Isolation and characterization of undersulphated chondroitin-4-sulphate from normal human plasma.

The present study was undertaken in order to characterize further the glycosaminoglycans of normal human plasma. Coagulation factor IX concentrate prepared from undiluted plasma by DEAE-Sephadex chromatography was used as the starting material. The concentrate was subjected to proteolytic treatment with papain and pronase, deproteinised with trichloroacetic acid, dialysed and passed through an AG 1 X 2 anion-exchange column. Glycosaminoglycans were eluted stepwise from the column with NaC1. The sole glycosaminoglycan obtained was an undersulphated chondroitin-4-sulphate which was identified by chemical analyses, digestibility with testicular hyaluronidase, electrophoretic behaviour and infrared spectrum. Gel-exclusion chromatography indicated a molecular weight of 17 000 for the compound. The undersulphated chondroitin-4-sulphate was calculated to represent at least 80% of the macromolecular glycosaminoglycans present in normal human plasma and to occur in a concentration of approx. 3 mg hexuronate per 1 of plasma.

Acid glycosaminoglycans (mucopolysaccharides) in the differential diagnosis of pleural effusion.

Pleural fluid glycosaminoglycans (GAG) in 64 patients with various diseases were isolated by anion-exchange chromatography after proteolysis, and characterised by spectrophotometric, electrophoretic and enzymatic techniques. GAG concentrations ranged from 7 to 1178 microng hexuronate/ml pleural fluid. The highest values (1178, 161 and 160 micron/ml) were found in patients with diffuse mesothelioma. Over 90% of the pleural fluid GAG consisted of hyaluronic acid (HA) in these patients. In other types of pleural effusion the relative HA content varied from 42 to 70% of the total GAG. Determination of pleural fluid HA consequently appears extremely valuable in the diagnosis of the form of mesothelioma producing HA. The mean GAG concentration of pleural fluid was significantly higher in tuberculous pleurisy than in hydrothorax (P less than 0.01), secondary malignant pleural effusion (P less than 0.0005) and idiopathic pleurisy (Pless than 0.03). It was impossible to demonstrate definite correlations between GAG and protein, and GAG and glucose concentrations of pleural fluid.

Serum baseline hyaluronate and disease activity in rheumatoid arthritis.

The baseline serum hyaluronate (HA) concentration from samples obtained five to seven hours after mobilization of the patient was quantified using a radiometric 125I-HA binding method in 58 patients with rheumatoid arthritis and compared with several clinical and laboratory parameters by means of stepwise multiple linear regression. In the age- and sex-adjusted model, the variables with independent predictive value for serum hyaluronate concentration were the erythrocyte sedimentation rate (ESR) and the joint score index measuring the extent of the synovial inflammation. The estimated synovial mass index and C-reactive protein values did not improve the fit of the model after ESR and joint score were entered, and were left out from the multiple regression equation. When ESR and joint score were studied in univariate regression analysis with serum hyaluronate, the coefficients were r = 0.492 and r = 0.397, and the P values were P less than 0.001 and P less than 0.005, respectively. It was concluded that the baseline hyaluronate level in serum is closely related to the synovitic activity of the rheumatoid inflammation and that measurement of serum hyaluronate is of value when the activity and extent of synovial inflammation is being assessed in patients with rheumatoid arthritis.

Fetal outcome in women with primary Sjögren's syndrome. A retrospective case-control study.

OBJECTIVE: To study fetal outcome in women with primary Sjögren's syndrome (SS) compared to that in women with systemic lupus erythematosus (SLE) and healthy women, and to study the possible association of fetal loss with anticardiolipin antibodies (aCL) and antibodies to SS-A/Ro and SS-B/La in women with primary SS. METHODS: A retrospective analysis of the fetal outcome in 55 pregnancies in 21 patients with primary SS compared to that in 100 pregnancies in 42 patients with SLE and 94 pregnancies in 42 healthy women matched for age, parity and the onset of the autoimmune disease with respect to pregnancy. IgG-, IgM- and IgA-aCL were determined by a cofactor-dependent ELISA and antibodies to SS-A/Ro and SS-B/La by ELISA using human recombinant antigens and affinity-purified antigens. RESULTS: Of all the 55 pregnancies in patients with primary SS, 8 (15%) occurred after the onset of primary SS symptoms. Eleven (20%) of the 55 pregnancies ended in fetal loss. The relative risk (RR) for fetal loss in patients with primary SS was 2.7 (95% CI 1.1-6.5; p = 0.023), and after the exclusion of the patient with four spontaneous abortions it was 2.0 (0.7-5.3; p = 0.18). In SLE the level of risk was 2.2 (0.9-5.0; p = 0.065). Fetal loss in patients with primary SS was not associated with elevated levels of anticardiolipin antibodies (aCL) or autoantibodies to SS-A/Ro or SS-B/La. Newborns of mothers with primary SS were not more premature or growth retarded than newborns of healthy women, but the absolute and the relative birth weights of the newborns of mothers with SLE was significantly lower than in healthy controls (P < 0.001 and P < 0.0001, respectively). CONCLUSION: We conclude that the majority of pregnancies in women with primary SS occur before the onset of the disease and that these women have an increased risk of fetal loss, which is not associated with elevated levels of ACL or antibodies to SS-A/Ro or SS-B/La. The risk of fetal loss in primary SS is similar to that in women with SLE, but fetal growth retardation appears to be more common in SLE than in primary SS.

Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate.

The ability of reactive oxygen species produced by triggered neutrophilic leukocytes, hypoxanthine/xanthine oxidase (HX/XAO), hydrogen peroxide, and hypochlorous acid/myeloperoxidase (HOCl/MPO) systems to degrade hyaluronate (HA) in human synovial fluid (SF) and purified umbilical cord HA was compared by measuring the molecular weight distribution of HA using high-performance liquid chromatography with a size-exclusion column. The exposure of noninflammatory SF to phorbol myristic acetate (PMA)-activated neutrophils or to hydrogen peroxide (H2O2) caused depolymerization of SF HA to the degree corresponding to that found in rheumatoid SFs. When HX/XAO was used as radical generator, the molecular weight of SF HA decreased from 3.42 x 10(6) to 1.40 x 10(4) daltons with concomitant decrease of SF viscosity to 36% from the original value. The HOCl/MPO system caused no depolymerization of SF HA, even at very high unphysiological HOCl concentrations that induced the precipitation of SF HA together with SF proteins. This effect was found to be comparable to conventional mucin clot formation in SF. However, purified human umbilical cord HA was easily depolymerized with HOCl/MPO or with H2O2, but these effects were sensitive to the hydroxyl radical scavenger mannitol and iron chelator desferrioxamine, indicating that the formation of reactive hydroxyl radical (OH.) is likely to participate in these reactions. Thus we conclude that in inflammatory SF HA is mainly depolymerized by OH. produced by decomposition of H2O2 catalyzed by iron, free or locally bound to HA itself. In contrast to what has been reported earlier, HOCl/MPO only depolymerizes purified umbilical cord HA (in a hydroxyl radical-dependent manner) but does not depolymerize HA in SF. As a matter of fact, HOCl/MPO has a scavenging action on SF HA by consuming H2O2 and thus preventing the formation of reactive hydroxyl radicals.

Spontaneous and in vitro activation of synovial fluid and peripheral blood lymphocytes in rheumatoid arthritis.

Peripheral blood (PB) and synovial fluid (SF) were compared in parallel samples in rheumatoid arthritis (RA). The kinetics of in vitro T-cell activation was assessed in phytohemagglutinin (PHA) stimulated PB or SF mononuclear cell cultures on days 0, 1, 3 and 5. The early lymphocyte activation as assessed by interleukin-2 receptor expression was faster in SF than in PB cell cultures. In particular, IFN-gamma secretion was higher in SF than in PB cell cultures (p less than 0.01). Accordingly, lymphocyte major histocompatibility complex (MHC) locus II antigen expression was higher in SF than in PB cell cultures (53 +/- 7% vs. 21 +/- 5%; p less than 0.01). Our results suggest that lymphocytes, which are particularly effective producers of IFN-gamma when stimulated in vitro are sequestered in the diseased joints in RA.

Cathepsin G and elastase in synovial fluid and peripheral blood in reactive and rheumatoid arthritis.

The purpose of the study was to evaluate the involvement of serine proteinases cathepsin G and elastase on pathomechanisms in synovial fluid (SF) of patients with reactive (ReA) and rheumatoid, (RA) arthritis. Cathepsin G, elastase, and their endogenous inhibitors alpha1-antichymotrypsin (alpha1-ACT) and alpha1-proteinase inhibitor (alpha1-PI) were identified immunohistochemically from SF and peripheral blood (PB) of patients with ReA and RA. Cathepsin G and elastase activities in SF and PB were measured spectrophotometrically. Dot-immunostaining was used to identify cathepsin G, elastase, but also alpha1-ACT and alpha1-PI from SF and PB. Cathepsin G and elastase-like activities (IU/I) were slightly elevated in ReA SF compared to the corresponding peripheral blood values (11.4 +/- 9.2 vs 4.8 +/- 1.7, NS, and 5.1 +/- 2.8 vs 2.3 +/- 2.2, NS), which was similar to what was seen in RA (16.4 +/- 6.2 vs 0.53 +/- 0.4, p < 0.05, and 6.51 +/- 1.8 vs 1.22 +/- 0.58, p < 0.05). Although some samples did not contain cathepsin G and/or elastase-like activities, all samples contained immunoreactive enzyme, but also alpha1-ACT and alpha1-PI. In ReA SF, in contrast to monocytes, all polymorphonuclear (PMN) cells contained cathepsin G and elastase. Cathepsin G and elastase activities correlated with each other (r = 0.78, p < 0.05) suggesting PMN / primary granules as their likely source. There was a closer association between the cathepsin G or elastase and SF leukocyte count in ReA than in RA. In ReA and RA SF elevated cathepsin G and elastase activities are detected compared to activity levels in PB suggesting local production mainly from PMNs. The co-existence of highly cellular SF and cathepsin G and elastase activity in the documented presence of endogenous inhibitors in ReA SF together with the, known, usually self-remitting clinical course of ReA, suggest a brisk and even exaggerated local PMN serine proteinase release; sparing of joints does not seem to be due to lack or inhibition of PMN responses but rather to a successful down-regulation or cessation of the responses initially elicited.

Urinary excretion of glycosaminoglycans in malignant diseases of the haemopoietic and lymphatic tissues.

A study has been made of the urinary excretion of glycosaminoglycans (GAG) in 50 patients with malignancies, including 6 patients with acute myeloid leukaemia (AML), 11 with chronic myeloid leukaemia (CML), 10 with chronic lymphatic leukaemia (CLL), 10 with multiple myeloma (MM), 7 with Hodgkin's disease and 6 with mycosis fungoides (MF). The total urinary GAG were isolated by precipitation with cetyltrimethyl-ammoniumbromide (CTAB), and assayed in terms of their hexuronic acid content. A statistically highly significant increase in the excretion of total GAG was observed in all the disorders studied, except Hodgkin's disease, the highest value being seen in myeloid leukaemia (ML). Constant amounts of non-dialysable urinary GAG were electrophoresed in 0.5 M lithium acetate on cellulose acetate strips, and stained with alcian blue. The densitometric tracing derived from the electrophoresis strips were analysed with a Du Pont Curve Resolver. The electrophoretic data suggested the existence of a qualitative deviation in GAG excretion in CLL and in MF, in that patients with these diseases excreted on an average larger than normal amounts of slowly migrating GAG fractions. Pooled crude urinary GAG material from patients with CLL, MF, AML and CML and from control subjects was further purified and subjected to analytical studies. These indicated that a similar qualitative urinary GAG distribution exists in ML and in controls, whereas the urinary GAG in CLL and MF patients contained relatively more dermatan sulphate (DS, in terms of iduronate) than those of the controls.

Acid glycosaminoglycans in plasma. I. Determination.

Plasma glycosaminoglycans (GAG) were isolated from 10 ml of plasma by a modification of the method of Calatroni et al. (3). DE-52 anion-exchange cellulose was used in the isolation of the fraction operationally defined as free GAG. Chondroitin sulphate and heparin added to plasma were quantitatively recovered in this fraction. After proteolysis with papain the fraction operationally defined as bound GAG was isolated using anion-exchange resin AG 1 X 2. GAG were measured as hexuronate with the m-hydroxydiphenyl method of Blumenkrantz and Asboe-Hansen (7) which was superior to various modifications of the carbazole/borate carbazole procedures. In 15 healthy females and in 15 healthy males the concentrations of the free GAG (mean +/- S.D., expressed as microgram per 10 ml of plasma) were: 12.2 +/- 2.8 and 16.8 +/- 3.8 (P less than 0.001); of the bound GAG 40.4 +/- 7.7, and 40.2 +/- 11.6; and of the total GAG 52.7 +/- 9.0 and 57.0 +/- 10.4, respectively. With the isolation procedures used, plasma GAG were obtained in sufficient quantity for their electrophoretic characterization. Assay of plasma GAG can be performed with satisfactory accuracy and precision within two days by the present method. In clinical chemistry its application to the study of proteoglycan and GAG metabolism in various diseases may prove valuable.

Amyloidosis.

Developments concerning amyloidosis associated with rheumatic diseases or often causing musculoskeletal symptoms are reviewed. The pathogenesis, clinical manifestations, diagnosis, and therapy of amyloid A, amyloid light-chain, and amyloid beta 2-microglobulin amyloidosis are discussed from the standpoint of a clinical rheumatologist. The biology of the precursor protein serum amyloid A (SAA) has been extensively studied, and a new assay for SAA has been developed. In amyloid beta 2-microglobulin amyloidosis, modified beta 2-microglobulin may function as a pathogenic factor that initiates an inflammatory reaction in which macrophages produce cytokines that induce osteoclastogenesis and bone resorption. Further experience with serum amyloid P component scintigraphy in the diagnosis and monitoring of amyloidosis has accumulated. Guidelines for the dosage of colchicine in the treatment of amyloidosis associated with familial Mediterranean fever have been published. In amyloid light-chain amyloidosis, intensive chemotherapy in combination with bone marrow transplantation or autologous stem-cell infusion has potential therapeutic significance.

Glycosaminoglycan metabolism in experimental osteoarthritis caused by immobilization. The effects of different periods of immobilization and follow-up.

Glycosaminoglycan metabolism at different developmental stages of osteoarthritis provoked by immobilization was studied in rabbits. Tissue concentrations and the specific radioactivity of glycosaminoglycans (GAG) after a long immobilization (IZ) period (12 weeks) without follow-up, and a moderate IZ time (4-7 weeks) with a long follow-up period (average 22 weeks) were compared with the results obtained after a short IZ time (17 days). In tibial weight-bearing cartilage the specific 35S-activity had increased and the galactosamine, glucosamine and uronic acid concentrations had decreased in all the IZ groups examined. In the marginal cartilage, the menisci and the collateral ligament, the specific radioactivity and GAG concentrations had decreased or were normal after a long IZ with or without follow-up, although they were markedly elevated after a short IZ time. THe depletion of GAG correlated roughly with the IZ and follow-up times, except for periodically immobilized rabbits. In long-term "chronic" disease the specific radioactivities and GAG concentrations were depressed on the contralateral, non-IZ sides. The changes in the non-IZ knee are discussed. In studies of experimental osteoarthritis it is important to determine the developmental stage of the disease to be studied.