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As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier. [ABSTRACT FROM AUTHOR]

Glycosylation is an important post-translational modification of proteins, contributing to protein function, stability and subcellular localization. Fungal immunomodulatory proteins (FIPs) are a group of small proteins with notable immunomodulatory activity, some of which are glycoproteins. In this study, the impact of glycosylation on the bioactivity and biochemical characteristics of FIP-nha (from Nectria haematococca) is described. Three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were constructed and expressed in Pichia pastoris to study the functionality of the specific N-glycosylation on amino acid N5 and N39. Their protein characteristics, structure, stability and activity were tested. WT and mutants all formed tetramers, with no obvious difference in crystal structures. Their melting temperatures were 82.2 °C (WT), 81.4 °C (N5A), 80.7 °C (N39A) and 80.1 °C (N5+39A), indicating that glycosylation improves thermostability of rFIP-nha. Digestion assays showed that glycosylation on either site improved pepsin resistance, while 39N-glycosylation was important for trypsin resistance. Based on the 3D structure and analysis of enzyme cleavage sites, we conclude that glycosylation might interfere with hydrolysis via increasing steric hindrance. WT and mutants exerted similar bioactivity on tumor cell metabolism and red blood cells hemagglutination. Taken together, these findings indicate that glycosylation of FIP-nha impacts its thermostability and digestion resistance. [ABSTRACT FROM AUTHOR]

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell‐based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1‐3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo‐ and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2‐5). [ABSTRACT FROM AUTHOR]

Background: 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. Methods: Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to inhibition immunoassays using the IMMULITE® 2000 XPi. Serum-specific IgE (sIgE) to seed storage allergens and pathogenesis-related protein 10 (PR10) allergens were determined and used for molecular multicomponent allergen correlation analyses with observed clinical symptoms and obtained inhibition data. Results: IgE cross-reactivity was observed in all patients. Hazelnut extract was a strong inhibitor of cashew nut sIgE (46.8%), while cashew nut extract was less able to inhibit hazelnut extract (22.8%). Peanut extract showed the least inhibition potency. Moreover, there are strong indications that a birch pollen sensitisation to Bet v 1 might play a role in the observed symptoms provoked upon ingestion of cashew nut and hazelnut. Conclusions: By applying an adjusted working protocol, the IMMULITE® technology can be used to perform inhibition assays to determine the risk of sIgE cross-reactivity between very different food components. [ABSTRACT FROM AUTHOR]

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 clone six putative PR10 genes. In addition, PR10 protein expression in raw cashew nuts was confirmed by immunoblotting and liquid chromatography–mass spectrometry (LC–MS/MS) analyses. An in silico allergenicity assessment suggested that all identified cashew PR10 proteins are potentially allergenic and may represent three different isoallergens. [ABSTRACT FROM AUTHOR]

Significant efforts are necessary to introduce new dietary protein sources to feed a growing world population while maintaining food supply chain sustainability. Such a sustainable protein transition includes the use of highly modified proteins from side streams or the introduction of new protein sources that may lead to increased clinically relevant allergic sensitization. With food allergy being a major health problem of increasing concern, understanding the potential allergenicity of new or modified proteins is crucial to ensure public health protection. The best predictive risk assessment methods currently relied on are in vivo models, making the choice of endpoint parameters a key element in evaluating the sensitizing capacity of novel proteins. Here, we provide a comprehensive overview of the most frequently used in vivo and ex vivo endpoints in murine food allergy models, addressing their strengths and limitations for assessing sensitization risks. For optimal laboratory‐to‐laboratory reproducibility and reliable use of predictive tests for protein risk assessment, it is important that researchers maintain and apply the same relevant parameters and procedures. Thus, there is an urgent need for a consensus on key food allergy parameters to be applied in future food allergy research in synergy between both knowledge institutes and clinicians. [ABSTRACT FROM AUTHOR]

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 technique. Allergen cross-reactivity patterns between Anacardiaceae species were subsequently examined by SDS-PAGE and immunoblot inhibition, and IgE-reactive allergens were identified by LC-MS/MS. Results: From the 56 subjects analysed, 36 were positive on dot blot for cashew nut (63%). Of these, 50% were mono-sensitised to cashew nuts, 19% were co-sensitised to Anacardiaceae species, and 31% were co-sensitised to tree nuts. Subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. In pink peppercorn, putative albumin- and legumin-type seed storage proteins were found to cross-react with serum of cashew nut-sensitised subjects in vitro. In addition, a putative luminal binding protein was identified, which, among others, may be involved in cross-reactivity between several Anacardiaceae species. Conclusions: Results demonstrate the in vitro presence of IgE cross-sensitisation in children towards multiple Anacardiaceae species. In this study, putative novel allergens were identified in cashew, pistachio, and pink peppercorn, which may pose factors that underlie the observed cross-sensitivity to these species. The clinical relevance of this widespread cross-sensitisation is unknown. [ABSTRACT FROM AUTHOR]

Abstract: The protein content and allergen composition was studied of cashews from 8 different origins (Benin, Brazil, Ghana, India, Ivory Coast, Mozambique, Tanzania, Vietnam), subjected to different in‐shell heat treatments (steamed, fried, drum‐roasted). On 2D electrophoresis, 9 isoforms of Ana o 1, 29 isoforms of Ana o 2 (11 of the acidic subunit, 18 of the basic subunit), and 8 isoforms of the large subunit of Ana o 3 were tentatively identified. Based on 1D and 2D electrophoresis, no difference in allergen content (Ana o 1, 2, 3) was detected between the cashews of different origins (P > 0.5), some small but significant differences were detected in allergen solubility between differently heated cashews. No major differences in N‐ and C‐terminal microheterogeneity of Ana o 3 were detected between cashews of different origins. Between the different heat treatments, no difference was detected in glycation, pepsin digestibility, or IgE binding of the cashew proteins. [ABSTRACT FROM AUTHOR]

Food allergy is a major health problem of increasing concern. The insufficiency of protein sources for human nutrition in a world with a growing population is also a significant problem. The introduction of new protein sources into the diet, such as newly developed innovative foods or foods produced using new technologies and production processes, insects, algae, duckweed, or agricultural products from third countries, creates the opportunity for development of new food allergies, and this in turn has driven the need to develop test methods capable of characterizing the allergenic potential of novel food proteins. There is no doubt that robust and reliable animal models for the identification and characterization of food allergens would be valuable tools for safety assessment. However, although various animal models have been proposed for this purpose, to date, none have been formally validated as predictive and none are currently suitable to test the allergenic potential of new foods. Here, the design of various animal models are reviewed, including among others considerations of species and strain, diet, route of administration, dose and formulation of the test protein, relevant controls and endpoints measured. [ABSTRACT FROM AUTHOR]

Approximately 70% of birch pollen allergic patients in Europe experience hypersensitivity reactions to Immunoglobulin E (IgE) cross-reactive food sources. This so-called pollen-food syndrome (PFS) is defined by allergic symptoms elicited promptly by the ingestion of fruits, nuts, or vegetables in these patients. So far, in the literature, less attention has been given to Bet v 1 cross-reactive symptoms caused by pear (Pyrus communis). In the Netherlands, pears are widely consumed. The primary objective of this study was to measure the type and severity of allergic symptoms during pear challenges in birch pollen allergic patients, with a positive history of pear allergy, using two different pear varieties. Fifteen patients were included, skin prick test (SPT), prick-to-prick test (PTP), specific Immunoglobulin E (sIgE), and single-blind oral challenges were performed with two pear (Pyrus communis) varieties: the 'Cepuna' (brand name Migo®) and the 'Conference' pears. All patients were sensitized to one or both pear varieties. A total of 12 out of 15 participants developed symptoms during the 'Cepuna' food challenge and 14/15 reacted during the 'Conference' challenge. Challenges with the 'Cepuna' pears resulted in less objective symptoms (n = 2) in comparison with challenges with 'Conference' pears (n = 7). Although we did not find significance between both varieties in our study, we found a high likelihood of fewer and less severe symptoms during the 'Cepuna' challenges. Consequently selected pear sensitized patients can try to consume small doses of the 'Cepuna' pear outside the birch pollen season. [ABSTRACT FROM AUTHOR]

Abstract: The human sweet taste receptor is a heterodimer comprised of the class C G protein‐coupled receptor (GPCR) subunits TAS1R2 and TAS1R3. A wide collection of sweet tasting compounds and modulators of sweet taste interact with this receptor. Although TAS1R2/TAS1R3‐mediated signaling is well‐studied, the molecular basis for its desensitization remains unclear while such knowledge would signify a profound step forward in understanding the mechanism behind sweet taste perception and taste modulation. In this work, the possible involvement of β‐arrestin in downstream signaling was investigated. A stable clonal Human Embryonic Kidney (HEK)‐derived cell line containing the PathHunter™ GPCR technology was developed, in which β‐arrestin‐mediated endosomal receptor internalization can be monitored by ligand‐induced enzyme complementation of β‐galactosidase (β‐gal). Stimulatory responses and antibody‐specific receptor detection indicated that the TAS1R2/TAS1R3 receptor is endogenously expressed in this clonal cell line. Natural sugars (including fructose, glucose, sucrose, maltose, maltitol and mannitol) and artificial sweeteners (acesulfame‐K and sucralose) stimulated enzyme complementation activity in a concentration dependent manner. Besides, we observed that the assay detected modification of sugar induced cell responses by sweetness enhancers. These results combined implicate that TAS1R2/TAS1R3 receptor desensitization by internalization is most likely mediated by β‐arrestin‐induced endocytosis. This assay approach, making use of naturally expressed TAS1R2/TAS1R3 receptors and required co‐factors, further allows effective screening for and development of novel high potency non‐caloric sweeteners, sweet taste modulators or optimal blends with enhanced sweet taste. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the immunological relevance of modifications of food protein structure due to thermal processing. We investigated the uptake of β-lactoglobulin, treated with 3 different processing methods, by THP-1 macrophages: wet heating (60 °C in solution) and high- or low-temperature (130 °C or 50 °C, respectively) dry heating, combined with either of 8 types of saccharides or without saccharide. The processing method that was applied significantly affected the uptake of BLG by THP-1 macrophages, while the type of saccharide only had an influence in high-temperature dry heated samples. A set of physicochemical parameters of processed samples was determined, to determine the samples' molecular weight, hydrophobicity, amyloid-like structure, surface charge and secondary structure. Analysis of protein structure alterations indicated the uptake to be linked to the wet heating processing method and percentage of α-helix structure, amyloid-like structures, polymers, and hydrophobicity. We hypothesize that both amyloid-like structures and molecular weight were related to the increased hydrophobicity and therefore postulate that the exposure of hydrophobic regions is the leading physicochemical characteristic for the observed uptake of wet heated BLG samples by THP-1 macrophages. This work demonstrates how differential thermal processing of foods, through protein modification, can have an impact on its interaction with the immune system. Unlabelled Image • The physico-chemical properties of β-lactoglobulin, under 3 different thermal processing conditions, were compared. • Hydrophobicity, aggregation and amyloid-like structure, appeared to promote the uptake of β-lactoglobulin. • The type of saccharide used for glycation has less influence on the measured properties of β-lactoglobulin than expected. • Uptake of β-lactoglobulin by THP-1 macrophages was related to structural changes. [ABSTRACT FROM AUTHOR]

Ever since the implementation of microfluidics in the biomedical field, in vitro models have experienced unprecedented progress that has led to a new generation of highly complex miniaturized cell culture platforms, known as Organs-on-a-Chip (OoC). These devices aim to emulate biologically relevant environments, encompassing perfusion and other mechanical and/or biochemical stimuli, to recapitulate key physiological events. While OoCs excel in simulating diverse organ functions, the integration of the immune organs and immune cells, though recent and challenging, is pivotal for a more comprehensive representation of human physiology. This comprehensive review covers the state of the art in the intricate landscape of immune OoC models, shedding light on the pivotal role of biofabrication technologies in bridging the gap between conceptual design and physiological relevance. The multifaceted aspects of immune cell behavior, crosstalk, and immune responses that are aimed to be replicated within microfluidic environments, emphasizing the need for precise biomimicry are explored. Furthermore, the latest breakthroughs and challenges of biofabrication technologies in immune OoC platforms are described, guiding researchers toward a deeper understanding of immune physiology and the development of more accurate and human predictive models for a.o., immune-related disorders, immune development, immune programming, and immune regulation., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Fungal immunomodulatory proteins (FIPs) are a group of proteins found in fungi, which are extensively studied for their immunomodulatory activity. Currently, more than 38 types of FIPs have been described. Based on their conserved structure and protein identity, FIPs can be classified into five subgroups: Fve-type FIPs (Pfam PF09259), Cerato-type FIPs (Pfam PF07249), PCP-like FIPs, TFP-like FIPs, and unclassified FIPs. Among the five subgroups, Fve-type FIPs are the most studied for their hemagglutinating, immunomodulating, and anti-cancer properties. In general, these small proteins consist of 110-125 amino acids, with a molecular weight of ~13 kDa. The other four subgroups are relatively less studied, but also show a noticeable influence on immune cells. In this review, we summarized the protein modifications, 3-dimensional structures and bioactivities of all types of FIPs. Moreover, structure-function relationship of FIPs has been discussed, including relationship between carbohydrate binding module and hemagglutination, correlation of oligomerization and cytokine induction, relevance of glycosylation and lymphocyte activation. This summary and discussion may help gain comprehensive understanding of FIPs' working mechanisms and scope future studies., (Copyright © 2020 Liu, Bastiaan-Net and Wichers.)

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 clone six putative PR10 genes. In addition, PR10 protein expression in raw cashew nuts was confirmed by immunoblotting and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. An in silico allergenicity assessment suggested that all identified cashew PR10 proteins are potentially allergenic and may represent three different isoallergens., (© 2020 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Exposure of food products to small-intestinal-like Caco-2 cells, combined with a gene expression based response analysis can be a valuable tool to classify potential bioactive effects of food homogenates. In order to study changes in gene expression upon food exposure, a robust set of stably expressed genes is required for normalization. Here we present a set of reference genes suitable for RT-qPCR that has been validated for exposure studies with the intestinal-like Caco-2 cell line. This study identified ribosomal phosphoprotein P0 (RPLP0) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as best reference genes. The set can be extended with β-2-microglobulin (B2M), splicing factor 3A, subunit 1 (SF3A1), and mitochondrial ribosomal protein L19 (MRPL19). Food homogenates did provoke responses in the Caco-2 cells, as was demonstrated by changed expression of NAD(P)H Quinone dehydrogenase 1 (NQO1), Claudin 4 (CLDN4), Nitric Oxide Synthase 2 (NOS2), and ATP-binding cassette, subfamily B, member 1 (ABCB1) in the same experiment. Results indicate that: i) natural food homogenates can exert effects in Caco-2 cells, and ii) stability in expression of the reference genes is not due to a lack of response of the Caco-2 cells.