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Evaluation of the complement split product C3dg in synovial fluid as a possible diagnostic guide in inflammatory arthropathies.

Analyses of C3 split products with C3d specificity (C3dg) were performed in 157 synovial fluids (SF) of 129 patients with various known joint diseases. Considerable overlap between all inflammatory infectious, and crystal induced disease categories confines the value of such measurements as a diagnostic guide to only a few clinical situations. In seronegative as well as in seropositive rheumatoid arthritis no correlation between SF C3d levels and erythrocyte sedimentation rate could be found.

Arthritis↗

Accelerated clearance of albumin from the osteoarthritic knee: implications for interpretation of concentrations of "cartilage markers" in synovial fluid.

OBJECTIVE: The magnitude of articular cartilage destruction or repair in osteoarthritic (OA) joints has been deduced in some studies from the synovial fluid (SF) concentration of proteins derived from the extracellular matrix of the cartilage, without regard to the low grade synovitis that is often present in this disease. We examined the clearance kinetics of albumin, as a surrogate for cartilage derived proteins, from OA and control joints in an established canine model. METHODS: Twelve weeks after the left anterior cruciate ligament of 6 normal dogs was transected, 131I albumin (RISA) was injected into the contralateral (control) knee. Surface radioactivity was monitored for 7 h, and SF was then aspirated to determine the SF RISA concentration and leukocyte count, and calculate the volume of distribution (VD) and clearance of RISA. One week later, RISA was injected into the cruciate deficient knee and these measurements were repeated, then the intensity of synovial inflammation and severity of cartilage changes of OA in both knees were assessed. RESULTS: Synovitis and articular cartilage ulceration were seen only in the cruciate-deficient OA knee, in which the mean SF leukocyte count (1570/mm3), thickness of the synovial intima (mean = 30 microns), and severity of synovial mononuclear cell infiltration (mean = 13,500 cells/mm2) significantly exceeded those in the contralateral knee (850/mm3, 13 microns, 1400 cells/mm2; p < or = 0.01 in each case). In each dog, RISA VD in the OA knee was higher than in the contralateral knee (mean = 9.2 +/- 3.6 and 3.7 +/- 0.9 ml, respectively; p = 0.008) and RISA clearance rate in the OA knee exceeded that in the contralateral knee (3.8 +/- 1.5 and 1.4 +/- 0.3 microliters/min, respectively; p = 0.009). CONCLUSION: Accelerated clearance of protein from the OA joint with low grade synovitis could significantly affect the SF concentration of cartilage derived proteins. Therefore, inferences about the effect of a therapeutic agent on cartilage metabolism based upon changes in the concentration of a protein in serial samples of OA SF may be misleading unless protein clearance kinetics have been determined.

Animals↗

"Milwaukee shoulder"--association of microspheroids containing hydroxyapatite crystals, active collagenase, and neutral protease with rotator cuff defects. II. Synovial fluid studies.

Hydroxyapatite crystals in spheroid-shaped masses 1.9-15.6 micrometer in diameter were found in 12 of 13 synovial fluids obtained from the shoulder joints of 4 patients with rotator cuff tears and glenohumeral osteoarthritis. Two of 16 control joint fluids also showed these particles. Collagen types I, II and III were identified in the joint fluid pellets from 3 of the 4 patients, and fibers with typical collagen periodicity were also seen on transmission electronmicroscopy. Collagenase and neutral protease activities were found in fluids from 5 joints in 3 patients, whereas active collagenase was found in only 1 of 10 fluids from rheumatoid arthritis patients and in none of 3 fluids from patients with osteoarthritis. Neutral protease activities were present in several rheumatoid joint fluids. These findings are compatible with the hypothesis of an enzymatic release of hydroxyapatite crystals from the synovium and endocytosis by synovial macrophage-like cells with subsequent crystal-stimulated release of collagenase and neutral protease into the joint fluid, completing a pathogenetic cycle.

Aged↗

Elevated salivary and synovial fluid beta2-microglobulin in Sjogren's syndrome and rheumatoid arthritis.

Beta2-Microglobulin is normally present in low concentrations in serum and other bodily fluids. By use of a radioimmunoassay, elevated concentrations of beta2--microglobulin were found in saliva and synovial fluid from patients with Sjogren's syndrome and rheumatoid arthritis, autoimmune inflammatory diseases that attack and destroy the salivary glands and articular tissues, respectively. Elevated beta2-microglobulin concentrations decreased in the saliva of two patients who simultaneously showed a clinical response to systemic treatment. Measurement of beta2-microglobulin in inflammatory fluids may offer a simple method of quantifying local activity in autoimmune states.

Arthritis, Rheumatoid↗

[Interleukin-6 activity in synovial fluids of temporomandibular joint distrubance syndrome].

Using the IL-6-dependent mouse hybridoma cell line KD 83, IL-6 biological activity in synovial fluids of the patients with Temporomandibular Joint Disturbance Syndrome (TMJDS) was tested. The results show that IL-6 level is high in 13 cases of 18 patients with TMJ degenerative changes and 5 cases of 12 patients with TMJ Disc Displacement. IL-6 level is less than 100 U/ml in all patients with Masticatory Muscle Disorder (59.4 +/- 29.4 U/ml). In addition, it has been found that TMJDS tends to have acute and chronic stages and IL-6 is probably related to acute stage of the patients.

Adolescent↗

Synovial fluid crystals derived from erythrocyte degradation products.

We report 5 patients with bloody effusions containing crystals not previously reported to occur in synovial fluid. In one patient, rectangular hemoglobin-like crystals were found inside red cells previously observed elsewhere. In the other 4 cases, golden brown rhomboid crystals with positive elongation appeared to be hematoidin, a degradation production of hemoglobin, which has been found in nonarticular areas of old hemorrhage. In one patient, hematoidin crystals were identified lying in macrophage vacuoles. Both types of crystals are potential sources of confusion with known pathogenic crystals.

Aged↗

Synovial fluid mononuclear cells exhibit a spontaneous HLA-DR driven proliferative response.

We have tested the hypothesis that the spontaneous proliferation of synovial fluid mononuclear cells (SFMC) observed during in vitro culture is due to a mechanism analogous to the autologous mixed lymphocyte reaction (AMLR). Thus by observing the effect of addition of cyclosporin A, monoclonal anti-HLA-DR antibody, and recombinant interleukin 2 (rIL-2) on the spontaneous proliferation of SFMC from patients with rheumatoid arthritis (RA) and a wide range of seronegative spondyloarthritides (SN), we have demonstrated that this phenomenon is: (i) a T cell response, (ii) inhibited by the addition of cyclosporin A or monoclonal anti-HLA-DR antibody, (iii) enhanced by the addition of rIL-2 and (iv) the rIL-2 enhancement of the spontaneous proliferation is inhibited by a monoclonal anti-HLA-DR antibody. These observations suggest that the spontaneous proliferation of SFMC is at least in part an HLA-DR driven, IL-2 dependent event analogous to the AMLR, and support the concept derived from immunohistological studies of an important role for an antigen-presenting cell/T cell interaction occurring in the synovial membrane in inflammatory joint diseases such as RA.

Arthritis, Rheumatoid↗

[Macroglobulin alpha-2 in synovial fluid: relationship with reactants of the acute phase of rheumatoid arthritis].

Metalloproteinases (e.g. collagenase, elastase, stromelysin) are present in large amount in synovial fluid (SF) during rheumatoid arthritis (RA) and are actively involved in articular tissue damage. alpha 2-Macroglobulin (alpha 2M) functions as a "molecular trap" for proteinases and is considered the major inhibitor of metalloproteinases. We found increased concentrations of alpha 2M in SF of RA patients, significantly related to acute phase reactants, local inflammatory parameters and joint damage. The alpha 2M ratio between, RA SF and control SF, was found higher than between RA serum and control serum, indicating a selective localization and activity of alpha 2M in inflamed joint. The relationship between alpha 2M and the inflammatory parameters, including IL-6, is discussed.

Acute Disease↗

Highly increased levels of tumor necrosis factor-alpha and other proinflammatory cytokines in psoriatic arthritis synovial fluid.

OBJECTIVE: To investigate the potential role of cytokines in psoriatic arthritis (PsA) by assessing the profiles of the proinflammatory cytokines in synovial fluid (SF) of PsA in comparison with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, and IL-8 were measured in SF using ELISA. RESULTS: Levels of TNF-alpha, IL-1beta, and IL-8 were significantly higher in PsA SF than in OA SF, although lower than in RA SF. No difference was detected in the IL-6 levels between PsA and RA SF, both of which were much higher than in OA SF. CONCLUSION: The pattern of expression of proinflammatory cytokines seen in PsA is similar to that in RA. Since PsA is also a destructive arthropathy, cytokines, in particular TNF-alpha and IL-1, may be principle factors in joint destruction.

Antirheumatic Agents↗

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.

Chromatography, High Pressure Liquid↗

Free radicals and inflammation: protection of synovial fluid by superoxide dismutase.

Enzymatically genierated superoxide radical. by reactitng with hydrogen peroxide to prduce the hydroxyl radical, depolymerized puirified hyaluronic acid and bovine synovial flulid. Since phagocytizing polymorphonuclear leukocytes produce superoxide radicals, this reac-tion is sutggested and shown to be quantitatively feasible as the ini vivo mechanism of synovial fluid degradation in anl inflamed joint. Superoxide dismutase, and catalase protect synaovial fluid against such degradation in vitro.

Animals↗

Beta2-microglobulin-containing IgG complexes in sera and synovial fluids of rheumatoid arthritis and systemic lupus erythematosus patients.

The occurrence and composition of complexed beta2-microglobulin (beta2m) in sera and synovial fluids of rheumatoid arthritis and systemic lupus erythematosus patients and control persons was investigated. Beta2m-containing complexes were detected in immune complex (IC)-enriched fractions isolated by precipitation with 3% polyethylene glycol 6000, in the macromolecular peaks after Sephadex G-200 gel filtration, and in IC desorbed from solid-phase Clq. Beta2m complexes were demonstrated also after precipitation of redissolved PEG-insoluble material by anti-human beta2m serum or isolation of the complexes by use of Sephadex-anti-beta2m. IgG was co-isolated with beta2m on Sephadex-anti-beta2m and free beta2m inhibited the binding of IgG to Sephadex anti-beta2m, indicating that IgG was present in the complexed beta2m. Analysis by SDS-polyacrylamide gradient electrophoresis under reducing conditions indicated that the purified beta2m complexes contained IgG and beta2m.

Antigen-Antibody Complex↗

Inhibition of interleukin-1alpha-induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid.

OBJECTIVE: To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS: COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS: IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION: MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Animals↗

Mass spectrometric quantitation of muramic acid, a bacterial cell wall component, in septic synovial fluids.

This is the first report describing the use of gas chromatography-mass spectrometry for detection of muramic acid in infected synovial fluid (SF). Muramic acid is a ubiquitous component of bacterial cell walls, and it has been proposed that it could serve as a chemical marker for the presence of live bacteria or bacterial debris in rheumatoid joints. Our goal was to determine whether muramic acid was present at detectable levels in septic SF, since this would serve as a positive control for studies of reactive and rheumatoid arthritis. Muramic acid was found to be present at levels of less than 250-1,700 ng/ml in 12 septic SF samples (10 of which were culture positive for Staphylococcus aureus and 1 each for Escherichia coli and Streptococcus pneumoniae). Among these samples, those containing low bacterial colony counts did not contain detectable muramic acid. Muramic acid was also not detected in any SF samples from 20 control patients. We conclude that muramic acid can be used as a marker for the presence of bacterial peptidoglycan in SF. With further lowering of gas chromatography-mass spectrometry detection limits, determination of the quantities of bacterial debris present in joints of patients with rheumatoid or reactive arthritis will be attainable.

Cell Wall↗

Detection of stromelysin in synovial fluid and serum from patients with rheumatoid arthritis and osteoarthritis.

Stromelysin levels were measured using a one-step sandwich immunoassay in synovial fluid (SF) obtained from 31 patients with rheumatoid arthritis (RA) (31 samples) and 13 patients with osteoarthritis (OA) (13 samples) and in serum from 81 patients with RA (106 samples), 12 with OA (14 samples), 12 with gouty arthritis (gout) (14 samples), and 8 with osteoporosis (OP) (14 samples) to identify differences in the levels in these diseases as well as correlations with clinical parameters in RA. SF stromelysin levels were significantly higher in RA than in OA, and rose with increasing joint destruction in the former. No significant correlations were found between the SF stromelysin level in RA and various clinical parameters, except for the volume of SF which showed a correlation. Serum levels of stromelysin were highest in RA, gout, OA, and osteoporosis in decreasing order, and in RA were correlated with the Steinbrocker Stage. A significant correlation was also found between the serum stromelysin level and number of swollen joints, and correlations with the Lansbury index, ESR, CRP, WBC and Plt. The stromelysin level in SF was thought to be a useful parameter of local joint involvement and that in serum of the severity of systemic joint inflammation.

Aged↗

Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis.

We performed a genotypic characterization of seven strains of Mycoplasma fermentans which have been isolated from the synovial fluid of patients with rheumatoid arthritis (n = 2), spondyloarthropathy (n = 1), and unclassified arthritis (n = 4). We compared them to three reference strains (strains PG18 and K7 and incognitus strain) and to a clinical isolate from the urethra of a patient with nongonococcal urethritis. The characterization methods included electrophoresis of native DNA, arbitrarily primed PCR, and restriction fragment length polymorphism analysis following conventional and pulsed-field gel electrophoresis. Southern blot analysis with a probe internal to an insertion sequence was performed with the restriction products produced by the last two techniques. No extrachromosomal DNA sequences were detected. The M. fermentans strains identified by these methods did not present a unique profile, but they could be separated into two main categories: four articular isolates were genetically related to PG18 and the three other isolates, the urethral isolate, and the incognitus strain were related to K7. We also looked for the presence of the bacteriophage MAV1 (associated with the arthritogenic property of Mycoplasma arthritidis in rodents) in the M. fermentans strains. MAV1 DNA was not detected in either the clinical isolates or the reference strains of M. fermentans.

Adult↗

A molecular dynamics study of the three-dimensional model of human synovial fluid phospholipase A2--transition state mimic complexes.

Different modes of binding of transition state mimics: amide, phosphonate and difluoro ketone, to human synovial fluid phospholipase A2 (HSF PLA2) are studies by molecular dynamics simulations computed in solvent. The results are analysed in the light of primary binding sites. Hydrogen bonding interaction plays an important role for amino acids such as Gly32, Val30, and Glu55, apart from the well known active site residues viz Asp48, Gly25, Gly29, Gly31, His27, His47, Lys62, Phe23, Asn114 and Tyr112. In addition, the hydrogen bonding interaction between Sn-1 tetrahedral phosphonate group of amide and difluoro ketone inhibitors and crystallographic water molecules (H2O 523, H2O 524 and H2O 401) seems to have a significant role. Many of the active site charged residues display considerable movement upon ligand binding. The structural effects of ligand binding were analyzed from RMS deviations of C alpha in the resulting energy-minimized average structures of the receptor-ligand complexes. The values of the RMS deviations differ among the HSF PLA2s, in a pattern that is not the same for the three complexes. This suggests that ligands with different pharmacological efficacies induce different types of conformational changes of the receptor. Our active-orientation model is, at least qualitatively, consistent with experimental data and should be useful for the rational design of more potent inhibitors.

Amides↗

Synovial fluid transforming growth factor beta inhibits dendritic cell-T lymphocyte interactions in patients with chronic arthritis.

OBJECTIVE: To examine whether rheumatoid synovial fluid (SF) inhibits dendritic cell (DC) expression of the CD80 and CD86 costimulator molecules and contributes to SF T lymphocyte hyporesponsiveness. METHODS: Cell-free rheumatoid SF was tested for its effect on DC-stimulated autologous/allogeneic mixed lymphocyte reactions and for its effect on DC surface antigen expression, as assessed by flow cytometry. Blocking monoclonal antibodies were used to identify the SF cytokines that inhibited DC-T lymphocyte interactions. RESULTS: Low concentrations of SF (2.5%) could inhibit DC-mediated autologous and allogeneic T lymphocyte proliferation. This inhibitory effect could be reversed by neutralizing transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2), but not by IL-12, in the SF. Hyaluronic acid, IL-6, IL-10, and tumor necrosis factor alpha were not associated with SF inhibition. In vitro culture alone and crosslinking with the CD40 ligand up-regulated DC CD80/CD86 expression and costimulator function, and this was not affected by inclusion of SF. In the presence of SF, DC clustered with autologous T lymphocytes showed decreased CD80 and CD86 expression, and variable CD80/CD86 decreases were observed on DC clustered with allogeneic T lymphocytes. CONCLUSIONS: TGFbeta in SF appears to suppress T lymphocyte function, which may affect both signaling to DC and the induction of DC costimulator function.

Antibodies, Monoclonal↗