Advancing the allergenicity assessment of new proteins using a text mining resource.

With a society increasingly demanding alternative protein food sources, new strategies for evaluating protein safety issues, such as allergenic potential, are needed. Large-scale and systemic studies on allergenic proteins are hindered by the limited and non-harmonized clinical information available for these substances in dedicated databases. A missing key information is that representing the symptomatology of the allergens, especially given in terms of standard vocabularies, that would allow connecting with other biomedical resources to carry out different studies related to human health. In this work, we have generated the first resource with a comprehensive annotation of allergens' symptomatology, using a text-mining approach that extracts significant co-mentions between these entities from the scientific literature (PubMed, ∼36 million abstracts). The method identifies statistically significant co-mentions between the textual descriptions of the two types of entities in the literature as indication of relationship. 1,180 clinical signs extracted from the Human Phenotype Ontology, the Medical Subject Heading terms of PubMed together with other allergen-specific symptoms, were linked to 1,036 unique allergens annotated in two main allergen-related public databases via 14,009 relationships. This novel resource, publicly available through an interactive web interface, could serve as a starting point for future manually curated compilation of allergen symptomatology.

In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Bile salts enhance the susceptibility of the peach allergenic lipid transfer protein, Pru p 3, to in vitro gastrointestinal proteolysis.

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein's sequence which were linked by disulphide bonds into structures of ~ 5-6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates.

A European-Japanese study on peach allergy: IgE to Pru p 7 associates with severity.

BACKGROUND: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. METHODS: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. RESULTS: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73-0.74). Pru p 3 tended to be a risk factor in South Europe only. CONCLUSIONS: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone.

Future of service member monitoring: the intersection of biology, wearables and artificial intelligence.

While substantial investment has been made in the early identification of mental and behavioural health disorders in service members, rates of depression, substance abuse and suicidality continue to climb. Objective and persistent measures are needed for early identification and treatment of these rising health issues. Considerable potential lies at the intersection of biology, wearables and artificial intelligence to provide high accuracy, objective monitoring of mental and behavioural health in training, operations and healthcare settings. While the current generation of wearable devices has predominantly targeted non-military use cases, military agencies have demonstrated successes in monitoring and diagnosis via off-label uses. Combined with context-aware and individualised algorithms, the integration of wearable data with artificial intelligence allows for a deeper understanding of individual-level and group-level mental and behavioural health at scale. Emerging digital phenotyping approaches which leverage ubiquitous sensing technology can provide monitoring at a greater scale, lower price point and lower individual burden by removing the need for additional body-worn technology. The intersection of this technology will enable individualised strategies to promote service member mental and physical health, reduce injury, and improve long-term well-being and deployability.

A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour.

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.

Early introduction of peanut reduces peanut allergy across risk groups in pooled and causal inference analyses.

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 < .0001) among children randomized to consume peanut from early infancy. A protective effect was present across all eczema severity groups, irrespective of enrollment sensitization to peanut, and across different ethnicities. Earlier age of introduction was associated with improved effectiveness of the intervention. In the pooled PP analysis, peanut consumption reduced the risk of PA by 98% (p < .0001). A causal inference analysis confirmed the strong PP effect (89% average treatment effect relative risk reduction p < .0001). A multivariable causal inference analysis approach estimated a large (100%) reduction in PA in children without eczema (p = .004). CONCLUSION: We demonstrate a significant reduction in PA with early peanut introduction in a large group of pooled, randomized participants. This significant reduction was demonstrated across all risk subgroups, including children with no eczema. Furthermore, our results point to increased efficacy of the intervention with earlier age of introduction.

An in vitro protocol to characterise the resistance of food proteins to intestinal digestion.

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 ß-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 sensitization - and hence contribute to future development of a more effective allergenicity risk assessment process.

Identification of allergen encoding sequences in a novel food ingredient from Moringa oleifera leaves.

Alternative sources of edible proteins are required to feed the world's growing population, such as Moringa oleifera leaves, a protein source with a balanced amino acid composition. Since Moringa leaf proteins is a novel food in the EU and UK, an assessment of their potential allergenicity of is required. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential.

Brain activity changes associated with pain perception variability.

Pain perception can be modulated by several factors. Phenomena like temporal summation leads to increased perceived pain, whereas behavioral conditioning can result in analgesic responses. Furthermore, during repeated, identical noxious stimuli, pain intensity can vary greatly in some individuals. Understanding these variations is important, given the increase in investigations that assume stable baseline pain for accurate response profiles, such as studies of analgesic mechanisms. We utilized functional magnetic resonance imaging to examine the differences in neural circuitry between individuals displaying consistent pain ratings and those who experienced variable pain during a series of identical noxious stimuli. We investigated 63 healthy participants: 31 were assigned to a "consistent" group, and 32 were assigned to a "variable" group dependent on pain rating variability. Variable pain ratings were associated with reduced signal intensity in the dorsolateral prefrontal cortex (dlPFC). Furthermore, the dlPFC connectivity with the primary somatosensory cortex and temperoparietal junction was significantly reduced in variable participants. Our results suggest that investigators should consider variability of baseline pain when investigating pain modulatory paradigms. Additionally, individuals with consistent and variable pain ratings differ in their dlPFC activity and connectivity with pain-sensitive regions during noxious stimulation, possibly reflecting the differences in attentional processing and catastrophizing during pain.

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

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.

Assessing protein digestibility in allergenicity risk assessment: A comparison of in silico and high throughput in vitro gastric digestion assays.

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 ß-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.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development.

Peanut Can Be Used as a Reference Allergen for Hazard Characterization in Food Allergen Risk Management: A Rapid Evidence Assessment and Meta-Analysis.

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.

Improving Severity Scoring of Food-Induced Allergic Reactions: A Global "Best-Worst Scaling" Exercise.

BACKGROUND: There is no current consensus on assigning severity to food-induced allergic reactions, for example, to assess the efficacy of allergen immunotherapy. Existing severity scores lack the capability to discriminate between non-anaphylaxis reactions of different severities. Attempts are ongoing to develop a more discriminatory score, which should ideally be data-driven and validated in multiple cohorts. OBJECTIVE: To undertake an exercise using best-worst scaling (BWS) to define a potential gold standard against which severity scoring of food-induced allergic reactions can be refined. METHODS: We undertook a global survey to better understand how health care professionals rate the severity of food-induced allergic reactions, using BWS methodology. Respondents were given a number of patient case vignettes describing real-world allergic reactions and asked to select the pair that, in their opinion, reflected the maximum difference in severity. Responses were then modeled and a preference score (representing severity) determined for each scenario. Scenarios were also scored using existing published scoring systems and the scores compared with the BWS score using Spearman r correlation and Cohen kappa. Given the differences in definitions of anaphylaxis globally, we also evaluated differences in BWS ranking depending on the geographical location of respondents. RESULTS: We received 334 complete responses, 183 (55%) from Europe and 65 (20%) from North America. Perception of severity of some reactions appeared to be affected by geographical location. The comparison of BWS ranking with current grading systems identified significant issues that varied from one grading system to another, such as prominence to some symptoms (eg, vomiting) that skew grading when using scoring systems not designed for food allergy. In general, current scoring systems poorly discriminate against more mild symptoms and often overestimate their severity. CONCLUSIONS: These data provide a methodology free of user scale bias to help define a potential, consensus-driven gold standard that can be used to guide and validate the development of improved grading systems to score food-induced allergic symptoms and highlight areas for education where there is the potential to miscategorize severity.

The impact of a baked muffin matrix on the bioaccessibility and IgE reactivity of egg and peanut allergens.

Baked matrices, such as muffin, may help to promote tolerance to food allergens by modifying allergen structure, digestibility, and capacity to stimulate the immune responses. However, the impact of the muffin matrix on the bioaccessibility of allergens in the gastrointestinal tract is not well understood. Muffin containing egg and peanut was subjected to in vitro oral-gastro-duodenal digestion. During gastric digestion, the majority of the egg allergen Gal d 2 and the peanut allergens Ara h 1 and 3 were not bioaccessible. Subsequent duodenal digestion increased allergen bioaccessibility with Gal d 2 and the peanut allergen Ara h 2 proving highly resistant to digestion. The IgE reactivity of bioaccessible peanut allergens was retained to a greater extent than that of egg allergens after oral-gastric digestion. The starch and gluten-rich muffin matrix modifies allergen bioaccessiblity in a manner more similar to baked matrices such as bread, than low water activity matrices such as cookies.

The efficacy of pulsed ultraviolet light processing for table and hatching eggs.

In the United States, every year an average of 287.1 eggs are consumed per person, and over 14.1 billion eggs are set in hatchery incubators to produce chicks destined for the egg and meat bird industries. By reducing the microbial load on eggs, food-borne-associated outbreaks can be reduced while good chick health is maintained. Pulsed ultraviolet (PUV) light system delivers an energy-intense broad spectrum (100-1,100 nm) pulse derived from a xenon flashlamp. In recent years, PUV light has been shown to reduce microbial pathogens on the surface of shell eggs by using a static PUV light system. In this study, shell eggs were surface inoculated with Escherichia coli or Enterococcus faecium and treated with PUV light using a modified egg candling conveyor that provided complete rotation of eggs under a flashlamp. Pulsed UV light treatment inactivated both microbial strains, with greater energy resulting in a greater germicidal response (P < 0.05). Treatments of 1.0, 2.4, 3.1, and 4.9 J/cm2 resulted in microbial reductions (Log10 CFU/cm2) of 3.83, 4.26, 4.28, and 4.62 for E. coli and 2.04, 3.12, 3.11, and 3.82 for E. faecium, respectively. This study also evaluated the effects of PUV light treatment of hatching eggs (commercial Leghorn hybrids) on both embryo and chick growth parameters. Using the same system, 4 replicates of 125 fertile eggs per rep were treated with 0 (control), 4.9, 24.4, or 48.8 J/cm2 of PUV light. After processing, eggs were placed in a commercial incubator under normal incubation conditions. There was no significant effect of the PUV light treatment on percent fertility, hatchability, or hatch (P > 0.05). Furthermore, there were no significant effects on posthatch observations, including livability and average bird weight at hatch or at 42 d of age (P > 0.05). In conclusion, this study supports the application of PUV light as an effective antimicrobial intervention for both table and hatching eggs.

Using data from food challenges to inform management of consumers with food allergy: A systematic review with individual participant data meta-analysis.

BACKGROUND: Eliciting doses (EDs) (eg, ED01 or ED05 values, which are the amounts of allergen expected to cause objective symptoms in 1% and 5% of the population with an allergy, respectively) are increasingly being used to inform allergen labeling and clinical management. These values are generated from food challenge, but the frequency of anaphylaxis in response to these low levels of allergen exposure and their reproducibility are unknown. OBJECTIVE: Our aim was to determine (1) the rate of anaphylaxis in response to low-level peanut exposure and (2) the reproducibility of reaction thresholds (and anaphylaxis) at food challenge. METHODS: We conducted a systematic review and individual participant data meta-analysis of studies that reported at least 50 individuals with peanut allergy reacting to peanut at double-blind, placebo-controlled food challenge (DBPCFC) and were published between January 2010 and September 2020. Risk of bias was assessed by using National Institute for Clinical Excellence methodologic checklists. RESULTS: A total of 19 studies were included (covering a total of 3151 participants, 534 of whom subsequently underwent further peanut challenge). At individual participant data meta-analysis, 4.5% (95% CI, 1.9% to 10.1%) of individuals reacted to 5 mg or less of peanut protein with anaphylaxis (moderate heterogeneity [I2 = 57%]). Intraindividual thresholds varied by up to 3 logs, although this variation was limited to a half-log change in 71.2% (95% CI, 56.2% to 82.6%) of individuals. In all, 2.4% (95% CI, 1.1% to 5.0%) of patients initially tolerated 5 mg of peanut protein but then reacted to this dose at subsequent challenge (low heterogeneity [I2 = 16%]); none developed anaphylaxis. CONCLUSION: Around 5% of individuals reacting to an ED01 or ED05 level of exposure to peanut might develop anaphylaxis in response to that dose. This equates to 1 and 6 anaphylaxis events per 2500 patients exposed to an ED01 or ED05 dose, respectively, in the broader population of individuals with peanut allergy.