A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

Interlaboratory evaluation of a cow's milk allergy mouse model to assess the allergenicity of hydrolysed cow's milk based infant formulas.

This study describes two phases of a multi-phase project aiming to validate a mouse model for cow's milk allergy to assess the potential allergenicity of hydrolysed cow's milk based infant formulas (claim support EC-directive 2006/141/E). The transferability and the discriminatory power of this model was evaluated in 4 research centers. Mice were sensitized by oral gavage with whey or extensively hydrolysed whey (eWH) using cholera toxin as an adjuvant. Whey-specific antibodies, mMCP-1 levels, anaphylactic shock symptoms, body temperature and the acute allergic skin response were determined upon whey challenge. In phases I and II, all 4 centers detected elevated levels of whey-specific IgE/IgG1 in whey sensitized animals. Elevated levels of mMCP-1, anaphylactic symptoms, body temperature drop and acute allergic skin response were scored upon whey challenge in 3 out of 4 research centers. In contrast, none of the evaluated parameters were elevated in eWH orally exposed groups. The cow's milk allergy mouse model is capable to distinguish the sensitizing capacity of complete or hydrolysed cow's milk protein. The model uses straightforward parameters relevant to food allergic responses and can be effectively transferred between different laboratories. We propose this mouse model as a new strategy for the screening of new hypoallergenic cow's milk formulas.

Evaluation of scientific criteria for identifying allergenic foods of public health importance.

Identification of allergenic foods of public health importance should be based on well-defined criteria. Björkstén et al. (2008) proposed that the criteria should assess the evidence for an IgE mechanism, the reaction, the potency and the severity of the effect of the food and its prevalence. This study evaluated the application of the proposed criteria based on published reports. Publications were selected from two databases to test whether the descriptions for ranking the level of evidence for each criterion were unambiguous and covered the full range of levels of evidence regarding seven foods, five known to be allergenic and two negative controls. The options available to rank the quality of evidence were appropriate but needed refinement to improve clarity and conceptual value. The criteria were helpful to assess known IgE-dependent allergens, and to exclude the non-allergenic substances. The criteria framework discriminated between papers with high, moderate and low quality of evidence. The advantage of using the proposed criteria is to make the decision-making process and rationale explicit. The framework helps to identify gaps in knowledge and to uncover the level of heterogeneity of the evidence thus guiding research and providing a basis for sound risk management decisions.

Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract.

Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcepsilonRI was developed. Our objective was to evaluate the degranulation of three cell-lines expressing either the alpha-(RBL-hEI(a)-2B12 and RBL-30/25cells) or alpha-, beta-, and gamma-subunits (RBL SX-38) of the human FcepsilonRI by beta-hexosaminidase release. Purified human IgE and serum-derived polyclonal IgE from peanut-allergic subjects following challenge with anti-IgE or peanut protein extract, respectively, were utilized. Robust degranulation was induced in all three: RBL-30/25 (84%), -hEI(a)-2B12 (54%), SX-38 (94%), respectively, using purified IgE+anti-human IgE. Good release (18%, 40-45%, and 65%, respectively) occurred for one peanut-allergic subject+peanut extract with all cell-lines. With serum from three other peanut-allergic subjects, no beta-hexosaminidase release occurred with RBL-hEI(a)-2B12 cells+peanut extract, while only serum from one subject induced good degranulation, 30% and 60%, respectively, with RBL-30/25 and RBL SX 38 cells. Consistent degranulation with a potent food allergen (peanuts) was not observed. The assay's utility in safety assessment, predictive value and reproducibility for evaluating the cross-reactivity of proteins with allergens needs further investigation with additional proteins and well-characterized sera.