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In vitro models that mimic intestinal mucosal tissue inflammation and assess the sensitizing capacity of food proteins are essential for understanding food allergy mechanisms and improving safety assessments. Current 2D models lack spatial epithelial‐immune cell interactions, including dendritic cell (DC) migration and DC–T cell crosstalk. Intestine‐on‐a‐chip (IoC) models are used for infections and inflammatory bowel disease (IBD) but are not yet widely used in food allergy research. Here, a 3D immunocompetent IoC model is presented using extrusion‐based bioprinting and melt electrowriting (MEW). The system integrates human intestinal epithelial cells (Caco‐2) seeded on half‐pipe‐shaped MEW scaffolds and co‐cultured with hydrogel‐embedded monocyte‐derived DCs (moDCs) inside the printed device. Subsequent moDC–T cell interactions are studied separately in a hydrogel‐embedded system. IoCs exhibited leak‐tight epithelial barriers comparable to transwell‐like systems, while demonstrating higher metabolic and brush border enzyme activity, and lower LDH leakage. After food allergens (peanut, milk, and egg), non‐allergen (Rubisco) or pro‐inflammatory stimuli (toxin A and LPS) exposure, distinguishable effects on epithelial barrier integrity and moDC driven Th1/Th2 immune responses are observed. The IoC model presents a significant step toward 3D in vitro systems that mimic the intestinal mucosa's compartments to study food allergen sensitization and inflammatory diseases. The immunocompetent Intestine‐on‐a‐Chip (IoC) is developed using melt electrowritten (MEW) half‐pipes. Intestinal epithelial cell (IEC) culture is optimized in the MEW‐based IoC, followed by co‐culture with monocyte‐derived dendritic cells (moDCs) and T cells in 3D hydrogels. This model supports IECs with a porous membrane, and provides a base for mimicking the step‐wise immune response as well as facilitating cell–cell interactions.

Background Tumour-associated macrophages (TAMs) are associated with poor clinical outcome in cancer patients, making them key targets in immunotherapy. TAMs mainly consist of anti-inflammatory M2 macrophages promoting tumour growth, metastasis and immunosuppression. Fungal immunomodulatory protein from Nectria haematococca (FIP-nha) is a potential anti-tumour agent, notably inhibiting tumour cells and exerting immunomodulatory activity on macrophages. However, the anti-tumour activity of FIP-nha via TAMs modulation is still unclear. Here, the immunomodulatory activity of rFIP-nha on M1 and M2 macrophages was explored, as well as its anti-tumour activity via macrophage modulation, using M2 macrophages as TAM representatives, through an intricate co-culture design with A549 lung cancer cells. Methods THP-1-monocyte derived M2 macrophages were treated with rFIP-nha and analysed phenotypically. Subsequently, rFIP-nha treated M2 macrophages were co-cultured with A549 cells to investigate anti-tumour activity via macrophage modulation. Results rFIP-nha exposure decreased macrophages phagocytosis activity, enhanced IL-1β, IL-12 and IL-10 cytokine secretion, and modulated specific cell surface markers for M2 (CD163 and CD206) towards those that are typical for M1 (CD68 and CD80) polarization. rFIP-nha treated M2 macrophages polarized towards a tumour inhibitory phenotype which, when co-cultured with A549 cells, resulted in greater reduction of tumour cell proliferation compared to A549 cells co-cultured with M2 or M1 macrophages. Moreover, rFIP-nha exacerbated tumour inhibition and cell death. Conclusion rFIP-nha inhibited tumour growth in two distinct ways, by targeting tumour cells directly and by directing macrophage polarization towards a tumour inhibitory phenotype. This dual bioactivity makes FIP-nha interesting as an additive support in cancer immunotherapy.

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.

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.

Fungal immunomodulatory proteins (FIPs) are small proteins from fungi with considerable immunomodulatory activity. FIP-nha ( Nectria haematococca ) contains two glycosylation sites at positions N5 and N39, and displays a high thermostability and notable anti-tumour activity. However, FIP-nha’s immunomodulatory activity on macrophages and the associated mechanism remain unclear. In this study, three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were recombinantly expressed in Pichia pastoris . To test the impact on FIP-nha’s immunomodulatory activity, the phagocytotic activity, cytokine secretion, and gene expression of THP-1 macrophages were investigated. rFIP-nha and its mutants reduced macrophage phagocytosis, and induced IL-1β, IL-12 and IL-10 cytokine secretion significantly, indicating that the protein confers a pro-inflammatory behaviour on THP-1 macrophages. However, there were no obvious differences among the different glycan mutants, indicating that the observed activation mechanisms are likely glycosylation-independent. Furthermore, to study the immunomodulatory mechanism, four kinds of inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) were tested at transcriptional level. AIM2 was found to be 10-fold upregulated. Then, THP1-KO-ASC cells and AIM2 related inhibitors showed that IL-1β release induced by rFIP-nha is ASC signalling pathway dependent. Taken together, these findings suggest that rFIP-nha activates THP-1 macrophages in a pro-inflammatory way by activating the AIM2 inflammasome.

Food security is under increased pressure due to the ever-growing world population. To tackle this, alternative protein sources need to be evaluated for nutritional value, which requires information on digesta peptide composition in comparison to established protein sources and coupling to biological parameters. Here, a combined experimental and computational approach is presented, which compared seventeen protein sources with cow’s whey protein concentrate (WPC) as the benchmark. In vitro digestion of proteins was followed by proteomics analysis and statistical model-based clustering. Information on digesta peptide composition resulted in 3 cluster groups, primarily driven by the peptide overlap with the benchmark protein WPC. Functional protein data was then incorporated in the computational model after evaluating the effects of eighteen protein digests on intestinal barrier integrity, viability, brush border enzyme activity, and immune parameters using a bioengineered intestine as microphysiological gut system. This resulted in 6 cluster groups. Biological clustering was driven by viability, brush border enzyme activity, and significant differences in immune parameters. Finally, a combination of proteomic and biological efficacy data resulted in 5 clusters groups, driven by a combination of digesta peptide composition and biological effects. The key finding of our holistic approach is that protein source (animal, plant or alternative derived) is not a driving force behind the delivery of bioactive peptides and their biological efficacy.

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. 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.

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.

The activity of α-amylases is frequently determined using a single-point assay at 20 °C. Previous work within INFOGEST “Working Group 5 - Starch digestion and amylases” identified significant interlaboratory variation with this protocol. The current study aimed to evaluate the repeatability (intralaboratory precision) and reproducibility (interlaboratory precision), measured as coefficients of variation (CVs), of a newly optimized protocol version based on four time-point measurements at 37 °C. Human saliva (a pool from ten healthy adults) and three porcine enzyme preparations (two pancreatic α-amylases and pancreatin) were tested in 13 laboratories across 12 countries and 3 continents. Assay repeatability for each lab remained below 20% for all test products and the overall repeatability was below 15%, ranging between 8 and 13% for all products. Reproducibility was greatly improved with interlaboratory CVs ranging from 16 to 21%, i.e. up to four times lower than with the original method. Five laboratories repeated the same assay at 20 °C, and the amylolytic activity of each product increased by 3.3-fold (± 0.3) from 20 to 37 °C. The newly optimized protocol is henceforth recommended to ensure precise determinations of α-amylase activity levels and to facilitate comparisons across different studies.

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. 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. 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. 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 cosensitised 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 crossreactivity between several Anacardiaceae species. Results demonstrate the in vitropresence 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 crosssensitivity to these species. The clinical relevance of this wide spread crosssensitisation is unknown.