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Fungal immunomodulatory proteins (FIPs) have been investigated for their use as potential natural derived anti-tumor molecules. However, the stability of FIPs is critical for their preparation and storage. In this study, the correlation between thermal stability and protein structural features of rFIP-nha, with significant anti-tumor activity, has been evaluated. For comprehensive analysis, FIP-nha and its homologues FIP-gmi, FIP-fve, and LZ-8 were all recombinantly expressed in E. coli. In solution, rFIP-nha and rFIP-gmi formed tetramers; rFIP-fve and rLZ-8 appeared as dimers. Their melting temperatures were 85.1 °C, 77.8 °C, 66.5 °C, and 64.4 °C, respectively. Accordingly, their cytotoxicity was also temperature dependent. To investigate the underlying mechanism of their thermostability, we solved the crystal structure of FIP-nha. Detailed structure analysis, molecular dynamic simulation and mutagenesis studies indicated that a higher thermostability was correlated to higher oligomerization states, larger interface area, and more interactions. The structure property studies indicate that Y12, D61 and Y108 were critical for oligomerization and high thermostability of rFIP-nha, but the dimeric and tetrameric states of rFIP-nha exert similar cytotoxicity on A549 cells. Taken together, these findings reveal that thermostability of FIPs was dependent on their oligomerization state, and correlated with their cytotoxicity. • The crystal structure of rFIP-nha has been determined. • Tetrameric rFIPs have a higher thermostability compared to dimeric rFIPs. • FIP-nha Y12, D61 and Y108 were critical for oligomerization and high thermostability. • Dimeric and tetrameric states of rFIP-nha exert similar cytotoxicity on A549 cells. [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]

[Display omitted] • In vitro gastric digestion models were set up representing different infant ages. • Gastric digestion of milk proteins increased with increasing infant age. • Unheated skim milk formed tight clots only at one-month-old infant conditions. Gastric digestion conditions change during infancy from newborn towards more adult digestion conditions, which can change gastric digestion kinetics. However, how these changes in gastric digestion conditions during infancy affect milk protein digestion has not been investigated. Therefore, we aimed to investigate milk protein digestion with static in vitro gastric digestion models representing one-, three- and six-month-old infants. With increasing age, gastric clots and soluble proteins were digested more extensively, which may partly be attributed to the looser gastric clot structure. Larger differences with increasing age were found for heated than unheated milk proteins, which might be caused by the presence of denatured whey proteins. Taken together, these findings show that gastric milk protein digestion increases during infancy. These in vitro gastric digestion models could be used to study how milk protein digestion changes with infant age, which may aid in developing infant formulas for different age stages. [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]

Edible insects are currently being evaluated as an alternative and more sustainable protein source for humans. The introduction of new food sources can lead to development of novel allergies. Because in the Western world, insects are unlikely to be consumed raw, it is important to know how processing and in vitro digestion might influence their allergenicity. Three edible mealworm species ( Tenebrio molitor , Zophobas atratus and Alphitobius diaperinus ) subjected to processing and in vitro digestion were analysed for IgE cross-reactivity. Immunoblot and MALDI-MS/MS analyses revealed that IgE from crustaceans or House dust mite (HDM) allergic patients showed cross-reactivity to mealworm tropomyosin or α-amylase, hexamerin 1B precursor and muscle myosin, respectively. Heat processing as well as in vitro digestion did diminish, but not eliminate, HDM or tropomyosin IgE cross-reactivity. Results show that individuals allergic to HDM or crustaceans might be at risk when consuming mealworms, even after heat processing. [ABSTRACT FROM AUTHOR]

Abstract: In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity. [Copyright &y& Elsevier]

To investigate intestinal health and its potential disruptors in vitro, representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However, the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently, the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge, IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model, while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately, comparison to in vivo transcriptomics data showed limited similarities, which could be explained by essential differences in the study setups. Altogether, hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro. [ABSTRACT FROM AUTHOR]

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]

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]

Food allergen sensitization is characterized by a type 2 immune response towards a specific food protein and can adversely affect the patient after re-exposure of this food allergen. The gut, skin, and lymph nodes (including immune cells) are interconnected key organs in food allergen sensitization. Gut and skin organ-on-a-chip (OoC) devices have been developed with an immune component to recapitulate the 3D epithelial cell–immune cell crosstalk, although specialized gut–immune–skin OoC models to study food protein sensitizing allergenicity capacity are not available yet. The inclusion of compartmentalized innate and adaptive immune cells is crucial to mimic the immune cascade in the gut–immune–skin axis in a stepwise manner. Engineering a gut–immune–skin axis OoC can better evaluate food allergen sensitization in the future and advance mechanistic insight. The global population is growing, rapidly increasing the demand for sustainable, novel, and safe food proteins with minimal risks of food allergy. In vitro testing of allergy-sensitizing capacity is predominantly based on 2D assays. However, these lack the 3D environment and crosstalk between the gut, skin, and immune cells essential for allergy prediction. Organ-on-a-chip (OoC) technologies are promising to study type 2 immune activation required for sensitization, initiated in the small intestine or skin, in interlinked systems. Increasing the mechanistic understanding and, moreover, finding new strategies to study interorgan communication is of importance to recapitulate food allergen sensitization in vitro. Here, we outline recently developed OoC platforms and discuss the features needed for reliable prediction of sensitizing allergenicity of proteins. [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]

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]

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]

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]