Platelet-Derived Extracellular Vesicles as Lipid Carriers in Severe Allergic Inflammation.

The resolution of inflammation is a complex process that is critical for removing inflammatory cells and restoring tissue function. The dysregulation of these mechanisms leads to chronic inflammatory disorders. Platelets, essential cells for preserving homeostasis, are thought to play a role in inflammation as they are a source of immunomodulatory factors. Our aim was to identify key metabolites carried by platelet-derived extracellular vesicles (PL-EVs) in a model of allergic inflammation. PL-EVs were isolated by serial ultracentrifugation using platelet-rich plasma samples obtained from platelet apheresis from severely (n = 6) and mildly (n = 6) allergic patients and non-allergic individuals used as controls (n = 8). PL-EVs were analysed by a multiplatform approach using liquid and gas chromatography coupled to mass spectrometry (LC-MS and GC-MS, respectively). PL-EVs obtained from severely and mildly allergic patients and control individuals presented comparable particle concentrations and sizes with similar protein concentrations. Strikingly, PL-EVs differed in their lipid and metabolic content according to the severity of inflammation. L-carnitine, ceramide (Cer (d18:0/24:0)), and several triglycerides, all of which seem to be involved in apoptosis and regulatory T functions, were higher in PL-EVs from patients with mild allergic inflammation than in those with severe inflammation. In contrast, PL-EVs obtained from patients with severe allergic inflammation showed an alteration in the arachidonic acid pathway. This study demonstrates that PL-EVs carry specific lipids and metabolites according to the degree of inflammation in allergic patients and propose novel perspectives for characterising the progression of allergic inflammation.

A method based on plateletpheresis to obtain functional platelet, CD3+ and CD14+ matched populations for research immunological studies.

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.

Work-related anaphylaxis to wasp sting.

OBJECTIVE: To describe the frequency and epidemiological features of wasp venom allergy in the workplace. METHODS: Retrospective review of 98 adult patients (age 18-65) who suffered an anaphylactic reaction to a wasp sting. Patients were asked about reactions during working hours. Personal history of atopy and previous wasp stings as well as the season, month and type of locality (urban or rural) at the moment of the sting were recorded. Serum-specific IgE levels to venoms from Vespula, Polistes and Apis were measured. RESULTS: Eighteen patients (18%) suffered a reaction to wasp venom during working hours. The average age was 37.4 years, 89% were men and 94% had a personal history of atopy. All patients but one reported more than three previous stings, the last sting occurring at least 1 year previously in 61%. Previous systemic reactions had occurred in 17%. Gardening was the most frequently reported occupation (39%). Most reactions occurred during the summer season (61%) and took place in rural areas (56%). Serum-specific IgE was positive to Vespula in all patients, Polistes in 78%. In the 80 cases occurring outside of working hours, the mean age was 40.6, the male/female ratio was 35/45 and 23% of these patients were atopic. CONCLUSION: Most anaphylactic reactions were not work related. Gardeners were the most frequently involved workers. Workplace anaphylactic reactions showed higher prevalences of atopy (94%) compared to those outside working hours (22%).