Anaphylaxis to Sunflower Seed with Tolerance to Sunflower Oil: A Case Report

Sunflower seeds (Helianthus annuus) are an uncommon source of allergy; however, some cases of allergy to sunflower seeds have been reported. Sunflower seed sensitization occurs to storage proteins (2S albumins) and lipid transfer proteins (LTPs). A 46-year-old female presented three allergic reactions within minutes of consuming sunflower seeds. A prick-to-prick test indicated a positive reaction only to sunflower seeds and a negative reaction to other nuts, such as almond, hazelnut, pistachio, cashew, peanut, macadamia, sesame, and walnut. Prick-to-prick and oral provocation tests of sunflower oil were performed, and a negative result was obtained. The patient was prescribed a 0.3 mg epinephrine autoinjector device for emergency intramuscular administration. The patient is currently under avoidance of sunflower seed but eats food cooked in sunflower seed oil. Based on this case, we should recognize that sunflower seeds have the potential to cause severe anaphylaxis, which indicates tolerance to sunflower oil. An accurate and fast diagnosis allows timely recommendation to practice strict avoidance of sunflower seeds, thus reducing the possibility of recurrence of an anaphylactic reaction.

Characterization of Relevant Biomarkers for the Diagnosis of Food Allergies: An Overview of the 2S Albumin Family

2S albumins are relevant and often major allergens from several tree nuts and seeds, affecting mainly children and young people. The present study aims to assess how the structural features of 2S albumins could affect their immunogenic capacity, which is essential to comprehend the role of these proteins in food allergy. For this purpose, twelve 2S albumins were isolated from their respective extracts by chromatographic methods and identified by MALDI-TOF mass-spectrometry. Their molecular and structural characterization was conducted by electrophoretic, spectroscopic and in silico methods, showing that these are small proteins that comprise a wide range of isoelectric points, displaying a general high structure stability to thermal treatment. Despite low amino acid sequence identity, these proteins share structural features, pointing conformational epitopes to explain cross-reactivity between them. Immunoblotting with allergic patients’ sera revealed those possible correlations between evolutionarily distant 2S albumins from different sources. The availability of a well-characterized panel of 2S albumins from plant-derived sources allowed establishing correlations between their structural features and their allergenic potential, including their role in cross-reactivity processes.

Comparison of Two Strategies to Generate Antigen-Specific Human Monoclonal Antibodies: Which Method to Choose for Which Purpose?

Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively ‘ARHGDIB’) as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB.

Onset of nut allergy in a pediatric cohort: clinical and molecular patterns. AFRUSEN study

Background: Nut allergy is a growing problem, but little is known about its onset in children. Objective: Characterize the onset of nut allergy in Southern European children. Methods: Consecutive patients up to 14years of age who presented to allergy departments with an initial allergic reaction to peanut, tree nut, or seed were included. An allergy workup including clinical history, food challenge, SPT, and whole-extract sIgE and ImmunoCAP 112-ISAC assay were performed. Results: Of the 271 children included, 260 were first diagnosed with nut allergy at age 6.5years, 11.8(±21.2SD) months after the index reaction. The most common culprit nuts at onset were walnut (36.5%), peanut (28.5%), cashew(10.4%), hazelnut (8.5%), pistachio (5.4%), and almond (5%). Peanut-allergy onset was more frequent in children ≤6y.o. and walnut in those >6y.o. (p=0.032). In 65% of cases, the allergic reaction occurred the first time the patient consumed the nut, and 35% of reactions were anaphylactic. Overall, nut polysensitization was detected by SPT in 64.9% of patients, though this rate was lower among walnut- (54.7%) and peanut (54.1%)-allergic children (p<0.0001). Sensitization to 2S albumins was predominant (75%), specially Jug r1 (52.8%), whereas LTPs was less relevant (37%). Conclusion: In our population, the onset of nut allergy occured around 6years of age, slightly later than that reported in Anglo-Saxon countries. Walnut was the main trigger, followed by peanut and 2S albumins storage proteins, especially Jug r1, are the most relevant allergens. This study will help guide management and may contribute to preventive strategies in pediatric nut allergy.

Predicting the allergenicity of legume proteins using a PBMC gene expression assay

Background Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. Results PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine). Conclusions The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Multispecies Identification of Oilseed- and Meat-Specific Proteins and Heat-Stable Peptide Markers in Food Products

Consumer demand for both plant products and meat products enriched with plant raw materials is constantly increasing. Therefore, new versatile and reliable methods are needed to find and combat fraudulent practices in processed foods. The objective of this study was to identify oilseed species-specific peptide markers and meat-specific markers that were resistant to processing, for multispecies authentication of different meat and vegan food products using the proteomic LC-MS/MS method. To assess the limit of detection (LOD) for hemp proteins, cooked meatballs consisting of three meat species and hemp cake at a final concentration of up to 7.4% were examined. Hemp addition at a low concentration of below 1% was detected. The LOD for edestin subunits and albumin was 0.9% (w/w), whereas for 7S vicilin-like protein it was 4.2% (w/w). Specific heat-stable peptides unique to hemp seeds, flaxseed, nigella, pumpkin, sesame, and sunflower seeds, as well as guinea fowl, rabbit, pork, and chicken meat, were detected in different meat and vegan foods. Most of the oilseed-specific peptides were identified as processing-resistant markers belonging to 11S globulin subunits, namely conlinin, edestin, helianthinin, pumpkin vicilin-like or late embryogenesis proteins, and sesame legumin-like as well as 2S albumins and oleosin isoforms or selected enzymic proteins.