Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

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).

New applications of advanced instrumental techniques for the characterization of food allergenic proteins.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Nanoarchitectures for efficient IgE cross-linking on effector cells to study amoxicillin allergy.

BACKGROUND: Amoxicillin (AX) is nowadays the ß-lactam that more frequently induces immediate allergic reactions. Nevertheless, diagnosis of AX allergy is occasionally challenging due to risky in vivo tests and non-optimal sensitivity of in vitro tests. AX requires protein haptenation to form multivalent conjugates with increased size to be immunogenic. Knowing adduct structural features for promoting effector cell activation would help to improve in vitro tests. We aimed to identify the optimal structural requirement in specific cellular degranulation to AX using well-precised nanoarchitectures of different lengths. METHOD: We constructed eight Bidendron Antigens (BiAns) based on polyethylene glycol (PEG) linkers of different lengths (600-12,000 Da), end-coupled with polyamidoamine dendrons that were terminally multi-functionalized with amoxicilloyl (AXO). In vitro IgE recognition was studied by competitive radioallergosorbent test (RAST) and antibody-nanoarchitecture complexes by transmission electron microscopy (TEM). Their allergenic activity was evaluated using bone marrow-derived mast cells (MCs) passively sensitized with mouse monoclonal IgE against AX and humanized RBL-2H3 cells sensitized with polyclonal antibodies from sera of AX-allergic patients. RESULTS: All BiAns were recognized by AX-sIgE. Dose-dependent activation responses were observed in both cellular assays, only with longer structures, containing spacers in the range of PEG 6000-12,000 Da. Consistently, greater proportion of immunocomplexes and number of antibodies per complex for longer BiAns were visualized by TEM. CONCLUSIONS: BiAns are valuable platforms to study the mechanism of effector cell activation. These nanomolecular tools have demonstrated the importance of the adduct size to promote effector cell activation in AX allergy, which will impact for improving in vitro diagnostics.

New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7.

BACKGROUND: Ole e 7 is a nonspecific lipid transfer protein (nsLTP) from olive pollen, one of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no evidence of cross-reactivity, which explains this observed co-sensitization. Therefore, the purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. METHODS: A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. Inhibition assays were performed to determine the existence of cross-reactivity between both nsLTPs. Allergic response was analyzed ex vivo (basophil activation test) and in vitro (RBL-2H3 mast cell model). RESULTS: Common IgG and IgE epitopes were identified between both allergens. IgE-binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. CONCLUSIONS: Our results suggest that Ole e 7 could play a new role as primary sensitizer in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.

Peptide Glycodendrimers as Potential Vaccines for Olive Pollen Allergy.

Olive pollen is one of the most important causes of respiratory allergy, with Ole e 1 being the most clinically relevant sensitizing allergen. Peptide-based vaccines represent promising therapeutic approaches, but the use of adjuvants is required to strengthen the weak immunogenicity of small peptides. We propose the use of dendrimeric scaffolds conjugated to the T cell immunodominant epitope of Ole e 1 (OE109-130) for the development of novel vaccines against olive pollen allergy. Four dendrimeric scaffolds containing an ester/ether with nine mannoses, an ester succinimidyl linker with nine N-acetyl-glucosamine units or nine ethylene glycol units conjugated to OE109-130 peptide were designed, and their cytotoxicity, internalization pattern, and immunomodulatory properties were analyzed in vitro. None of the dendrimers exhibited cytotoxicity in humanized rat basophil (RBL-2H3), human bronchial epithelial Calu-3, and human mast LAD2 cell lines. Confocal images indicated that mannosylated glycodendropeptides exhibited lower colocalization with a lysosomal marker. Moreover, mannosylated glycodendropeptides showed higher transport tendency through the epithelial barrier formed by Calu-3 cells cultured at the air-liquid interface. Finally, mannosylated glycodendropeptides promoted Treg and IL10+Treg proliferation and IL-10 secretion by peripheral blood mononuclear cells from allergic patients. Mannosylated dendrimers conjugated with OE109-130 peptide from Ole e 1 have been identified as suitable candidates for the development of novel vaccines of olive pollen allergy.

Profilin-mediated food-induced allergic reactions are associated with oral epithelial remodeling.

BACKGROUND: In areas of high exposure to grass pollen, allergic patients are frequently sensitized to profilin, and some experience severe profilin-mediated food-induced reactions. This specific population of patients is ideal to study the relationship between respiratory and food allergies. OBJECTIVE: We sought to determine the role of oral mucosal epithelial barrier integrity in profilin-mediated allergic reactions. METHODS: Thirty-eight patients with profilin allergy stratified into mild or severe according to their clinical history and response to a profilin challenge test and 6 nonallergic subjects were recruited. Oral mucosal biopsies were used for measurement of CD11c, CD3, CD4, tryptase, claudin-1, occludin, E-cadherin, and vascular endothelial growth factor A levels; Masson trichrome staining; and POSTN, IL33, TPSAB, TPSB, and CMA gene expression analysis by using quantitative RT-PCR. Blood samples were used for basophil activation tests. RESULTS: Distinct features of the group with severe allergy included the following: (1) impaired epithelial integrity with reduced expression of claudin-1, occludin, and E-cadherin and decreased numbers of epithelial cells, which is indicative of acanthosis, higher collagen deposition, and angiogenesis; (2) inflammatory immune response in the mucosa, with an increased number of CD11c+ and CD4+ infiltrates and increased expression of the cytokine genes POSTN and IL33; and (3) a 10-fold increased sensitivity of basophils to profilin. CONCLUSIONS: Patients with profilin allergy present with significant damage to the oral mucosal epithelial barrier, which might allow profilin penetration into the oral mucosa and induction of local inflammation. Additionally, severely allergic patients presented with increased sensitivity of effector cells.

Delineation of the Olive Pollen Proteome and Its Allergenome Unmasks Cyclophilin as a Relevant Cross-Reactive Allergen.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree (Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.

Disposable Amperometric Immunosensor for the Detection of Adulteration in Milk through Single or Multiplexed Determination of Bovine, Ovine, or Caprine Immunoglobulins G.

This paper reports the first immunoplatforms for the detection of adulteration in milk with milk or colostrum from other animals. The developed electrochemical bioplatforms allow the reliable determination of immunoglobulins G (IgGs) from cows, sheeps, or goats. They rely on sandwiching each animal species-specific IgGs with selective antibody pairs [unconjugated and conjugated with horseradish peroxidase (HRP)] onto magnetic microbeads (MBs) used as solid supports and amperometric transduction with the H2O2/hydroquinone (HQ) system at disposable electrodes. The immunoplatforms allow achieving limits of detection (LODs) of 0.74, 0.82, and 0.66 ng mL-1 for bovine, ovine, and caprine IgGs, respectively, which are lower than those obtained with conventional enzyme-linked immunosorbent assay (ELISA) methodologies and in 2-5 times shorter time. The bioplatforms were successfully applied to the determination of the individual content of the target IgGs in milk samples of different animals (cow, sheep, and goat) and type (colostrum, raw, and pasteurized), without matrix effect and after just a sample dilution. They were also applied to the detection of adulteration with milks from other animals at levels below than those required by the European legislation (1.0%, v/v). The possibility to detect milk adulteration with colostrum using a strategy based on the measurement of the total content of the three target IgGs in raw milks is also demonstrated. Multiplexing platforms were constructed to be used in routine surveillance of milk. They are able to provide in a single run and in just 30 min relevant information regarding the milk sample including its animal origin, the undergone heat treatment, and whether it was adulterated with milk or colostrum from other species.

Effect of processing technologies on the allergenicity of food products.

Heat treatment has been used since ancient times for food processing, first to ensure the safety of food and its storage, but also to transform its characteristics (in its raw form) and obtain new textures, flavors, or novel foods. However, the transformation experienced by food components when heated, or processed, can dramatically affect the allergenicity of food, either reducing or increasing it. To date, most of the articles published dealing with the changes in the potential allergenicity of food are focused on heat treatment and the Maillard reaction. However, it is also important to give prominence to other group of new technologies developed nowadays, such as high-pressure processing, microwaves and food irradiation. These techniques are not likely to replace traditional processing methods, but they are becoming attractive for the food industry due to different reasons, and it is expected in the near future to have different products on the market processed with these new technologies at an affordable cost. Moreover, other biochemical modifications, particularly enzymatic cross-linking of proteins, have attracted wide-spread attention and will be considered as well in this review, because of its great opportunities to induce protein modification and thus affect food allergenicity. Together with the effect of processing of food allergens, this review will place special attention on gastroduodenal digestion of processed allergens, which directly affects their allergenicity.

Anaphylaxis induced by a drug containing lysozyme and papain: influence of papain on the IgE response.

BACKGROUND: This paper reports the case of an egg-allergic pediatric patient who, once desensitized to egg following a successful rush oral immunotherapy protocol, could also tolerate Lizipaina®, a drug containing lysozyme (LYS) and papain, which had previously caused him a severe allergic reaction. Because the LYS amount that elicited the anaphylactic reaction (5 mg) was much lower than that tolerated during a double-blind placebo-controlled food challenge (corresponding to approximately 60 mg of LYS), the possibility that the presence of papain could increase the allergenic potential of LYS was investigated. METHODS: Lizipaina, LYS and LYS hydrolyzed with papain were analyzed by SDS-PAGE under reducing and nonreducing conditions, and Western blotting of sera from egg-allergic patients was performed in order to detect IgE-binding fragments. Finally, sequence identification of the IgE-reactive bands was carried out by MALDI-TOF/TOF. RESULTS: The SDS-PAGE pattern of LYS treated with papain under nonreducing conditions showed the presence of intact LYS that partially disappeared following reduction with ß-mercaptoethanol, releasing IgE-reactive fragments as determined by Western blotting. MALDI-TOF/TOF revealed that papain degraded LYS, giving rise to three IgE-binding fragments: LYS (22-129), LYS (34-96) and LYS (62-128) that likely remained linked through the disulfide bonds present in the LYS molecule. CONCLUSION: The combined administration of LYS with proteolytic enzymes such as papain may have developed a severe allergic reaction in the patient studied, underlining the importance of considering all the components and their interactions when drugs are to be consumed by allergic persons.

Electrochemical bioplatform to manage alpha-gal syndrome by tracking the carbohydrate allergen in meat.

This work presents the first bioplatform described to date for the determination of galactose-α-1,3-galactose (α-Gal), a non-primate mammalian oligosaccharide responsible for almost all cases of red meat allergy. The bioplatform is based on the implementation of an indirect competitive immunoassay and enzymatic labeling with the enzyme horseradish peroxidase (HRP) built on the surface of magnetic microparticles (MBs) and amperometric transduction on screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The target α-Gal competed with biotinylated α-Gal immobilized on the surface of neutravidin-modified MBs for the limited immunorecognition sites of a detection antibody enzymatically labeled with an HRP-conjugated secondary antibody. The resulting magnetic immunoconjugates were trapped on the surface of the SPCE working electrode and amperometric transduction was performed, providing a cathodic current variation inversely proportional to the concentration of α-Gal in the analyzed sample. The developed biotool was optimized, characterized and applied with satisfactory results to the determination of the target allergen in different samples of raw and processed meats.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

BACKGROUND: Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. METHODS: Epithelial activation and/or barrier effects upon exposure to 2-50 µg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 µg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. RESULTS: Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. CONCLUSIONS: Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Molecular Approaches for Food Protein Allergenicity Assessment and the Diagnosis and Treatment of Food Allergies.

Food allergy, an adverse immune reaction triggered by commonly innocuous food proteins, is a health problem that affects millions of people worldwide (around 10% of the global population), and the most recent reports suggest its increasing progression [...].

Egg yolk lipids induce sensitization to egg white proteins in a mouse model without adjuvant and exacerbate Th2 responses to egg white in cells from allergic patients.

This study evaluates the influence of egg lipid fractions in the induction of allergic sensitization to egg white (EW) proteins, using a mouse model of orally adjuvant-free induced allergy. Egg triglycerides (TG) and phospholipids (PL), and to a higher extent the whole egg lipid fraction (EL), induced allergy to EW proteins characterized by increased EW-specific IgG1. EL also increased EW-specific IgE. The administration to mice of a mixture of EW and EL increased the intestinal expression of Il33, Il25, and Tslp, the secretion of IL-33 and IL-6, the expansion of group 2 innate lymphoid cells, the regulation of Gata3, Il4 and Il13, dendritic cell (DC) activation and expression of DC molecules that drive Th2 differentiation. TG promoted the absorption of proteins through the intestinal epithelium, enhancing local Th2 responses, while PL favoured the delivery of antigens to the Peyer's Patches. This differential modulation of the site of absorption of egg proteins determined the different behaviour of TG and PL. Egg yolk lipids also induced activation of Th2-inducing innate responses on intestinal human cells in vitro and enhanced adaptive Th2 functions through the activation of DCs in egg-allergic subjects.

Is There Evidence of Health Risks From Exposure to Micro- and Nanoplastics in Foods?

The human health impact of exposure to micro (MP) and nanoplastics (NP) from food remains unknown. There are several gaps in knowledge that prevent a complete risk assessment of them. First, the fact that some plastics may be chemically harmful, either directly toxic themselves or because they absorb and carry other components, which makes these particles may possess 3 types of hazards, physical, chemical and biological. In addition, the levels at which toxic effects may occur are unknown and there is a lack of studies to estimate the levels to which we are exposed. Plastic particles can induce physical stress and damage, apoptosis, necrosis, inflammation, oxidative stress and immune responses, which could contribute to the development of diseases such as cancer, metabolic disorders, and neurodevelopmental conditions, among others. In addition, they may have effects on other pathologies that have not yet been studied, such as food allergy, where they could act modifying the digestibility of food allergens, increasing intestinal permeability, promoting an intestinal inflammatory environment or causing intestinal dysbiosis, which could promote food allergen sensitization. However, given the limited information on the presence of MP and especially NP in food, further research is needed to estimate whether they could amplify the risk of allergic sensitization to food proteins and to elucidate the risk to human health.

Caco-2 Cell Response Induced by Peptides Released after Digestion of Heat-Treated Egg White Proteins.

The heat treatment of food proteins induces structural modifications that influence their interaction with human fluids and cells. We aimed to evaluate the Caco-2 cell response induced by peptides produced after digestion of heat-treated egg white proteins. In vitro digestion of ovalbumin (OVA), ovomucoid (OM), and lysozyme (LYS), untreated or previously heated, was performed. The digestibility of proteins and the response of Caco-2 cells exposed to peptides (<10 kDa) generated during digestion were evaluated. Intact OVA and LYS persisted after the digestion of native proteins, whereas OM was completely hydrolysed. A heat treatment at 65 °C for 30 min did not alter the digestibility of OVA, whereas at 90 °C for 3 min, protein degradation was favoured. The digestibility of OM and LYS was not affected by heat treatment. Peptides derived from OVA and OM digestion induced IL-6 and IL-8 production. OVA and LYS digestion promoted the expression of Tslp, and Il6 and Il33, respectively. A heat treatment prior to OVA digestion reduced IL-6 production and Tslp expression. It was concluded that heat treatments can reduce the release of OVA-derived peptides, but not OM and LYS, with proinflammatory activity during digestion.

IgE-Binding and Immunostimulating Properties of Enzymatic Crosslinked Milk Proteins as Influenced by Food Matrix and Digestibility.

Dairy foods are essential in the diet, although in some susceptible individuals they may cause allergy to cow's milk proteins. Therefore, alternative methods are sought to reduce their allergenicity. Transglutaminase (TG) is widely used in dairy products mainly to improve texture. Although it has been claimed that TG can be used to modify the digestibility and allergenicity of foods, its impact within a real matrix has been rarely studied. The aim of this work was to assess the allergenic potential of crosslinked skim milk (SM), milk casein fraction (CN), and whey protein (WP). To this purpose, inhibition ELISA with sera from milk allergic patients, in vitro activation tests of mouse mast cells and splenocytes, and simulated gastrointestinal digestion assays were performed. The results showed that cross-linking increased the binding of IgE to WP, but decreased IgE-binding to SM and CN. However, no differences were observed in the ability of cross-linked proteins to induce mast cell degranulation compared to native proteins. The cross-linking of SM and CN reduced Th2 cytokine release from the splenocytes of sensitized mice. All TG-treated samples exhibited more resistance to in vitro digestion than the untreated proteins and the human IgE binding capacity after digestion was higher. In conclusion, TG treatment of milk proteins does not reduce the risk of eliciting allergic symptoms in cow's milk allergic patients.

In vitro simulated semi-dynamic gastrointestinal digestion: evaluation of the effects of processing on whey proteins digestibility and allergenicity.

The effect of thermal processing on digestibility of milk proteins should be better understood as this can greatly affect their immunoreactivity. The aim of this study was to evaluate the effects of thermal processing and lactosylation on digestibility and allergenicity, by comparing non heat-treated with industrially processed whey proteins. A semi-dynamic model was used to mimic the kinetics of digestion, and ELISA inhibition tests against human specific serum IgE were performed on the mass-spectrometry characterized products. A quicker gastric digestion of the industrially treated sample produced a lower immunogenic response in comparison with the raw sample, where intact conformational epitopes remained. In later digests, greater IgE reactivity was shown in the heat treated product, probably due to the release of linear epitopes, while at intestinal level the immunogenic response was negligible. Moreover, transepithelial transport of a reported ß-lactoglobulin-derived allergen, KIDALNENVLVL, produced during digestion was assayed. It was found that the epitope-belonging peptide could be transported through the cell monolayer, both in the native and mono-lactosylated forms, with a favored passage of the native peptide.