Proteins retained with hyaluronic acid during ultrafiltration of synovial fluid.

The identity of the proteins associated with hyaluronic acid after ultrafiltration of bovine synovial fluid was examined. With discontinuous filtration, alpha2 macro- and IgM globulins were retained in hyaluronic complex both from synovial fluid and from a similar mixture of hyaluronic acid and serum. With continuous filtration and stirring to prevent formation of gels, protein was retained with hyaluronic acid in similar proportions. This protein showed immunologic identity with serum albumin, but differed in amino acid composition. After comparison with results of gel filtration, it was concluded that the identity of the retained proteins can be determined by the concentration of hyaluronic acid without variation in other ionic conditions.     

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.     

Characterization of a bovine synovial fluid lubricating factor. III. The interaction with hyaluronic acid.

Although hyaluronate is not a boundary lubricant in cartilaginous and latex:glass bearings, a distinct interaction with purified synovial lubricating factor (PSLF) was demonstrated by three means: 1) enhancement of lubricating ability in an artificial test system; 2) viscometry; 3) electron microscopy. The interaction was of a physical rather than a specific chemical type; it varied with the degree of purification of PSLF and of hyaluronate. The interaction accounts for retention of the relatively small PSLF molecule (approximately 280 kDa) with the synovial mucin on a 0.22 microns filter. The data provide evidence that hyaluronate and PSLF act synergistically in the boundary lubricating activity of animal joints.     

Serum hyaluronic acid in patients with disseminated neoplasm.

Hyaluronic acid concentrations were measured by a laser nephelometric assay in serum samples from 50 patients with advanced disseminated neoplasm and 50 healthy controls matched for age and sex. The identity of hyaluronic acid was confirmed by a combination of electrophoretic and enzymatic techniques. The mean serum hyaluronic acid concentration for the control group was 1.09 mg/l, with a range of 0-4 mg/l. The mean concentration for patients with neoplastic disease was 10.38 mg/l, with a range of 0-100 mg/l. Sixty two per cent of the patients with disseminated neoplasm had serum hyaluronic acid concentrations above the control range. There was no correlation between the increased concentration of hyaluronic acid and tumour type, serum bilirubin, serum alkaline phosphatase, or serum urea concentrations. There was a higher incidence of hypercalcaemia in patients with increased hyaluronic acid concentrations, but the correlation between hyaluronic acid and calcium concentrations was not significant. In view of the possible role of hyaluronic acid in cellular differentiation and morphogenesis the finding of increased hyaluronic acid concentrations in patients with advanced neoplastic disease may be of fundamental importance in cancer biology.     

Disrupted homeostasis of synovial hyaluronic acid and its associations with synovial mast cell proteases of rheumatoid arthritis patients and collagen-induced arthritis rats

Hyaluronic acid (HA) is the main component of the extracellular matrix (ECM) of joints, and it is important for a lubricating joint during body movement. Degradation is the main metabolic process of HA in vivo. Hyaluronidases (HAase) were known for HA degradation. The inflammation-induced HA rapid-metabolism can reduce HA viscosity and concentration in joints. Mast cells (MC) containing their specific proteases were found in synovium tissue. It is unclear if MC-proteases could be involved in HA degradation pathways. This study aims to explore the correlations between HA concentration vs mast cell proteases, or matrix metalloproteinase-2/9 (MMP-2/9) and to investigate the association of MC-specific proteases with disrupted synovial HA homeostasis in rheumatoid arthritis (RA) or collagen-induced arthritis rats. The synovial fluid samples from no-RA and RA patients were collected; the collagen-induced arthritis (CIA) rat model was established; HA concentration and the activities of MC-protease and MMP-2/9 in the samples were detected, and the correlations were analyzed. In vitro interaction experiment was carried out by mixing MC-proteases with HA to observe the degradation speed. The HA concentrations in synovial fluids were decreased in RA patients and CIA rats compared with those in no-RA subjects or normal rats respectively. The activities of mast cell proteases in synovial fluids were increased and positively correlated with MMP-9, but negatively correlated with HA concentrations. In vitro study, the addition of MC-chymase and tryptase promoted the speed in HA degradation. MC-proteases may influence HA degradation pathway.

     

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.     

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.     

Elevated angiogenin levels in synovial fluid from patients with inflammatory arthritis and secretion of angiogenin by cultured synovial fibroblasts

Angiogenesis is a key process in the pathogenesis of inflammatory arthritis. Angiogenin is one of the most potent inducers of neovascularization in experimental models in vivo. To look for evidence that angiogenin is involved in inflammatory joint disease, we examined plasma and synovial fluid (SF) samples from rheumatology patients and synovial fibroblast cell culture supernatants. Angiogenin levels were determined by radioimmunoassay and ELISA. Plasma angiogenin concentrations ranged from 96 to 478 ng/ml, with no significant difference between patients and normal controls. In SF, angiogenin concentrations were significantly higher in patients with acute or chronic synovitis (rheumatoid arthritis (RA): median, 104 ng/ml; range 13-748, n = 14; crystal-induced arthritis (CIA): median, 149 ng/ml; range, 37-616, n = 14, and other chronic inflammatory arthritis: median, 42 ng/ml; range, 15-205; n = 9) than in the 18 patients with osteoarthritis (OA) (median, 20 ng/ml; range 8-116) (P < 0.0001, anova). Angiogenin levels in SF from RA patients in remission with secondary OA were similar to those achieved in primary OA, and decreased in parallel with the resolution of acute gout. Angiogenin protein was released by cultured synovial fibroblasts from OA and RA patients, and reached 1.18 ng/106 cells/day. These data suggest that angiogenin may mediate local inflammation in arthritis via effects on angiogenesis and leucocyte regulation.     

Lack of T cell oligoclonality in enzyme-digested synovial tissue and in synovial fluid in most patients with rheumatoid arthritis.

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.     

Ultrastructural changes in articular cartilage affected by a non-physiological pH value of the synovial fluid

The physiological pH-value of the synovial fluid (pH 7.2-7.4) is important for the integrity of normal articular cartilage. After the intraarticular injection of acid (pH 4.0) or alkaline (pH 10.0) isoosmolale buffer solutions into the knee joints of young rabbits ultrastructural changes are seen by the aid of transmission electron microscopy. The number of cell organelles in the chondrocytes (rough endoplasmic reticulum, Golgi-complex, mitochondria) is diminished and the number of intracytoplasmic filaments is increased. The intercellular matrix shows a looseness of the collagen fibre mesh work, swollen and split collagen fibres, the appearance of numerous coarse rounded structures with an electron dense envelop, and a loss of matrix granules. These results are interpreted as regressive changes, and they are discussed in the context of an activation of proteinases.     

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.     

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：     

Morphological changes induced by picolinic acid in cultured mammalian cells.

The morphological changes induced by picolinic acid treatment in cultured normal rat kidney (NRK) and BALB/3T3 cells, and SV40-transformed NRK and BALB/3T3 cells were accompanied by altered distribution of cytoskeletal elements. These changes were prevented by the divalent cation ionophore A-23187. Untreated transformed cells were predominately round or fusiform in shape while untreated normal cells were predominately flat; both normal and transformed cells contained relatively few microfilaments and microtubules. Culturing these cells in medium containing 3 mM picolinic acid induced microfilaments and microtubules to accumulate in bundles at the cell periphery. In transformed cells the agent also induced the formation of long narrow cytoplasmic processes which showed parallel arrays of microfilaments near the cortex and microtubules near the center. These changes were apparent within 8 to 12 hr and acquired maximal expression by 24 to 36 hr. Picolinic acid also enhanced the formation of substrate adhesion plaques and intercellular adherens junctions in transformed cells. In SV40-transformed cells but not in normal cells mitochondria showed characteristic toxic alterations. Most of the mitochondria were electron dense and elongated or showed enlargement, swelling or vacuolated areas after 48 hr of treatment. The mitochondrial changes clearly reflected a selective toxicity induced by picolinic acid in SV40-transformed cells. These results suggest that picolinic acid specifically interferes with systems that are important in the determination of cell shape and neutralizes some of the effects of transformation.     

Anorexia induced by toxohormone-L isolated from ascites fluid of patients with hepatoma.

To ascertain anorexigenic effect of toxohormone-L, a polypeptide extracted and purified from ascites of patients with hepatoma were infused into the rat third cerebroventricle. Food intake decreased on the first day after infusion of an optimum dose of 10.0 micrograms (p less than 0.05). The suppressive effect on feeding was linearly dose dependent (p less than 0.05). Meal size and latency to the first meal decreased in the 12-h dark period, and the first and the second 4-h cumulative blocks after infusion of a 10.0 micrograms dose (p less than 0.01 for each). The suppressive effects on total food intake and meal size were completely recovered within 24 h after infusion. Neither postprandial intermeal interval nor eating speed was affected. Periprandial drinking, a ratio of water intake to food intake, was not affected after infusion of 5.0 and 10.0 micrograms toxohormone-L. Infusion of a 10.0 micrograms dose showed no effect on ambulation. These findings suggest that anorexia and cachexia produced in cancer patients may essentially be due to the suppressive effect of toxohormone-L on food intake.     

阻塞性睡眠呼吸暂停患者体位改变致流体向头颈部迁移的研究

目的当人体由立姿或坐姿改变为睡眠时的卧姿时,下肢流体会向头颈部迁移,使得上气道变窄。研究体位改变时阻塞性睡眠呼吸暂停(obstructive sleep apnea,OSA)患者头颈部组织中流体重新分布的过程,对于理解流体迁移在OSA患者气道阻塞上扮演的角色具有重要的意义。本研究通过测量OSA重度患者随着体位改变,在不同时刻腿部流体量,以及颈部和腿部几何参量,验证迁移流体量随时间非线性变化的假设。方法对于9名睡眠呼吸暂停低通气指数AHI30的重度患者,进行整夜多导睡眠监测,记录睡眠状态及AHI的变化,并测量站立、刚刚仰卧、仰卧15 min,及早上起床前仰卧诸时刻的腿部流体量以及颈围、腿围、踝围等几何参量,分析迁移流体量随时间的变化规律。结果由立姿改为仰卧后,几乎有总迁移量1/5的流体立刻发生迁移;仰卧15 min后迁移量升至1/3。睡眠监测显示受试者进入睡眠时间为(301±183)min。通过数据曲线拟合可推算出,仰卧30 min后流体迁移量将升至总迁移量的1/2。结论在OSA重度患者中,体位改变后,向头颈部迁移的流体量随时间以强非线性规律变化。相比于整夜的流体迁移量,在患者进入睡眠前,主要的流体迁移过程已基本完成。     

A study of opioid peptides in synovial fluid and synovial tissue in patients with rheumatoid arthritis]

Authors have often experienced that psychological stress influences rheumatoid arthritis (RA). In addition, recent reports show a modulatory role for neuropeptide such as substance P in arthritis. These findings prompted us to study endogenous opioid peptides in RA, which are found mainly in the brain and have an effect on the central nervous system. We examined methionine-enkephalin (Met-enk), leucine-enkephalin (Leu-enk) and beta-endorphin (beta-end) in opioid peptides. We measured these peptides in plasma and synovial fluid samples obtained from 28 knees of 24 RA patients and the quantity in the synovial tissue of 13 knees. We also measured plasma and synovial fluid samples from patients with osteoarthritis of the knee and plasma samples from healthy candidates. Leu-enk and beta-end levels in synovial fluid were significantly higher than plasma levels only in RA. Larger quantity of Leu-end and beta-end were contained apparently in the synovial tissue than Met-enk. The synovial tissue with proliferative change tends to contain larger quantity of opioid peptides. These results indicate that the synovial tissue produces or secretes Leu-enk and beta-end and that opioid peptides are related to the degree of inflammation in RA.     

膝骨关节炎患者关节腔注射玻璃酸钠后血清及关节液中可溶性Fas蛋白的变化

背景：关节软骨细胞凋亡在骨关节炎的发病中起重要作用,Fas是肿瘤坏死因子受体家族成员,是细胞凋亡通路中的重要蛋白。目的：观察膝骨关节炎患者血清可溶性Fas蛋白水平的变化以及玻璃酸钠治疗对血清及关节液中可溶性Fas蛋白的影响。方法：选取64例膝骨关节炎患者和48名正常对照者,膝骨关节炎患者再随机分为常规治疗组和玻璃酸钠组。常规治疗组给予常规治疗,玻璃酸钠组在常规治疗的基础上,给予关节腔内注射玻璃酸钠2 mL,1次/周,连续5周。检测正常组及膝骨关节炎患者治疗前后血清及关节液中可溶性Fas蛋白水平的变化。结果与结论：与对照组相比,膝骨关节炎患者血清可溶性Fas蛋白水平明显增高,差异有显著性意义。非条件logistic回归分析表明,升高的血清可溶性Fas蛋白水平是膝骨关节炎发病的独立危险因子。治疗5周后,玻璃酸钠组的血清及关节液中的可溶性Fas蛋白水平较治疗前明显下降,差异有显著性意义;而常规治疗组的血清及关节液中的可溶性Fas蛋白水平与治疗前相比无明显变化。提示玻璃酸钠具有降低膝骨关节炎患者血清及关节液中可溶性Fas蛋白水平的作用。关键词：玻璃酸钠;膝骨关节炎;可溶性Fas蛋白;肿瘤坏死因子受体;回归分析doi:10.3969/j.issn.1673-8225.2012.03.041