[Show abstract][Hide abstract] ABSTRACT: Allergic sensitisation to food may occur through non-gastrointestinal routes such as via skin or lung. We recently demonstrated in mice that cutaneous or respiratory pre-exposures to peanut proteins on intact epithelia induce a Th2 priming and allow subsequent oral sensitization without the use of adjuvant. We then aimed to assess the impact of a similar pattern of exposure to another relevant food allergen, cows' milk.
The humoral and cellular immune response induced in BALB/cJ mice after repeated cutaneous applications on intact skin or after intranasal administration of cows' milk proteins was analysed. In order to assess the potential effect of the food matrix, we used either a purified major cows' milk allergen, β-lactoglobulin (BLG), or whole cows' milk containing the same amount of BLG. We then studied the impact of these pre-exposures on a subsequent oral exposure to milk in the presence or absence of the mucosal Th2 adjuvant, Cholera toxin (CT). Cutaneous applications of milk induced production of BLG-specific IgE and IgG1 in 5 and 8 mice out of 20 respectively, whereas purified BLG alone did not. Intranasal exposure to milk, but not to BLG, led to BLG-specific IgG1 production in 8 out of 20 mice. Notably, cutaneous pre-exposure to milk favours further oral sensitisation without CT, while intra-nasal pre-exposure to BLG prevents further experimental sensitisation.
Altogether, our results thus demonstrated that the immune response induced after non-gastrointestinal exposure to food depends on the allergen, the matrix and the route of exposure.
[Show abstract][Hide abstract] ABSTRACT: Precautionary allergen labelling (PAL) was introduced by the food industry to help manage and communicate the possibility of reaction from the unintended presence of allergens in foods. However, in its current form, PAL is counter-productive for consumers with food allergies. This review aims to summarise the perspectives of all the key stakeholders (including clinicians, patients, food industry and regulators), with the aim of defining common health protection and risk minimisation goals. The lack of agreed reference doses, has resulted in inconsistent application of PAL by the food industry and in levels of contamination that prompt withdrawal action by enforcement officers. So there is a poor relationship between the presence or absence of PAL and actual reaction risk. This has led to a loss of trust in PAL, reducing the ability of consumers with food allergies to make informed choices. The result has been reduced avoidance, reduced quality of life and increased risk-taking by consumers who often ignore PAL. All contributing stakeholders agree that PAL must reflect actual risk. PAL should be transparent and consistent with rules underpinning decision-making process being communicated clearly to all stakeholders. The use of PAL should indicate the possible, unintended presence of an allergen in a consumed portion of a food product at or above any proposed action level. This will require combined work by all stakeholders to ensure everyone understands the approach, and its limitations. Consumers with food allergy then need to be educated to undertake individualised risk assessments in relation to any PAL present. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging “omics” technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that “unintended” does not necessarily mean “harmful”. This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics.
Electronic supplementary material
The online version of this article (doi:10.1007/s11248-015-9867-7) contains supplementary material, which is available to authorized users.
Full-text · Article · Feb 2015 · Transgenic Research
[Show abstract][Hide abstract] ABSTRACT: The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event.
Electronic supplementary material
The online version of this article (doi:10.1007/s00204-014-1374-8) contains supplementary material, which is available to authorized users.
Full-text · Article · Oct 2014 · Archives of Toxicology
[Show abstract][Hide abstract] ABSTRACT: Background:
Recent data suggested that non-gastrointestinal exposure can lead to sensitisation to food allergens. We thus assessed the immune impact of respiratory or cutaneous exposure to peanut proteins on non-altered epithelium and investigated the effect of such pre-exposure on subsequent oral administration of peanut.
BALB/cJ mice were exposed to purified Ara h 1 or to a non-defatted roasted peanut extract (PE) by simple deposit of allergens solutions on non-altered skin or in the nostrils. Exposures were performed 6 times at weekly intervals. Pre-exposed mice then received intra-gastric administrations of PE alone or in the presence of the Th2 mucosal adjuvant cholera toxin (CT). The specific humoral and cellular immune response was assessed throughout the protocol.
Both cutaneous and respiratory exposures led to the production of specific IgG1. Local and systemic IL-5 and IL-13 production were also evidenced, demonstrating activation of specific Th2 cells. This effect was dose-dependent and most efficient via the respiratory route. Moreover, these pre-exposures led to the production of specific IgE antibodies after gavage with PE, whatever the presence of CT.
Cutaneous or respiratory exposures to peanut induce Th2 priming in mice. Moreover, pre-exposures promote further sensitisation via the oral route without the use of CT; this proposes a new adjuvant-free experimental model of sensitisation to food that may reflect a realistic exposure pattern in infants. These results also suggest that non-gastrointestinal peanut exposure should be minimised in high-risk infants, even those with non-altered skin, to potentially reduce allergic sensitisation to this major food allergen.
No preview · Article · Jul 2014 · International Archives of Allergy and Immunology
[Show abstract][Hide abstract] ABSTRACT: Experimental in silico, in vitro, and rodent models for screening and predicting protein sensitizing potential are discussed, including whether there is evidence of new sensitizations and allergies since the introduction of genetically modified crops in 1996, the importance of linear versus conformational epitopes, and protein families that become allergens. Some common challenges for predicting protein sensitization are addressed: (a) exposure routes; (b) frequency and dose of exposure; (c) dose-response relationships; (d) role of digestion, food processing, and the food matrix; (e) role of infection; (f) role of the gut microbiota; (g) influence of the structure and physicochemical properties of the protein; and (h) the genetic background and physiology of consumers. The consensus view is that sensitization screening models are not yet validated to definitively predict the de novo sensitizing potential of a novel protein. However, they would be extremely useful in the discovery and research phases of understanding the mechanisms of food allergy development, and may prove fruitful to provide information regarding potential allergenicity risk assessment of future products on a case by case basis. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute.
[Show abstract][Hide abstract] ABSTRACT: Cow's milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cow's milk β-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age.
Oral sensitization to cow's milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to αS1- and κ-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice.
Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.
No preview · Article · Dec 2012 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: The immunomodulatory potential of fragments derived from the cow's milk allergen bovine β-lactoglobulin (BLG) was assessed in a mouse model of oral tolerance (OT) [Adel-Patient, K.; Wavrin, S.; Bernard, H.; Meziti, N.; Ah-Leung, S.; Wal, J. M. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine β-lactoglobulin. Allergy2011, 66 (10), 1312-1321]. Native BLG (nBLG) and chemically denatured BLG (lacking S-S bridges, dBLG), products resulting from their hydrolysis using cyanogen bromide (CNBr) and some synthetic peptides, were produced and precisely characterized. CNBr hydrolysates correspond to pools of peptides of various sizes that are still associated by S-S bridges when derived from nBLG. nBLG, dBLG, and CNBr hydrolysate of nBLG efficiently prevented further sensitization. CNBr hydrolysate of dBLG was less efficient, suggesting that the association by S-S bridges of peptides increased their immunomodulatory potential. Conversely, synthetic peptides were inefficient even if covering 50% of the BLG sequence, demonstrating that the immunomodulatory potential requires the presence of all derived fragments of BLG and further supporting the use of partially hydrolyzed milk proteins to favor OT induction in infants with a risk of atopy.
No preview · Article · Oct 2012 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Despite a sequence homology of 90% between bovine and caprine β-caseins (CN), IgE antibodies from patients allergic to goat's milk (GM), but tolerant to cow's milk (CM), recognize caprine β-CN without cross-reacting with bovine β-CN. We investigated this lack of cross-reactivity by evaluating the IgE-reactivity toward peptides isolated from plasmin hydolysates of bovine and caprine β-CN.
The IgE-binding capacity of plasmin-derived peptides was evaluated with sera from 10 CM-allergic patients and 12 GM-allergic/CM-tolerant patients. In CM-allergic patients, IgE reactivity of caprine fragments (f29-107) and (f108-207), but not (f1-28), was similar to that of the bovine counterparts. In contrast, all bovine fragments were poorly recognized by IgE antibodies from GM-allergic/CM-tolerant patients. The peptide (f29-107) was generally the most immunoreactive fragment of caprine β-CN. By using synthetic peptides, the immunodominant IgE-binding epitope recognized by most GM-allergic/CM-tolerant patients was located in the caprine domain 49-79.
The restricted specificity of the IgE response toward the caprine β-CN in GM-allergic/CM-tolerant patients is mainly directed against the domain 49-79, which differs from its bovine counterpart by only three amino acid substitutions.
No preview · Article · Oct 2012 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.
Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.
1D (1)H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D (1)H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins.
[Show abstract][Hide abstract] ABSTRACT: 2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated.
Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71) or at Cys(73) -Cys(115) rendered Arg(16/20) or Arg(114) susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58) or at Cys(59) -Cys(107) led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products.
Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation.
No preview · Article · Apr 2012 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1.
Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein.
Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.
No preview · Article · Dec 2011 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ-free (GF) mice exhibit Th2-polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant.
β-lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ- and αS1-caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG-reactivation, IL-4, IL-5, IL-13 and IFN-γ productions by splenocytes were higher in GF mice than in CV mice. Heat-treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat-treatment increased protein immunogenicity and led to the production of anti-BLG and anti-κ-casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL-4, IL-5 and IL-13 in BLG-reactivated splenocytes from CV mice.
Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.
No preview · Article · Nov 2011 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut.
Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6.
Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.
[Show abstract][Hide abstract] ABSTRACT: Cow's milk (CM) allergy affects about 2% of infants. The allergenicity of dietary proteins, including those from CM, has been related to their digestibility although the generality of the link and its causality remains to be demonstrated. In this study we use an in vitro digestion system, to investigate the digestibility of β-lactoglobulin (blg) during gastrointestinal transit and to assess the impact of this process on blg allergenic reactivity in CM allergic children.
Blg digesta were prepared using an in vitro digestion protocol simulating either gastric digestion alone or followed by duodenal digestion with or without phosphatidylcholine (PC). Biochemical analysis of blg digesta was performed by SDS-PAGE and their concentration was measured by a sandwich ELISA. Assessment of their allergenic reactivity was done in vitro by EAST inhibition, specific basophil activation (basotest) and lymphocyte proliferation (PCNA-flow cytometry) assays using sera and cells from patients allergic to blg and in vivo by skin prick testing (SPT) of these patients.
Blg was only broken down to smaller peptides after gastro-duodenal digestion although a sizeable amount of intact protein still remained. Digestion did not modify the IgE binding capacity of blg except for gastro-duodenal digestion performed in the absence of PC. These results are consistent with the quantity of intact blg remaining in the digesta. Overall both gastric and gastroduodenal digestion enhanced activation of sensitized basophils and proliferation of sensitized lymphocytes by blg. However, there was a tendency towards reduction in mean diameter of SPT following digestion, the PC alone during phase 1 digestion causing a significant increase in mean diameter.
Digestion did not reduce the allergenic reactivity of blg to a clinically insignificant extent, PC inhibiting digestion and thereby protecting blg allergenic reactivity. SPT reactivity was reduced compared to blg immunoreactivity in in vitro tests.