S300 Abstracts J ALLERGY CLIN IMMUNOL JANUARY 2002 Q t O Relative Potency of the Major Peanut Allergens,Ara hl and Ara ! U h2, in a Functional Assay G William Palmer*, Donald A Dibbern*, Wesley Burks§, Gao' A Ban- non§, S Allan Bock~, Stephen C Dreskin* *University of Colorado, Den- ver, CO §University of Arkansas, Little Rock, AR ~,Iational Jewish Med- ical and Research Center, Denver, CO Rat basophilic leukemia (RBL)-SX38 cells (gift from J-P Kinet) express the alpha, beta, and gamma chains of the human high-affinity lgE receptor (Fc epsilon RI). Following sensitization with IgE from peanut-allergic human donors (total IgE> 100 kU per liter and/or peanut-specific IgE_>6 kU per liter), these cells can be triggered by exposure to anti-IgE or to very low concentrations of peanut allergens. We hypothesized that this assay would allow us to determine if individual sera from peanut-allergic patients react preferentially to Ara hl or Ara h2. We evaluated sera from 7 patients with severe reactions to peanuts. RBL-SX38 cells were labeled with 3H-sero- tonin and sensitized by overnight incubation with serum diluted 1:10 or greater and then washed. Anti-IgE or serial dilutions of purified Ara h I and Ara h2 were added to the sensitized cells and release of 3H-serotonin was measured. Degranulation by Ara h 1 or Ara h2 is expressed as net 3H-sero- tonin release relative to the release caused by anti-IgE. Compared to the release with anti-lgE (defined for this study as 100%), maximal signals with the purified peanut allergens were 71% _.+14% for Ara h2 and 55% -+ 19% for Ara hi. Individual sera responded optimally at doses ranging from 0.25 to 250 ng/ml for Ara h2 and from 2.5 to 2500 ng/ml for Ara hi. Six of the seven serum samples reacted preferentially to Ara h2 compared to Ara hi. Cells sensitized with sera from two of these patients responded only to Ara h2, with no release to Ara h 1. With the other four sera. both Ara h I and Ara h2 caused degranulation, but the sensitized cells responded to Ara h2 at 10-100 fold lower concentrations than to Ara hi. Serum from one patient reacted similarly to both Ara hl and Ara h2 at 0.25 ng/ml (70% compared with anti-IgE). This functional assay suggests that for the majority of these peanut-allergic patients, Ara h2 is more potent than Arah 1. 91 9 ProteinStructurePlaysa Critical Role in Peanut AllergenAra h 2 Stability and May Determine ImmunodominantIgE Binding Epitopes Moon Sen*, Randall Kopper*, Laurent Pons*, EC Abraham*, Wesley Burks§, Gary A Bannon§ *Arkansas Children's Hospital Research Insti- tute, Little Rock, AR §University of Arkansas, Little Rock, AR Hypersensitivity to peanuts is a reaction mediated by lgE antibodies in response to several peanut protein allergens. Among these allergenic pro- teins, Ara h 2 is one of the most commonly recognized allergens. Ara h 2 is a 17 kD protein that has 8 cysteine residues that could form up to 4 disulfide bonds. Circular dichroism studies showed substantial changes in the sec- ondary and tertiary structures of the reduced Ara h 2 protein as compared to the native molecule. In addition, the native, non-reduced Ara h 2 protein migrated in 12% SDS-PAGE as a doublet protein with an average molecu- lar weight of 12 kDa. In contrast, the reduced Ara h 2 migrated as a slightly larger doublet with an average moleculare weight of 17 kDa. These results support the circular dichroism measurements and provide a visual confir- mation of the importance disulfide bonds play in defining overall Ara h 2 structure. In order to assess the importance of secondary and tertiary struc- ture to the overall stability of the allergen to the action of proteases com- monly encountered in the human GI tract, native and reduced Ara h2 was exposed to pepsin, trypsin or chymotrypsin and the stability of the protein assessed by gel electrophoresis and immunoblotting. Upon treatment with trypsin, chymotrypsin, or pepsin, a number of relatively large fragments are produced which are resistant to further enzymatic digestion. The enzyme- treated allergen remains essentially intact despite the action of proteases until the fragments are dissociated when the disulfide linkages are reduced. The most prominent and stable fragment is a 10 kDa peptide. The 10 kDa Arah 2 peptide was also stable to sequential digestion by these enzymes as would be encountered in the human GI tract. The 10 kDa peptide contains intact IgE-binding epitopes and several potential enzyme cut sites that are protected from the enzymes by the compact structure of the protein. Amino acid sequence analysis of the resistant protein fragments indicates that they contain most of the immunodominant IgE-binding eptiopes. These results provide a link between allergen structure and the immunodominant IgE- binding epitopes within a population of food allergic individuals. 920 Linking ,gE Bindingof Peanuts to Maillard ReactionAdducts Si-Yin Chung, Elaine T Champagne USDA-ARS, Southern Regional Research Center, New Orleans, LA Maillard reaction adducts are protein-sugar reaction products that cross- link with proteins. One of these adducts is the advanced glycation end- product (AGE). Recently, we have demonstrated the presence of AGE adducts in raw and roasted peanuts, using polyclonal antibodies against AGE (Chung et al., J. Agric. Food Chem. 2001, 49, 3911-3916). Also, in that study, we showed that roasted peanut exhibited an increased level of IgE binding, which was accompanied by an increase of AGE adducts. This suggests a potential correlation between AGE adducts and IgE binding. To further determine the validity of this correlation, peanuts of different vari- eties and from different countries were examined in this study forAGE and lgE binding. AGE adducts were determined in a direct ELISA, using poly- clonal antibodies against AGE. IgE binding was determined in a competi- tive inhibition ELISA, using a pooled serum from patients allergic to peanuts. Data showed that the majority of roasted peanuts exhibited increased IgE binding, which coincided with an increased level of AGE adducts. It was concluded that there is a strong correlation between AGE adducts and lgE binding. The implication of this is that AGE adducts could be potential indicators of IgE binding. 921 Gastrointestinal Dysfunction in a Swine Model of Peanut Allergy Ricki M Helm*, Glenn T Furuta§, Joseph Steve Stanley*, Jianhui Ye*, Gael Cockrell*, Cathie Connaughton*, GaD' A Bannon*, Wesley Burks* *University of Arkansas, Little Rock, AR §Harvard Medical School, Boston, MA BACKGROUND: The neonatal pig model of peanut allergy mimics physical and immunologic characteristics of peanut allergy in humans. Pre- viously, we reported the physical features observed in the sensitized pigs challenged with peanut meal that included emesis, lethargy, diarrhea, hives, and respiratory distress. Immunologic assessment suggested an IgE-medi- ated response. AIM: To determine the morphometric and immunologic features of the gastrointestinal tract following peanut challenge. METHODS: Non-peanut sensitized and peanut-sensitized pigs were challenged with peanut meal by gastric gavage. Two hours following chal- lenge, some pigs underwent endoscopic evaluation and biopsy of the upper intestine and rectosigmoid region with a GIF-XQ40 gastroscope (Olympus America, Corp). These animals and additional animals were euthanized and the gastrointestinal tissue harvested and assessed for gross morphome- tric features. Tissues were sectioned for formalin fixation and mRNA isola- tion. Each part of the gastrointestinal tract was processed for hematoxylin and eosin staining and assessed by two observers, mRNA isolation was accomplished by Trizol extraction and RT-PCR performed for a variety of cytokines. RESULTS: The gastric and proximal small intestinal cavities of peanut sensitized and challenged pigs were nearly devoid of peanut meal two hours after challenge. This was in stark comparison to the unsensitized and challenged animals that had retained the peanut challenge dose. Endoscopy was performed in one challenged and one control pig. Endoscopic analysis revealed a loss of vascular pattern in the small intestinal mucosa of the chal- lenged pig but not the control. Histologic assessment identified a patchy distribution of vascular congestion within the gastric, small intestinal and colonic mucosa of the sensitized/challenged pig. The most remarkable findings were identified in the proximal small intestine where frank hemor- rhage and focal villus denudation was observed. In addition, submucosal congestion and goblet cell mucus depletion was identified in challenged pigs. Within the lamina propria, an expansion of lymphocytes and macrophages was present, without evidence of increased neutrophils or