Stine Kroghsbo

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

Background Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion.

Occurrence of the carcinogenic Bracken constituent ptaquiloside in fronds, topsoils and organic soil layers in Denmark

Bracken (Pteridium aquilinum (L.) Kuhn) is a common fern found on all continents except Antarctica. It is under suspicion of causing cancer among people who utilizes it as food. The main carcinogenic compound is thought to be the water-soluble compound ptaquiloside. Ptaquiloside-uptake may occur not only through food, but also via drinking water as ptaquiloside might leach from plant material. The purpose of the study was to identify environmental parameters that correlate with the ptaquiloside-content in fronds, and to quantify the amount of ptaquiloside in the soil environment. The ptaquiloside-content in fronds, Oi/Oe-, and Oa/A-horizons was quantified at end of the growth season at 20 sites in Denmark. The fronds had ptaquiloside-contents between 108 and 3795 microgg(-1). The Oi/Oe-horizons had contents between 0.09 and 7.70 microgg(-1), while Oa/A-horizons had contents between 0.01 and 0.09 microgg(-1). The ptaquiloside-content in the standing biomass, which could be transferred to the soil by the end of the growing season, ranged between 10 and 260 mgm(-2), with nine sites having ptaquiloside loads over 100 mgm(-2). The carbon-content in the O-horizon, the precipitation, the amount of Bracken-litter, the turnover rate and the size of Bracken-stands determined the ptaquiloside-content in the soil materials while the content in fronds was found to be a function of the frond-height and the light-exposure in the ecosystem.

Sensitization with 7S Globulins from Peanut, Hazelnut, Soy or Pea Induces IgE with Different Biological Activities Which Are Modified by Soy Tolerance

Background: It is not known why some foods sensitizing via the gastrointestinal tract are prevalent allergenic foods and others are not. Eating habits, processing, and the food matrix have been suggested to influence the allergenicity of a given food. Factors related to protein structure, such as stability to digestion, have also been suggested. 7S globulins from peanut, hazelnut, soy, and pea were studied to determine whether related proteins would induce a similar sensitization when removed from their ‘normal’ matrix. Methods: Brown Norway rats (soy tolerant or nontolerant) were immunized i.p. 3 times with 100 µg purified peanut, hazelnut, soy, or pea 7S without adjuvant. Sera were analyzed for specific antibodies by different ELISAs (IgG1, IgG2a, and IgE), inhibition ELISA, and rat basophilic leukemia cell assay. Results: The 4 related 7S globulins induced a response with an almost identical level of specific antibodies, but peanut 7S induced IgE of higher avidity than hazelnut and pea 7S which, again, had a higher avidity than IgE induced by soy 7S. Soy tolerance reduced the functionality of IgE without influencing antibody titers. Conclusions: Although the 4 7S globulins are structurally related allergens, they induce antibodies with different antigen-binding characteristics. Peanut 7S induces IgE of a higher avidity than hazelnut and pea 7S which, again, has a higher avidity than IgE induced by soy 7S. We also show that soy tolerance influences the function of antibodies to peanut 7S. These findings may help explain how antibodies of different clinical significances can develop in different individuals sensitized to the same allergen.

Experimental Parameters Differentially Affect the Humoral Response of the Cholera-Toxin-Based Murine Model of Food Allergy

Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle immunomodulating factors or factors effective during response initiation, this maximal response-based model is less suitable due to a lack of sensitivity. Therefore, in attempts to identify essential parameters to fine-tune the immune response towards a submaximal level, potentially more sensitive, we were interested in characterizing the individual effects of the parameters in the CT-based model: CT dose, antigen type and dose, and number of immunizations. Methods: BALB/c mice were orally sensitized weekly for 3 or 7 weeks with graded doses of CT and various food antigens (soy-trypsin inhibitor, ovalbumin or ovomucoid). Antigen-specific IgG1, IgG2a, IgA and IgE were monitored by ELISA. Results: The CT dose exerted a clear dose-dependent effect on the antigen-specific antibody response whereas the antigen dose tended to affect the kinetics of the developing response. Both the intensity and kinetics of the antibody response depended on the type of antigen and number of immunizations. Conclusions: The critical parameters of the CT-based murine allergy model differentially control the intensity and kinetics of the developing immune response. Adjustment of these parameters could be a key tool for tailoring the response to submaximal levels rendering the model potentially more sensitive for evaluating the effect of subtle immunomodulating factors that would be lost in the maximal response-based model.

Assessment of the Sensitizing Potential of Processed Peanut Proteins in Brown Norway Rats: Roasting Does Not Enhance Allergenicity

Background IgE-binding of process-modified foods or proteins is the most common method for examination of how food processing affects allergenicity of food allergens. How processing affects sensitization capacity is generally studied by administration of purified food proteins or food extracts and not allergens present in their natural food matrix. Objectives The aim was to investigate if thermal processing increases sensitization potential of whole peanuts via the oral route. In parallel, the effect of heating on sensitization potential of the major peanut allergen Ara h 1 was assessed via the intraperitoneal route. Methods Sensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN) rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-), heated (H-) or heat glycated (G-)Ara h 1. Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL) cell assay. Results In rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the lowest level of RBL degranulation. However, extract from roasted peanuts was found to be a superior elicitor of RBL degranulation. Process-modified Ara h 1 had similar sensitizing capacity as NAra h 1 but specific IgE reacted more readily with process-modified Ara h 1 than with native. Conclusions Peanut products induce functional specific IgE when dosed orally to BN rats. Roasted peanuts do not have a higher sensitizing capacity than blanched peanuts. In spite of this, extract from roasted peanuts is a superior elicitor of RBL cell degranulation irrespectively of the peanut product used for sensitization. The results also suggest that new epitopes are formed or disclosed by heating Ara h 1 without glucose.

Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

Background Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis. Objectives To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten. Methods High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay. Results Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of ‘new’ epitopes. Conclusions In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten.

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

BackgroundCharacterisation 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; β-lactoglobulin (BLG), α-lactalbumin (ALA) and β-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.MethodsSpecific 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.ResultsThe 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 > β-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > β-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.ConclusionsThis 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.

Roasting or heating increases elicitation capacity of peanut allergens but does not affect their sensitisation potential in a brown Norway rat model for food allergy

Background Allergenic potential of processed food allergens has primarily been studied by their IgE-binding capacity (elicitation). Roasting of peanuts has been shown to increase IgE-binding capacity. In this study we examined whether processing of whole peanuts or of the major peanut allergen Ara h 1 influenced the sensitisation potential. Methods Brown Norway rats were either dosed orally by gavage each day for 42 days with finely ground whole peanut products (blanched or roasted peanuts or peanut butter) mixed with water [~2 mg Ara h 1/rat/day] or immunised i.p. three times with 200 μg of native, heated or heat glycated Ara h 1. Sera obtained at sacrifice were analysed for specific IgG and IgE by ELISA and for biological functionality of IgE by rat basophilic leukaemia (RBL) assay. Results Processing was found to decrease solubility and thus extractability of Ara h 1 from peanut products. Aggregation state and secondary structure changes induced by heating of purified Ara h 1 were identical to those observed when Ara h 1 was heated in the presence of glucose. Although a significant anti-Ara h 1 IgE response was only found when dosing rats with roasted peanuts, examination of functional specific IgE by RBL assay showed that processing of peanuts did not influence sensitisation potential. However, extract from roasted peanuts was found to be a superior elicitor compared to extract from blanched peanuts irrespective of the peanut product used for sensitisation. Processing of purified native Ara h 1 did not influence the sensitisation capacity. Nonetheless, ELISA results indicated that new epitopes are formed or disclosed by heating of Ara h 1. Furthermore, IgG1-binding capacity was found to reflect whether rats were sensitised to native or processed Ara h 1 or dosed with blanched or roasted peanut products. Conclusion Roasted peanuts, either as such or as peanut butter, do not have a higher sensitisation capacity than blanched peanuts. This is supported by the finding that processmodified Ara h 1 has a similar sensitisation capacity as native Ara h 1. Yet, our results show that roasting increases elicitation capacity.

Linear versus conformational epitopes of three cow's milk allergens

Method Specific antibodies were raised in a Brown Norway (BN) rat model of food allergy. BN rats were dosed either (1) intraperitoneally (i.p.) with the purified native cow’s milk allergens with or without the use of Al(HO)3 as adjuvant or (2) orally with skimmed milk powder without the use of adjuvant. The specific IgG1 and IgE antibody responses were analysed against native and denatured allergens by means of different ELISAs.

Enzymatic hydrolysis of gluten proteins changes the dose-response of oral sensitization, but not the gluten epitopes

Background Wheat gluten proteins are very complex proteins containing hundreds of components present as monomers, oligomers and polymers that by definition are not soluble in water. To improve utilization, gluten proteins can be hydrolyzed by enzymes or acid. This increases solubility and provide proteins with new functional properties. During the last decade, cases of severe allergic reaction to hydrolyzed wheat proteins have been reported in subjects tolerant to wheat.