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Background: The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). Main body: The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. Conclusion: The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs. © 2017 The Author(s).

Background Allergic diseases are increasing world-wide, and according to the hygiene hypothesis may be related to a decreased exposure to environmental bacteria. Probiotic bacteria are recognized for their immunomodulating properties, and may benefit allergy patients. In vitro studies reveal immunomodulatory effects that are strain dependent. Differential immunomodulatory in vitro capacities cannot be extrapolated directly to in vivo efficacy. Thus, in vitro screening should preferably be followed by a comparative analysis of the selected immunomodulatory strains in an in vivo setting. Objective We selected five Lactobacillus strains on their IL-10-inducing capacity, and evaluated the immunomodulatory properties in birch-pollen-allergic subjects outside the hayfever season, with a reduction of IL-13 as the primary outcome. Methods A double-blind, placebo-controlled parallel study was performed in which 62 subjects with a proven birch-pollen allergy consumed one of five different probiotic yoghurts containing four Lactobacillus plantarum strains and one Lactobacillus casei strain or a placebo yoghurt. Blood samples were collected at the start and after 4 weeks. Several immune parameters were determined in serum and peripheral blood mononuclear cell cultures (PBMC) derived from these subjects. Results A decrease in birch-pollen-specific IgE was found for four probiotic strains. L. casei Shirota reduced the number of CD16+/CD56+ cells in peripheral blood mononuclear cells. For strain L. plantarum CBS125632, the decrease in IgE coincided with significant decreases in IL-5 and IL-13 production by aCD3/aCD28-stimulated PBMC cultures. Conclusion and Clinical Relevance Subjects with seasonal allergy can be used to determine immunomodulatory responses outside the pollen season within a 4-week study period. L. plantarum CBS125632 decreased several immune markers related to allergy, and may have the potential to alleviate the severity of seasonal allergy symptoms

The objective of this study was to assess the potential immunomodulatory effect of six Lactobacillus strains on human peripheral blood mononuclear cells (hPBMC) isolated from allergic patients. hPBMC from patients allergic to birch pollen or grass pollen were cultured in vitro in the presence or absence of selective bacterial strains. Cultures were left unstimulated or stimulated with aCD3/aCD28 or Bet v 1. After 1, 4 and 8 days, cells and culture supernatants were harvested and the effect on cellular proliferation and the supernatant levels of several cytokines was assessed. All strains had the ability to repress IL-13 production but did show a differential effect on IFN-¿ induction. Both strains B223 and B1697 showed a lower IFN-¿, IL-12 and TNF-a induction as compared with the other tested strains. Strain B633 showed the best proliferation-suppressive properties in aCD3/aCD28-stimulated cells. Suppression of the T-helper type 2 (Th2) cytokine induction and induction of the Th1 cytokine production by specific strains might be beneficial for allergic patients having a disturbed Th1/Th2 immune balance. Furthermore, hPBMC of patients with seasonal allergy outside the pollen season can be used to determine the immunomodulatory activities of probiotic bacteria

Allergic diseases such as allergic rhinitis, allergic asthma, atopic eczema and food allergy have become an increasing health problem world-wide, affecting between 20-30% of the total population. Peanut allergy (prevalence ~1%) is a common and persistent food allergy accounting for severe allergic reactions. Peanuts are often consumed after thermal processing (e.g. boiling, roasting) which can alter the protein structure and change its immunoreactivity and allergenicity. In vitro diagnostic testing, however, is generally performed using the native, unprocessed protein and more knowledge on the effect of processing on allergens is necessary to improve these diagnostic procedures. In addition, rationally designed processing could also lead to reduction of the allergen content in certain products and therefore be an effective food technological approach in allergy management. Another approach in allergy management is the use of immunomodulating foods, such as probiotics. There are indications that probiotics, e.g. specific lactic acid bacteria, could be beneficial for many conditions, including different clinical expressions of allergy. Chapter 1 gives an overview of several aspects of allergy with a focus on food allergy. Firstly the basic mechanism and the involved immune cells are discussed, after which the prevalence of food allergy in the context of the EuroPrevall project is described. Different food allergens are discussed with an emphasis on the allergens from peanut and different methods are described to assess the potential allergenicity of proteins under widely used processing conditions, including heating and the Maillard reaction. Lastly, different methods to prevent or treat allergies are discussed with a special emphasis on immunomodulation by lactic acid bacteria. This introduction chapter is concluded with the research aim and thesis outline. Section 1: Influence of processing on allergenicity of proteins In our first study, described in Chapter 2, Ara h 2/6 was purified from raw peanut and heated in solution (boiling) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanuts for comparison. Structural changes, the capacity to induce cell proliferation and cytokine production, and IgE-binding and IgE cross-linking capacity were evaluated. Although no effect of processing on T-cell reactivity was observed, heat-induced denaturation reduced the IgE-binding and cross-linking capacity. Interestingly, the soluble fraction of the Ara h 2/6 isolated from roasted peanuts retained the conformation and allergenic activity of the native protein. In Chapter 3 similar methods were used to assess the effect of heating and glycation on Ara h 1. Heating in solution, irrespective of their level of glycation, resulted in formation of aggregates having reduced IgE-binding and cross-linking capacity, while T-cell reactivity was retained. The soluble fraction of Ara h 1 isolated from roasted peanuts appeared to be highly denatured, formed more globular and smaller aggregates, and showed no evidence of glycation. However, these smaller aggregates retained IgE-binding capacity, unlike the aggregates formed after heating and glycating purified Ara h 1. These results could account for observed differences between boiled and roasted peanuts and suggest that other modifications than the Maillard reaction affect the allergenicity of Ara h 1. As peanuts are often consumed after roasting, the wet-thermal processing procedures, employed in the two previous described studies, were related to the effect of thermal treatment and Maillard reaction under low moisture conditions, which is described in Chapter 4. The extensive heating at low moisture resulted in hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble Ara h 1 formed large aggregates. Thermally treated Ara h 2/6 had both a lower IgE-binding and degranulation capacity compared to the native form, and the presence of glucose during heating partly counteracted both the decrease in IgE-binding and degranulation capacity. The IgE-binding capacity of Ara h 1 was also decreased; however, the basophil degranulation capacity increased significantly. This demonstrates the importance of including degranulation assays in addition to IgE-binding assays, when assessing allergenic potency of allergens. In addition, we here propose a role for large aggregates in the increased IgE-cross-linking capacity of individual allergens. Chapter 5 describes the effect of glycation on the immunoreactivity and basophil degranulation capacity of Cor a 11, the 7S globulin from hazelnut (and thus a homologue of Ara h 1). Three processing methods (heating at low moisture content at 37, 60 and 145°C) resulted in proteins with increasing degrees of glycation. Glycation at 37°C did not influence the specific IgG or IgE binding, while both were decreased after heating at 60°C and 145°C. However, heating at 145°C in the absence or presence of glucose resulting in the formation of aggregated structures, increased the basophil degranulation capacity of Cor a 11 using sera high in Cor a 11 specific IgE, but not when using sera from peanut allergic patients low in Cor a 11 specific IgE. Therefore, this study besides showing the importance of the use of a combination of tests also indicated the importance of using well-characterized sera as a source of IgE. In Chapter 6 we focused on the clinical features of all our clinically well-defined peanut allergic patients of which immune cells and sera were used for the previously described studies. In addition, soy allergic patients were included and an extensive IgE profile was determined for all patients. Gly m 4 (Bet v 1 homologue from soy) sensitization was suggested to be an important indicator of severe soy allergy in the soy allergic patients, while in peanut allergic patients sensitization to allergens from soy and pea extract nor Gly m 5 and 6 was found to have a good diagnostic specificity. This is likely due to the presence of clinically non-relevant cross-reactivity between peanut-specific IgE and homologues soy and pea components. Section 2: Immunomodulation by Lactobacillus strains In the first in vitro study, described in Chapter 7, initially 51 Lactobacillus strains were screened of which 8 were selected and tested for their immunomodulating effects on peripheral blood mononuclear cells (PBMC) of healthy donors. All tested Lactobacillus strains were capable of inducing the production of IL-1β, IL-10, IFN-γ and TNF-α. Clear strain-specific effects were observed with L. plantarum strains showing significantly higher induction capacity of IFN-γ, IL-12 and TNF-α compared with L. acidophilus strains. We therefore concluded that especially L. plantarum strains are promising candidates in IgE-mediated allergy by their stimulation potential of the T-cell response toward a putative Th1 response. As healthy subjects, in contrast to allergic individuals, are assumed to finely regulate the Th1/Th2 balance by inducing sufficient Treg cell activity, immunomodulatory effects of six selected Lactobacillus strains were investigated on PBMC of pollen-allergic patients in Chapter 8. All strains could modulate PBMC to induce innate cytokine production and in addition, all strains had the ability to repress IL-13 production. Again a differential effect on IFN-γ and IL-12 induction was observed. In addition, one strain could extensively suppress proliferation induced by anti-CD3/anti-CD28 stimulation. Specific strains that were able to suppress the Th2 cytokine induction and induce Th1 cytokines might be beneficial for allergic patients. Effects found in vitro cannot directly be extrapolated to in vivo and therefore, in Chapter 9, we performed an in vivo screening including five Lactobacillus strains. Blood samples were collected before and after a 4-week intervention with probiotics from all 62 birch-pollen-allergic patients included. Four strains caused a decrease in birch-pollen-specific IgE and for one specific strain this coincided with significant decreases in IL-5 and IL-13 and an increase in IL-10 production by anti-CD3/anti-CD28 stimulated PBMC cultures and might therefore have the potential to alleviate seasonal allergy symptoms. The last chapter, Chapter 10, gives an overview of the most important results of this thesis and discusses the research limitations and future research perspectives. We hypothesize the role of protein aggregation in allergenicity and we elaborate on the importance of a proper stepwise approach to realize selection of a proper lactic acid strain for in vivo human testing. In conclusion, this thesis showed that processing effects can have profound and specific effects on the structure and the allergenicity of relevant allergens. However, to test putative effects on allergenicity, IgE-binding tests only are not sufficient and mediator release assays are important to include, particularly when testing aggregated proteins. These results might have consequences for the proper diagnosis of food allergy in daily practice. Finally, as effects of lactic acid bacteria are strain specific, a proper pre-selection of candidate strains is important to choose the most promising strains for clinical testing. In our in vivo screening, one strain, L. plantarum CBS125632, was found to be promising because of its desired immunomodulatory activity to test in a follow-up trial to reduce symptoms of birch-pollen allergy.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins

Initially the resistance to digestion of two cow's milk allergens, β-casein, and β-lactoglobulin (β-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both β-casein and β-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of β-casein digestion in the low-protease assay were slower, β-Lg being pepsin resistant. During duodenal digestion, β-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins. © 2009 Elsevier Inc.

Background: A major drawback of oral immunotherapy for food allergy is the possibility of severe side-effects. We assessed both safety and efficacy of a low allergenic hydrolysed egg (HydE) preparation used in a double-blind placebo-controlled randomized study in egg allergic children. Methods: In a pilot multicentre study, 29 egg allergic patients (aged 1–5.5 years) were administered daily for 6 months 9 g HydE or placebo in a blinded, randomized manner. Safety was verified by oral food challenge to assess tolerance towards HydE at the start and efficacy by an open oral food challenge (OFC, primary outcome) at the end. Additionally, changes in basophil activation and specific IgE and IgG4 were assessed. Results: All egg allergic patients randomized to HydE (n = 15) tolerated the full dose at day 1 and received the maintenance dose from the start at home. No statistically significant difference was observed on the final OFC (36% and 21% had a negative OFC in the treatment and placebo groups, respectively). Specific IgG4 levels increased, while both CD203c+ and CD63+ basophils decreased significantly more over time in the treatment than in the placebo group. Conclusions: HydE can be regarded as a safe, low allergenic product to use in children allergic to egg. Although not significant, HydE given for 6 months increased numerically the proportion of patients becoming tolerant to egg. HydE induced a modulation of the immune response towards better tolerance. A longer treatment period and/or a higher dose may improve the clinical outcome and should be evaluated. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd