and C1,2C epitope (COL2-3/4C(short)). The primary outcome was the mean change in serum and urine C1,2C/C2C ratio in the glucosamine and placebo groups from baseline to final (flare or Week 24) visit. Linear regression analyses were conducted to adjust for potential confounders. Due to non-normal distributions, the data were log-transformed (lnC1,2C/C2C). Secondary outcomes included comparison of mean change scores at final visit compared to baseline for serum and urine C1,2C and C2C in the 2 treatment groups and in Flare versus No-Flare groups. RESULTS: Baseline and final visit samples were available in 130 subjects for serum analysis and 126 subjects for urinalysis. No significant difference was seen between placebo and glucosamine groups in the serum C1,2C/C2C ratio, with a mean (SD) change from baseline to final visit of 0.8 (27.8) and -0.1 (1.8), respectively (mean difference 0.9; 95% CI -6.0, 7.7, p = 0.80). Similarly, no differences between treatment groups were seen for mean change in urine C1,2C/C2C (p = 0.82), or for mean change in C2C or C1,2C. In linear regression analysis, after adjustment for sex, radiographic severity, baseline lnC1,2C/C2C ratio, WOMAC function, and flare status, treatment was not a significant predictor of final serum or urine lnC1,2C/C2C ratio. When those who experienced flare were contrasted with those without flare, there was a nonsignificant trend toward a difference in mean baseline to final visit change score for serum C1,2C/C2C ratio (p = 0.12). In addition, in the multivariable linear regression analysis, flare status showed a borderline association with final visit serum lnC1,2C/C2C ratio (p = 0.16). CONCLUSION: No statistically significant effect of glucosamine sulfate on type II collagen fragment levels in serum or urine was observed for knee OA over 6 months. Further research is necessary to elucidate which biopathologic systems, if any, are affected by glucosamine treatment. While collagen degradation products may be of value in predicting progression, at least as defined by clinical flare, a larger dataset would be needed to prove this.

Effect of oral glucosamine on cartilage degradation in a rabbit model of osteoarthritis.

OBJECTIVE: To determine whether oral glucosamine alleviates cartilage degradation in an animal model of osteoarthritis (OA). METHODS: The effect of 8 weeks of daily oral glucosamine hydrochloride on degeneration of articular cartilage was evaluated in rabbits in which anterior cruciate ligament transection (ACLT) was performed to induce OA. Animals were treated with glucosamine (n = 16) or a placebo (n = 16) and necropsied at 11 weeks. Seven unoperated rabbits served as controls. The articular cartilage was evaluated macroscopically and histologically and analyzed for total type II collagen and glycosaminoglycan (GAG) content. RESULTS: Histologic analysis revealed that loss of proteoglycan, based on Safranin O-fast green staining, was significantly reduced in the lateral tibial plateau cartilage of ACL-transected limbs in the glucosamine group compared with ACL-transected limbs in the placebo group, with a similar, but not significant, trend for the lateral femoral condylar cartilage. Likewise, macroscopic analysis of cartilage showed that the lateral tibial plateau alone had a significantly lower rate of disease in the glucosamine group, which was consistent with the results of the independent histologic assessment. However, no significant treatment effect was detected when composite histologic scores were analyzed. A significant reduction in GAG content was observed in the femoral condyles of placebo-treated ACL-transected joints, but not in the same region of glucosamine-treated ACL-transected joints, compared with their respective contralateral unoperated joints. CONCLUSION: Oral administration of glucosamine had a detectable, site-specific, partial disease-modifying effect in this model of OA. From a clinical perspective, the administration of glucosamine did not prevent fibrillation and/or erosions of the articular cartilage in all of the treated animals, and no effects were detected in the medial joint compartments.

Cartilage biomarkers in ankylosing spondylitis: relationship to clinical variables and treatment response.

OBJECTIVE: Ankylosing spondylitis (AS) is a progressive disease in which chronic inflammation can lead to extensive new bone formation throughout the spine. At present, few measures of the activity or extent of the disease are available. In this study, we sought to determine whether markers of cartilage synthesis and degradation could provide such quantitative measures. METHODS: Serum samples from 23 patients receiving infliximab treatment for AS were obtained at baseline and at weeks 2, 6, 14, and 22. Patients were stratified with respect to joint involvement and baseline levels of inflammatory markers, and responders were defined according to the Assessments in Ankylosing Spondylitis 20% criteria. Serial measurements of interferon-gamma, tumor necrosis factor alpha, transforming growth factor beta (TGFbeta), interleukin-10 (IL-10), and IL-1 were done at each time point. The following biomarkers were measured by enzyme-linked immunosorbent assay: the proteoglycan aggrecan 846 epitope, a marker of cartilage turnover; C-propeptide of type II collagen (CPII), a biosynthesis marker; and the Col2-3/4(long mono) (C2C) and Col2-3/4(short) (C1-2C) neoepitopes, reflecting collagen cleavage of type II collagen and type I/type II collagen, respectively. RESULTS: At baseline, patients with AS demonstrated significant elevations in serum levels of CPII, the 846 epitope, and the CPII-to-C2C (CPII:C2C) ratio (but not C2C or C1-2C) compared with normal controls. Of the biomarkers examined, only CPII:C2C showed a correlation with the C-reactive protein (CRP) level. Among the biomarker-cytokine relationships, TGFbeta demonstrated a trend toward a positive correlation with the 846 epitope. CONCLUSION: In AS, elevated serum levels of CPII and the 846 epitope may be related to biosynthetic turnover of hyaline cartilage and the intervertebral discs but may also reflect progressive bone formation as a result of endochondral ossification. The correlation of the CPII:C2C ratio with CRP suggests that the CPII:C2C ratio might prove to be a useful marker of disease activity in AS.

Immunoassays for collagens in chondrocyte and cartilage explant cultures.

Quantitative immunoassays have been developed to measure the content, degradation, and synthesis of types II and IX collagens in hyaline cartilages. Some of these assays and their applications are described in this chapter. These and other assays are commercially available. The applications of these assays are discussed with examples from recent publications.

Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis.

OBJECTIVE: To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA). METHODS: Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4C(long mono), or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses. RESULTS: Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4C(long mono) and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4C(long mono) was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4C(long mono) was elevated at 12 weeks after surgery. CONCLUSION: Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4C(long mono) and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.

The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases.

A monoclonal antibody has been developed which recognizes a neoepitope in type II collagen which is generated by the intrahelical cleavage of collagenases. Antibody reactivity is directed at the carboxyl-terminus of the TCA or 3/4 piece of the degraded alpha1(II) chain. Reactivity is dependent upon hydroxylation of proline. Evidence is provided suggesting that epitope binding involves the recognition of a conformational neoepitope. Using an ELISA, we show that this neoepitope can be detected in the urines and sera of nonarthritic persons and patients with rheumatoid arthritis (RA). An increased content is observed in the sera and urines of patients. The assay may be of value in studying cartilage type II degradation both in vitro and in vivo such as in those with arthritis.

Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans: relationship to mediators of systemic and local inflammation.

OBJECTIVE: To determine in vivo the extent of damage to, and changes in turnover of, articular cartilage type II collagen (CII) and the proteoglycan aggrecan following the onset of inflammatory arthritis in humans, and to examine the hypothesis that there are direct relationships between cartilage biomarkers of damage/turnover and clinical, histologic, and molecular markers of inflammation. METHODS: Synovial fluid (SF) and synovial membrane (SM) were obtained by arthroscopy, and a synovitis score was determined, in 32 patients with rheumatoid arthritis (RA) (13 with early untreated disease, 19 with established disease), 18 with psoriatic arthritis (PsA), and 10 with osteoarthritis (OA). Systemic disease activity markers were recorded, and SM CD3+ T cells, CD4+ T cells, CD68+ macrophages, and lining layer hyperplasia were quantified. SF levels of tumor necrosis factor alpha (TNFalpha), interleukin-10 (IL-10), matrix metalloproteinase 1 (MMP-1), MMP-3, Col2-3/4C(Long mono) neoepitope (C2C) (reflecting collagenase cleavage of cartilage CII), C-propeptide of type II procollagen (PIICP) (a biosynthesis marker), keratan sulfate (KS), and the 846 epitope of aggrecan (turnover) were measured by enzyme-linked immunosorbent assay or radioimmunoassay. RESULTS: Levels of cartilage degradation products in early RA or early PsA were not elevated above levels in OA, although in early inflammatory arthritis, TNFalpha and MMP-1 levels were similar to those observed in late inflammatory disease and higher than those in OA. PIICP was reduced in early RA. Correlations were observed between the SF C2C neoepitope level and the Health Assessment Questionnaire score, C-reactive protein level, plasma viscosity, synovitis score, and SF TNFalpha and MMP-1 levels. KS epitope content was reduced in direct relation to SM macrophage infiltration in the sublining and lining layers and in the presence of elevated SF MMP-3. Both SF MMP-1 and SF MMP-3 levels correlated with CD4+ T cell infiltration and lining layer hyperplasia in the SM, and MMP-1 levels correlated with lining layer CD68 levels, but TNFalpha and IL-10 levels did not. CONCLUSION: Except for CII synthesis, there were no significant changes in extracellular matrix turnover of aggrecan or CII in the early stages of human inflammatory arthritis. However, the direct correlation between the increases in TNFalpha and MMP-1 production and collagen degradation suggests that collagenase cleavage of cartilage collagen is related to the activities of TNFalpha and MMP-1. The reduction in CII synthesis in early RA may contribute to the developing pathology, since a lack of synthesis of this molecule would inhibit maintenance of cartilage matrix.

The pathobiology of focal lesion development in aging human articular cartilage and molecular matrix changes characteristic of osteoarthritis.

OBJECTIVE: To determine if early focal lesions seen in aging exhibit molecular changes in the extracellular matrix that are similar to those seen in osteoarthritis (OA) and to examine the interrelationships between matrix degradation and synthesis and how they relate to cartilage turnover. METHODS: Condylar cartilage was obtained postmortem from lesion-free joints and from the lesion (where present as well as) from areas adjacent to and remote from the lesion of 31 knees without signs of joint injury (damage to ligaments or menisci). Cartilage was graded histologically and assayed for type II collagen and proteoglycan aggrecan glycosaminoglycan (GAG) contents and turnover (specifically, type II collagen denaturation and its cleavage by collagenase), type II collagen synthesis (C-propeptide [CPII] content), and aggrecan turnover (846 epitope content). To study the degradation of aggrecan reflected by the release of GAG, we cultured cartilage samples from 10 knees. RESULTS: The more degenerated cartilage from the lesion and adjacent area exhibited significantly more collagen cleavage by collagenase than did cartilage remote from the lesion. Type II collagen denaturation and synthesis were also significantly elevated in the lesion and adjacent cartilage, but neither cleavage nor denaturation correlated with synthesis. Type II collagen content decreased with increasing degeneration, with the lowest levels present in the lesion. Collagen content was indirectly related to denaturation and cleavage adjacent to and remote from the lesion and to denaturation within the lesion. Collagen cleavage and denaturation adjacent to and remote from the lesion were directly interrelated. Cartilage from the lesion contained significantly less GAG than did cartilage adjacent to and remote from the lesion. Aggrecan turnover (846 epitope) was also elevated in both the lesion and adjacent cartilage, whereas GAG release was elevated only in the lesion. GAG and 846 epitope contents were interrelated only at sites remote from the lesion. There was also a direct correlation between collagen and GAG contents in the lesion and in adjacent sites. This correlation was also seen between collagen synthesis (CPII) and the 846 epitope. CONCLUSION: These results demonstrate that lesions seen in aging exhibit molecular changes in matrix turnover similar to those seen in OA articular cartilage at arthroplasty, but not in healthy normal aging cartilage. The direct relationships between type II collagen cleavage and denaturation and the inverse relationship between type II collagen content and cleavage or denaturation implicate collagenase activity and damage to collagen in this loss of collagen during lesion development. The lack of correlation of the increased synthesis with the degradation or content of type II collagen indicates that these aspects of turnover are not coordinated in the pathologic state. However, the direct relationship between collagen and GAG contents in and adjacent to the lesion illustrates the structural interrelationships of collagen and proteoglycan aggrecan molecules. These results suggest that these focal lesions represent the development of early OA and that this involves the progressive damage to articular cartilage surrounding the lesion as part of the process of the development of idiopathic OA.

Molecular changes in human osteoarthritic cartilage after 3 weeks of oral administration of BAY 12-9566, a matrix metalloproteinase inhibitor.

OBJECTIVE: To determine the effect of BAY 12-9566, a matrix metalloproteinase inhibitor, on articular cartilage metabolism in patients with osteoarthritis (OA). METHODS: Thirty-five patients with OA were randomized to receive oral daily dosing of BAY 12-9566 (25, 100, or 400 mg) or placebo for 3 weeks prior to knee surgery. Cartilage samples were obtained at surgery and examined for markers of proteoglycan aggrecan turnover (846 epitope, a putative synthesis marker, and keratan sulfate epitope content) and type II collagen synthesis (C-propeptide content), cleavage by collagenase (COL 2-3/4C short), denaturation, and content (COL2-3/4m epitope). BAY 12-9566 concentrations were measured by HPLC in serum, synovial fluid, and cartilage. RESULTS: Comparisons between study drug and placebo treatments revealed that at the 100 mg dose there was a significant increase in the 846 epitope (p = 0.012). Total type II collagen content was also higher at this dosage (p = 0.012). Alterations in collagen degradation and synthesis were not detected. CONCLUSION: BAY 12-9566 at daily doses of 100 mg significantly altered proteoglycan turnover, resulting in a cartilage composition reflected by the content of the 846 epitope that is more characteristic of a young growing individual. The increase in this epitope may signify increased matrix synthesis. The increase in type II collagen content was unexpected, since there was no other evidence for altered collagen turnover. However, increased matrix assembly would also be indicated by this increased content.