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Specific Objective #1: Determine the NOAEL and the LOAEL of plant derived food proteins with high vs. low/no allergenic potential in this model. In this study, we will evaluate oral elicitation potencies of four model food proteins, two with high allergenic potential (hazelnut, and cashew nuts) and two with low/no allergenic potential (pigeon pea and pinto bean). We have established all the methodology required for these experiments in our laboratory. Using standard toxicological methods we will establish dose-response curves for these food proteins and determine the NOAEL and LOAEL and compare them to the human data. <P>Specific Objective #2: Determine the immune, clinical and physiological responses to isolated pure food proteins with high vs. low/no allergenic potential. One critical challenge faced in predicting allergenic potential of novel proteins used in GE foods is to test the hazard of novel pure proteins in isolation. Here we will determine whether the our model can distinguish purified allergenic protein (Cor a 9 major allergen in hazelnut) from purified non allergen (gamma protein, a major protein in pigeon pea). We have detailed protocols in place for this work. <P>Specific Objective #3: To determine the effect of extrusion processing on hazelnut allergenicity in vivo in our mouse model. The extrusion processed hazelnut protein will be prepared with the assistance of Dr. Perry Ng (Professor, FSHN). We will conduct these studies in two parts: first we will evaluate the sensitization capacity of extrusion processed hazelnuts. In the second part, we will evaluate the oral elicitation capacity of extrusion processed hazelnut protein. These studies are described briefly below. <P>Expected results and outputs. A positive outcome from this research (Objectives #1, 2) will be further validation of our mouse model for allergenicity hazard identification of novel dietary proteins such as pesticidal proteins used in GE foods. Successful results from Objectives 3 studies will provide us specific processing conditions for potential application to produce hypoallergenic and/or non allergenic hazelnut protein.
Non-Technical Summary:<br/>
The long-term goal of this umbrella project is to develop and validate methods to assess allergenic potential of novel foods including processed foods, functional foods and in particular genetically engineered (GE) foods, a nationally identified need by EPA, NIH, FDA and USDA. This research is linked to food safety and therefore has direct relevance to the mission of the AgBioResearch. While about 175 different types of foods have been documented to trigger an allergic reaction in sensitized subjects, 90% of food allergies are due to 8 major food types: Chicken egg, cow's milk, soybean, wheat, peanut, tree nuts, fish and shellfish. Notably, as reviewed recently, improved methods for predicting allergenicity of food proteins are critically needed. A promising approach is to test if a dietary protein might induce allergic reactions in a validated food allergy mouse model. However, a validated mouse model of food allergy is not available at present to evaluate allergenicity of novel foods. Consequently, development and validation of mouse models for assessment of allergenic potential of novel foods was identified as a critical need by experts. During the past 9 years, with the generous support of funding from the US EPA (two major grants), and the MAES/ABR we have made significant contribution to advancing this area of research. The objectives 1 and 2 of the proposed research, further advances this critical area of research. Besides direct application for assessment of allergenic potential of food, an animal model would have several other critical applications. First, a food allergy model should help elucidate the molecular mechanisms of allergic responses to food-derived proteins that are poorly understood at present. Second, development of effective therapeutic and preventive methods is also critically dependent on the availability of an in vivo disease model. Third, the role of environmental toxicants/dietary factors in induction/exacerbation/prevention of food allergy can be investigated using this model. Finally, this model can also be used to evaluate the impact of food processing on food allergenicity, focus of objective 3 of this proposal. In recognition of these outstanding needs and potential applications, our goal (next 5 years) is to further characterize and validate the mouse model for assessment of allergenic potential of novel foods such as genetically engineered foods and processed foods.
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Approach:<br/>
Food proteins. All standardized food proteins will be purchased from Greer laboratories (Greer Labs, Lenoir, NC, USA). Isolation and purification of Cor a 9 from hazelnut and gamma protein from pigeon pea protein extract. We will use the previously published biochemical methods for isolation and purification of these proteins. We have estimated that 50 grams each of Cor a 9 and gamma protein would be adequate to complete the proposed studies. We will ensure purity by HPLC, sequencing, SDS-PAGE and LPS analyses. We have worked together previously in isolation and sequencing of hazelnut and sesame allergens. Mice, transdermal sensitization and bleeding. All mice (adult female, 6 weeks age) will be purchased from The Jackson Lab (Bar Harbor, Maine, USA). All animal procedures are approved by the Michigan State University Animal Protocol review board. Transdermal exposure protocol is described by us before. Measurement of food protein specific IgE antibodies in the plasma. A novel ELISA based method to measure food protein specific IgE antibodies in mouse previously reported by our lab will be used here. Oral food protein vs. vehicle challenge and clinical scoring of systemic analysis. Four days after the 4th transdermal exposure, mice will be orally challenged with either food protein or vehicle (0.5 mL/mouse) and observed for clinical signs of systemic anaphylaxis for 60 minutes by 3 individuals in a blinded manner according to the method described previously. Measurement of rectal temperature. Rectal temperature will be measured using a temperature probe (Yellow Springs Instrument Co., Yellow Springs, OH, USA) before and at 30 minutes after oral challenge with food protein vs. vehicle . Measurement of mouse mast cell protease 1. We have optimized the ELISA based assay for this purpose in the lab. Extrusion processing: Shelled skinned raw hazelnuts will be purchased from commercial sources. A portion of the hazelnuts will be ground via a hammer mill to pass through a 2 mm sieve. The non ground and ground hazelnuts will be stored at -20 C till use. Extrusion: Extrusion processing will be carried out according to Chang and Ng (2009) with an APV co-rotating, twin screw extruder, with a barrel diameter of 19 mm and a barrel length to diameter (L/D) ratio of 25:1 (Model MP 19T2-25, APV Baker, Grand Rapids, MI, USA). Extruded samples will be stored in a freezer at -20 C until analyses or use as oral test material.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Food allergies are growing both in prevalence as well as severity in the USA and many other countries. In particular, they can trigger life-threatening systemic reactions. Long term goal of this project is to develop and validate methods to predict life-threatening allergic reactions to food proteins. Towards this goal, we have developed a novel mouse model of food allergy that uses transdermal exposure to allergens to sensitize mice followed by oral exposure to allergens to elicit systemic allergic reactions. This model simulates the most-severe forms of human food allergies such as life-threatening systemic anaphylactic reactions. Currently, mechanisms underlying life-threatening allergic reactions to foods are not fully understood. Using funding from two major US EPA grants we have been working on this problem.
<br/>Our major accomplishments during the reporting year of 2012 are: 1) Optimizing different protocols for sensitization followed by oral elicitation of reaction to develop dose-response curves for oral hazelnut induced severe systemic reactions in this model; 2) Evaluation of mechanisms underlying walnut extract induced life-threatening reactions; 3) Characterizing immune cell phenotype and memory responses in hazelnut allergy mouse model.
<br/>PARTICIPANTS: Dr. Elizabeth Gardner, Associate Professor, Food science and Human Nutrition, Dr. Robert J Tempelman, Professor, Animal Science, Statistics and probability, Food Science and Human Nutrition, Michigan State University: Role: Co-PI on the US EPA funded project RD833133; Consultants: Dr. James Pestka, Professor, Food Science and Human Nutrition, Michigan State University Dr. Gale Strasburg, Professor, Food Science and Human Nutrition, Michigan State University Dr. Maurice Bennink, Professor, Food Science and Human Nutrition, Michigan State University; Collaborator: Dr. Jack Harkema, Professor,PDI,Michigan State University.
<br/>TARGET AUDIENCES: Not relevant to this project.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Two abstracts were presented during the American Association of Immunologists (AAI), annual meeting held during May 2012 in Boston. One manuscript accepted for publication; one under revision.