We have found evidence that points to the association of severe allergic inflammation with platelet functions alteration, together with reduced protein synthesis, and switch of immune cells to aerobic glycolysis.

Background Sublingual allergen‐specific immunotherapy (SLIT) intervention improves the control of grass pollen allergy by maintaining allergen tolerance after cessation. Despite its widespread use, little is known about systemic effects and kinetics associated to SLIT, as well as the influence of the patient sensitization phenotype (Mono‐ or Poly‐sensitized). In this quest, omics sciences could help to gain new insights to understand SLIT effects. Methods 47 grass‐pollen‐allergic patients were enrolled in a double‐blind, placebo‐controlled, multicenter trial using GRAZAX® during 2 years. Immunological assays (sIgE, sIgG4, and ISAC) were carried out to 31 patients who finished the trial. Additionally, serum and PBMCs samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were allocated in Mono‐ or Poly‐sensitized groups, excluding patients allergic to epithelia. Individuals were compared based on their treatment (Active/Placebo) and sensitization level (Mono/Poly). Results Kinetics of serological changes agreed with those previously described. At two years of SLIT, there are scarce systemic changes that could be associated to improvement in systemic inflammation. Poly‐sensitized patients presented a higher inflammation at inclusion, while Mono‐sensitized patients presented a reduced activity of mast cells and phagocytes as an effect of the treatment. Conclusions The most relevant systemic change detected after two years of SLIT was the desensitization of effector cells, which was only detected in Mono‐sensitized patients. This change may be related to the clinical improvement, as previously reported, and, together with the other results, may explain why clinical effect is lost if SLIT is discontinued at this point.

No abstract

Background: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. Methods:Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC-MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex.Results: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype.

Background In previous studies with peripheral blood cells, platelet factors were found to be associated with severe allergic phenotypes. A reliable method yielding highly concentrated and pure platelet samples is usually not available for immunological studies. Plateletpheresis is widely used in the clinics for donation purposes. In this study, we designed a protocol based on plateletpheresis to obtain Platelet‐Rich Plasma (PRP), Platelet‐Poor Plasma (PPP) as well as CD3+ and CD14+ cells matched samples from a waste plateletpheresis product for immunological studies. Methods Twenty‐seven subjects were voluntarily subjected to plateletpheresis. PRP, PPP and blood cell concentrate contained in a leukocyte reduction system chamber (LRSC) were obtained in this process. CD3+ and CD14+ cells were isolated from the LRSC by density‐gradient centrifugation and positive magnetic bead isolation. RNA was isolated from PRP, CD3+ and CD14+ cell samples and used for transcriptomic studies by Affymetrix. PRP and PPP samples were used for platelet protein quantification by multiplex assays. Results A reliable high yield method to obtain matched samples of PRP, PPP, CD3+ and CD14+ from a single donor for RNA and protein analyses has been designed. The RNA quality indicators (RQI) routinely used for other cell types were not suitable for platelet RNA characterization. Despite this, the platelet RNA was valid for transcriptomic studies by Affymetrix, as platelet transcripts obtained in our previous studies were confirmed in PRP samples. Platelet samples were enriched in platelet factors as determined in protein multiplex analysis. Conclusions We have developed a method that yields not only high content and pure platelet samples from a single donor but also CD3+ and CD14+ matched samples that can be used for RNA and protein analyses in immunological studies.