Diagnostic value of synovial fluid analysis in children with reactive arthritis

We report on three children with pauciarticular arthritis in whom the clinical picture and serology were compatible with both arthritis reactive to infection with Yersinia or Salmonella and with Lyme arthritis. Results of analysis of synovial fluid by polymerase chain reaction for enterobacterial or borrelial sequences were negative. Immunofluorescence with specific antibodies revealed the presence of amorphous enterobacterial antigens in synovial fluid cells. Since this staining did not reveal enterobacterial morphology, we infected synovial fluid cells of two children with juvenile rheumatoid arthritis in vitro with Yersinia or Salmonella. After 24 h typical rods were observed, but after about 1 week amorphous antigen similar to what had been found in the three patients was seen. In cases of reactive arthritis with ambiguous results of serological testing the diagnosis may be confirmed by demonstration of enterobacterial antigens in synovial fluid.     

IgE-containing immune complexes in synovial fluid of patients with rheumatoid arthritis

Summary The prevalence and composition of IgE-containing immune complexes in paired synovial fluid and serum of 42 patients with classical or definite rheumatoid arthritis were studied. IgE-containing immune complexes were found in 15/42 synovial fluids; 15 sera were also positive. The correlation between serum and synovial fluid complexed IgE levels was high (r=0.77). The mean ratio of synovial fluid/serum levels was 1.96, i.e. significantly higher than 0.33, the synovial fluid/serum ratio for alpha-2-macroglobulin (molecular weight 820 kD), which was taken as high molecular weight control protein (p<0.0001). Apart from IgE in immune complex form, monomeric IgE was also significantly higher in synovial fluid compared to serum (ratio = 2.94). Other constituents which could be found in the immune complexes, i.e. anti-IgE antibodies, rheumatoid factors and anticollagen antibodies, were also higher in synovial fluid than predicted. Our results suggest intra-articular production of IgE-containing complexes in the synovial fluid, in addition to possible exudation of the complexes from the serum. These findings provide further evidence for the role of IgE-containing immune complexes in rheumatoid synovitis.     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

Protease inhibitors in rheumatoid synovial fluid

Summary Paired plasma and synovial fluids from 17 patients with seropositive rheumatoid arthritis were examined for electrophoretic homogeneity/heterogeneity and enzymic inhibitory capacity of the protease inhibitors. The high degree of saturation (90%) of the polyvalent protease inhibitor ₂-macroglobulin in the rheumatoid synovial fluid contrasts sharply with the low saturation of ₁-antitrypsin. The inhibitory reactivity of the non-complexed fraction of both of these dominating antiproteases was retained (85–90%). Thus, a selective inactivation of synovial ₁-antitrypsin could not be demonstrated. ₁-Anti-chymotrypsin revealed electrophoretic homogeneity in all synovial fluids. Electrophoretic heterogeneity of the plasmin inhibitor antiplasmin was detected in the majority of synovial fluids indicating plasmin activation. The existence of a protease-antiprotease imbalance in the rheumatoid joint was indicated by the high degree of saturation of ₂-macroglobulin and a cleavage of C3 in rheumatoid synovial fluids.     

Arthritis diagnosis based upon the near-infrared spectrum of synovial fluid

Synovial fluid aspirates have been characterized by measuring their visible/near-infrared spectra (400–2500 nm). The hypothesis tested in this study is that the spectra contain sufficient information to serve as an aid in the diagnosis and/or staging of arthritic disorders. The concentrations of all major constituents are carried implicitly in the spectra, and in this sense this approach is similar in spirit to conventional synovial fluid analysis. The distinguishing feature of this method is that we have not converted the raw data (spectra) explicitly to analytical information. Rather, we have used automated pattern recognition methods to identify significant characteristics of the spectra themselves. A total of 109 spectra were measured and split into three classes according to the disease (osteoarthritis, rheumatoid arthritis, or spondyloarthropathy) affecting the patient from whom the synovial fluid sample was taken. An automated classification method was then trained by correlating features derived from these spectra to the clinical diagnoses. The robustness of the classification was validated using the leave-one-out cross-validation method, i.e., by training on all but one of the spectra and using the resulting model to predict the classification for the spectrum that is left out. The result derived by following this procedure for each of the spectra was that 105 of the 109 predicted classifications correctly matched the clinical diagnosis. These results suggest that the near-infrared spectrum of synovial fluid is sufficient to allow diagnosis of the disease affecting the joint from which the aspirate is drawn.     

Comparison of the effects of different anti-inflammatory drugs on synovial fluid prostanoid concentrations in patients with rheumatoid arthritis

Summary The effects of one-day treatment with nine nonsteroidal anti-inflammatory drugs and prednisolone on human synovial fluid concentrations of prostanoids were studied. The doses were calculated so as to be approximately equipotent according to clinical experience and the recommendations of the manufacturs. Most of the drugs used reduced clearly PGE₂ and TxB₂ levels in synovial fluid, but only a slight diminution in 6-keto-PGF₁ values was found. Carprofen, diclofenac, indomethacin, naproxen and tolfenamic acid reduced significantly the synovial fluid PGE₂ concentrations. Diclofenac and indomethacin also reduced significantly the synovial TxB₂ concentrations.     

Induction of a lymphotoxin-like mediator in peripheral blood and synovial fluid lymphocytes by incubation with synovial fluid from patients with rheumatoid arthritis

Summary A significantly increased spontaneous cell-mediated cytotoxicity (SCMC) has been reported in synovial fluid lymphocytes (SFL) as compared to peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA) and that of normal controls [1–3]. To determine whether this increased SCMC activity is due to the production of a lymphokine and related to the production of a lymphotoxin (LT)-like mediator, PBL from normal controls and PBL and SFL from RA patients were incubated either with a human melanoma cell line (IGR 3) or with cell-free synovial fluid (SF) from RA patients. The SF and the cell-free supernatants of the different cultures were tested for LT activity by estimation of inhibition of DNA synthesis of HeLa cell monolayers and they were added to a SCMC assay system using normal PBL and IGR 3 as target.In the supernatants from cocultures of either PBL from controls or PBL and SFL from RA patients with IGR 3 cells, there was no significant difference in LT activity. An LT-like mediator was observed in the supernatants of all lymphocytes cocultured with SF, whereas SF alone and supernatants of lymphocytes alone exhibited little or no LT activity. In a control experiment, LT induction was not observed when normal lymphocytes were cultured with the serum of RA patients. Absorption of the culture supernatants with an insolubilised goat anti-human Ig did not remove LT activity. The demonstrated release of an LT-like mediator from lymphocytes incubated with SF might be one contributing mechanism to the inflammatory joint reaction in RA patients.     

Food allergy and seronegative arthritis: report of two cases

The link between food allergy and arthritis is still a matter for debate. Here we report two cases of patients suffering from arthritis sustained by food allergy. Diagnosis was performed on the basis of a 2-week elimination diet followed by an open and double-blind challenge test which was repeated three times. Both patients had a previous medical history of food allergy/intolerance. As the number of patients with joint complaints sustained by food allergy is very small it makes no sense to put all patients on diet. A previous medical history of food intolerance is one of the main reasons to start the long and difficult path towards a diagnosis of food allergy. Received: 3 July 2000 / Accepted: 17 January 2001     

Measurement of colony-stimulating factors in synovial fluid: potential clinical value

In this study, 100 synovial fluid (SF) samples from patients with a variety of arthritides were assayed for levels of colony-stimulating factors (CSFs) using a human bone-marrow bioassay and enzyme immunoassays for granulocyte (G-) and granulocyte-macrophage (GM-) CSFs. GM-CSF was found more frequently in samples from rheumatoid arthritis (RA) subjects (49%) than in non-RA samples (29%). Absence of GM- but not G- or bioassay CSFs characterised samples from subjects with psoriatic arthritis and ankylosing spondylitis (n=14). There was strong evidence of an antagonistic relationship between levels of G- and GM-CSFs in samples from RA patients, an effect independent of drug treatment. However, treatment with non-steroidal anti-inflammatory agents (NSAIDs) may affect reported CSF concentrations: G-CSF levels were significantly lower in samples from subjects not taking NSAIDs. These results suggest that SF-CSF estimations using commercially available assays could provide useful diagnostic clues for clinicians, but careful interpretation is warranted particularly in patients on long-term NSAID treatment.     

Methods of assessing the synovial fluid cell count

Summary We have compared a rapid side-room testing strip and an automated cell counter with the conventional haemocytometer counting chamber as methods for assessing the synovial fluid cell count. The testing strip was shown to be very sensitive in detecting esterases derived from granulocytes, but to the experienced clinician it offered little clinical advantage over naked-eye judgement. The automated counter provides a fast and reliable alternative to the haemocytometer and in situations where an accurate cell count is required it could replace the haemocytometer.     

Comparison of microheterogeneity of alpha-1-acid-glycoprotein in serum and synovial fluid from rheumatoid arthritis patients

Summary Microheterogeneity of alpha-1-acid glycoprotein was studied using affinity immunoelectrophoresis with concanavalin A as a ligand in the samples of serum and synovial fluid obtained at the same time from 22 patients with rheumatoid arthritis. Individuals with intercurrent infection or other illnesses were excluded from the study. The results were expressed as reactivity coefficient (RC). Disease activity was evaluated by Mallya-Mace Activity Score, Lansbury Joint Index and laboratory tests. In most of the studied samples the glycosylation pattern was similar, composed of a nonreactive variant and 2 reactive (the first and the second) with Con A variants. In seven samples of synovial fluid an extra third peak representative of the strongly reactive one with Con A fraction was observed. It was the cause of the remarkable elevation of AGP-RC. Moreover, the level of IgM and IgA RF was higher in the synovial fluid derived from these patients — with the presence of the third peak in AFF-EP with Con A — than in those without the considered fraction.     

Comparison of blood and synovial fluid lymphocyte subsets in rheumatoid arthritis and osteoarthritis

Summary Immunoregulatory T-cell deficiency is thought to underlie pathogenesis of rheumatoid arthritis (RA) as a systemic autoimmunopathy. The aim of this study was a simultaneous analysis of peripheral blood and synovial lymphocyte subsets (Ly-SS) of RA patients as compared to patients with locally active osteoarthritis (OA). Peripheral blood Ly-SS and paired synovial fluid Ly-SS from 87 RA patients were analysed by two dimensional flow cytometry (Simulset Becton Dickinson) as compared to 15 OA patients. The control group consisted of 32 healthy subjects. The peripheral blood analysis from RA and OA patients revealed a significant decrease of CD8+T-cells and increase of CD4+:CD8+ ratio when compared to the control group. The blood of RA patients showed a significant increase of HLA DR+ and IL 2R+T cells as compared to OA group. The synovial fluid from RA and OA patients showed a significant increase of CD3+, CD8+, HLA DR+ T-cells and decrease of CD4+:CD8+ ratio and CD19+ cells in comparison to the peripheral blood. This study shows, that the OA T-cell system seems not to be activated in peripheral blood in opposition to RA patients. Synovial fluid Ly-SS in OA, however, showed only quantitative but not qualitative differences. OA seems to be mainly a local inflammatory response depending on T-cells, when lymphocyte T activity in blood is diminished.     

Identification of citrullinated cellular fibronectin in synovial fluid from patients with rheumatoid arthritis

Abstract

Objectives. Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients.

Methods. Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification.

Results. Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF.

Conclusions. Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.     

Soluble CD21 in sera and synovial fluid of arthritic patients

Soluble CD21 (sCD21) is the ectodomain of the CD21 glycoprotein released by shedding from the cellular membrane. The ectodomain of CD21 is capable of binding complement fragments, Epstein-Barr virus (EBV) and CD23. Functionally sCD21 can activate monocytes and abrogate B-cell/follicular dendritic cell interaction, thereby inhibiting antibody production by antigen primed B cells. Levels of sCD21 vary in several clinical conditions. Here we analyzed sCD21 in synovial fluids and sera in arthritic patients. sCD21 concentrations were consistently lower in synovial fluids compared to paired sera samples from the same patients. In contrast to healthy donors, sCD21 levels are significantly reduced in rheumatoid arthritis patients sera. Potential causes and consequences of the data are discussed.     

类风湿关节炎活动期滑液中葡萄糖6-磷酸异构酶水平及与抗环瓜氨酸肽抗体的关系

目的 检测葡萄糖-6-磷酸异构酶(GPI)在类风湿关节炎(RA)活动期膝关节滑液中的含量,并探讨滑液中GPI与抗环瓜氨酸肽(CCP)抗体的关系.方法 用酶联免疫吸附法(ELISA)检测22例RA活动期患者和37例骨关节炎活动期患者滑液中GPI和抗CCP抗体的浓度.组间比较采用t检验,相关性分析采用Spearman相关分析.结果 RA活动期患者膝关节滑液中的GPI浓度高于骨关节炎活动期患者[(9.6±8.4)和(0.9±1.8)μg/ml],差异有统计学意义(P＜0.01);RA活动期患者与骨关节炎活动期患者滑液的抗CCP抗体浓度分别为(14.61±18.64)和(1.42:±0.09) U/ml,差异有统计学意义(P＜0.01);RA活动期患者膝关节滑液中GPI含量和抗CCP抗体浓度呈正相关(r=0.447,P=0.037).结论 GPI在RA活动期患者滑液中高表达,可能与RA发生发展过程中慢性滑膜炎、骨质破坏有关.GPI与抗CCP抗体呈正相关,两者可作为RA实验室诊断的参考指标.     

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

Summary 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.     

Evidence for activation of platelets in the synovial fluid from patients with rheumatoid arthritis

Summary Platelets were isolated by gel filtration from paired samples of peripheral blood and synovial fluid (SF) aspirated from inflamed knee joints from 20 adult patients with rheumatoid arthritis (RA) as well as from peripheral blood obtained from 20 healthy subjects. The platelets from the three different sources were investigated for quantitative differences in the number of two distinct types of intracellular storage organelles using immunofluorescence staining for platelet factor 4 (PF4) and labelling with the fluorescent substance mepacrine (MC). The number of PF4-stained organelles per cell was the same in the peripheral normal and RA platelets. This number was distinctly lower in the SF platelets. The peripheral and SF platelets from the RA patients had the same number of MC-labelled organelles. This number was distinctly lower than in the normal cells. The results suggest that the peripheral RA platelets had been activated to liberate serotonin and other substances from one type of organelles, and that the SF platelets had been activated to an additional liberation of PF4 from another such type. Liberated PF4, serotonin, and other substances from SF platelets may, in several ways, contribute to the inflammatory responses of RA.     

他克莫司对类风湿关节炎关节滑膜液淋巴细胞协同刺激分子的作用

目的研究他克莫司对类风湿关节炎（RA）患者关节滑膜液淋巴细胞协同刺激分子的作用．并初步探讨其免疫抑制的机制。方法分离培养RA关节滑膜液淋巴细胞，经100nmol/L的他克莫司处理后．用流式细胞仪检测T淋巴细胞协同刺激分子CD28、CD154（CD40L）和B淋巴细胞协同刺激分子CD80、CD86、CD40的表达。同时检测T、B淋巴细胞活化标志CD69、CD25、HLA—DR和CD71的表达．用酶联免疫吸附法（ELISA）检测淋巴细胞培养上清Th1细胞分泌的细胞因子白细胞介素（IL）-2、干扰素（IFN）-γ和Th2细胞分泌的细胞因子IL-6、IL-10的水平，并设不加他克莫司处理的为对照组。结果经他克莫司处理后。T淋巴细胞的CD154（CD40L）和B淋巴细胞的CD86表达阳性率低于对照组（P〈0．05），而T淋巴细胞的CD28和B淋巴细胞的CD80、CD40表达阳性率与对照组相比差异无统计学意义；T、B淋巴细胞的HLA—DR表达阳性率明显低于对照组（P〈0．01），而CD69、CD25、CD71表达阳性率与对照组相比差异无统计学意义；淋巴细胞培养上清Th1细胞分泌的细胞因子IL-2、IFN-γ和Th2细胞分泌的细胞因子IL-6、IL-10的水平与对照组相比均显著下降（P〈0．01）。结论他克莫司能明显抑制RA关节滑膜液淋巴细胞的活化，降低Th1细胞因子和Th2细胞因子的分泌水平，这种作用可能是通过下调淋巴细胞的协同刺激分子的表达而实现。     

Evaluation of a recombinant antigen enzyme-linked immunosorbent assay (ELISA) in the diagnostics of antinuclear antibodies (ANA) in children with rheumatic disorders

The reliability and accuracy of ELISAs for the detection of circulating ANA in children with rheumatic diseases has recently been questioned. In this study we evaluated an allegedly superior ELISA method using recombinant antigens in a paediatric population with various rheumatic conditions and compared it to a conventional Hep-2 IFA assay. Sera from 123 children (204 blood samples) were simultaneously tested by conventional ANA immunofluorescence on Hep-2 cells (ANA-IFA) and recombinant antigen ELISA (rELISA). There were 44 children with systemic lupus erythematosus (SLE), 29 with juvenile rheumatoid arthritis (JRA), eight with mixed connective tissue disease (MCTD), eight with reactive arthritis, five with juvenile fibromyalgia syndrome, three with dermatomyositis (JDMS) and 31 with other diagnoses.  Thirty-five children (27%) had a positive Hep-2 result, which remained undetected by ELISA (P <0.002). Almost all of these children had significant IFA titres above 1:160. The major discrepancies were observed in children with JRA and SLE. There was no titre correlation between the two assays and the rELISA’s OD readings were not linear.  The ELISA using recombinant antigens was not useful for the detection of serum ANA in children with rheumatic diseases due to a high rate of false negative results. These data concur with recent reports about the lack of reliability of ELISAs using non-recombinant antigens. Received: 2 May 2001 / Accepted: 10 September 2001     

类风湿关节炎滑膜组织血管内皮生长因子转录和转录后的研究

目的 研究类风湿关节炎(RA)患者血清、滑液及滑膜组织中血管内皮生长因子(VEGF)的表达水平，探讨其在RA致病中的作用。方法 应用双抗夹心酶联免疫吸附测定（ELISA)法分别测定RA患者血清、滑液中VEGF蛋白水平；采用半定量反转录-聚合酶链反应(RT-PCR)法和Northern blot检测滑膜组织VEGF mRNA表达水平。以骨关节炎(OA)病人及因外伤截肢的正常人作对照。结果 RA患者血清、滑液中VEGF蛋白水平显著高于对照组；RA滑膜组织VEGF mRNA表达水平也明显高于对照组。结论 RA患者滑膜组织VEGF的过度表达，引起血管增生，是引起RA发病的重要因素。