Synovial fluid calcium pyrophosphate dihydrate crystals and alizarin red positivity: analysis of 3000 samples.

Three thousand synovial fluids (1312 patients: chronic pyrophosphate arthropathy (CPA), 41%; osteoarthritis (OA), 12%; rheumatoid arthritis (RA), 16%) were examined for crystals, including calcium pyrophosphate dihydrate (CPPD), by polarized microscopy (score 0-3); calcific particles, by alizarin red positivity (ARP; 0-3); and total cell count. For 1150 fluids, local joint inflammation was assessed as 'active' or 'inactive' using a summated score of six clinical variables. CPPD and ARP scores did not correlate, but each showed positive correlation with age (P less than 0.01, P less than 0.02 respectively). Pseudogout had the highest mean CPPD score (P less than 0.001); intermittent CPPD positivity (range 8-100%) was seen in serially aspirated CPA joints, and there was no difference in CPPD positivity or score between active and inactive CPA. ARP was most frequent in OA subsets (72% of CPA, 46% of OA, 31% of RA; P less than 0.001). ARP was more frequent in active than inactive OA (P less than 0.05) but showed no association with inflammation in CPA or RA. Cell counts were higher in RA and pseudogout compared to OA and CPA, and in active compared to inactive RA. No correlation was found between ARP or CPPD scores and cell count. Cholesterol crystals were uncommon (0.2%) and showed no disease or joint predilection. In arthritic joints, CPPD and calcific particles particularly associate with the OA process and ageing. CPPD may contribute to acute and other calcific particles to chronic inflammation in OA.

Measurement of the 'fast' or complexed form of alpha 2 macroglobulin in biological fluids using a sandwich enzyme immunoassay.

A sandwich enzyme immunoassay has been developed for measuring the 'fast' or complexed form of alpha 2 macroglobulin using a complex-specific monoclonal antibody. The working range of this assay is 1.5-15 micrograms/l and is suitable for use with various biological fluids. Using this assay the normal plasma levels were found to range from 4.2 mg/l to 14.4 mg/l (0.17%-0.70% of total alpha 2 macroglobulin) with a mean value of 7.6 mg/l +/- 2.6 (0.37% +/- 0.12% of total alpha 2 macroglobulin). Elevated levels were seen in plasma samples taken on the day of admission from patients with acute pancreatitis and in some synovial fluid samples from patients with various arthritides.

Immune complexes and rheumatoid factors in canine arthritides.

Thirty two domestic dogs with naturally occurring polyarthritis were investigated to determine the contribution of autoimmunity in the pathological mechanisms. Comparisons were made with canine infective arthritis (12 dogs), osteoarthritis (32), and osteoarthritis secondary to rupture of the cranial cruciate ligament (19). Rheumatoid factors, immune complexes, and complement fixation (C1q binding) were measured in sera and synovial fluids. Compared with normal dogs (32), dogs with rheumatoid arthritis (RA) had increased serum and synovial fluid immune complexes and rheumatoid factors. Increases were generally also seen in dogs with other arthropathies, however. Rheumatoid factors were higher in sera than in synovial fluids. Rheumatoid factors correlated with immune complex levels and complexed rheumatoid factor only in the group of dogs with RA. Both rheumatoid factors and immune complexes may contribute to the pathogenesis of canine RA but are considered to arise as a result of non-specific inflammatory mechanisms in the non-rheumatoid groups.

[Measurement of keratan sulfate levels in synovial fluid in joint diseases]. / Merení hladiny keratan sulfátu v synoviální tekutine u kloubních onemocnení.

The authors describe their own modification of the ELISA method for the assessment of the epitope of keratate sulphate (KS) in synovial fluid. Considerably elevated values of KS were found in patients with gouty arthritis and reactive arthritis, as compared with patients with rheumatoid arthritis. The KS levels in the exudate of patients with osteoarthritis were significantly higher than in patients with rheumatoid arthritis. In patients with rheumatoid arthritis a negative relationship was found between the KS level in the exudate and the activity of the disease. A relationship was found between KS in the exudate and the interleukin-1 level in the exudate. Finally the authors discuss the importance of assessment of cartilage fragments in body fluids by immunochemical methods and future development trends of this discipline.

Joint capsular stiffness in knee arthritis. Relationship to intraarticular volume, hydrostatic pressures, and extensor muscle function.

Increased intraarticular hydrostatic pressure (Pia) may inhibit juxtaarticular muscle function, obstruct blood supply to joint structures and promote anoxic joint destruction in chronic arthritis. Joint capsular stiffness together with synovial fluid volume determines Pia at rest. Seventeen knee joints with effusive arthritis and different degrees of radiological cartilage involvement in 13 patients with chronic arthritis were examined. Since capsular elastance was difficult to standardize, we introduce a measure of joint capsular stiffness where the intraarticular volume yielding a pressure of 50 mm Hg (V50) is used. After normalization of injected volumes according to the V50, pressure volume curves became similar. Intraarticular hydrostatic pressure and maximal voluntary isometric extensor torque were measured simultaneously, while altering the intraarticular fluid volume in 9 knee joints. In 5 of these, quantified electromyography (EMG) of the vastus medialis and lateralis portion of the quadriceps muscle was also monitored. Progressive inhibition of extensor torque and EMG was found as the intraarticular pressure volume was increased in both intact and destroyed joints. No difference in inhibition was found for the 2 portions of quadriceps muscle tested. Increased intraarticular hydrostatic pressure Pia levels between 200 and 1150 mm Hg were observed during maximal voluntary activation of extensor muscles. The reproducibility was good for all variables studied. In a few instances evidence of intraarticular compartmentalization was found at low volumes. We conclude that the V50 is a convenient expression of capsular stiffness. Furthermore, increasing Pia caused by joint effusion inhibits knee extensor muscle function and impairs synovial blood flow. Awareness of these relations will facilitate more rational therapeutic approaches in chronic arthritis.

Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.

Interleukin-6 in synovial fluid from patients with arthritis.

Synovial fluid and serum from patients with rheumatoid arthritis, other inflammatory arthritides, and traumatic arthritis were assayed for the presence of interleukin-6 (IL-6) by means of an IL-6-dependent mouse hybridoma cell line. The cytokine was detected in all the samples of synovial fluid (range 50-22000 U/ml). IL-6 in synovial fluid was positively correlated (r = 0.58, P = 0.03) with the erythrocyte sedimentation rate in patients with inflammatory arthritis. In serum, the concentration of IL-6 was slightly elevated in some patients with rheumatoid arthritis. The results demonstrate that IL-6 is released into synovial fluid in joints affected by arthritis, and there appears to be an association between the levels of IL-6 and disease activity.

[Pathomechanism of calcium phosphate arthropathy based on a case]. / A kalcium-pirofoszfát arthropathia pathomechanizmusáról esetünk alapján.

Authors describe the case of a 43 years old male patient with complaints and restriction of motions since years, localized on the talocrural joint. In the background of the disease, responding inadequately to the therapy, calcium pyrophosphate depositions were found in the joint tissues. As the pathomechanism of the disease is even presently not cleared authors summarize our present knowledge referring to this problem.

Intra-articular apatite deposition in mixed connective tissue disease: crystallographic and technetium scanning characteristics.

An acute arthritis in a patient with mixed connective tissue disease (MCTD) was found to be associated with intra-articular deposition of carbonated hydroxyapatite crystals. A technetium hydroxymethylene diphosphonate bone scan showed intense uptake in the delayed phase scan of the affected joints. Synovial fluid analysis demonstrated uptake of the radiopharmaceutical drug directly onto the crystals.

Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease.

Synovial fluid samples (139) from 121 patients with rheumatoid arthritis, osteoarthritis, pseudogout, chronic pyrophosphate arthritis, gout, and reactive arthritis were analysed for cartilage proteoglycan components. Keratan sulphate (KS) epitope was determined by a competitive radioimmunoassay, and total sulphated glycosaminoglycans (S-GAG) were determined after papain digestion by a specific dye binding assay. Increased concentration of both KS epitope and S-GAG were found in synovial fluid from joints with acute inflammatory arthropathy (gout, pseudogout, and reactive arthritis). Analysis of consecutive samples from the same joint at different stages showed that the concentration of KS epitope or total S-GAG varied with acute inflammatory activity. In samples from patients with chronic conditions during active and inactive inflammatory phases concentrations were much lower and not distinguishable among these disease groups. The detection of raised concentration of proteoglycan components may reflect the rapid depletion or greatly increased turnover of proteoglycan in the articular cartilage during acute inflammation in the joint. This did not appear to be sustained in most patients with chronic joint diseases.

Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum.

Synovial fluids from 6 of 12 patients with rheumatoid arthritis (RA) and from 3 of 11 patients with reactive arthritis contained measurable levels of tumor necrosis factor alpha (TNF alpha). Seven of 12 sera from RA patients contained TNF alpha, while only 1 of those from reactive arthritis patients was positive. Gamma-interferon was detected in the synovial fluids and sera of only the RA patients. Tumor necrosis factor beta was not detected in any sera or synovial fluids. RA patients with detectable TNF alpha had higher erythrocyte sedimentation rates and synovial fluid leukocyte counts.

Crystal identification in human synovial fluids. Methods and interpretation.

Gout is largely solved, both from diagnostic and therapeutic standpoints. Acute gout is easily suppressed and joint destruction can be prevented and at least reversed by lowering the serum uric acid level with relatively safe and very effective drugs. But the arthritides associated with the calcium-containing crystals remain untreatable by other than symptomatic or surgical means. If we had a method or a drug to remove CPPD or BCP crystal deposits from joints, would it make any difference in the severity of the arthritis? Which of the paradigms shown in Figure 5 holds for these crystals? If joint damage directly follows crystal deposition as in gout, then crystal removal should prove prophylactic. The unusual pattern of joint degeneration associated with polyarticular CPPD crystal deposition and the initial appearance of CPPD crystals in radiographically normal cartilage favors this idea. But radiologic chondrocalcinosis appearing in knees subjected years before to meniscectomy but not in the contralateral knees suggests that crystal deposition, in these cases at least, is secondary to trauma or surgery. If degeneration of cartilage precedes crystal deposition, as it probably does in the case of BCP crystals, then crystal removal may not be particularly helpful. Dieppe and his colleagues proposed that the calcium crystals provide a positive feedback (amplification) loop. This represents the minimalistic view of their importance. The biologic consequences of the calcium crystal deposition diseases are now being explored at the molecular level. Much more data are needed before more than speculative answers to the questions posed here can be formulated. Calcium crystal deposition is more common in older persons. The degenerative and destructive arthropathies associated with them will predictably become increasingly common as our population ages.

Human arthritic synovial fluid influences proteoglycan biosynthesis and degradation in organ culture of bovine nasal cartilage.

The influence of synovial fluid and serum from patients with inflammatory joint disease on proteoglycan metabolism was studied in organ culture of bovine nasal cartilage. Proteoglycan biosynthesis, i.e. incorporation of [35S]-sulphate, was reduced after addition of synovial fluid from rheumatoid arthritis and reactive arthritis patients. Also some rheumatoid arthritis sera but no reactive arthritis serum reduced the biosynthesis compared to control sera. Proteoglycan degradation, i.e. release of proteoglycans prelabelled with [35S]-sulphate, as well as release of proteoglycans determined by chemical methods, was highest under the influence of rheumatoid arthritis synovial fluid. This effect appears to represent an activity truly stimulating degradation, since added control serum did not prevent the effect. The lowest proteoglycan degradation was observed in culture medium only. Addition of synovial fluid compared to addition of control serum did not increase proteoglycan degradation in freeze-killed cartilage indicating that the effect requires living cells. The findings are consistent with the presence in synovial fluid of mediators stimulating the chondrocytes both to activate proteoglycan degradation and to reduce proteoglycan biosynthesis.

The detection and initial characterization of colony-stimulating factors in synovial fluid.

In this study which included 16 patients with inflammatory or non-inflammatory arthropathies, human granulocyte-macrophage colony-stimulating activity was detected in synovial fluid. This was attributable to the presence of colony-stimulating factor(s) (CSF), as a direct action on human bone marrow progenitor cells was demonstrated using clone transfer experiments. Samples of synovial fluid also stimulated the growth of murine macrophage colonies and induced differentiation in the murine myelomonocytic leukemia cell line, WEHI-3B(D+), which are characteristic properties of human macrophage-CSF or granulocyte-CSF respectively. These findings and the results of preliminary fractionation procedures suggested that the colony-stimulating activity in synovial fluid was not explicable by the presence of any one of the well-characterized human CSF acting in isolation. This provides a new insight into the pathogenesis of inflammatory arthropathies and supports the hypothesis that CSF have important roles in vivo in addition to the regulation of haemopoiesis.

Cell receptors to sulphated polysaccharides in the acute and chronically inflamed synovial joint.

Receptors to sulphated polysaccharides have recently been discovered on "free" joint fluid cells and synovial membrane cells in the normal joint. A search for these receptors on cells was made in rabbits with acute and chronic adjuvant inflammatory arthritis in an attempt to further elucidate their role in joint homeostasis. These experiments demonstrated a significant increase in cell numbers within the joint. Receptor activity was most marked on macrophages found free within the synovial fluid. It is postulated that exogenous cells may be important in the process of joint destruction and are outside the control of the normal joint regulatory mechanisms. The endogenous cell population, which exhibits receptor activity, may be responding to the process of joint destruction by proliferation as a secondary phenomenon.

Total and free ketoprofen in serum and synovial fluid after intramuscular injection.

Free and total ketoprofen levels in serum and synovial fluid were determined in 37 patients after a single intramuscular injection of ketoprofen, 100 mg. Free drug was separated by equilibrium dialysis. Ketoprofen was assayed by HPLC. Ketoprofen penetrated into the joints rapidly and significant concentrations were found at 15 minutes. The equilibrium time was about 3 1/2 hours. The AUC for total ketoprofen was greater in serum than in synovial fluid. On the other hand, the free fraction AUC in the serum and synovial fluid were quite similar. The mean residence time in the joint was about three times that in the systemic circulation. Ketoprofen was strongly bound to proteins and the percentage of free ketoprofen was not significantly different between serum and synovial fluid. These results provide a possible explanation for duration of the therapeutic effect of ketoprofen despite the short elimination half-life from the serum.