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There is an increasing demand for rapid, affordable, in-field screening methods for low molecular weight (LMW) compound detection. Anti-idiotypes (Ab2s) are biologically derived surrogates that can replace LMW compounds and their protein conjugates in immunoassays. Substitution with anti-idiotypes can improve assay standardisation, reduce cost, and contribute to environmental safety. Their application has been limited by difficult generation processes and varied effects on assay performance. This review examines a recent resurgence in the use of Ab2s within LMW compound detection, driven by the application of phage display and nanobodies. The methods used for Ab2 production are critically discussed and their potential role in improving LMW compound immunoassays is highlighted. Finally, forward-looking ideas for the production of anti-idiotypes are provided, along with barriers to their generation.

Alergia; Anafilaxia; Asma Allergy; Anaphylaxis; Asthma Al·lèrgia; Anafilaxi; Asma Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the “EAACI Molecular Allergology User's Guide” (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

International audience; Developing reliable methodologies for detecting and quantifying allergens in processed food commodities iscrucial to support food business operators in allergen risk assessment and properly implementing precautionaryallergen labels whenever required to safeguard the health of allergic consumers. Multiple Mass Spectrometry(MS) methods have been developed so far and applied for single and multi-allergen detection in foods, generatinga heterogeneous literature on this topic, with little attention paid to the extraction and the digestion steps, crucialin delivering accurate allergen measurements.This investigation carried out within an international consortium specifically built up to convey a prototypeMS based reference method, reports on the first part of the method development, namely the optimization of thesample preparation protocol for six allergens detection (cow’s milk, hen’s egg, soy, peanut, hazelnut, andalmond) in chocolate. The latter was chosen as model complex food matrix, having a high lipid and polyphenolcontent.Different steps of the sample preparation protocol have been taken into consideration: (i) sampling, (ii)composition of the extraction buffer, (iii) protein purification, (iv) protein enzymatic digestion, (v) peptidepurification and pre-concentration, and some experiments were carried out by two independent laboratories andtwo different MS platforms to provide a first assessment of the robustness of the method under development.Fifty target peptides were monitored in multiple reaction monitoring mode and validated in different labora-tories to trace the six allergenic ingredients in the incurred chocolate and the best performing protocol for samplepreparation was identified. This work paves the way of the forthcoming full analytical validation of a prototypereference method for MS-based allergen quantification.

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a "gluten-free" claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 °C) or sonication at 60 °C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDS-PAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 °C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food.

SARS-CoV-2 infection in children results in a wide range of clinical outcomes. Paediatric Multisystem Inflammatory syndrome temporally associated with COVID-19(PIMS-TS) occurs weeks after a SARS-CoV-2 infection, and results in severe illness. This protocol describes a study to fully characterize the circulating proteome of children who have PIMS-TS, the proteome of healthy children who have previously been infected with SARS-CoV-2 and the proteome of febrile children with a confirmed invasive infection. Orthogonal proteomic techniques will be utilized to provide a deep proteomic characterization.

Background: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high-throughput assays limits the clinical utility. We aimed to develop a high-throughput basophil activation test based on human progenitor cell-derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases. Methods: Progenitor cell-derived basophils were differentiated from CD34+ progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed. Results: Following passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose-dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut-sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r2 =.49, p =.001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC20) correlated with PCBAT AUC (r2 =.33, p =.016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r2 =.59, p 2 =.57, p =.001) and IgE to Ara h1 (r2 =.55, p =.007), but not with sIgE to whole peanut or Ara h2. All peanut-sensitized but tolerant subjects showed no reaction to peanut on PCBAT. Conclusion: Progenitor cell-derived basophils activation test is a high-throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing.

Background: The Learning Early About Peanut allergy (LEAP) study has shown the effectiveness of early peanut introduction in prevention of peanut allergy (PA). In the Enquiring About Tolerance (EAT) study, a statistically significant reduction in PA was present only in per-protocol (PP) analyses, which can be subject to bias. Objective: The aim of this study was to combine individual-level data from the LEAP and EAT trials and provide robust evidence on the bias-corrected, causal effect of early peanut introduction. Method: As part of the European Union-funded iFAAM project, this pooled analysis of individual pediatric patient data combines and compares effectiveness and efficacy estimates of oral tolerance induction among different risk strata and analysis methods. Results: An intention-to-treat (ITT) analysis of pooled data showed a 75% reduction in PA (p

In vitro digestion tests provide data on the form in which dietary proteins maybe presented to the gut mucosal immune system, one of many strands of evidence used in allergenicity risk assessment. A 96-well plate format in vitro intestinal digestion protocol has been developed with a high and low enzyme activity test executed at pH 6.5 and 8.0. It was applied to the systematic analysis of test proteins (including six allergens and one non-allergenic comparator) which were either completely resistant to pepsinolysis or gave rise to large persistent fragments following in vitro gastric digestion. Digestion was monitored using SDS-PAGE and densitometry. Proteins resistant to pepsin were also resistant to intestinal digestion irrespective of the protocol applied and gave rise to large persistent digestion fragments. In contrast persistent fragments from pepsin digestion were readily digested. Bile salts enhanced the digestibility of two highly resistant proteins, lysozyme ad β-lactoglobulin, changing the rank order of protein digestibility. Intestinal digestion tests that include bile salts provide a more physiologically relevant system for future investigation into how digestion products may influence the balance between tolerance and sensitisation - and hence contribute to future development of a more effective allergenicity risk assessment process.

A multiplex lateral flow immunoassay (LFA) has been developed to detect the primary marine biotoxin groups: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, and diarrhetic shellfish poisoning toxins. The performance characteristics of the multiplex LFA were evaluated for its suitability as a screening method for the detection of toxins in shellfish. The marine toxin-specific antibodies were class-specific, and there was no cross-reactivity between the three toxin groups. The test is capable of detecting all three marine toxin groups, with working ranges of 0.2-1.5, 2.5-65.0, and 8.2-140.3 ng/mL for okadaic acid, saxitoxin, and domoic acid, respectively. This allows the multiplex LFA to detect all three toxin groups at the EU regulatory limits, with a single sample extraction method and dilution volume. No matrix effects were observed on the performance of the LFA with mussel samples spiked with toxins. The developed LFA uses a simple and pocket-sized, portable Cube Reader to provide an accurate result. We also evaluated the use of this Cube Reader with commercially available monoplex lateral flow assays for marine toxins.

The susceptibility of a novel food protein to digestion in the pepsin resistance test is widely used to inform the allergenicity risk assessment process. However, it does not model the variation in the intragastric environment found in vivo. Consequently a 96-well plate format in vitro gastric digestion protocol has been developed with a high and low pepsin activity test executed at pH 1.2, 2.5, 5.5 and 6.5. It was used to analyse seven allergens (from milk, egg, peach and peanut) and two non-allergens (cytochrome c and zein). Digestion was monitored using SDS-PAGE and densitometry. In silico predictions were not confirmed experimentally for most of the proteins studied. Proteins were ranked according to half-life and showed susceptibility to digestion was related to the stability of protein structure and protein solubility rather than allergenicity per se. Highly digestible proteins, such as β-casein and Ara h 1, generated abundant resistant fragments Mr 3.5 kDa in the low pepsin activity test which could be immunologically significant within the context of allergenicity risk assessment for susceptible groups such as infants. The high- and low pepsin activity tests used in this study provided complementary data to support allergenicity risk assessment and used only 10 mg protein.

Despite high levels of need, many young people who experience health issues do not seek, access or receive support. Between May and November 2021, using semi-structured interviews, we explored the perspectives of 51 young people (aged 13–14) from two schools who had taken part in a novel online health and wellbeing screening programme, the Digital Health Contact (DHC). One school delivered the DHC during home-learning due to COVID-19 restrictions, whilst the other delivered it in school when restrictions were lifted. The DHC was seen as a useful approach for identifying health need and providing support, and had high levels of acceptability. Young people appreciated the online format of the DHC screening questionnaire and thought this facilitated more honest responses than a face-to-face approach might generate. Completion at home, compared to school-based completion, was perceived as more private and less time-pressured, which young people thought facilitated more honest and detailed responses. Young people’s understanding of the screening process (including professional service involvement and confidentiality) influenced engagement and responses. Overall, our findings afford important insights around young people’s perspectives of participating in screening programmes, and highlight key considerations for the development and delivery of health screening approaches in (and out of) school.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral–gastro–duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20–30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

Scope: The Caco2/HT29-MTX co-culture system is widely used as a cell model of the intestinal epithelium. Although the gut epithelium plays an important role in the uptake of free fatty acids and the resynthesis of triglycerides the lipid distribution profile of the co-culture system is not well understood. Desorption electrospray ionization (DESI) is a mass spectrometry (MS) technique which has been widely used to study the main classes of lipid molecules on different tissue surfaces. This has been used to map lipid species and their distribution in Caco2 and HT29-MTX co-culture system. Methods and results: Caco2 and HT29-MTX cells were seeded on coverslips either singly or as cocultures in ratios of 75:25 and 50:50. Cells were cultured for 21 days before MS imaging using a DESI source in both the positive and negative ionization modes. The identity of selected lipids was confirmed in negative and positive ionisation modes using tandem MS. Although many lipids were common to both cell lines, there were distinctive patterns in the lipidomes. Thus, the lipidome of Caco2 cells was more heterogeneous and rich in cholesterol esters and triglycerides whilst HT29-MTX cells has a distinctive lipidome relating to phosphatidylethanolamines, phosphatidylinositols and odd chain lipids, including C17 fatty acids. Conclusion: DESI-MSI has shown that Caco2 and HT29-MTX cells have distinctive lipidomes which are still evident when the cells are cocultured. It has potential to both allow further validation of these widely used cell models and provide insights into how dietary components may modify lipid metabolism in future.

Pure oats are generally accepted to be safe for most celiac patients, and consumption of oats provides advantageous dietary fibers. However, oats can be contaminated by gluten proteins from wheat, barley, and/or rye. The analytical challenge lies in the reliability of the quantification method and how to maintain the contamination level under a gluten-free food threshold of 20 mg/kg. In this study, we investigated barley-spiked oat flour samples at four levels using four gluten ELISA kits. The largest recovery variance was with the R5 kit that gave 5-6 times overestimation; the G12 kit cross-reacted with oat proteins and gave 4-5 times overestimation at all spiked levels. The Total Gluten and Morinaga kits gave satisfactory recoveries. Total barley hordeins were isolated and characterized to be used as a common calibrator in all four kits aiming at harmonizing the results and to test the kits' performance. Immunoblotting of total hordein isolate revealed that Total Gluten and Morinaga antibodies provided an overall detection, while R5 and G12 antibodies recognized specific hordein groups leading to a larger difference when wheat and barley were used as the calibrant. Calibration with total hordein isolate corrected the overestimation problem and decreased the variability between the four gluten kits.

Regional and national legislation mandates the disclosure of “priority” allergens when present as an ingredient in foods, but this does not extend to the unintended presence of allergens due to shared production facilities. This has resulted in a proliferation of precautionary allergen (“may contain”) labels (PAL) that are frequently ignored by food-allergic consumers. Attempts have been made to improve allergen risk management to better inform the use of PAL, but a lack of consensus has led to variety of regulatory approaches and nonuniformity in the use of PAL by food businesses. One potential solution would be to establish internationally agreed “reference doses,” below which no PAL would be needed. However, if reference doses are to be used to inform the need for PAL, then it is essential to characterize the hazard associated with these low-level exposures. For peanut, there are now published data relating to over 3000 double-blind, placebo-controlled challenges in allergic individuals, but a similar level of evidence is lacking for other priority allergens. We present the results of a rapid evidence assessment and meta-analysis for the risk of anaphylaxis to a low-level allergen exposure for priority allergens. On the basis of this analysis, we propose that peanut can and should be considered an exemplar allergen for the hazard characterization at a low-level allergen exposure. Resumen: La legislación regional y nacional exige la divulgación de alérgenos "prioritarios" cuando están presentes como ingrediente en los alimentos, pero esto no se extiende a la presencia involuntaria de alérgenos debido a instalaciones de producción compartidas. Esto ha dado lugar a una proliferación de etiquetas de precaución para alérgenos ("pueden contener") (PAL) que los consumidores alérgicos a los alimentos suelen ignorar. Se han hecho intentos para mejorar la gestión del riesgo de alérgenos para informar mejor el uso de PAL, pero la falta de consenso ha llevado a una variedad de enfoques regulatorios y a la falta de uniformidad en el uso de PAL por parte de las empresas alimentarias. Una posible solución sería establecer “dosis de referencia” acordadas internacionalmente, por debajo de las cuales no se necesitaría PAL. Sin embargo, si se van a utilizar dosis de referencia para informar la necesidad de PAL, entonces es esencial caracterizar el peligro asociado con estas exposiciones de bajo nivel. Para el maní, ahora hay datos publicados relacionados con más de 3000 desafíos doble ciego controlados por placebo en individuos alérgicos, pero falta un nivel similar de evidencia para otros alérgenos prioritarios. Presentamos los resultados de una evaluación rápida de la evidencia y un metanálisis del riesgo deanafilaxia a una exposición a alérgenos de bajo nivel para alérgenos prioritarios. Sobre la base de este análisis, proponemos que el cacahuete puede y debe considerarse un alérgeno ejemplar para la caracterización del peligro en una exposición a un alérgeno de bajo nivel. Instituto de Investigación de Tecnología de Alimentos Fil: Turner, Paul J. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Patel, Nandinee. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Ballmer-Weber, Barbara K. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Ballmer-Weber, Barbara K. Clínica de Dermatología y Alergología. Kantonsspital; Suiza. Fil: Baumert, Joe L. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Blom, W. Marty. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Brooke-Taylor, Simon. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Brough, Helen. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Brough, Helen. King's College London. Departamento de Alergia Pediátrica; Reino Unido. Fil: Campbell, Dianne E. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Campbell, Dianne E. Tecnologías DBV. Montrouge; Francia. Fil: Chen, Hongbing. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Chinthrajah, R. Sharon. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Crevel, René W.R. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Dubois, Anthony E.J. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Ebisawa, Motohiro. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Elizur, Arnon. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Elizur, Arnon. Universidad de Tel Aviv. Facultad de Medicina Sackler. Departamento de Pediatría; Israel. Fil: Gerdts, Jennifer D. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Gowland, M. Hazel. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Houben, Geert F. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Hourihane, Jonathan O.B. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Knulst, André C. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: La Vieille, Sébastien. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: López, María Cristina. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Mills, E.N. Clare. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Polenta, Gustavo Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación Tecnología de Alimentos; Argentina. Fil: Polenta, Gustavo Alberto. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Purington, Natasha. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Said, María. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Sampson, Hugh A. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Sampson, Hugh A. Escuela de Medicina Icahn. División de Alergia e Inmunología Pediátricasen. Nueva York. Estados Unidos de América. Fil: Schnadt, Sabine. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Södergren, Eva. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Södergren, Eva. ThermoFisher Scientific; Suecia. Fil: Taylor, Stephen L. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Remington, Benjamin C. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido. Fil: Remington, Benjamin C. Grupo BV. Consultoría Remington; Holanda.

Background: the heterogeneity and lack of validation of existing severity scores for food allergic reactions limit standardization of case management and research advances. We aimed to develop and validate a severity score for food allergic reactions. Methods: following a multidisciplinary experts consensus, it was decided to develop a food allergy severity score (FASS) with ordinal (oFASS) and numerical (nFASS) formats. oFASS with 3 and 5 grades were generated through expert consensus, and nFASS by mathematical modeling. Evaluation was performed in the EuroPrevall outpatient clinic cohort (8232 food reactions) by logistic regression with request of emergency care and medications used as outcomes. Discrimination, classification, and calibration were calculated. Bootstrapping internal validation was followed by external validation (logistic regression) in 5 cohorts (3622 food reactions). Correlation of nFASS with the severity classification done by expert allergy clinicians by Best-Worst Scaling of 32 food reactions was calculated. Results: oFASS and nFASS map consistently, with nFASS having greater granularity. With the outcomes emergency care, adrenaline and critical medical treatment, oFASS and nFASS had a good discrimination (receiver operating characteristic area under the curve [ROC-AUC]>0.80), classification (sensitivity 0.87–0.92, specificity 0.73–0.78), and calibration. Bootstrapping over ROC-AUC showed negligible biases (1.0 × 10 −6–1.23 × 10 −3). In external validation, nFASS performed best with higher ROC-AUC. nFASS was strongly correlated (R 0.89) to best-worst scoring of 334 expert clinicians. Conclusion: FASS is a validated and reliable method to measure severity of food allergic reactions. The ordinal and numerical versions that map onto each other are suitable for use by different stakeholders in different settings.

The severity of allergic reactions to foods can vary markedly. Little is known of variations in reaction severity within or between individuals or the effects of cofactors.We examined the effects of sleep deprivation and exercise and repeat challenges on the severity and patterns of allergic reactions to peanut.In a randomized crossover study, adults with peanut allergy underwent 3 open peanut challenges in random order: with exercise after each dose, with sleep deprivation preceding challenge, and with no intervention. The primary outcome was eliciting dose, reported elsewhere. Reaction severity was a secondary outcome, evaluated using a weighted log-transformed numerical severity score. Analyses estimated the difference in severity between nonintervention challenge and challenges with exercise or sleep deprivation, adjusting for challenge order and using the highest dose tolerated by each individual across all their challenges. Symptom pattern reproducibility was assessed by comparing symptom sequences using pairwise sequence alignment to obtain a percentage match in symptom pattern.Eighty-one participants (mean age 25 y) completed at least 1 postbaseline challenge. Sleep deprivation, but not exercise, significantly increased severity score by 48% (95% CI 12%-84%; P = .009) compared with no intervention. A 38% increase in severity was observed between the first and the last postbaseline challenge (95% CI 1%-75%; P = .044). The average pairwise match of symptoms within individuals was 82.4% and across individuals was 78.3%.A novel severity score demonstrates that sleep deprivation and repeated challenges increase reaction severity. Understanding factors affecting severity is essential for effective risk management. We also show that symptom patterns in repeat peanut challenges are similar within and between individuals.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.