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Background: In immune cell models, macrophages are one of the most frequently used cell types. THP-1 cells are often used as model to study macrophage function, however they may act differently from primary human monocyte derived macrophages (MDMs). Methods: In this study, we investigated the intrinsic baseline differences between the human macrophage cell line THP-1 and human primary MDMs. Additionally, we studied the difference in response to treatment with long-chain polyunsaturated fatty acids (LCPUFAs): well-described immunomodulators. Results: Although the amount of cells that phagocytose were similar between the cell types, primary MDMs consumed significantly more E. coli bioparticles compared to THP-1 macrophages. In M1 macrophages, IL-12 secretion was almost fifty times higher by primary MDMs compared to THP-1 macrophages, thereby increasing the IL-12/IL-10 ratio. Despite this, the IL-12 secretion by THP-1 M1 macrophages was higher that the secretion of IL-10, thereby showing that it is still a suitable M1 type. Cytokine profiles differed between primary MDMs and THP-1 M1 and M2 macrophages. In response to LCPUFAs, primary M1 MDMs and THP-1 M1 macrophages were alike. Interestingly, primary M2 MDMs secreted less IL-10 and CCL22 when treated with LCPUFAs, whereas THP-1 M2 macrophages secreted more IL-10 when treated with LCPUFAs and showed no difference in CCL22 secretion. Conclusions: In conclusion, in an M1 setting, both THP-1 and primary MDMs are suitable models. However, when interested in M2 models, the model choice highly depends on the research question.

[EN] In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 mu g) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol., The authors thank the European Commission COST Action FA1005 INFOGEST and INRAE for financing the meetings and conferences that enable the 21 laboratories to network and organise the work presented in this article. We also acknowledge the 'Healthy and Safe Food System (KB37) ' knowledge base program of the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) , with grant number KB-37-001-007 for funding of this work performed at Wageningen Food & Biobased Research. We are grateful to Dr Sawsan Amara (CEO of Lipolytech SA., Marseille, France) for her generous gift of RGE and to Drs Jan Ludemann, Anja Jensen, Olaf Friedrich and Claus Middelberg from Nordmark Arzneimittel GmbH & Co. KG (Uetersen, Germany) for their generous gift of Nordmark pancreatin. Finally, we thank Drs Maria Fatima Cabral and Judite Costa from iBET for their technical support and discussion.

In vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with targeted enzyme activities as described in in vitro digestion protocols, it is important to determine the activities of enzyme preparations using validated methods. The purpose of this inter-laboratory study within the INFOGEST network was to test the repeatability and reproducibility of lipase assays using the pH-stat technique for measuring the activities of gastric and pancreatic lipases from various sources. Twenty-one laboratories having different pH-stat devices received the same protocol with identical batches of RGE and two pancreatin sources. Lipase assays were performed using tributyrin as a substrate and three different amounts (50, 100 and 200 mu g) of each enzyme preparation. The repeatability results within individual laboratories were satisfactory with coefficients of variation (CVs) ranging from 4 to 8% regardless of the enzyme amount tested. However, the inter laboratory variability was high (CV > 15%) compared to existing standards for bioanalytical assays. We identified and weighted the contributions to inter-laboratory variability of several parameters associated with the various pH-stat equipment used in this study (e.g. reaction vessel volume and shape, stirring mode and rate, burette volume for the automated delivery of sodium hydroxide). Based on this, we established recommendations for improving the reproducibility of lipase assays using the pH-stat technique. Defining accurate and complete recommendations on how to correctly quantify activity levels of enzyme preparations is a gateway to promising comparison of in vitro data obtained from different laboratories following the same in vitro digestion protocol., European Commission COST Action [FA1005 INFOGEST]; INRAE; 'Healthy and Safe Food System (KB37) ' knowledge base program of the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) [KB-37-001-007], The authors thank the European Commission COST Action FA1005 INFOGEST and INRAE for financing the meetings and conferences that enable the 21 laboratories to network and organise the work presented in this article. We also acknowledge the 'Healthy and Safe Food System (KB37) ' knowledge base program of the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) , with grant number KB-37-001-007 for funding of this work performed at Wageningen Food & Biobased Research. We are grateful to Dr Sawsan Amara (CEO of Lipolytech SA., Marseille, France) for her generous gift of RGE and to Drs Jan Ludemann, Anja Jensen, Olaf Friedrich and Claus Middelberg from Nordmark Arzneimittel GmbH & Co. KG (Uetersen, Germany) for their generous gift of Nordmark pancreatin. Finally, we thank Drs Maria Fatima Cabral and Judite Costa from iBET for their technical support and discussion.

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.

Bøgh et al., 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., The authors are all part of the COST Action FA1402 entitled: Improving Allergy Risk Assessment Strategy for New Food Proteins (ImpARAS). LOM is supported by Swiss National Foundation grants (Project Numbers: CRSII3_154488 and 310030-144219) and European Union research grants.

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.

35 Pág. Centro de Biotecnología y Genómica de Plantas, 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).

International audience; 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.