ASSESSMENT OF ALLERGENIC POTENTIAL OF FOOD

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0192201
• Project No.: MICL02023
• Project Start Date: May 1, 2012
• Project End Date: Apr 30, 2017

Project Director
Gangur, V.

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Food Science

Non Technical Summary
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.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1210	1090	25%
711	1219	1090	25%
723	1412	1090	10%
723	1413	1090	10%
723	1414	1090	10%
723	1853	1090	10%
723	5010	1090	10%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
1414 - Lentil; 1210 - Filbert; 1219 - Edible tree nuts, general/other; 1853 - Sesame; 5010 - Food; 1412 - Peas (dry); 1413 - Peas (fresh, fresh-processed);

Field Of Science
1090 - Immunology;

Keywords

food safety, food allergens, food allergy, mouse model, anaphylaxis,

allergenicity, hazard assessment, food toxicology

Goals / Objectives
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. 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. 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. 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.

Project Methods
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.

Progress 05/01/12 to 04/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-doctoral reseach associate, PhD greaduate student and two undergraduate students were trained. Results were presented by the PI during the American Association of Immunologists Annual meeting 2017 in Washington DC. How have the results been disseminated to communities of interest?Results were presentedduring the American Association of Immunologists Annual meeting 2017 in Washington DC. Research grant proposals were prepared and submitted to USDA and NIH using the data from this projectas preliminary studies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Food-induced adverse reactions such as food allergies are a growing public health problem in the USA and many other countries. Development and characterization of animal models of food allergywill help in elucidating mechanisms of disease development. In addition, validated models of food allergy are vaulable tools toassess the allergenic potenital of novel foods including genetically engineered foods. During this project we have successfully estiablished novel mouse models of food allergy and characterized the responses to a number ofallergenic foods.During this reportingyear we futhercharacterized the immune response to wheat proteins. High quality salt-soluble and alcohol-soluble wheat proteins were prepared and characterized from soft-wheat. Groups of mice were injected with these proteins and immune and allergicrespones were characterized. Wheat protein specific IgG1 and IgE antibodyresponses werequantified using ELISA based methods. Systemic anaphylaxis to injection with wheat proteins were determined using hypothermia shock as a quantifiable readout of allergencitiy. Mucosal mast cell degranulation was monitored by measurig plasma levels of murine mucosal mast cell protease-1 protein. Spleen tissues were collected and immune biomarmerresponses were measured. Our analyses demonstrated that: (i) alcohol-soluble wheat protein elicited stronger IgG1responses and IgE (allergenic) responses than that of the salt-soluble wheat proteins; (ii) alcohol-soluble wheat protein elicited stronger anaphylaxis and mucosal mast cell degranulation response; and (iii) alcohol-soluble and salt-soluble wheat proteins elicited severalcommon as well as some disntictbiomarkers in the spleen. These data provide the first direct evidence that wheat proteinfractions that differ in solubility alsodiffer ineliciting immune and allergicreactions in genetically identical mice.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Venugopal Gangur, Yining Jin, Sarah Ebaugh, Anna Martens, Eric Olson and Perry KW Ng. Distinct immune and clinical responses to alcohol-soluble vs. salt-soluble wheat proteins in mice. J Immunol May 1, 2017, 198 (1 Supplement) 194.7;

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Post-Doctoral Research Associate and three research assistants trained Opportunity to present the paper at theImmunology conference Presentation at the Michigan Wheat Association How have the results been disseminated to communities of interest?Presentation of results from this work during the annual meeting of Amercian Association of Immunologists; Presentation of the work at the Michigan Wheat Association What do you plan to do during the next reporting period to accomplish the goals?Evaluate allergenicity and validate the model for other food proteins

Impacts
What was accomplished under these goals? Both the prevalence and severity of food-induced allergic reactions are growing at an alarming rate for reasons that are incompletely understood. Wheat is identified as a major red-flag allergenic food in many countries including the USA. Although animal models are very useful to study wheat allergy, a mouse model of hypersensitivity to salt-soluble wheat protein (SSWP)--more common type of wheat allergens--is unavailable. Here we tested the hypothesis that SSWP from durum (Carpio) wheat will elicit allergic response in BALB/c mice. High quality SSWP from durum (Carpio) wheat was prepared, characterized and used in the study. Pups were weaned onto and maintained on a plant protein-free diet. Group of mice (n=5/group) received five i.p. injections with SSWP plus alum as an adjuvant. Blood was collected and used in measurement of the specific (S) IgE, IgG1, and IG2a antibody levels and the total plasma IgE (TIgE) concentration. Upon intraperitoneal challenge with SSWP, mice were monitored for hypothermia shock response (HSR). Blood collected before vs. after challenge was used in the measurement of mucosal mast cell protease (mMCP)-1 response. Durum wheat SSWP elicited time-dependent robust SIgE, SIgG1 as well SIgG2a antibody responses. Sensitized mice also exhibited significant elevation of TIgE levels. Upon challenge with SSWP, sensitized mice exhibited marked HSR by 20 to 30 minutes post-challenge. Furthermore, SSWP challenged mice showed significant elevation of circulating levels of mMCP-1 confirming IgE antibody mediated anaphylactic reaction. In summary, we report a novel mouse model of immediate hypersensitivity to SSWP for the first time (Gangur et al 2016). In another study, we reported the impact of extrusion processing on immune stimulating properties of hazelnut allergen in a mouse model. We demonstrated that extrusion processing significantly reduces the mucosal mast cell degranulation capacity of hazelnut protein (Ortiz et al 2016).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Gangur V, Yining J, Ebaugh S, Secord J, Ng KWP, Olson E. A novel mouse model of hypersensitivity to salt-soluble wheat protein. The Journal of Immunology May 1, 2016 vol.196 (1 Supplement) 123.6.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ortiz T, Para R, Gonipeta B, Reitmeyer M, He Y, Srkalovic I, Ng PK, Gangur V. Effect of extrusion processing on immune-activation properties of hazelnut protein in a mouse model. Int J Food Sci Nutr. 2016 Sep;67(6):660-9. doi: 10.1080/09637486.2016.1191445. Epub 2016 Jun 2. PubMed PMID: 27251648.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate student and graduate student training How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Evaluate allergenicity and validate the model for other food proteins

Impacts
What was accomplished under these goals? Food allergies are the leading cause of life-threatening allergic reactions with incompletely understood mechanisms. We previously reported a novel mouse model of near-fatal hazelnut (HN) allergy that involves transdermal sensitization followed by oral elicitation of allergic reactions. Here we studied the cardiac mast cell and cardiac tissue responses during oral nut induced allergic reaction in this mouse model. Oral allergen challenge (OAC) of transdermal sensitized mice results in hypothermia, hypotension, tachycardia and rapid elevation of circulating mMCP-1. The pathology analysis of small intestine found significant expansion of mMCP-1+ MMCs and mMCP-4+ CTMCs. The pathology analysis of cardiac tissues showed very little mMCP-1 expression, but marked mMCP-4 expression. Furthermore, repeated OAC resulted in significant expansion of mMCP-4+ cardiac MCs in both the pericardium and the myocardium. Protein array analysis revealed significant elevation of cardiac IL-6 and CCR1/3 and CXCR2 signaling chemokines upon oral elicitation compared to sensitization alone. These results demonstrate that: (i) besides the intestine, cardiac mast cells and the cardiac tissue respond during oral nut induced allergic reaction; and (ii) repeated oral elicitation of reaction is associated with cardiac mMCP-4+ mast cell expansion and elevation of cardiac IL-6, and CCR1/3 and CXCR2 signaling chemokines. In another study we evaluated the allergenicity of egg white protein extract (EWPE) vs purified egg white protein ovalbumin (OVA) in a novel transdermal sensitization model without adjuvants such as alum/cholera toxin/super-antigen that are commonly used in mouse models of food allergy. Groups of mice received transdermal exposure (TDE) to EWPE for four weeks. Four TDEs were sufficient to elicit robust SIgE Ab responses. Upon oral challenge (OC) EWPE elicited near-fatal clinical signs of anaphylaxis associated with rapid hypothermia reactions. We then evaluated allergenicity of purified OVA using this model. Surprisingly, OVA 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 depend on whether protein extracts or pure protein from the same food source is used in model development.

Publications

• Type: Other Status: Published Year Published: 2015 Citation: Gangur V, Ortiz T, Parvataneni S, Gonipeta B, He Y, Thi S, Nolkemper M. Different allergenicity profile of egg white protein extract vs. purified ovalbumin in an adjuvant-free mouse model (HYP7P.267) The Journal of Immunology May 1, 2015 vol.194 (1 Supplement) 191.15
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Gonipeta B, Para R, He Y, Srkalovic I, Ortiz T, Kim E, Parvataneni S, Gangur V. Cardiac mMCP-4+ mast cell expansion and elevation of IL-6, and CCR1/3 and CXCR2 signaling chemokines in an adjuvant-free mouse model of tree nut allergy. Immunobiology. 2015 May;220(5):663-72. PMID: 25499102
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Gonipeta B, Kim E, Gangur V. Mouse models of food allergy: how well do they simulate the human disorder? Crit Rev Food Sci Nutr. 2015;55(3):437-52. PMID: 24915373.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Regulatory agencies, biotech and food industry, academic scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Research assistants trained Presentation of results at the conference Invited lecture at the US FDA, CFSAN How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Evaluate allergenicity and validate the model for other food proteins

Impacts
What was accomplished under these goals? Life-threatening nut allergy is a growing public health problem in many countries including the USA. Causes underlying this alarming trend are incompletely understood and methods are urgently needed to prevent such immune reactions. Here, we tested the hypothesis that extrusion processing of hazelnuts will reducein vivoallergenicity using an adjuvant-free mouse model of hazelnut allergy established in our lab. Groups of mice received transdermal exposure (TDE) to extrusion processed hazelnut protein (EHNP) versus raw hazelnut protein (RHNP) extract preparations. Mice were evaluated for systemic IgE antibody responses (sIgER), hypothermia shock response (HSR), and mucosal mast cell responses by measuring plasma mouse mast cell protease-1 (mMCP-1) levels. Results showed that EHNP elicited a reduced sIgER compared to RHNP after 4 TDE but similar responses after 5 TDE. However, EHNP induced less HSR compared to RHNP upon oral challenge. Both protein preparations induced similar HSR upon i.p., challenge. Finally, we measured plasma mMCP-1 levels after oral and i.p., injection challenges. We found that EHNP elicited reduced mMCP-1 response compared to RHNP upon both oral as well as i.p., challenges. This is the first study demonstrating the utility of a mouse model to evaluate the effect of food processing onin vivofood allergenicity in general and hazelnut in particular.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ortiz T, Para R, Gonipeta B, Ravi R, Reitmeyer M, He Y, Srkalovic I, Ng P, Gangur V Effect of extrusion processing on in vivo allergenicity of hazelnut in an adjuvant-free mouse model (HYP7P.313). The Journal of Immunology May 1, 2014vol.192 (1 Supplement)119.28
• Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: ORTIZ, T. EFFECT OF EXTRUSION PROCESSING ON IN VIVO ALLERGENICITY OF HAZELNUT PROTEIN EXTRACT IN AN ADJUVANT-FREE MOUSE MODEL. MS THESIS. MICHIGAN STATE UNIVERSITY, 2014.
• Type: Other Status: Other Year Published: 2014 Citation: Invited Lecture: Gangur V. Threshold doses for food allergens: utility of a mouse model. Joint Institute of Food Safety and Applied Nutrition (JIFSAN)/US FDA/University of Maryland, Washington DC. Visiting Program, April 6-7th, 2014.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Regulatory agencies, biotech and food industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One post-doctoral research associate, one Graduate student and two undergraduate students had the opportunity to get research training and professional development during this time. PI was invited to present the research as a Workshop Speaker Seminar at a major workshop organized by the International Life Sciences Institute, in Washington DC. How have the results been disseminated to communities of interest? Presentation during conferences as listed above. What do you plan to do during the next reporting period to accomplish the goals? Evaluate allergenicity and determine threshold doses for other food proteins is planned.

Impacts
What was accomplished under these goals? Food allergies are a significant public health problem because they trigger life-threatening systemic allergic reactions. We have previously reported a novel mouse model that uses transdermal exposure (TDE) to nut protein for sensitization followed by oral challenge (OC) to elicit systemic reactions. Here we evaluated the utility of this model to determine the oral threshold doses for hazelnut (HN). Groups of mice (n=8-10/group) were sensitized with 2, 3, 4 or 5 TDE to HN or saline, followed by OC with HN to induce systemic anaphylaxis. The data demonstrated that a minimum of 4 TDE to HN and a definition of >/=1 o C drop in rectal temperature (RT) at 30 minutes post OC, ensures 100% positive responses and <10% false positive responses. We then evaluated threshold oral elicitation doses in three scenarios: (1) homogeneous 4 TDE to allergen, followed by repeated OC with different doses of HN at weekly intervals; 2) homogeneous 4 TDE to allergen, followed by single OC per dose per group; and 3) heterogeneous (i.e., 4, 5 or 6) TDE with HN followed by single/repeated OC to simulate the human exposure conditions that are generally heterogeneous. The percent responder curves were established for each case and the NOAEL and LOAEL (25 and 50% responses) were estimated. The NOAELs were </=125 mg/Kg; the 25% response LOAELs ranged from 270-312 mg/Kg; and the 50% response LOAELs ranged from 362-412 mg/Kg.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Gangur V et al. Evaluation of a mouse model of food allergy for determination of threshold oral elicitation doses. J Immunology, 190, 62.11 (Abstract).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Gangur V et al. Evaluation of a mouse model of food allergy for determination of oral elicitation threshold doses of hazelnut. International Life Sciences Institute, Health and Environmental Sciences Institute, Washington DC, May 7-8, 2013 (Invited workshop speaker seminar) (http://www.hesiglobal.org/files/19-Gangur-Mouse%20Model-Hazelnut-May%202013.pdf).

Progress 01/01/12 to 12/31/12

Outputs
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. 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. 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. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
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.

Publications

• Gangur V, Gonipeta B, Kim E, Parvataneni S. Mechanism of walnut induced anaphylaxis-like shock reaction in mice. J Immunol May 2012, 188; 175.1(AAI Annual Meeting, Boston, May 4-8, 2012).
• Gangur V, Gonipeta, B, Parvataneni S, Duriancik D, Kim E, Gardner E. Analysis of T and B cell phenotype and effector memory T cell response in a mouse model of tree nut allergy. J Immunol May 2012, 188: 125.10 (AAI Annual Meeting, Boston, May 4-8, 2012).

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Food allergy is a major global public health problem not only due to increased recent prevalence but also increasing severity of the reactions with potential for fatality. Reasons for this alarming trend are incompletely understood. There is concern that novel genetically engineered foods might sensitize susceptible individuals for food allergy. Therefore, methods to predicting potential allergenicity of a novel food before introduction into the food chain, is a critical need. Long term goal of this project is to develop and validate methods to predict food allergenicity. We have developed a novel adjuvant-free mouse model to study allergenicity of food proteins. Using funding from US EPA during 2006-2010, we have demonstrated the positive and negative predictive value of this mouse model. In 2011, we started the newly funded US EPA project. Focus of this project is to determine the threshold oral elicitation doses for food proteins in this mouse model. Our major accomplishments during this year are: 1) Establishment of the oral elicitation dose-response curves for hazelnut protein in our mouse model using three different sensitization protocols; 2) determination of the effect of boiling on oral elicitation dose-response curves for hazelnut; 3) demonstration that measurement of MMCP1 in plasma serves as a quantifiable bio-maker of oral reactions in this model; 4) development of a novel infrared transdermal scanning thermometry as a useful non-invasive method to quantify oral reactions in this model; and 5) optimization of a method to prepare pigeon pea protein extract for use in the project. An expected significant outcome from this project will be a validated adjuvant-free mouse model for allergenicity hazard identification of novel foods. PARTICIPANTS: 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. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
One abstract was presented during the American Association of Immunologists (AAI), annual meeting held during May 2011. Two manuscripts and one abstract are in preparation.

Publications

• Gangur V, Parvataneni S, Gonipeta B, Kim EJ. Walnut extract induces near fatal anaphylactoid shock via the complement C3 dependent pathway in naive mice. AAI Annual Meeting, San Francisco, 2011, May 16 (Poster presentation)Abstract Number 1060737.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Currently there is an ongoing epidemic of food allergies for reasons that are incompletely understood. There is growing concern that novel foods introduced into the food chain might sensitize susceptible individuals for potentially fatal food allergies. Therefore, predicting food protein allergenicity before a novel food is introduced into the food chain is critical. During this project, we have been developing novel methods towards predicting food protein allergenicity. A major problem is that a validated animal model is not available at present. We have continued to develop and validate mouse models to address this significant global problem. During this project we have developed novel adjuvant-free mouse models to study allergenicity of hazelnut, cashew nut, milk and sesame seed proteins. We have tested validity of some of these models by comparing their immunological characters to those of human disorder. We have published our findings in peer reviewed journals and presented our data during major immunology conferences. We submitted grant proposals using the data as preliminary studies. Our long-term goals are to understand the mechanism of food protein allergenicity and to evaluate the suitability of the mouse model for predicting allergenicity of novel food proteins (e.g., GMO proteins, pesticidal proteins used in GE foods etc). PARTICIPANTS: 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 Consultants: Dr. James Pestka, Professor, Food Science and Human Nutrition, Michigan State University Dr. Gale Strasburg, Professor and Chairperson, 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, National Food Safety and Toxicology center,Michigan State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The data generated during this project on hazelnut, sesame seed, cashew nut and milk proteins have been published in major peer reviewed journals. We also presented the results during the American Association of Immunologists, Annual Meetings during 2009 (Seattle), and during 2010 (Baltimore). The data was used to prepare two major grant proposals to federal agencies during 2010 (USDA, US EPA). The grant proposal to US EPA has been funded ($424,000/3 yrs).

Publications

• Gangur V, Parvataneni S, Gonipeta B. A mouse model of food induced acute allergic angioedema, AAI Annual Meeting, Baltimore, 2010, May 7-11 Poster presentation, May 10)
• Gangur V, Gonipeta B, Parvataneni S. Long-term characteristics of immune response to milk protein in an adjuvant-free mouse model, AAI Annual Meeting, Baltimore, 2010, May 7-11 (Poster presentation, May 10)
• Gonipeta B, Parvataneni S, Paruchuri P, Gangur V. Long-term characteristics of hazelnut allergy in an adjuvant-free mouse model. Int Arch Allergy Immunol. 2010,152 (3): 219-225 (PMID: 20145410).
• Parvataneni S, Birmingham NP, Gonipeta B, Gangur V. Dominant, non-MHC genetic control of food allergy in an adjuvant-free mouse model. Int J Immunogenet. 2009, Oct; 36(5):261-7 (PMID: 19624800).
• Kelly C, Gangur V. Sex-disparity in food allergy: evidence from the PubMed database. J Allergy. Volume 2009, Article ID 159845, 7 doi:10.1155/2009/159845

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Potential allergenicity of genetically engineered (GE) foods is a major international public health concern. Currently, experts in the field suggest the use of animal models to assess allergenicity hazard of novel food proteins. However a validated animal model is not available at present. We continue our efforts to address this significant problem. In 2007 we published a novel adjuvant-free mouse model of food allergy using hazelnut as a model allergenic tree nut (Birmingham et al 2007 Int Archives Allergy and Immunology). During 2008-09, we completed testing cashew nut and milk proteins in this model. This work was published recently. We continued further characterization of this model to test how it compares to the human disorder. Our long-term goal is to evaluate the suitability of this model for predicting allergenicity of novel food proteins including pesticidal proteins used in GE foods. PARTICIPANTS: 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 Consultants: Dr. James Pestka, Professor, Food Science and Human Nutrition, Michigan State University Dr. Gale Strasburg, Professor and Chairperson, 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, National Food Safety and Toxicology center, Michigan State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Data generated during 2008-2009 on cashew nut allergenicity is now published in the journal of International Archives Allergy and Immunology. Data on milk protein allergenicity is now published in the Journal of Dairy Science. We presented the data as a poster during the American Association of Immunologists, Annual Meeting, Seattle, 2009. We presented invited seminars at the International Life Sciences Institute, Health and Environmental Sciences Institute, Washington DC., during a workshop focused on assessment of allergenic potential of genetically engineered foods. We also presented an invited seminar during a symposium at the Society of Toxicology Annual Meeting, Baltimore, 2009. This project has received funding support from the US EPA.

Publications

• B. Gonipeta, S. Parvataneni, R. J. Tempelman and V. Gangur An adjuvant-free mouse model to evaluate the allergenicity of milk whey protein J. Dairy Sci. 2009. 92:4738-4744.
• Sitaram Parvataneni, Babu Gonipeta, Rob Tempelman, Venu Gangur. Development of an adjuvant-free mouse model of cashew nut allergy. Int Arch Allergy Immunol 2009;149:299-304. Babu Gonipeta, Sitaram Parvataneni, Venu Gangur. Immune response and clinical reaction to food proteins with high vs. low/no allergenic potential in a mouse model. Abstract presented during the American Association of Immunologists Annual Meeting, Seattle, May, 2009

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Potential allergenicity of genetically engineered (GE) foods is a major international public health concern. An expert committee of the FAO/WHO has proposed a decision tree approach for assessment of allergenic potential of such GE foods. Another method termed as weight of evidence approach has also been used for this purpose. Both methods propose the use of animal model for hazard assessment of GE foods for allergenicity. However a validated animal model is not available at present. We have focused our efforts on addressing this problem of international significance. In 2007 we published a novel adjuvant-free mouse model of food allergy using hazelnut as a model allergenic tree nut (Birmingham et al 2007 Int Archives Allergy and Immunology). Since then, we have been testing additional human dietary proteins for allergenicity in this model. During 2007-2008, we completed testing cashew nut and milk proteins in this model. Our long-term goal is to determine the positive and negative predictive value of this model for testing allergenicity of GE foods. PARTICIPANTS: 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 Consultants on the EPA funded project: Dr. James Pestka, Professor, Food Science and Human Nutrition, Michigan State University Dr. Gale Strasburg, Professor and Chairperson, 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, National Food Safety and Toxicology center,Michigan State University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Data generated during 2007-2008 on cashew nut allergenicity was accepted for publication in a peer reviewed journal. Data on milk protein allergenicity was presented as a poster during the American Association of Immunologists, Annual Meeting, San Diego, 2008. We were invited to present a seminar about our work by the International Life Sciences Institute, Health and Environmental Sciences Institute, Washington DC., during a recent workshop focused on assessment of allergenic potential of genetically engineered foods. This project has received funding support from the US EPA.

Publications

• Sitaram Parvataneni, Babu Gonipeta, Rob Tempelman, Venu Gangur. Development of an adjuvant free mouse model of cashew nut allergy. International Archives of Allergy and Immunology 2008 (In Press).
• Babu Gonipeta, Sitaram Parvataneni, Venu Gangur. Immune response and clinical reactions to milk protein in an adjuvant free mouse model of food allergy. Abstract presented during the American Association of Immunologists Annual Meeting, San Diego, 2008.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Potential allergenicity of genetically engineered (GE) foods is a major public health concern at present. An expert committee of the FAO WHO has proposed a decision tree approach for assessment of allergenic potential of such GE foods. This approach includes the use of animal model as one of the methods for hazard assessment. However a validated animal model is not available for this purpose. We have focused our efforts on addressing this current critical problem of major international significance. During this reporting period we have developed and published a novel adjuvant-free mouse model of food allergy using hazelnut as a model allergenic tree nut. Major features of this model include induction of systemic allergic response to transdermal allergen exposure and clinical signs of systemic anaphylaxis in response to oral food allergen challenge. We plan to validate this model by testing a large panel of human allergenic and non-allergenic dietary proteins. We also plan to study mechanism underlying food allergy in this model. Some of these data have been published (Birmingham et al 2007; Gangur et al 2007). Thus, we continue to make significant progress on this project focused on assessment of allergenic potential of food. PARTICIPANTS: Dr. Robert J Tempelman, Professor, Animal Science, Statistics and probability, Food Science and Human Nutrition, Michigan State University Dr. James Pestka, Professor, Food Science and Human Nutrition, Michigan State University Dr. Gale Strasburg, Professor and Chairperson, Food Science and Human Nutrition, Michigan State University Dr. Maurice Bennink, Professor, Food Science and Human Nutrition, Michigan State University Dr. Jack Harkema, Professor, National Food Safety and Toxicology center, Michigan State University

Impacts
We have developed and characterized an adjuvant-free mouse model for tree nut allergy for the first time. Some of the results obtained during 2006-2007 were presented as a poster during a major scientific meeting (American Association of Immunologists, Annual Meeting, Miami Beach, 2007). One paper related to this work was published in 2007. Based on these results, the US EPA has funded a research project.

Publications

• Neil Patrick Birmingham, Sitaram Parvataneni, Hanem M. Ahmed Hassan, Jack Harkema, Sridhar Samineni, Lalitha Navuluri, Caleb James Kelly, Venu Gangur. An Adjuvant-Free Mouse Model of Tree Nut Allergy Using Hazelnut as a Model Tree Nut International Archives of Allergy and Immunology 2007,144, 203-210
• Venu Gangur, Sitaram Parvataneni, Neal Birmingham, John Fyolek, Caleb Kelly and Hanem Hassan Characterization of allergen driven type-2 cytokine response in an adjuvant-free mouse model of tree nut allergy, Critical role of IL-4 The Journal of Immunology, 2007, 178, 37.9.

Progress 01/01/06 to 12/31/06

Outputs
An expert committee of the FAO WHO has proposed a decision tree approach for assessment of allergenic potential of foods. This approach includes the potential use of animal model as one of the methods. However a validated animal model is not available. Our effort supported by MAES has been to aid FAO, WHO decision tree approach by developing a novel method using mouse as a model system. During the last year we have further worked on the food allergenicity mouse model. We have demonstrated that the model exhibits features of human food allergy when used with two allergenic foods, hazelnut and sesame seed protein. Two key features of this model are systemic allergic response following transdermal exposure to food proteins and clinical signs of systemic anaphylaxis following oral food allergen challenge. We plan to further characterize this model to make it more economical and to elucidate the underlying immune mechanisms. Some of these data have been published (Navuluri et al 2006, Int Archives Allergy Immunology). Thus, we continue to make significant progress on this project focused on developing a mouse based system for evaluating food allergenicity.

Impacts
We have been able to develop a mouse model with features similar to some aspects of human food allergy. The results we have obtained during 2005-2006 were presented as two posters during a major scientific meeting (American Association of Immunologists, Annual Meeting, Boston, 2006). One paper related to this work was published in 2006. Based on these findings, we have received one major grant from US EPA.

Publications

• Lalitha Navuluri, Sitaram Parvataneni, Hanem Ahmed, Neil P Birmingham, Sridhar Samineni, Kelly J Caleb, Venu Gangur. Allergic and anaphylactic response to sesame seeds in mice. Identification of Ses i 3 and basic subunit of 11s globulins as allergens. Int Arch Allergy Immunol. 2006, 140(3), 270. Epub 2006 May 11
• Venu Gangur, Neil P Birmingham, Sitaram Parvataneni, Sridhar Samineni, Lalitha Navuluri, Caleb K James. Characterization of an adjuvant free mouse model of tree nut induced systemic anaphylaxis. Essential role of Stat6. American Association of Immunologists, Immunology 2006, Boston, May 15, 2006. J. Immunol. Vol 176, Suppl. To April 1, 2006. Abstract 145.2. S287.
• Sitaram Parvataneni, Lalitha Navuluri, Hanem Ahmed, Venu Gangur, Neil P Birmingham, Sridhar Samineni, Caleb K James. Transdermal exposure to sesame seed protein results in systemic IgE response, CD4 dependent Type 2 cytokine activation and sensitization for systemic anaphylaxis in mice. American Association of Immunologists, Immunology 2006, Boston, May 15, 2006. J. Immunol. Vol 176, Suppl. To April 1, 2006, Abstract 145.3, S287.

Progress 01/01/05 to 12/31/05

Outputs
Dr. Gangur started this project in 2001 fall. Major accomplishments during 2001-2004 have been reported in the previous two annual reports. An expert committee of the FAO WHO has recently (2001) proposed a decision-tree approach for assessment of allergenic potential of foods. This approach includes use of animal models as one of the methods. Our effort supported by MAES has been to aid FAO WHO decision tree approach by developing a novel method using mouse as a model system. During the last few years we have further worked on the food allergenicity mouse model using the novel enzyme linked immunoassay (ELISA) based method for measuring food specific IgE antibodies in mouse blood that we have described (Birmingham et al 2003 J Immunological Methods). During the past year, we have tested the utility of a gene knockout mouse for optimizing immune markers of food allergy. We have also examined suitability of the method for other allergenic foods such as hazelnuts. We also optimized the Type 2 cytokine responses as potential readouts for food allergic responses in mice. Some of these data have been published (Birmingham et al 2005, Int Archives Allergy & Immunology). In essence, we continue to make considerable progress in developing a mouse based system for potential future use for evaluating food allergenicity.

Impacts
Our results are expected to lead to methods to assess alleregenic potential of novel foods such as genetically modified foods. The results we have obtained during 2005 were presented during a major scientific meeting (FASEB Meeting, 2005) and three manuscripts were published. One paper received world-wide publicity via media (News Papers, Journals, online web sites and TV agencies).

Publications

• Venu Gangur, Caleb K James and Lalitha Navuluri. Sesame Allergy: A growing food allergy of global proportions? Annals of Allergy, Asthma and Immunology (2005) 95(1):4-11.
• Neil P Birmingham, Lalitha Navuluri, Sridhar Samineni, Caleb K James and Venu Gangur Hazelnut Allergy: Evidence that hazelnut can directly elicit specific IgE antibody response via activating Type-2 cytokines in mice. Int Archives Allergy & Immunol (2005) 137(4):295-302.
• Karmaus Wilfried & Venu Gangur. Does allo-immune reactivity play a role in the prenatal programming of childhood allergy? Clinical & Exp. Allergy (2005) 35(4): 405-7.
• Neil P Birmingham, Lalitha Navuluri, Sridhar Samineni, Caleb K James and Venu Gangur Hazelnut can directly elicit specific IgE antibody and Type-2 cytokine response in mice. Poster Presented: FASEB Meeting (2005), San Diego.

Progress 01/01/04 to 12/31/04

Outputs
Dr. Gangur started this project in 2001 fall. Major accomplishments during 2001-2002-2003 have been reported in the previous two annual reports. An expert committee of the FAO/WHO has recently (2001) proposed a decision-tree approach for assessment of allergenic potential of foods. This approach includes use of animal models as one of the methods. Our effort supported by MAES has been to aid FAO/WHO decision tree approach by developing improved methods using mouse as a model system. During the last year we have further worked on the food allergenicity mouse model using the novel enzyme-linked immunoassay (ELISA) based method for measuring food specific IgE antibodies in mouse blood that we have described (Birmingham et al 2003 J Immunological Methods). We have been testing the utility of a gene knockout mouse for optimizing immune markers of food allergy that may be potentially used for assessment of allergenicity of novel foods. We have also been examining whether the ELISA based method is suitable for other allergenic foods. Currently, work is in progress on testing the possibility of using Type-2 cytokines as potential readouts for food allergy in mice. We have been able to optimize various Type-2 cytokine assays for this purpose during the last year. In essence, we have made considerable progress in developing a mouse based system for potential future use for evaluating food allergenicity.

Impacts
Impact Improved methods to assess allergenicity of novel foods is a recognized need. The novel methods that we expect to develop may potentially assist in assessment of alleregenicity of novel foods such as genetically modified foods. The results we have obtained during 2003-2004 were presented during. Major scientific meeting (International Immunology Congress, Montreal, 2004) and one more poster will be presented during the Annual Meeting of American Association of Immunologists in 2005. One manuscript was submitted for publication that is under revision for re-submission.

Publications

• Publications during 2004 Campbell JD, Gangur V, Simons FE, HayGlass KT. Allergic humans are hyporesponsive to a CXCR3 ligand-mediated Th1 immunity-promoting loop (2004). FASEB J 18(2):329-31.
• Venu Gangur. Food Allergy: A Synopsis (2004) In: Handbook of Food Science; Ed: Y.H. Hui; Publisher: CRC Press/Marcel Dekker Inc. (In Press).
• Venu Gangur, Neil P Birmingham, Lalitha Navuluri, Sridhar Samineni and James Caleb (2004). Hazelnut allergy: Evidence that hazelnut can directly elicit specific IgE anitbodies via activating Type-2 cytokines. Poster presentation due 2005. FASEB Conference. Submitted

Progress 01/01/03 to 12/31/03

Outputs
Major accomplishments during 2002 have been reported in the previous annual report dated 01/12/2003. During the last year we have applied the methodologies that were established during 2002 to address an important problem as outlined in the project: development of methods to assess allergenic potential of food. An expert committee of the FAO/WHO has recently (2001) proposed a decision-tree approach for assessment of allergenic potential of foods. This approach includes use of animal models as one of the methods. Our effort supported by MAES has been to aid FAO/WHO decision tree approach by developing a novel method using mouse as a model system. We are reporting here a major accomplishment during the past year in MAES supported project. This relates to the development of a novel enzyme-linked immunoassay (ELISA) based method for measuring food specific IgE antibodies in mouse blood. Food specific IgE antibodies are thought to be central mediators of most food allergic reactions. However, there was no in vitro method available to measure food specific IgE antibody in mouse system. Passive cutaneous anaphylaxis (PCA) assay has been a gold standard method to measure allergen-specific IgE antibody (ASIgE Ab) levels in allergy mouse models. Many factors including stringent guidelines for laboratory animal use make PCA a difficult choice. Therefore, alternative methods are needed that can be readily applied for measurement of specific IgE antibody levels in mouse serum. Consequently, we developed this novel ELISA-based method that is more sensitive in comparison to PCA, IgE isotype-specific (because it has little cross-reactivity with IgG1 or IgG2a isotype) and highly reproducible (<10% inter- or intra-assay variation). Furthermore, we demonstrated the utility of this assay to measure specific IgE Ab against a variety of food extracts including chicken egg, peanut, almond, filbert/hazelnut and sweet potato. We have published these significant results in a major peer-reviewed journal, Journal of Immunological Methods. In addition one abstract was published in FASEB Journal. Currently, experiments are underway to test the utility of gene knockout mouse for optimizing immune markers of food allergy that may be potentially used for assessment of allergenicity of novel foods.

Impacts
Improved methods to assess allergenicity of novel foods is a recognized need. The novel method that we have developed may potentially assist in assessment of alleregenicity of novel foods such as genetically modified foods. The significance of our results is evidenced by publication in a major peer-reviewed journal. In addition, we have received a large number of reprint requests of our paper from around the world. Based on this work we have received invitations for book chapters (from Marcel Dekker; American Chemical Society & FDA) and requests for assistance with regard to use of this novel method by industry (Bayer).

Publications

• Neil Birmingham, Sandhya P, S. Thanesvorakul, Bill Stefura, Kent HayGlass and Venu Gangur (2003) An ELISA based method for measurement of food specific IgE antibody in mouse serum: An alternative to the passive cutaneous anaphylaxis assay. Journal of Immunological Methods 275: 89-98.
• Venu Gangur, Neil Birmingham, S. Thanesvorakul and S. Joseph (2003). CCR3 and CXCR3 as drug targets for allergy: Principles and Potential. Current Drug Targets:Inflammation & Allergy 2, 53-62.
• Neil Birmingham, Sandhya P, S Thanesvorakul, Bill Stefura, Kent HayGlass and Venu Gangur. Food Allergy: An ELISA based method for measurement of food specific IgE antibody in mouse serum. FASEB J 2003, 17(7): C253 (AB#121.21).
• Neil P Birmingham and Venu Gangur. Food Allergy: Hazelnuts elicit specific IgE antibody response in mice. FASEB J 2003, 17(7): C252 (AB#121.20).
• Neil P Birmingham and Venu Gangur. Immune response to hazelnuts in mice: Comparison of antigenic vs. allergenic cross-reactivity with foods and aeroallergens. NFSTC 5th year poster presentations. NFSTC/CVM, MSU, October 8, 2003.
• Venu Gangur, and Carol Wruble (2003) Food Allergy Facts, Michigan State University Extension. Extension quarterly media packet.
• Venu Gangur, and Carol Wruble (2003) Peanut Allergy; Michigan State University Extension, Extension quarterly media packet.

Progress 01/01/02 to 12/31/02

Outputs
Dr. Gangur started this project ONE year ago. Major accomplishments in the FIRST year of the project are briefly described below. 1. Setting up a Food Allergy & Immunology Research Laboratory at the Michigan State University. Dr. Gangur has established a state-of-the art Food Allergy & Immunology Research Laboratory at the Michigan State University in the past year. He has established the lab virtually from scratch as only the lab space was provided. This lab was earlier a food chemistry research lab and therefore had to be reorganized extensively. He setup the lab starting from ordering basic lab equipment such as pH meter, balances etc. He worked extensively identifying highly competitive and appropriate source for purchase of each of the general equipment as well as the specialty item relevant to his food allergy research. He spent lot of time organizing the laboratory clean up and updating the basic laboratory furniture (such as student working chairs, desks, lab carts etc). He has also established a cell culture laboratory in the department for general use by the Faculty. A summer student assisted in this process. 2.Following are the major scientific accomplishments in the past year. i) Dr. Gangur established the following Immunological Research Technologies in his new research lab in the past year: (A) Established an ultra-sensitive assay system based on ELISA for measuring food allergen specific IgE Ab as an alternative to animal based, widely used, PCA (passive cutaneous anaphylaxis) method. He was successful in demonstrating suitability of this assay for a number of food types including peanut, hazelnut and chicken egg allergens. This work has been submitted for publication. (B) Methods for quantitation of total serum IgE, IgG1 and IgG2a levels. (C) Methods for quantitation of food specific IgG1 and IgG2a antibody levels against the following food types: Chicken egg, peanut, almond, hazelnut, walnut, soybean, coffee and sweet Potato. (D) Dr. Gangur has established the differential leukocyte assay system, which is used to determine the relative proportion of various immune cells in the peripheral blood. He is especially excited for being able to apply this assay system to determine accurately the number of eosinophils (one major type of immune cells that cause food allergy) in mice that were sensitized to food allergens. (ii) Establishment of a mouse model for molecular studies on tree-nut allergy. Dr. Gangur has been working on developing a mouse model for food allergy with a focus on tree-nut allergy. He has been successful in identifying several critical factors (such as type of mice strain to use etc.,) towards this major goal. He is planning to use this system to address the research proposed in the MAES umbrella project. Please note that some of major accomplishments listed above are scientifically highly significant. This is evidenced by the publication of some these data in major professional journals (please see publications list). Furthermore, these accomplishments have established a strong foundation to further pursue the proposed research objectives for the second and subsequent years of the MAES umbrella project.

Impacts
This was the FIRST year of the project.

Publications

• Gangur, V., Birmingham, N.P. and Thanesvorakul, S. 2002. Chemokines in health and disease. Vet. Immunol. Immunopathol. 86:127-136.
• Birmingham, N.P., Thanesvorakul, S. and Gangur, V. 2002. Relative immunogenicity of commonly allergenic foods versus rarely allergenic and non-allergenic foods in mice. J Food Prot. Vol. 65, No.12, 1988-91pp.
• Birmingham, N.P., Thanesvorakul, S. and Gangur, V. 2002. Food allergy: Relative antigenicity of common vs. rarely allergenic foods. FASEB J 16(5): A1240 (AB#931.11).