## 7 Animal Studies Supporting the Use of Heated Proteins as Immunotherapeutic Agents  
There has been some work in animal models of allergy describing the effect on ingestion of heated egg proteins, but little about heated milk proteins. [PERSON] [64] demonstrated that heat treatment of ovalbumin and ovomucoid reduces the allergen partly by changing the digestibility of the protein. Egg-sensitized C3H/HeJ mice were challenged with either untreated or heated ovalbumin or ovomucoid. Oral challenge with the unheated protein induced anaphylaxis in all of the mice, but not when challenged with the heated proteins. Even when challenged intraperitoneally, the majority of the mice only displayed mild reactions. In a separate experiment, mice fed heated ovalbumin had minimal T-cell proliferation in the mesenteric lymph nodes, or Peyer's Patches, compared with those consuming unheated ovalbumin, suggesting that heating ovalbumin affects the intestinal absorption of the intact form capable of triggering effector cells and T-cells. In this same paper, [PERSON] [64] also reported that heated ovalbumin and ovomucoid still retained the ability to bind to serum from egg allergic children on immunoblotting, indicating that the epitopes were still intact.  
[PERSON] [73] used a mouse model of SOTI to show that heated egg protein (ovomucoid) is able to desensitize egg allergic mice as effectively as unheated egg protein. C3H/HeJ mice sensitized to egg were treated with increasing doses of egg white or heated or native ovomucoid. Mice were challenged with ovalbumin or ovomucoid either one day or two weeks after the end of the desensitization period. At day 1, significantly fewer of the SOTI treated mice experienced anaphylaxis when compared with controls; however, when the mice were re-challenged at two weeks after ceasing SOTI, the previously desensitized mice were no longer protected from anaphylaxis. Mice given heated ovomucoid were completely protected against anaphylaxis compared with controls, as were mice given a higher dose egg white treatment compared with a low dose treatment. Antigen specific IL-13, IL-10 and IFNy responses were all significantly lower in SOTI treated mice compared with controls, and this difference remained two weeks post treatment. Whole genome microarray analysis of jejunum of sensitized control mice compared with those who were treated with SOTI indicated that administration of SOTI may lead to the regulation of a subset of genes in the intestine, including some expressed by intestinal epithelial cells. They postulated that the reduction in both the IL-10 and IL-13 after the SOTI indicated that the effect was related to an overall suppression rather than a shift in TH1/Th2 response.  
As discussed, human studies investigating the ingestion of heated cow's milk and heated egg in allergic individuals demonstrated that this was associated with decreased skin test seal diameters and allergen-specific IgE levels and increased allergen-specific IgG4 levels [8,74]. Further studies are required to further quantify the immune changes that occur with ingestion of heated proteins to enable us to understand if ingestion of heated egg or heated cow's milk affects the natural history of egg and cow's milk allergies when compared with strict avoidance. Other important information that is required includes identification of diagnostic tests to accurately predict reactions to heat treated proteins and target potential candidates for this form of therapy, development of optimal protocols and evaluation of their efficacy and safety.