High-mannose glycans from Schistosoma mansoni eggs are important for priming of Th2 responses via Dectin-2 and prostaglandin E2.

The parasitic helminth Schistosoma mansoni is a potent inducer of type 2 immune responses by stimulating dendritic cells (DCs) to prime T helper 2 (Th2) responses. We previously found that S. mansoni soluble egg antigens (SEA) promote the synthesis of Prostaglandin E2 (PGE2) by DCs through ERK-dependent signaling via Dectin-1 and Dectin-2 that subsequently induces OX40L expression, licensing them for Th2 priming, yet the ligands present in SEA involved in driving this response and whether specific targeting of PGE2 synthesis by DCs could affect Th2 polarization are unknown. We here show that the ability of SEA to bind Dectin-2 and drive ERK phosphorylation, PGE2 synthesis, OX40L expression, and Th2 polarization is impaired upon cleavage of high-mannose glycans by Endoglycosidase H treatment. This identifies high-mannose glycans present on glycoproteins in SEA as important drivers of this signaling axis. Moreover, we find that OX40L expression and Th2 induction are abrogated when microsomal prostaglandin E synthase-1 (mPGES) is selectively inhibited, but not when a general COX-1/2 inhibitor is used. This shows that the de novo synthesis of PGE2 is vital for the Th2 priming function of SEA-stimulated DCs as well as points to the potential existence of other COX-dependent lipid mediators that antagonize PGE2-driven Th2 polarization. Lastly, specific PGE2 inhibition following immunization with S. mansoni eggs dampened the egg-specific Th cell response. In summary, our findings provide new insights in the molecular mechanisms underpinning Th2 induction by S. mansoni and identify druggable targets for potential control of helminth driven-Th2 responses.

Congruence and Complementarity of Differential Mobility Spectrometry and NMR Spectroscopy for Plasma Lipidomics.

The lipid composition of lipoprotein particles is determinative of their respective formation and function. In turn, the combination and correlation of nuclear magnetic resonance (NMR)-based lipoprotein measurements with mass spectrometry (MS)-based lipidomics is an appealing technological combination for a better understanding of lipid metabolism in health and disease. Here, we developed a combined workflow for subsequent NMR- and MS-based analysis on single sample aliquots of human plasma. We evaluated the quantitative agreement of the two platforms for lipid quantification and benchmarked our combined workflow. We investigated the congruence and complementarity between the platforms in order to facilitate a better understanding of patho-physiological lipoprotein and lipid alterations. We evaluated the correlation and agreement between the platforms. Next, we compared lipid class concentrations between healthy controls and rheumatoid arthritis patient samples to investigate the consensus among the platforms on differentiating the two groups. Finally, we performed correlation analysis between all measured lipoprotein particles and lipid species. We found excellent agreement and correlation (r > 0.8) between the platforms and their respective diagnostic performance. Additionally, we generated correlation maps detailing lipoprotein/lipid interactions and describe disease-relevant correlations.

Changes in Plasma Lipid Levels Following Cortical Spreading Depolarization in a Transgenic Mouse Model of Familial Hemiplegic Migraine.

Metabolite levels in peripheral body fluids can correlate with attack features in migraine patients, which underscores the potential of plasma metabolites as possible disease biomarkers. Migraine headache can be preceded by an aura that is caused by cortical spreading depolarization (CSD), a transient wave of neuroglial depolarization. We previously identified plasma amino acid changes after CSD in familial hemiplegic migraine type 1 (FHM1) mutant mice that exhibit increased neuronal excitability and various migraine-related features. Here, we aimed to uncover lipid metabolic pathways affected by CSD, guided by findings on the involvement of lipids in hemiplegic migraine pathophysiology. Using targeted lipidomic analysis, we studied plasma lipid metabolite levels at different time points after CSD in wild-type and FHM1 mutant mice. Following CSD, the most prominent plasma lipid change concerned a transient increase in PGD2, which lasted longer in mutant mice. In wild-type mice only, levels of anti-inflammatory lipid mediators DPAn-3, EPA, ALA, and DHA were elevated 24 h following CSD compared to Sham-treated animals. Given the role of PGs and neuroinflammation in migraine pathophysiology, our findings underscore the potential of monitoring peripheral changes in lipids to gain insight in central brain mechanisms.

Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring.

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

The lipid profile for the prediction of prednisolone treatment response in patients with inflammatory hand osteoarthritis: The HOPE study.

Objective: To explore the use of lipidomics for prediction of prednisolone treatment response in patients with inflammatory hand osteoarthritis. Design: The Hand Osteoarthritis Prednisolone Efficacy (HOPE) study included patients (n â€‹= â€‹92) with symptomatic inflammatory hand osteoarthritis, fulfilling the ACR criteria. The present analyses comprised only patients randomized to prednisolone treatment (10 â€‹mg daily, n â€‹= â€‹40). Response to prednisolone treatment was defined according to the OARSI-OMERACT responder criteria at six weeks. Baseline blood samples were obtained non-fasted. Lipid species were quantified in erythrocytes with the Lipidyzer™ platform (Sciex). Oxylipins were analyzed in plasma using an in-house LC-MS/MS platform. Elastic net regularized regression was used to predict prednisolone treatment response based on common patient characteristics alone and including the patients' lipid profile. ROC analyses with 1000 bootstrapped area under the curve (AUC) was used to determine the discriminatory accuracy of the models. Results: Among included patients, 78% fulfilled the OARSI-OMERACT responder criteria. From the general patient characteristics, elastic net selected baseline hand function as only predictor of treatment response, with an AUC of 0.78 (0.56; 0.97). Addition of lipidomics resulted in an AUC of 0.92 (0.78; 0.99) and 0.85 (0.65; 0.98) for inclusion of the Lipidyzer™ platform and oxylipin platform, respectively. Conclusion: Our results suggest that the patients' lipid profile may improve the discriminative accuracy of the prediction of prednisolone treatment response in patients with inflammatory hand osteoarthritis compared to prediction by commonly measured patient characteristics alone. Hence, lipidomics may be a promising field for biomarker discovery for prediction of anti-inflammatory treatment response.

Magnetic Beads for Desalting of Monoclonal Antibodies and Antibody-Drug Conjugates.

Characterization of antibody-drug conjugates (ADCs) using mass spectrometry (MS) is important in drug discovery and formulation development and as part of the quality control processes. To facilitate electrospray ionization (ESI) and produce high-quality mass spectra, common components of storage solutions for monoclonal antibodies (mAbs) and ADCs, such as nonvolatile phosphate-buffered saline (PBS), should be replaced before analysis. Centrifugal spin-type kits are extensively used for the desalting or buffer-exchange of mAbs and ADCs samples. The commercially available kits commonly require at least 100 µL of a sample at 1 mg/mL for optimal recovery. However, most ESI-MS based analyses require no more than 25 µg of protein for triplicate injection. In this study, we present a novel method for desalting of ADCs and mAbs building on the SP3 approach with nonfunctionalized carboxylate coated magnetic beads without affinity ligands. The analytes bind to the hydrophilic beads upon the addition of organic solvent, and various solutions of volatile salts or acids can be used in the elution step. The optimized protocol allowed for 88% recovery of ADC at a 25 µL sample volume and 90% recovery at 100 µL. More than 90% of the salts were removed using a process of 20 min. The intra- and interday precision showed little variation with an RSD of 1% and 2%, respectively. The compatibility of this new workflow with low sample volumes is highly valuable since a smaller fraction of the sample is wasted for analysis of the expensive analytes, without compromising recovery.

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/m2), 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.