Raised chondroitin sulfate epitopes and hyaluronan in serum from rheumatoid arthritis and osteoarthritis patients.

OBJECTIVES: Serum hyaluronan (HA) and chondroitin sulfate (CS) epitopes WF6 and 3B3 (+) were determined to investigate disease association in patients with osteoarthritis (OA), rheumatoid arthritis (RA) and healthy controls. METHODS: Specific assays for HA and CS epitopes WF6 and 3B3 (+) were established and applied to a cross-sectional study of serum samples from patients (96 OA, 57 RA and 50 healthy controls). RESULTS: Both CS epitopes were increased in serum of many OA and RA patients and average levels were significantly above in healthy controls. In contrast serum HA was increased in RA, but only in few OA patients. CONCLUSIONS: CS epitopes WF6 and 3B3 (+) are raised in serum of patients with both OA and RA and were thus distinct from serum HA. The results suggest that OA may be detected systemically as well as RA. The range of levels of CS epitopes detected in OA and RA was wide and correlation with any aspect of disease activity is yet to be determined.

A quantitative method to detect fucoidan in human plasma using a novel antibody.

We have developed an antibody-based method to assess plasma uptake of a proprietary Undaria-derived fucoidan galactofucan sulfate (GFS(TM)) after oral ingestion by human volunteers. Fucoidans have high-molecular-weights but exert biological effects in experimental animals after oral intake. By using a novel antibody raised against sulfated polysaccharides, we carried out a competitive ELISA to quantitate GFS in plasma samples from healthy volunteers who ingested 3 g/day of whole Undaria containing 10% GFS fucoidan, purified 75% GFS fucoidan, or 3 g of a nonsulfated placebo polysaccharide over 12 days. Increased reactivity to the novel antibody, as measured against preingestion levels, was detected at all time points. Assuming the measured material to be intact GFS, the concentration detected (median) was 4.002 and 12.989 mg/l when 3 g of 10% or 75% pure fucoidan was ingested orally over a period of 12 days, respectively. High-molecular-weight fucoidan can be detected in plasma using an ELISA competitive assay based on a novel antibody to sulfated polysaccharides.

Correlation between serum hyaluronan and disease activity and severity in Thai patients with rheumatoid arthritis.

Serum hyaluronan (HA) concentration was quantified using an ELISA-based assay with biotinylated hyaluronan binding proteins, and correlated with the clinical and laboratory variables in 100 consecutive rheumatoid arthritis (RA) patients (mean +/- SD age and duration of disease of 50.1 +/- 12.5 and 7.9 +/- 6.6 years respectively). Thirty-four patients received prednisonole at an average dose of 5.0 mg/day. The correlations were good between the serum HA level and the joint swollen scores (r = 0.26, p = 0.04), joint space narrowing scores (r = 0.25, p = 0.03), joint erosion scores (r = 0.26, p = 0.03), and erythrocyte sedimentation rate (r = 0.31, p < 0.01) in RA patients who did not take prednisolone. These correlations were diminished in those who received prednisolone, although their disease was more severe. It might be possible that corticosteroids could decrease inflammation of the joint, thus interfering with the correlations. It was concluded that the serum HA level is a useful marker for the activity and severity of disease in patients with RA.

Oral polymeric N-acetyl-D-glucosamine and osteoarthritis.

Many patients with arthritis are using alternative modes of therapy, including nutritional supplements, to treat their arthritis. Most patients never tell their doctors that they are taking alternative medications, and few doctors even ask about such activities. Over-the-counter supplements are expensive and consume large amounts of patients' healthcare dollars. Glucosamine has been widely touted as being an effective arthritis treatment. The authors designed and undertook a study to test the efficacy of a polymer of N-acetyl-D-glucosamine (NAG), or POLY-Nag, in a double-blind, placebo-controlled study in patients with osteoarthritis. Results indicate that POLY-Nag may be useful in treating patients with osteoarthritis.

Engagement of CD147 molecule-induced cell aggregation through the activation of protein kinases and reorganization of the cytoskeleton.

CD147 is a broadly expressed cell surface molecule of the immunoglobulin superfamily whose expression is up-regulated upon T cell activation. Engagement of CD147 by CD147 monoclonal antibodies (mAbs) has been shown to induce homotypic aggregation of U937 cells. To study intracellular signal transduction induced by the engagement of CD147 molecules, protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors were used to inhibit cell aggregation. The results indicated that a PKC inhibitor, sphingosine, and a PTK inhibitor, herbimycin A, inhibited CD147 mAb-induced cell aggregation in a dose-dependent manner. In contrast to herbimycin A, a PTK inhibitor, genistein, enhanced cell aggregation. This discrepancy may be due to the differential effect of herbimycin A and genistein on the target cells. Effect of actin filament polymerization blocking agent, cytochalasin B, was also studied and it was found that cytochalasin B completely inhibited CD147 mAb-induced cell aggregation. This result implied that U937 cell aggregation induced by CD147 mAbs is associated with cytoskeleton reorganization. Thus, our observations suggest that cell aggregation induced by the engagement of CD147 with specific mAbs depend upon the activation of protein kinases and a functional cytoskeleton.

Isolation and characterization of lectin from Thai marine crab (Scylla serrata) with binding specificity to sialoglycoconjugates and its application.

A lectin from Thai marine carb (Scylla serrata) hemolymph has been isolated and purified by affinity column chromatography and preparative electrophoresis. The amino acid composition and 10 amino-terminal residues have been deduced, and its reactivities have been studied using a biotin labeling technique. A method for the determination of sialoglycoconjugates in human serum is described using this lectin. The principle is based on the reaction between the sialoglycoconjugates and biotinylated lectin. The bovine submaxillary mucin (BSM) is immobilized on polystyrene microplate. The unknown sample or sialoglycoconjugate (BSM equivalent) standards, together with excess biotinylated purified lectin (B-lectin), are then added. The B-lectin that binds to the immobilized BSM is then incubated with the peroxidase-conjugated monoclonal antibiotin antibody, and the color that develops after the addition of enzyme substrate is determined by light absorption using a microplate reader. The assay is not only convenient and reliable, but also capable of measuring sialoglycoconjugates in solution at the submicrogram level. It was used in determining the sialoglycoconjugates in human serum from normal subjects and samples positive for carcinoembryonic antigen.

Aflatoxin exposure is higher in vegetarians than nonvegetarians in Thailand.

Aflatoxin-albumin (AFB-albumin) adducts and hepatitis B markers (anti-HBs, and anti-HBc) were measured in vegetarians and nonvegetarians from Chiang Mai, Thailand. The AFB-albumin adduct levels were detected in 62% (37 of 60) of the vegetarian samples and 22% (22 of 100) of nonvegetarians. Somewhat higher levels were detected in vegetarians sera collected in the summer than in the winter, although this difference was not statistically significant. Subjects who were hepatitis B surface antigen (HBsAg)-positive had slightly higher AFB-albumin adduct levels than subjects who had evidence of past exposure (anti-HBc-positive) or no HB virus infection. This study indicated that vegetarians may have a higher frequency of aflatoxin exposure than nonvegetarians. Thai vegetarians consume various vegetables, grains, peanut, soybean, and fermented products, which have been reported to be sources of aflatoxin.

Antimutagenicity of lemon grass (Cymbopogon citratus Stapf) to various known mutagens in salmonella mutation assay.

Lemon grass (Cymbopogon citratus Stapf) was extracted with 80% ethanol. The extract was not found to be mutagenic in the Salmonella mutation test with or without metabolic activation. However, the extract was found to possess antimutagenic properties towards chemical-induced mutation in Salmonella typhimurium strains TA98 and TA100. Mutagenicity of AFB1, Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, IQ, MNNG and AF-2, was inhibited by the extract of lemon grass in a dose-dependent manner, but no effect was found on the mutagenic activity of benzo[a]pyrene.

Pentosan polysulphate and glycosaminoglycan polysulphate stimulate the synthesis of hyaluronan in vivo.

Pentosan polysulphate (PPS) and glycosaminoglycan polysulphate (GAGPS) were examined for their ability to alter hyaluronan synthesis in vivo. The inflamed rat subcutaneous air pouch model was used for the study. PPS or GAGPS injected into the air pouch at a dose of 2.5 mg/kg daily for 7 days resulted in higher molecular weight hyaluronan in the pouch fluid compared with control non-drug-treated pouch fluid. The quantity of the hyaluronan was increased by PPS, but not by GAGPS. We concluded that both drugs could be beneficial in the treatment of inflammatory arthritides in which a decrease in normal synovial hyaluronan concentration and molecular weight occurs.

A new sandwich-ELISA method for the determination of keratan sulphate peptides in biological fluids employing a monoclonal antibody and labelled avidin biotin technique.

A new sandwich-ELISA for the determination of keratan sulphate peptides in biological fluids is described. The technique involves binding a commercially available monoclonal antibody against keratan sulphate to microtitre plates, adding the unknown keratan sulphate antigen then interacting the keratan sulphate-antibody complex with a biotin-monoclonal antibody conjugate which was also specific for keratan sulphate peptides. Alkaline phosphatase conjugated streptravidin was then added and the amount which bound to the biotin was determined by measuring the release of chromogen from an added chromogenic substrate. Using this assay, keratan sulphate peptides in biological fluids within the range 10-1000 ng/ml could be quantitated. This method was found to be more sensitive than presently used techniques. The intra- and inter-assay coefficients of variation were 11% and 13%, respectively.

A method for the quantitation of hyaluronan (hyaluronic acid) in biological fluids using a labeled avidin-biotin technique.

An improved method for the detection and quantitation of hyaluronan (hyaluronic acid) (HA) in biological fluids is described. The principle on which the method is based is that HA binds strongly to a biotinylated HA-binding protein (B-HABP) which was prepared from cartilage proteoglycans. HA was immobilized on polyvinyl chloride plates which had been precoated with poly-L-lysine. The unknown sample or HA standards together with excess B-HABP are then added. The B-HABP that binds to the immobilized HA is then incubated with the enzyme-conjugated avidin (e.g., alkaline phosphatase), and the color which develops on addition of enzyme substrate (e.g., p-nitrophenyl phosphate) is determined by light absorption using a microtitration plate reader. The assay is not only convenient and reliable but is capable of measuring HA in solution at the picogram level. The assay was used to determine HA levels in human sera and synovial fluid taken from volunteers and patients with rheumatoid arthritis and osteoarthritis.

A monoclonal antibody that recognizes 2,3-, 2,6-, and 4,6-disulphate ester ring substitution in pyranose-containing polysaccharides. Its production, characterization and application for the quantitation of pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphate and chondroitin sulphate E.

A method is described for the preparation of a monoclonal antibody (MAb) which binds specifically to polysaccharides which contain 2,3-, 2,6- and 4,6-disulphate ester pyranose ring substitution. Such molecules include the semisynthetic heparinoids, pentosan polysulphate (PPS), dextran sulphate (DS) and glycosaminoglycan polysulphates (GAGPS), as well as the naturally occurring polysulphated polysaccharides, chondroitin sulphate E, and the 2,6-disulphated galactoses of carrageenans. The antibody (MAb 5-B-10) did not significantly cross-react with other sulphated polysaccharides such as heparin, heparan sulphate, the chondroitin sulphates, A, B, C or D, or keratan sulphate. No cross-reactivity was found with non-sulphated polysaccharides or polyanions including hyaluronic acid, xylan, or DNA. MAb 5-B-10 was characterized as IgM and kappa light chains, and was to develop an amplified enzyme-linked immunosorbent inhibition assay (ELISIA) to detect and quantitate some of the polysulphated polysaccharides in biological fluids. Using this assay, the lower limits of detection of these compounds in serum were in the order of 50 ng/ml; however 50% inhibition was obtained between 200-500 ng/ml. The intra- and inter-assay coefficients of variation for PPS were 4.2 +/- 2.8 and 16.7 +/- 13.8% respectively. The MAb 5-B-10 and the ELISIA were used to determine the levels of PPS in plasma of three healthy non-fasted volunteers for up to 120 min post-intravenous infusion (1.0 mg/kg). The results obtained compared favourably with kinetic data reported by others using a competitive binding assay method for this drug.

Pentosan polysulfate (Cartrophen) prevents the hydrocortisone induced loss of hyaluronic acid and proteoglycans from cartilage of rabbit joints as well as normalizes the keratan sulfate levels in their serum.

The intraarticular injection of a sterile solution of 25 mg hydrocortisone succinate into rabbit knee joints once a week for 8 weeks reduced the levels of proteoglycans and hyaluronic acid (HA) in articular cartilage. In contrast, the keratan sulfate (KS) peptide levels present in sera of these animals were elevated relative to a saline treated control group. By injecting 25 mg hydrocortisone succinate combined with pentosan polysulfate (Cartrophen) (5 mg) into rabbit joints over the 8 week period, the loss of proteoglycans and HA from articular cartilage was abrogated and serum KS-peptides were restored, with time, to within control levels. These findings suggest that intraarticular administration of high dose hydrocortisone succinate to rabbits has a deleterious effect on articular cartilage HA and proteoglycan metabolism but this effect can be attenuated by co-intraarticular administration of Cartrophen.

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids.

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 +/- 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.

Application of an enzyme-linked immunosorbent-inhibition assay to quantitate the release of KS peptides into fluids of the rat subcutaneous air-pouch model and the effects of chondroprotective drugs on the release process.

The effects of the chondroprotective agents (Arteparon, SP-54 and DH40J) on the release of proteoglycan degradation products (as keratan sulphate peptide fragments) from articular cartilage implanted into rat subcutaneous air pouches have been investigated by using an enzyme-linked immunosorbent-inhibition assay (ELISIA). The ELISIA technique was capable of quantitating the keratan sulphate peptides (KS peptides) in fluids within the range of 100-2,000 ng/ml by using the monoclonal antibody line 1/20/5-D-4 and human articular cartilage KS peptides as standard reagents. It was found that the levels of KS peptides present in the air-pouch fluid of rats treated with the chondroprotective drugs was significantly less than in fluid aspirated from the pouches of non-drug-treated control animals. On the basis of these findings we suggest that the assessment of KS peptide by ELISIAs may provide a useful means of monitoring proteoglycan breakdown products in biological fluids (e.g. synovial fluids or blood) and for evaluating the effects that antiarthritic drugs may have on this process.