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In silico methods are an integral part of the allergenicity risk assessment process, both for novel foods and newly expressed proteins in Genetically Modified Organisms (GMOs) for food use. They help to identify novel proteins that have the potential to cause cross-reactive food allergies in the existing allergic population. Many of the tools developed rely on databases of curated allergen sequences but the curation process does not necessarily consider whether an allergen is clinically relevant and plays a role in triggering an IgE-mediated reaction. In order to fill this gap in our knowledge, a systematic review of allergens identified in a range of foods is planned. The foods include those for which food allergen labelling is mandated in the European Union and the UK, other foods such as fruit, which are known to cause IgE-mediated food allergies in at least 0.5% of the European population and foods of low allergenicity such as rice. The approach taken includes a Population-Outcome (PO) approach in order to address the primary research question “What scientific knowledge (evidence) is there that clinical manifestation(s) of IgE-mediated allergic reaction(s) are caused by ingestion of a food?”. A modified Population-Exposure-Comparator-Outcome (PECO) approach will be taken to address the secondary research question “Which food protein molecules are recognised by serum-IgE from individuals allergic to foods (identified by addressing the primary question) and are responsible for causing an IgE-mediated adverse reaction to those foods?” A protocol for executing the systematic review has been developed together with grading criteria and risk of bias analysis. The research process will be fully documented to allow the search to be assessed and reproduced as per PRISMA guidelines.

Insects or insect-derived products have been used as feed for livestock industries for a long time because their highly nutritional value. With the rapid increase of the human population on this planet, an increasing amount of insects or insect derivatives has been applied as food. However, there are limited studies reporting the effect of insects and their components on human health, particularly on intestinal health. In this thesis, we investigated the interaction of insect-derived preparations, and insect derivative chitin, with intestinal in vitro models to identify potential beneficial health effects. We firstly studied the bactericidal impact of in vitro digested insect-derived fractions on the growth of Clostridium perfringens, revealing that insect-derived fractions can suppress the growth of C. perfringens of and this effect was attribute to the presence of antimicrobial peptides in the digestion of insect-derived fractions. Furthermore, we extend our research to investigate the effect of the digests on the intestinal immune barrier function using in vitro models to mimic small intestinal-like epithelial cells and residing macrophages. The results showed that digests of insect-derived fractions can reduce the barrier damage of bacterial toxins and modify immune responses of small intestinal-like Caco-2 cells. To extend on the immunomodulatory findings, we exposed a set of in vitro models representing the epithelium, immune cells and microbiota to pure chitin with different physicochemical properties. We observed that small chitin fractions led to production of a set of cytokines by macrophages via a multi-receptor and clathrin-mediated endocytosis. This immunomodulatory activity of chitin makes it a promising agent for subjects in need of immune support. Additionally, chitin has also been described to be beneficial for our health in a broader perspective. Therefore, we reviewed literature regarding the reported beneficial effects of chitin, and its derivati

Immune-globulin E (IgE)-mediated food allergy is characterized by a variety of clinical entities within the gastrointestinal tract, skin and lungs, and systemically as anaphylaxis. The default response to food antigens, which is antigen specific immune tolerance, requires exposure to the antigen and is already initiated during pregnancy. After birth, tolerance is mostly acquired in the gut after oral ingestion of dietary proteins, whilst exposure to these same proteins via the skin, especially when it is inflamed and has a disrupted barrier, can lead to allergic sensitization. The crosstalk between the skin and the gut, which is involved in the induction of food allergy, is still incompletely understood. In this review, we will focus on mechanisms underlying allergic sensitization (to food antigens) via the skin, leading to gastrointestinal inflammation, and the development of IgE-mediated food allergy. Better understanding of these processes will eventually help to develop new preventive and therapeutic strategies in children.

Tree nut-allergic individuals are often sensitised towards multiple nuts and seeds. The underlying cause behind a multi-sensitisation for cashew nut, hazelnut, peanut and birch pollen is not always clear. We investigated whether immunoglobulin E antibody (IgE) cross-reactivity between cashew nut, hazelnut and peanut proteins exists in children who are multi-allergic to these foods using a novel IMMULITE®-based inhibition methodology, and investigated which allergens might be responsible. In addition, we explored if an allergy to birch pollen might play a role in this co-sensitisation for cashew nut, hazelnut and peanut. Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to i

Dry heating of cow’s milk protein, as applied in the production of “baked milk”, facilitates the resolution of cow’s milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow’s milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

Proteins from cashew nut can elicit mild to severe allergic reactions. Three allergenic proteins have already been identified, and it is expected that additional allergens are present in cashew nut. pathogenesis-related protein 10 (PR10) allergens from pollen have been found to elicit similar allergic reactions as those from nuts and seeds. Therefore, we investigated the presence of PR10 genes in cashew nut. Using RNA-seq analysis, we were able to identify several PR10-like transcripts in cashew nut and clo

Heat processing is a common procedure in producing cow’s milk products in industry, amongst others to extend their shelf life. Thermal processing is reported to influence a protein’s structure and potentially its immunogenicity. This thesis describes the impact of various types of processing on structural characteristics of ß-lactoglobulin (BLG), as the major allergen in cow’s milk allergy, and on the immune response of antigen presenting cells (APCs) in particular. These cells were chosen as they are the first immune cells to come in contact with the exogenous food protein and subsequently initiate further immune responses, including the sensitization step of the allergic reaction. Next to BLG, thyroglobulin and lysozyme were studied in the thesis to estimate whether generally applicable conclusions could be drawn on the relationship between structural modifications of proteins and the subsequent immune responses.This thesis studied a comprehensive set of parameters that were considered to be relevant for this question, from a protein’s accessibility to intestinal APCs after processing, its physicochemical structural modifications to its uptake by APCs and further immune responses. To generate a range of structural modifications, including unfolding, aggregation and glycation, three different heating methods were used. Reducing saccharides were also included in the heating process to induce glycation, which quite commonly occurs in milk processing and is expected to be important for structural modifications and immunogenicity of the protein. The methods were wet-heating (W; heating in solution at 60 °C for 3 days) which led to aggregation of protein, high-temperature dry-heating (H; heating at 130 °C for 10 minutes) for advanced glycation and low-temperature dry-heating (L; heating at 50 °C for 9 hours) for mild glycation.In Chapter 2, the changes of the accessibility of BLG to intestinal immune cells after different processing methods was studied. The intestine is

Cashew nut allergy has been recognized as a severe tree nut allergy amongst (Dutch) children and young adults and its prevalence seems to be increasing. For clinical diagnosis of an allergy, it is essential to know the causative agents in the food product causing the allergic symptoms.In this thesis entitled ‘Cracking the cashew nut: strategies to identify and characterize novel allergens’, we aimed to apply innovative strategies and technologies to identify and characterize putative allergenic proteins in cashew nut, to broaden the current knowledge on cashew nut allergens beyond those already known (Ana o 1, Ana o 2 and Ana o 3). Our knowledge of cashew nut proteins that can trigger an allergic reaction is currently very limited, especially compared to other nuts or seeds in which the allergen repertoire has been researched much more widely. Using several different strategies, we evidenced that additional allergens must be present in cashew nuts, which presumably contribute to the elicitation of allergic symptoms in cashew nut allergic patients. Knowledge of newly identified cashew nut proteins provides a basis for further research to extend clinical diagnostic tests and treatments currently available for cashew nut allergy.Chapter 2 includes an opinion on the use of current in vivo and ex vivo endpoints in murine food allergy models and their suitability for evaluating the sensitizing capacity of protein concentrates and/or food products. An overview is given of the best predictive risk assessment methods and endpoint parameters currently relied on in in vivo food allergy models with a focus on milk, egg and peanut allergens, addressing their strengths and limitations for assessing sensitization risks. Findings indicated that, although the current available models are suitable for studying the pathophysiology of food allergy, they still couldn’t predict the magnitude of the allergic potential of a particular allergen. Thus, there is still a strong need to better

Immune-globulin E (IgE)-mediated food allergy is characterized by a variety of clinical entities within the gastrointestinal tract, skin and lungs, and systemically as anaphylaxis. The default response to food antigens, which is antigen specific immune tolerance, requires exposure to the antigen and is already initiated during pregnancy. After birth, tolerance is mostly acquired in the gut after oral ingestion of dietary proteins, whilst exposure to these same proteins via the skin, especially when it is inflamed and has a disrupted barrier, can lead to allergic sensitization. The crosstalk between the skin and the gut, which is involved in the induction of food allergy, is still incompletely understood. In this review, we will focus on mechanisms underlying allergic sensitization (to food antigens) via the skin, leading to gastrointestinal inflammation, and the development of IgE-mediated food allergy. Better understanding of these processes will eventually help to develop new preventive and therapeutic strategies in children.

Background: Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach: In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions: The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches. A© 2019 Elsevier Ltd

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1β, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot

Background: The double-blind, placebo-controlled food challenge test (DBPCFC) is the gold standard in cashew nut allergy. This test is costly, time consuming and not without side effects. Analysis of IgE reactivity to cashew nut components may reduce the need for food challenge tests. Methods: In a prospective and multicentre study, children with suspected cashew nut allergy underwent a DBPCFC with cashew nut. Specific IgE to cashew nut and to the components Ana o 1, 2 and 3 were determined. A skin prick test (SPT) with cashew nut extract was performed. The association between the outcome of the food challenge test and specific IgE to Ana o 1, 2 and 3 was assessed with logistic regression analyses, unadjusted and adjusted for other diagnostic variables. Discriminative ability was quantified with a concordance index (c). Results: A total of 173 children (103 boys, 60%) with a median age of 9 years were included. About 79% had a positive challenge test outcome. A steep rise in the risk of a positive challenge was observed for specific IgE to each individual component Ana o 1, 2 and 3 with estimated risks up to approximately 100%. Median values of Ana o 1, 2, 3 were 1.29 kU/l (range 0–100 kU/l), 4.77 kU/l (range 0–100 kU/l) and 8.33 kU/l (range 0–100 kU/l) respectively and varied significantly (p < 0.001). Specific IgE to Ana o 1, 2 and 3 was better distinguished between cashew-allergic and tolerant children (c = 0.87, 0.85 and 0.89, respectively) than specific IgE to cashew nut or SPT (c = 0.76 and 0.83, respectively). Conclusion: The major cashew nut allergens Ana o 1, 2 and 3 are each individually predictive for the outcome of food challenge tests in cashew-allergic children.

Background: Cashew nut, pistachio nut and mango belong to the Anacardiaceae family and are botanically related. Therefore, cashew nut sensitised children are frequently advised to eliminate cashew nuts and pistachio nuts from their diet. The ‘Improvement of Diagnostic mEthods for ALlergy assessment (IDEAL trial number NTR3572) study showed that cashew nut sensitised children were co-sensitised to pistachio nut in 98% of cases and to mango in 21% of cases. The aim of this follow-up study to IDEAL is to assess the clinical relevance of co-sensitisation to pistachio nut and mango in cashew nut sensitised children. Methods: Children were recruited from the study: ‘Improvement of Diagnostic mEthods for ALlergy assessment (IDEAL trial number NTR3572). Inclusion criterion for the IDEAL study was sensitization to cashew nut as demonstrated by either SPT or sIgE, and a clinical history of reactions to cashew nuts or no previous (known) exposure. Sensitized children who were tolerant to cashew nuts were excluded. Inclusion criterion for this IDEAL follow-up study was co-sensitization to pistachio nut, regardless the result of the DBPCFC with cashew nut. In this follow-up study a double-blind placebo-controlled food challenge with pistachio nut and an open food challenge with mango were performed. Results: Twenty-nine children (mean age of 11.6 years, 62% male) were included. Pistachio nut sensitisation was clinically relevant in only 34% of cashew-sensitised children and only 31% of cashew challenge positive children. None of the children was challenge positive to mango. Conclusion: Although co-sensitisation between cashew nut and pistachio nut was observed in 98%, pistachio nut sensitisation was only clinically relevant in 34% of the children. Therefore, a challenge test with pistachio nut is recommended in children with cashew nut and pistachio nut sensitisation.

Cashew nut allergy can be a severe food allergy of which the prevalence appears to be increasing. The aim of this thesis was a comprehensive molecular and serological characterisation of the cashew nut allergens Ana o 1, 2 and 3 for improved diagnosis and characterisation of patient populations. Chapter 1 in this thesis provides background information on cashew nuts, allergy, the allergens Ana o 1, 2 and 3, the effect of heat treatments on cashew nut proteins, the digestibility of cashew nut proteins, cross-reactivity between cashew nut proteins and other tree nuts, and the detection of cashew nut in food products. Subsequently, in Chapter 2, a review is presented on the topic of epithelial protein and allergen transport. This review describes multiple pathways of intestinal protein transport, sums up existing experimental data concerning protein and peptide transport, and presents different methods to study this. Interestingly, the pathway of (allergenic) protein transport can differ between sensitized and non-sensitized persons. In sensitized persons, protein transport occurs transcellularly via enterocytes, and paracellularly with the involvement of mast cells, while in non-sensitized persons microfold cells and enterocytes are considered most important. In the next three chapters, cashew nut allergens were studied. Cashew nut allergy and cashew nut allergens were chosen because of a high number of undiagnosed cashew nut allergic children reported at the children’s hospital “Kinderhaven”, in Rotterdam, an outpatient clinic that is involved in this study. Chapter 3 describes a protocol for the purification of Ana o 1, 2 and 3 from cashew nuts. Ana o 1 and 3 were purified by protein extraction, salt precipitation and filtering over a 30kDa molecular weight membrane. Ana o 2 was purified by protein extraction followed by gel filtration chromatography. These purified proteins were characterised by SDS-PAGE, western blot, glycoprotein stain, and protein identification

Non-digestible polysaccharides (NDPs) are considered as important ingredients to support health. Among these health effects, immunomodulatory effects raised interests in the past decade. The intestine is the primary organ that interact with NDPs. The intestinal epithelial cells (IECs) form a dynamic physical barrier and together with associated immune cells determine for a large part our immune homeostasis. Studying the direct interaction between NDPs and intestinal and immune cells could help us to uncover the mechanism by which NDPs exert immunomodulatory effects and how NDPs can differ in this activity. In this thesis, we investigated the immunomodulatory effects of NDPs through interaction with intestinal immune cells using in vitro methods in order to characterise the NDPs and preselect NDPs with differential activity for further in vivo evaluations. The intestinal immune barrier is formed by various IECs and immune cells, which are introduced and their specific functions discussed in Chapter 1. NDPs could interact directly with both IECs and immune cells that sample in or from the lumen. The majority of IECs are enterocytes and most relevant immune cells responsible for sampling in the lumen have been characterised as macrophages, which leads us to focus on these cell types by in vitro approaches. In addition, basic information on NDPs and current status on health effects of NDPs both in vitro and in vivo are discussed. In Chapter 2, the direct response of IEC to NDPs stimulation was investigated. IECs form the largest surface of the body that, with a crucial role as barrier also, perform a role in signalling towards immune cells. We used 21-day transwell cultured Caco-2 to resemble the small intestinal enterocytes that form largest part of this intestinal layer. We first characterized the chemical composition of five NDPs which revealed different mono sugar composition, linkages of backbone and side chains and a wide range of MW (from 17 KDa to 2100 KDa). The

Background: The double-blind, placebo-controlled food challenge test (DBPCFC) is the gold standard in cashew nut allergy. This test is costly, time consuming and not without side effects. Analysis of IgE reactivity to cashew nut components may reduce the need for food challenge tests. Methods: In a prospective and multicentre study, children with suspected cashew nut allergy underwent a DBPCFC with cashew nut. Specific IgE to cashew nut and to the components Ana o 1, 2 and 3 were determined. A skin prick test (SPT) with cashew nut extract was performed. The association between the outcome of the food challenge test and specific IgE to Ana o 1, 2 and 3 was assessed with logistic regression analyses, unadjusted and adjusted for other diagnostic variables. Discriminative ability was quantified with a concordance index (c). Results: A total of 173 children (103 boys, 60%) with a median age of 9 years were included. About 79% had a positive challenge test outcome. A steep rise in the risk of a positive challenge was observed for specific IgE to each individual component Ana o 1, 2 and 3 with estimated risks up to approximately 100%. Median values of Ana o 1, 2, 3 were 1.29 kU/l (range 0-100 kU/l), 4.77 kU/l (range 0-100 kU/l) and 8.33 kU/l (range 0-100 kU/l) respectively and varied significantly (p <0.001). Specific IgE to Ana o 1, 2 and 3 was better distinguished between cashew-allergic and tolerant children (c = 0.87, 0.85 and 0.89, respectively) than specific IgE to cashew nut or SPT (c = 0.76 and 0.83, respectively). Conclusion: The major cashew nut allergens Ana o 1, 2 and 3 are each individually predictive for the outcome of food challenge tests in cashew-allergic children.

Background: Cashew nut, pistachio nut and mango belong to the Anacardiaceae family and are botanically related. Therefore, cashew nut sensitised children are frequently advised to eliminate cashew nuts and pistachio nuts from their diet. The ‘Improvement of Diagnostic mEthods for ALlergy assessment (IDEAL trial number NTR3572) study showed that cashew nut sensitised children were co-sensitised to pistachio nut in 98% of cases and to mango in 21% of cases. The aim of this follow-up study to IDEAL is to assess the clinical relevance of co-sensitisation to pistachio nut and mango in cashew nut sensitised children. Methods: Children were recruited from the study: ‘Improvement of Diagnostic mEthods for ALlergy assessment (IDEAL trial number NTR3572). Inclusion criterion for the IDEAL study was sensitization to cashew nut as demonstrated by either SPT or sIgE, and a clinical history of reactions to cashew nuts or no previous (known) exposure. Sensitized children who were tolerant to cashew nuts were excluded. Inclusion criterion for this IDEAL follow-up study was co-sensitization to pistachio nut, regardless the result of the DBPCFC with cashew nut. In this follow-up study a double-blind placebo-controlled food challenge with pistachio nut and an open food challenge with mango were performed. Results: Twenty-nine children (mean age of 11.6 years, 62% male) were included. Pistachio nut sensitisation was clinically relevant in only 34% of cashew-sensitised children and only 31% of cashew challenge positive children. None of the children was challenge positive to mango. Conclusion: Although co-sensitisation between cashew nut and pistachio nut was observed in 98%, pistachio nut sensitisation was only clinically relevant in 34% of the children. Therefore, a challenge test with pistachio nut is recommended in children with cashew nut and pistachio nut sensitisation.