Adjuvant-free Model Research Articles

Analysis of transdermal sensitization and oral anaphylaxis using salt-soluble protein extracts from diploid wheats in an adjuvant-free mouse model

Abstract Wheat allergies are growing at an alarming rate for reasons that are not well understood. The commonly consumed wheats belong to the AABB and AABBDD genotypes. The AA wheat is less commonly consumed, and the DD wheat is not commercially available. It is unknown at present whether the diploid wheats elicit allergic reactions in mice. Here we tested the hypothesis that the salt-soluble protein extracts (SSPEs) from AA and DD wheats will induce food allergy in Balb/cJ male mice in an adjuvant-free model. We grew the DD wheat at our university and used it in this study. The AA wheat was obtained from www.einkorn.com. Balb/cJ male mice were bred and maintained on a plant protein-free diet. Mice were exposed to SSPEs or saline via skin once a week for nine weeks. SSPE-specific IgE (sIgE) was measured using an ELISA. After sensitization, mice were orally challenged with SSPEs or saline to elicit a hypothermia shock response (HSR). We found that both AA- and DD-SSPEs elicited significant and comparable levels of sIgE responses upon skin exposures. Oral challenge with DD-SSPE but not saline in DD-SSPE sensitized elicited a mild HSR lasting up to only 15 minutes. In contrast, oral challenge with AA-SSPE but not saline in AA-SSPE sensitized mice elicited a severe HSR lasting for 30 minutes. These data together suggest that the diploid wheats induce comparable sensitization, but DD wheat is less potent in inducing oral anaphylaxis compared to the AA wheat.

Allergic and anaphylactic responses to alcohol-soluble wheat gluten in female versus male Balb/cJ mice in an adjuvant-free model

Abstract Wheat is a major allergenic food capable of eliciting life-threatening anaphylaxis. Food allergies including wheat allergy are growing globally, and therefore warrant further investigation. While animal studies are being conducted to advance our understanding of wheat allergy, it is unknown whether female versus male mice differ in their allergic responses to wheat gluten. Here we tested the hypothesis that alcohol-soluble wheat gluten (ASWG) when applied over the skin will sensitize both female and male mice for oral hypothermia shock response in an adjuvant-free mouse model. Balb/cJ mice were produced and kept on a plant protein-free diet during this study. Groups of adult mice (n = 8–10) were exposed to durum wheat ASWG via the skin (once a week for nine weeks), without an adjuvant using our previously reported transdermal exposure (TDE) method. In females, TDE with the ASWG elicited a significant ASWG-specific IgE antibody response. However, in males, TDE with the ASWG did not elicit a significant ASWG-specific IgE antibody response. Oral challenge of ASWG, but not vehicle, elicited significant hypothermia shock response in ASWG skin-sensitized female mice. In contrast, no significant hypothermia shock response was observed in males upon oral challenge with ASWG or vehicle. These data demonstrate that female mice are more susceptible than males to develop an allergic response and oral anaphylaxis to ASWG in this model.

Mechanisms of Wheat Allergenicity in Mice: Comparison of Adjuvant-Free vs. Alum-Adjuvant Models.

Wheat protein is considered a major type of food allergen in many countries including the USA. The mechanisms of allergenicity of wheat proteins are not well understood at present. Both adjuvant-based and adjuvant-free mouse models are reported for this food allergy. However, it is unclear whether the mechanisms underlying wheat allergenicity in these two types of models are similar or different. Therefore, we compared the molecular mechanisms in a novel adjuvant-free (AF) model vs. a conventional alum-adjuvant (AA) model of wheat allergy using salt-soluble wheat protein (SSWP). In the AF model, Balb/cJ mice were sensitized with SSWP via skin exposure. In the AA model, mice were sensitized by an intraperitoneal injection of SSWP with alum. In both models, allergic reactions were elicited using an identical protocol. Robust IgE as well as mucosal mast cell protein-1 responses were elicited similarly in both models. However, an analysis of the spleen immune markers identified strikingly different molecular activation patterns in these two models. Furthermore, a number of immune markers associated with intrinsic allergenicity were also identified in both models. Since the AF model uses skin exposure without an adjuvant, the mechanisms in the AF model may more closely simulate the human wheat allergenicity mechanisms from skin exposure in occupational settings such as in the baking industry.

Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity

summary In the absence of extrinsic adjuvants, purified Amb a 1 induced a strong Th2-polarized immune response in mice. This high allergenic activity seems to be linked to the allergen’s susceptibility to endolysosomal proteolysis.

Pathways of immediate hypothermia and leukocyte infiltration in an adjuvant-free mouse model of anaphylaxis

Conflicting results have been obtained regarding the roles of Fc receptors and effector cells in models of active systemic anaphylaxis (ASA). In part, this might reflect the choice of adjuvant used during sensitization because various adjuvants might differentially influence the production of particular antibody isotypes. We developed an "adjuvant-free" mouse model of ASA and assessed the contributions of components of the "classical" and "alternative" pathways in this model. Mice were sensitized intraperitoneally with ovalbumin at weekly intervals for 6weeks and challenged intraperitoneally with ovalbumin 2weeks later. Wild-type animals had immediate hypothermia and late-phase intraperitoneal inflammation in this model. These features were reduced in mice lacking the IgE receptor FcεRI, the IgG receptor FcγRIII or the common γ-chain FcRγ. FcγRIV blockade resulted in a partial reduction of inflammation without any effect on hypothermia. Depletion of monocytes/macrophages with clodronate liposomes significantly reduced the hypothermia response. By contrast, depletion of neutrophils or basophils had no significant effects in this ASA model. Both the hypothermia and inflammation were dependent on platelet-activating factor and histamine and were reduced in 2 types of mast cell (MC)-deficient mice. Finally, engraftment of MC-deficient mice with bone marrow-derived cultured MCs significantly exacerbated the hypothermia response and restored inflammation to levels similar to those observed in wild-type mice. Components of the classical and alternative pathways contribute to anaphylaxis in this adjuvant-free model, with key roles for MCs and monocytes/macrophages.

Different allergenicity profile of egg white protein extract vs. purified ovalbumin in an adjuvant-free mouse model (HYP7P.267)

Abstract Food allergies are the leading cause of life-threatening allergic reactions with incompletely understood mechanisms. Here we initially evaluated the allergenicity of egg white protein extract (EWPE) in a novel transdermal sensitization model without adjuvants such as alum/cholera toxin/super-antigen that are commonly used in mouse models of egg allergy. Groups of mice received transdermal exposure (TDE) to EWPE (1 mg/exposure/week) for four weeks. Sensitization was measured by specific IgE antibody (SIgE Ab) levels in plasma using ELISA. Systemic anaphylaxis was assessed upon oral challenge (OC) with EWPE using clinical scoring method, and hypothermia reactions. Four TDEs were sufficient to elicit robust SIgE Ab responses. Upon OC (15 mg/mouse), EWPE elicited near-fatal clinical signs of anaphylaxis associated with rapid hypothermia reactions. We then evaluated allergenicity of purified ovalbumin (OVA) using this model. Groups of mice received TDE to OVA at 4 different doses. Robust OVA SpIgE responses were observed upon TDE. Surprisingly, sensitized mice did not exhibit significant clinical symptoms or hypothermia reactions upon OC with a wide dose range. Nevertheless, robust anaphylaxis was evident upon i.p., injection with OVA. These data suggest that mechanisms of oral egg protein-induced reactions in adjuvant-free model likely depends on whether protein extracts or pure protein from the same food source (egg) is used in model development.