Reproducibility of targeted lipidome analyses (Lipidyzer) in plasma and erythrocytes over a 6-week period

It is essential to measure lipid biomarkers with a high reproducibility to prevent biased results. We compared the lipid composition and inter-day reproducibility of lipid measurements in plasma and erythrocytes. Samples from 42 individuals (77% women, mean age 65 years, mean body mass index (BMI) 27 kg/m(2)), obtained non-fasted at baseline and after 6 weeks, were used for quantification of up to 1000 lipid species across 13 lipid classes with the Lipidyzer platform. Intraclass correlation coefficients (ICCs) were calculated to investigate the variability of lipid concentrations between timepoints. The ICC distribution of lipids in plasma and erythrocytes were compared using Wilcoxon tests. After data processing, the analyses included 630 lipids in plasma and 286 in erythrocytes. From these, 230 lipids overlapped between sample types. In plasma, 78% of lipid measurements were reproduced well to excellently, compared to 37% in erythrocytes. The ICC score distribution in plasma (median ICC 0.69) was significantly better than in erythrocytes (median ICC 0.51) (p-value < 0.001). At the class level, reproducibility in plasma was superior for triacylglycerols and cholesteryl esters while ceramides, diacylglycerols, (lyso)phosphatidylethanolamines, and sphingomyelins showed better reproducibility in erythrocytes. Although in plasma overall reproducibility was superior, differences at individual and class levels may favor the use of erythrocytes.Clinical epidemiolog

Do high molecular weight adiponectin levels associate with radiographic progression in early rheumatiod arthritis and hand osteoarthritis?

Pathophysiology and treatment of rheumatic disease

Lipid metabolism of leukocytes in the unstimulated and activated states

Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting-edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently, differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the Lipidyzer (TM) platform to obtain a comprehensive overview of leukocyte-related lipid metabolism in the resting and activated states. First, we tested the linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+), we shed light on leukocyte-specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at the resting state, PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ cells and PMNs, whereas CD14+ cells did not show significant changes. By exploring the fatty acid content of the significantly upregulated lipid classes, we mainly found increased concentrations of very long and polyunsaturated fatty acids. Our results indicate the usefulness of the Lipidyzer (TM) platform for studying cellular lipid metabolism. Its application allowed us to explore the lipidome of leukocytes.Proteomic

Fc gamma receptor binding profile of anti-citrullinated protein antibodies in immune complexes suggests a role for FcγRI in the pathogenesis of synovial inflammation

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). Here, we studied binding of ACPA-IgG immune complexes (IC) to individual Fc gamma receptors (FcγR) to identify potential effector mechanisms by which ACPA could contribute to RA pathogenesis. METHODS: ACPA-IgG1 and control IgG1(IgG1 depleted of ACPA-IgG1) were isolated from plasma and synovial fluid (SF) of RA patients by affinity chromatography using CCP2 peptides. Subsequently, IC were generated using fluorescently labelled F(ab’)2 fragments against the F(ab’)2 region of IgG, or by using citrullinated fibrinogen. IC were incubated with FcγR-transfected CHO cell lines or neutrophils from healthy donors. FcγR binding of IC was analysed by flow cytometry in the presence or absence of specific blocking antibodies. RESULTS: ACPA-IgG1 IC predominantly bound to FcγRI and FcγRIIIA on FcγR-transfected CHO cell lines, while much lower binding was observed to FcγRIIA and FcγRIIB. ACPA-IgG1 IC showed reduced binding to FcγRIIIA compared to control IgG1 IC, in line with enhanced ACPA-IgG1 Fc core-fucosylation. Neutrophils activated in vitro to induce de novo expression of FcγRI showed binding of ACPA-IgG IC, and blocking studies revealed that almost 30% of ACPA-IgG IC binding to activated neutrophils was mediated by FcγRI. CONCLUSIONS: Our studies show that ACPA-IgG1 IC bind predominately to activating FcγRI and FcγRIIIA, and highlight FcγRI expressed by activated neutrophils as relevant receptor for these IC. As neutrophils isolated from SF exhibit an activated state and express FcγRI in the synovial compartment, this IC-binding could contribute to driving disease pathogenesis in RA.Drug Delivery Technolog

Hyaluronidase treatment of synovial fluid is required for accurate detection of inflammatory cells and soluble mediators

Background Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis. Methods SF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry. Results In all patients, there was less variation in IgG, 17-HDHA, leukotriene B-4 (LTB4), and prostaglandin E-2 (PGE(2)) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7-85% (median 22%) and 7-88% (median 23 %), respectively. Conclusions Homogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.Orthopaedics, Trauma Surgery and Rehabilitatio

Association between weight or Body Mass Index and hand osteoarthritis: a systematic review

Objective: To investigate the association between weight or Body Mass Index (BMI) and the development of hand osteoarthritis (OA). Methods: Systematic review of observational studies. Medical databases were searched up to April 2008. Articles which presented data on the association between weight and hand OA were selected. The qualities of these studies were then assessed by two independent reviewers using a 19 criteria scoring syst

Targeted lipidomics reveals activation of resolution pathways in knee osteoarthritis in humans

Proteomic

Translation of clinical problems in osteoarthritis into pathophysiological research goals

Osteoarthritis (OA) accounts for more disability among the elderly than any other disease and is associated with an increased mortality rate. The prevalence in Europe will rise in the future since this continent has a strongly ageing population and an obesity epidemic; obesity and age both being major risk factors for OA. No adequate therapeutic options, besides joint replacement, are available, although they are greatly needed and should be acquired by adequate research investments. However, the perspective on OA from a researcher's point of view is not always aligned with the perspective of a patient with OA. Researchers base their views on OA mainly on abnormalities in structure and function while patients consider OA as a collection of symptoms. In this viewpoint paper, we discuss the possibility of translating the most important clinical problems into pathophysiological research goals to facilitate the translation from bench to bedside and vice versa. This viewpoint is the outcome of a dialogue within the 'European League Against Rheumatism study group on OA' and People with Arthritis/Rheumatism across Europe (PARE) representatives