A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Approaches to assess IgE mediated allergy risks (sensitization and cross-reactivity) from new or modified dietary proteins.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks.

Can we identify patients at risk of life-threatening allergic reactions to food?

Anaphylaxis has been defined as a 'severe, life-threatening generalized or systemic hypersensitivity reaction'. However, data indicate that the vast majority of food-triggered anaphylactic reactions are not life-threatening. Nonetheless, severe life-threatening reactions do occur and are unpredictable. We discuss the concepts surrounding perceptions of severe, life-threatening allergic reactions to food by different stakeholders, with particular reference to the inclusion of clinical severity as a factor in allergy and allergen risk management. We review the evidence regarding factors that might be used to identify those at most risk of severe allergic reactions to food, and the consequences of misinformation in this regard. For example, a significant proportion of food-allergic children also have asthma, yet almost none will experience a fatal food-allergic reaction; asthma is not, in itself, a strong predictor for fatal anaphylaxis. The relationship between dose of allergen exposure and symptom severity is unclear. While dose appears to be a risk factor in at least a subgroup of patients, studies report that individuals with prior anaphylaxis do not have a lower eliciting dose than those reporting previous mild reactions. It is therefore important to consider severity and sensitivity as separate factors, as a highly sensitive individual will not necessarily experience severe symptoms during an allergic reaction. We identify the knowledge gaps that need to be addressed to improve our ability to better identify those most at risk of severe food-induced allergic reactions.

Objective eliciting doses of peanut-allergic adults and children can be combined for risk assessment purposes.

BACKGROUND: To improve food labelling strategies, information regarding eliciting doses (EDs) and the effect of patient characteristics on these EDs is necessary. OBJECTIVE: To establish EDs for objective and subjective symptoms and analyse the effect of sensitization levels and other patient characteristics on threshold distribution curves (TDCs). METHODS: Threshold data from 100 adults and 262 children with a positive food challenge were analysed with interval-censoring survival analysis (ICSA) and fitted to a TDC from which EDs could be extracted. Possible influencing factors were analysed as covariates by ICSA. A hazard ratio (HR) was calculated in case of a significant effect. RESULTS: TDCs for both objective and subjective symptoms were significantly different between adults and children (P < 0.001). Objective ED05 values, however, were comparable (2.86 mg peanut protein in adults and 6.38 mg in children). Higher levels of sIgE to Ara h 2 and peanut extract were associated with a larger proportion of patient groups reacting to a dose increase with objective symptoms (adults and children) or subjective symptoms (adults, in children a trend). Age had a similar effect in children (HR 1.05 for objective symptoms and 1.09 for subjective symptoms). Gender had no effect on TDCs. CONCLUSION AND CLINICAL RELEVANCE: Subjective and objective TDCs were different between adults and children, but objective ED05 values were comparable, meaning that threshold data from children and adults can be combined for elaboration of reference doses for risk assessment. Higher sIgE levels to Ara h 2 and peanut extract were associated with a larger proportion of both patient groups to react to a certain dose increase.

Unintended allergens in precautionary labelled and unlabelled products pose significant risks to UK allergic consumers.

BACKGROUND: Allergens in food may pose a risk to allergic consumers. While there is EU regulation for allergens present as an ingredient, this is not the case for unintended allergen presence (UAP). Food companies use precautionary allergen labels to inform allergic individuals of a potential risk from UAPs. This study investigates the risk of an allergic reaction within the milk-, wheat-, hazelnut- and peanut-allergic populations when ingesting UK foods across multiple product categories with and without precautionary allergen labelling. METHODS: Allergen risk assessment using probabilistic techniques enables the estimation of the residual risk after the consumption of a product that unintentionally contains an allergen. RESULTS: Within this selection of UK products, the majority that tested positive for an allergen contained a concentration of allergen predicted to cause a reaction in >1% of the allergic population. The concentrations of allergens measured were greater than the VITAL(®) 2.0 action levels and would trigger precautionary allergen labelling. This was found for products both with and without precautionary allergen labelling. CONCLUSIONS: The results highlight the need for the food industry and regulators to adopt a transparent, risk-based approach for the communication of the risk associated with potential cross-contact that could occur in the processing facility or production chain.

Precautionary allergen labelling: perspectives from key stakeholder groups.

Precautionary allergen labelling (PAL) was introduced by the food industry to help manage and communicate the possibility of reaction from the unintended presence of allergens in foods. However, in its current form, PAL is counterproductive for consumers with food allergies. This review aims to summarize the perspectives of all the key stakeholders (including clinicians, patients, food industry and regulators), with the aim of defining common health protection and risk minimization goals. The lack of agreed reference doses has resulted in inconsistent application of PAL by the food industry and in levels of contamination that prompt withdrawal action by enforcement officers. So there is a poor relationship between the presence or absence of PAL and actual reaction risk. This has led to a loss of trust in PAL, reducing the ability of consumers with food allergies to make informed choices. The result has been reduced avoidance, reduced quality of life and increased risk-taking by consumers who often ignore PAL. All contributing stakeholders agree that PAL must reflect actual risk. PAL should be transparent and consistent with rules underpinning decision-making process being communicated clearly to all stakeholders. The use of PAL should indicate the possible, unintended presence of an allergen in a consumed portion of a food product at or above any proposed action level. This will require combined work by all stakeholders to ensure everyone understands the approach and its limitations. Consumers with food allergy then need to be educated to undertake individualized risk assessments in relation to any PAL present.

Frequency, severity and causes of unexpected allergic reactions to food: a systematic literature review.

Food allergic patients have to deal with an avoidance diet. Confusing labelling terms or precautionary labels can result in misinterpretation and risk-taking behaviour. Even those patients that strictly adhere to their diet experience (sometimes severe) unexpected allergic reactions to food. The frequency, severity and causes of such reactions are unknown. The objective of this review was to describe the frequency, severity and causes of unexpected allergic reactions to food in food allergic patients aged > 12 years, in order to develop improved strategies to deal with their allergy. A systematic review was carried out by two researchers, in six electronic databases (CINAHL, Cochrane, EMBASE, Medline, Psychinfo and Scopus). The search was performed with keywords relating to the frequency, severity and causes of unexpected allergic reactions to food. This resulted in 24 studies which met the inclusion criteria; 18 observational and six qualitative studies. This review shows that knowledge about the frequency of unexpected reactions is limited. Peanut, nuts, egg, fruit/vegetables and milk are the main causal foods. Severe reactions and even fatalities occur. Most reactions take place at home, but a significant number also take place when eating at friends' houses or in restaurants. Labelling issues, but also attitude and risky behaviour of patients can attribute to unexpected reactions. We conclude that prospective studies are needed to get more insight in the frequency, severity, quantity of unintended allergen ingested and causes of unexpected allergic reactions to food, to be able to optimize strategies to support patients in dealing with their food allergy. Although the exact frequency is not known, unexpected reactions to food occur in a significant number of patients and can be severe. For clinical practice, this means that patient education and dietary instructions are necessary.

Application of scientific criteria to food allergens of public health importance.

Scientific criteria for identifying allergenic foods of public health importance (Björkstén, B., Crevel, R., Hischenhuber, C., Løvik, M., Samuels, F., Strobel, S., Taylor, S.L., Wal, J.-M., Ward, R., 2008. Criteria for identifying allergenic foods of public health importance. Regulatory Toxicology and Pharmacology 51(1), 42-52) have been further refined to incorporate an assessment of the strength of available scientific evidence (van Bilsen, J.H., Ronsmans, S., Crevel, R.W., Rona, R.J., Przyrembel, H., Penninks, A.H., Contor, L., Houben, G.F., 2011. Evaluation of scientific criteria for identifying allergenic food of public health importance. Regulatory Toxicology and Pharmacology 60, 281-289). A multi-disciplinary group was invited to critically test the refined approach. They independently evaluated selected publications on coconut, soy and/or peanut allergy, scored them using the newly developed level of evidence criteria, and debated proposed approaches for combining and utilising the scores to measure the overall impact of an allergen in public health impact assessments. The evaluation of selected publications using the modified criteria produced a relatively consistent result across the experts. These refined criteria were judged to be a way forward for the identification of allergenic foods of public health importance, and for prioritisation of allergen risk management and future data gathering. The debate to combine available evidence when assessing whether an allergenic food is of sufficient public health importance to warrant active management led to proposals on how to weight and combine evidence on allergen severity, potency and prevalence. The refined criteria facilitate a debate to find a meaningful sequence of steps to summarise the available information in relation to a food allergen.

Evaluation of scientific criteria for identifying allergenic foods of public health importance.

Identification of allergenic foods of public health importance should be based on well-defined criteria. Björkstén et al. (2008) proposed that the criteria should assess the evidence for an IgE mechanism, the reaction, the potency and the severity of the effect of the food and its prevalence. This study evaluated the application of the proposed criteria based on published reports. Publications were selected from two databases to test whether the descriptions for ranking the level of evidence for each criterion were unambiguous and covered the full range of levels of evidence regarding seven foods, five known to be allergenic and two negative controls. The options available to rank the quality of evidence were appropriate but needed refinement to improve clarity and conceptual value. The criteria were helpful to assess known IgE-dependent allergens, and to exclude the non-allergenic substances. The criteria framework discriminated between papers with high, moderate and low quality of evidence. The advantage of using the proposed criteria is to make the decision-making process and rationale explicit. The framework helps to identify gaps in knowledge and to uncover the level of heterogeneity of the evidence thus guiding research and providing a basis for sound risk management decisions.

Concentrations of undeclared allergens in food products can reach levels that are relevant for public health.

Food products can become contaminated with food allergens due to cross-contact. Precautionary 'may contain' labelling may alert to the possible presence of an allergen, but guidance for such labelling is lacking. As a result, allergy information on the packaging may not be reliable and allergic consumers might be at risk of allergic reactions after consuming unlabelled, but indeed contaminated, products. Recently, a cow's milk protein allergic patient experienced a severe allergic reaction to a dark chocolate product containing undeclared milk proteins. This case induced the authors to investigate to what extent allergen concentrations of unlabelled products reach levels that are of public health relevance. The concentrations of milk proteins in the complaint sample and a collection of products of other batches and brands purchased from different stores were analysed. Together with appropriate threshold and food consumption data, the risks of allergic reactions and the severity of these reactions within the adult milk-allergic population were determined using probabilistic risk assessment techniques. The results show that milk protein concentrations in unlabelled products reach levels that may elicit allergic reactions in up to 68% of the adult allergic consumers. Therefore, concentrations of allergens in unlabelled products could reach levels that are of public health relevance. Application of probabilistic risk assessment can be an aid in revealing the public health consequences of undeclared allergens in food, in risk management decision-making and developing guidance in terms of risk-based concentration limits for precautionary labelling.

Approaches to risk assessment in food allergy: report from a workshop ''developing a framework for assessing the risk from allergenic foods".

A workshop was organised to investigate whether risk assessment strategies and methodologies used in classical/conventional toxicology may be used for risk assessment of allergenic foods, to discuss the advantages and limitations of different approaches and to determine the research needed to move the area forward. Three possible approaches to safety assessment and risk assessment for allergenic foods were presented and discussed: safety assessment using NOAEL/LOAEL and uncertainty factors, safety assessment using Benchmark Dose and Margin of Exposure (MoE), and risk assessment using probabilistic models. The workshop concluded that all the three approaches to safety and risk assessment of allergenic foods should continue to be considered. A particular strength of the MoE and probabilistic approaches is that they do not rely on low-dose extrapolations with its inherent issues. Probabilistic modelling is considered to be the most promising approach for use in population risk assessment (which is a particular focus for risk managers). For all approaches, further improvement of input data is desirable, particularly data on consumption patterns/food choices in food allergic consumers, data on minimum eliciting doses and data that can be used to evaluate whether the whole population at risk has been modelled accurately. Specific research topics were identified.

Prevention of work-related airway allergies; summary of the advice from the Health Council of the Netherlands.

The Health Council of the Netherlands published a report in which the best procedure and method for recommending health-based occupational exposure limits (OELs) for inhaled allergens were identified by evaluating the scientific state of the art. Many respiratory disorders in the workplace arise from inhalation of substances which can cause allergy. To protect workers against respiratory allergy, various preventive measures are taken, one of them being reduction of exposure by setting legally binding standards. These are based on health-based OELs that specify a level of exposure to an airborne substance, a threshold level, below which it may reasonably be expected that there is no risk of adverse health effects. The Council is of the opinion that an OEL should prevent against allergic sensitization, as sensitization plays a crucial biological role and is a prerequisite for the development of allergy. Furthermore, the Council considers it most likely that the exposure level below which no allergic sensitization develops for most allergens is so low, that OELs are difficult to set with the current knowledge and technical feasibilities. An alternative approach is to accept exposure, which carries a small predefined risk in developing allergic sensitization. In addition, it is worth considering periodic screening of exposed workers on allergic sensitization, because timely intervention can prevent worse. The feasibility of periodic screening and what else is needed to comply with the most important criteria, should however be judged case-by-case.

Probabilistic risk assessment model for allergens in food: sensitivity analysis of the minimum eliciting dose and food consumption.

Previously, TNO developed a probabilistic model to predict the likelihood of an allergic reaction, resulting in a quantitative assessment of the risk associated with unintended exposure to food allergens. The likelihood is estimated by including in the model the proportion of the population who is allergic, the proportion consuming the food and the amount consumed, the likelihood of the food containing an adventitious allergen and its concentration, and the minimum eliciting dose (MED) distribution for the allergen. In the present work a sensitivity analysis was performed to identify which parts of the model most influence the output. A shift in the distribution of the MED reflecting a more potent allergen, and an increase in the proportion of the population consuming a food, increased the number of estimated allergic reactions considerably. In contrast, the number of estimated allergic reactions hardly changed when the MEDs were based on a more severe response, or when the amount of food consumed was increased. Development of this work will help to generate a more accurate picture of the potential public health impact of allergens. It highlights areas where research is best focused, specifically the determination of minimum eliciting doses and understanding of the food choices of allergic individuals.

Risk assessment and food allergy: the probabilistic model applied to allergens.

In order to assess the risk of unintended exposure to food allergens, traditional deterministic risk assessment is usually applied, leading to inconsequential conclusions as 'an allergic reaction cannot be excluded'. TNO therefore developed a quantitative risk assessment model for allergens based on probabilistic techniques resulting in a more exhaustive risk assessment and more detailed information. By now, this approach is recognized as the future approach in allergen risk assessment. A case study (hazelnut proteins in chocolate spread) is presented as a proof of concept.

Transfer of chemicals from feed to animal products: The use of transfer factors in risk assessment.

The human risk assessment of feed contaminants has often been hampered by a lack of knowledge concerning their behaviour when consumed by livestock. To gain a better understanding of the transfer of contaminants from animal feed to animal products, a meta-analysis of public literature was made. Data concerning feed contaminant concentrations, feeding periods, residue levels in animal products, and other parameters were gathered and recorded. For each case a 'transfer factor', which was defined as the ratio of the concentration of a chemical in an animal product to the concentration of the chemical in animal feed, was calculated. Scientifically founded transfer factors were calculated and analysed for groups of chemicals based on their contaminant classes or physicochemical properties. These database-derived transfer factors enable a more accurate risk assessment in the case of a feed contamination, and enable rapid risk management decision-making and/or intervention.

[Food additives as a cause of medical symptoms: relationship shown between sulfites and asthma and anaphylaxis; results of a literature review]. / Voedseladditieven als oorzaak van medische klachten: alleen voor sulfiet verband met astma en anafylaxie aangetoond; resultaten van een literatuuronderzoek.

OBJECTIVE: To determine if a causal connection exists between food additives and various medical complaints. DESIGN: Literature study. METHOD: Medline over the period January 1966-January 1999 was searched for articles on the following substances not containing protein and lactose: monosodium glutamate (MSG), sulfites, azo-dyes (tartrazine, sunset yellow, azorubin, amarant, cochineal red), benzoates, sorbates, butylated hydroxyanisole/butylated hydroxytoluene (BHA/BHT), parabens, cinnamon and vanilla, in combination with key words regarding food and side effects. Of those studies purporting to demonstrate an effect, only double-blind randomized placebo-controlled studies with oral challenge were assessed further, unless the complaint was anaphylaxis. Of studies not demonstrating an effect the design was assessed. RESULTS: Only for sulfites as causative agents of asthma and anaphylaxis, methodologically adequate studies demonstrating a causal connection could be found. For azo-dyes, benzoates, MSG, sorbates and BHA/BHT, no link with medical symptoms was demonstrable. For parabens, cinnamon and vanilla there were insufficient or inadequate data to justify a conclusion.

Comparison of antibody responses to hen's egg and cow's milk proteins in orally sensitized rats and food-allergic patients.

BACKGROUND: No adequate enteral sensitization models are available to study food allergy and the allergenicity of food proteins. To further validate an enteral brown Norway (BN) rat sensitization model under development, we studied specific protein recognition to determine whether a comparable pattern of proteins is recognized by the rat immune system and the human immune system. METHODS: The animals were exposed to either ovalbumin as a positive reference control, hen's egg-white-protein extract, or a cow's milk preparation by daily gavage dosing (0.5, 1, 2.5, 5, 10, or 15 mg protein per rat/day) for 9 weeks. No adjuvants were used during the sensitization studies. The specificities of antibodies against hen's egg-white proteins or cow's-milk proteins in sera from orally sensitized rats and food-allergic patients were studied and compared by immunoblotting. RESULTS: The IgG and IgE antibodies to hen's egg-white proteins and cow's-milk proteins present in sera from orally sensitized rats and food-allergic patients showed a comparable pattern of protein recognition. CONCLUSIONS: Upon daily intragastric exposure to food allergens, the specificities of the induced antibody responses in the BN rat resemble those found in food-allergic patients. These studies add further support to the hypothesis that the BN rat may provide a suitable animal model for food allergy research and research on the allergenicity of food proteins.

Humoral and cellular immune responses in different rat strains on oral exposure to ovalbumin.

No adequate enteral sensitization models are available to study food allergy and allergenicity of food proteins. Using a previously described oral sensitization protocol to sensitize Brown Norway rats (BN) to food proteins, the influence of genetically-based strain-specific characteristics of the immune system on the outcome of oral sensitization studies was investigated. BN, Hooded Lister (HL), Piebald Virol Glaxo (PVG) and Wistar rats were daily administered 1 mg of ovalbumin (OVA) by gavage dosing for 42 days without the use of an adjuvants. The highest OVA-specific IgG responses were detected in the BN rats followed by Wistar, HL and PVG rats. OVA-specific IgE responses were only detectable in the BN rats. The cellular immune response was examined by determination of delayed-type hypersensitivity (DTH) reactions in the animals. The response was most pronounced in the HL and Wistar rats. PVG and BN rats showed comparable DTH responses but the responses were significantly weaker than those observed in HL and Wistar rats. It was concluded that the genetic make-up of different rat strains influences the outcome of oral sensitization studies. In addition, using the described oral sensitization protocol, the BN rat seems to be the most suitable strain for inducing oral sensitization.

An oral sensitization model in Brown Norway rats to screen for potential allergenicity of food proteins.

We developed an oral sensitization protocol for food proteins for the rat. Young Brown Norway (BN) rats were exposed to 1 mg ovalbumin (OVA) by daily gavage dosing for 42 days without the use of an adjuvant. OVA-specific IgE and IgG responses were determined by ELISA. On an oral challenge with OVA some clinical symptoms of food allergy-like effects on the respiratory system, blood pressure, and permeability of the gastrointestinal barrier were studied. In addition, BN rats were orally exposed to a total hen egg white protein (HEW) extract and cow's milk (CM) and the specificities of induced antibody responses were compared with the specificities of antibodies in sera from egg- and milk-allergic patients using immunoblotting. Animals orally exposed to the allergens developed specific IgE and IgG antibodies which recognized the same proteins compared with antibodies from egg- or CM-allergic patients. Among the various clinical symptoms of food allergy, gut permeability was increased after an oral challenge. In addition, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. The results obtained show that the Brown Norway rat is a suitable animal model for inducing specific IgG and IgE responses on daily intragastric dosing of OVA without the use of an adjuvant. Moreover, local immune-mediated effects on oral challenge are observed. The observation that enterally exposed BN rats and food-allergic patients demonstrate antibody responses to a comparable selection of proteins on exposure to different protein mixtures (HEW and CM) further supports the suitability of the BN rat as an animal model for food allergy research and for the study of the allergenicity of (novel) food proteins.

Immune-mediated effects upon oral challenge of ovalbumin-sensitized Brown Norway rats: further characterization of a rat food allergy model.

Although several in vivo antigenicity assays using parenteral immunization are operational, no full validated enteral models are available to study food allergy and allergenicity of food proteins. To further validate a developed enteral Brown Norway (BN) rat food allergy model, systemic and local immune-mediated reactions were studied upon oral challenges. The animals were exposed to ovalbumin (OVA) by daily gavage dosing (1 mg OVA/rat/day) for 6 weeks, without the use of an adjuvant, or by intraperitoneal injections with OVA together with AL(OH)3. Subsequently, effects on breathing frequency, blood pressure, and gastrointestinal permeability were investigated upon an oral challenge with 10 to 100 mg OVA in vivo. In both parenterally and orally sensitized rats, an increase in gut permeability (increased passage of beta-lactoglobulin as bystander protein) was determined between 0.5 and 1 h after an oral OVA challenge was given. An oral challenge with OVA did not induce a clear effect on the respiratory system or blood pressure in the majority of the animals. However, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. Upon oral challenge with OVA of orally and parenterally sensitized animals, local effects were observed in all animals whereas systemic effects were observed at a low frequency, which reflects the situation in food allergic patients after an oral challenge. These studies show that the BN rat provides a suitable animal model to study oral sensitization to food proteins as well as immune-mediated effects after oral challenge with food proteins.