The role of skin inflammation, barrier dysfunction, and oral tolerance in skin sensitization to gluten-derived hydrolysates in a rat model.

BACKGROUND: Adverse reactions to wheat-containing skin care products have been linked to food allergy development. OBJECTIVES: To determine the role of skin barrier dysfunction and inflammation in sensitization to gluten-derived hydrolysates via the skin in Brown Norway rats with and without oral tolerance to wheat. METHODS: Skin barrier defect was induced by mechanical disruption, and skin inflammation was induced by topical application of SLS or MC903. Unmodified, enzyme hydrolyzed, or acid hydrolyzed gluten products were applied to the skin three times per week for 5 weeks. Subsequently, rats were orally gavaged with unmodified gluten. RESULTS: Wheat-naïve rats were readily sensitized to gluten hydrolysates via the skin. Skin barrier defect and skin inflammation had little effect on the skin sensitization and hydrolysate-specific IgE levels. Oral administration of unmodified gluten promoted the production of unmodified gluten-specific IgE in rats sensitized via the skin. Sensitization through intact skin, disrupted skin barrier, or inflamed skin was unable to break tolerance to unmodified gluten in rats on a wheat-containing diet. CONCLUSIONS: Mechanical skin barrier disruption and skin inflammation play a limited role in experimental skin sensitization to gluten-derived hydrolysates.

The use of aluminium hydroxide as adjuvant modulates the specific antibody response-A Brown Norway rat study with native and denatured cow's milk allergens.

There is a need for efficient methods to treat food allergy; however, no immunotherapeutic method has yet been satisfactory due to the high rate of unpredictable severe reactions and the limited efficacy. Therefore, modified versions of food allergens have been suggested as alternatives to the parent proteins for immunotherapy. The aim of the study was to compare the inherent allergenicity of the native and denatured version of the cow's milk proteins ß-lactoglobulin and α-lactalbumin, and to study the impact of the use of Al(OH)3 as an adjuvant. Brown Norway rats were immunized intraperitoneally with either native or denatured ß-lactoglobulin or α-lactalbumin, with or without the use of Al(OH)3 as adjuvant. Antibody responses were analysed in various ways by means of different ELISAs. Both the immunogenicity and the sensitizing capacity of the cow's milk allergens were influenced by their globular folding, with the native version being more allergenic than the denatured counterpart. The native folded proteins mainly raised antibodies against conformational epitope, whereas the denatured versions predominantly raised antibodies against linear epitopes. Most interestingly, the study showed that the use of Al(OH)3 , besides increasing immunogenicity and sensitizing capacity of the cow's milk allergens, caused a modification of the specificity of the antibodies raised against the native version of the proteins. Adsorption of the native forms of the allergens to Al(OH)3 caused a significant greater proportion of antibodies raised against linear epitopes, stressing that the adsorption induced a partly unfolding of the proteins. This may have implications for IT safety and efficacy.

An Animal Model for Wheat Allergy Skin Sensitisation: A Comparative Study in Naive versus Tolerant Brown Norway Rats.

BACKGROUND: Allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food, leading to the assumption that food allergy sensitisation may occur through the skin. Concerns have been raised regarding the safety of use of personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy through the skin, and even cause an abrogation of an already established oral tolerance. OBJECTIVE: To establish an animal model for food allergy skin sensitisation and compare the sensitising capacity of an unmodified and an acid-hydrolysed gluten product via slightly damaged skin in naïve versus tolerant rats. METHODS: Gluten products were applied on the slightly damaged skin of naïve or tolerant Brown Norway (BN) rats without adjuvant 3 times per week for 3 or 5 consecutive weeks. The effect of the skin applications was evaluated by means of different ELISAs and immunoblotting. RESULTS: A robust animal model was developed for food allergy skin sensitisation. In naïve rats, both gluten products were able to induce a statistically significant level of specific antibodies and sensitise through the skin, but in the wheat-tolerant rats, only the acid-hydrolysed gluten was able to sensitise through the skin, albeit at a level much lower than in the naïve rats. Results showed that new epitopes had been developed as a result of acid hydrolysis but original epitopes were maintained. This may explain why only the acid-hydrolysed gluten could induce specific antibody responses in the tolerant animals. CONCLUSIONS: This study showed that it is possible to sensitise BN rats through slightly damaged skin, and that the sensitising capacity is heavily influenced by the tolerance status of their immune system and the degree of modification of the wheat products.

Preclinical Brown Norway Rat Models for the Assessment of Infant Formulas in the Prevention and Treatment of Cow's Milk Allergy.

BACKGROUND: Infant formulas (IFs) based on hydrolysed cow's milk proteins are central in the management of cow's milk allergy (CMA) in infants and small children. New IF compositions with improved prevention and treatment properties are needed, along with appropriate preclinical animal models, to evaluate these properties before introduction into humans. OBJECTIVES: We aimed to develop preclinical models for the assessment of the primary preventive and desensitising capacity of cow's milk IF in allergy-prone, high-IgE responder Brown Norway rats. METHOD: Preventive capacity was assessed in cow's milk-naïve rats given a 2- or 4-week regimen of whey-based extensively hydrolysed IF (eHF), partially hydrolysed IF (pHF), or intact ß-lactoglobulin (BLG) ad libitum in drinking bottles, followed by intraperitoneal (i.p.) immunisation with BLG. Desensitising capacity was assessed in orally BLG-sensitised rats after a 3- or 6-week regimen of eHF, pHF, or intact BLG administration in drinking bottles, followed by i.p. challenge with BLG. Primary preventive and desensitising capacity were analysed by serum BLG-specific IgG1 and IgE. RESULTS: The preventive regimens did not induce detectable BLG-specific IgG1 or IgE in cow's milk-naïve rats. A preventive regimen consisting of pHF or BLG, but not eHF, induced complete tolerance to BLG, as demonstrated by the absence of BLG-specific IgE following i.p. immunisation. Desensitising regimens had a limited effect on BLG-specific IgG1 or IgE when comparing sensitised rats before and after treatment. Challenge with BLG (i.p.) increased BLG-specific IgE in all treatment regimens except for in the BLG group, suggesting a limited desensitising capacity of IF based on hydrolysates and a need for the presence of intact allergen for desensitisation. CONCLUSIONS: The presented models highlight that different mechanisms are at play in the induction of de novo tolerance to cow's milk proteins and the desensitisation of CMA. Different IF products may be needed for the primary prevention and treatment of CMA.

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3-8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.

The sensitising capacity of intact ß-lactoglobulin is reduced by co-administration with digested ß-lactoglobulin.

BACKGROUND: It is generally believed that protein hydrolysis in the gastrointestinal tract decreases the allergenicity of food allergens. However, it remains unknown if specific properties of digestion products determine whether a sensitisation or tolerogenic immune response will develop. We sought to examine the sensitising capacity of the cow's milk allergen ß-lactoglobulin (BLG) and digestion products thereof in a Brown Norway (BN) rat model. METHODS: Intact BLG was digested in an in vitro model simulating the gastro-duodenal digestion process and subsequently fractionated by gel permeation chromatography. BN rats were dosed with either PBS, 200 µg of intact BLG, 30 µg of intact BLG, 200 µg of partially digested BLG, 200 µg of digested BLG, or with 200 µg of a fraction of large complexes or a fraction of small complexes. Sera from BN rats were analysed for specific antibodies and avidity was measured. RESULTS: BLG partly resisted the digestion process. However, the BLG molecules that did not survive the digestion process were rapidly broken down to peptides of sizes less than Mr 4,500. Specific antibody responses revealed that both 200 and 30 µg of intact BLG had immunogenic as well as sensitising capacity, while digested BLG could not induce any specific antibodies. Most importantly, while intact BLG showed a significant sensitising capacity when administered alone, this sensitising capacity was significantly reduced when co-administered with digested BLG. CONCLUSIONS: Co-immunisation of intact BLG with digested BLG reduces the sensitising capacity of intact BLG, which could result from tolerogenic mechanisms induced by the digestion products.

Risk of consuming products with or without precautionary wheat or gluten labelling for persons with coeliac disease.

Persons with Coeliac disease (CD) need to keep a lifelong diet without gluten and therefore need to know which foods are safe to eat. The objective of this study was to analyse ordinary foods products without gluten containing ingredients (PWG) with or without the precautionary allergen statement (PAL) "May contain wheat/gluten" or similar wording and use these data to perform a probabilistic risk assessment. Foods with and without PAL were analysed for gluten. Inputs for the risk assessment were analytical results, daily food consumption and data from a 90-days challenge study in coeliac patients (Catassi et al., 2007).20/77 products with PAL and 10/51 without PAL had gluten ≥5 mg/kg 3/77 with PAL and 3/51 without PAL had gluten ≥20 mg/kg. A coconut cookie brand was outlier with 421 mg gluten/kg. The likelihood of developing mucosal damage in the CD population when eating the same PWG for 90 days in addition to certified gluten-free products was low (0.2-3.2%) with little difference between with or without PAL. Including the coconut cookie results increased the risk to 3.8-9.2%. Caution should be taken in connection to foods where there is an obvious chance of contamination.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Gluten tolerance prevents oral sensitization with enzymatic or acid hydrolyzed gluten: A study in Brown Norway rats.

BACKGROUND: There are several reports describing allergy to hydrolyzed wheat products. After a large outbreak in Japan it was established that sensitization was caused by skin contact with acid hydrolyzed gluten in soap. It is still not clear if other forms of hydrolyzed gluten may sensitize, and if the skin is the only relevant route of sensitization in humans and to what extent oral tolerance to wheat play a role. OBJECTIVES: The aim of the present study was to examine if wheat-tolerant rats may be sensitized via the oral or i.p. route when exposed to gluten, enzymatic or acid hydrolyzed gluten. METHODS: Brown Norway rats, tolerant to wheat, were dosed by three i.p. injections without adjuvant or by oral gavage daily for 35 days with the three gluten products, respectively. Sera were analyzed by ELISA for specific IgG1 and IgE. In addition inhibition and avidity ELISAs were performed. Results were compared to a similar study in rats naïve to wheat. RESULTS: More than half the animals had measurable IgG1 at the start of the dosing period. I.p. immunization resulted in significant specific IgG1 and IgE to the antigen used for immunization but significantly lower than in naïve rats. The results of inhibition and avidity ELISA's indicate that the underlying tolerance to epitopes common to the three products influences the immune response. Oral dosing did not induce significant changes in response to either gluten or the hydrolyzed gluten product used for dosing. CONCLUSIONS: The study shows that i.p. immunization with the three products can break the underlying tolerance to wheat. Exposure by the oral route to enzymatic or acid hydrolyzed gluten is very unlikely to break an already established tolerance to gluten and induce sensitization.

Defining the targets for the assessment of IgE-mediated allergenicity of new or modified food proteins.

Many food innovations rely on the introduction and use of new or modified proteins. New or modified food proteins may lead to major health risks due to their inherent potential to cause food allergy. Currently, the pre-market allergenicity assessment for new or modified food proteins and protein sources relies on methods for identifying allergenic hazards based on characteristics of known allergens. However, there is no general consensus on the allergenicity parameters to use and the criteria that should apply for the evaluation and decisions to be made. In this paper, we propose that the strategy for allergenicity risk assessment of new or modified food proteins and the methodologies applied should be governed by the risk management questions to be answered, reflected in the information needed by risk managers to enable their informed decision making. We generated an inventory of health outcome-related assessment parameters and criteria potentially important for risk management decision-making and we discuss the implications of selecting different optional criteria (e.g. cut-off values) for what could be accepted as safe with regards to the health outcomes in the (at risk) population. The impact of these various options on both method development and risk management practices was investigated.

The impact of structural integrity and route of administration on the antibody specificity against three cow's milk allergens - a study in Brown Norway rats.

BACKGROUND: Characterisation of the specific antibody response, including the epitope binding pattern, is an essential task for understanding the molecular mechanisms of food allergy. Examination of antibody formation in a controlled environment requires animal models. The purpose of this study was to examine the amount and types of antibodies raised against three cow's milk allergens; ß-lactoglobulin (BLG), α-lactalbumin (ALA) and ß-casein upon oral or intraperitoneal (i.p.) administration. A special focus was given to the relative amount of antibodies raised against linear versus conformational epitopes. METHODS: Specific antibodies were raised in Brown Norway (BN) rats. BN rats were dosed either (1) i.p. with the purified native cow's milk allergens or (2) orally with skimmed milk powder (SMP) alone or together with gluten, without the use of adjuvants. The allergens were denatured by reduction and alkylation, resulting in unfolding of the primary structure and a consequential loss of conformational epitopes. The specific IgG1 and IgE responses were analysed against both the native and denatured form of the three cow's milk allergens, thus allowing examination of the relative amount of linear versus conformational epitopes. RESULTS: The inherent capacity to induce specific IgG1 and IgE antibodies were rather similar upon i.p. administration for the three cow's milk allergens, with BLG = ALA > ß-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > ß-casein. Co-administration of SMP and gluten had a great impact on the specific antibody response, resulting in a significant reduced amount of antibodies. Together results indicated that most antibodies were raised against conformational epitopes irrespectively of the administration route, though the relative proportions between linear and conformational epitopes differed remarkably between the allergens. CONCLUSIONS: This study showed that the three-dimensional (3D) structure has a significant impact on the antibodies raised for both systemic and orally administered allergens. A remarkable difference in the antibody binding patterns against linear and conformational epitope was seen between the allergens, indicating that the structural characteristics of proteins may heavily affect the induced antibody response.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.