EFFECT OF EXTRUSION PROCESSING ON IN VIVO ALLERGENICITY OF HAZELNUT PROTEIN EXTRACT IN AN ADJUVANT-FREE MOUSE MODEL By Tina Consetta Ortiz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Food Science – Master of Science 2014 ABSTRACT EFFECT OF EXTRUSION PROCESSING ON IN VIVO ALLERGENICITY OF HAZELNUT PROTEIN EXTRACT IN AN ADJUVANT-FREE MOUSE MODEL By Tina Consetta Ortiz Life-threatening nut allergy is a growing public health problem in many countries including the United States. 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 reduce in vivo allergenicity using an adjuvant-free mouse model of hazelnut allergy established in our laboratory. Groups of mice received transdermal exposure (TDE) to soluble extrusion processed hazelnut protein (EHNP) versus soluble raw hazelnut protein (RHNP) extract preparations. Mice were evaluated for systemic IgE antibody response (sIgER), hypothermia shock response (HSR), and mucosal mast cell response. 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 IP challenge. Finally, we measured plasma mouse mast cell protease-1 (mMCP-1) levels after oral and IP injection challenges. We found that EHNP elicited reduced mMCP-1 response compared to RHNP upon both oral as well as IP injection challenges. These results suggest that it is possible to use extrusion processing to reduce in vivo allergenicity of hazelnut in a mouse model. This is also the first study demonstrating the utility of a mouse model to evaluate the effect of food processing on in vivo food allergenicity in general and hazelnut in particular. Dedicated to my loving partner MATT & Our children SPENCER & KAITLYN iii ACKNOWLEDGMENTS This work could not have been accomplished without the help and support of many individuals. First and foremost, I would like to thank Dr. Venu Gangur for being my teacher, mentor, advisor, and friend. He has not only taught me to be an effective researcher, but he has taught me the value of finding balance in life. I am eternally grateful for the wisdom that he’s shared with me and for his unwavering support during my time at MSU. I would like to also thank my committee members, Dr. Perry K.W. Ng and Dr. Sungjin Kim who so kindly shared with me their extensive knowledge and expertise in their fields of study and who have supported me throughout my journey. I also thank all of the lab members that I’ve been so fortunate to work with throughout the last two years: Babu Gonipeta, Radhakrishna Para, Yingli He, Ines Srkalovic and Mike Reitmeyer. I would like to thank Ramasamy Ravi for his assistance with extrusion processing and Heather Dover and Dr. Cheryl Rockwell for their assistance with Western blotting. I thank my friends in Dr. Pestka’s lab and Dr. Linz’s lab for their support. I would also like to thank MSU Department of Food Science & Human Nutrition, MSU College of Agriculture & Natural Resources, MSU Ag Bio Research, and the US EPA for their financial support. I thank my family for their constant support and words of encouragement. Finally, I would like to especially thank my stepmother and best friend, Dr. Deirdre Ortiz, who has supported me, guided me, taught me, and loved me throughout my academic career. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix KEY TO ABBREVIATIONS ........................................................................................................ xi CHAPTER 1: Introduction ............................................................................................................. 1 1.1 Statement of the Problem .......................................................................................................... 6 1.2 Rationale for the Research ........................................................................................................ 7 1.3 Overall Goal and Hypothesis .................................................................................................... 7 1.4 Specific Objectives.................................................................................................................... 8 1.5 Scope of the Research ............................................................................................................... 8 CHAPTER 2: Review of Literature ................................................................................................ 9 2.1 Adverse Reactions to Food Proteins: Immune and Non-Immune Disorders ............................ 9 2.2 Hypersensitivity Disorders: Gell-Coombs Classification ....................................................... 10 2.3 Mechanisms of Hypersensitivity Reactions to Foods ............................................................. 13 2.4 Food Processing and its Effect on Food Protein Allergenicity ............................................... 17 2.5 Hazelnut as a Major Allergenic Food ..................................................................................... 21 2.6 Effect of Food Processing on Hazelnut Allergenicity ............................................................ 24 2.7 An Adjuvant-free Mouse Model of Food Allergy .................................................................. 29 CHAPTER 3: Materials and Methods .......................................................................................... 32 3.1 Preparation of Defatted Hazelnut Flour .................................................................................. 32 3.2 Extrusion Processing............................................................................................................... 32 3.3 Protein Extraction ................................................................................................................... 35 3.4 Protein Concentration ............................................................................................................. 35 3.5 SDS-PAGE ............................................................................................................................. 37 3.6 Animals ................................................................................................................................... 37 3.7 Sensitization Method .............................................................................................................. 38 v 3.8 Blood Sample Collection ........................................................................................................ 39 3.9 Analysis of IgE Antibody Response ....................................................................................... 39 3.10 Analysis of IgG1 and IgG2a Antibody Response ................................................................. 40 3.11 Analysis of Hypothermia Shock Response ........................................................................... 41 3.12 Analysis of Mast Cell Degranulation .................................................................................... 41 3.13 Western Blot Analysis .......................................................................................................... 42 3.14 Randomized Controlled Study Design.................................................................................. 43 3.14.1 Objective 1 ......................................................................................................................... 43 3.14.2 Objective 2.1 ...................................................................................................................... 43 3.14.3 Objective 2.2 ...................................................................................................................... 46 3.14.4 Objective 3.1 ...................................................................................................................... 46 3.14.5 Objective 3.2 ...................................................................................................................... 48 CHAPTER 4: Results ................................................................................................................... 52 4.1 Analysis of Raw and Extrusion Processed Hazelnut Protein Extracts Using SDS-PAGE ..... 52 4.2 Characterization of Hazelnut-Specific Systemic IgE Antibody Responses in BALB/c Mice Upon Transdermal Exposure to Raw Versus Extrusion Processed Hazelnut Protein Extract ...... 56 4.3 Characterization of Hazelnut-Specific Systemic IgG1 Antibody Responses in BALB/c Mice Upon Transdermal Exposure to Raw Versus Extrusion Processed Hazelnut Protein Extract ...... 59 4.4 Characterization of Hazelnut-Specific Systemic IgG2a Antibody Responses in BALB/c Mice Upon Transdermal Exposure to Raw Versus Extrusion Processed Hazelnut Protein Extract...... 59 4.5 Identification of IgE Antibody Binding Proteins in Raw and Extrusion Processed Hazelnut Protein Extracts Using Western Blot Analysis ............................................................................. 63 4.6 Analysis of Hypothermia Shock Response in BALB/c Mice Upon Oral Challenge With Raw Versus Extrusion Processed Hazelnut Protein Extract ................................................................. 66 4.7 Analysis of Hypothermia Shock Response in BALB/c Mice Upon Systemic Challenge With Raw Versus Extrusion Processed Hazelnut Protein Extract ......................................................... 67 4.8 Characterization of Mucosal Mast Cell Response in BALB/c Mice Upon Oral Challenge With Raw Versus Extrusion Processed Hazelnut Protein Extract ................................................ 69 4.9 Characterization of Mucosal Mast Cell Response in BALB/c Mice Upon Systemic Challenge With Raw Versus Extrusion Processed Hazelnut Protein Extract ................................................ 72 CHAPTER 5: Discussion.............................................................................................................. 76 CHAPTER 6: Summary and Future Direction ............................................................................. 85 vi APPENDICES .............................................................................................................................. 89 Appendix A ................................................................................................................................... 90 Protein Profile from an Alternative Extraction Method ............................................................... 90 Appendix B ................................................................................................................................... 92 Vehicle Control Data for Hazelnut-specific Systemic IgE: Bar Graphs....................................... 92 Appendix C ................................................................................................................................... 94 Vehicle Control Data for Hazelnut-specific Systemic IgG1: Bar Graphs .................................... 94 Appendix D ................................................................................................................................... 96 Vehicle Control Data for Hazelnut-specific Systemic IgG2a: Bar Graphs .................................. 96 Appendix E ................................................................................................................................... 98 Vehicle Control Data for Mucosal Mast Cell Protease (mMCP)-1 Levels After Oral Challenge 98 Appendix F.................................................................................................................................. 100 Vehicle Control Data for Mucosal Mast Cell Protease (mMCP)-1 Levels After Systemic Challenge .................................................................................................................................... 100 REFERENCES ........................................................................................................................... 102 vii LIST OF TABLES Table 1 List of allergenic foods identified by the US FDA, CFIA and the EU .............................. 3 Table 2 List of allergenic tree nuts as identified by the US FDA, CFIA and the EU ..................... 4 Table 3 Major characteristics that distinguish mucosal and connective tissue mouse mast cells 16 Table 4 Effect of common food processing methods on food allergenicity: a summary ............. 22 Table 5 Allergenic proteins from hazelnut: a summary................................................................ 23 Table 6 Thermal stability of hazelnut allergens ............................................................................ 25 Table 7 Quantification of effect of extrusion processing on in vivo allergenicity of hazelnut protein based on clinical reactivity in an adjuvant-free mouse model of food allergy ................. 75 viii LIST OF FIGURES Figure 1 Gel and Coombs classification of hypersensitivity disorders ......................................... 11 Figure 2 Preparation of defatted hazelnut flour from shelled hazelnuts ....................................... 33 Figure 3 The co-rotating twin-screw extruder (APV Baker MPF-19/25) used in this study........ 34 Figure 4 Preparation of protein extract from raw and extruded defatted hazelnut flour .............. 36 Figure 5 Approach used to conduct the study described for Objective 1 ..................................... 44 Figure 6 Approach used to conduct the study described for Objective 2.1 .................................. 45 Figure 7 Approach used to conduct the study described for Objective 2.2 .................................. 47 Figure 8 Approach used to conduct the study described for Objective 3.1 .................................. 49 Figure 9 Approach used to conduct the study described for Objective 3.2 .................................. 50 Figure 10 Summary of the study design to evaluate the impact of extrusion-processing on in vivo allergenicity of hazelnut protein extract in an adjuvant-free mouse model .................................. 51 Figure 11 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts under non-reducing conditions.............................................................. 53 Figure 12 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts under reducing conditions ..................................................................... 55 Figure 13 Systemic IgE antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves ................................................................................................................. 57 Figure 14 Systemic IgE antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis............................................................................................................. 58 Figure 15 Systemic IgG1 antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves ................................................................................................................. 60 ix Figure 16 Systemic IgG1 antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis............................................................................................................. 61 Figure 17 Systemic IgG2a antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves ................................................................................................................. 62 Figure 18 Systemic IgG2a antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis............................................................................................................. 64 Figure 19 The IgE-binding hazelnut protein analysis by Western blot method ........................... 65 Figure 20 Comparison of hypothermia shock response upon oral challenge to raw hazelnut protein (RHNP) versus extrusion processed hazelnut protein (EHNP) extracts........................... 68 Figure 21 Comparison of hypothermia shock response upon IP challenge to raw hazelnut protein (RHNP) versus extrusion processed hazelnut protein (EHNP) extracts ....................................... 70 Figure 22 Analysis of mucosal mast cell degranulation responses upon oral challenge with raw versus extrusion-processed hazelnut protein extract..................................................................... 71 Figure 23 Analysis of mucosal mast cell degranulation responses upon IP challenge with raw versus extrusion-processed hazelnut protein extract..................................................................... 73 Figure 24 Summary of major findings from this study................................................................. 89 Figure 25 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts prepared by an independent published method (Cucu and others 2011): analysis under reducing conditions ............................................................................................... 91 Figure 26 Systemic IgE antibody responses in vehicle control mice ........................................... 93 Figure 27 Systemic IgG1 antibody responses in vehicle control mice ......................................... 95 Figure 28 Systemic IgG2a antibody responses in vehicle control mice ....................................... 97 Figure 29 Analysis of mucosal mast cell degranulation responses upon oral challenge with vehicle control ............................................................................................................................... 99 Figure 30 Analysis of mucosal mast cell degranulation responses upon IP challenge with vehicle control ......................................................................................................................................... 101 x KEY TO ABBREVIATIONS BSA Bovine Serum Albumin EHNP Extrusion Processed Hazelnut Protein ELISA Enzyme-linked Immunosorbent Assay HN Hazelnut HSR Hypothermia Shock Response Ig Immunoglobulin IL Interleukin IP Intraperitoneal mMCP-1 Mouse Mast Cell Protease-1 MW Molecular Weight OC Oral Challenge RHNP Raw Hazelnut Protein SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TDE Transdermal Exposure TGP 30 mM Tris-HCl, 50% v/v Glycerin and 0.2% Phenol xi CHAPTER 1: Introduction Food allergies are an undesirable immune response to food proteins that lead to a potentially lethal disease. This type of allergy is typically associated with the production of IgE antibodies specific to food proteins called food allergens (Sampson 2001; Boyce and others 2011). Allergen specific IgE antibodies are capable of activating effector immune cells, such as mast cells, resulting in mediator release and consequent local and/or systemic disease symptoms. Locally, the clinical consequences of these reactions include symptoms such as hives, nausea, vomiting, and diarrhea. However, food allergy is also capable of inducing multiple organ systemic reaction known as anaphylaxis, a life-threatening reaction that requires emergency medical attention. Food allergies affect both children and adults with an estimated prevalence of 8% among children and 5% among adults (Gupta and others 2011; Sicherer and Sampson 2014). Food allergies result in a number of symptoms ranging from mild localized symptoms to lifethreatening anaphylaxis. According to an expert panel on food allergy, IgE-mediated food allergies are associated with gastrointestinal symptoms such as nausea, vomiting and diarrhea; oral allergy syndrome; skin conditions such as acute urticaria, angioedema, and atopic dermatitis; as well as respiratory complications indicated in anaphylactic reactions (Boyce and others 2011). Anaphylaxis may also present with dangerous cardiovascular symptoms. In addition, it has been reported that nearly 40% of children with food allergies have had severe life-threatening allergic reactions in the past with the most severe reactions being to foods such as tree nuts or peanuts (Gupta and others 2011). Furthermore, both the 1 prevalence and the severity of food allergies are reportedly growing at an alarming rate (Branum and Lukacs 2009; Sicherer and Sampson 2014). In addition, the economic burden from food allergies is estimated at $24.8 billion per year for children alone (Gupta and others 2013). Thus, food allergies are a growing and significant public health concern in the United States. Other countries, including Australia and the European Union, are reporting a similar trend (Osborne and others 2011; Nwaru and others 2014). According to the United States Food and Drug Administration (US FDA), there are 8 major allergenic foods that are responsible for 90% of food allergic reactions (2006). These “red flag” allergenic foods are: cow’s milk, chicken eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeans. Notably, other regulatory agencies from Canada and the European Union (EU) include additional foods besides those 8 foods in their regulations (Table 1). In contrast, US FDA has identified more tree nuts in its list compared to Canada and the EU (Table 2). Nevertheless, hazelnut (also known as filbert) is considered an allergenic tree nut by all of these agencies. These major allergens are regulated in the United States under the Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004 (US FDA 2006). According to this law, foods containing any of these allergens must be properly identified on the food label. Absence of such labeling is a leading cause of Class I recalls (Gendel and Zhu 2013). There is currently no effective method to prevent food allergy other than complete avoidance of exposure to the food allergen. Thus, individuals afflicted with this potentially fatal condition are instructed by clinicians to avoid foods that contain the allergen. The currently enforced zero tolerance policy can be difficult to implement and accidental ingestion can and 2 Table 1 List of allergenic foods identified by the US FDA, CFIA and the EU Country Allergenic Foods Reference * US FDA Milk, Eggs, Fish, Crustacean shellfish, Tree nuts, United States Peanuts, Wheat, Soybeans Food and Drug Administration 2006 § † CFIA Eggs, Milk, Mustard, Peanuts, Seafood (fish, crustaceans, shellfish), Sesame, Soy, Sulphites, Tree nuts, Wheat EU Cereals containing gluten: i.e. wheat, rye, barley, oats, spelt, kamut Crustaceans, Eggs, Fish, Peanuts, Soybeans, Milk (including lactose), Tree nuts, Celery, Mustard, Sesame seeds, Sulphur dioxide and sulphites > 10mg/kg * United States Food and Drug Administration § Canadian Food Inspection Agency † Official Journal of the European Union 3 Canadian Food Inspection Agency 2014 Official Journal of the European Union 2003 (Directive 2003/89/EC) Table 2 List of allergenic tree nuts as identified by the US FDA, CFIA and the EU * US FDA §CFIA †EU TREE NUT Almond + + + Beech nut + Brazil nut + + + Butternut + Cashew + + + Chestnut (Chinese, American, European, Seguin) + Chinquapin + Coconut + Filbert/Hazelnut + + + Ginko nut + Hickory nut + Lichee nut + Macadamia nut/Bush nut/Queensland nut + + + Pecan + + + Pine nut/Pinon nut + + Pili nut + Pistachio + + + Sheanut + Walnut (English, Persian, Black, Japanese, + + + California), Heartnut * United States Food and Drug Administration 2011 § Canadian Food Inspection Agency 2012 † Official Journal of the European Union 2003 4 does occur (Atkins and Bock 2009). Highly sensitized individuals are especially at high risk of potentially fatal anaphylactic reaction, even at low doses (Cianferoni and others 2013). Therefore, other measures besides avoidance to prevent such a life-threatening condition are desperately needed. There are few therapeutic options for food allergies such as sublingual immunotherapy (SLIT), oral immunotherapy (OIT), and epicutaneous immunotherapy (EPIT) that are intended to desensitize food allergic individuals by inducing tolerance to the allergen (Berin 2014; Sato and others 2014). However, since these therapies involve exposure to the natural food allergen, they can potentially be dangerous (Enrique and other 2005; Keet and others 2012). Vaccination methods in experimental models have also been studied in an effort to reduce the risk of anaphylaxis. These methods include a DNA-based vaccination that suppresses the IgE antibody-mediated immune response in a murine model and therefore prevents anaphylactic hypersensitivity in response to β-galactosidase (Horner and others 2000). Another study used vaccination with genetically engineered hypoallergenic birch pollen proteins to produce protective IgG antibodies and reduce IgE antibody production in birch pollen-allergic patients (Niederberger and others 2004). Another recent vaccination method used modified vaccinia virus Ankara-encoding ovalbumin and was investigated for its potential to suppress IgE antibody production in BALB/c mice (Bohnen and others 2013). However, these vaccination methods are not available for many allergenic foods and their success has been limited. For instance, a Phase I study of EMP-123, a vaccination against peanut allergy, 5 resulted in frequent allergic reactions among human participants with 20% of participants having anaphylactic responses (Wood and others 2013). Other potential methods include the use of food processing to reduce allergenicity (Sathe and others 2005). Some common methods used to process foods include baking, dry roasting, frying, boiling, blanching, steaming, extrusion, microwaving, and irradiation. Some studies explore the use of processing methods to make hypoallergenic foods. The underlying principle is that processing can disrupt protein structure by causing events such as denaturation, partial unfolding, aggregation, gelation, and chemical modification (Davis and Williams 1998). It is proposed that such changes in protein structure could potentially alter the ability of food proteins to cause allergic reactions (Besler and others 2001). 1.1 STATEMENT OF THE PROBLEM Little is known about the effect of food processing on tree nut allergenicity in vivo and the effect of extrusion processing on in vivo allergenicity of tree nuts, such as hazelnuts, is entirely unknown. Tests on in vivo allergenicity include human studies and animal models. In human studies, the double-blind placebo-controlled food challenge (DBPCFC) has long been the gold standard for in vivo testing (Sampson 1999; Asero and others 2007; Pettersson and others 2014). However, as previously stated, it is potentially dangerous to expose foodallergic humans to allergens that trigger anaphylaxis and therefore few human studies have been conducted to assess the effects of processing on hazelnut allergy using DBPCFC (Hansen and others 2003; Worm and others 2009). These studies are further described in Chapter 2 (page 26 and 28). Mouse models have often been used in immunological studies and they provide a more ethical and cost-efficient method in which to study food allergy. 6 However, most mouse models utilize adjuvants and / or intraperitoneal (IP) injection challenges and therefore may not adequately simulate the human condition (Gonipeta and others 2013). In our study, we used an adjuvant-free mouse model to evaluate the effect of extrusion processing on hazelnut allergenicity in vivo upon oral challenge. 1.2 RATIONALE FOR THE RESEARCH Results from previous studies in the literature provide a strong rationale to evaluate the effect of food processing on in vivo allergenicity of tree nuts in general and hazelnut in particular. Previous studies demonstrate altered (increased or decreased) allergenicity after heat treatment. Some in vitro studies demonstrate that processing methods may decrease the allergenicity of food proteins (Masthoff and others 2013). Others have indicated that processing may increase allergenicity (Maleki and others 2000). Therefore, it is critical to study the effects of processing on food allergens and to examine the potential change in allergenicity in vivo. The outcome of this study may provide insight into making hypoallergenic hazelnut proteins as well as insight into whether processing may increase allergenicity. The aim of this study was to address this critical gap in knowledge. 1.3 OVERALL GOAL AND HYPOTHESIS The overall goal was to compare in vivo allergenicity of soluble raw versus extrusion processed hazelnut protein extract in a mouse model of food allergy. The hypothesis was tested that extrusion processing will reduce in vivo allergenicity of soluble raw hazelnut protein in a mouse model of food allergy. 7 1.4 SPECIFIC OBJECTIVES There were three objectives in this study: 1. To evaluate whether soluble extrusion processed hazelnut protein extract elicits a reduced systemic IgE antibody response compared to soluble raw hazelnut protein extract 2. To test whether soluble extrusion processed hazelnut protein extract induces less hypothermia shock response than soluble raw hazelnut protein extract in hazelnutsensitized mice 3. To test whether soluble extrusion processed hazelnut protein extract elicits reduced mucosal mast cell degranulation response compared to soluble raw hazelnut protein extract in hazelnut-sensitized mice 1.5 SCOPE OF THE RESEARCH This research is limited to extractable soluble proteins from raw hazelnuts and extrusion processed hazelnuts. Soluble proteins were studied because most food allergens are generally thought to be glycoproteins that are water-soluble (O’Neil and others 2011). This research is limited to the use of a previously published mouse model of food allergy (Birmingham and others 2007). Many, but not all, clinical reactions can be assessed in currently available mouse models (Gonipeta and others 2013). Overall conclusions from this work must be viewed in the context of a mouse system and not necessarily the human situation. 8 CHAPTER 2: Review of Literature 2.1 ADVERSE REACTIONS TO FOOD PROTEINS: IMMUNE AND NON-IMMUNE DISORDERS Food associated adverse reactions are categorized as immune or non-immune disorders. Immune disorders can be further categorized as IgE-mediated or non-IgE-mediated while non-immune mediated food disorders are generally referred to as intolerances (Ortolani and Pastorello 2006). Most food allergies are an immune reaction associated with the production of IgE antibodies against food proteins (Sampson 1999). IgE-mediated food allergy causes symptoms such as immediate gastrointestinal (GI) hypersensitivity, skin related conditions such as acute urticaria and angioedema, respiratory conditions such as allergic asthma, and systemic lifethreatening anaphylaxis (Sabra and others 2003; Boyce and others 2011). In addition, some pollen-allergic individuals produce IgE antibodies that can recognize food proteins and lead to oral allergy syndrome (OAS). OAS is a form of localized IgE-mediated reaction that causes itching and swelling of the lips, mouth, and throat (Kondo and Urisu 2009). It is caused by the cross-reactivity between pollen and foods such as fruits, vegetables, and nuts. Some OAS symptoms may also present in other cross-reactive syndromes such as latex-fruit syndrome. This condition occurs when latex-allergic individuals react to eating fruits such as banana, kiwi, and peach due to cross-reacting epitopes (Wagner and Breiteneder 2002). Eosinophilic esophagitis (EOE) is a mixed IgE- and non-IgE-mediated food-induced disorder characterized by an abundance of eosinophils in the esophageal mucosa. It causes 9 inflammation in the esophagus leading to symptoms such as heartburn, dysphagia, and other esophageal impairment (Raheem and others 2014). Other food related immune disorders are not IgE-mediated but are cell mediated and include diseases such as celiac disease (also known as gluten-sensitive enteropathy) and food proteininduced enterocolitis (FPIES). Celiac disease is an autoimmune disorder that occurs as an immunological response to gluten proteins in wheat, barley, and rye. It causes GI responses including abdominal pain and diarrhea that can lead to serious complications such as malabsorption and failure to thrive (Green and Cellier 2007). FPIES also causes GI symptoms and can lead to acute dehydration, hypotension, and sepsis. The mechanism of FPIES is not completely understood but it is known to be induced by food proteins and it occurs without the production of food-specific IgE antibodies (Fiocchi and others 2014). Other food-induced adverse conditions are non-immune related disorders. These generally refer to intolerances such as lactose intolerance. Lactose intolerance occurs when an individual is deficient in lactase, a β-galactosidase enzyme necessary to hydrolyze the dissacharide lactose into its monosaccharide subunits, glucose and galactose. If lactose is not hydrolyzed, it enters the large intestines and is fermented by bacteria, causing symptoms such as bloating and diarrhea (Ortolani and Pastorello 2006). 2.2 HYPERSENSITIVITY DISORDERS: GELL-COOMBS CLASSIFICATION Hypersensitivity disorders occur when innocuous substances induce deleterious immunological responses. These responses are distinguished based on the Gell-Coombs classification of hypersensitivity reactions summarized in Figure 1. Type I immediate 10 Figure 1 Gel and Coombs classification of hypersensitivity disorders. Various hypersensitivity reactions mediated by antibody and cellular mechanisms are shown in the figure. (Figure source: Murphy and others 2008). 11 hypersensitivity reactions are IgE antibody-mediated responses, type II and III hypersensitivity reactions are IgG antibody-mediated responses, and type IV hypersensitivity reactions are cell-mediated delayed-type hypersensitivity reactions (Murphy and others 2008). Type I immediate hypersensitivity reactions are generally associated with allergies, such as food allergies, and occur within minutes after exposure to an allergen (Galli and others 2008). This type of reaction occurs when IgE antibodies are produced against an allergen and bind to receptors on the surface of mast cells. Upon subsequent exposure, allergens bind to these receptor-bound IgE antibodies. This signals mast cells to release preformed mediators that precipitate allergic reactions such as anaphylaxis (Murphy and others 2008). Type II hypersensitivity reactions are mediated by IgG antibodies that are specific to selfantigens or drugs that have bound to host cell surfaces or receptors. In the case of drugs, such as penicillin, antibodies attach to the host cells targeting these cells for destruction by phagocytic immune cells. This leads to host tissue damage that can occur within minutes to hours of exposure (Murphy and others 2008). Type III hypersensitivity reactions occur when IgG antibodies specific to soluble antigens form immune complexes that trigger the inflammatory response. Once antigen-specific IgG production is induced, subsequent reactions can occur within 3-10 hours of exposure to antigen (Ghaffar 2010). Type II and III hypersensitivity reactions are thought not to be associated with reactions to foods. 12 Type IV delayed-type hypersensitivity reactions occur 48-72 hours after exposure to antigen and can cause food-specific chronic diseases. They are cell-mediated and further categorized by the type of T lymphocyte involved. T helper 1 (Th1) cells and cytotoxic T lymphocytes (CTL) are indicated in Celiac disease. The disease occurs as a result of direct destruction of enterocytes by CTLs (Sollid and Jabri 2005). T helper 2 (Th2) cells are involved in both an early-phase (type I immediate hypersensitivity) and a late-phase response. The early-phase results in the production of IgE antibodies and mast cell activation while the late-phase response occurs as a result of mediators released by Th2 cells during the early-phase reaction. This reaction causes chronic allergic inflammation symptoms such as asthma and allergic rhinitis (Galli and others 2008). 2.3 MECHANISMS OF HYPERSENSITIVITY REACTIONS TO FOODS Most food allergies are type I immediate hypersensitivity reactions that are IgE- and mast cell-mediated (Sabra and others 2003; Sicherer and Sampson 2010). Food allergy consists of two phases: sensitization and oral elicitation. Sensitization occurs when individuals produce food-specific IgE antibodies to food allergens. The elicitation phase occurs upon subsequent oral exposure to the allergen, when the sensitized individual exhibits clinical reactions as a result of mediators that are released from mast cells. The immunological process of sensitization occurs when antigen-presenting cells (APCs), such as dendritic cells, deliver food proteins to T lymphocytes. T lymphocytes recognize processed food protein fragments bound to major histocompatibility complex (MHC) class II on APCs. In response, T cells receive polarizing signals to differentiate into either T helper 1 (Th1) or T helper 2 (Th2) cells that secrete various cell-signaling molecules (cytokines and 13 chemokines) and lead to differing humoral responses. In mice, Th1 cells produce IFN-γ that helps B cells to produce antibodies such as IgG2a while Th2 cells are characterized by the production of three specific sets of cytokines: IL-4, IL-5, and IL-13; and helps the production of IgE and IgG1 antibodies by B cells (Romagnani 1997; Adel-Patient and others 2000). Increased IgG2a responses are considered as anti-allergic responses in mouse models (Lewkowich and others 2004). Food allergies are mostly associated with the production of IgE antibodies. Among the cytokines secreted by Th2 cells, IL-4 is an essential cytokine in signaling B cells to class switch antibody production from the IgM / IgG isotype to the IgE isotype. The half-life of IgE antibodies is about 2 days, the shortest of any immunoglobulin isotype. IgE antibodies bind to mast cells, basophils, or eosinophils via a cell surface receptor, FcεRI, which binds the constant region of IgE antibodies with high-affinity (Sicherer and Sampson 2010; Stone and others 2010). When bound to IgE, the FcεRI high-affinity receptors are expressed for the life of the cell, which can be months for mast cells or days for basophils (Stone and others 2010). Although mast cells, basophils, and eosinophils can all participate in food allergic reactions, mast cells and basophils are considered major players in near-fatal systemic anaphylaxis (Kawakami and Galli 2002). In addition, mast cells are the predominant initial responders in the intestinal mucosa and are further discussed below (Bischoff 1996). Mast cells are prototypic allergy-mediating immune cells because they function within the innate immune system by releasing secretory granules that contain active preformed mediators in response to activation signals such as cross-linking of cell surface FcεRI when receptor-bound IgE antibodies bind allergen via their variable regions. Mast cells are present 14 within both connective and mucosal tissues. Once an individual produces IgE antibodies and their mast cells are loaded with IgE, they are considered sensitized. Subsequent exposure to a food allergen causes the clinical elicitation phase of the reaction. The binding of allergen molecules to the surface-bound IgE on mast cells crosslinks cell surface receptors, FcεRI, and sends a signal to the mast cell to degranulate. The release of active preformed mediators as well as newly synthesized mediators as a result of this signal is responsible for the clinical symptoms of the type I immediate hypersensitivity response (Ghaffar 2010). Within their granules, mast cells store toxic substances such as histamine, a short-acting amine that causes smooth muscle contraction leading to diarrhea, vomiting, and the constriction of airways during an anaphylactic reaction. Histamine is pro-inflammatory and causes vasodilation, vascular permeability and enhances mucus production, increased heart rate, and flushing (Castells 2006). In general, mast cells are classified into two types, connective tissue mast cells (CTMC) and mucosal mast cells (MMC). The CTMCs reside in the skin and other areas of tissues close to the external environment while MMCs are predominantly found in mucosal tissues such as the mucosa of the gastrointestinal tract. These cells can be distinguished based on differing characteristics such as those listed in Table 3. For instance, CTMCs release more histamine than MMCs, contain different proteoglycans and lipid mediators as well as different proteases (Metcalfe and others 1997; Welle 1997; Moon and others 2010). Two pathways of anaphylaxis have been described in the mouse system. The classic pathway is mediated by IgE antibodies binding to mast cells via the high-affinity receptor, FcεRI 15 Table 3 Major characteristics that distinguish mucosal and connective tissue mouse mast cells Mucosal Mast Cell Connective Tissue Mast Cell Preformed mediators Histamine < 1 pg / cell ≥ 15 pg / cell Proteoglycans Chondroitin sulfate Heparin di-B, A, E Chondroitin sulfate E Mouse mast cell proteases (mMCP) 1, 2 3, 4, 5, 6, 7 Carboxypeptidase A Mediators synthesized de novo Leukotrienes (LT) C4 + (Metcalfe and others 1997; Welle 1997; Moon and others 2010) 16 - (Sabra and others 2003; Sicherer and Sampson 2010). However, an alternative pathway has also been described, which is mediated by IgG1 antibodies, low affinity FcγRIII, macrophages, and platelet activating factor (PAF) (Miyajima and others 1997; Finkelman 2007). Both pathways are Th2-mediated and involve the production of IgG1 antibodies. Therefore, it is necessary to distinguish between these pathways in mouse models. A landmark paper published recently reported that mMCP-1, a MMC-specific marker, can distinguish IgE- from IgG1-mediated anaphylaxis (Khodoun and others 2011). 2.4 FOOD PROCESSING AND ITS EFFECT ON FOOD PROTEIN ALLERGENICITY As previously stated, most food allergies are attributed to an IgE-mediated response to food proteins. Therefore, the effect of processing on allergenicity is thought to be directly related to the changes in protein structure that can occur during various processes (Wal 2003). Many of these are thermal processes such as roasting (i.e. dry heat processing) whereas other processes such as autoclaving, extrusion, blanching, boiling, and steaming incorporate water (i.e. wet heat processing) (Sathe and others 2005). Extrusion is a common type of food processing method that combines both thermal and mechanical energy to produce products such as texturized vegetable proteins, breakfast cereals, snack foods, and pasta (Chang 2008). Co-rotating twin-screw extruders are the most preferred type and offer a low cost, high throughput option for mixing, heating, shearing and forming products (Ding and others 2006). Raw material and moisture are fed into the barrel of the extruder at specified rates. The controller chooses the screw configuration and speed to determine the applied shear force on the material as well as the temperature profile to which 17 the material will be subjected. The final product exits the extruder barrel through a die with a predetermined size and shape also chosen by the operator (Frame 1994; Chang 2008). Materials that undergo extrusion processing contain components such as proteins, simple sugars, starch, dietary fiber, lipids, and other vitamins and nutrients (Singh and others 2007). Such components may undergo chemical and structural changes as a result of the thermal and mechanical energy applied during the extrusion process. In particular, proteins undergo structural changes such as denaturation, partial unfolding, aggregation, gelation, and chemical modification as a result of thermal processes (Davis and Williams 1998). These changes in protein structure could potentially alter the ability of food proteins to cause allergic reactions (Besler and others 2001). Allergens contain epitopes; distinct sections of the proteins that are recognized by the immune system and are capable of generating an immune response (Sampson 2004). Epitopes may be linear or conformational in nature. Linear epitopes are made up of sequential stretches of amino acids while conformational epitopes are composed of amino acids from more than one area of the protein that make up a unique molecular motif recognized by the immune system when the protein is in its native folded state. Proteins may contain multiple linear and conformational epitopes, or a combination of the two. Linear epitopes are unlikely to be affected by heat treatment alone but may be susceptible to processes that cause hydrolysis or chemical modification (Sathe and others 2005). However, thermal processing can break disulfide bonds that hold proteins within their native threedimensional structure and can therefore disrupt conformational epitopes. This disruption may lead to changes in allergenicity (Sathe and Sharma 2009). 18 One potential change that may occur is the formation of an allergoid. An allergoid is formed through the modification of the allergenic protein such that a decrease or elimination of allergenicity occurs. Allergoids have potential use by clinicians that provide immunotherapy services. For example, in an effort to produce a hypoallergenic birch pollen allergen for specific immunotherapy (SIT), Kahlert and others generated a recombinant Bet v 1 variant capable of stimulating T lymphocytes but that had reduced IgE binding and associated basophil activity (2008). In addition, allergoids may also be useful to produce hypoallergenic food products and food processing methods are of interest in their production. Many studies have shown that dry roasting or the Maillard reaction reduces hazelnut allergenicity (Müller and others 2000; Wigotzki and others 2000; Pastorello and others 2002; Hansen and others 2003; Worm and others 2009; Iwan and others 2011; Cucu and others 2012). Boiling has been shown to reduce the allergenicity of some legumes such as kidney bean, black gram, and peanut (Álvarez-Álvarez and others 2005; Kasera and others 2012). Pulse UV-light was shown to reduce peanut allergy (Chung and others 2008). Noorbakhsh and others showed that pistachio nut allergenicity is reduced by steam-roasting (2010). Another potential consequence of food processing, and one that can have a deleterious effect is the production of neoallergens through the unfolding and refolding of proteins that can occur during processes such as thermal treatments. These neoallergens contain epitopes not present in the native proteins and can cause an increase in allergenicity. For example, it has been reported that roasting and the Maillard reaction both increase IgE binding to peanuts as a result of novel epitopes formed during processing (Maleki and others 2000; Gruber and others 2005). 19 Furthermore, the presence of heat stable linear and conformational epitopes may render thermal processes useless in changing the allergenicity of such proteins. Studies have shown that blanching, roasting, autoclaving and microwaving have no effect on almond allergenicity (Roux and others 2001; Venkatachalam and others 2002). In addition, Su and others showed that γ-irradiation followed by autoclaving, dry or oil roasting, blanching, or microwaving had no effect on the major allergenic proteins in almond, cashew, or walnut (2004). Boiling, extrusion, and microwave heating also had no effect on lupine allergenicity (Álvarez-Álvarez and others 2005). Extrusion processing was used in this study because it has not been extensively studied in regard to allergens. Two in vitro studies have been conducted to test the effect of extrusion processing on allergenicity. Extrusion processing was found to have no major impact on lupine allergens (Álvarez-Álvarez and others 2005). However, the major allergen in texturized soy protein was eliminated (Franck and others 2002). To date, no in vivo studies have been conducted to assess the effect of extrusion processing on the allergenicity of tree nuts such as hazelnuts. Food processing has the potential to decrease or eliminate allergenicity and produce hypoallergenic foods. The potential of thermal processes to alter allergenicity is associated with the denaturation and restructuring of proteins during heat treatment (Mills and others 2009). However, the effect of different processing methods on different foods varies greatly. Changes in allergenicity, or lack thereof, are dependent on the intrinsic characteristics of the allergenic proteins within the food and the food processes applied. Therefore it is critical for researchers to explore the potential effects of thermal processing on allergenicity of specific 20 food proteins in order to better understand how to mitigate life-threatening reactions caused by food allergies as well as the consequences in regard to the formation of neoallergens. The effect of processing methods on the allergenicity of some foods is summarized in Table 4. 2.5 HAZELNUT AS A MAJOR ALLERGENIC FOOD Hazelnut is a common tree nut frequently used by the food industry. Hazelnuts are low in saturated fat, high in unsaturated fat, naturally cholesterol free and contain vitamins, nutrients, and antioxidants, making them an ideal ingredient choice for food companies (AgMRC 2013; USDA 2014). In fact, evidence suggests that routine consumption of tree nuts may decrease the risk of coronary heart disease by as much as 35% (Kris-Etherton 2008; Orem and others 2013). Hazelnuts are often used for flavorings and incorporated into baked goods, snack foods, savory dishes and many processed foods. Tree nut allergy is reported by more than 1% of the US population and the prevalence and severity of this condition is increasing, especially among children (Branum and Lukacs 2009; Boyce and others 2011; Sicherer and Sampson 2010; Jackson 2013). While hazelnuts are typically considered a healthy food, they are also known to cause persistent food allergy in some individuals and lead to severe reactions, particularly among hazelnut-allergic children (Gonipeta and others 2010; Gupta and others 2011). Table 5 shows the allergenic proteins of hazelnut that have been characterized to date. Hazelnuts contain many allergenic proteins and they are classified into two types: pollendependent and pollen-independent. Hazelnut allergens that share homology with birch pollen and cause OAS are Cor a 1 and Cor a 2 (Lüttkopf and others 2002). Inhaled allergens that 21 Table 4 Effect of common food processing methods on food allergenicity: a summary Food Processing Method Effect on References Food Tested Allergenicity Dry roasting Almond ↔ [Maleki 2000; Müller 2000; Cashew ↔ Wigotzki 2000; Roux 2001; Hazelnuts ↓ Pastorello 2002; Peanut ↑ Venkatachalam 2002; Su Pistachio ↔ 2004; Worm 2009; Walnut ↔ Noorbakhsh 2010] Roasting / Maillard reaction Hazelnuts ↓ [Hansen 2003; Gruber 2005; Peanuts ↑ Iwan 2011; Cucu 2012] Oil roasting Almond ↔ [Su 2004] Cashew ↔ Walnut ↔ Frying Almond ↔ [Su 2004] Cashew ↔ Walnut ↔ Boiling Kidney bean ↓ [Álvarez-Álvarez; 2005; Black gram ↓ Kasera 2012] Lupine ↔ Peanut ↓ Blanching Almond ↔ [Roux 2001; Venkatachalam Cashew ↔ 2002; Su 2004] Walnut ↔ Microwaving Almond ↔ [Venkatachalam 2002; Su Cashew ↔ 2004; Álvarez-Álvarez 2005] Lupine ↔ Walnut ↔ Pulsed UV-Light Peanut ↓ [Chung 2008] Extrusion Lupine ↔ [Álvarez-Álvarez 2005; Soy ↓ Wilson 2005] γ-irradiation Almond ↔ [Venkatachalam 2002; Su Cashew ↔ 2004; Kasera 2012] Walnut ↔ Kidney bean ↔ Black gram ↔ Peanut ↔ Autoclaving Almond ↔ [Roux 2001; Venkatachalam Cashew ↔ 2002; Su 2004; ÁlvarezHazelnuts ↓ Álvarez 2005; López 2012] Lupine ↓ Walnut ↔ High pressure processing Hazelnuts ↔ [López 2012] Steam-roasting Pistachio nut ↓ [Noorbakhsh 2010] Heat + γ-irradiation Egg ↓ [Kim 2002] ↔ No impact; ↑ increased allergy; ↓ decreased allergy 22 Table 5 Allergenic proteins from hazelnut: a summary Hazelnut Protein MW (kDa) Reference Cor a 1a Bet v 1 homologue 18 [Hirschwehr 1992; Lüttkopf 2002; Pastorello 2002; López 2012] a Cor a 2a Profilin; Bet v 2 homologue 14 [Hansen 2003; Hirschwehr 1992] Cor a 8b nsLTP 9 [López 2012; Schocker 2004] Cor a 9b Acidic Subunit Basic Subunit 11 S globulin/legumin Cor a 11b 7S globulin/vicilin Cor a 12 Oleosin 17 Cor a 13 Oleosin 14-16 [Akkerdaas 2006; Willison 2014] Cor a 14 2S albumin 15-16 [Akkerdaas 2006; Willison 2014] [Beyer 2002; López 2012] 35 25 47-48 Pollen-dependent Pollen-independent b 23 [Lauer 2004; López 2012; Pastorello 2002] [Akkerdaas 2006; Garino 2010; Willison 2014] cause this type of reaction are considered class 2 food allergens (Sampson 2004). Pollen-independent hazelnut allergens induce class I food allergies. These allergens have cross-reactivity potential with peanuts and other tree nuts due to structural similarities of allergenic epitopes within plant protein families (de Leon and others 2003). The widespread use of hazelnuts in the food industry, its cross-reactivity with other allergens, and the increasing prevalence of tree nut allergy in general mean that it is imperative to develop strategies to attenuate the potential for tree nuts such as hazelnuts to cause anaphylaxis. 2.6 EFFECT OF FOOD PROCESSING ON HAZELNUT ALLERGENICITY Hazelnuts (HN) contain numerous allergenic proteins that vary in linear and conformational epitopes as well as thermal stability. Therefore, the impact of food processing methods as well as time and temperature profiles has differing effects. An overview of HN proteins and what is known about their susceptibility to thermal processing is provided in Table 6. Roasting is a typical thermal food processing method used to process tree nuts such as HNs. Consequently, many of the studies conducted have focused on the effect of roasting on HN allergenicity. Müller and others tested patient sera in vitro and characterized proteins that cross-reacted with birch pollen (2000). They used raw HN and roasted HN at 140 °C for 20, 30, or 40 minutes. A reduction in protein bands and IgE-binding patterns was observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, 24 Table 6 Thermal stability of hazelnut allergens Protein Bet v 1 homologue Thermal Stability HL1, 2, 3, 4, 6 Cor a 2 Profilin; Bet v 2 homologue HL1, 2 [Pastorello 2002; Worm 2009] Cor a 8 Non-specific lipid transfer protein (nsLTP) HR1, 5 HL6 [Wigotzki 2000; Pastorello 2002; López 2012] Cor a 9 11 S globulin/legumin HR4 HL6 [Cucu 2012; López 2012] Cor a 11 7S globulin/vicilin HR1 HL4, 6 [Müller 2000; Cucu 2012; López 2012] Hazelnut Allergen Cor a 1 HL: Heat labile HR: Heat resistant 1 Roasting at 140 °C up to 40 min 2 Roasting at > 170 °C for 15 min 3 Roasting at 144 °C 4 Maillard reaction at 70 °C for 48 hrs 5 Roasting at 185 °C for 15 min 6 Autoclaving up to 138 °C for 30 min 25 Reference [Müller 2000; Pastorello 2002; Hansen 2003; Worm 2009; Cucu 2012; López 2012] particularly at the 40 minute time point. Immunoblotting was performed with sera from 27 HN-allergic patients or 28 children allergic to both HN and birch pollen and partial Nsequencing was used to characterize major allergens. They determined that a cross-reactive 17-18 kDa protein known to be a Bet v 1 homologue was eliminated after roasting. They also identified a heat-resistant 48 kDa protein that could contribute to pollen-independent HN allergy. Pastorello and others conducted a double-blind, placebo-controlled food challenge (DBPCFC) and characterized allergenic proteins in raw HN and HN roasted at 140 °C for 40 minutes (2002). However, the DBPCFC was conducted with only raw HN (Ortolani and others 2000). Sera from 65 of the DBPCFC-positive patients plus 7 patients with a history of anaphylaxis were used in this study. Allergenicity was determined by IgE immunoblotting, cross-reactivity was assessed with immunoblotting inhibition, and characterization with partial N-terminal sequencing. Major allergens were identified as an 18 kDa Bet v 1 homologue that was eliminated during roasting and a heat-resistant 9 kDa lipid transfer protein responsible for anaphylaxis in some patients. Other major allergens identified were: a 32 kDa 2S albumin, a 35 kDa legumin, and a 47 kDa sucrose-binding protein. Hansen and others tested the effect of raw versus roasted (140 °C, 40 minutes) HN extracts on 17 birch pollen-allergic patients in a DBPCFC (2003). Most patients (16/17) were sensitized to Cor a 1, 7/17 to Cor a 2, and none to Cor a 8. Patients were orally challenged with successive doses of 0-10 g or 0-18.2 g of raw or roasted HN and mild oral allergy syndrome (OAS) symptoms were observed. Patients were evaluated for allergic reaction using skin prick test (SPT), histamine release, hazelnut-specific IgE binding, and enzyme 26 allergosorbent test (EAST) inhibition. They reported that roasting decreased but did not eliminate HN allergenicity in all birch pollen-allergic patients. Worm and others also conducted an in vivo DBPCFC with raw HN and roasted HN at 144°C (2009). Doses were successively administered in the following pattern: 0.01-0.02-0.03-0.050.1-0.2-0.4-1.0-2.5-5.0-10.0 g with 15 minutes between doses. Patients were evaluated for clinical reactions, positive skin prick tests, and basophil reactivity. Higher threshold levels were observed when patients were exposed to roasted HN. In addition, 88 of 90 patients had positive SPT to raw HN while >50% of patients had negative SPT to roasted HN. There was also a marked reduction of basophil reactivity to roasted HN. They concluded that roasting decreases hazelnut allergenicity. Wigotzki and others assessed the effect of HN allergenicity in vitro when HN was heated to temperatures of 100-185 °C for 15 minutes and 100 °C for times of 15-90 minutes (2000). They also used microwave treatment of 630 W for 10 minutes. Tests were conducted using sera from 19 hazelnut-allergic patients. EAST, EAST inhibition, SDS-PAGE, and immunoblotting were used to evaluate allergenicity. Major allergens of 18 kDa and 14 kDa were heat labile at temperatures exceeding 170 °C. A protein less than 14 kDa was stable to dry heat up to 185 °C and no effect on HN allergenicity was observed after heating to 100 °C for up to 90 minutes or after microwaving. Iwan and others studied the in vitro effect of the Maillard reaction on the HN allergen Cor a 11 (2011). HN extract was incubated with glucose for 7 days at 37 °C, 3 days at 60 °C, or 20 minutes at 145 °C. They used pooled sera from 8 HN-allergic patients, competitive ELISA, 27 immunoblotting and a mediator release assay with rat basophilic leukemia (RBL) cells to assess the effect on the Cor a 11 protein. No changes were observed at 37 °C. Notably, while they saw a decrease in IgE binding at 60 and 145°C, basophil activity was unchanged at these temperatures. Cucu and others evaluated the impact of the Maillard reaction on HN allergenicity using a basophil activation test (BAT) (2012). HN protein extract was incubated with glucose and with or without wheat proteins in a 70 °C water bath for 48 hours. SDS-PAGE was used to assess the HN protein profile. Blood samples from a total of 10 patients were used in the BAT. They reported that wheat did not contribute to major changes in basophil activity. In addition, the Maillard reaction eliminated basophil activity in 4 patients with OAS while a decrease in basophil activity was seen in some of the other 6 patients with a history of systemic reactions. They concluded that some patients may still react to HN proteins after processing in the presence of glucose. López and others studied the effect of high-pressure (HHP) processing and autoclaving on hazelnut allergenicity in vitro and characterized allergenic proteins (2012). Protein extract was subjected to: autoclaving at 121 °C for 15 or 30 minutes, autoclaving at 138 °C for 15 or 30 minutes, and pressures of 300, 400, 500, or 600Mba. SDS-PAGE and structural analysis and modeling were used to characterize HN allergens. Sera from 15 patients with HNspecific IgE antibodies were used for immunoblotting. IgE binding to Cor a 1, Cor a 8, Cor a 9, and Cor a 11 decreased after all autoclaving treatments. They concluded that HHP processing had no effect on HN allergenicity whereas all autoclaving treatments decreased in vitro HN allergenicity. 28 In general, much of the research assessing the effects of food processing on HN allergenicity has been limited to in vitro studies. While these studies afford us valuable data and lay the foundation for future research, it is crucial to evaluate the effects of food processing in vivo to develop a better understanding of the role that food processing plays in allergenicity. Furthermore, as demonstrated in the study by Iwan and others, a decrease in IgE does not necessarily translate to a decrease in degranulation capacity of basophils (2011). The mechanism of food allergy is complex and specific biomarkers alone cannot accurately predict clinical reactivity. This thesis describes our efforts to evaluate the effects of extrusion processing on hazelnut allergenicity in vivo using a mouse model of hazelnut allergy. 2.7 AN ADJUVANT-FREE MOUSE MODEL OF FOOD ALLERGY This study utilizes an adjuvant-free mouse model of food allergy previously developed in our laboratory using hazelnut as a model tree nut (Birmingham and others 2007). In this model, transdermal exposure of hazelnut protein was found to induce the Th2 response leading to IgE production and subsequent sensitization in BALB/c mice. Antibody responses (IgE, IgG1, and IgG2a) are assessed in this model using an ELISA-based method also previously developed in our laboratory (Birmingham and others 2003). Clinical reactivity is then evaluated by measurement of rectal temperatures post oral challenge. Severe systemic anaphylaxis as evidenced by immediate hypothermia shock response and pathological changes in the gut tissue upon oral hazelnut protein challenge are among the major features of this model that simulates the near-fatal human allergic reactions to hazelnut. The genetic strain and gender of mouse used for animal studies is an important determinate of their immune response to food allergens (Morafo and others 2003; Gonipeta and others 29 2013). Female BALB/c mice are used in this model of food allergy because they are predisposed to the development of the Th2 immune response and sensitization as a result of food allergen exposure (Kimber and others 2003; Gonipeta and others 2013). Another advantage of using BALB/c mice is that they exhibit hypothermia upon induction of systemic anaphylaxis that can be easily measured by rectal thermometry (Birmingham and others 2007; Gonipeta and others 2010). In this model, BALB/c mice are repeatedly exposed to food allergens via the transdermal route of sensitization. Mice often develop tolerance when orally exposed to food allergens while transdermal exposure induces sensitization and ultimately systemic anaphylaxis upon oral challenge (Strid and others 2005; Gonipeta and others 2013). In addition, the skin is thought to be an important route of sensitization to allergens in humans (Hudson 2006; Callard and Harper 2007; Berin and Sampson 2013). Therefore, sensitization using this model may mimic a known route of human exposure to allergens. Adjuvant-free models of food allergy more closely represent the human disease because of the natural route of sensitization (Berin and Mayer 2009). The use of adjuvants, such as cholera toxin, is sometimes used in mouse models of food allergy (Gonipeta and others 2013). Cholera toxin is a toxic protein produced by Vibrio cholerae and is used in animal models as an adjuvant co-administered with antigen to stimulate the immune system (Bharati and Ganguly 2011). Cholera toxin is also known to induce IgE and IgG1 production in mice (Helm 2002). However, the relevance of adjuvants to human food allergies is unknown (Gonipeta and others 2013). Furthermore, adjuvant-based models are not considered highly suitable for evaluating allergenicity of novel foods including processed foods and genetically 30 modified foods. For example, it is very difficult to elucidate the effects of processing on food allergens in such adjuvant-based models due to exaggerated impact of the adjuvants on the immune system in such models. There are various routes of exposure used in mouse models to assess the clinical response to food allergens, such as intragastric gavage and intraperitoneal injection challenges (Gonipeta and others 2013). However, models that use oral challenge are desirable because most human food-allergic reactions occur after oral exposure to the allergen. Animal models are a useful way in which to assess food allergy in vivo. The most desirable models are those that best simulate the human disease (Helm 2002). The mouse model used in this study has many desirable characteristics that relate to the human situation. These include the use of an optimal genetic strain and gender, sensitization without the use of an adjuvant, and oral elicitation route to assess clinical reactivity and precise quantitation of the allergic reactions using hypothermia shock response and elevation of mucosal mast cell mediator level in the plasma after oral allergen challenge. 31 CHAPTER 3: Materials and Methods 3.1 PREPARATION OF DEFATTED HAZELNUT FLOUR Hazelnuts (HN) used in this study were donated by Diamond Foods (Stockton, CA) (large hazelnut premium quality; lot number 11302D312S; 01030000228). HN shells were removed and 2.2 kg of raw HNs were ground into flour using an Osterizer 12 speed blender (Boca Raton, FL). HNs were then defatted as shown in Figure 2. HN flour was defatted by submerging in hexanes (95% n-hexane {J.T. Baker, St. Louis, MO}) overnight. Hexanes and HN oil were decanted the following day. Fresh hexanes were added to the HN flour and left at room temperature for 2 hours. Hexanes and HN oil were again decanted and approximately 2 kg of defatted HN flour was air dried overnight. Defatted HN flour was then stored in an airtight bag at 4 °C and used in this study for raw hazelnut protein extraction and extrusion processing. 3.2 EXTRUSION PROCESSING Hazelnut flour was extruded using an APV Baker MPF-19/25 twin-screw extruder (Staffordshire, England) shown in Figure 3. The screw configuration used was: 8D Twin, 7x30° FKP (forward kneading paddle), 4D Twin, 4x60° FKP, 4x30° RKP (reverse kneading paddle), 2D Twin, 6x60° FKP, 4x30° RKP, 1D Single, 7x90° P, 2D Single where 1 Diameter (D) equals 19 mm and 1 paddle (P) equals 0.25D. The temperature profile was 147-124-10162-60 °C. HN flour was extruded at a screw speed of 150 rpm and feed rate of 16.42 g/min with a feed moisture of 31.6%. The sample was collected over the course of 5 minutes at 140 °C, 20 psi, and 14.7% torque. The sample was collected from a 4 mm circular die opening. The extruded HN sample was stored at -70 °C until protein extraction was conducted. 32 Figure 2 Preparation of defatted hazelnut flour from shelled hazelnuts. The figure shows the process used in making defatted hazelnut flour from ground raw hazelnuts. 33 Figure 3 The co-rotating twin-screw extruder (APV Baker MPF-19/25) used in this study. Left panel shows the entire twin-screw extruder; middle panel shows the closed barrel and 4mm die from which samples were collected during processing; right panel shows the internal compartment containing the screws and partially processed hazelnut flour after extrusion was complete. 34 3.3 PROTEIN EXTRACTION Extruded HN was ground into a powder with a mortar and pestle. Protein was extracted from defatted raw HN flour or defatted extruded HN flour with 30 mM Tris-HCl, pH 8.0 as depicted in Figure 4. Tris-HCl solution was made by dissolving UltraPure™ Tris base (Invitrogen, Carlsbad, CA) in double distilled water and adding hydrochloric acid to reach pH 8.0. Extraction was conducted by combining 4 g defatted raw HN flour or 4 g defatted extruded HN flour with 20 mL Tris-HCl (pH 8.0) buffer and incubating overnight at 4 °C. Initial filtration was performed with Whatman 90 mm circle cellulose filter papers (grade 1 medium flow) to extract soluble HN protein. Soluble HN protein extract was then syringe filtered under a biological safety hood with a MILLEX® - HA (sterile) syringe driven filter unit (pore size 0.45 µm) (EMD Millipore, Billerica, MA). Samples were stored in sterile microcentrifuge tubes as 1 mL aliquots at 4 °C. 3.4 PROTEIN CONCENTRATION Protein concentration was determined using the Lowry-Folin method (Lowry and others 1951). Albumin from bovine serum (BSA) (Sigma-Aldrich, St. Louis, MO) was used to generate standards of 0-100µg/ml. The reaction was carried out with 0.5% CuSO4 (Mallinckrodt, Dublin, Ireland) and 1% Na Citrate (Fisher Scientific, Hampton, New Hampshire) added to 0.1 N NaOH (J.T. Baker) and 2% Na2CO3 (J.T. Baker). Folin & Ciocalteu’s Phenol Reagent (Sigma-Aldrich, St. Louis, MO) was added to standards and samples. Plates (96-well) were read at 750 nm in the BIO-TEK Synergy HT (Winooski, VT) plate reader. The BIO-TEK KC4 v3.1 program (Winooski, VT) was used in conjunction with the BIO-TEK reader for generating data from plates. Samples were tested at 3 dilutions in quadruplet and the average from 6 data points was used to determine concentration. 35 Figure 4 Preparation of protein extract from raw and extruded defatted hazelnut flour. The figure shows the process used in making hazelnut protein extracts from raw and extruded defatted hazelnut flour. 36 3.5 SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted with BIO-RAD Mini-PROTEAN®TGX 10% precast gels or 15% Tris-HCl BIO-RAD Ready Gel® precast gels. Gels were electrophoresed under reducing or non-reducing conditions. Reducing and non-reducing sample buffers consisted of 250 mM Tris-HCl (pH 6.8), 10% SDS, 30% glycerin, and 0.02% bromophenol. In addition, reducing sample buffer also contained 10% 2-Mercaptoethanol (Life Technologies, Carlsbad, CA) and all reduced samples were heated to 100 °C for 5 minutes and allowed to cool prior to electrophoresis. A total volume of 20 µL of protein extract was loaded into wells at a concentration of 6 or 10 µg. Gels were electrophoresed in a BIO-RAD Mini PROTEAN 3 cell apparatus (Hercules, CA) and running buffer of 0.1% SDS (Sigma-Aldrich, St. Louis, MO), 0.3% Tris and 1.44% Glycine (both from Invitrogen, Carlsbad, CA). Running times were optimized to 95 minutes for 10% precast gels and 71 minutes for the 15% precast gel and running conditions of 100 volts and 0.01 - 0.03 amps. Gels were stained with BIO-RAD Coomassie Brilliant Blue R250 and destained with a mixture of 10% methyl alcohol (J.T. Baker, Mallinckrodt, St. Louis, MO) and 10% acetic acid, glacial (EMD Millipore, Billerica, MA). 3.6 ANIMALS Female BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at age 6 weeks. Mice were allowed to rest for 1 week prior to beginning experimentation. All experiments were conducted under the authorization of Michigan State University (MSU) Institutional Animal Care and Use Committee (IACUC). 37 Mice were housed in irradiated (sterile) 100% PET cages (Innovive San Diego, CA) with sterile aspen chip bedding, 4-5 mice per cage with a 12 hour light/dark cycle. No special dietary requirements were provided for this study. Mice were fed 8640 Teklad rodent diet and acidified water (Harlan Laboratories, Indianapolis, IN). 3.7 SENSITIZATION METHOD Sensitization of mice was conducted via transdermal exposure (TDE) based on an adjuvantfree mouse model of food allergy developed in our laboratory (Birmingham 2007). Hair was clipped from the back of mice 1 week after arrival and as needed throughout the experiments. Frequency of sensitization varied for mice that were to be orally challenged versus mice that would be challenged via intraperitoneal (IP) injection as described below. Mice that were orally challenged were sensitized with 1 mg of soluble raw hazelnut nut protein (RHNP) or extrusion processed hazelnut protein (EHNP) extract in 100μL TGP vehicle (30 mM Tris-HCl, 50% v/v Glycerin and 0.2% Phenol (Sigma-Aldrich, St. Louis, MO). Control mice were exposed to 100 μL TGP vehicle alone. TDE was accomplished by applying 100 μL of RHNP, EHNP or vehicle to the back of mice once per week for 7 weeks and patching overnight with sterile latex free heavy-duty fabric bandages (Meijer, Grand Rapids, MI). Patches were removed approximately 24 hours after each exposure. Mice that were challenged via IP injection were sensitized with 1mg of soluble raw hazelnut nut protein (RHNP) or extrusion processed hazelnut protein (EHNP) extract in 100μL TGP vehicle. Control mice were exposed to 100 μL TGP vehicle alone. TDE was accomplished by applying 100 μL of RHNP, EHNP or vehicle to the back of mice once per week for 3 weeks 38 and patching overnight with sterile latex free heavy-duty fabric bandages (Meijer, Grand Rapids, MI). Patches were removed approximately 24 hours after each exposure. 3.8 BLOOD SAMPLE COLLECTION Blood samples were collected from mouse saphenous vein approximately 1 week after arrival (prior to experimentation) and a minimum of every other week after beginning experimentation. Samples were collected in heparin-coated Microvette capillary tubes (Sarstedt, Nümbrecht, Germany) and centrifuged at approximately 9754 x g for 10 minutes. Blood plasma was extracted and placed in microcentrifuge tubes. In addition, 5 μL from each sample was pooled for each set of mice and used in all antibody testing. Samples were stored at -70 °C. 3.9 ANALYSIS OF IgE ANTIBODY RESPONSE Hazelnut (HN)-specific systemic IgE response (sIgER) was measured using an indirect ELISA-based method developed in our laboratory (Birmingham and others 2003). Commercial HN protein extract (Greer Laboratories, Lenoir, NC) was diluted in sodium bicarbonate coating buffer, pH 9.6, and used to coat plates at a concentration of 5 mg/ml. The total volume of all reagents was 50 μL/well except for blocking that was 80 μL/well. Blocking was performed with 5% gelatin from porcine skin, for electrophoresis, type A (Sigma-Aldrich, St. Louis, MO) Wash buffer consisted of 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), PBS solution, pH 7.4, and 0.04% NaN3 (Sigma-Aldrich, St. Louis, MO). Detection was carried out with biotin rat anti-mouse antibody (BD Pharmingen, San Diego, CA) and alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in dilution buffer (0.085% BSA, 0.05% Tween 20 / 39 PBS, pH 7.4). Phosphatase substrate (Sigma-Aldrich, St. Louis, MO) was dissolved in substrate buffer (MgCl2, Diethanolamine, and HCl added to reach pH 9.8). Plates (96-well) were read at 405-690 nm 2 hours after adding substrate using an ELISA plate reader BIOTEK Synergy HT (Winooski, VT). The BIO-TEK KC4 v3.1 program (Winooski, VT) was used in conjunction with the BIO-TEK reader for generating data from ELISA plates. Microsoft Excel 2010 was utilized for data and statistical analysis. 3.10 ANALYSIS OF IgG1 AND IgG2a ANTIBODY RESPONSE Hazelnut (HN)-specific systemic IgG1 and IgG2a responses were measured using an indirect ELISA-based method developed in our laboratory (Birmingham and others 2003). Commercial HN protein extract (Greer Laboratories, Lenoir, NC) was diluted in sodium bicarbonate coating buffer, pH 9.6, and used to coat plates at a concentration of 20 ug/ml. The total volume of all reagents was 50 μL/well except for blocking that was 80 μL/well. Blocking was performed with 0.17% BSA in phosphate buffered saline (PBS) solution. Wash buffer consisted of 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), PBS solution, pH 7.4, and 0.04% NaN3 (Sigma-Aldrich, St. Louis, MO). Detection was carried out with biotin rat anti-mouse antibody (BD Pharmingen, San Diego, CA) and alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in dilution buffer (0.085% BSA, 0.05% Tween 20 / PBS, pH 7.4). Phosphatase substrate (SigmaAldrich, St. Louis, MO) was dissolved in substrate buffer (MgCl2, Diethanolamine, and HCl added to reach pH 9.8). Plates (96-well) were read at 405-690 nm 1 hour after adding substrate using an ELISA plate reader BIO-TEK Synergy HT (Winooski, VT). The BIOTEK KC4 v3.1 program (Winooski, VT) was used in conjunction with the BIO-TEK reader 40 for generating data from ELISA plates. Microsoft Excel 2010 was utilized for data and statistical analysis. 3.11 ANALYSIS OF HYPOTHERMIA SHOCK RESPONSE Mice that were sensitized to 1 mg RHNP or EHNP extract once per week for 7 weeks were orally challenged with 10 mg of RHNP or EHNP extract in 100 μL Tris-HCl buffer, pH 8.0, respectively. Control mice that were sensitized to 100 μL TGP vehicle were fed 100 μL of 30 mM Tris-HCl buffer. Rectal temperatures were taken just prior to each challenge, as well as 10, 20, and 30 minutes post exposure to allergen or Tris-HCl buffer. Physitemp Thermalert TH-5 (The Sea Ranch, CA) was used for measuring rectal temperatures. Mice that were sensitized to 1 mg RHNP or EHNP extract once per week for 3 weeks were injected via IP route with 1 mg soluble RHNP or EHNP extract in 50 μL Tris-HCl buffer, pH 8.0, respectively. Control mice were injected with 50 μL of 30 mM Tris-HCl buffer. Rectal temperatures were taken just prior to each challenge, as well as 10, 20, and 30 minutes post exposure to allergen or Tris-HCl buffer. Physitemp Thermalert TH-5 (The Sea Ranch, CA) was used for measuring rectal temperatures. 3.12 ANALYSIS OF MAST CELL DEGRANULATION Blood plasma samples collected 45 minutes post oral or IP injection challenges were screened for mouse mast cell protease-1 (mMCP-1). A sandwich ELISA kit, Mouse MCPT-1 (mMCP-1) ELISA Ready-SET-Go! ® (eBioscience, San Diego, CA) was used to detect mMCP-1. The sensitivity of the assay was 120 pg/mL and quantification was possible as a 41 recombinant protein was provided in the kit to produce a standard curve with a range of 12015,000 pg/mL. 3.13 WESTERN BLOT ANALYSIS SDS-PAGE was conducted with BIO-RAD Mini-PROTEAN®TGX 10% precast gel. The gel was electrophoresed under reducing conditions. Reducing buffer consisted of 250 mM TrisHCl (pH 6.8), 10% SDS, 30% glycerin, and 0.02% bromophenol, and 10% 2Mercaptoethanol (Life Technologies, Carlsbad, CA). Samples were heated to 100 °C for 5 minutes and allowed to cool prior to electrophoresis. A total volume of 20 µL of protein extract was loaded into wells at a concentration of 50 µg/well. Immediately after SDS-PAGE was completed, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Pall Corporation, Port Washington, NY). Transfer buffer consisted of 0.3% Tris, 1.44% Glycine (both from Invitrogen, Carlsbad, CA), and 20% methyl alcohol (J.T. Baker, Mallinckrodt, St. Louis, MO). Proteins were transferred for 60 minutes at 90 volts. Blocking of the PVDF membrane was performed with 3% BSA in phosphate buffered saline with Tween 20 (PBST). PBST consisted of 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) in PBS solution, pH 7.4. PBST was used for all subsequent rinsing and washing steps. Samples were added and incubated at 4 °C overnight. Detection was carried out with biotin rat anti-mouse antibody (BD Pharmingen, San Diego, CA) and horseradish peroxidase avidin D (Vector Labs, Burlingame, CA) each diluted in PBST. Electrochemiluminescence (ECL) 42 solution (VWR International, Radnor, PA) was then added and the membrane was read with the LI-COR Odyssey® FC (Lincoln, NE). 3.14 RANDOMIZED CONTROLLED STUDY DESIGN 3.14.1 Objective 1 The first objective of this study was to evaluate whether soluble extrusion processed hazelnut protein (EHNP) extract elicits a reduced HN-specific systemic IgE antibody response (sIgER) compared to soluble raw hazelnut protein (RHNP) extract. Control mice (n = 9) were sensitized via transdermal exposure (TDE) to 100 μL of TGP vehicle (30 mM Tris-HCl, 50% v/v Glycerin and 0.2% Phenol (Sigma-Aldrich, St. Louis, MO) once per week for 7 weeks. A second set of mice (n = 5) received TDE to RHNP extract once per week for 7 weeks at a dose of 1 mg/TDE in 100 μL/mouse. A third set of mice (n = 4) received TDE to EHNP extract once per week for 7 weeks at a dose of 1 mg/TDE in 100 μL/mouse. The approach to Objective 1 is described in Figure 5. Blood plasma was collected from mice approximately 1 week after arrival and prior to experimentation (Pre sample) and a minimum of every other week after beginning experimentation. The Pre sample and blood plasma collected after TDE 4 and 5 were evaluated for HN-specific sIgER. 3.14.2 Objective 2.1 The first part of the second objective of this study was to test whether soluble extrusion processed hazelnut protein extract induces less hypothermia shock response than soluble raw hazelnut protein extract in hazelnut-sensitized mice upon oral challenge (OC). The approach to Objective 2.1 is described in Figure 6. Control mice (n = 9) that received TDE to 100 μL 43 Figure 5 Approach used to conduct the study described for Objective 1. The figure shows the approach for the vehicle control group (top panel), raw hazelnut protein exposed group (middle panel), and the extrusion-processed hazelnut protein exposed group (bottom panel). 44 Figure 6 Approach used to conduct the study described for Objective 2.1. The figure shows the approach for the vehicle control group (top panel), raw hazelnut protein exposed group (middle panel), and the extrusion-processed hazelnut protein exposed group (bottom panel). 45 of TGP vehicle control once per week for 7 weeks received OC with 100 μL of 30 mM TrisHCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg RHNP extract in100μL/mouse. A third set of mice (n = 4) that received TDE to EHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg EHNP extract in 100μL/mouse. 3.14.3 Objective 2.2 The second part of the second objective of this study was to test whether soluble extrusion processed hazelnut protein extract induces less hypothermia shock response than soluble raw hazelnut protein extract in hazelnut-sensitized mice upon intraperitoneal (IP) injection, a method of systemic challenge. The approach to Objective 2.2 is described in Figure 7. Control mice (n = 5) that received TDE to 100 μL of TGP vehicle control once per week for 3 weeks were challenged via intraperitoneal (IP) injection with 50 μL of 30 mM TrisHCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/ mouse were challenged via IP route with 1 mg RHNP extract in 50μL/mouse. A third set of mice (n = 5) that received TDE to EHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/mouse were challenged via IP route with 1 mg EHNP extract in 50μL/mouse. 3.14.4 Objective 3.1 The first part of the third objective of this study was to test whether the ability of soluble extrusion processed hazelnut protein extract elicits reduced mucosal mast cell degranulation response compared to soluble raw hazelnut protein extract in hazelnut-sensitized mice upon 46 Figure 7 Approach used to conduct the study described for Objective 2.2. The figure shows the approach for the vehicle control group (top panel), raw hazelnut protein exposed group (middle panel), and the extrusion-processed hazelnut protein exposed group (bottom panel). 47 OC. The approach to Objective 3.1 is described in Figure 8. Mice were sensitized to vehicle control, RHNP, or EHNP and orally challenged as described in Objective 2.1 (page 43). Blood samples were collected from mice 45 minutes post OC and analyzed for mMCP-1. 3.14.5 Objective 3.2 The second part of the third objective of this study was to test whether the ability of soluble extrusion processed hazelnut protein elicits reduced mucosal mast cell degranulation response compared to soluble raw hazelnut protein in hazelnut sensitized mice upon IP injection. The approach to Objective 3.2 is described in Figure 9. Mice were sensitized to vehicle control, RHNP, or EHNP and challenged via IP injection as described in Objective 2.2 (page 46). Blood samples were collected from mice 45 minutes post IP challenge and analyzed for mMCP-1. This study was designed to test the hypothesis that extrusion processing will reduce in vivo allergenicity of soluble raw hazelnut protein extract in a mouse model of food allergy. A summary of the study design is described in Figure 10. Briefly, raw and extruded HN protein extracts were prepared and protein profiles were evaluated in vitro. Using a mouse model of food allergy, antibody responses and allergen challenges were conducted in vivo. Samples from mice sensitized and orally challenged with RHNP or EHNP extracts were incubated with proteins that had been transferred to a PVDF membrane to test binding of antibody to specific HN proteins in vitro (Western blot). 48 Figure 8 Approach used to conduct the study described for Objective 3.1. The figure shows the approach for the vehicle control group (top panel), raw hazelnut protein exposed group (middle panel), and the extrusion-processed hazelnut protein exposed group (bottom panel). 49 Figure 9 Approach used to conduct the study described for Objective 3.2. The figure shows the approach for the vehicle control group (top panel), raw hazelnut protein exposed group (middle panel), and the extrusion-processed hazelnut protein exposed group (bottom panel). 50 Figure 10 Summary of the study design to evaluate the impact of extrusion-processing on in vivo allergenicity of hazelnut protein extract in an adjuvant-free mouse model. The figure shows the in vitro and in vivo methods used to evaluate allergenicity in this study. 51 CHAPTER 4: Results 4.1 ANALYSIS OF RAW AND EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACTS USING SDS-PAGE Soluble raw hazelnut protein (RHNP) and extrusion processed hazelnut protein (EHNP) extracts were filtered and protein content measured using the Lowry-Folin method (Lowry and others 1951). To ensure accuracy of protein estimation, samples were tested in quadruplicates in three two-fold dilutions using the Lowry-Folin method. Bovine serum albumin (BSA) was used to construct standard curves to quantify protein content. Protein content was determined by two individuals independently. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then conducted to compare RHNP and EHNP protein profiles. Initial experiments were conducted to optimize the loading volume, running conditions, and gel percent and used in analysis. RHNP and EHNP were used in 10% SDS-PAGE analysis at two concentrations, 6 and 10 μg/lane. The result of analysis under non-reducing conditions in the 10% gel is shown in Figure 11. There were 6 major bands and 3 minor bands identified in the RHNP extract. The molecular weight (MW) estimation of the major bands in the RHNP extract corresponded to 170, 51-55, 42-48, 39, 31-32, and < 15 kDa while the MW of the minor bands corresponded to 129, 36-37, and 1416 kDa. In contrast, there were only 4 major bands and 5 minor bands in the EHNP extract. The MW estimation of the major bands in the EHNP extract corresponded to 170, 51-55, 48, and < 15 kDa while the minor bands corresponded to 129, 39, 36-37, 31-32, and 14-16 kDa. Notably, proteins in the range of 129, 42-47, 39, 36-37, and 31-32 were either not detectable or were very faint in the EHNP extract. There was also a slightly decreased intensity of the 52 Figure 11 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts under non-reducing conditions. Protein content was quantified in quadruplicates by the Lowry-Folin method and indicated amount was loaded in 20µL per lane. Protein was electrophoresed in a 10% gel, lanes 1 and 4) marker; 2) 6µg RHNP; 3) 10µg RHNP; 5) 6µg EHNP; 6) 10µg EHNP. 53 170 kDa band and an increase in intensity of the band of 14-16 kDa. However, protein bands of 51-55, 48, and < 15 kDa were comparable to the RHNP extract. Results from the analysis under reducing conditions with 2-Mercaptoethanol in the 10% gel are shown in Figure 12. There were 5 major bands and 5 minor bands identified in the RHNP extract. The MW estimation of the major bands in the RHNP extract corresponded to 51-55, 42-48, 39, 31-32, and < 15 kDa while the MW of the minor bands corresponded to 170, 129, 120, 36-37, and 14-16 kDa. In contrast, there were only 3 major bands and 4 minor bands in the EHNP extract. The MW estimation of the major bands in the EHNP extract corresponded to 51-55, 48, and < 15 kDa while the MW of the minor bands corresponded to 39, 36-37, 3132, and 14-16 kDa. Notably, proteins in the range of 170, 129, 120, 39, 36-37, and 31-32 were either not detectable or were very faint in the EHNP extract. There was also an increase in intensity of the 14-16 kDa band. However, proteins in the range of 51-55, 48, and < 15 kDa were comparable to the RHNP extract. The higher molecular weight band, 170 kDa was present in RHNP under non-reducing conditions, but had nearly disappeared when reduced and reduction resulted in the enhancement of bands in the 31-39 kDa range. A second method of protein extraction was tested using a previously published protocol (Cucu and others 2011). A SDS-PAGE was conducted to evaluate the protein profile of these RHNP and EHNP extracts (Appendix A). The protein profile was not different from the results shown in Figures 11 and 12. Therefore, protein extracted with Tris-HCl buffer, pH 8.0 was used in all subsequent testing. 54 Figure 12 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts under reducing conditions. Protein content was quantified in quadruplicates by the Lowry-Folin method and indicated amount was loaded in 20µL per lane. Protein reduced with 2-Mercaptoethanol and electrophoresed in a 10% gel, lane 1 and 4) marker; 2) 6µg RHNP; 3) 10µg RHNP; 5) 6µg EHNP; 6) 10µg EHNP. 55 4.2 CHARACTERIZATION OF HAZELNUT-SPECIFIC SYSTEMIC IgE ANTIBODY RESPONSES IN BALB/C MICE UPON TRANSDERMAL EXPOSURE TO RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Tests were conducted to determine whether EHNP extract elicits reduced hazelnut-specific systemic IgE antibody responses (sIgER) relative to RHNP extract in our mouse model. RHNP and EHNP extracts were quantified using the Lowry-Folin method and used in the transdermal sensitization protocol (Lowry and others 1951). Three groups of adult BALB/c female mice were pre-bled to collect plasma samples prior to exposure. Mice then received transdermal exposure (TDE) to 1 mg of RHNP or EHNP extract or TGP vehicle once per week for 7 weeks. Blood samples were collected after 4 and 5 TDE and plasma was used in hazelnut-specific IgE antibody analysis. A previously optimized ELISA based method was used to measure IgE antibody levels using serial dilution of plasma. As expected, none of the mice had hazelnut-specific IgE antibodies in the pre-exposure plasma sample (Figure 13A, 14A, 14B). Upon 4 TDE to RHNP and EHNP, marked IgE responses were noted (Figure 13B). Using the linear portion of the titration curve, we compared the statistical difference between the two sets of data. RHNP elicited significantly higher levels of IgE antibody responses compared to EHNP (Figure 14C and 14D). However, results from analysis of plasma samples collected after 5 TDE demonstrated that both RHNP and EHNP elicited comparable IgE antibody responses (Figure 13C) and there was no significant difference between the RHNP and EHNP in eliciting IgE antibody responses (Figure 14E and 14F). There were no detectable hazelnut-specific IgE antibodies in the blood samples from vehicle control mice prior to exposure or after 4 and 5 TDE to vehicle (Appendix B). 56 Figure 13 Systemic IgE antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves. The y-axis shows hazelnut (HN)-specific systemic IgE (sIgE) antibody (Ab) levels in BALB/c mice before (Pre) (A) and after 4 transdermal exposures (4R) (B) and 5 transdermal exposures (5R) (C) to raw versus extrusion processed hazelnut protein extract. The X-axis shows plasma dilutions used in ELISA analysis. 57 Figure 14 Systemic IgE antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis. The y-axis shows hazelnut (HN)-specific systemic IgE (sIgE) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to raw versus extrusion processed hazelnut protein extract. The X-axis shows two plasma dilutions, 1/640 (A, C, E) and 1/1280 (B, D, F) used in ELISA analysis. Student’s t-Test: * indicates significant difference, p < 0.05. 58 4.3 CHARACTERIZATION OF HAZELNUT-SPECIFIC SYSTEMIC IgG1 ANTIBODY RESPONSES IN BALB/C MICE UPON TRANSDERMAL EXPOSURE TO RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Tests were conducted to determine whether EHNP extract elicits reduced hazelnut-specific systemic IgG1 antibody responses relative to RHNP extract in our mouse model. As expected, none of the mice had hazelnut-specific IgG1 antibodies in the pre-exposure plasma sample (Figure 15A). Upon 4 TDE to RHNP and EHNP, marked IgG1 responses were noted (Figure 15B). Using the linear portion of the titration curve, we compared the statistical difference between the two sets of data. RHNP elicited significantly higher levels of IgG1 antibody responses compared to EHNP (Figure 16C and 16D). In addition, marked IgG1 responses were noted upon 5 TDE to RHNP and EHNP (Figure 15C). Using the linear portion of the titration curve, we compared the statistical difference between the two sets of data. RHNP elicited significantly higher levels of IgG1 antibody responses compared to EHNP (Figure 16E and 16F). There were no detectable hazelnut-specific IgG1 antibodies in the blood samples from vehicle control mice prior to exposure or after 4 and 5 TDE to vehicle (Appendix C). 4.4 CHARACTERIZATION OF HAZELNUT-SPECIFIC SYSTEMIC IgG2a ANTIBODY RESPONSES IN BALB/C MICE UPON TRANSDERMAL EXPOSURE TO RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Here we tested whether EHNP extract elicits reduced hazelnut-specific systemic IgG2a antibody responses relative to RHNP extract in our mouse model. As expected, none of the mice had any hazelnut-specific IgG2a antibodies in the pre-exposure plasma sample (Figure 17A). Upon 4 TDE to RHNP and EHNP, marked IgG2a responses were noted (Figure 17B). 59 Figure 15 Systemic IgG1 antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves. The y-axis shows hazelnut (HN)-specific systemic IgG1 (sIgG1) antibody (Ab) levels in BALB/c mice before (Pre) (A) and after 4 transdermal exposures (4R) (B) and 5 transdermal exposures (5R) (C) to raw versus extrusion processed hazelnut protein extract. The X-axis shows plasma dilutions used in ELISA analysis. 60 Figure 16 Systemic IgG1 antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis. The y-axis shows hazelnut (HN)-specific systemic IgG1 (sIgG1) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to raw versus extrusion processed hazelnut protein extract. The X-axis shows two plasma dilutions, 1/20000 (A, C, E) and 1/40000 (B, D, F) used in ELISA analysis. Student’s t-Test: * indicates significant difference, p < 0.05. 61 Figure 17 Systemic IgG2a antibody responses to raw versus extrusion-processed hazelnut protein extract: titration curves. The y-axis shows hazelnut (HN)-specific systemic IgG2a (sIgG2a) antibody (Ab) levels in BALB/c mice before (Pre) (A) and after 4 transdermal exposures (4R) (B) and 5 transdermal exposures (5R) (C) to raw versus extrusion processed hazelnut protein extract. The X-axis shows plasma dilutions used in ELISA analysis. 62 Using the linear portion of the titration curve, we compared the statistical difference between the two sets of data. RHNP elicited significantly higher levels of IgG2a antibody responses compared to EHNP (Figure 18C and 18D). In contrast, EHNP elicited significantly higher levels of IgG2a after 5 TDE (Figure 18E and 18F). There were no detectable hazelnutspecific IgG2a antibodies in the blood samples from vehicle control mice prior to exposure or after 4 and 5 TDE to vehicle (Appendix D). 4.5 IDENTIFICATION OF IGE ANTIBODY BINDING PROTEINS IN RAW AND EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACTS USING WESTERN BLOT ANALYSIS In order to identify the specific allergens (that is, IgE antibody binding proteins) in RHNP versus EHNP extracts, Western blot analysis was performed using anti-IgE antibodies. Western blot experimental conditions were optimized with the help of collaborators and used in this study. RHNP and EHNP extracts were resolved in a 10% SDS-PAGE gel under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane. Plasma samples from RHNP-sensitized mice were used in developing the membrane. Results showed 2 major proteins (33-35 and 38 kDa) and 3 minor proteins (43, 25, and 14-16 kDa) bound to IgE in the RHNP sample (Figure 19). In contrast, in EHNP sample, 2 major proteins (43 and 39 kDa) and 1 minor protein (33-35 kDa) bound to IgE. Notably, proteins in the range of 33-35, 25, and 14-16 kDa that bound to IgE in RHNP were either absent or a marked reduction in intensity was observed in EHNP samples. However, there was an increase in intensity of the 43 kDa band in the EHNP. 63 Figure 18 Systemic IgG2a antibody responses to raw versus extrusion-processed hazelnut protein extract: statistical analysis. The y-axis shows hazelnut (HN)-specific systemic IgG2a (sIgG2a) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to raw versus extrusion processed hazelnut protein extract. The X-axis shows two plasma dilutions, 1/250 (A, C, E) and 1/500 (B, D, F) used in ELISA analysis. Student’s t-Test: * indicates significant difference, p < 0.05. 64 Figure 19 The IgE-binding hazelnut protein analysis by Western blot method. The raw (lane 2) and extruded hazelnut protein (lane 3) extracts were resolved in a 10% SDS-PAGE gel under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane. The IgE-binding proteins were identified using labeled anti-mouse IgE antibody as described in the text. Lane 1) molecular weight marker. 65 4.6 ANALYSIS OF HYPOTHERMIA SHOCK RESPONSE IN BALB/C MICE UPON ORAL CHALLENGE WITH RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Mice were orally challenged to evaluate hypothermia shock in response to raw versus extruded hazelnut protein extracts. Control mice (n = 9) that received TDE to 100 μL of TGP vehicle control once per week for 7 weeks received OC with 100 μL of 30 mM TrisHCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg RHNP extract in100μL/mouse. A third set of mice (n = 4) that received TDE to EHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg EHNP extract in 100μL/mouse. Rectal temperatures were taken prior to OC and 10, 20, and 30 minutes post OC. Rectal temperatures taken from vehicle control, RHNP, and EHNP groups of mice prior to OC (Pre) were comparable (Figure 20A). At 10, 20, and 30 minutes post OC, temperatures taken from the RHNP and EHNP groups of mice were significantly lower than temperatures taken from the control mice. At 10 minutes post OC, temperatures taken from the RHNP group of mice were significant lower than temperatures taken from the EHNP group of mice. Temperatures taken from the RHNP group of mice at 20 and 30 minutes were still lower than temperatures taken from the EHNP group of mice. However, the difference between the RHNP and EHNP groups of mice at 20 and 30 minutes was not found to be significantly different as assessed by Student’s t-test. The change in rectal temperatures (Δ °C) was then evaluated as shown in Figure 20B. At 10, 20, and 30 minutes, there was a significantly greater drop observed in the RHNP group 66 versus the control group. At 10 minutes, there was a significantly greater drop in the EHNP group versus the control group but there was no significant difference between EHNP and control groups at 20 and 30 minutes. At 10 and 20 minutes, there was a significantly greater change in temperature observed in the RHNP group versus the EHNP group. 4.7 ANALYSIS OF HYPOTHERMIA SHOCK RESPONSE IN BALB/C MICE UPON SYSTEMIC CHALLENGE WITH RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Mice were challenged via intraperitoneal (IP) injection, a method of systemic challenge to evaluate hypothermia shock in response to raw versus extruded hazelnut protein extracts. Control mice (n = 5) that received TDE to 100 μL of TGP vehicle control once per week for 3 weeks were challenged via intraperitoneal (IP) injection with 50 μL of 30 mM TrisHCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/ mouse were challenged via IP route with 1 mg RHNP extract in 50μL/mouse. A third set of mice (n = 5) that received TDE to EHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/mouse were challenged via IP route with 1 mg EHNP extract in 50μL/mouse. Rectal temperatures taken from vehicle control, RHNP, and EHNP groups of mice prior to IP injection (Pre) were comparable (Figure 21A). At 10, 20, and 30 minutes post IP injection, temperatures taken from the RHNP and EHNP groups of mice were significantly lower than temperatures taken from the control mice. At 10, 20, and 30 minutes post IP injection, there was no significant difference in temperatures taken from the RHNP and EHNP groups of mice as assessed by Student’s t-test. 67 A B Figure 20 Comparison of hypothermia shock response upon oral challenge to raw hazelnut protein (RHNP) versus extrusion processed hazelnut protein (EHNP) extracts. Groups of mice were orally challenged with 10 mg of RHNP, EHNP or 100 μL TGP vehicle control to test for systemic shock reaction by rectal thermometry. (A) Actual rectal temperature; (B) change in temperature from pre to 30 minutes post oral feeding. * Student’s t-test, p<0.05. 68 The change in rectal temperatures (Δ °C) was then evaluated as shown in Figure 21B. At 10, 20, and 30 minutes, there was a significantly greater drop observed in the RHNP and EHNP groups versus the control group. At 10, 20, and 30 minutes, there was no significant difference observed in temperatures taken from the RHNP and EHNP groups of mice. 4.8 CHARACTERIZATION OF MUCOSAL MAST CELL RESPONSE IN BALB/C MICE UPON ORAL CHALLENGE WITH RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Mice were evaluated for mucosal mast cell response post oral challenge by measuring mouse mast cell protease-1 (mMCP-1), a biomarker of IgE-mediated anaphylaxis. Control mice (n = 9) that received TDE to 100 μL of TGP vehicle control once per week for 7 weeks received OC with 100 μL of 30 mM Tris-HCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg RHNP extract in100μL/mouse. A third set of mice (n = 4) that received TDE to EHNP extract once per week for 7 weeks at a dosage of 1 mg/TDE in 100 μL/mouse received OC with 10 mg EHNP extract in 100μL/mouse. Blood plasma samples were collected prior to (Pre) and 45 minutes post oral challenge (After OC) and were screened for mouse mast cell protease-1 (mMCP-1) (Figure 22). As expected, there was little to no mucosal mast cell response prior to oral challenge (Pre) and no significant difference was observed between RHNP and EHNP groups at this time point. The mucosal mast cell response induced after mice were orally fed the allergen to which they were sensitized was significantly higher in the RHNP versus the EHNP group. There was little to no mucosal mast cell response in the vehicle control group (Appendix E). 69 A B Figure 21 Comparison of hypothermia shock response upon IP challenge to raw hazelnut protein (RHNP) versus extrusion processed hazelnut protein (EHNP) extracts. Groups of mice (n=5/group) were challenged with 1 mg of RHNP, EHNP or 50 μL vehicle via IP route to test for systemic shock reaction by rectal thermometry. (A) Actual rectal temperature; (B) change in temperature from pre to 30 minutes post IP injection. * Student’s t-test, p<0.05. 70 Figure 22 Analysis of mucosal mast cell degranulation responses upon oral challenge with raw versus extrusion-processed hazelnut protein extract. Figure shows mucosal mast cell protease (mMCP)-1 level in the blood before (Pre, left panel) and after (right panel) oral challenge (OC) induced by raw hazelnut protein (RHNP) versus extrusion-processed hazelnut protein (EHNP) extracts. The plasma samples were analyzed by ELISA method using a commercial mMCP-1 quantitation kit. Student’s t-Test: * indicates significant difference, p<0.05. 71 4.9 CHARACTERIZATION OF MUCOSAL MAST CELL RESPONSE IN BALB/C MICE UPON SYSTEMIC CHALLENGE WITH RAW VERSUS EXTRUSION PROCESSED HAZELNUT PROTEIN EXTRACT Mice were evaluated for mucosal mast cell response post oral challenge by measuring mouse mast cell protease-1 (mMCP-1), a biomarker of IgE-mediated anaphylaxis. Control mice (n = 5) that received TDE to 100 μL of TGP vehicle control once per week for 3 weeks were challenged via intraperitoneal (IP) injection with 50 μL of 30 mM Tris-HCl/mouse. A second set of mice (n = 5) that received TDE to RHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/ mouse were challenged via IP route with 1 mg RHNP extract in 50μL/mouse. A third set of mice (n = 5) that received TDE to EHNP extract once per week for 3 weeks at a dose of 1 mg/TDE in 100μL/mouse were challenged via IP route with 1 mg EHNP extract in 50μL/mouse. Blood plasma samples were collected prior to (Pre) and 45 minutes post IP injection challenge (After IP) and were screened for mouse mast cell protease-1 (mMCP-1) (Figure 23). As expected, there was little to no mucosal mast cell response prior to IP injection challenge (Pre) and no significant difference was observed between RHNP and EHNP groups at this time point. The mucosal mast cell response induced after mice were injected with the allergen to which they were sensitized was significantly higher in the RHNP versus the EHNP group. There was little to no mucosal mast cell response in the vehicle control group (Appendix F). 72 Figure 23 Analysis of mucosal mast cell degranulation responses upon IP challenge with raw versus extrusion-processed hazelnut protein extract. Figure shows mucosal mast cell protease (mMCP)-1 level in the blood before (Pre, left panel) and after (right panel) IP challenge (OC) induced by raw hazelnut protein (RHNP) versus extrusion-processed hazelnut protein (EHNP) extracts. The plasma samples were analyzed by ELISA method using a commercial mMCP-1 quantitation kit. Student’s t-Test: * indicates significant difference, p<0.05. 73 Data from HSR and mMCP-1 from EHNP- versus RHNP-sensitized mice were quantifiable and the percent reduction in allergenicity was calculated and is reported in Table 7. Oral challenge conducted on EHNP-sensitized / challenged mice resulted in a reduction in HSR of 38% compared to RHNP-sensitized / challenged mice. However, systemic challenge (IP injection) did not have an effect on HSR. In addition, a reduction in mMCP-1 occurred after both OC and IP injection challenges. After OC, EHNP elicited a 53% lower level of mMCP1 than RHNP while IP injection of EHNP elicited a 36% lower level of mMCP-1 than RHNP. Notably, while EHNP reduced mast cell degranulation after both OC and IP injection challenges, it reduced HSR only after OC. Mice challenged with EHNP or RHNP elicited a similar HSR response after IP injection. 74 Table 7 Quantification of effect of extrusion processing on in vivo allergenicity of hazelnut protein based on clinical reactivity in an adjuvant-free mouse model of food allergy Reduction of in vivo allergenicity of raw hazelnut protein Allergen challenge model extract by extrusion processing as assessed by: Hypothermia Shock Response* mMCP-1** Oral challenge 71% 47% IP challenge 13% 64% * Reduction in response was calculated using data of rectal temperature change at 20 minutes post challenge; ** Reduction in response was calculated using data of plasma mMCP-1 levels determined by ELISA method. 75 CHAPTER 5: Discussion There is currently no cure for food allergy and those afflicted with the condition have no choice but to practice complete avoidance. This can be a difficult undertaking as it can be socially and nutritionally disadvantageous and the possibility of accidental ingestion can be frightening for food allergic individuals. One potential method for reducing allergenicity focuses on the production of hypoallergenic foods by utilizing food-processing methods. This study was conducted to evaluate the impact of extrusion processing on hazelnut (HN) allergenicity in vivo using a novel adjuvant-free mouse model that was previously developed in our laboratory (Birmingham and others 2007). There are seven novel findings from this study: (i) extrusion processing of defatted HN flour resulted in significant changes in the soluble protein profile, such as reduction in the soluble proteins in the molecular weight (MW) range of 129, 42-47, 39, 36-37, and 31-32 kDa, without significant changes in the proteins in the MW range of 51-55, 48, or the band < 15 kDa; however, it increases content of protein in the 14-16 kDa range; (ii) transdermal exposure (TDE) of adult female BALB/c mice with extrusion processed hazelnut protein (EHNP) extract elicits significantly reduced early (4 TDE) hazelnut-specific systemic IgE antibody response (sIgER); however, continued exposure results in later (5 TDE) sIgER that is not markedly different from those of raw hazelnut protein (RHNP) exposed mice; (iii) EHNP is significantly less immunogenic as measured by IgG1 antibody responses at both early (4 TDE) and later (5 TDE) responses; (iv) RHNP elicits significantly higher IgG2a antibody responses than EHNP at early (4 TDE) response however, EHNP elicits 76 significantly higher IgG2a antibody response at later (5 TDE) response; (v) extrusion processing results in marked reduction in IgE-binding proteins (i.e., allergens) in the MW range 33-35, 25, and 14-16 kDa; however, EHNP caused increased binding to the 43 kDa protein; (vi) oral challenge of sensitized mice with EHNP elicits significantly reduced hypothermia shock response (HSR) compared to RHNP; however, systemic IP challenge of sensitized mice with EHNP elicits HSR that are comparable to RHNP; and (vii) oral as well as systemic challenge of sensitized mice with EHNP, as opposed to RHNP, elicits significantly reduced release of mouse mast cell protease-1 (mMCP-1). The hypothesis was tested that extrusion processing will reduce in vivo allergenicity of soluble RHNP in a mouse model of food allergy. Several lines of evidence obtained from various experiments support this overall hypothesis and are discussed below: Both SDS-PAGE and Western blot analysis together demonstrate marked and selectively reduced IgE-binding proteins suggesting reduced soluble allergen content in the range of 3147 and 129 kDa. However, IgE binding-proteins specific for 48-55 and < 15 kDa were not affected by extrusion processing. In contrast, extrusion processing increased protein content in the MW range of 14-16 kDa. These results together suggest 31-47 kDa and 129 kDa protein allergens but not 48-55 or < 15 kDa allergens in HN are sensitive to temperature / pressure conditions used in extrusion processing on IgE-binding proteins in HN. Other studies report that some IgE-binding proteins in HN are heat labile while others are heat stable (Müller and others 2000; Wigotzki and others 2000; Pastorello and others 2002; López and others 2012). These studies did not evaluate the effect of extrusion processing. Results 77 from this study shows that attenuation of HN allergens by extrusion-processing is likely due to a combination of thermal and mechanical shear forces on the protein structures. Together, these results show that it is possible to selectively attenuate specific HN allergens by extrusion processing (our study) or other processing conditions (previous studies). In this study we noted that extrusion-processed HN protein profile in SDS-PAGE analysis was qualitatively different compared to the profile of RHNP. Although the identical quantity of total protein was loaded per lane in the SDS gels, the protein content for EHNP samples appears lower compared to RHNP in the gels. It is noteworthy that SDS analysis shows proteins between 15-170 kDa range. Therefore, proteins outside this range, even if present in our protein extracts, are not expected to be visible. It is possible that extrusion-processing induces protein aggregation and / or protein degradation. Such products are not expected to be visible in the SDS analysis. However, they would be expected to be present in the total protein extracts. The total protein content of the extracts was repeatedly quantified by two individuals independently to ensure accuracy using the Lowry-Folin method. Furthermore, EHNP and RHNP were prepared and tested using an additional published method to assess quality (Appendix A). As evident, the difference in the protein profile observed was consistent. For these reasons, it is unlikely that the differences observed on the SDS gels between EHNP and RHNP is in the quantity of protein that was loaded per lane. A major strength of this study is that it is the first study to evaluate the effect of food processing in general and extrusion processing in particular on in vivo allergic responses to HN using a mouse model of food allergy. We used sIgER as readout of in vivo allergic responses because IgE production and consequent sensitization of mast cells is the first 78 essential step for development of HN allergy (Sabra and others 2003; Sicherer and Sampson 2010). Although extrusion processing results in lower initial IgE responses in vivo, continued exposure to EHNP results in significant sIgER that are comparable to RHNP. These results suggest that even though proteins in the range of 31-47 kDa are markedly reduced in EHNP, other proteins present in the extruded preparation can elicit robust IgE antibody responses. Consequently, when we measure sIgER (that reflects overall IgE response to all HN proteins), the ability of RHNP and EHNP extract to elicit IgE responses becomes similar. The second major strength of this study is that, in addition to examining changes in the HN protein profile (by SDS-PAGE and Western blot analysis) and their ability to elicit sIgER upon extrusion processing, we also evaluated their ability to elicit other antibody responses. We evaluated IgG1 and IgG2a antibody isotype responses. The underlying reason is that IgG1 antibody responses represent general immunogenicity of food proteins and it has been previously used in comparing food proteins for their immunogenicity (Birmingham and others 2003; Dearman and Kimber 2005). Our finding that EHNP elicits reduced IgG1 responses suggests reduced immunogenicity of the EHNP extract. In contrast to IgE and IgG1 that are associated with Th2 immune responses in mice, IgG2a antibody responses are associated with Th1 (in particular, IFN-γ cytokine production) responses that are generally associated with anti-allergic or protective immune responses (Mosmann and Coffman 1989; Tighe and others 2000; Johansen and others 2005). Our finding that EHNP elicits enhanced IgG2a responses imply that immune responses in vivo may be shifting from Th2 dominated response (that is IgE / IgG1 dominated response that occurs with RHNP) towards anti-allergic Th1 responses with enhanced IgG2a production. 79 The enhancement of IgG2a response is considered a desirable effect in general and in our study in particular because it is associated with overall reduced allergenicity of HN protein by extrusion processing. Furthermore, enhanced IgG2a responses also provide evidence that the total protein content was not lower in the EHNP extract as compared to the RHNP extract. The third major strength of this study is evaluating in vivo response of sensitized host to oral challenge (OC) with RHNP versus EHNP. The double-blind placebo-controlled food challenge (DBPCFC) has long been the gold standard for in vivo testing (Sampson 1999; Asero and others 2007; Pettersson and others 2014). While there are no such studies that have tested the effect of food processing on allergenicity in mouse models, there are only two studies evaluating the ability of processed HN protein to elicit food allergic reactions upon oral administration in humans. Hansen and others reported that roasted HN (< 7 g), when administered to 17 birch pollen-sensitized subjects 14-65 years old, elicited reduced symptoms associated with oral allergy syndrome (OAS) (2003). The temperature used in roasting of the HN protein preparation was 140 °C and was sufficient to reduce allergenicity upon OC. Worm and others reported that roasted HN (0.01-10 g), when administered to birch-pollen- or hazelnut-sensitized subjects, elicited reduced frequency of OAS symptoms, skin itching, erythema, and rhinoconjunctivitis (2009). The roasting temperature used in roasting of the HN protein preparation was 144 °C and was sufficient to reduce allergenicity upon OC. Our finding that extrusion processed HN protein (140 °C, 20 psi) elicits significantly reduced HSR upon OC in mice is consistent with reduced allergenicity in vivo. 80 The fourth major finding is that we studied a potential underlying mechanism to explain why there was a difference in in vivo HSR between RHNP and EHNP. For this, we evaluated the release of mucosal mast cell (MMC) specific biomarker, mMCP-1, release upon OC and systemic IP challenge. We used mMCP-1 because it has been recently shown to serve as a useful biomarker to distinguish IgE mediated anaphylaxis from IgG1 mediated anaphylaxis in mouse models (Khodoun and others 2011). In mice, both IgE and IgG1 antibodies can participate in anaphylaxis upon protein challenge (Miyajima and others 1997; Finkelman 2007). Therefore it was critical to evaluate this mechanism in this model. Thus, our finding that reduced allergenicity is associated with markedly reduced release of mMCP-1 from mucosal mast cells is highly significant. This explains the underlying mechanism that EHNP elicits reduced shock responses because of reduced MMC response upon OC with EHNP. This reduced potency of EHNP to elicit MMC response was also confirmed in the IP challenge study. Furthermore, changes in circulating mMCP-1 level upon OC also confirm that we are studying IgE-medicated activation of MMCs and not IgG1-mediated anaphylaxis. Since IgE-mediated reactions are more relevant for human HN allergy, our study demonstrates that this model is similar to human IgE-mediated HN allergy and that it can be used as a pre-clinical tool to evaluate the effect of processing on food proteins in such IgEmediated allergic reactions. Development of useful biomarkers for assessment of changes in food protein allergenicity upon food processing is highly desirable. Here, we evaluated two biomarkers to assess impact of processing on HN protein allergenicity: sIgER and mMCP-1 measurement. Our data demonstrate that sIgER distinguishes EHNP from RHNP only at early, but not later, 81 time points and is therefore limited in its utility. However, mMCP-1 is very useful as a biomarker both in OC as well as systemic IP challenge models. Currently, methods used to evaluate the allergenicity of processed foods include in vitro methods such as inhibition ELISAs and immunoblot analysis (Masthoff and others 2013). These methods are based on the principle of evaluating changes in the binding of IgE antibodies to native food proteins versus processed food proteins. Typically, serum / plasma from allergic subjects is used in these methods. While these in vitro methods are attractive because of the technical advantages such as time and ease, they face the challenge of interpretation. For instance, they will not be able to inform how much reduction in IgE binding in such tests are required to predict lack of clinical reactions upon OC. In contrast, in vivo methods such as OC studies are considered gold standards but have been rarely used primarily because of risk of anaphylaxis and expenses involved. As an alternative, mouse models such as the one we have used are desirable in vivo methods for pre-clinical evaluation of allergenic potential of modified foods such as genetically modified foods as well as processed foods (Birmingham and others 2007; Ladics and Selgrade 2009). Our pilot study presented here provides evidence of this concept. There are few limitations to this study. First, this study was conducted in a mouse model. Consequently, it is imperative the overall conclusion that extrusion processing reduced allergenicity must be viewed in the context of a mouse system and not in a human situation. Further studies using samples from HN allergic subjects and double-blind placebo-controlled human OC studies are necessary to make a final conclusion as to the usefulness and relevance of these findings to human situations. Second, since this was a pilot study, the 82 effect of extrusion processing was examined using a limited combination of temperature / pressure processing conditions. Additional studies with more temperature / pressure combinations will reveal the broader effect, dose-responses, and evaluate whether there is a potential to reduce allergenicity further (beyond 47-71% reduction observed in this study). Finally, we evaluated the effect of extrusion processing on soluble proteins. Therefore, the impact in the context of whole food and, in particular, the sensory characteristics of the food containing such extruded HN proteins must be evaluated for consumer acceptance should such hypoallergenic HN proteins be used as a food ingredient. Additionally, it is noteworthy that extrusion processing may cause some proteins to aggregate and form insoluble proteins. We do not know if this happened in our studies but there was no evidence for the formation of higher MW proteins in SDS-PAGE or Western blot analysis. Notably, the scope of the present study was limited to studying the effect of processing on soluble proteins from HNs. We studied only soluble proteins because it is generally thought that protein allergens are largely aqueous soluble proteins in water and buffers (O’Neil and others 2011). In conclusion, the results of this study demonstrate that it may be possible to decrease hazelnut allergenicity through the use of extrusion processing. It also provides further evidence that food-processing techniques may be useful to ameliorate the probability that food allergy will cause anaphylaxis. However, the variability in the type of allergenic proteins and the dose required for sensitization complicates efforts to develop a method that will reduce or eliminate allergenicity. Foods may also contain multiple potential allergens, as is often the case with processed foods, and while specific processing techniques may 83 decrease or eliminate the allergenicity of one allergen, it may have no effect or even an adverse effect on other allergens within the final product. Continuing research in this area is essential to better understand the impact and potential benefits of processing on food allergy. 84 CHAPTER 6: Summary and Future Direction Food allergies are an adverse immune response to food proteins associated with the production of IgE antibodies specific to food proteins (allergens) (Sampson 2001; Boyce and others 2011). These IgE antibodies are capable of activating effector immune cells, such as mast cells, resulting in mediator release and consequent local reactions and/or lifethreatening systemic anaphylaxis. Food allergies affect approximately 8% of children and 5% of adults in the United States with the most severe symptoms among children occurring as a result of tree nut or peanut allergy (Gupta and others 2011; Sicherer and Sampson 2014). Furthermore, food allergies are a growing and significant public health concern in the United States as well as other countries (Branum and Lukacs 2009; Osborne and others 2011; Sicherer and Sampson 2014; Nwaru and others 2014). Food processing may attenuate food allergy because it has the potential to impact protein structure and therefore, the ability of the immune system to recognize and respond to food allergens (Besler and others 2001; Sathe and others 2005). Some common methods used to process foods include baking, dry roasting, frying, boiling, blanching, steaming, extrusion, microwaving, and irradiation. The underlying principle is that processing can disrupt protein structure by causing events such as denaturation, partial unfolding, aggregation, gelation, and chemical modification (Davis and Williams 1998). It is proposed that such changes in protein structure could potentially alter the ability of food proteins to cause allergic reactions (Besler and others 2001). 85 Animal models are a useful way in which to assess food allergy in vivo and the most desirable models are those that best simulate the human disease (Helm 2002). The mouse model used in this study has desirable characteristics such as the use of an optimal genetic strain and gender, route of sensitization without the use of an adjuvant, and oral elicitation route to assess clinical reactivity. In view of the mechanism of food allergy discussed in Chapter 2 (pages 13-17), four biomarkers were used in this study to confirm IgE-mediated anaphylaxis in vivo in our mouse model of food allergy: i) food-specific IgE was tested as an indicator of sensitization to HN allergens, ii) IgG1 was tested as an indicator of immunogenicity and because this type of antibody is produced during the Th2 response that leads to IgE production, iii) IgG2a, an indicator of the Th1 response, was evaluated because it is generally associated with antiallergic or protective immune responses and, iv) mouse mast cell protease (mMCP)-1 was measured because this protease is specific to mouse mucosal mast cells (MMC) and has been reported as a useful biomarker to distinguish IgE- from IgG1-mediated anaphylaxis. In this pilot study, we evaluated the potential utility of an adjuvant-free mouse model of food allergy to assess the effect of extrusion processing on in vivo allergenicity of hazelnut (HN) protein. Our hypothesis was that soluble extrusion processed hazelnut protein (EHNP) will exhibit reduced allergenicity. Our results demonstrate that reduced in vivo allergenicity of EHNP upon oral challenge (OC) in our model are associated with reduction in the MW range of 25-40 kDa allergen content, reduced overall immunogenicity, variable systemic IgE responses (sIgER), enhanced IgG2a responses and reduced MMC degranulation responses. 86 Furthermore, these results demonstrate that both hypothermia shock response (HSR) and mMCP-1 levels are precisely quantifiable and useful to evaluate the changes in HN protein allergenicity upon extrusion processing. This study demonstrates for the first time the preclinical utility of an adjuvant-free mouse model to evaluate the in vivo allergenicity of processed HN protein. This model may also be useful to evaluate the impact of other types of food processing on in vivo allergenicity. Future animal studies using IFN-γ knockout mice may be useful to assess the impact of food processing techniques, such as extrusion, on food allergy. IFN-γ is known to have an inhibitory effect on the Th2 response (Gavett and others 1995). Therefore, one could hypothesize that without IFN-γ production there may be a marked increase in the induction of IgE antibodies by Th2 cells and / or a decrease in the production of anti-allergic IgG2a in EHNP-sensitized mice. In addition, this could support the use of IL-12, a regulator of the Th1 response, as an immunotherapeutic agent to treat food allergies (Barnes 2000). Other studies using samples from HN allergic human subjects are necessary to make a final conclusion as to the usefulness and relevance of our findings to human situations. In vitro studies such as inhibition ELISAs and immunoblotting with human samples will confirm the IgE-binding potential in HN-allergic humans to raw versus extruded HN proteins. Additionally, double-blind placebo-controlled human studies are necessary to confirm the clinical reactivity observed in our mouse model. 87 Since this was a pilot study, the effect of extrusion processing was examined using a limited combination of processing conditions. Additional studies with more temperature / pressure combinations will reveal the broader effect, dose-responses, and evaluate whether there is a potential to reduce allergenicity further. In addition, the impact in the context of whole food and, in particular, the sensory characteristics of the food containing such extruded HN proteins must be evaluated for consumer acceptance should such hypoallergenic HN proteins be used as a food ingredient. In conclusion, the results of this study demonstrate extrusion processing may reduce hazelnut allergenicity. The results of this study are depicted in Figure 24. These results also provide further evidence that food-processing techniques may be useful to ameliorate the probability that food allergy will cause anaphylaxis. However, further studies are necessary to confirm the findings from this study and the applicability to humans who suffer from food allergy, and hazelnut allergy in particular. 88 Figure 24 Summary of major findings from this study. The overall effect of extrusionprocessing on in vitro and in vivo hazelnut allergenicity observed in this study are illustrated. ↔ No impact; ↑ increased; ↓ decreased. 89 APPENDICES 89 APPENDIX A PROTEIN PROFILE FROM ALTERNATIVE EXTRACTION METHODS 90 Figure 25 SDS-PAGE protein profile of raw hazelnut protein (RHNP) versus extruded hazelnut protein (EHNP) extracts prepared by an independent published method (Cucu and others 2011): analysis under reducing conditions. Protein content was quantified in quadruplicates by the Lowry-Folin method and indicated amount was loaded in 20µL per lane. Protein reduced with 2-Mercaptoethanol and electrophoresed in a 10% gel, lane 1 and 5) marker; 2) 6µg EHNP; 3) 6µg RHNP; 4) 10µg EHNP; 6) 10µg RHNP. 91 APPENDIX B VEHICLE CONTROL DATA FOR HAZELNUT-SPECIFIC SYSTEMIC IGE: BAR GRAPHS 92 Figure 26 Systemic IgE antibody responses in vehicle control mice. The y-axis shows hazelnut (HN)-specific systemic IgE (sIgE) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to vehicle. The X-axis shows two plasma dilutions, 1/640 (A, C, E) and 1/1280 (B, D, F) used in ELISA analysis. 93 APPENDIX C VEHICLE CONTROL DATA FOR HAZELNUT-SPECIFIC SYSTEMIC IGG1: BAR GRAPHS 94 Figure 27 Systemic IgG1 antibody responses in vehicle control mice. The y-axis shows hazelnut (HN)-specific systemic IgG1 (sIgG1) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to vehicle. The X-axis shows two plasma dilutions, 1/640 (A, C, E) and 1/1280 (B, D, F) used in ELISA analysis. 95 APPENDIX D VEHICLE CONTROL DATA FOR HAZELNUT-SPECIFIC SYSTEMIC IGG2A: BAR GRAPHS 96 Figure 28 Systemic IgG2a antibody responses in vehicle control mice. The y-axis shows hazelnut (HN)-specific systemic IgG2a (sIgG2a) antibody (Ab) levels in BALB/c mice before (Pre) (A, B) and after 4 transdermal exposures (4R) (C, D) and 5 transdermal exposures (5R) (E, F) to vehicle. The X-axis shows two plasma dilutions, 1/640 (A, C, E) and 1/1280 (B, D, F) used in ELISA analysis. 97 APPENDIX E VEHICLE CONTROL DATA FOR MUCOSAL MAST CELL PROTEASE (MMCP)-1 LEVELS AFTER ORAL CHALLENGE 98 Figure 29 Analysis of mucosal mast cell degranulation responses upon oral challenge with vehicle control. Figure shows mucosal mast cell protease (mMCP)-1 level in the blood before (Pre, left panel) and after (right panel) oral challenge (OC) induced by vehicle control. 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