Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Ara h 2 and Ara h 6 sensitization predicts peanut allergy in Mediterranean pediatric patients.

BACKGROUND: Peanut allergy (PA) management was improved by the introduction of molecular allergology, but guidelines for Mediterranean patients are lacking. We aimed at evaluating peanut component-resolved diagnosis as a diagnostic and prognostic tool in children from Southern France. METHODS: In 181 pediatric patients, PA diagnosis was founded on medical history, skin prick testing, serum-specific IgE to Arachis hypogea extract and components, Pru p 4, and plant carbohydrates, and oral food challenge. Allergen microarray was also performed in 68 of these patients. RESULTS: In peanut-allergic children (n = 117), IgE to Ara h 6 were most prevalent (64%), followed by Ara h 2 (63%), Ara h 1 (60%), and Ara h 9 (52%). Ara h 6 was the best predictor of PA. The second best predictor was the ratio of Ara h 2 IgE to peanut IgE (cutoff 0.113). Persistent childhood PA was associated with complex molecular profiles. Comparison of singleplex and microarray results showed poor concordance for Ara h 2 and Ara h 9. CONCLUSION: Ara h 6 and Ara h 2 are the best predictors of PA at diagnosis in Mediterranean pediatric patients. Ara h 1, Ara h 8, and molecular complexity are associated with PA persistence.

Possible alternative strategies to implement basophil activation testing in multicentric studies.

The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin- and EDTA-anticoagulated whole blood samples followed both BAT workflows: "collect, store, process & analyze" or "collect, process, store & analyze". Storage temperatures of 18-25 °C or 2-8 °C and preservation times from 0 to 7 days were considered. Interleukin-3 was also evaluated. With the "collect, store, process & analyze" workflow, heparin-anticoagulated blood and 18-25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin-3 had no role in enhancing basophil reactivity after storage. Conversely, the "collect, process, store & analyze" workflow demonstrated that either heparin- or EDTA-anticoagulated blood can be processed and kept up to 7 days at 18-25 °C or 2-8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The "collect, store, process & analyze" workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The "collect, process, store & analyze" workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation.

BACKGROUND: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. OBJECTIVES: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. METHODS: A dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors (<24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 °C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. RESULTS: This streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low intra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 5 µL of blood per point could be used. CONCLUSIONS: A whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required.

Tomosyn functions as a PKCδ-regulated fusion clamp in mast cell degranulation.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins mediate membrane fusion critical for vesicular transport and cellular secretion. Mast cells rely on SNARE-mediated membrane fusion for degranulation stimulated by crosslinking of immunoglobulin E (IgE) bound to the Fcε receptor (FcεRI). We investigated the mechanisms downstream of receptor activation that control degranulation. We found that the SNARE binding protein tomosyn-1 (also known as STXBP5) inhibited FcεRI-stimulated degranulation of mast cells. After mast cell activation, tomosyn-1 was phosphorylated on serine and threonine residues, dissociated from the SNARE protein syntaxin 4 (STX4), and associated with STX3. We identified PKCδ as the major kinase required for tomosyn-1 threonine phosphorylation and for regulation of the interaction with STXs. Incubation with high IgE concentrations increased tomosyn-1 abundance in cultured mast cells. Similarly, in basophils from allergic patients with high amounts of serum IgE, the abundance of tomosyn-1 was increased as compared to that in patients with normal IgE concentrations. Our findings identified tomosyn-1 as an inhibitor of mast cell degranulation that required PKCδ to switch its interaction with STX partners during fusion. We suggest that the IgE-mediated increase in tomosyn-1 abundance in allergic patients may represent a counterregulatory mechanism to limit disease development.