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Accelerating the induction of tolerance to cow’s milk (CM) reduces the burden of cow’s milk allergy (CMA). In this randomised controlled intervention study, we aimed to investigate the tolerance induction of a novel heated cow milk protein, the iAGE product, in 18 children with CMA (diagnosed by a paedriatric allergist). Children who tolerated the iAGE product were included. The treatment group (TG: n = 11; mean age 12.8 months, SD = 4.7) consumed the iAGE product daily with their own diet, and the control group (CG: n = 7; mean age 17.6 months, SD = 3.2) used an eHF without any milk consumption. In each group, 2 children had multiple food allergies. The follow-up procedures consisted of a double-blind placebo-controlled food challenge (DBPCFC) with CM t = 0, t = 1 (8 months), t = 2 (16 months), and t = 3 (24 months). At t = 1, eight (73%) of 11 children in the TG had a negative DBPCFC, versus four out of seven (57%) in the CG (BayesFactor = 0.61). At t = 3, nine of the 11 (82%) children in the TG and five of seven (71%) in the CG were tolerant (BayesFactor = 0.51). SIgE for CM reduced from a mean of 3.41 kU/L (SD = 5.63) in the TG to 1.24 kU/L (SD = 2.08) at the end of intervention, respectively a mean of 2.58 (SD = 3.32) in the CG to 0.63 kU/L (SD = 1.06). Product-related AEs were not reported. CM was successfully introduced in all children with negative DBPCFC. We found a standardised, well-defined heated CM protein powder that is safe for daily OIT treatment in a selected group of children with CMA. However, the benefits of inducing tolerance were not observed.

Dry heating of plant proteins occurs during production of plant-based infant formula. To study the effect of dry heating on physicochemical changes and in vitro infant digestion of soy and pea proteins, these proteins were mild dry heated (60 °C) for 6 h and 48 h, in the presence or absence of glucose. In this study, we found that with extended dry heating, the degrees of glycation increased in the presence of glucose. After 48 h of dry heating, the degree of gastric hydrolysis decreased from 2.7% to 0.2% for soy protein and from 2.8% to 0.4% for pea protein, compared to non-treated proteins. Such resistance to gastric digestion was probably due to the extensive protein aggregation induced by glycation. However, the intestinal digestion was less affected, as the degree of hydrolysis was similar at the end of intestinal digestion, ranging from 31.0% to 35.4% for soy protein and 36.7% to 48.5% for pea protein. This may be due to the broad range of proteases in pancreatin negating the effect of glycation. By contrast, for the glucose-free samples, no glycation happened and only limited insoluble aggregates were formed during dry heating. Despite this aggregation, the variation in dry heating duration did not lead to different digestibility. Therefore, 60 °C of mild dry heating will influence the gastric digestion of glucose-containing samples, but will not influence the final intestinal digestibility of all the samples with or without glucose.

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible β-glucans (oatβG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived β-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived β-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses. Electronic supplementary material The online version of this article (10.1007/s00262-020-02707-4) contains supplementary material, which is available to authorized users.

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.

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.

The introduction of baked milk products in cow’s milk (CM) allergic children has previously been shown to accelerate induction tolerance in a selected group of children. However, there is no standardized baked milk product on the market. Recently, a new standardized, heated and glycated cow’s milk protein (HP) product was developed. The aim of this study was to measure safety and tolerability of a new, well characterized heated CM protein (HP) product in cow’s milk allergic (CMA) children between the age of 3 and 36 months. The children were recruited from seven clinics throughout The Netherlands. The HP product was introduced in six incremental doses under clinical supervision. Symptoms were registered after introduction of the HP product. Several questionnaires were filled out by parents of the children. Skin prick tests were performed with CM and HP product, sIgE to CM and α-lactalbumin (Bos d4), β-lactoglobulin (Bos d5), serum albumin (Bos d 6), lactoferrin (Bos d7) and casein (Bos d8). Whereas 72% percent (18 out of 25) of the children tolerated the HP product, seven children experienced adverse events. Risk factors for intolerance to the HP product were higher skin prick test (SPT) histamine equivalent index (HEP) results with CM and the HP product, higher specific IgE levels against Bos d4 and Bos d8 levels and Bos d5 levels. In conclusion, the HP product was tolerated by 72% of the CM allergic children. Outcomes of SPT with CM and the HP product, as well as values of sIgE against caseins, α-lactalbumin, and β-lactoglobulin may predict the tolerability of the HP product. Larger studies are needed to confirm these conclusions.

The introduction of baked milk products in cow’s milk (CM) allergic children has previously been shown to accelerate induction tolerance in a selected group of children. However, there is no standardized baked milk product on the market. Recently, a new standardized, heated and glycated cow’s milk protein (HP) product was developed. The aim of this study was to measure safety and tolerability of a new, well characterized heated CM protein (HP) product in cow’s milk allergic (CMA) children between the age of 3 and 36 months. The children were recruited from seven clinics throughout The Netherlands. The HP product was introduced in six incremental doses under clinical supervision. Symptoms were registered after introduction of the HP product. Several questionnaires were filled out by parents of the children. Skin prick tests were performed with CM and HP product, sIgE to CM and α-lactalbumin (Bos d4), β-lactoglobulin (Bos d5), serum albumin (Bos d 6), lactoferrin (Bos d7) and casein (Bos d8). Whereas 72% percent (18 out of 25) of the children tolerated the HP product, seven children experienced adverse events. Risk factors for intolerance to the HP product were higher skin prick test (SPT) histamine equivalent index (HEP) results with CM and the HP product, higher specific IgE levels against Bos d4 and Bos d8 levels and Bos d5 levels. In conclusion, the HP product was tolerated by 72% of the CM allergic children. Outcomes of SPT with CM and the HP product, as well as values of sIgE against caseins, α-lactalbumin, and β-lactoglobulin may predict the tolerability of the HP product. Larger studies are needed to confirm these conclusions.

Advanced glycation end products (AGEs) can be present in food or be endogenously produced in biological systems. Their formation has been associated with chronic neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. The implication of AGEs in neurodegeneration is related to their ability to bind to AGE-specific receptors and the ability of their precursors to induce the so-called “dicarbonyl stress”, resulting in cross-linking and protein damage. However, the mode of action underlying their role in neurodegeneration remains unclear. While some research has been carried out in observational clinical studies, further in vitro studies may help elucidate these underlying modes of action. This review presents and discusses in vitro methodologies used in research on the potential role of AGEs in neuroinflammation and neurodegeneration. The overview reveals the main concepts linking AGEs to neurodegeneration, the current findings, and the available and advisable in vitro models to study their role. Moreover, the major questions regarding the role of AGEs in neurodegenerative diseases and the challenges and discrepancies in the research field are discussed.

Scope β‐lactoglobulin (BLG) is a major cow milk allergen encountered by the immune system of infants fed with milk‐based formulas. To determine the effect of processing on immunogenicity of BLG, this article characterized how heated and glycated BLG are recognized and internalized by APCs. Also, the effect of heat‐induced structural changes as well as gastrointestinal digestion on immunogenicity of BLG is evaluated. Methods and results The binding and uptake of BLG from raw cow milk and heated either alone (BLG‐H) or with lactose/glucose (BLG‐Lac and BLG‐Glu) to the receptors present on APCs are analyzed by ELISA and cell‐binding assays. Heated and glycated BLG is internalized via galectin‐3 (Gal‐3)and scavenger receptors (CD36 and SR‐AI) while binding to the receptor for advanced glycation end products (R AGE) does not cause internalization. Receptor affinity of BLG is dependent on increased hydrophobicity, β‐sheet exposure and aggregation. Digested glycated BLG maintained binding to sRAGE and Gal‐3 but not to CD36 and SR‐AI, and is detected on the surface of APCs. This suggests a mechanism via which digested glycated BLG may trigger innate (via RAGE) and adaptive immunity (via Gal‐3). Conclusions This study defines structural characteristics of heated and glycated BLG determining its interaction with APCs via specific receptors thus revealing enhanced immunogenicity of glycated versus heated BLG., Heat treatment of bovine milk antigen bovine β‐lactoglobulin (BLG) generates ligands for RAGE, Gal‐3, CD‐36 and SR‐AI as a result of structural changes and glycation. Glycated BLG is less sensitive to digestion and maintains its binding to RAGE and Gal‐3. Post‐digestion retained immunogenicity of glycated BLG may be mediated via RAGE as a signaling pathway, and via Gal‐3 facilitating internalization.

Allergen recognition and processing by antigen presenting cells is essential for the sensitization step of food allergy. Macrophages and dendritic cells are both phagocytic antigen presenting cells and play important roles in innate immune responses and signaling between the innate and adaptive immune system. To obtain a model system with a homogeneous genetic background, we derived macrophages and dendritic cells from THP-1 monocytes. The difference between macrophages and dendritic cells was clearly shown by differences in their transcription response (microarray) and protein expression levels. Their resemblance to primary cells was analyzed by comparison to properties as described in literature. The uptake of β-lactoglobulin after wet-heating (60°C in solution) by THP-1 derived macrophages was earlier reported to be significantly increased. To analyse the subsequent immune response, we incubated THP-1 derived macrophages and dendritic cells with native and differently processed β-lactoglobulin and determined the transcription and cytokine expression levels of the cells. A stronger transcriptional response was found in macrophages than in dendritic cells, while severely structurally modified β-lactoglobulin induced a more limited transcriptional response, especially when compared to native and limitedly modified β-lactoglobulin. These results show that processing is relevant for the transcriptional response toward β-lactoglobulin of innate immune cells.

We studied several sustainable alternative protein concentrates and a whey protein concentrate as reference, to determine their protein composition, digestibility and quality using the harmonized INFOGEST static protocol. The proteins concentrates were analyzed to determine their conversion factor, degree of hydrolysis, true ileal digestibility, in vitro digestible indispensable amino acid score (IVDIAAS) and total absorbable amino acids and total essential amino acids. The results revealed that whey, blood plasma concentrate and yeast protein concentrate have a high mean true ileal indispensable amino acid in vitro digestibility (91.1–85.8%), closely followed by corn, pea, potato and proteins derived from lesser meal worms (ranging between 82.5 and 77.9%). Mycoprotein gave a much lower score in the mass balance, so that its other results could not be interpreted as being reliable. Whey, potato, blood plasma and yeast protein concentrates ranked highest in IVDIAAS (between 119 and 97.2), followed by lesser meal worm and pea (between 73.8 and 57.8) with corn protein concentrate having the lowest IVDIAAS due to underrepresentation of lysine. The method and data presented in this paper can form a start for further applying the INFOGEST in vitro digestion protocol to evaluate protein quality.

For the determination of the binding of heated cow’s milk whey proteins such as β-lactoglobulin to the receptors expressed on immune cells, inhibition ELISA with the soluble form of the receptor for advanced glycation end products (sRAGE) and scavenger receptor class B (CD36) has been successfully used in the past. However, binding to heated and glycated caseins in this read-out system has not been tested. In this study, inhibition ELISA was applied to measure the binding of cow’s milk casein alone, as well as all milk proteins together, which underwent differential heat treatment, to sRAGE and CD36, and we compared those results to a dot blot read out. Moreover, binding to sRAGE and CD36 of differentially heated milk protein was measured before and after in vitro digestion. Casein showed binding to sRAGE and CD36, independent from the heat treatment, in ELISA, while the dot blot showed only binding to high-temperature-heated milk protein, indicating that the binding is not related to processing but to the physicochemical characteristics of the casein. This binding decreased after passage of casein through the intestinal phase.

Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20–25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.

Scope Chitin, the most abundant polysaccharide found in nature after cellulose, is known for its ability to support wound healing and to lower plasma‐oxidized low‐density lipoprotein (LDL) levels. Studies have also revealed immunomodulatory potential but contradicting results are often impossible to coalesce through usage of chitin of different or unknown physicochemical consistency. In addition, only a limited set of cellular models have been used to test the bioactivity of chitin. Methods and Results Chitin is investigated with well‐defined physicochemical consistency for its immunomodulatory potency using THP‐1 macrophages, impact on intestinal epithelial barrier using Caco‐2 cells, and fermentation by fecal‐derived microbiota. Results show that chitin with a degree of acetylation (DA) of ≈83%, regardless of size, does not affect the intestinal epithelial barrier integrity. Large‐sized chitin significantly increases acetic acid production by gut microbiota without altering the composition. Exposure of small‐sized chitin to THP‐1 macrophages lead to significantly increased secretion of IL‐1β, IL‐8, IL‐10, and CXCL10 in a multi‐receptor and clathrin‐mediated endocytosis dependent manner. Conclusions These findings indicate that small‐sized chitin does not harm the intestinal barrier nor affects SCFA secretion and microbiota composition, but does impact immune activity which could be beneficial to subjects in need of immune support or activation., This study indicates that small‐sized chitin does not harm the intestinal barrier but does support the production of beneficial microbial metabolites and supports immune activity through receptor‐mediated endocytosis.

Dry heating of cow's milk protein in the presence of the milk sugar lactose leads to a loss of solubility and digestibility. Most studies that investigated the loss of solubility in milk protein powders suggested that, besides structural changes, hydrophobic interaction, hydrogen bonds, disulphide bonds, and Maillard reaction-induced crosslinking are responsible for this. However, little is known about the direct contribution of these inter- and intramolecular interactions on loss of solubility and protein digestibility. Low temperature (60 °C) and high temperature (130 °C) dry heating of cow's milk protein in the presence of lactose was applied after which both the soluble and insoluble fractions were analysed with SDS-PAGE and LC-MS/MS. The Maillard reaction was monitored by quantification of Ne-carboxymethyllysine, Ne-carboxyethyllysine, and pentosidine with LC-MS/MS. Loss of solubility was analysed with solvent solubility tests. Protein hydrolysis after simulated infant in vitro digestion, and after hydrolysis with single enzymes, was monitored using SDS-PAGE and the o-phthaldialdehyde assay. The results indicated that caseins are the main proteins that become insoluble upon dry heating, independent of the heating temperature. The decreased solubility of low temperature dry heated cow's milk protein is induced by hydrogen bonds and hydrophobic interactions and did not impair protein hydrolysis. At the same time, covalent protein crosslinking is an important determinant in protein solubility and digestibility of high temperature dry heated cow's milk protein.

Impact of processing on immunogenicity of food proteins has clearly been demonstrated, but the underlying mechanisms are still unclear. In our previous study, the uptake of the cow’s milk protein β-lactoglobulin (BLG) by THP-1 macrophages varied after applying different processing methods and was positively correlated with hydrophobicity and aggregation. Here we applied the same 3 processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry heating in absence or presence of reducing sugars (i.e. glucose, lactose or galacto-oligosaccharide) to lysozyme and thyroglobulin, which have different pI or molecular weight compared to BLG, respectively. Uptake of the soluble fraction was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Heat-treatment-mediated increases in uptake did thus not induce a response in our read-out systems. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

Chitin and chitosan have been recognised for their beneficial health effects since the 1980s. Over the past few decades, numerous studies and several clinical trials have been performed which demonstrated that these compounds can reduce body weight and cardiovascular disease (CVD), improve wound healing, but can also modulate the immune system and demonstrate antifungal and antibacterial activity. In particular, weight reduction and improvement of cardiovascular status are interesting targets as the prevalence of obesity and CVD are increasing. Both diseases are associated with various pathological disorders, including diabetes and hypertension and put a strain on healthcare costs and capacity. In general, lifestyle‐based interventions such as the oral intake of chitin and/or chitosan are becoming increasingly popular as these are easily integrated into current treatments and improve the self‐assertiveness of patients. Of interest, but beyond the scope of this chapter, is the use of chitin‐glucans as supplements in cosmetics. Several studies have demonstrated that chitin‐glucan reduced wrinkling and skin‐ageing, suggesting an interaction between the chitin‐glucan and cells of the epidermis. Although these effects may not be attributed to chitin alone, this does demonstrate the strong biological potential of chitin on cells of the human body. In this chapter, scientific literature has been reviewed that demonstrated the beneficial health effects of chitin and chitosan from an immunomodulatory point‐of‐view. First, we provide an overview of in vitro studies that offer in‐depth mechanistic insights, followed by describing preclinical animal studies. Finally, we list various human intervention trials that most clearly demonstrate the beneficial health effects of chitin and chitosan. Furthermore, we purposely discriminate between data on chitin and chitosan as they are chemically distinct and therefore possibly demonstrate unique effects on health

Dry heating of cow&rsquo, s milk protein, as applied in the production of &ldquo, baked milk&rdquo, facilitates the resolution of cow&rsquo, 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&rsquo, s milk protein powder was heated at low temperature (60 &deg, C) and high temperature (130 &deg, 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.

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.

Heat treatment is a commonly applied unit operation in the processing of β-lactoglobulin containing products. This does, however, influence its structure and thereby impacts its activity and digestibility. We describe how various heat-treatments of β-lactoglobulin change the digestibility using a modified version of the current consensus INFOGEST protocol. Additionally, protein was investigated for its translocation over the intestinal epithelial barrier, which would bring them in contact with immune cells. The extent of gastric digestibility was higher when the protein structure was more modified, while the influence of glycation with lactose was limited. Translocation studies of protein across Caco-2 cell monolayers showed a lower translocation rate of protein heated in solution compared to the others. Our study indicates that structural modifications after different heat-treatments of β-lactoglobulin increase in particular gastric digestibility and the translocation efficiency across intestinal epithelial cells.

Background: Severe allergic reactions, including anaphylaxis, occur during oral food challenges (OFCs) and the first-line treatment of anaphylaxis is epinephrine.Objective: To evaluate the percentage of anaphylactic reactions treated with epinephrine during OFCs and to identify associated factors for the administration of epinephrine.Methods: Children who underwent an OFC with peanut, hazelnut, cow's milk, hen's egg, or cashew nut from 2005 through 2015 in the Netherlands were evaluated. Children with reactions meeting the criteria for anaphylaxis according to the European Academy of Allergy and Clinical Immunology guidelines for food allergy and anaphylaxis were included. Children with an anaphylactic reaction treated with vs without epinephrine were compared. Possible factors associated with the administration of epinephrine, such as age, sex, symptoms consistent with asthma, history of an allergic reaction to the tested allergen, and symptom types during the anaphylactic reaction, were evaluated using logistic regression analysis.Results: Eighty-three children in clinical and research settings (43% boys; median age, 7 years; range, 1-17) who met the criteria for anaphylaxis were included in this study. Thirty-two of 83 children (39%) with anaphylaxis were treated with epinephrine. Respiratory symptoms during the OFC were treated significantly more often with epinephrine than gastrointestinal symptoms (P = .01).Conclusion: Only 39% of children with anaphylaxis, according to the guideline criteria, were treated with epinephrine during the OFC and most of these children had respiratory symptoms. There is need for an easy-to-use international guideline for the treatment of allergic symptoms during OFCs. (C) 2017 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Introduction: Adoptive T-cell treatments of solid cancers have evolved into a robust therapy with objective response rates surpassing those of standardized treatments. Unfortunately, only a limited fraction of patients shows durable responses, which is considered to be due to a T cell-suppressive tumor microenvironment (TME). Here we argue that naturally occurring β-glucans can enable reversion of such T cell suppression by engaging innate immune cells and enhancing numbers and function of lymphocyte effectors. Areas covered: This review summarizes timely reports with respect to absorption, trafficking and immune stimulatory effects of β-glucans, particularly in relation to innate immune cells. Furthermore, we list effects toward well-being and immune functions in healthy subjects as well as cancer patients treated with orally administered β-glucans, extended with effects of β-glucan treatments in mouse cancer models. Expert opinion: Beta-glucans, when present in food and following uptake in the proximal gut, stimulate immune cells present in gut-associated lymphoid tissue and initiate highly conserved pro-inflammatory pathways. When tested in mouse cancer models, β-glucans result in better control of tumor growth and shift the TME toward a T cell-sensitive environment. Along these lines, we advocate that intake of β-glucans provides an accessible and immune-potentiating adjuvant when combined with adoptive T-cell treatments of cancer.

Melanin is the main pigment responsible for the color of human skin, hair and eye. Its biosynthesis requires three melanogenic enzymes, tyrosinase (TYR), and the tyrosinase-related proteins TYRP1 and TYRP2. The difficulty of isolating pure and homogeneous proteins from endogenous sources has hampered their study, and resulted in many contradictory findings regarding their physiological functions. In this review, we summarize recent advances on the structure and function of TYR and TYRPs by virtue of the crystal structure of human TYRP1, which is the first available structure of a mammalian melanogenic enzyme. This structure, combined with tyrosinase structures from other lower eukaryotes and mutagenesis studies of key active site residues, sheds light on the mechanism of TYR and TYRPs. Furthermore, a TYRP1-based homology model of TYR provides a high-quality platform to map and analyze albinism-related mutations, as well as the design of specific antimelanogenic compounds. Finally, we provide perspectives for future structure/function studies of TYR and TYRPs.

Dietary non-digestible polysaccharides (NDPs) might promote intestinal health via immuno-modulation. Immunomodulatory effects of NDP are most likely brought about by antigen processing cells such as macrophages that populate the intestine, although the mechanisms are still poorly understood. We validated the in vitro model of M1 and M2 macrophages to mimic the intestinal inflammatory and tolerant macrophages using literature and microarray-derived gene markers. All these markers were used to characterise the macrophage phenotype following NDP stimulation. This identified an alternative subset, termed M(NDP), which commonly modulated a set of 126 genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function. This gene-based analysis for macrophage subsets provides an additional tool to characterise NDP bioactivity for their in vivo potential.

International audience; Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase-like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys-rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor-like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism-related mutations of TYRP1 affect its stability or activity

Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1β and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1β, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.

Water extracts from Pleurotus ostreatus containing no statins showed 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitory activity (in vitro) that might be due to specific water-soluble polysaccharides (WSPs); when isolated and deproteinized, increasing concentrations of the WSP extract induced higher inhibition. The WSP extract contained mainly β-glucans, mannogalactans, and glycogen (e.g., α-glucans), although derivatives or fragments with lower molecular weights (between 14 and 3.5 kDa) were present and were able to induce the inhibitory activity. The extract contained more β-(1→3)-glucans than β-(11→3),(11→6)-glucans, and they partially survived digestion and managed to pass through Caco2 cell monolayers to the lower compartment after in vitro digestion and transport experiments. The WSP might also modulate Caco2 membrane integrity.

Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protege Study (ISRCTN14236739; www.isrctn.com) we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained males (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% Wmax the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose/rhamnose solution was ingested. Markers of energy metabolism, lactulose/rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated one week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (one hour plasma lactulose/rhamnose ratio), serum interleukin-6, interleukin-10, fibroblast growth factor-21, and muscle creatine kinase levels were only significantly increased compared to rest during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be due to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise. This article is protected by copyright. All rights reserved

The effect of glycation and aggregation of thermally processed &beta, lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity &lt, 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (&lt, 73 &deg, C) and high temperatures (&gt, 90 &deg, C) in the presence or absence of the milk sugar lactose. N&epsilon, (carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow&rsquo, s milk BLG.

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.

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.

Food-derived peptides are known to possess inhibitory activity against the dipeptidyl-peptidase IV (DPP-IV) enzyme, a target in the management of type 2 diabetes. While proteins from commonly consumed food commodities have been investigated as precursors of DPP-IV-inhibiting peptides, studies on novel protein sources, such as those from insects, are sparse. This research aimed to determine if DPP-IV inhibitors can be generated upon in vitro digestion or enzymatic hydrolysis of lesser mealworm protein isolate and concentrate. Treatment of the proteins with digestive enzymes and proteases generated hydrolysates with varying potency, thermolysin being the most effective at releasing active peptides (IC 50 = 0.63 and 0.60 mg mL −1 for the isolate and concentrate). Ultrafiltration of the thermolysin-treated hydrolysates did not significantly improve the potency. This study shows that DPP-IV inhibitors can be generated from lesser mealworm protein and provides insight on the potential of insects to serve as functional food ingredients.

Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE receptor (sRAGE). Samples consisting of mixtures of whey protein and lactose were heated at 130 °C. An in vitro infant digestion model was used to study the influence of heat treatment on the digestibility of whey proteins. The amount of sRAGE-binding ligands before and after digestion was measured by an ELISA-based sRAGE-binding assay. Water activity did not significantly affect the extent of digestibility of whey proteins dry heated at pH 5 (ranging from 3.3 ± 0.2 to 3.6 ± 0.1% for gastric digestion and from 53.5 ± 1.5 to 64.7 ± 1.1% for duodenal digestion), but there were differences in cleavage patterns of peptides among the samples heated at different pH values. Formation of sRAGE-binding ligands depended on the formation of aggregates and was limited in the samples heated at pH 5. Moreover, the sRAGE-binding activity of digested sample was changed by protease degradation and correlated with the digestibility of samples. In conclusion, generation of sRAGE-binding ligands during extensive heat treatment of whey protein/lactose mixtures is limited in acidic heating condition and dependent on glycation and aggregation.

In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. N&epsilon, carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, &beta, lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to &beta, lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of &beta, sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified &beta, lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

Scope: Salmonellosis is a prevalent food-borne illness that causes diarrhea in over 130 million humans yearly and can lead to death. There is an urgent need to find alternatives to antibiotics as many salmonellae are now multidrug resistant. As such, specific beneficial bacteria and dietary fibers can be an alternative as they may prevent Salmonella Typhimurium (STM) infection and spreading by strengthening intestinal barrier function.Methods and Results: We tested whether immune active long-chain inulin-type fructans and/or L. acidophilus W37, L. brevis W63, and L. casei W56 can strengthen barrier integrity of intestinal Caco-2 cells in the presence and absence of a STM. Effects of the ingredients on intestinal barrier function were first evaluated by quantifying transepithelial electric resistance (TEER) and regulation of gene expression by microarray.Only L. acidophilus had effects on TEER and modulated a group of 26 genes related to tight-junctions. Inulin-type fructans, L. brevis W63 and L. casei W56 regulated other genes, unrelated to tight- junctions. L. acidophilus also had unique effects on a group of six genes regulating epithelial phenotype toward follicle-associated epithelium. L. acidophilus W37 was therefore selected for a challenge with STM and prevented STM-induced barrier disruption and decreased secretion of IL-8.Conclusion: L. acidophilus W37 increases TEER and can protect against STM induced disruption of gut epithelial cells integrity in vitro. Our results suggest that selection of specific bacterial strains for enforcing barrier function may be a promising strategy to reduce or prevent STM infections.

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

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.