Augmented plasma and tissue kallikrein like activity in synovial fluid of patients with inflammatory articular diseases

We studied some of the components of the kininogen-kallikrein-kinin system, simultaneously, in plasma and synovial effusions of patients with inflammatory articular diseases. Plasma and tissue kallikrein like activity and kininogen levels were evaluated. Active plasma and tissue kallikreins in plasma and synovial fluid were detected by their amidase activity upon specific chromogenic substrates. Kininogen levels were determined by a bioassay. Both specific amidase activity of plasma and tissue kallikreins were augmented in synovial effusions in relation to their own plasma activity. Kininogen levels in synovial fluid tended to be diminished in relation to plasma, however statistical significance was not reached. The consumption of kininogen is probably related to kinin production. This finding together with increased activities of plasma and tissue kallikreins reinforce the involvement of kinins in pathogenesis of inflammatory articular diseases.accepted by W. B. van den Berg     

骨性关节炎关节液中白细胞介素6和基质金属蛋白酶3的表达

 背景：已有实验证实膝骨关节炎的发病与炎症细胞因子有密切的关系。目的：研究骨性关节炎患者关节液中白细胞介素6和基质金属蛋白酶3水平及其相关性,以及两者与骨性关节炎严重程度之间的相关性。方法：收集关节穿刺治疗的骨性关节炎患者关节液标本78份,应用ELISA法检测标本中白细胞介素6和基质金属蛋白酶3质量浓度,并对患者骨性关节炎严重程度进行HSS评分,采用Pearson相关分析探讨白细胞介素6、基质金属蛋白酶3和美国纽约特种外科医院(HSS)评分两两之间的相关性。结果与结论：骨性关节炎患者膝关节液中白细胞介素6为(13.1±5.7) μg/L,基质金属蛋白酶3为(989.3±429.5) μg/L, HSS为(56.4±12. 0)分。Pearson相关分析显示白细胞介素6、基质金属蛋白酶3的表达水平呈显著性正相关关系(r=0.70,P < 0.001);白细胞介素6和基质金属蛋白酶3均与关节美国纽约特种外科医院(HSS)评分均呈负相关关系(r=-0.70,r=-0.75,P < 0.001)。结果显性关节液中白细胞介素6和基质金属蛋白酶3水平之间,以及两者与骨性关节炎严重程度之间均呈正相关,提示测定骨性关节炎患者关节液中白细胞介素6和基质金属蛋白酶3水平对早期诊断和治疗具有重要意义。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Supravital staining of synovial fluid with Testsimplets.

We explored the use of Testsimplet (TS) in synovial fluid (SF) analysis. TS is a glass slide coated with a dry mixture of methylene blue and cresyl violet, which in contact with one drop of SF provides a stained fresh preparation. We applied the TS to the study of 159 SFs of patients with different rheumatic diseases. In those SFs of patients with crystal-associated diseases, the crystal search was performed both on unstained preparations and with TS. TS was as good as the Wright's and Papanicolaou stain in characterizing SF cells, lupus erythematosus cells, and detection of occasional bacteria. TS allowed a better visualization of Reiter's cells, cartilage fragments, synovial villi, fat droplets, and fibrin. Crystals were identified in every TS of those patients with crystal-associated diseases. TS is a rapid and reproducible method of SF supravital staining. Crystals are well preserved for simultaneous examination with compensated polarized light.     

关节液中纳米颗粒的测量对关节疾病诊断的意义

 目的：探讨不同状态下关节液中纳米颗粒与关节疾病种类的相关性。方法：抽取正常人、膝关节骨性关节炎（KOA）和滑膜炎（KTS）患者的关节液，利用准弹性激光散射技术测定关节液中纳米颗粒的粒度大小及其分布，利用相分析电泳光散射技术测定关节液纳米颗粒的Zeta电位，并采用相关分析方法分析纳米颗粒的粒度、Zeta电位与疾病的相关性。结果：KOA和KTS关节液纳米颗粒的平均粒度和Zeta电位都分别显著大于正常对照（P<0.01），粒度和Zeta电位分布曲线比正常对照组的宽（P<0.01）。关节液中纳米颗粒的平均粒度（r_p=0.7972，P<0.01）、Zeta电位（r_p=0.6319，P<0.01）与关节的疾病种类存在很好的相关性。结论：关节液纳米颗粒的粒度和Zeta电位与关节疾病存在显著的相关关系, 可建立起一个关节疾病早期诊断的检测方法。     

Correlation of changes in pain intensity with synovial fluid adenosine triphosphate levels after treatment of patients with osteoarthritis of the knee with high-molecular-weight hyaluronic acid

We sought to determine whether a clinical association exists between osteoarthritis (OA)-associated knee pain and adenosine triphosphate (ATP) levels in synovial fluid (SF). A total of 28 patients with 28 primary OA knees were included. They routinely received intra-articular injection of high-molecular-weight hyaluronic acid (HA) once weekly for 5 weeks (treated group). Eight patients without knee pain who had undergone an operation for anterior or posterior cruciate ligament reconstruction 2 years ago were also examined (control group). SF and blood ATP concentrations, total amount of ATP, total SF volume, and Visual Analogue Scale (VAS) scores in all patients were measured and we compared pre-treatment values with those 1 week after the final treatment. We evaluated the correlation of change in total ATP (??ATP) and change in VAS score (??VAS), ??VAS and change in SF volume (??SF), and ATP concentration in SF and blood. In the treated group, SF ATP concentration, total amount of ATP, SF volume, and VAS score were all significantly lower post-treatment than pre-treatment (p = 0.0005, 0.0003, 0.0022, and < 0.0001, respectively). In treated group, ??VAS was significantly associated with ??ATP (r = 0.56, p = 0.0032), ??SF was significantly associated with ??VAS (r = 0.78, p < 0.0001), and total amount of SF ATP and SF volume at pre-treatment were significantly higher than the control group (p < 0.0001, p < 0.0001) We demonstrated an association between SF ATP level changes and OA knee pain, which should facilitate a further understanding of OA pain mechanisms.     

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP⁺ and VNR⁺ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.     

Immunohistochemical localization of metallothionein in synovial tissue of patients with chronic inflammatory and degenerative joint disease

 Metallothioneins (MTs) are low-molecular-weight cytosolic proteins, which are thought to participate in metal homeostasis and protection against metal toxicity and oxidative stress. MT synthesis can be induced by a variety of inflammatory mediators and antirheumatic drugs, and high levels of MT have been implicated in resistance of cells to some antirheumatic drugs. We studied the expression and localization of MT in synovial tissue samples from patients with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or osteoarthritis (OA) by quantitative immunohistochemistry. Immunostaining for MT was detected in a large number of intimal lining cells in most of the investigated synovial tissue samples (75%). In a smaller proportion of samples (42%), some of the fibroblast-like cells of the subsynovial layer were also MT positive. Immunostaining and double-staining experiments with antibodies against monocyte-, macrophage- and leucocyte-associated antigens suggested that most of the MT-positive cells were intimal fibroblast-like cells and subsynovial fibroblasts. However, there were no statistically significant differences in the intensity of staining for MT between the rheumatic diseases and OA at the single-cell level. Thus, MT is expressed in synovial tissue and may participate in homeostatic and protective functions. The interindividual variability in the expression of MT in synovial tissue may be related to the therapeutic efficacy of the gold compounds and chemotherapeutic antirheumatic drugs sequestered by MT. Received: 25 July 1997 / Accepted: 23 March 1998     

Neuropeptides and interleukin-6 in human joint inflammation. Relationship between intraarticular substance P and interleukin-6 concentrations

Plasma and synovial fluid concentrations of interleukin-6 (IL-6), using an enzyme-linked immunosorbent assay, as well as immunoreactive levels of calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal peptide (VIP) were measured in 18 patients with rheumatoid arthritis and 20 with osteoarthritis of the knee. The concentrations of IL-6 were elevated in both plasma and synovial fluids from patients with rheumatoid arthritis whereas higher levels of substance P-, CGRP- and VIP-like immunoreactivities were found in the synovial fluid, but not in plasma, from patients with rheumatoid arthritis when compared with those in osteoarthritis. Furthermore, IL-6 and substance P levels in synovial fluid were significantly correlated both in rheumatoid arthritis and osteoarthritis patients. Our data seem to support the idea of an important role shared by neuropeptides and IL-6 in the pathogenesis of human inflammatory joint disease.     

关节滑液检测在关节假体周围感染诊断中的研究进展

关节假体周围感染(periprosthetic joint infection,PJI)是人工关节置换术后可能发生的一种严重并发症,其导致的严重后果,无论对于医生还是患者来说,都很难接受。目前,由于多种不确定因素的存在,PJI诊断的准确性较低。传统的血清学检查、影像学检查有一定的价值,但是易受全身情况的影响,导致特异性不高。为了正确诊断关节假体周围感染,不同学科的研究人员采用各种不同的方法进行了大量的诊断研究,并取得了丰富的成果。近年来,关节滑液炎性标志物检测、分子生物学方法等被研究证实具有较高的敏感性和特异性。因此,关节滑液CRP、-防御素、白细胞酯酶、PCR技术等被广泛研究,期望能从中找到诊断关节假体周围感染的特异性指标,提高临床诊断的准确性。     

Elevated chemerin levels in synovial fluid and synovial membrane from patients with knee osteoarthritis

To test the serum, synovial fluid and synovial membrane levels of the adipokine chemerin in patients with knee osteoarthritis (KOA) and investigate their relationships with the severity of articular cartilage damage and synovitis. According to the American College of Rheumatology criteria for diagnosis of osteoarthritis (OA), 30 cases with OA diagnoses (OA group) were selected from patients who underwent arthroscopic surgery in our hospital from June 2013 to February 2014. Another 30 cases with other knee joint diseases (non-OA group) were included as controls. The synovial fluid and serum levels of chemerin were assayed by ELISA, and the synovial membrane level of chemerin was assayed by the immunohistochemical method. The severity of the knee articular cartilage damage and synovitis-related pathological changes were evaluated by arthroscopy using the Outerbridge and Ayral scores, respectively. The synovial fluid and synovial membrane levels of chemerin in the OA group were higher than those in the non-OA group. Statistically significant differences were found between the two groups in the synovial fluid and synovial membrane levels of chemerin (P < 0.05). The synovial fluid and synovial membrane levels of chemerin were positively correlated with the serum level of high-sensitivity C-reactive protein (HS-CRP), Outerbridge score and Ayral score in the OA group. The synovial fluid and synovial membrane levels of chemerin are increased in KOA patients and are positively correlated with the severity of KOA.     

Mucin from rheumatoid arthritis synovial fluid enhances interleukin-6 production by human peripheral blood mononuclear cells

The carbohydrate chains represented by mucins (MUCs) are expressed by a variety of normal and malignant secretory epithelial cells and induce a variety of immunoreactions. To find new mucins related to the pathogenesis of rheumatoid arthritis (RA), we examined high-molecular-weight molecules inducing cytokines on human peripheral blood mononuclear cells (PBMCs) in synovial fluid from affected joints. We found a high-molecular-weight substance that induces interleukin 6 production on PBMCs in RA synovial fluid on gel filtration. MUC-1 was present in the resulting fractions, although they had been purified by CsCl density gradient centrifugation. We also found that MUC-1 was expressed on synovial cells and infiltrating inflammatory mononuclear cells on the sublining layer and lymphoid follicles in RA synovial tissues. CD68-positive superficial synovial cells colocalized with MUC-1 and CD68-positive macrophages were in contact with MUC-1-positive mononuclear cells. These findings imply that mucins, including MUC-1, may be related to immunoinflammatory reactions in the pathogenesis of RA.     

Interrelationships between interleukin (IL)-1, IL-6 and IL-8 in synovial fluid of various arthropathies

High levels of many cytokines, including interleukin (IL)-1, IL-6 and IL-8, were found in various arthropathies suggesting that they play a role in the pathogenesis of disease, although their relationship with the type and activity of disease is still not clear. The synovial fluid (SF) of 24 patients with rheumatoid arthritis (RA), 19 with psoriatic arthritis (PA) and 33 with osteoarthritis (OA) was analyzed for IL-1, IL-6 and IL-8. The highest concentration of the three cytokines was found in the SF of RA. IL- detectable levels (>-20 pg/ml) were observed in 8/24 (33.3%) patients with RA, in one patient with PA but in no patient with OA.IL-6 (mean±SD) (1610.37±1781.65 pg/ml) was higher in RA than in PA (672.47±867.40 pg/ml,p=0.043) and OA (89.45±120.52 pg/ml,p=0.0001). IL-8 (1042.72±698.64 pg/ml) was higher in RA than in PA (660.36±625.11 pg/ml,p=0.03) and OA (89.9±45.88 pg/ml,p=0.0001). A correlation between IL-1, IL-6 and IL-8 was found in RA. In all patients a correlation between IL-6 and IL-8 levels was found; moreover, these two cytokines were associated with SF indices of inflammation, such as white blood cells (WBC) count and total protein (TP) concentration.Out findings suggest that these interrelationships play a role in the evolution of more severe erosive arthropathy such as RA.     

急性颅脑损伤后脑脊液中IL-6和IL-8的变化及临床意义

目的研究急性颅脑损伤后脑脊液中白细胞介素-6（IL-6）和白细胞介素-8（IL-8）含量的变化规律,探讨其含量变化与损伤程度、预后的关系。方法对54例急性颅脑损伤患者以GCS评分分组,分别检测不同时段脑脊液中IL-6、IL-8的含量。结果急性颅脑损伤后第1天脑脊液中IL-6、IL-8含量显著升高,第3天降至最低（P〈0.01）。轻、中型颅脑损伤组脑脊液中IL-6含量与重型颅脑损伤组相比无显著差异（P〉0.05）;而脑脊液中IL-8含量在两组间有显著性差异（P〈0.01）。脑脊液中IL-6、IL-8含量在存活组和死亡组间有显著性差异（P〈0.01）。结论IL-6、IL-8参与了颅脑损伤的病理生理过程,对指导治疗、判断伤情和预后有重要意义。     

Regulatory effects of IL-13 on synovial fluid macrophages and blood monocytes from patients with inflammatory arthritis.

Activated macrophages are central to the destructive processes of chronic inflammatory arthritis. In this study, it was hypothesized that IL-13, a product predominantly of 'Th2-type' lymphocytes, may be used therapeutically to down-regulate monocyte/macrophage activities at sites of chronic inflammation. Synovial fluid mononuclear cells were isolated from 12 patients with chronic inflammatory arthritis. Peripheral blood mononuclear cells (PBMC) were isolated at the same time as synovial fluid cells from all 12 patients. IL-13 significantly inhibited lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production by mononuclear cells from peripheral blood, but not synovial fluid. In contrast, IL-13 inhibited LPS-induced IL-1 beta production by all cells, and as a positive response to IL-13, CD23 expression was increased on both cell populations. Blood monocytes cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF responded to IL-13 in a manner similar to that detected for synovial fluid-derived cells, with suppression of LPS-induced IL-1 beta, but not TNF-alpha, production. In all experiments, the responses to IL-13 were very similar to those detected to IL-4, but differed from those measured with IL-10. Thus, the responses to IL-13 by synovial fluid cells and cultured monocytes are not equal to those of blood monocytes. The similar responses to IL-4 and IL-13 support claims of a common element for signalling from the IL-4 and IL-13 receptors. Furthermore, the activity of a common receptor chain may be altered by monocyte activation and differentiation.     

IL-1ra ELISA: reduction and alkylation of synovial fluid eliminates interference by IgM rheumatoid factors

IL-1 and a specific receptor antagonist of IL-1, IL-1ra, may play important roles in the pathophysiology of rheumatoid arthritis and in other types of inflammatory synovitis. Measurement of IL-1ra in synovial fluids and in other body fluids may lead to a greater understanding of its possible activity as a modulator of the immune and inflammatory systems in vivo. Therefore, a modified sandwich ELISA was developed to measure IL-1ra protein concentration in synovial fluids. The antibodies used in this ELISA were polyclonal and derived from rabbits hyperimmunized with human recombinant IL-1ra. IgM rheumatoid factors within synovial fluid resulted in false elevation of determined IL-1ra by the sandwich ELISA through binding of the primary and secondary antibodies. Reduction and alkylation of synovial fluid samples before application to the ELISA plate eliminated the interference caused by > 2000 μg/ml IgM rheumatoid factor (latex agglutination titer of 1/5,120). This ELISA was specific for IL-1ra; there was no detection of IL-1, IL-1β, or lysozyme. The sensitivity of this ELISA was <200 pg/ml, making it a useful assay for the accurate measurement of synovial fluid IL-1ra protein concentration.     

Effects of detomidine HCl on the biochemical and cytological properties of synovial fluid after administration into the radiocarpal joint in horses

Detomidine may induce analgesia when administered at peripheral sites, e.g., at joint cavities or around nerves. The purpose of this study was to document the effects of detomidine HCl on biochemical and cytological properties of synovial fluid in horses. Five horses underwent clinical examination for lameness and flexion test to document limb health prior to acceptance to the study. Synovial fluids were taken from both radiocarpal joints, and 20 μg/kg detomidine HCl diluted in 3 ml saline and 3.2 ml saline was injected randomly to joints at day 0. Synovial fluid collection from the joints was performed at days 1, 2, and 7 after injection. Twenty-five-meter straight-line trot was used to assess lameness at days 1, 2, and 7 post detomidine injection. Total white blood cells (WBC), cytological examination, total protein, specific gravity, alkaline phosphates (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CK), glucose, and quality of mucin clot were measured. No detectable lameness was observed throughout the study. Mucin clot quality test revealed no significant differences between various sampling times (P>0.05). Cytological examination of synovial fluid of horses showed no significant differences between various sampling times for total WBC and also various types of leukocytes (P>0.05). The amount of total protein, specific gravity, ALP, CK, glutamic–pyruvic transaminase (GPT), and glucose revealed no significant differences between various sampling times between test and control groups (P>0.05). The only significant difference was seen at day 7 for AST between test and control groups (P<0.05).     

膝骨关节炎时关节滑液中炎症相关物质的表达

背景：膝骨关节炎的主要改变是关节软骨面的退行性病变和继发性的骨质增生,病变的机制还不明确,但现已有实验证实膝骨关节炎的发病与炎症相关物质有密切的关系。 目的：分析炎症相关物质在膝骨关节炎发病机制中的作用。 方法：按美国风湿病学分会制定的诊断标准选择膝骨关节炎患者60例,来自因外伤截肢或半月板损伤性手术治疗的患者(排除膝关节内损伤)60例作为对照组,抽取两组患者膝关节液,采用酶联免疫吸附试验方法检测白细胞介素1β、白细胞介素6、白细胞介素8、白细胞介素10、肿瘤坏死因子α、碱性成纤维细胞生长因子、骨桥蛋白水平,采用一氧化氮检测试剂盒检测一氧化氮水平,按TBA荧光法测定过氧化脂质浓度。 结果与结论：膝骨关节炎患者表达高水平白细胞介素1β、白细胞介素6、白细胞介素8、白细胞介素10、肿瘤坏死因子α、碱性成纤维细胞生长因子、骨桥蛋白、一氧化氮和过氧化脂质水平均明显高于对照组;这些细胞因子及一氧化氮、过氧化脂质与膝骨关节炎的病变呈正相关。结果提示这些炎症相关物质确实参与了人膝骨关节炎的发病进程。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Evaluation of soluble IL-6 receptor concentration in serum and epithelial lining fluid from patients with interstitial lung diseases.

We measured soluble IL-6 receptor (sIL-6R) levels in serum and bronchoalveolar lavage fluids (BALF) from patients with interstitial pneumonia of unknown etiology (IP) (n = 17), sarcoidosis (n = 8) and normal control subjects (n = 10), to investigate its role in pulmonary diseases. Soluble IL-6R was determined by an ELISA. The volume of epithelial lining fluid (ELF) in BALF was estimated using an urea method. We found that levels of sIL-6R in serum, BALF, and ELF from patients with IP or sarcoidosis were significantly higher than those from normal subjects. Furthermore, levels of sIL-6R in BALF or ELF were significantly correlated with those of albumin, indicating that sIL-6R, together with albumin, may enter ELF as a result of the increased permeability caused by pulmonary inflammation. Thus most of the sIL-6R in ELF would be from serum, and relatively small amounts of it might be produced locally. However, sIL-6R levels in ELF, but neither serum nor BALF, were significantly correlated with levels of C-reactive protein in patients with IP. These results suggest that both systemic and local production of sIL-6R are increased, and raised sIL-6R is involved in the modulation of systemic and local inflammatory responses in patients with IP and sarcoidosis.     

雌激素与IL-6、IL-8在卵巢癌细胞中的调节作用

目的 探讨雌激素与IL-6、IL-8在卵巢癌细胞中的交互调节作用及作用机制.方法 选择兼有雌激素受体(estrogen receptor,ER)及IL-6、IL-8受体表达的卵巢癌细胞系CAOV-3和OVCAR-3作为研究模型,分别探讨17B-雌二醇(estradiol,E2)对IL-6、IL-8及其受体表达的作用以及IL-6、IL-8对EB表达及ER转录活性的作用.结果 一方面E2不仅可经NF-κB途径促进卵巢癌细胞IL-6、IL-8分泌,而且还对二者受体的表达具有一定的调节作用.E2诱导的促IL-6、IL-8分泌作用可被其受体阻断剂他莫昔芬(tamoxifen,Txf)完全阻断.另一方面在无雌激素的条件下,IL-6、IL-8能上调卵巢癌细胞Erα表达及下调ERB表达,且还能分别通过丝裂原活化蛋白激酶(MAPK)信号通路和Src活化增强卵巢癌细胞ER的转录活性,该作用可被Txf完全封闭.结论 雌激素与IL-6、IL-8两种细胞因子在卵巢癌细胞中交互调节,由此通过产生的放大信号通路促进卵巢癌的生长和发展.     

Cartilage extracellular matrix metabolism differs in serum and synovial fluid

Most cartilage explant culture studies assume conventional serum-supplemented growth media are biologically equivalent to the natural synovial fluid which baths cartilage in vivo. Few studies have systematically compared the effects of serum versus synovial fluid in culture. To address this assumption we conducted a series of studies to determine if cartilage matrix synthesis is significantly different in serum-based versus synovial fluid-based media. Normal bovine cartilage explants were cultured in DMEM either alone or supplemented with bovine serum or bovine synovial fluid. Matrix synthesis was measured with radiolabeling techniques. We then compared responses to insulin-like growth factor I (IGF-I, a stimulator of matrix synthesis), and interleukin-1 (IL-1, an inhibitor of matrix synthesis). We observed significantly lower matrix synthesis activity in synovial fluid versus serum. Caution shoud be used in extrapolating studies of cartilage grown in media supplemented with serum rather than synovial fluid.