Multisystem inflammatory syndrome in children (MIS‐C) is a rare, but severe complication of coronavirus disease 2019 (COVID‐19). It develops approximately 4 weeks after severe acute respiratory ...syndrome coronavirus 2 (SARS‐CoV‐2) infection and involves hyperinflammation with multisystem injury, commonly progressing to shock. The exact pathomechanism of MIS‐C is not known, but immunological dysregulation leading to cytokine storm plays a central role. In response to the emergence of MIS‐C, the European Academy of Allergy and Clinical Immunology (EAACI) established a task force (TF) within the Immunology Section in May 2021. With the use of an online Delphi process, TF formulated clinical statements regarding immunological background of MIS‐C, diagnosis, treatment, follow‐up, and the role of COVID‐19 vaccinations. MIS‐C case definition is broad, and diagnosis is made based on clinical presentation. The immunological mechanism leading to MIS‐C is unclear and depends on activating multiple pathways leading to hyperinflammation. Current management of MIS‐C relies on supportive care in combination with immunosuppressive and/or immunomodulatory agents. The most frequently used agents are systemic steroids and intravenous immunoglobulin. Despite good overall short‐term outcome, MIS‐C patients should be followed‐up at regular intervals after discharge, focusing on cardiac disease, organ damage, and inflammatory activity. COVID‐19 vaccination is a safe and effective measure to prevent MIS‐C. In anticipation of further research, we propose a convenient and clinically practical algorithm for managing MIS‐C developed by the Immunology Section of the EAACI.
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.
We describe a platelet isolation method based on plateletpheresis that allows to obtain pure, highly concentrated and functional platelet samples from a single donor. Additionally, matched CD3+ and CD14+ populations can be obtained from a waste product of plateletpheresis procedure. All the samples are suitable for omics studies.
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.
Conclusions
This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies.
This study analyzes the lipidomic and transcriptomic profile of platelets in patients with mild and severe allergy. The lipidomic profile of platelets form severe allergic patients shows high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins and low levels of arachidonic acid. Transcriptomics reveals a higher expression of platelet activation genes and the alteration of IL‐17, MAPK, TLR, and NLR pathways in the severe phenotype, which was validated by P‐Selectin and IL‐17 AF protein quantification.Abbreviations: ALOX12, arachidonate 12‐lipoxygenase; CD40L, CD40 ligand; CPM, counts per millon; IL, interleukin; ITGB3, integrin subunit beta 3; −Log(P), −Logarithm in base 10 of P value; MAPK, mitogen‐activated protein kinase; NLR, NOD‐like receptor; PPBP, pro‐platelet basic protein; SELP, selectin P; TLR, Toll‐like receptor
The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays ...and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.
Key message
Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second ...pollination and may keep away herbivores until seed maturation.
The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of
Pru p 3
was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.
Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose ...complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.
The prevalence of allergies is increasing since mid twentieth century; however the underlying causes of this increase are not fully clear. Understanding the mechanism by which a harmless protein ...becomes an allergen provides us with the basis to prevent and treat these diseases. Although most studies on allergen immunogenicity have traditionally focused on structural properties of the proteins, it is increasingly clear that allergenicity cannot be determined only based on structural features of the allergenic proteins. In fact, allergens do not encounter human facings as isolated molecules but contained in complex mixtures of proteins, carbohydrates and lipids, such as pollen grains or foods. As a result, attention has lately been directed to examine whether allergen-associated molecules exhibit immune-regulatory properties. The present review aims to illustrate some examples of how non-protein molecules accompanying the allergen can modulate allergic responses.
Emerging evidence suggests that platelets, cytoplasmic fragments derived from megakaryocytes, can no longer be considered just as mediators in hemostasis and coagulation processes, but as key ...modulators of immunity. Platelets have received increasing attention as the emergence of new methodologies has allowed the characterization of their components and functions in the immune continuum. Platelet activation in infectious and allergic lung diseases has been well documented and associated with bacterial infections reproduced in several animal models of pulmonary bacterial infections. Direct interactions between platelets and bacteria have been associated with increased pulmonary platelet accumulation, whereas bacterial-derived toxins have also been reported to modulate platelet function. Recently, platelets have been found extravascular in the lungs of patients with asthma, and in animal models of allergic lung inflammation. Their ability to interact with immune and endothelial cells and secrete immune mediators makes them one attractive target for biomarker identification that will help characterize their contribution to lung diseases. Here, we present an original review of the last advances in the platelet field with a focus on the contribution of platelets to respiratory infections and allergic-mediated diseases.
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