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Wheat Bioactives and Immune Function

Permanent Link: http://ufdc.ufl.edu/UFE0042724/00001

Material Information

Title: Wheat Bioactives and Immune Function
Physical Description: 1 online resource (62 p.)
Language: english
Creator: BONARD,SUZANNA P
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: BRAN -- CELL -- DELTA -- FERULIC -- GAMMA -- IMMUNITY -- T -- WHEAT
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Polyphenols found in the diet are an emerging area of interest in immunity. They have been shown to have multiple beneficial effects when consumed. Whole wheat bran cereal contains many polyphenols, the most abundant of them being ferulic acid. Ferulic acid is a phenolic acid that is found bound to the fiber portion of whole wheat bran cereal. We believe that this compound has immune modifying abilities. ?? T cells are a specific group of T cells that are first responders in an immune response. These T cells have been shown to be modified by certain dietary compounds such as L- theanine from tea, polysaccharides in yamoa and compounds in grapes. Our study aimed to determine whether daily consumption of whole wheat bran cereal would modify ?? T cells by priming them to respond at a faster rate when encountering a pathogen. Peripheral blood mononuclear cells (PBMCs) were cultured with PHA-L, IL-15 and IL-2 for 10 days. The percentage of ?? T cells in the total lymphocyte population was determined at baseline and after 21 days of bran consumption by flow cytometry. Consuming the wheat bran cereal resulted in a 70% increase in ?? T cell numbers compared to baseline. These results suggest that there are certain compounds in the whole wheat bran cereal that are able to prime ?? T cells. In order to determine if ferulic acid was, in fact, the bioactive of interest in the whole wheat bran cereal, and whether it was bioavailable, a Caco-2 cell line was used to determine absorption. We simulated digestion of the whole wheat bran cereal and used 21-day differentiated Caco-2 cells to simulate an intestinal model. The digesta was incubated with Caco-2 cell monolayers for two, three and four hours in the apical chambers of a Transwell? plate. The basolateral chamber contents were analyzed by HPLC-DAD analysis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SUZANNA P BONARD.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042724:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042724/00001

Material Information

Title: Wheat Bioactives and Immune Function
Physical Description: 1 online resource (62 p.)
Language: english
Creator: BONARD,SUZANNA P
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: BRAN -- CELL -- DELTA -- FERULIC -- GAMMA -- IMMUNITY -- T -- WHEAT
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Polyphenols found in the diet are an emerging area of interest in immunity. They have been shown to have multiple beneficial effects when consumed. Whole wheat bran cereal contains many polyphenols, the most abundant of them being ferulic acid. Ferulic acid is a phenolic acid that is found bound to the fiber portion of whole wheat bran cereal. We believe that this compound has immune modifying abilities. ?? T cells are a specific group of T cells that are first responders in an immune response. These T cells have been shown to be modified by certain dietary compounds such as L- theanine from tea, polysaccharides in yamoa and compounds in grapes. Our study aimed to determine whether daily consumption of whole wheat bran cereal would modify ?? T cells by priming them to respond at a faster rate when encountering a pathogen. Peripheral blood mononuclear cells (PBMCs) were cultured with PHA-L, IL-15 and IL-2 for 10 days. The percentage of ?? T cells in the total lymphocyte population was determined at baseline and after 21 days of bran consumption by flow cytometry. Consuming the wheat bran cereal resulted in a 70% increase in ?? T cell numbers compared to baseline. These results suggest that there are certain compounds in the whole wheat bran cereal that are able to prime ?? T cells. In order to determine if ferulic acid was, in fact, the bioactive of interest in the whole wheat bran cereal, and whether it was bioavailable, a Caco-2 cell line was used to determine absorption. We simulated digestion of the whole wheat bran cereal and used 21-day differentiated Caco-2 cells to simulate an intestinal model. The digesta was incubated with Caco-2 cell monolayers for two, three and four hours in the apical chambers of a Transwell? plate. The basolateral chamber contents were analyzed by HPLC-DAD analysis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by SUZANNA P BONARD.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042724:00001


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1 WHEAT BIOACTIVES AND IMMUNE FUNCTION By SUZANNA P. BONARD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 201 1

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2 2011 Suzanna P. Bonard

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3 To my parents, Richard and Linda Bonard, my aunt, Terri Bonard and my grandparents, Dick and Dawn Bonard

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4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Susan S. Percival for her guidance and wisdom throughout my graduate career. I also want to thank Dr. Anne Mathews and Dr. Joseph Larkin III for their time and input on my supervisory committee. I appreciate the invaluable assistance Dr. Cheryl A. Rowe has given me in the lab oratory I am grate ful to my entire family for their endless supp ort and always believing in me, no matter what. Finally, I would like to express my heartfelt thank s to Jason McAninley for h is strength and love throughout.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 13 Introduction ................................ ................................ ................................ ............. 13 Immunity ................................ ................................ ................................ ................. 13 Innate Immunity ................................ ................................ ................................ 14 Acquired Immunity ................................ ................................ ............................ 14 Whole Wheat Bran ................................ ................................ ................................ .. 16 2 WHOLE WHEAT BRAN CEREAL AND ITS EFFECT ON HUMAN GAMMA DELTA T CELLS ................................ ................................ ................................ ..... 22 Overview ................................ ................................ ................................ ................. 22 T Cells ................................ ................................ ................................ ................ 22 Materials and Methods ................................ ................................ ............................ 25 Subjects and Study Design ................................ ................................ .............. 25 Reagents ................................ ................................ ................................ .......... 26 Sample Collection ................................ ................................ ............................ 26 Serum Collection ................................ ................................ .............................. 27 PBMC Is olation ................................ ................................ ................................ 27 ................................ ................................ ...................... 28 Flow Cytometry ................................ ................................ ................................ 28 Statistical Analysis ................................ ................................ ............................ 29 Results ................................ ................................ ................................ .................... 30 Discussion ................................ ................................ ................................ .............. 30 3 BIOAVAILABILITY OF FERULIC ACID FROM A WHOLE WHEAT BRAN CEREAL USING A CACO 2 CELL MODEL ................................ ............................ 35 Overview ................................ ................................ ................................ ................. 35 Materials and M ethods ................................ ................................ ............................ 36 In Vitro Digestion ................................ ................................ .............................. 36 Reagents ................................ ................................ ................................ .... 36

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6 Oral digestion ................................ ................................ ............................. 37 Gastric digestion/intestinal digestion ................................ .......................... 37 Caco 2 Bioavailability Intestinal Model ................................ ............................. 37 Reagents ................................ ................................ ................................ .... 37 Cell culture ................................ ................................ ................................ 38 In Vitro bioavailability study ................................ ................................ ........ 38 Analysis ................................ ................................ ................................ ...... 39 Results ................................ ................................ ................................ .................... 39 APPENDIX: HUMAN PBMC STUDY DOCUMENTS ................................ ..................... 41 Internal Review Board Documents ................................ ................................ ......... 41 Flyer for Recruiting Participants ................................ ................................ ....... 41 Inclusion/Exclusion Questionnaire ................................ ................................ .... 42 Informed Consent ................................ ................................ ............................. 43 Final Questionnaire ................................ ................................ ................................ 56 LIST OF REFERENCES ................................ ................................ ............................... 58 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 62

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7 LIST OF TABLES Table page 1 1 r esearch ................................ ................................ ............................... 19 2 1 Percent of CD3+lymp ............................... 32 2 2 Demographics and compliance of study participants. ................................ ......... 34 2 3 Responses from the final questionnaire in number of participants ..................... 34

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8 LIST OF FIGURES Figure page 1 1 Structure and linkage of an arabinoxylan. ................................ .......................... 20 1 2 Basic structure of ferulic acid. ................................ ................................ ............. 20 1 3 Diagram of a wheat kernel (26). ................................ ................................ ......... 21 2 1 Percent baseline (Draw A) and 21 days of consumption (Draw B) ................................ 32 2 2 Example of gated lymphocyte data from FlowJo software. ................................ 33 3 1 Increasing trans epithelial electrical resistance (TEER) measured in Ohms. .... 40

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9 LIST OF ABBREVIATION S ANOVA Analysis of Variance ATCC American Type Culture Col lection Alpha Beta B cell B Lymphocyte BMI Body Mass Index C Celsius CD Cluster Designation CO 2 Carbon Dioxide DAD Diode Array Detector FBS Fetal Bovine Serum FITC Fluorescein Isothiocyanate Gamma D elta H 2 O Water HCl Hydrochloric Acid HEPES 4 (2 hydroxyethy l) 1 piperazineethanesulfonic acid HPLC High Performance Liquid Chromatography IL Interleukin IRB Internal Review Board MEM Minimum Essential Medium mg Milligram mL Milliliter mM Mil l imolar mm Millimeter NaCl Sodium C hloride

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10 NaHCO 3 Sodium Bicarbonate NaN 3 Sodium Azide NaOH Sodium Hydroxide ng Nanogram O 2 Oxygen Ohms PAMP Pathogen A ssociated M olecular P attern PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PE Phycoerythrin PHA L Phytohemagglutinin Leukocyte PRR Pattern R ecognition R eceptor rpm Revolutions Per Minute RPMI Roswell Park Memori al Institute RT Room Temperature SD Standard Deviation SST Serum Separation Tubes T cell T Lymphocyte TCR T C ell Receptor TEER Trans Epithelial E lectrical R esistance U Units Microgram Microliter Micromolar

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11 Abstract of Thesis Presented to th e Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science WHEAT BIOACTIVES AND IMMUNE FUNCTION By Suzanna P. Bonard May 2011 Chair: Susan S. Percival Major: Food Science and Human Nutrition Polyphenols found in the diet are an emerging area of interest in immunity. They have been shown to have multiple beneficial effects when consumed. Whole wheat bran cereal contains many polyphenols the most abundant of them being fer ulic acid. Ferulic acid is a phenolic acid that is found bound to the fiber portion of whole wheat bran cereal. We believe that this compound has immune modifying abilities s in an immune response. These T cells have been shown to be modified by certain dietary compounds such as L theanine from tea, polysaccharides in yamoa and compounds in grapes Our study aim ed to determine whether daily con sum ption of whole wheat bran cereal w ould mod encountering a pathogen. Peripheral blood mononuclear cells (PBMCs) were cultured with PHA L, IL 15 and IL 2 for 10 days. The percentage in the total lymphocyte population was de termined at baseline and after 21 days of bran consumption by flow cytometry. Consuming the wheat bran cereal resulted in a 70 % increase in numbers compared to baseline. These results suggest that there are certain compounds in the whole wheat br an c ereal that are able to prime

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12 In order to determine if ferulic acid was in fact the bioactive of interest in the whole wheat bran cereal and whether it was bioavailable, a Caco 2 cell line was used to determine absorption. We simulated di gestion of the whole wheat bran cereal and used 21 day differentiated Caco 2 cells to simulate an intestinal model. The digesta was incubated with Caco 2 cell monolayer s for two, three and four hours in the apical chamber s of a Transwell plate The basola teral chamber contents were analyzed by HPLC DAD analysis.

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13 CHAPTER 1 BACKGROUND I ntroduction The human immune system is an intricate web of cellular interactions. Many factors can influence the immune system, such as health, genetics and nutrition. One up a nd coming area of research in immunity is how nutrition can affect certain cells of the immune system; specifically, the effect T cell. t heanine from tea, condensed tannins from apples, proanthocyanadins and anthocyanadins from grapes and polysaccharides from yamoa (1 6) Currently, the research concerning food components and their interaction with t he immune system is limited. Epidemiological studies have shown that an increase in consumption of whole grain foods is associated with lower risks for some chronic diseases like heart disease, diabetes and even some cancers (7, 8) Whole wheat bran is a whole grain that is emerging as an interest in the area of functional food components that effect immunity. Its main polyphenolic compound is ferulic acid, which is found bound to arabinoxylose, omponent. Our study aim was to show that ferulic acid, a bioactive in whole whea t bran cereal is able to contribute to functional immunity by in creasing T cell proliferation. Immunity According to Roitt et al (9) t he immune system is basically made up of two parts: the acquired or adaptive immune system and the innate immune system. Although they are comprised of different c ells and components, they are reliant on each other to make

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14 the immune system function as a whole. In order for an immune response to be mounted, a pathogen must be recognized. This recognition can involve b oth the innate and acquired immune systems Inn ate Immunity The innate immune system is non specific and is one of the first lines of defense. I t does not have any memory of previously encountered pathogens. Some of the main responsibilities of the innate immune system include : recruitment of ce lls to areas of infection, presentation of antigens to cells of the adaptive immune system, production of cytokines which lead to the activation of T and B cells, and activation of the complement cascade. T hese responses occur when pattern recognition rec eptors (PRRs) on innate cells recognize pathogens or stress indicators based on their pathogen associated molecular patterns (PAMPs). These molecular patterns are found only on pathogens, and not on Leukocytes, or white blood cells, are the main cells involved in the innate immune response These cells include natural killer cells, mast cells, basophils, eosinophils and phagocytic cells such as macrophages and neutrophils. The p hagocytic cells have the responsibility of engulfing the pathogen and then either digesting and presenting the antigen or killing the pathogen. This leads to an inflammatory response which triggers signaling cascades that are able to increase the cytotoxic functions of immune cells (10) Acquired Immunity The acquired immune system is differentiated from the innate immune system by two key aspects: specificity and memory. The acquired immune system is extremely specific for individual pathogens and its response improve s each time it encounte rs the

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15 Lymphocytes are the key cel ls of the acquired immune system involved in pathogen recognition and therefore the initiation of the acquired immune response. The two mai n lymphocytes are the T cell and the B cell. B oth types of cells origina te from stem cells, but the T cell s matur e in the thymus while the B cell s matu re in the bone marrow. There are several different types of T cells in the body O ne type called T help er cells is involved in interaction with B cells helping them to differentiate and produce antibodies and coordinating the phagocytic activity of macrophages. Another type of T cell the cytotoxic T cell, destroy s cells of the body that have become infec ted wi th viruses or tumor cells. The other groups of T cells are the memory, natural killer and regulatory T cells In order to react T cells must be able to recognize antigens. This recognition of antigens is accompl ished through a T cell receptor, comm only referred to as a TCR and non TCR molecules, such as Toll like Receptors (TLRs) on the surface of the T cell After recognition, T cells secrete cytokines to enhance their respons e to the invader A specific subset of T cells the T cells will be discussed in more detail in Chapter 2. B cells are the other type of lymphocyte involved in the humoral immune respons e. Their primary reactions in an immune response include : generating antibodies to antigens acting as antigen presenting cells and devel oping into memory cells. Each B cell in the body exists to r ecognize a single antigen by a specific surface receptor on that particular antigen. B cells use their B cell receptors (BCR) to recognize pathogens.

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16 After recognition, the B cell multiplies and d ifferentiates in order to secrete antibodies that bind to the antigen (9) An immune response is necessary for eradication of pathogens in the body, but a prolonged immune response can be detrimental. Inflammation is an integral p art of this response, but chronic inflammation can affect the body in a negative manner. Heart disease is one result of chronic inflammation. For this reason, it is important for cells of the immune response not to expand and proliferate until a pathogen i s encountered. On the other hand, if these cells could be pathogenic challenge, immune cells might be able to respond at a faster rate (9) Whole Wheat Bran Guidelines released in 2010, half of all grains consumed should be whole grains with a minimum of three servings of whole grains a day. ground, cracker or flaked caryopsis, w hose principal anatomical components the starchy endosperm, germ and bran are present in the same relative proportions as Traditionally Americans over consume the recommended amount of grains, so the USDA does not advise increased consumption of grains, but rather substituting whole grains for refined grains that are already included in the diet. Benefits from consuming whole grains include protection against cardiovascular disease and a decreased incidence of type II diabetes (11) Refined grains are processed in a way that removes the bran and germ portion of the grain, leaving only the endosperm. Whole wheat grains are not proce ssed and are therefore mainly left intact Whole wheat products contain all three parts of the wheat kernel; the germ, endosperm and the bran. Many of the derived protective health

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17 benefits from whole grains a re attributed to the phenolic content of the outer bran layer. With refined grains, this layer is removed therefore negating most of the protective effects whole grains can have (12) Phenolic acids are produced i n plants in response to stress conditions like pathogen invasions and ultraviolet radiation. They act as antioxidants in the plants, scavengers of free radicals which are produced under stress conditions The amount of phenolic acids in plants can depend on several factors such as processing, and growing and storage conditions (13) The two main classes of phenolic acids are hydroxybenzoic acids and hydroxycinnamic acids. The hydroxycinnamic acids include p coumaric, sinapic, ca ffeic and ferulic acids. Most of these compounds do not exist in the free form in plants, but are either bound to polysaccharides or exist as esters (14) The major polyphenol in w hole wheat bran is ferulic acid, which is found in the outer bran layer. The bran laye r consists of two parts the aleurone and the pericarp. T he greatest amount of ferulic acid is found in the aleurone portion (15) Ferulic acid is synthesized from phenylalanine in plants and is a derivative of cinnamic acid (16) Other compounds that are found in whole wheat bran include cinnamic acids, benzoic acids, amino phenolics, quinines and many other derivatives of these compounds. Most of these compounds are not found in abundance in fruits and vegetables, so the only way to get a significant amou nt from the diet is to consume grains ( 17) Some of the ferulic acid is not bound and therefore can be digested by the small intestine. However, most of the ferulic acid contained in whole wheat bran is in the insoluble, bound form, and made accessible to the body by the microflora in the gut (18) The ferulic acid is bound to arabinoxylose whol

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18 The arabinoxylose fiber component consists of a polysaccharide, xylan backbone with arabinose residues linked to the xylan backbone. The ferulic acid attaches to the arabinose residues. These components of the whole wheat br an can be resistant to digestion in the small intestine and pass, largely intact in to the large gut. In the gut, they are fermented by the microflora a process which produce s short chain fatty acids (SCFA). The microflora of the large gut play an import ant role in releasing the ferulic acid and other polyphenols attached to the fiber component of the whole wheat bran. The SCFAs produced by the microflora help to provide fuel for colonic cells, maintaining the colonic mucosa. The fermentation process also helps to lower the pH of the gut and has been linked to lower cholesterol levels in the serum (8)

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19 Table 1 1 cell Research Intervention Measurement Reference Model Design Safflower Oil Berger, 1993 (19) Mice splenic T cells Feeding Mistletoe heat treated (Prenyl Phosp hate) proliferation Fischer, 1996 (20) Human PBMCs ACC Alkyl Amines proliferation & IL 2 secretion Bukowski, 1999 (2) Human PBMCs ACC Green Tea (L theanine) T cell proliferation Kamath, 2003 (1) Human PBMCs Feeding Fruit/Vegetable Concentrate proliferation Nantz, 2006 (21) Human PBMCs Feeding Uncaria tomentosa) Dong Quai ( Angelica sinensis) Apple ( Malus domestica) Yamoa ( Futumia elastica ) cell activation (IL Holderness, 2007 (22) Human and Bovine PBMCs ACC Prenyl Pyrophosphates Isoprenylphosphate (IPP) Holderness, 2007 (22) cells ACC Unripe Apple Peel (Polyphenol) activation (IL Morita, 2007 (23) Bovine PBMCs ACC L theanine and EGCG T cell proliferation Graff, 2007 (5) Human PBMCs Feeding Cocoa Rowe, 2007 (24) Rat lymphocytes Feeding Yamoa (polysaccharide) cell activation (IL Ramiro Puig, 2008 (25) Bovine and human PBMCs and mouse cells ACC Grapes ber Rowe, 2011 (26) Human PBMCs Feeding ACC: Added to Cells in Culture.

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20 A B Figure 1 1. Structure and li nkage of an arabinoxylan. The A designates a (1 4) linked xylan backbone. The B designates an arabinose xylan linkage. Figure 1 2. Basic structure of ferulic acid.

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21 Figure 1 3 Diagram of a wheat kerne l (27)

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22 CHAPTER 2 WHOLE WHEAT BRAN CEREAL AND IT S EFFECT ON HUMAN GAMMA DELTA T CELLS Overview Most research on wheat bran and immunity focuses on its antioxidant activity in reduc ing oxidati ve da mage in the body. My study aimed to show that whole wheat bran and function in the body. We believe d that the structure of the specific polyphenol ferulic acid, in the who le wheat bran m ight act as a This pseudo PAMP c ould a quicker immune response and less damage to the body in the form of chronic inflammation. T Cells the gap between innate and acquired immunity since they have attributes that relate to both systems (28) They act as the sentinels of the immune system and their response to a pathogen invasion is much faster than most other immune cells (29) they are able to rearrange to form a memory phenotype T hey also develop in the thymus along with CD8 + T cells. These characteristics place them in the acquired immune system. On the other hand, their TCR can also act as a PRR allowing them to recognize PAMPs. p hagocyte s (30) This set of characteristics place them in the innate immune syste m With these combined attributes, incl uding surveillance, a top priority of the immune system, form an important part of the immune system as a whole

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23 to CD8 + T cells They have a TCR like CD8 + T cells, but one that is composed of and ch ains instead of and chains (31) 10%) of circulating T cells, but comprise a large majority of T cells present in tissues of the intestines and epithelia (31) In developing fetuses, numbers of produced tend to surpass those of their CD8 + counterparts (10, 32) After birth, CD8 + T cells dominate while migrate to e pithelial linings; mainly located in the reproductive tract, the gut and the lungs (10, 32) We can also encou as they migrate through the body to get to where they are needed. include the ability to lys e cells via perforin or Fas ligan d dependent pathways and the secretion of cytokines and chemokines in order to recruit monocytes or macrophages and neutrophils to the site of inflammation (31) The main difference between CD8 + T cells lie in their ability to sense pathogens. Conventional CD8 + T cells recognize pathogens by peptides that are processed and presented by major histocompatibility complexes (MHC) on the outside of antigen presenting cells. with pathogens and can rapidly expand without the usual T cell mechanisms (30, 31) not express the MHC complex recognition molecules CD4 or CD8, their recognition of antigen is MHC independent. PAMPs, phosphoantigens or non peptide, lipid antigens (31) also express toll like receptors (TLR s ) on the ir surface Usually, TLR are expressed in innate cells as a PRR. without the presence of antigen presenting cells (31)

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24 to 10 fold in response to microbial invasions such as Salmonella typihurium, Listeria monoctogenes and Escherichia coli (33 35) This led to the discovery of a ell antigens called alkylamines. These antigens are characterized by one to five carbon length straight or branched chain s with only a single, primary amine group (2) These microbial alkylamines are secreted by m ultiple human pathogens, but they are also found in human secretions such as breast milk and vaginal secretions. The metabolism of L theanine, found in tea, produces ethylamine, another alkylamine, which has also been shown to cause (1) Bacteria in the large gu t are also capable of producing alkylamines (2) These secretions of alkylamines in the bod T cells to diff erentiate between normal condition s and pathogenic condition s in the body In 2005, Hedges et al showed that bovine directly to PAMPs even in the absence of APCs. The inflammatory response seen with direct stimulation of stimulated with known antigens sponded to the crude phLPS challenge by upregulating several chemokines and activation genes (36) In order to support our hypothesis, we conducted a human nutritional intervention study. ls are able to be primed by ferulic acid, o r another wheat bioactive, in whole wheat bran cereal. from cultured PBMCs, would proliferate at a greater rate after being exposed to the bioactive

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25 components in whole wheat bran cereal. The bioactive components would act as Materials and Methods Subjects and Study Design Healthy male (n= 18 ) and female subjects (n= 25 ), aged 18 43, were recruited to participate in a 21 day, pilot study at the University of Florida. These subjects were recru ited via flyer, and word of mouth on the University of Florida campus and the surrounding Gainesville, FL community. Subjects were asked a series of questions concerning their health in order to include or exclude them from the study. Exclusion factors i ncluded: having a chronic health problem, high blood pressure, being a vegan, not allowing blood draws and having a BMI of more than 25. A total of 43 subjects were registered to participate in this study, but one subject dropped out after 20 days and one subject did not return for the final blood draw. All subjects were informed and their consent obtained. The study was approved by the Internal Review Board at the University of Florida on May 16, 2010. Subjects remained in communication with the enrolling graduate assistant by email and telephone throughout the study The study was conducted from May through June of 2010. At the initial fasting blood draw, subjects were assigned a number from 1to 43 There were two treatment groups. One group was given on e serving (50 grams) of a whole wheat bran cereal and the other group was given two servings (100 grams) of cereal to consume per day. The cereal was a whole wheat bran cereal provided by Randomization occurred at the first blood dra w. The first 22 people to come in for the baseline blood draw were assigned to group one, and asked to consum e one serving of cereal daily The next 21 people to come in were assigned to group two, and asked to

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26 consum e two servings of cereal. The baseline blood draw occurred after an overnight fast and the subjects were given a continental breakfast after the blood draw. The subjects were instructed to consume the bag ( s ) of cereal throughout the day, and to drink a good deal of water They were asked to r eturn for the final fasting blood draw on day 22 and to bring any uneaten cereal bags with them At the final blood draw, subjects returned to the clinical laboratory had their blood drawn and complete d a final questionnaire (Table 2 3) The final questio nnaire asked questions pertaining to side effects, compliance, satiety and supplement use. Overall compliance was assessed using self reported data and counting left over bags of cereal. Reagents RPMI 1640 complete with L g lutamine and phenol red was used as cell culture media (Mediatech, Manassas, VA). HEPES buffer at 25mM (Mediatech, Manassas, VA), g entamycin at 50g/mL (Gibco, Grand Island, NY), p enicillin at 100 U/mL, s treptomycin at 100g/mL, f ungizone at 0.25g/mL and 2 Mercaptoethanol at 50M (Sigma Aldrich, St. Louis, MO) were added to prepare the RPMI 1640 complete. Recombinant human IL 2 (BD Pharmogen) and recombinant human IL 15 (Biosource, Camarillo, CA) were used as cytokines Anti human CD3 conjugated to phycoerythrin ( PE ) and anti human TCR conjugated to fluorescein isothiocyanate ( FITC ) were the antibodies used for flow cytometry staining (eBioscience, Sand Diego, CA ). Sample Collection Blood samples were collected at baseline and after 21 days of consumption of the cereal Blood was drawn i nto one 10 mL sodium heparin coated tube for PBMC isolation and one 10 mL SST tube for serum collection (BD Vacutainer, BD Franklin Lakes, NJ). The tubes for PBMCs were kept at room temperature (RT) and serum tubes were kept

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27 on ice (4C) and in the dark until processing. All tubes were processed within 1 2 hours of collection. Serum Collection The serum tubes were centrifuged for 10 minutes at 4 C in a Jouan CR412 centrifuge at 1562g to collect the serum. S erum (400 l) was added to tubes containing 1.6m L RPMI 1640 (Cellgro; Mediatech, Herndon, VA) complete medium (100,000U/L penicillin; 100mg/L streptomycin; 0.25mg/L fungizone; 50mg/L gentamicin; 2mM/L l glutamine; 25mM/L HEPES buffer) to prepare culture medium with 20% autologous serum for each subject. The tubes were stored at 4 C until used. PBMC Isolation Peripheral blood mononuclear cells were isolated from blood in the heparinized tube using a polysaccharide gradient. The level of the blood in the tube was marked using a permanent marker. The conten ts of the entire tube was then poured into a 50 mL conical tube and diluted 1:1 with 0.9% sterile NaCl using the level marked on the original blood tube. Using aseptic techniques and a 10 mL serological pipette, 6 mL of the diluted blood was layered over 3 Laboratories Ltd., Burlington, NC) cell separation medium contained in one 15 mL conical tube. The gradient tubes were centrifuged with out a brake for 20 minutes at 20 C in the Hettich Rotina 420R centrifuge at 591g A fine tip, sterile transfer pipette was used to remove the PBMC band from the gradient tube to a 15mL conical tube containing 10mL RPMI 1640 complete media The tubes were centrifuged for 10 minutes at 4C in a Jouan CR412 centrifuge at 391g to remove the gradient medium The medi um was poured off and the pellet gently vortexed. Ten mL of RPMI 1640 complete were added to the same tube and the cell

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28 pellets resuspended for a second wash. After the second wash, the medium was poured off and the PBMC pellets re suspended in 2 mL of RPMI 1640 complete. The PBMCs were counted at a 1:1000 dilution (10 l cells + 10 mL Isoton) i n the Coulter Z1 S Particle Counter (Beckman Coulter, Brea, CA). Complete RPMI 1640 medium was added to each tube to obtain 2 x 10 6 cells /mL One mL (2 x 10 6 ) of the PBMC suspension was transferred to a snap cap tube and centri fuged for 10 minutes at 4C in a Hettich Rotina 420 R centrifuge at 316g The pellets were then resuspended in 1.0 mL RPMI 1640 complete containing 20% autologous serum. Cell Proliferation First, 0.5 mL (1 x 10 6 PBMC) of each PBMC suspension in RPMI 1640 with 20% autologous serum was seeded in one well of a 24 well plate Then, 0.5 mL of a n RPMI 1640, IL 2 (2ng/mL), PHA L (20g/mL) and IL 15 (2ng/mL) solution was adde d to each well. Final concentrations were as follows: PHA L, 10g/mL; IL 2, 1ng/mL and IL 15, 1 ng/mL. The plates were incubated in a humidified CO 2 incubator at 37C for 10 days. On day 6, 250l of a 1X PHA L/IL 2/IL 15 solution was added to each w ell. On day 10, cells and supernatants from each well w ere harvested and transferred to a snap cap tube for staining for flow cytometry. Flow Cytometry The remaining 0.5 mL (1 x 10 6 PBMC) of PBMC suspension from th cell proliferation protocol was centrifuged to obtain a pelle t and resuspended in 0.2 mL of f low cytometry s taining b uffer (PBS + 0.1% NaN 3 + 2% FBS). To each tube 10l of PE conjugated anti human CD3+ and FITC conjugated anti huma n T cell surface marker TCR antibodies were added The cells were incubated at 4 o C for 30 minutes Cells were washed with 1 mL wash buffer (PBS + 0.1% NaN 3 ) and centrifuged ( 300g, 10 min,

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29 4 o C). To fix the cells, they were resuspended in 0.5 mL 1% paraformaldehyde and r efrigerated until they could be read on the BD FACSort instrument within 48 hours in the Flow Cytometry laboratory of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Cells were harveste d on day 10 processed as above. Data was analyzed using FlowJo version 7.6.1software. The population was gated to exclude debris and the result ing population was determined to be the lymphocyte population. Quadrants were established for CD3 positive, CD 3 negative, TCR positive TCR negative cells. Percentages of TCR positive cells were determined from the gated l ymphocyte population and expressed as the standard error of the mean. Statistical Analysi s All statistics were performed using SigmaPlot version 11.0, Systat Software. Two way repeated measures ANOVA ( two factor repetition) was performed for treatment and time b oth Draw A and B, and repeated for cultured cells (Day 10) for both Draw A and B The Student Newman Keuls method was used for significant differences. No significant differences were noted for subjects consuming one serving or two servings, so all subject s were grouped into the one serving treatment group. After noting significances, another two way repeated measures ANOVA (two factor repetition) was repeated for time and culture status (Cultured or Uncultured). The Student Newman Keuls method was used for significant differences.

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30 Results (Table 2 1, Figure 2 1) There was a significant subjects that consumed whole wheat bran cereal for 2 1 days, with a P value of less than 0.001. There were about 70% more circulating ls in subjects after consuming whole wheat bran for 21 days. There was also a significant increase in ten bran cereal for 21 days, with a P value of less than 0.001. s proliferated about 20% better after consuming whole wheat bran for 21 days. Discussion These results indicate that there is a compound (s) in the whole wheat bran cereal that is ac ting as a priming agent for the T cells. This compound is allowing the T cells to be maintained in a ready state for when a pathogen invasion does occur but not fully activated This ready state is ideal, because if the T cells were completely ac tivated by the compound, a state of chronic inflammation would occur, which would be harmful to the body. This compound is acting as a pseudo PAMP, causing recognition by the T cells. This recognition is perhaps allowing the body to create more mRNA for the proliferation of T cells. When a pathogen does invade the body, the mRNA can be translated into T cells at a faster rate than before the mRNA was created. The data indicates that the baseline T cells were able to proliferate after 10 days of culture. Ideally, the T cells should not be able to proliferate in culture before consumption of the wheat. therefore, would not have the ability to proliferate after stimulation. I believe these

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31 re sults were seen because of the lack of control for diet in the study. Table 1 1 shows T cells. Participants in the study could have regularly consumed some of these foods, such as cranberries or drink tea on a regular basis. In the future, human nutritional intervention studies looking at T cells should control for diet in the participants. Although we did not see a significant difference between the subjects who consumed one serving and subjects who consumed two servings, the data did approach significance with a P value of 0.063. The data might have been more significant if more participants were used. The compliance might have been a contributing factor as well. According to the final questionnaires the participants filled out, overall compliance in the study was around 94% for both groups, which is satisfactory (Table 2 2, Table 2 3). About 73% of the participants experienced an increase in satiety from the whol e wheat bran cereal. The cereal contains a large amount of fiber which could account for this side effect. This might have made consuming two entire servings of the cereal difficult. We believe that the compound in the whole wheat bran cereal that is actin g as the priming agent could be ferulic acid or its side group, arabinoxylose This phenolic acid makes up the greatest quantity of phenolic compounds in whole wheat bran and would be a logical starting place. In order to determine if ferulic acid could be the bioactive another experiment was performed with details being found in Chapter 3.

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32 Table 2 1. Percent of CD3+ lymphocyte population t hat are Baseline 21 Days Uncultured Day 0 6.39 0.71 10.88 0.80 Cultured Day 10 17.90 0.76 21.80 0.97 Time P Value: < 0.001 Culture State P Value: < 0.001 Interaction P Value: 0.686 Figure 2 1. Percentage of cultured and uncultured cells in CD3+ population at baseline (Draw A) and 21 days of consumption (Draw B) Cultured cells represent the isolated PBMCs after 10 days in culture. Different l etters above the bars indicate significa n t differences (p value <0.001) Data is presented as mean SEM.

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33 A B C D Figure 2 2 Example of gated l ymphocyte data from FlowJo software. A) Draw A, Day 0 B) Draw A, Day 10 C) Draw B, Day 0 D) Draw B, Day 10.

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34 Table 2 2. Demographics and compliance of study participants. 1 Serving (50 grams) 2 Servings (100 grams) Sex Male 10 8 Female 1 3 1 2 Ag e (years) 26. 3 5.47 26.0 6.82 Compliance 9 4.8 % 93.3% Two subjects in the two serving group consumed only one bag a day so they were added to the one serving group. Table 2 3. Responses from the final questionnaire in number of participants 1 S erving 2 Servings Experienced Side Effects Yes 12 Yes 7 No 11 No 11 Took Supplements Yes 0 Yes 0 No 23 No 18 Changes in Satiety Yes 15 Yes 15 No 8 No 3 Weight Loss/Gain Yes 3 Yes 5 No 20 No 13 Co nsumed All Cereal Yes 15 Yes 11 No 8 No 7 I ncrease in Hunger Yes 1 Yes 0 No 22 No 18 Supplements not including a multi vitamin.

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35 CHAPTER 3 BIOAVAILABILITY OF FERULIC ACID FROM A WHOLE WHEAT BRAN CEREAL USING A CACO 2 CELL MODEL O verview Caco 2 cells are a continuous line of human colon ad enocarcinoma cells that spontaneously differentiate after forming a confluent monolayer in culture. This monolayer is structurally simil ar to the human small intestine epithelium because of the enterocyte like differentiation that occurs in culture. The C aco 2 cells express transporters and morphology very similar to human intestinal mucosa like tight junctions and transport enzymes. This similarity allows the Caco 2 cells to be used as a simple in vitro mode l for intestinal absorption (37) Caco 2 cells have been shown to transport ions, sugars and peptides when grown as monolayers on filters such as Corning Transwell inserts. We aim ed to show that ferulic acid, a phenolic acid, is also transported across the m onolayer. Several studies have been done showing the transport of ferulic acid across a Caco 2 monolayer (14, 38 40) but none with a digested wheat cereal. In 1998, a study was conducted by Bourne and Rice Evans th at had participants ingest a single bolus of fresh tomatoes to examine the bioavailability of ferulic acid in vivo The investigators looked at the amount of ferulic acid excreted in the urine and the peak time for maximal recovery. Ferulic acid was found to have a peak time for maximal excretion of 7 hours and the total amount of free ferulic acid excreted was 11 25% of that ingested. This study provided evidence that ferulic acid is bioavailable (41) Kern et al showed in 2003 that the in vitro differentiated Caco 2 cell model is able to metabolize dietary hydroxycinnamates. This metabolism occurs th rough expression of phase I and phase II enzymes. These enzymes have hydrolase activity that break down the esters

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36 of the dietary hydroxycinnamates and diferulic acids, allowing transport through the monolayer (39) In 2008, LaParra et al. analyzed both raw and cooked common beans ( Phaseolus vulgaris ) for their phenolic content. They then performed an in vitro digestion on the beans and used the Caco 2 model to determine bioaccessibility of individual po lyphenols. Ferulic acid was found to be bioaccesible from cooked beans (42) According to Janicke et al, the mechanics of absorption of ferulic acid in the GI tract depend on the food that is consumed and the matrix carrying the ferulic acid (38) Clifford et al also specifies that wheat bran can provide up to 50 mg of ferulic acid for every 10 grams of wheat bran consumed (43) In order to demonstrate that ferulic acid is able to be a bsorbed in the body, we u sed a Caco 2 cell intestinal cell model. We simulated digestion of the whole wheat bran cereal and incubated the sample in the apical side of a Transwell We hypothesize that if the ferulic acid is able to be freed from the fiber component of the cereal a fter digestion, th e n the ferulic acid will be absorbed by the Caco 2 cell monolayer and be transported across the monolayer into the basolateral compartment of the Transwell plate Materials and Methods In Vitro Digestion Simulated digestion of the whole wheat bran cereal was performed. Reagents Salivary amylase at 100 U/L (Sigma Aldrich, St. Louis, MO) and NaCl at 15.4 mM (Fisher Scientific Pittsburgh, PA ) were used in the oral digestion phase Hydrochloric acid at 1 M and 0.1 M (Fisher Scientific Pi ttsburgh, PA ), porcine pepsin at

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37 10 mg/ml (Sigma Aldrich, St. Louis, MO), sodium bicarbonate at 1M and 0.1M (Fisher Scientific Pittsburgh, PA ), ox bile extract at 2.4 mg/ml (Fisher Scientific Pittsburgh, PA ), porcine pancreatin at 0.4 mg/ml (Sigma Aldric h, St. Louis, MO) and sodium hydroxide at 1 M (Fisher Scientific Pittsburgh, PA ) were used for the gastric/intestinal digestion phase. Oral d igestion One package (50 g) a mortar and pestle to sim ulate mastication. A portion of the ground cereal 10g, was added to 50 mL of s aline solution (9 g NaCl in 1 L of H 2 O) in a beaker to homogenize the cereal further. salivary amylase (10L, 100 U/L) was added to the sample and incubated for 10 minutes. Gastric d igestion/ intestinal d igestion The orally digested sample was acidified by adding 1 M HCl (pH 2.0). Porcine pepsin, at a final concent ration of 10 mg/mL, in 0.1 M HCl was added to the sample. The sample was After incubation, the sample was immediately adju sted to pH 6.0 by adding 1 M NaHCO 3 Ox bile extract, at a final concentration of 2.4 mg/mL, and porcine pancreatin, at a final concentration o f 0.4 mg/mL, in 0.1 M NaHCO 3 were added to the sample. The pH was then adjusted to 7.0 using 1 M NaOH added dropwise. Caco 2 Bioavailability Intestinal Model Reagents Minimum Essential Medium (MEM) with L glutamine and phenol red was used as cell culture medium (Mediatech, Manassas, VA). Gentamycin at 50g/mL (Gibco, Grand Island, NY), p enicillin at 100 U/mL, s treptomycin at 100 g/mL, f ungizone at 0.25g/mL

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38 (Sigma Aldrich, St. Louis, MO), non essential amino acids at 1% (v/v) ( Mediatech, Manassas, VA ), s odium pyruvate at 1% (v/v) ( Hyclone, Logan, UT ) and heat inactivated f etal b ovine s erum at 15% ( Invitrogen, Carlsbad, CA ) were added to the Minimum Essential Medium to make it complete. Trypsin EDTA solution at 0.25 % / 2.21 mM EDTA (Mediatech, Manassas, V A) was used to remove the cells from the cell culture dishes. Plastic cell cultu re t reated dishes and collagen coated Transwell COL inserts for culturing the cells. Cell culture The human colon carcinoma cell line Caco 2 was obtained from the American Type Culture Collection (ATCC) (Rockville, MD) at passage 29. The cells were maintained in 100 cm 2 7.5 % CO 2 atmosphere in complete MEM. After seeding, the cells were passed at 85% confluency usually every 4 to 5 days. The MEM was changed every 3 days. In Vitro bioavailability study Caco 2 cells were seeded in 12 well plates with Transwells at 2.6 x 10 5 cells/cm 2 The cells were allowed to grow and differentiate for a p eriod of 21 days. The medium (MEM complete) was changed in both the apical and basolateral co mpartments the day after seeding, every other day for the first seven days, and then, the apical compartments were changed every day and the basolateral compartment every other day. To eva luate the integrity of the cell monolayer, the transepithelial elec trical resista nce (TEER) was measured using an EVOM 2 instrument ( World Precision Instruments Sarasota FL, USA) The TEER was measured four days after seeding and

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39 every four days thereafter. Monolayers with TEER values exceeding 250 2 were used for t he bioavailability experiment. added to the apical side of the T ranswells and 1.5 mL of HBSS (pH 7.4, 37) was added to the basolateral chamber The cells were incubated for 2, 3 and 4 hours and the basolateral chamber contents collected. Methanol was added 1:1 to the samples and kept at 4 C for 16 hours in order to precipitate out the protein. Samples were then centrifuged ( 29,338g 4 C, 15 min) and filtered through a 0.22 m filter. Analysis The digesta collected from both chambers were analyzed using reverse phase HPLC DAD. Ferulate compounds were separated using a Luna C18 (2) column (25 cm x 4.6 mm i.d., 5 mm; Phenomenex). A gradient profile was achieved by using solven t A (10% aqueous acetonitrile (v/v) and TFA to 1 mM) and solvent B (40% aqueous acet onitrile (v/v) and TFA to 1 mM). Results The differentiated Caco 2 cell monolayers used in this experiment showed TEER values above 250 after an average time period of one week. The measurements took into account a blank of value 120 (Figure 3 1) The results indicate a monolayer was formed after a week, however, according to the literature, Caco 2 cell monolayer s would only be differentiated after a period of 19 21 days o f culture (44) The results from the HPLC analysis are not shown.

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40 Figure 3 1 Increasing t rans epithelial electrical resistance (TEER) measured in Ohms A blank of 120 Ohms was used. Plate 1 was incubated for a total of 2 1 days. Plate 2 was incubated for 7 days but became contaminated Plate 3 was incubated for 10 days.

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41 APPENDIX HUMAN PBMC STUDY DOCUMENTS Internal R eview B oard Documents The documents included in the appendix are the IRB 02 approved flyer, for recruitment purposes, the incl usion/exclusion questionnaire and the informed consent. Flyer for R ecruiting P articipants RESEARCH PARTICIPANTS WANTED To take part in a Nutrition Study of the Effect of Wheat Bioactives on the Immune System If you are between the ages of 21 and 50 and are generally healthy, you may qualify to take part in this study examining the potential health benefits of a wheat bran cereal. Here is what you will do if you quali fy: Eat one (50 grams) or two (100 grams) boxes of cereal a day for 3 weeks. Make two (2) visits to the University of Florida, Dept. of Food Science and Nutrition. Give a sample of blood on each visit. Have your blood pressure measured at each visit. Complete a final questionnaire at the end of 3 weeks. Total time commitment will be approx. 4 hours. Compensation up to $100.00. Interested? Please call (352)392 1991 x255 and ask about Cereal Study Eligibility.

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42 Inclusion/Exclusion Q uestionnaire Inclusion/Exclusion Evaluation P.I. Dr. Susan S. Percival Introductor y Statement: will be doing at the University of Florida in the Department of Food Science and Human Nutrition. The purpose of the study is to evaluate the effects of a whole grain, wheat bran cereal on human immune function. Participation in this study would last 21 days and will require 2 visits to our lab. You will be required to consume one serving (50 grams) or two servings (100 grams) of a whole grain, wheat bran cereal d aily for 21 days and come into the lab for blood draws between 8:00a.m. 9:00a.m. after fasting overnight. To see if you might qualify for this study, I will need to ask you some questions about your health history and present condition. [For female partic ipants] If you Questions: 1. Will you be in Gainesville during the entire Summer A term of 2010? Yes 2. Are you between the ages of 18 and 50 years old? Yes 3. What is your current height and weight? [Determine BMI from information] 18 35 (BMI over 35 is not acceptable) 4. Do you have any ongoing or chronic illness or infection? No

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43 5. Are you on any of the following: antihypertensive medication, immunosuppressive drugs, antibiotics or chronic use of NSAIDS? No 6. Do you take any dietary supplements besides a multi vitamin? If yes, are you willing to refrain from taking them during the 21 days of the study? Yes 7. During the study will you consume no more than 2 glasses of alcoholic beverages per day? Yes hank you. You qualify for participation in the study. [An appointment location, time and date are set] Suzanna will send you an information email to confirm your participation in the study, along with your first appointment time and study ank you. We Informed Consent INTRODUCTION Name of person seeking your consent:_____Suzanna Bonard__________________ Place of employment & position:___________FSHN Gradu ate Student___________ This is a research study of how wheat bioactives may benefit immune function in humans. Could participating in this study offer any direct benefits to you? Yes, as described on page 4. Could participating cause you any discomforts or are there any risks to you? Yes, as described on page 3. Please read this form which describes the study in some detail. I or one of my co workers will also describe this study to you and answer all of your questions. Your

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44 participation is entirely volunt ary. If you choose to participate you can change your mind at any time and withdraw from the study. You will not be penalized in any way or lose any benefits to which you would otherwise be entitled if you choose not to participate in this study or to with draw. If you have questions about your rights as a research subject, please call the University of Florida Institutional Review Board (IRB) office at (352) 846 1494. If you decide to take part in this study, please sign this form on page 10. GENERAL INFORM ATION ABOUT THIS STUDY 1. Name of Participant ("Study Subject") ____________________________________________________________________ 2. What is the Title of this research study? Wheat Bioactives and Immune Function 3. Who do you call if you have questi ons about this research study? Dr. Susan S. Percival work: 352 392 1991 x217 cell: 352 562 9670 email: percival@ufl.edu 4. Who is paying for this research study? The sponsor of this study is Kelloggs Corporate Citizen's Fund 5. Why is this research study being done? The purpose of this research study is to evaluate the effects of wheat bioactives on human immune function and to determine if wheat bioactives provide antioxidant protection to DNA.

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45 You are being asked to be in this research study because you are a healthy individual between the ages of 21 and 50. WHAT CAN YOU EXPECT IF YOU PARTICIPATE IN THIS STUDY? 6. What will be done as part of your normal clinical care (even if you did not participate in this research study)? Nothing will be done as part of your normal clinical care because you are a healthy volunteer and therefore do not have any normal clinical care. Because this study is not related to your normal clinical care, your physician will not be informed that you are taking part in this study. 7. What will be done only because you are in this research study? This is a 3 week long study. At the beginning of the study, you will first complete the initial paperwork which includes this informed consent form and the form required to pay you, which s hould take about 10 minutes. A trained phlebotomist will obtain a sample of venous blood (four teaspoons total in two tubes) from you for an immune assessment, if you decide to take part in this study. You will be randomly assigned to consume either one se rving (50 grams) or two servings (100 grams) of wheat cereal. You are required to fast a minimum of 6 hours prior to the blood draw and it is preferable if you fast overnight. 1. You will be given a 3 week supply of your assigned cereal serving. For the fi rst 4 days you will consume in the morning and at night. By the 5th day you will

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46 consume each serving in its entirety. Water consumption should be maximized as well. 2. After 3 weeks of consuming the cereal, you will be asked to return to have a traine d phlebotomist obtain a sample of venous blood (four teaspoons total in two tubes) from you for immune assessment. You are required to fast a minimum of 6 hours prior to the blood draw and it is preferable if you fast overnight. 3. Your blood pressure will be measured at both of your two (2) visits to the lab. If on your first visit, if either one of your blood pressure values are high, defined as 140/90, you will not be allowed to participate. If you have any questions now or at any time during the study, please contact Dr. Susan S. Percival in question 3 of this form. 8. How long will you be in this research study? The total time commitment for this research is estimated to be 4 hours, over a 3 week period. Each of the two blood draw sessions (first day an d at the end of 3 weeks) is expected to last no more than 1 hour (equivalent to 2 hours total). You will be required to come to the Food Science and Human Nutrition building for the blood draws. You will consume either one serving (50 grams) or two serving s (50) of cereal every day for 3 weeks. The time to eat the cereal daily and to fill in the final questionnaire is expected to total approximately 2 hours. 9. How many people are expected to take part in this research study? 40 people are needed to partici pate in this research study. WHAT ARE THE RISKS AND BENEFITS OF THIS STUDY AND WHAT ARE YOUR OPTIONS?

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47 10. What are the possible discomforts and risks from taking part in this research study? There are no risks associated with consuming one to two servings of cereal a day. The increased fiber may cause some discomfort, at first, in the form of gastrointestinal distress. This may include, but is not limited to bloating, cramps, and gas. To minimize the discomfort of high fiber, we ask the participant to gradu ally increase their intake of the high fiber cereal over 4 days. We also recommend that participants drink a lot of water to help with the digestion of the fiber. The risks of drawing blood from a vein include discomfort at the site of puncture; possible b ruising and swelling around the puncture site; rarely, an infection; and, uncommonly, faintness from the procedure. An overnight fast is required and may cause physical discomfort, however we will n Other possible risks to you may include: Researchers will take appropriate steps to protect any information they collect about you. However, there is a slight risk that information about you could be revealed inappropriately or accidentally. Depending on t he nature of the information, such a release could upset or embarrass you, or possibly affect your insurability or employability. Questions 17 21 in this form discuss what information about you will be collected, used, protected, and shared. This study may include risks that are unknown at this time.

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48 Participation in more than one research study or project may further increase the risks to you. If you are already enrolled in another research study, please inform Dr. Susan S. Percival (listed in question 3 o f this consent form) or the person reviewing this consent with you before enrolling in this or any other research study or project. Throughout the study, the researchers will notify you of new information that may become available and might affect your dec ision to remain in the study. If you wish to discuss the information above or any discomforts you may experience, please ask questions now or call the PI or contact person listed on the front page of this form. 11a. What are the potential benefits to you f or taking part in this research study? You may or may not personally benefit from participating in this study. You may experience better immune health if the wheat bioactive works as we predict. 11b. How could others possibly benefit from this study? Consu ming the wheat bran cereal may result in benefits such as stronger immunity. 11c. How could the researchers benefit from this study? In general, presenting research results helps the career of a scientist. Therefore, Dr. Susan S. Percival may benefit if th e results of this study are presented at scientific meetings or in scientific journals. Dr. Percival does not receive any compensation, monetary or otherwise, from the sponsor outside of the funding for this research. 12. What other choices do you have if you do not want to be in this study?

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49 The option to taking part in this study is doing nothing. If you do not want to take part in this study, tell the Principal Investigator or her assistant and do not sign this Informed Consent Form. You have been invited to participate in this research project because you qualify as a member of the generally healthy population. The investigators associated with this project may or may not teach in your college or be associated with courses for which you are enrolled or mi ght be expected to register in the future. Your participation in this study is voluntary and any decision to take part or not to participate will in no way affect your grade or class standing. If you believe that your participation in this study or your de cision to withdraw from or to not participate in this study has improperly affected your grade(s), you should discuss this with the dean of your college or you may contact the IRB office. 13a. Can you withdraw from this study? You are free to withdraw your consent and to stop participating in this study at any time. If you do withdraw your consent, you will not be penalized in any way and you will not lose any benefits to which you are entitled. In addition, you hav e the right to refuse to answer any specific question that you do not want to answer. If you decide to withdraw your consent to participate in this study for any reason, please contact Dr. Susan S. Percival at 352 392 1991 ext. 217 or study coordinator at 352 293 1991 ext. 255. They will tell you how to stop your participation safely. If you have any questions regarding your rights as a research subject, please call the Institutional Review Board (IRB) office at (352) 846 1494.

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50 13b. If you withdraw, can inf ormation about you still be used and/or collected? If you withdraw, no new information will be collected about you. However, information that was already collected may still be used and disclosed to others if the researchers have relied on it to complete a nd protect the validity of the research. 13c. Can the Principal Investigator withdraw you from this study? You may be withdrawn from the study without your consent for the following reasons: You are unable to keep appointments, complete a final questionnai re or take the study capsules as directed, or the study is cancelled by the Food and Drug Administration (FDA), the National Institutes of Health (NIH), the company supplying the study treatment, and/or other administrative reasons. You may also be withdra wn from the study if you have a change in your medical health status including blood pressure. WHAT ARE THE FINANCIAL ISSUES IF YOU PARTICIPATE? 14. If you choose to take part in this research study, will it cost you anything? It will not cost you anythin g to take part in this study. The grain cereal will be provided at no cost to you while you are participating in this study. The Sponsor will pay for all activities provided as part of your participation in this study. There will be no cost to you. If you receive a bill related to this study, please contact Dr. Susan S. Percival at 352 392 1991 x 217 or the study coordinator at 352 392 1991 x 255 15. Will you be paid for taking part in this study?

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51 You will receive compensation for taking part in this study. We will pay you $25 for participation in each of the two blood draws, and $50 for completing the final questionnaire, totaling $100. You will receive payment after the study is completed. Please allow between 4 8 weeks after the completion of the study fo r payment. If you are paid for taking part in this study, your name and social security number will be reported to the appropriate University administrative personnel for purposes of making and recording the payment. The information will be used for the pu rpose of payment and will be kept confidential. You are responsible for paying income taxes on any payments provided by the study. If you are a University of Florida employee, taxes will be deducted from your payment which will be added to your regular pay check. If the payments total $600 or more, the University must report the amount you received to the Internal Revenue Service (IRS). 16. What if you are injured because of the study? If you are injured as a direct result of your participation in this study any resulting medical expenses will be billed to you or your insurance provider. You will be responsible for any deductible, co insurance, or co payments. Some insurance companies may not cover costs associated with research studies. Please contact your insurance company for additional information. No additional compensation is offered. The Principal Investigator and others involved in this study may be University of Florida employees. As employees of the University, they are protected under state law, wh ich limits financial recovery

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52 for negligence. Please contact the Principal Investigator listed in question 3 of this form if you experience an injury or have questions about any discomforts that you experience while participating in this study. 17. How wi ll your health information be collected, used and shared? If you agree to participate in this study, the Principal Investigator will create, collect, and use private information about you and your health. This information is called protected health informa tion or PHI. In order to do this, the Principal Investigator needs your authorization The following section describes what PHI will be collected, used and shared, how it will be collected, used, and shared, who will collect, use or share it, who will have access to it, how it will be secured, and what your rights are to revoke this authorization. Your protected health information may be collected, used, and shared with others to determine if you can participate in the study, and then as part of your partici pation in the study. This information can be gathered from you or your past, current or future health records, from procedures such as physical examinations, x rays, blood or urine tests or from other procedures or tests. This information will be created b y receiving study treatments or participating in study procedures, or from your study visits and telephone calls. More specifically, the following information may be collected, used, and shared with others:

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53 prescribed ionnaires. This information will be stored in locked filing cabinets or on computer servers with secure passwords, or encrypted electronic storage devices. Some of the information collected could be included in a "limited data set" to be used for other res earch purposes. If so, the limited data set will only include information that does not directly identify you. For example, the limited data set cannot include your name, address, telephone number, social security number, photographs, or other codes that l ink you to the information in the limited data set. If limited data sets are created and used, agreements between the parties creating and receiving the limited data set are required in order to protect your identity and confidentiality and privacy. 18. Fo r what study related purposes will your protected health information be collected, used, and shared with others? Your PHI may be collected, used, and shared with others to make sure you can participate in the research, through your participation in the res earch, and to

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54 evaluate the results of the research study. More specifically, your PHI may be collected, used, and shared with others for the following study related purpose(s): To evaluate the effects of wheat bioactives on human immune function and to det ermine if grain bioactives provide antioxidant protection to DNA. Once this information is collected, it becomes part of the research record for this study. 19. Who will be allowed to collect, use, and share your protected health information? Only certain people have the legal right to collect, use and share your research records, and they will protect the privacy and security of these records to the extent the law allows. These people include the: nd research staff associated with this project. study related treatment or procedures peopl e who are responsible for looking after the rights and welfare of people taking part in research). 20. Once collected or used, who may your protected health information be shared with? Your PHI may be shared with: Corporate Ci tizen's Fund

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55 that are responsible for overseeing research, such as the Food and Drug Administration, the Department of Health and Human Services, and the Office of Human Research Protections encies who are responsible for overseeing public health concerns such as the Centers for Disease Control and federal, state and local health departments Malcolm Randall VA Medical Center (Gainesville) yment Otherwise, your research records will not be released without your permission unless required by law or a court order. It is possible that once this information is shared with authorized persons, it could be shared by the persons or agencies who rece ive it and it would no longer be protected by the federal medical privacy law. 21. If you agree to take part in this research study, how long will your protected health information be used and shared with others? Your PHI will be used and shared with other s for up to three (3) years after the study ends. If you withdraw your permission for the use and sharing of your protected health information, then your information will be removed from the database You are not required to sign this consent and authoriza tion or allow researchers to collect, use and share your PHI. Your refusal to sign will not affect your treatment, payment, enrollment, or eligibility for any benefits outside this research study.

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56 However, you cannot participate in this research unless you allow the collection, use and sharing of your protected health information by signing this consent and You have the right to review and copy your protected health information. However, we can make this available only after the study is finished. You can revoke your authorization at any time before, during, or after your participation in this study. If you revoke it, no new information will be collected about you. However, information that was already collected may still be used and shared with others if t he researchers have relied on it to complete the research. You can revoke your authorization by giving a written request with your signature on it to the Principal Investigator. Final Questionnaire Subject # ___ Thank you once again for participating in our Wheat study. Please answer the following questions as completely as possible. 1. During the study, did you experience any side effect(s) that might be attributed to the cereal? Yes___ No___ If yes, please explain:____ ________________________________________ 2. During the study, did you take any dietary supplement(s) other than a vitamin/mineral? Yes___ No___ If yes, what type(s) and how often:__________________________________

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57 3. During the study, did you notice any changes in satiety (feeling of fullness) after consuming the cereal? Yes___ No___ If yes, how did this effect your usual eating habits:______________________ 4. During the study, did you experience any weight loss or weight gain? Yes___ No___ 5. Did you consume the cere al we provided to you daily, for the entire 3 weeks? Yes___ No___ 6. If you missed eating the cereal, approximately how many days did you miss? ______________________________________________________________ 7. During the study, did you find yourself feeling more hungry than usual? Yes___ No ___ 8. Any additional information you would like us to know, or any comments regarding this study? ______________________________________________________________ ______________________________________________________________

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58 LI ST OF REFERENCES 1. Kamath AB, Wang L, Das H, Li L, Reinhold VN, Bukowski JF. Antigens in tea beverage prime human Vgamma 2Vdelta 2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses. Proc Natl Acad S ci U S A. 2003 May 13;100:6009 14. 2. Bukowski JF, Morita CT, Brenner MB. Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity. Immunity. 1999 Jul;11:57 65. 3. Holderness J, Hedges JF, Daughenbaugh K, Kimmel E, Graff J, Freedman B, Jutila MA. Response of gammadelta T Cells to plant derived tannins. Crit Rev Immunol. 2008;28:377 402. 4. Percival SS. Grape consumption supports immunity in animals and humans. J Nutr. 2009 Sep;139:1801S 5S. 5. Graff JC, Jutila MA. Differential regulation of CD11b on gammadelta T cells and monocytes in response to unripe apple polyphenols. J Leukoc Biol. 2007 Sep;82:603 7. 6. Graff JC, Kimmel EM, Freedman B, Schepetkin IA, Holderness J, Quinn MT, Jutila MA Hedges JF. Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo. Int Immunopharmacol. 2009 Oct;9:1313 22. 7. WHO. Diet Nutrition and the Prevention of Chronic Diseases. Genev a: World Health Organization; 2003. 8. Slavin J. Why whole grains are protective: biological mechanisms. Proc Nutr Soc. 2003 Feb;62:129 34. 9. Roitt I, Brostoff J, Male D. Immunology. 4 ed. London: Times Mirror International Publishers Limited; 1996. 10. B onneville M, O'Brien RL, Born WK. Gammadelta T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol. 2010 Jul;10:467 78. 11. Agriculture USDo. Report of the Dietary Guidelines Advisory Committee on the Dietary Gui delines for Americans; 2010. 12. Anson NM, Selinheimo E, Havenaar R, Aura AM, Mattila I, Lehtinen P, Bast A, Poutanen K, Haenen GR. Bioprocessing of wheat bran improves in vitro bioaccessibility and colonic metabolism of phenolic compounds. J Agric Food Ch em. 2009 Jul 22;57:6148 55.

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59 13. Naczk M, Shahidi F. Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J Pharm Biomed Anal. 2006 Aug 28;41:1523 42. 14. Poquet L, Clifford MN, Williamson G. Transport and metabolism of ferulic acid through the colonic epithelium. Drug Metab Dispos. 2008;36:190 7. 15. Mateo Anson N, van den Berg R, Havenaar R, Bast A, Haenen GR. Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.). J Agric Food Chem. 2008 Jul 23;56:5589 94. 16. Liu R, Adom K. Whole Grain Phytochemicals and Antioxidant Activity. In: Marquart L, Jacobs D, McIntosh G, Poutanen K, Reicks M, editors. Whole Grains and Health. Ames, Iowa, USA: Blackwell Publishing Professional; 2007. p. 185 208. 17. Adom KK, Sorrells ME, Liu RH. Phytochemicals and antioxidant activity of milled fractions of different wheat varieties. J Agric Food Chem. 2005 Mar 23;53:2297 306. 18. Adom KK, Liu RH. Antioxidant activity of grains. J Agric Food Chem. 2002 Oct 9; 50:6182 7. 19. Berger A, German JB, Chiang BL, Ansari AA, Keen CL, Fletcher MP, Gershwin ME. Influence of feeding unsaturated fats on growth and immune status of mice. J Nutr. 1993 Feb;123:225 33. 20. Fischer S, Scheffler A, Kabelitz D. Activation of human gamma delta T cells by heat treated mistletoe plant extracts. Immunol Lett. 1996 Sep;52:69 72. 21. Nantz MP, Rowe CA, Nieves C, Jr., Percival SS. Immunity and antioxidant capacity in humans is enhanced by consumption of a dried, encapsulated fruit and veg etable juice concentrate. J Nutr. United States; 2006. p. 2606 10. 22. Holderness J, Jackiw L, Kimmel E, Kerns H, Radke M, Hedges JF, Petrie C, McCurley P, Glee PM, et al. Select plant tannins induce IL 2Ralpha up regulation and augment cell division in g ammadelta T cells. J Immunol. 2007 Nov 15;179:6468 78. 23. Morita CT, Jin C, Sarikonda G, Wang H. Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev. Denmark; 2007. p. 59 76.

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60 24. Rowe CA, Nantz MP, Bukowski JF, Percival SS. Specific formulation of Camellia sinensis prevents cold and flu symptoms and enhances gamma,delta T cell function: a randomized, doub le blind, placebo controlled study. J Am Coll Nutr. United States; 2007. p. 445 52. 25. Ramiro Puig E, Perez Cano FJ, Ramos Romero S, Perez Berezo T, Castellote C, Permanyer J, Franch A, Izquierdo Pulido M, Castell M. Intestinal immune system of young rat s influenced by cocoa enriched diet. J Nutr Biochem. United States; 2008. p. 555 65. 26. Rowe CA, Nantz MP, Nieves C, Jr., West RL, Percival SS. Regular consumption of concord grape juice benefits human immunity. J Med Food. 2011 Jan Feb;14:69 78. 27. Com pany CB. Wheat Bran. [cited 2011 January, 12]; Available from: 28. Jutila MA, Holderness J, Graff JC, Hedges JF. Antigen independent priming: a transitional response of bovine gammadelta T cells to infection. Anim Health Res Rev. 2008 Jun;9:47 57. 29. M eresse B, Cerf Bensussan N. Innate T cell responses in human gut. Semin Immunol. 2009 Jun;21:121 9. 30. Wu Y, Wu W, Wong WM, Ward E, Thrasher AJ, Goldblatt D, Osman M, Digard P, Canaday DH, Gustafsson K. Human gamma delta T cells: a lymphoid lineage cell c apable of professional phagocytosis. J Immunol. 2009 Nov 1;183:5622 9. 31. Beetz S, Wesch D, Marischen L, Welte S, Oberg HH, Kabelitz D. Innate immune functions of human gammadelta T cells. Immunobiology. 2008;213:173 82. 32. Casetti R, Martino A. The plas ticity of gamma delta T cells: innate immunity, antigen presentation and new immunotherapy. Cell Mol Immunol. 2008 Jun;5:161 70. 33. Hara T, Mizuno Y, Takaki K, Takada H, Akeda H, Aoki T, Nagata M, Ueda K, Matsuzaki G, et al. Predominant activation and exp ansion of V gamma 9 bearing gamma delta T cells in vivo as well as in vitro in Salmonella infection. J Clin Invest. 1992 Jul;90:204 10. 34. Jouen Beades F, Paris E, Dieulois C, Lemeland JF, Barre Dezelus V, Marret S, Humbert G, Leroy J, Tron F. In vivo and in vitro activation and expansion of gammadelta T cells during Listeria monocytogenes infection in humans. Infect Immun. 1997 Oct;65:4267 72.

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61 35. De Libero G, Casorati G, Giachino C, Carbonara C, Migone N, Matzinger P, Lanzavecchia A. Selection by two pow erful antigens may account for the presence of the major population of human peripheral gamma/delta T cells. J Exp Med. 1991 Jun 1;173:1311 22. 36. Hedges JF, Lubick KJ, Jutila MA. Gamma delta T cells respond directly to pathogen associated molecular patte rns. J Immunol. 2005 May 15;174:6045 53. 37. Hilgers AR, Conradi RA, Burton PS. Caco 2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm Res. 1990 Sep;7:902 10. 38. Janicke B, Onning G, Oredsson SM. Differential effects of f erulic acid and p coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco 2. J Agric Food Chem. 2005 Aug 24;53:6658 65. 39. Kern SM, Bennett RN, Needs PW, Mellon FA, Kroon PA, Garcia Conesa MT. Characterization of me tabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco 2 cells. J Agric Food Chem. 2003 Dec 31;51:7884 91. 40. Konishi Y, Shimizu M. Transepithelial Transport of Ferulic Acid by Monocarboxylic Acid Transporter in Cac o 2 Cell Monolayers. Bioscience, Biotechnology, and Biochemistry. 2003;67:856 62. 41. Bourne LC, Rice Evans C. Bioavailability of ferulic acid. Biochem Biophys Res Commun. United States: 1998 Academic Press.; 1998. p. 222 7. 42. Laparra JM, Glahn RP, Mill er DD. Bioaccessibility of phenols in common beans ( Phaseolus vulgaris L.) and iron (Fe) availability to Caco 2 cells. J Agric Food Chem. 2008 Nov 26;56:10999 1005. 43. Clifford MN. Chlorogenic acids and other cinnamates nature, occurrence, dietary burd en, absorption and metabolism. J Sci Food Agric. 2000;80:1033 45. 44. Hubatsch I, Ragnarsson EGE, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco 2 monolayers. Nat Protocols. 2007;2:2111 9.

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62 BIOGRAPHICAL SKETCH Suzanna Perry Bonard was born in Palm Beach Garde ns, Florida. Suzanna grew up in Martin County Florida and attended Suncoast Community High School in Riviera Beach, Florida. She pursued a Bachelor of Science in food science and human nutrition with an emphasis on nutritional sciences at the University of Florida after high school and received her degree in 2008. In the fall of 2009, Suzanna enrolled as a working toward her degree, she studied under Dr. Susan S. Percival. Suzanna graduated with her Master of Science in spring of 2011. In her free time, Suzanna enjoys spending time with friends and family and reading. After graduation, she intends to pursue a Doctor of Dental Surgery degree