Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL)

Osteoclasts are bone-resorbing cells that are derived from haemopoietic precursors, including cells present in peripheral blood. The recent identification of RANKL [receptor activator of nuclear factor (NF)-kappaB ligand], a new member of the tumour necrosis factor ligand superfamily that has a key role in osteoclastogenesis, has allowed the in vitro generation of osteoclasts in the absence of cells of the stromal/osteoblast lineage. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL and human macrophage colony-stimulating factor form osteoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood, we have selected CD14(+) cells from PBMC to examine their osteoclastogenic potential and their expression of novel members of the tumour necrosis factor superfamily involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrated mRNA expression of receptor activator of NF-kappaB, but no expression of RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three factors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days with human macrophage colony-stimulating factor and soluble RANKL generated osteoclasts and showed extensive bone resorption. Similar numbers of osteoclasts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was significantly less intra-assay variability with CD14(+) cells, suggesting the absence of stimulatory/inhibitory factors from these cultures. The ability of highly purified CD14(+) cells to generate osteoclasts will facilitate further characterization of the phenotype of circulating osteoclast precursors and cell interactions in osteoclastogenesis.     

Circulating levels of osteoprotegerin and receptor activator of NF-kappaB ligand in patients with chronic renal failure.

BACKGROUND: Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of nuclear factor-kappaB ligand (RANKL). OPG and RANKL have been shown to be important regulators of osteoclastogenesis. The aim of this study was to investigate the relationship between the OPG-RANKL system and bone mineral metabolism in patients with chronic renal failure (CRF). METHODS: Serum OPG, RANKL, osteocalcin, cross-linked c-telopeptide of type I collagen (ICTP), intact parathyroid hormone (PTH), bone alkaline phosphatase and cystatin C levels were measured in 40 chronic hemodialysis male patients and 32 age- and sex-matched healthy controls. Their lumbar spine bone mineral density (LS-BMD) was measured by dual energy X-ray absorptiometry. RESULTS: Serum OPG, RANKL, PTH, bone alkaline phosphatase and cystatin C levels were significantly increased in patients with CRF. Serum OPG was positively correlated to serum RANKL and cystatin C. Positive correlations were found between serum RANKL and cystatin C and ICTP. LS-BMD was significantly lower in patients with CRF than in controls. In patients with CRF, LS-BMD was inversely correlated to serum RANKL and cystatin C, whereas it was positively correlated to serum OPG. CONCLUSIONS: The OPG-RANKL system is involved in the pathogenesis and regulation of bone turnover in CRF. Circulating levels of OPG and RANKL may be useful markers to assess turnover renal osteopathies.     

High levels of synovial fluid osteoprotegerin (OPG) and increased serum ratio of receptor activator of nuclear factor-kappa B ligand (RANKL) to OPG correlate with disease severity in patients with primary knee osteoarthritis

ObjectiveEvaluation of serum and synovial fluid OPG and sRANKL in 37 patients with primary knee osteoarthritis.Design and methodOPG and sRANKL were measured using ELISA.ResultsOPG, sRANKL and sRANKL/OPG were increased in osteoarthritis patients' serum. Synovial OPG was higher than serum OPG, while sRANKL/OPG was higher in the serum; both correlated with disease severity.DiscussionRANKL/OPG pathway is implicated in the pathogenesis of knee osteoarthritis being a suitable target for therapeutic intervention.     

Fish oil supplementation decreases serum soluble receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio in female patients with rheumatoid arthritis

ObjectiveSoluble receptor activator of nuclear factor-kappa B ligand (sRANKL) to osteoprotegerin ratio is designated as a bone metabolism equation in many rheumatologic disorders and would be modified with fish oil (FO) supplementation.Design and methodsEighty-three females with rheumatoid arthritis were divided randomly to 40 and 43 patients treated with (1 g/day) or without FO for 3 months accompanied with conventional drugs, respectively. Osteoprotegerin, sRANKL, tumor necrosis factor alpha (TNFα) serum levels were measured before and after treatment.ResultsSerum levels of osteoprotegerin increased, although sRANKL, TNFα and sRANKL/osteoprotegerin ratio decreased with FO therapy.A significant positive correlation was observed between sRANKL/osteoprotegerin ratio and TNFα levels (r = 0.327, p = 0.040) in the FO-treated group.ConclusionsFO could decrease the inflammatory response by lowering of serum TNFα levels and sRANKL/osteoprotegerin ratio.     

RNA干扰技术抑制成骨细胞核激活因子受体配体基因对破骨细胞生成的影响

目的:利用RNA干扰技术抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator of NF-κB,RANKL)的表达,观察成骨细胞RANKL/骨保护素(osteoprotegerin,OPG)比值变化,探讨成骨细胞与破骨细胞生成的相关性。方法:实验于2006-05/10在四川大学华西医院移植免疫实验室(国家重点实验室)完成。①实验材料:体外化学合成4条RANKL序列特异性小干扰RNA(small interfering RNA,siRNA),用Lipofectin2000TM转染成骨细胞。②实验方法:采用荧光实时定量聚合酶链反应检测RANKL mRNA的表达,筛选出最有效的干扰序列。③实验分组:分为最有效的siRNA转染组、阴性对照组和空白对照组,每组设5个平行样本。采用免疫组织化学染色检测成骨细胞中RANKL、OPG蛋白的表达,观察RANKL/OPG比值变化。④实验评估:采用成骨细胞、破骨细胞共培养技术观察以上3组转染后的成骨细胞对破骨细胞生成的影响。结果:①4种siRNA敲除RANKL基因后mRNA的表达:4种siRNA转染成骨细胞后,相对空白对照组,siRNA4分子对RANKLmRNA的抑制作用最为明显,达到89%。siRNA1,siRNA3对RANKLmRNA的抑制作用大于50%,而siRNA2对RANKLmRNA的也有相当的抑制作用。②各组成骨细胞中RANKL和OPG蛋白水平:RANKL和OPG的阳性表达均显示为棕黄色颗粒,RANKL主要分布于成骨细胞的胞浆和胞膜,OPG主要分布于成骨细胞的胞浆。siRNA4转染组RANKL表达(平均积分光密度值)低于空白对照组(分别为3.05±2.17,11.31±1.26),差异有显著性意义(P<0.01);阴性对照组与空白对照相比,差异无显著性意义(P>0.05)。siRNA4转染组、阴性对照组OPG表达与空白对照组比较,差异均无显著性意义(P>0.05)siRNA4转染组RANKL/OPG比值低于空白对照组(分别为0.12±0.03,0.45±0.04),差异有显著性意义(P<0.01)。③转染的成骨细胞对破骨细胞生成的影响:各组共培养形成的破骨细胞形态与分离的成熟破骨细胞相似,为不规则形,胞浆红染,并有伪足形成。siRNA4转染组破骨细胞形成低于空白对照组、阴性对照组[分别为(12±2),(31±7),(28±5)个/孔],差异有显著性意义(P<0.01);阴性对照组与空白对照相比,差异无显著性意义(P>0.05)。结论:RNA干扰沉默RANKL表达可下调成骨细胞RANKL/OPG比值,抑制破骨细胞的生成。     

Analytical,biochemical and clearance considerations of soluble urokinase plasminogen activator receptor (suPAR) in healthy individuals

BackgroundSoluble urokinase plasminogen activator receptor (suPAR) is an emerging marker of cardiovascular disease burden. Appropriate assessment of assay performance and reference interval are required to enable interpretation of results to facilitate its clinical application.MethodssuPAR was measured using the suPARnostic® ELISA in 155 healthy volunteers. Assay performance was assessed for anticoagulant effect, recovery, interference, linearity and cross-reactivity. The identity of immunoreactive suPAR was confirmed by size-exclusion HPLC. To establish anatomical sites of release and uptake, we measured suPAR in regional samples from subjects undergoing cardiac catheterization.ResultsThe median concentration of suPAR was 2.1 ng/mL (IQR:1.7–2.3) in health. In comparison with EDTA, suPAR measurements were affected by lithium heparin (>10% change) and increased with serum usage. suPAR reactivity also increased in the presence of haemolysis (10 g/L), but was suppressed with urokinase and lipids (4 g/L). In multiple regression analyses, suPAR associated independently with body weight, NT-proBNP and MR-proADM (P = .03) for healthy individuals. Regional plasma sampling showed lower suPAR concentrations in the coronary sinus and renal vein compared with concentrations in femoral arterial samples. Immunoreactive circulating suPAR species had M_r of 10–39 kDa.ConclusionThe suPARnostic® assay performs acceptably for a clinical assay but is limited in the presence of high levels of hemolysis, lipids and urokinase. We provide the first evidence for the heart and kidneys as organs of suPAR clearance in humans. Additional investigations are warranted to determine whether there is a need to compare the marker performance of differing circulating forms of suPAR.     

可溶性尿激酶型纤溶酶原激活物受体(suPAR)在脓毒症中的研究进展

可溶性尿激酶型纤溶酶原激活物受体(soluble urokinase plasminogen activator receptor,suPAR)是表达于多种免疫活化细胞表面的尿激酶型纤溶酶原激活物受体(urokinase plasminogen activator receptor,uPAR)的可溶形式.在炎症或感染的过程中,血清中suPAR的水平可反映机体免疫系统的激活程度.目前,已有多项研究揭爪,suPAR作为一种新型的炎症标志物,在脓毒症的早期诊断、预后评估、严重程度分级以及指导治疗等方面具有一定的价值.鉴于该标志物预测脓毒症患者预后情况的准确性,临床医生也许可以利用suPAR对脓毒症患者进行早期的危险分级,从而为今后开展个体化的治疗提供依据.     

Identification of a soluble form of a ligand for the lymphocyte homing receptor

Lymphocytes are engaged in constant trafficking from the blood into secondary lymphoid tissues, such as peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP). The initial step in this process is the binding of lymphocytes to high endothelial venules (HEV), and in the case of trafficking of cells to the PLN, it is required that they bear the L-selectin surface receptor. Using a chimeric protein, combining the extracellular domains of L-selectin with a human immunoglobulin (Ig) G1 Fc region (L-selectin-IgG), we have probed the expression of ligands for this receptor on HEV and in cell lysates. Two sulfated glycoproteins of 50 and 90 kD have been identified in lysates from PLN and MLN, but not PP. Here we show that the 50-kD molecule is secreted in organ cultures in vitro and is present in the blood of normal animals. Indeed, normal serum inhibits lymphocyte binding to HEV by approximately 50% in an in vitro assay. This inhibitory activity can be removed by passage of the serum over an L-selectin-IgG column and has a molecular mass of approximately 50 kD. We speculate on the possible reasons for secretion of a homing receptor ligand.     

同种异体肾移植排斥患者血清sFas和sFasL水平的临床观察

目的观察肾同种移植急性排斥患者血清可溶性Fas(sFas)和sFas配体(sFasL)的水平及临床意义.方法采用酶联免疫吸附试验(ELISA)分别对健康对照组及实验组透析前后sFas、sFasL进行检测.结果对照组sFas为(256.8±72.0)ng/L,sFasL为(227.9±65.9)ng/L;实验组透析前后sFas分别为(1 225.7±467.6)ng/L、(1 225.8±464.0)ng/L,sFasL分别为(227.9±147.2)ng/L、(226.9±109.6)ng/L.实验组与对照组的sFas比较差异有显著性(P＜0.01),而sFasL比较差异无显著性(P＞0.05).结论 sFas在排异的病理反应过程中参与了细胞凋亡的抑制,透析并不能改善Fas-FasL介导的细胞凋亡.     

Novel enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay) for anti-thyroglobulin IgG in human serum

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in human serum is described. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. After eliminating nonspecific IgG in test serum by washing, the complex was eluted from the polystyrene ball with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG 7-chain) Fab'-horse-radish peroxidase conjugate. The present enzyme immunoassay was 1,000–3,000-fold more sensitive than the conventional enzyme immunoassay, in which a thyroglobulin-coated polystyrene ball was incubated with serum containing anti-thyroglobulin IgG and subsequently with rabbit (anti-human IgG 7–chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in all patients with Graves' disease and chronic thyroiditis.     

Risk assessment in sepsis: a new prognostication rule by APACHE II score and serum soluble urokinase plasminogen activator receptor

ABSTRACT: INTRODUCTION: Early risk assessment is the mainstay of management of patients with sepsis. APACHE II is the golden standard prognostic stratification system. A prediction rule is developed aimed to improve prognostication by APACHE II with the application of serum suPAR (soluble urokinase plasminogen activator receptor). METHODS: A prospective study cohort enrolled 1914 patients with sepsis including 62.2% with sepsis and 37.8% with severe sepsis/septic shock. Serum suPAR was measured in samples drawn after diagnosis by an enzyme-immunoabsorbent assay; in 367 patients sequential measurements were performed. After ROC analysis and multivariate logistic regression analysis a prediction rule for risk was developed. The rule was validated in a double-blind fashion by an independent confirmation cohort of 196 sepsis patients, predominantly severe sepsis/septic shock patients, from Sweden. RESULTS: Serum suPAR remained stable within survivors and non-survivors for 10 days. Regression analysis showed that APACHE II [greater than or equal to] 17 and suPAR [greater than or equal to] 12 ng/ml were independently associated with unfavorable outcome. Four strata of risk were identified: i) APACHE II <17 and suPAR <12 ng/ml with mortality 5.5%; ii) APACHE II < 17 and suPAR [greater than or equal to] 12 ng/ml with mortality 17.4%; iii) APACHE II [greater than or equal to] 17 and suPAR <12 ng/ml with mortality 37.4%; and iv) APACHE II [greater than or equal to] 17 and suPAR [greater than or equal to] 12 ng/ml with mortality 51.7%. This prediction rule was confirmed by the Swedish cohort. CONCLUSIONS: A novel prediction rule with four levels of risk in sepsis based on APACHE II score and serum suPAR is proposed. Prognostication by this rule is confirmed by an independent cohort.     

同种异体肾移植排斥患者血清sFas和sFasL水平的临床观察

目的　观察肾同种移植急性排斥患者血清可溶性Fas(sFas)和sFas配体(sFasL)的水平及临床意义。 方法　采用酶联免疫吸附试验(ELISA)分别对健康对照组及实验组透析前后sFas、sFasL进行检测。结果　对 照组sFas为(256.8±72.0)ng/L,sFasL为(227.9±65.9)ng/L;实验组透析前后sFas分别为(1225.7±467.6) ng/L、(1225.8±464.0)ng/L,sFasL分别为(227.9±147.2)ng/L、(226.9±109.6)ng/L。实验组与对照组的 sFas比较差异有显著性(P<0.01),而sFasL比较差异无显著性(P>0.05)。结论　sFas在排异的病理反应过 程中参与了细胞凋亡的抑制,透析并不能改善Fas FasL介导的细胞凋亡。     

Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein

OBJECTIVE: Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high-sensitivity C-reactive protein (hsCRP) has recently attracted increased interest. The purpose of our study was to determine reference intervals, variability caused by sampling time, biological variation and stability after repeated freeze-thaw cycles of circulating levels of OPN, OPG, total sRANKL and hsCRP. MATERIAL AND METHODS: Plasma OPN and plasma OPG concentrations were determined with sandwich ELISA; serum total sRANKL concentration was determined using a two-site sandwich ELISA; and hsCRP was analysed by turbidimetry in 300 Danish blood donors (183 M and 117 F) with a median age of 43 years (range 18-64 years). Variability due to biological variation and sampling time was studied in serial samples from 38 healthy subjects. RESULTS: The 95th percentiles in the donors were 76 microg/L for OPN, 4.2 pmol/L for OPG, 40.2 nmol/L for total sRANKL and 12 mg/L for hsCRP. The overall medians for both genders were 51 microg/L, 2.2 pmol/L, 0.66 nmol/L and 1.0 mg/L, respectively. We found a significant correlation between hsCRP and OPN (rho = 0.173; p<0.003). The biological within-subject variations were calculated to be 8.2 % for OPN, 8.8% for total sRANKL and 50% for hsCRP. CONCLUSIONS: Reference intervals have been established with a high analytic performance for OPN and an acceptable analytic performance for OPG and total sRANKL. The study revealed low biological variation for OPN and total sRANKL and high biological variation for hsCRP.     

不同程度阿尔茨海默病患者血清可溶性Fas及其配体水平的变化

目的探讨阿尔茨海默病(AD)患者血清可溶性Fas(s Fas)及其配体(s Fas L)水平的变化。方法选取AD患者100例,以54例老年疾病(包括糖尿病17例、高血压18例、高血脂15例、其它内分泌疾病4例)患者和46名健康体检者分别作为老年疾病对照组和正常对照组。采用酶联免疫吸附试验(ELISA)检测所有受检者血清s Fas、s Fas L水平,使用临床痴呆评定量表(CDR)、简易智能精神状态量表(MMSE)、Hachinski缺血指数量表(HIS)评定AD患者认知功能。结果 AD患者血清s Fas、s Fas L水平明显高于老年疾病对照组和正常对照组(P0.01);随着病程加重,其血清s Fas、s Fas L水平逐渐升高。AD患者血清s Fas与年龄、病程和MMSE评分呈正相关(r=0.856、0.834、0.799,P0.01),与Hachinski评分呈负相关(r=-0.714,P0.01);血清s Fas L与年龄、病程呈正相关(r=0.863、0.857,P0.01),与MMSE评分、HIS评分呈负相关(r=-0.801、-0.745,P0.01)。结论 AD患者存在血清s Fas、s Fas L水平异常,提示AD患者在发病过程中可能存在神经细胞凋亡,且血清s Fas、s Fas L水平可能与患者的智能水平、脑缺血程度有关。     

Immunoassay for estrogen receptor does not detect inactivated receptor

Accurate quantification of estrogen receptor (ER) is essential for optimal clinical characterization of individual cases of breast cancer. If breast tumors are mishandled, the relatively labile ER protein may lose its steroid-binding capacity (become inactivated) and not be measurable by the routine steroid-binding assay. We tested whether the commercial enzyme immunoassay of Abbott Laboratories could quantify inactivated ER. Samples of powdered breast tumors from humans were exposed to various temperature and homogenization conditions known to inactivate ER, and any remaining ER was quantified by both the immunoassay and the steroid-binding assay. For all inactivation conditions tested, the two assay methods detected the same proportions of remaining ER. We conclude that the inactivation reaction for ER also alters one or both of the antigenic site(s) necessary for the immunoassay. Hence, for breast tumors mishandled to the extent of inactivating ER, the immunoassay offers no advantage over the more conventional steroid-binding assay for quantifying any remaining ER.