Increased binding behaviour to enterocytes and initiation of signalling pathways could trigger the epimmunome and influence the sensitization capacity of food proteins. in a food allergy mouse model. led to the formation of Ara h 2 oligomers in solution. Heated protein exhibited a significantly higher immunogenic potential as determined by IgG and IgE serum antibody levels as well as IL-2 and IL-6 release by splenocytes. In human Caco-2/TC7 cells, Ara h 2 incubation led to a response in immune- and stress signalling related pathway components at the RNA level, whereas heated allergen induced a stress-response only. We suggest from this peanut allergen example that LY 3200882 food processing may change the molecular immunogenicity and modulate the interaction capacity of food allergens with the intestinal epithelium. Increased binding behaviour to enterocytes and initiation of signalling pathways could trigger the epimmunome and influence the sensitization capacity of food proteins. in a food allergy mouse model. We report here that besides its molecular properties, heating as one of the most important food processing methods influences not only the IgE triggering potential of peanut allergens  but also affects the consecutive immune response. MATERIALS AND METHODOLOGY Protein Production Coding sequences of Ara h 2 and Cyp c 1 [13, 14] were cloned into a pET26b vector (Novagen, Madison, Wisconsin) lacking the PelB signal peptide (pET26b plain). PDCD4 was identified as structurally Ara h 2 related control protein using the DALI search engine (http://ekhidna.biocenter.helsinki.fi/dali_server/)  and cloned (cDNA clone IMAGp998L1512389Q; Imagenes, Berlin, Germany) into pET26b plain. Bl21(DE3) (Cyp c 1, PDCD4) and Rosetta-Gami-II(DE3) (Ara h LY 3200882 2) bacteria (both strains from Novagen) were used for recombinant production of 6x His-tagged proteins [14, 16] and purified under native conditions using NiNTA superflow columns (Qiagen, Hilden, Germany). Natural Ara h 2 was purified from roasted or raw, unshelled peanuts (obtained from a local market, originally imported and distributed by Heuschen & Schrouf, Landgraaf, The Netherlands) as described elsewhere . Allergen purity was determined by SDS-PAGE and subsequent LY 3200882 protein staining. Purified proteins were dialysed against H2Odest and stored at ?80C. Thermal Processing Purified proteins were thermally processed following a protocol previously used to assess the effect of heat treatment and the Maillard reaction on IgE binding from human peanut allergic patients to Ara h 2 . Briefly, proteins were heated at 200g/ml in 20% PBS in Safe Lock tubes (Eppendorf) in a Thermomixer Comfort (Eppendorf) for 90min at 99C under shaking. Sample protein content was analyzed by SDS-PAGE/silver staining. For kinetic analyses by circular dichroism analysis, heating was conducted for 5, 10, 15, 30, 45, 60, or 90 minutes. Before use, frozen Ara h 2 was warmed to 37C and mixed for solution of cryo-precipitates. Cell Culture Human Caco-2/TC7 cells were a kind gift of Monique Rousset (INSERM, Paris, France). Cells were cultured and differentiated as previously described using cell culture plastic from Corning (Amsterdam, The Netherlands) . ELISA Assays Allergen specific ELISA for detection of specific antibodies in human or mouse serum was performed as previously described [18, 19]. Collection of patient sera was approved by the ethics committee of the Medical University of Vienna and performed according to ethical guidelines. Informed consent was obtained from all patients. For cell ELISA experiments, Caco-2/TC7 cells were differentiated in 96 well flat bottom plates for at least 10 days after confluence. Cells were then kept on ice and washed with cold PBS and blocked 1h with 1% BSA. Next, 50g recombinant allergen or control protein was added onto the cells in triplicates and incubated on ice for 90min. Bound protein was detected using biotinylated Penta-His-Conjugate (Qiagen) and HRP conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pennsylvania) and TMB substrate (R&D Systems, Minneapolis, Minnesota). Fluorescent allergen binding analysis Allergens and control proteins were fluorescently labelled using Rabbit Polyclonal to DGKZ the DyeLight 488 Labelling Kit (Pierce). Caco-2/TC7 cells were differentiated in 8 well TissueTek Chamber slides (Nunc) and incubated with 10g labelled protein for 90min on ice, washed, fixed with 3.7% formaldehyde and nuclear-stained for 30min at RT with Hoechst dye followed by mounting. Allergen binding to cells was analyzed by fluorescence microscopy using an Axio Observer.Z1 inverted microscope /AxioVision software (Zeiss, Jena, Germany). Structural Analysis Dynamic light scattering (DLS): DLS measurements were carried out in a MRC Crystallization Plate (96-well SBS format, Swissci AG, Switzerland) sealed with SmartSeal (Greiner, Kremsmnster, Austria) using a Spectro Light 500 plate reader (Molecular.