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Cranberry Polyphenols Down-Regulate the Toll-Like Receptor 4 Pathway and Nuclear Factor-Kappa B Activation, While Still ...

Permanent Link: http://ufdc.ufl.edu/UFE0041362/00001

Material Information

Title: Cranberry Polyphenols Down-Regulate the Toll-Like Receptor 4 Pathway and Nuclear Factor-Kappa B Activation, While Still Enhancing Tumor Necrosis Factor Alpha Secretion in HL-60 Cells
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Muller, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: associated, cranberry, health, hl60, ikappab, immunity, interleukin, kinase, like, lipopolysaccharide, neutrophil, plant, polyphenol, proanthocyanidin, receptor, thesis, tlr, toll
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: Plant polyphenols have been studied extensively for their effects on immune responses. Tea and grape polyphenols have received a great deal of attention in this area of study, while the cranberry, known for its uncommon proanthocyanidin linkages and high antioxidant activity, has been given little of this attention. In this study, six cranberry polyphenol fractions were examined for antioxidant activity, their ability to increase total inhibitor of kappa B alpha (IkappaB alpha), decrease phosphorylated IkappaB alpha (pIkappaB alpha) and decrease interleukin-1 receptor-associated kinase (IRAK) 4, a signaling protein in the toll-like receptor (TLR) pathway, protein expression and to decrease tumor necrosis factor alpha (TNF alpha) secretion in a lipopolysaccharide (LPS)-stimulated neutrophil model. The six fractions included proanthocyanidins, anthocyanins, and other polyphenols from the presscake, as well as the presscake concentrate and proanthocyanidins and a mixture of polyphenols from the juice. HL-60 cells were used to study differentiation and were also differentiated with all-trans retinoic acid for use as a neutrophil model. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays were used to measure antioxidant activity. Differentiation was assessed via the reduction of cytochrome C by superoxide anion production in response to phorbol myristate acetate. Protein levels of total and pIkappaB alpha and IRAK4 were determined using the Western blot technique and an enzyme-linked immunosorbent assay (ELISA) was run to measure TNF? secretion. The antioxidant activity assays showed that the proanthocyanidin-rich presscake and juice fractions and the mixed-polyphenol-enriched juice fraction had the highest activity. These three fractions were used in the protein experiments. None of the fractions caused differentiation in the HL-60 cells at 75 microgram/mL. In the neutrophil like cells treated with LPS, the juice and presscake proanthocyanidin-rich fractions significantly reduced the amount of pI?B? protein present, while the presscake proanthocyanidin-treated cells showed reduced IRAK4 levels compared to untreated cells stimulated with LPS. The proanthocyanidin-rich fractions from the presscake and the juice also significantly increased total I?B? protein levels compared to untreated, LPS-stimulated cells. Interestingly, TNF alpha secretion was approximately 10 times higher in fraction-treated cells compared to the LPS control cells. These results suggest that the proanthocyanidin-rich cranberry fractions can prevent the extensive inflammatory response mediated by the release of nuclear factor kappa B (NF-kappaB) from IkappaB. One way these polyphenols accomplish this is likely through interfering with the TLR4 pathway as evidenced by the reduced IRAK4 expression. However, the increase in TNF alpha suggests that the polyphenol-rich fractions studied may also up-regulate additional, possibly more targeted, inflammatory pathways to aid in coping with the pathogenic onslaught.
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 Catherine Muller.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041362:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041362/00001

Material Information

Title: Cranberry Polyphenols Down-Regulate the Toll-Like Receptor 4 Pathway and Nuclear Factor-Kappa B Activation, While Still Enhancing Tumor Necrosis Factor Alpha Secretion in HL-60 Cells
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Muller, Catherine
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: associated, cranberry, health, hl60, ikappab, immunity, interleukin, kinase, like, lipopolysaccharide, neutrophil, plant, polyphenol, proanthocyanidin, receptor, thesis, tlr, toll
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: Plant polyphenols have been studied extensively for their effects on immune responses. Tea and grape polyphenols have received a great deal of attention in this area of study, while the cranberry, known for its uncommon proanthocyanidin linkages and high antioxidant activity, has been given little of this attention. In this study, six cranberry polyphenol fractions were examined for antioxidant activity, their ability to increase total inhibitor of kappa B alpha (IkappaB alpha), decrease phosphorylated IkappaB alpha (pIkappaB alpha) and decrease interleukin-1 receptor-associated kinase (IRAK) 4, a signaling protein in the toll-like receptor (TLR) pathway, protein expression and to decrease tumor necrosis factor alpha (TNF alpha) secretion in a lipopolysaccharide (LPS)-stimulated neutrophil model. The six fractions included proanthocyanidins, anthocyanins, and other polyphenols from the presscake, as well as the presscake concentrate and proanthocyanidins and a mixture of polyphenols from the juice. HL-60 cells were used to study differentiation and were also differentiated with all-trans retinoic acid for use as a neutrophil model. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays were used to measure antioxidant activity. Differentiation was assessed via the reduction of cytochrome C by superoxide anion production in response to phorbol myristate acetate. Protein levels of total and pIkappaB alpha and IRAK4 were determined using the Western blot technique and an enzyme-linked immunosorbent assay (ELISA) was run to measure TNF? secretion. The antioxidant activity assays showed that the proanthocyanidin-rich presscake and juice fractions and the mixed-polyphenol-enriched juice fraction had the highest activity. These three fractions were used in the protein experiments. None of the fractions caused differentiation in the HL-60 cells at 75 microgram/mL. In the neutrophil like cells treated with LPS, the juice and presscake proanthocyanidin-rich fractions significantly reduced the amount of pI?B? protein present, while the presscake proanthocyanidin-treated cells showed reduced IRAK4 levels compared to untreated cells stimulated with LPS. The proanthocyanidin-rich fractions from the presscake and the juice also significantly increased total I?B? protein levels compared to untreated, LPS-stimulated cells. Interestingly, TNF alpha secretion was approximately 10 times higher in fraction-treated cells compared to the LPS control cells. These results suggest that the proanthocyanidin-rich cranberry fractions can prevent the extensive inflammatory response mediated by the release of nuclear factor kappa B (NF-kappaB) from IkappaB. One way these polyphenols accomplish this is likely through interfering with the TLR4 pathway as evidenced by the reduced IRAK4 expression. However, the increase in TNF alpha suggests that the polyphenol-rich fractions studied may also up-regulate additional, possibly more targeted, inflammatory pathways to aid in coping with the pathogenic onslaught.
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 Catherine Muller.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Percival, Susan S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041362:00001


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1 CRANBERRY POLYPHENOLS DOWNREGULATE THE TOLL LIKE RECEPTOR 4 PATHWAY AND NUCLEAR FACTORKAPPA B ACTIVATION, WHILE STILL 60 CELLS By CATHERINE E. MULLER A THESIS PRESENT ED 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 2010

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2 2010 Catherine E. Muller

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3 To my parents, Bruce and Marie Muller, and my grandparen ts, John and Madeline Tuite

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4 ACKNOWLEDGMENTS I thank Dr. Susan S. Percival for her endless support and enthusiasm as an advisor I thank Dr. Liwei Gu, Dr. Bobbi LangkampHenken, and Dr. Joseph Larkin III for their time and guidance on my supervisory committee. I thank Mrs. Meri Nantz and Dr. Cheryl Rowe for their willingness to show me the ropes in the lab.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LI ST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRAC T ................................................................................................................... 12 CHAPTER 1 BACKGROUND ...................................................................................................... 14 Introduction ............................................................................................................. 14 The Immune System ............................................................................................... 14 Cells of the Immune System ............................................................................. 14 TollLike Receptors .......................................................................................... 15 Plant Pol yphenols ................................................................................................... 16 Anthocyanins and Proanthocyanidins .............................................................. 16 Anti inflammatory Characteristics of Polyphenols ............................................ 17 Cranberry ................................................................................................................ 23 Cranberry and Periodontal Disease ................................................................. 23 Cranberry and Immunity ................................................................................... 25 HL 60 Cells ............................................................................................................. 27 2 ANTIOXIDANT ACTIVITY OF CRANBERRY POLYPHENOLS, INCLUDING ANTHOCYANINS AND PROANTHOCYANIDINS .................................................. 30 Introduction ............................................................................................................. 30 Materials and Methods ............................................................................................ 31 DPPH ............................................................................................................... 31 ORAC ............................................................................................................... 32 Results .................................................................................................................... 32 DPPH ............................................................................................................... 32 ORAC ............................................................................................................... 33 Discussion .............................................................................................................. 33 3 PROLIFERATION AND DIFFERENTIATION OF HL 60 CELLS AFTER INCUBATION WITH CRANBERRY FRACTIONS .................................................. 37 Introduction ............................................................................................................. 37 Materials and Methods ............................................................................................ 37 HL 60 Cell Culture ............................................................................................ 37

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6 Proliferation Assay ........................................................................................... 38 Respiratory Burst Assay ................................................................................... 38 Results .................................................................................................................... 39 Proliferation ...................................................................................................... 39 Respiratory Burst .............................................................................................. 39 Discussion .............................................................................................................. 40 4 CYTOTOXICITY OF CRANBERRY FRACTIONS AND LIPOPOLYSACCHARIDE ON DIFFERENTIATED HL 60 CELLS .......................... 43 Introduction ............................................................................................................. 43 Materials and Methods ............................................................................................ 43 HL 60 Cell Differentiation ................................................................................. 43 Cytotoxicity ....................................................................................................... 43 Results .................................................................................................................... 44 HL 60 Cell Differentiation ................................................................................. 44 Cytotoxicity ....................................................................................................... 44 Discussion .............................................................................................................. 45 HL 60 Cell Differentiation ................................................................................. 45 Cytotoxicity ....................................................................................................... 45 5 TOTAL AND PHOSPHORYLATED INHIBITOR OF KAPPA B ALPHA AND INTERLEUKIN1 RECEPTOR ASSOCIATED KINASE 4 PROTEIN LEVELS IN HL 60 CELLS AFTER INCUBATION WITH CRANBERRY FRACTIONS AND LIPOPOLYSACCHARIDE ....................................................................................... 47 Introduction ............................................................................................................. 47 Materials and Methods ............................................................................................ 47 Results .................................................................................................................... 50 Discussion .............................................................................................................. 50 6 TUMOR NECROSIS FACTOR ALPHA SECRETION INCREASES WITH INCUBATION IN CRANBERRY FRACTIONS ........................................................ 54 Introduction ............................................................................................................. 54 Materials and Methods ............................................................................................ 54 Results .................................................................................................................... 55 Discussion .............................................................................................................. 56 7 CONCLUSION ........................................................................................................ 60 LIST OF REFERENCES ............................................................................................... 63 BIOGRAPHICAL SKETCH ............................................................................................ 70

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7 LIST OF TABLES Table page 2 1 Cranberry fraction origins and constituents ........................................................ 35 3 1 Cytotoxicity of HL 60 cells in 75 g/mL cranberry fractions ................................ 42

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8 LIST OF FIGURES Figure page 1 1 TLR4 pathway. ................................................................................................... 28 1 2 Basic structure of a flavonoid. ............................................................................. 28 1 3 Proanthocyanidin structure and linkage. ............................................................. 29 2 1 DPPH results for cr anberry Fractions A thr ough F ............................................. 35 2 2 ORAC results for cr anberry Fractions A through F ............................................. 36 3 1 Rate of cytochrome C reduction in HL60 cells treated with 75 g/mL cranberry fractions .............................................................................................. 41 4 1 Rate of cytochrome c reduction in ATRA treated cells versus untreated cells ... 46 4 2 Viabilit y of differentiated HL60 cells treated with various concentrations of six cranberry fractions and LPS. ......................................................................... 46 5 1 ment with cranberry fractions .............................................................................................................. 52 6 1 TNF secretion after 24hour incubation in cranberry fractions and LPS ........... 59

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9 LIST OF ABBREVIATION S g Microgram L Microliter M Micromolar ALT Alanine am inotransferase ANOVA Analysis of variance AST Aspartate aminotransferase ATRA Alltrans retinoic acid CD Cluster designation CT Catechin DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DP B B type dimeric procyanidin oligomers from cocoa DPBS Dulbeccos phosphate buffered saline DPPH 2,2 Diphenyl 1 picrylhydrazyl EC Epicatechin EGCG Epigallocatechin ELISA Enzyme linked immunosorbent assay ERK Extracellular signal related protein kinase GI Gastrointestinal Inhibitor of kappa B Inhibitor of kappa B alpha IFN Interferongamma IgA Immunoglobulin A IgG Immunoglobulin G

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10 IKK Inhibitor of kappa B kinase IL Interleukin IMDM Iscoves Modified Dulbeccos media IRAK4 Interleukin1 receptor associated kinase 4 IT Infected, treated IU Infected, untreated JNK C jun N terminal LBP Lipopolysaccharide binding protein LDH Lactate dehyrogenase LPS Lipopolysaccharide MAP Mitogenactivated protein mL Milliliter mM Millimolar MMP Matrix metalloproteinase MTT 3 (4,5 Dime thylthiazolyl 2) 2,5 diphenyltetrazolium bromide NF Nuclear factor kappa B ng Nanogram NK Natural killer nm Nanometer nM Nanomolar NT Non infected, treated NU Non infected, untreated ORAC Oxygen radical absorbance capacity PBS Phosphate buffered saline pg Picogram

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11 Phosphorylated inhibitor of kappa B PMA Phorbol myristic acid R ANTES Regulated upon activation normal T cell expressed and secreted T cell Thymocyte TLR Tolllike receptor TLR4 Tolllike receptor 4 Tumor necrosis factor alpha

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CRANBERRY POLYPHENOLS DOWNREGULATE THE TOLL LIKE RECEPTOR 4 PATHWAY AND N UCLEAR F ACTOR KAPPA B ACTIVATION, WHILE STILL ENHANCING TUMOR NECROSIS FACTOR SECRETION IN HL 60 CELLS By Catherine E. Muller May 2010 Chair: Susan S. Percival Major: Food Science and Human Nutrition Plant polyphenols have been studied extensively for their effects on immune responses. Tea and grape polyphenols have recei ved a great deal of attention in this area of study, while the cranberry, known for its uncommon proanthocyanidin linkages and high antioxidant activity, has been given little of this attention. In this study, six cranberry polyphenol fract ions were exami ned for antioxidant activity, their ability to increase total inhibitor of kappa B alpha and decrease interleukin1 receptor associated kinase (IRAK) 4, a signaling protein in the toll like receptor (TLR) pathway, protein expression and to decrease tumor necrosis lipopolysaccharide (LPS) stimulated neutrophil model. The six fractions included proanthocyanidins, anthocyanins and other polyphenols from the presscake, as well as the presscake concentrate and proanthocyanidins and a mixture of polyphenols from the jui ce. HL 60 cells were used to study differentiation and were also differentiated with all trans retinoic acid for use as a neutrophil model. The 2,2diphenyl 1 picrylhydrazyl (DPPH) and oxygen radical absorbance capacity

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13 (ORAC) assays were used to measure antioxidant activity Differentiation was assessed via the reduction of cytochrome C by superoxide anion production in and IRAK4 were determined using the Western blot technique an d an enzyme linked immunosorbent assay ( ELISA) The antioxidant activity assays s howed that the proanthocyanidinrich presscake and juice fractions and the mixed polyphenol enriched juice fraction had the highest activity. These three fractions were used in the protein experiments. None of the fractions caused differentiation in the HL60 cells at 75 g/mL. In the neutrophil like cells treated with LPS, the juice and presscake proanthocyanidinrich fractions significantly reduced the amount of p protein present, while the presscake proanthocyanidintreated cells showed reduced IRAK4 levels compared to untreated cells stimulated with LPS. The proanthocyanidinrich fractions from the presscake and the juice also significantly increased total I protein levels compared to untreated, LPSin fractiontreated cells compared to the LPS control cells. These results suggest that the proanthocyanidinrich cranberry fra ctions can prevent the extensive inflammatory response mediated by the release of nuclear factor kappa B ( NF ) through interfering with the TLR4 pathway as evidenced by the reduced IRAK4 expr ession. However suggests that the polyphenol rich fractions studied may also upregulate additional, possibly more targeted, inflammatory pathways to aid in coping with the pathogenic onslaught.

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14 CHAPTER 1 BACKGROUND Introduction Pla nts and their chemical constituents have enjoyed a great deal of attention for the health benefits they may impart. The c ranberry Vaccinium macrocarpon is no exception and has a rich history in research and in folk medicine. However, cranberrys impact on immunity has been largely overlooked in the laboratory. Due to the expanse of research on plant polyphenols, and the scarcity of work on cranberrys impact on immunity, a project to shed more light on this area was carried out. Based on the literature, it was hypothesized that polyphenol rich cranberry fractions which included proanthocyanidins, would be effective in encouraging differentiation of immature immune cells and that they would i nhibit the toll like receptor (TLR ) 4 and nuclear factor kappa B ( NF B ) inflammatory pathways and reduce inflammatory cytokine secretion in response to lipopolysaccharide ( LPS ) activation. The Immune System The immune system is the guardian of health. It is comprised of various cells througho ut the body that interact via chemical messengers, like cytokines and chemokines. Generally, these messengers affect immune cells by binding to receptors on the cell surface and inducing a signaling cascade which results in some effect on the cell, such as production of its own cytoki nes Specific immune cells and receptors are discussed in the sections below. Cells of the Immune System The immune system is composed of several different types of cells that function in innate or acquired immunity. The cells of the innate immune system include

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15 neutrophils, macrophages, basophils, mast cells, eosinophils, and natural killer (NK) cells. Innate immunity is a quick acting defense against a broad range of invaders, while acquired immunity offers a slower, more specific response. Innate imm une cells can migrate to the site of infection this action is called chemotaxis where they may use phagocytosis and respiratory burst to kill pathogens. Cytokines secreted by other immune cells prompt chemotaxis to the site of infection. This immune resp onse may cause local inflammation, but this is generally mild and brief. However, if there is chronic inflammation due to continual immune activation, it can negatively impact health. Heart disease is one of many diseases that is, at least in part, caused by inflammation. A review by Libby and Theroux [1] of the role of inflammation in heart disease can be consulted for more detailed information. Toll L ike Receptors To begin the immune response, a component of the bacterium or other foreign body must be recognized as nonself. Recognition of pathogens can occur through cellular receptors, like TLRs. TLRs are cell surface receptors that ultimately cause NF in 1997 by Medzhitov et al. [2 ]. They wer e named for their homology to the Toll receptors first found in Drosophila In Drosophila Toll mediates embryo development and immunity. In mammals, TLRs are thought to be related only to immunity. There are many proteins that aid TLR in transducing it s signal once an antigen has been presented to the receptor and a few of the key signaling proteins are shown in Figure 11 A detailed explanation of the pathway can be found in the review written by ONeill [ 3 ]. Ultimately, the pathway leads to the inh being phosphorylated, which causes the

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16 as a transcription factor for numerous genes. Some of the types of genes it encourages the transcription of include cytokines, acute phase proteins, cellular adhesion molecules, stress response proteins, growth factors, other transcription factors, enzymes, and cell surface receptors [ 4 ] Plant Polyphenols Polyphenols are a diverse group of compounds comm only found in plants. They include phenolics acids, benzoquinones, acetophenones, phenylpropenes, stilbenes, lignins, and flavonoids among other s [5 ]. Flavonoids have a threeringed structure pictured in Figure 12 Most commonly, they have a glucose, g alactose, rhamnose, xylose, or arabinose moiety attached to their main structure [ 6 ]. Flavonoids can also be broken down into classes based on their structure and are widespread in plants [ 5 ]. Two classes of flavonoids include anthocyanins and proanthocy anidins Anthocyanins and Proanthocyanidins Anthocyanins are responsible for red, blue, and p urple pigments in plants [6]. Proanthocyanidins are oligomers and polymers of flavan3 ols and generally have high molecular weights [ 5 7 ]. Catechin (CT) and e picatechin (EC) are the most common monomers of proanthocyanidins [ 7 ]. The monomers are linked either with one bond, the B type linkage, or with two bonds, the A type linkage, illustrated in Figure 1 3 A type linkages are relatively uncommon and have bee n characterized only in cranberries, avocado, plum, curry, cinnamon, and peanuts so far [ 8 ]. A type linkages have been shown to have anti viral effects against the human immunodeficiency virus and herpes simplex virus [ 9 ] and may be useful in preventing t he bacterial adhesi on of urinary tract infections [10,11]. The B type linkages of proanthocyanidins are ubiquitous in nature.

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17 Anti inflammatory Characteristics of Polyphenols A great deal of research has been devoted to the study of polyphenols from vario us plants. There is a large body of evidence for their efficacy to modulate the inflammatory process both in vitro and in vivo animal studies. This section includes a sampling of the literature that specifically involves plant polyphenols and their effec ts on inflammation. Several groups have looked at polyphenols and their anti inflammatory nature in in vitro situations [12 15] One set of experiments focused on 23 polyphenol compounds from various sources, which included several anthocyanin extracts [ 1 2 ] Researchers found that the polyphenols reduced nitric oxide production after RAW 264.7 macrophages were stimulated with LPS and interferongamma ( IFN ) Tumor necrosis factor alpha ( ) levels were increased by some polyphenols and decreased by A second study looked at KU812 cells, a cancerous cell line differentiated to mast cells, and pomegr anate polyphenols [ 13 ]. Cellular mRNA and interleukin ( IL ) 6 and IL8 levels decreased after the cells were incubated with phorbol myristic acid ( PMA ) and then treated with pomegranate extract high in polyphenols, especially anthocyanins. The pomegranate extact also inhibited the phosphorylation of cjun N terminal ( JNK ) and extracellular signal related protein kinase ( ERK ) the degradation of I B and the movement of NF B into the nucleus. Interestingly, another group [14 ] showed that MOLT 4 thymocytes ( T cell) precursor cells treated with quercetin and ellagic acid activated the mitogenactivated protein (MAP) kinases JNK1, JNK 2 and p38. M ackenzie et al. studied EC CT and B type dimeric procyanidin (DP B) and their effect on T cell NF B activation in response to PMA sti mulation [15]. It was determined that all three test

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18 flavonoids were transported into the cell and that EC and DP B wer e also found in the nucleus of Jurkat T cells The three test compounds significantly inhibited the binding of NF B to deoxyribonucleic acid ( DNA ) and the secretion of IL2 compared to the PMA control. I B phosphorylation was decreased in all three flavonoids compared to the PMA control and the inhibitor of kappa B kinase beta ( IKK ) was decreased in EC and DP B samples. There was a trend toward decreasing IKK phosphorylation in CT, but it was not significant due to a high standard error. Cellular ox idants decreased compared to the PMA control in cells incubated with EC, CT, and DP B. The authors also showed that the flavonoids could selectively inhibit the binding of NF B to DNA without interfering with the binding of other transcription factors l ike octamer transcription factor 1 or cyclic adenosine monophosphate response element binding protein. Polyphenol rich fractions of several cereals were examined for their effect on immune response markers in a study by lvarez et al [16 ].Female ICR mice were f ed a control diet of standard rodent diet or a diet of 80% standard diet and 20% wheat germ, buckwheat flour, fine rice bran, or wheat middlings fractions ad libitum for 5 weeks The cereal fractions were mixed with water and formed into biscuits. Control mice were also fed biscuits made from standard diet which were prepared in a similar fashion as the cereal biscuits. In mice fed the cereal enriched diet, lymphocyte chemotaxis and proliferation in response to Concanavalin A were increased beyond that of the normal chow control mice. Lymphocyte proliferation in response to LPS stimulation was higher in all cereal enriched diets except the wheat germ fed mice in comparison to the controls. IL 2 secretion was increased after stimulation in all treatment animals except those fed fine rice bran compared to the controls. TNF was significantly decreased

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19 after stimulation with LPS in all the the treatment fed mice, except those on the wheat germ diet. The oxidized glutathione and malondialdehyde leve ls in mice fed the treatment diets were lower than those of the control mice, while reduced glutathione levels did not change. Catalase activity in leukocytes was increased in the treatment fed mice, except for the wheat germ fed mice. This study demonst rated that polyphenols from cereals can also modulate immunity by influencing the immune responses, cytokines, and redox state of immune cells While th e data reported here are interesting there are some concerns about the comparison of the treatment diets to the control diets. Twen ty percent of the control diet (the biscuits made with the standard rodent diet ) had 12 vitamins and minerals that were not found in the treatment diets cereal containing biscuits. The vitamins and minerals that were present in the treatment biscuits were also at very different concentrations compared to the control biscuits. The cereal containing biscuits also had 1.5 to 14 times less carbohydrates and contained 75% to nearly 220% of the protein of the standard chow biscuit s. It would be most correct t o attribute the findings of this research to the cereal fractions rather than just the cereal polyphenols, since the control and treatment diets have some important differences. Another article reported the effects of curcumin, a spice polyphenol, on the formation of granulomas in a parasite infection in male CD1 albino mice [ 17] Mice infected with Schistosoma mansoni were treated with 400 mg/kg body weight of curcumin. The curcumin was partitioned into 16 injections, given twice a week for 8 weeks. After 8 weeks, worm and egg burdens were determine d as were number of leukocytes, hepatic granuloma size, hepatic enzyme acti vities, cytokine levels liver

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20 fibrosis level and antibodies against the parasite. The researchers found that the body weights of the infected, treated (IT) mice were similar to noninfected, untreated controls (NU) and noninfected, treated (NT) controls and that the infected, untreated (IU) mice had a significantly lower body weight compared to the other s. Liver and spleen weights of the IT group were lower than the IU group, but still higher than either NU or NT controls. The overall white blood cell counts were similar across all groups. Monocytes saw no change across groups, but neutrophils were muc h higher in both treatment groups compared to the controls. Eosinophils were higher in the IU group compared to the NU and NT controls and the IT mice. Lymphocytes were lowest in the IU mice and slightly higher in the IT mice compared to both control groups. The IT mice had 44% fewer parasites, on average, compared to the IU group. They also had nearly 31% fewer eggs per gram of liver and intestinal tissue compared to their untreated counterparts. Hepatic liver granuloma volume and hepati c collagen wer e decreased by 79% and 38.6%, respectively, the IT mice versus the IU mice. Alanine aminotransferase (ALT) activity was lower in the IT mice compared to the NU controls, but similar to the NT control. IU ALT activity was lower than the IT mice. Aspartat e aminotransferase (AST) activity in the IT group was similar to the NU controls, but lower than the NT controls. AST activity was further depressed in the IU mice compared to the IT mice. Gammaglutamyl transferase activity in the IT group was similar t o both controls. It was lowered in the IU group. Lactate dehydrogenase (LDH) activity was similar between the NU controls and the IT mice. However, the IT mice LDH activity was decreased compared to the NT controls. IU and IT mice had similar LDH activ ity, but the activity of the IU mice was still lower than both controls. Catalase activity was

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21 high in the IT group compared to all other groups. Serum IL 10 was high in both treatment groups compared to the controls, while IL12 was only high in the IU mice. TNF compared to controls. IL4 was not detected in the sera of any mice. Immunoglobulin G ( IgG ) and IgG1 levels against soluble worm antigen and soluble egg antigen were increas ed in the IT group compared to the IU group. IgG2a and immunogloblin M lev e ls against both antigens were similar in both treatment groups. This research illustrated that curcumin, a polyphenolic compound, can reduce the damage of an inflammatory response to a parasitic infestation, but can also aid in enhancing the efficacy of the immune response by reducing the parasitic load possibly due to the increased antibody levels. A search of the PubMed database using the terms polyphenols and immun provided only one human study result In this study [ 18 ] 7 male and 9 female participants between 20 and 22 years of age were placed on low polyphenol diets or a diet containing 200 g of purple sweet potato leaves per day. All participants were given a control low polyphenol diet for one week before alternating with two weeks of the low polyphenol diet again or the treatment diet containing purple sweet potato leaves. Both diets had similar carotene levels. Between the control diet and the treatment diet, participants ate the control diet for one week as a washout period. The urine polyphenol content of participants was measured after each intervention diet. Plasma carotene lymphocyt e proliferation, cytokine levels, NK cell activity, and salivary immunoglobulin A ( IgA ) were measured as well. Plasma carotene increased in both diets compared to baseline measurements, while plasma polyphenol levels decreased

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22 in the control diet and remained the same as baseline in the purple sweet potato leaves diet. However, urinary polyphenol levels were decreased in the control diet and increased in the treatment diet. Lymphocyte proliferation after s timulation with Concanavalin A was decreased co mpared to the baseline while on the control diet, but increased during the second week on the treatment diet. The levels of IL 2 and IL 4 secreted by periph eral blood mononuclear cells were increased only in the treatment diet compared to baseline. NK ce ll activity was decreased in the second week of the control diet and increased in the second week of the treatment diet compared to baseline. Salivary IgA decreased in the control group, but normalized on the treatment diet. The results are intriguing e specially because it is a human study. However, it had a small number of participants and certainly needs validation. It could also benefit from the measurement of different immune markers and the use of a purified polyphenol supplement instead of ingest ion of whole plant parts. The changes seen in the treatment group may be due to the polyphenols of the p urple sweet potato leaf, but they also could be due to other compounds in the leaves. Another human study looks at immunity in more detail, but has a m ore general focus on phytochemicals in fruits and vegetables instead of only polyphenols [ 19 ]. In this study law students were given a commercially available fruit and vegetable concentrate supplement for 11 weeks. T cell counts, cytokine production, lym phocyte DNA damage, blood antioxidant, vitamin C and carotenoid levels and reported illnesses were assessed. The number of T cells in the blood increased and lymphocyte DNA damage decreased in individuals taking the supplement compared to those taking the placebo. Vitamin C and carotenoid levels and oxygen radical absorbance capacity

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23 (ORAC) of the blood also increased compared to baseline. IFN levels decreased in stimulated cells in those taking the supplement compared to the placebo, while other cy tokines measured did not change between the two treatment groups. Overall, these results show that compounds in fruits and vegetables administered in capsule form can increase important immune parameters while still encouraging an anti inflammatory state. However, in this study, these results cannot be solely attributed to polyphenols. Cranberry Cranberries and their effect on urinary tract infections have been studied extensively [ 2025]. Theyve also been implicated in variations in cholesterol metabol ism [ 26,27 ] and as anti proliferative agents of various human cancer cell lines [ 28,29]. Cranberry and Periodontal Disease An emerging area of cranberry research focuses on how cranberry may affect the immune system. Most research in this field deals with the anti inflammatory capabilities of cranberry and its components in relation to gingival fibroblasts and macrophages [ 3033 ]. In 2006, Bodet et al. [ 33 ] published the first research to examine the anti inflammatory role of cranberry proanthocyanidins on immune cells stimulated with an inflammationinducing compound, like LPS. The researchers used the U937 leukemia cell line as a model of macrophage immune cells. The cells were differentiated to macrophagelike cells using PMA incubated with 10 to 5 0 g/mL of a proanthocyanidinrich cranberry fraction, lyophilized juice fraction, or epigallocatechin (EGCG) for 2 hours and then incubated with LPS for 24 hours before being assayed. Cell viability and the production of IL 6, IL 8, TNF regulated upon activation

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24 normal T cell expressed and secreted ( RANTES ) were measured. No cytotoxic effects were seen in any of the test compounds at the various concentrations used. TNF IL 6 levels were signific antly decreased in the 25 g/mL and 50 g/mL concentration of the proanthocyanidin fraction and in the 10 g/mL concentration of EGCG. Interestingly, IL incubated with 10 g/mL proanthocyanidin fraction. However, at 50 g/mL the ILlevels were similar to EGCG, the positive control. Only the proanthocyanidin fraction significantly decreased the IL8 levels when cells were stimulated with LPS. RANTES was significantly decreased in all t hree of the proanthocyandin concentrations and with the 10 g/mL EGCG. The lyophilized cranberry juice fraction had no significant effects on any of the cytokines that were tested. Another study from this same group [ 34] also examined the ability of cranberry proanthocyanidins to reduce the negative effects of bacterial LPS on gingival fibroblasts. These fibroblasts are sensitive to LPS and mount an inflammatory response to it that can damage healthy tissue when the response becomes chronic. In this study, the fibroblasts incubated with the proanthocyanidins secreted less IL6, IL8, and prostaglandin E2 and had lower cyclooxygenase II expression when the cells were treated with LPS. S eeram et al. [35 ] showed that cranberry and other berry anthocyanins als o inhibited cyclooxygenase II activity. The Bodet group has gone on to show that cranberry proanthocyanidins reduce the production and secretion of matrix metalloproteinase(MMP) 3 and MMP 9 in macrophages, MMP 3 in gingival fibroblasts as well as, reduc ing the activity of these two proteins and elastase in cells stimulated with LPS from periodontal pathogens [ 32]. The proanthocyanidins also decreased the

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25 expression of intracellular signaling molecules that were upregulated in fibroblasts treated with LPS. Expanding on this MMP data, another group of researchers studied how A type proanthocyanidins affect inflammatory markers [ 30]. MMP 1, 3, 7, 8, 9, and 13 levels were shown to significantly to decrease in cells treated with these A type proanthoc yanidins for two hours an d then incubated in LPS for 24 hours compared to cells that had no cranberry treatment. The NF reduced in macrophages treated with the fractions compared to those only having the LPS stimulation. Four protein kinases that were upregulated in the LPS control had significantly reduced levels when treated with the proanthocyanidins from cranberry. A recent study [31 ] showed that cranberry proanthocyanidins had a protective effect on macrophages that were exposed to bacterial cell wall components that normally significantly reduce cell viability. U937 cells were differentiated into macrophagelike cells using PMA A human oral epithelial cell line, GMSM K, was also used in this experiment. C ell w all from was prepared from Peptostreptococcus micros HG1251, a dental pathogen, and used to treat the differentiated U937 cells and oral epithelial cells. Cells were treated with 0, 5, 10, 25, 50 or 100 g/mL of cranberry fraction for 2 hours and then cul tured with the cell wall at 20 g/mL for 24 hours. Cell viability was measured by 3 (4,5 dimethylthiazolyl 2) 2,5 diphenyltetrazolium bromide ( MTT ) reduction A 50 g/mL concentration of the cranberry fraction resulted in a significant reduction of cell death in the U937 and GMSM K cells. Cranberry and Immunity While periodontal disease has been in the forefront of cranberry research in regards to immunity, there are other groups that are taking a more global look at

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26 immunity. This section will discuss the body of research done in relation to cranberry and systemic immunity. Hochman et al. [36 ] show that a nondialyzable fraction of cranberry juice slows in vitro growth and movement across membranes of murine lymphoma cells. This research group also test ed the effect of injected nondialyzable cranberry fractions on mice that had been inoculated with murine lymphoma cells. Eighty percent of the mice that were injected with the lymphoma cells and that did not receive treatment had tumor growth after 60 day s. No mice treated with the cranberry fraction had developed tumors after 100 days post inoculation. Furthermore, these cranberry fractiontreated mice had produced anti lymphoma antibodies Delehanty and colleagues examined [37] cranberry proanthocyani dins and their impact on TLR4 and the NF researchers investigated proanthocyanidins from red grape, cranberry juices and black tea and their ability to bind LPS, alter LPS endocytosis, prevent LPS from binding to TLR4, LPS binding protein (LBP) and cluster designation ( CD) 14, and reduce NF cranberry (dialyzable and nondialyzable), tea, and grape proanthocyanidins all bound LPS. The highest molecular weight cranberry proanthocyanidins bound LPS most effectively. The a verage degree of polymerization for this group of proanthocyanidins was 21. Since the fraction of dialyzable cranberry proanthocyanidins bound LPS the most efficiently, it was used exclusively in the rest of the experiments. These high molecular weight proanthocyanidins were shown to modestly reduce the binding of LPS to the cell surface and significantly reduce LPS endocytosis. However, overall endocytosis was not affected when cells were treated with cranberry proanthocyanidins.

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27 The proanthocyanidins did not inhibit the binding of LPS to LBP, but did inhibit the binding of LPS to CD14 at a concentration of 500 nM. Also at that concentration, proanthocyanidins nearly completely inhibited the interaction of LPS and the TLR4/MD 2 complex. The NF vity was also significantly reduced when cells were incubated with 10 nM high molecular weight proanthocyanidins. Together these studies discussed above suggest that cranberry proanthocyanidins have anti inflammatory properties. Each study used different methods, cell types, and proanthocyanidin concentrations during their research. More evidence is necessary to fully understand and validate these findings. The question of whether other cranberry polyphenols like anthocyanins, may have similar effects is also raised by this research. HL 60 Cells HL 60 cells are a human promyelocytic leukemia cell line. The cells were originally from a 36year old female with acute promyelocytic leukemia [ 38]. Approximately 5% to 10% of cells in culture will spontaneous differentiate, but cells can be pus hed into differentiation toward macrophageor neutrophil like cells using various chemicals. HL 60 cells are frequently used in research as models for both of these cell types [ 3945 ] It is advantageous to use a continuous cell line due to their ability for continuous growth, their relative hardiness, and their replacement of human donor s every time cells are needed. In my study HL 60 cells were differentiated to neutrophil like cells using alltrans retinoic acid ( ATRA) Breitman et al. first discussed the granulocytic differentiation of this cell line in th e presence of ATRA in 1980 [46]. While this discovery first excited medical doctors and cancer researchers, it has become important to the study of immunity due to the widespread use of the HL60 cell line.

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28 Figure11. TLR4 pathway. Figure 12 Basic structure of a flavonoid.

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29 A B Figure 13 Pro anthocyanidin structure and linkage. A) Structure of two monomers of a procyanidin with an A linkage between the 2position of the C ring of one monomer and the hydroxyl group on the 7position of the A ring of the second monomer and a second bond between the 4position of the C ring of one monomer and the 8position of the A ring of the second monomer [7] Th e second bond of the A linkage can also be between the 4position of the C ring of one monomer and the 6position of the A ring of a second monomer. (B) Structure of two monomers of a proanthocyanidin linked with a B linkage between the 4position of the C ring of one monomer and the 8position of the A ring of the second monomer [7]

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30 CHAPTER 2 ANTIOXIDANT ACTIVITY OF CRANBERRY POLYPHE NOLS, INCLUDING ANTHOCYANINS AND PRO ANTHOCYANIDINS Introduction Two methods were used to determine the antioxidant capacit y of six cranberry fractions. These included the 2,2diphenyl 1 picrylhydrazyl (DPPH) and oxygen radical absorbance capacity ( ORAC) methods. Both methods examine the ability to quench different types of radicals and, so, used together they are advantageous in exploring a compounds antioxidant status. DPPH the chemical, is a stable nitrogen free radical [ 47 ]. The DPPH assay measures the efficiency with which another chemical can subdue this radical. DPPH is deep purple in color when dissolved in methanol or ethanol, with the addition of a strong antioxidant, the color of the solution changes from purple to yellow. This change is measured spectrophotometrically and compared to a standard (e.g. ascorbic acid) at known concentrations The procedure below used 6 hydroxy 2,5,7,8 tetramethylchroman2 carboxylic acid, also known as T rolox, for the standard. Trolox is analogous to vitamin E and is water soluble. ORAC is commonly used in industry and research to quantify oxygen radical absorbance capacity. T his assay examines the ability of a compounds to quench a peroxyl radical. It was first reported by Cao and colleagues in 1993 [48]. In this assay, 2,2 a zobis(2amidinopropane) dihydrochloride (AAPH) produces peroxyl radi cals. Fluorescein los es fluoresc ence when it is oxidized by the peroxyl radical. If antioxidants are present they prev ent the oxidation of fluorescein and, therefore, the loss of fluorescence. The change in fluorescence can be measured with a plate reader and compared to a standard cur ve.

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31 Materials and Methods DPPH Six cranberry fractions were fractionated and provided by Ocean Spray Cranberries, Inc. (Lakeville Mass.). Table 21 presents what part of the cranberry each fraction came from and its major constituents. Fractions A C and F are soluble in methanol. They were dissolved in methanol (Fisher Scientific, Fair Lawn, NJ ) and compared with a T rolox standard curve (Fluka, Switzerland) in methanol Fraction B and D are water soluble and these were compared with a T rolox standard c urve in water. Fraction E is soluble in water and methanol. It was dissolved in water and compared with the T rolox standard curve in water alo ng with F ractions B and D. The final concentrations of T rolox were: 40 M, 30 M, 15 M, 5 M, 0.5 M, and 0 M. Fractions were incubated for 30 minutes in the dark at concentrations between 1 and 5 g/mL with a 0.216 mM final concentration of DPPH (Sigma, St. Louis, MO ). Following incubation, fractions along with their standards were plated in quadruplicate in a 96well plate. They were read at 517 nm using a SpectraMax 340PC 384 (Molecular Devices, Sunnyvale, Calif.) plate reader. Samples were compared to the standard curve to determine their T rolox equivalents /mg sample ) DPPH was run 3 times for F r actions A C D, E, and F and twice for B (due to the limited quantity of fraction B available). A one way analysis of variance ( ANOVA) (SigmaStat Version 9 Systat, San Jose, CA) was used to test for significant differences among the means of the log10 t ransformed data due to a failed normality test and an unequal variance. To test for which means differed, a Tukeys post hoc all pairwise analysis was used.

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3 2 ORAC Fractions A, B, C D, E, and F were dissolved in an acetone/water/acetic acid (Fisher Scienti fic) (70%: 29.5%:0.5%, v/v/v) extraction solvent at a concentration of 1 mg of fraction per mL extraction solvent. The samples were sonicated using a Microson sonicator (Misonix, Farmingdale, NY) for 3 short bursts of 5 seconds and all fra ctions went into solution. The final dilutions of each fraction tested were between 500 and 1700 ng/mL The samples were compared to a T rolox standard curve using the fol lowing final concentrations of T rolox to make the standard curve: 3.125 0.391 in a black Costar plate with a clear, flat bottom (Corning, Corning, NY) with 22.3 nM concentration of fluorescein (Sigma) and allowed to mix and incubate for 10 minutes at 37C in a SpectraMax Gemini XPS (Molecular Devices) fluorescent plate reader. Immediately afte r the incubation, AAPH (Wako, Richmond, VA) was added to each well, except for the control wells, at a final concentration of 19 mM and the plate was read. The plate reader was set at 485 nm for excitation and 530 nm for emission with 76 readings taken in 40 minutes. Three independent experiments were conducted. A one way ANOVA was used to test for significant differences among the means of the log10Results transform ed data due to a failed normality test and an unequal variance. To test for which means differed, a Tukeys post hoc all pairwise analysis was used. DPPH Fractions A and F showed the highest antioxidant capacity in the DPPH assay, which examines an electron transfer reac tion involving DPPH [49] Fraction D had the

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33 lowest antioxidant activity of all fractions tested. The P value for the difference between F ractio ns A and E is 0.038, while the P value for the difference between F ractions B and E is 0.001. All other significant differences have a P value of less than 0.001. The letters above the bars in Figure 2 1 denote significance. Fractions with the same letter are not statistically different from one another. ORAC In the ORAC assay, which inv olves a hydrogen transfer as part of the radical quenching [ 49 ] F ractions E and F showed the greatest antioxidant activity. Fraction B showed the lowest amount of activity. All differences between the fractions had a P value less than 0.001. The letter s above the bars in Figure 22 denote significance. Fractions with the same letter are not statistically different from one another. Disc u ssion Fractions A and F had the greatest ability to quench the nitrogen radical in the DPPH assay, while F ractions E and F were best at scavenging the peroxyl radical s in ORAC. Fractions A and F are both enriched in cranberry proanthocyanidins. However, Fraction A contains proanthocyanidins from the presscake, while Fraction Fs are from the juice. These two fractions have different DPPH and ORAC results, likely because the overall characteristics of the proanthocyanidins in these two components are different. The characteristics and amounts of polyphenols, in general, can vary between plants, growing seasons, and in response to environmental conditions. They also vary between different parts of the same plant. Unpublished research performed in Dr. Susan Percivals lab using peripheral blood mononuclear cells have suggested that the cranberry polyphenol fractions that have the most antioxidant activity tended to have more activity in cell culture

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34 experiments. The findings from these previous experiments were used to select F ractions A, E, and F for further study.

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35 Figure 21. DPPH results for cranberry F ractions A through F. The bars represent the mean mol Trolox equivalents per milligram sample and are shown with their standard deviation for 3 independent experiments (2 independent experiments for F raction B) for each fraction. Bars with the same letter above are not statistically different from one another. Table 21. Cranberry fraction origins and constituent s Fraction Major Component Portion of cranberry that was fractionated A Proanthocyanidins Presscake B Anthocyandins Presscake C Other phenolics Presscake D Whole presscake Presscake E Polyphenols Juice F Proanthocyandins Juice

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36 Figure 22 ORAC results for cranberry F ractions A through F Th e bars represent the mean mol T rolox equivalents per milligram sample and are shown with their standard deviation for 3 independent experiments for each fraction. Bars with the same letter above are not statistically different from one another.

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37 CHAPTER 3 PROLIFERATION AND DI FFERENTIATION OF HL 60 CELLS AFTER INCUB ATION WITH CRANBERRY FRACT IONS Introduction It was necessary to determine the highest concentration of each cranberry fraction that affected the cells without causing cell death. In metabolically active cells, cells h aving functional mitochondria, 3 (4,5 Dimethylthiazolyl2) 2,5 diphenyltetrazoli um bromide ( MTT ) is taken up and reduced [50 ]. The metabolism of MTT changes the color of the chemical from yellow to dark purple. This color change can be measured by a spectrophotometer to determine the extent of cell death. This assay does not distin guish between the routes of cell death (i.e. apoptosis versus necrosis), but does offer a quick way to compare cellular growth between treatments. To determine if differentiation has occurred in cells treated with cranberry fractions the production of superoxide anion in response to a stimulus was measured by the respiratory burst assay. Superoxide anion is usually produced by mature immune cells in response to a pathogen or stimulus, like phorbol myristic acid ( PMA ) Superoxide anion reduces cytochrome C and this can be measured spectrophotometrically [ 51] Immature or undifferentiated immune cells, like promyelocytes, do not secrete superoxide anion in response to a pathogen or stimuli. This kind of killing activity is one of the hallmarks of an immune response in mature immune cells. Materials and Methods HL 60 Cell Culture HL 60 cells (American Type Culture Collection, Manassas, VA) were grown in Iscoves Modified Dulbeccos media (IMDM) with L glutamine and HEPES (Lonza, Walkersville, MD) and 10% fet al bovine serum (Cellgro, Mediatech, Inc., Herndon, VA),

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38 100 IU/mL p enicillin (Cellgro), 100 g/mL s treptomycin (Cellgro), 0.25 g/mL a mphotericin B (Cellgro), and 50 g/mL gentamicin (Gibco, Invitrogen Corp., Grand Island, NY) Cells were kept between co ncentrations of 1.5 x 105 cells/mL and 1 x 106Prolifera tion Assay cells/mL The true passage number was unknown. When they were received from ATCC t here passage was assumed to be one. The viability was always equal to or greater than 94% unless otherwise stated. The proliferation assay, also called the MTT assay was adapted from the work of Mo sman [52 ]. HL 60 cells were incubated in a particular concentration of cranberry fraction that were dissolved in dimethyl sulfoxide ( DMSO ) (Sigma) and IMDM media for 48 hours. Each fraction was tested at several concentrations between 0.1 and 1000 g/mL At the end of incubation, a final concentration of 238 g/mL of MTT dissolved in phosphate buffered saline was added. Cells and MTT were allowed to incubate for four hours. T hen 100 L of a 0.04 normal acid isopropanol solution was added and the wells were mixed thoroughly for 5 minutes at room temperature. The acid isopropanol aided in dissolving the blue formazan crystals that had formed. The plate was read at 570 nm using a SpectraMax 340PC 384 and the reference, read at 630 nm, was subtracted from that. Two independent experiments were conducted Respiratory Burst Assay HL 60 cells at 4 x 105 cells/mL were incubated in a final concentration of 75 g/mL of one of the 6 cranberry fractions for 48 hours at 37C and 5% CO2. At the end of incubation, cells were washed with PBS twice. Cells were then counted and cytotoxicity was assessed via the trypan blue exclusion dye method. Cells were resuspended in Dulbe ccos phosphate buffered saline (DPBS) with calcium and magnesium (Cellgro)

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39 with glucose (1 g/L) (Sigma) at a concentration of 1 x 107A one way analysis of variance (ANOVA) was used to test for significant differences among the means of the viability data. To test for which means differed, a Student NewmanKeuls post hoc all pairwise analysis was used. A Kruskal Wallis oneway ANOVA was used to test for significant differences among the ranks of the respiratory burst data due to a failed normality test on the means. No post hoc analyses were used to interpret these data. cells/mL Plate cells in a clear 96well plate with flat bottomed wells along with 100 L of DPBS, 58.8 ng/mL of PMA (Si gma) and 1.1 mg/mL of ferricytochrome C from horse heart (Sigma). The plate was read immediately on a SpectraMax 340PC 384 plate reader at 550 nm, from which the absorption at 490 nm was subtracted, for 10 minutes with a read interval of 30 seconds. Thi s procedure was adapted from Babior et al. [51 ]. Three independent experiments were performed. Results Proliferation The MTT data did not show a dose dependent response in the HL60 cells to varying concentrations of the cranberry fractions. It appeared as though the color of the fractions may have given misleading data (data not shown) due to its absorbance being near the absorbance of the formazan crystals produced in the MTT assay. Therefore, conclusions cannot be made based on this data. Respiratory Burst There was no difference ( P value = 0.190) between any of the fractions or the control in the rate of cytochrome C reduction, as shown in Figure 31 The rate at which superoxide anion was produced, secreted, and allowed to reduce cytochrome C did not

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40 vary significan tly between any of the fractiontreated, PMA sti mulated cells and the control cells that were only stimulated with PMA. The viability of the cells treated with the cranberry fractions was assessed before cells were used in the respiratory burst assay and are presented in Table 31 Only F raction F res ulted in a significant change in viability compared to the untreated control cells. The average viability for cells incubated in F raction F was 46.4%, while the average viability for untreated cells was 82.9%. The P value for the difference between these two groups was less than 0.001. There was also a trend seen in F raction A treated cells for decreasing viability ( P value = 0.079) compared to the controls cells, but it did not approach significance. Discussion It was determined that the fractions, even at the low concentrations used, interfered with the spectrophotometric readings necessary for the assay The lack of reliable results for the MTT assay showed that the fractions have strong absorption near or at an important wavelength. A different metho d of determining cytotoxicity, the trypan blue dye exclusion method, was used. Mature immune cells use prooxidants, like superoxide anion, to kill invading pathogens. In vitro these cells can be induced to secrete superoxide anion by exposing them to PMA or some other stimulus. Superoxide release is an effective killing mechanism, but it is also a good example of why chronic inflammation can be so damaging to healthy tissue. The rate of superoxide aniondriven reduction of cytochrome C was used as an in dicator of superoxide anion production. This rate was similar between all fractiontreated cells and the controls cells, suggesting that no differentiation occurred during the treatment of HL60 cells with cranberry fractions.

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41 Figure 31. Rate of cyt ochrome C reduction in HL60 cells treated with 75 g/mL cranberry fractions

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42 Table 31 Cytotoxicity of HL 60 cells in 75 g/mL cranberry fractions Fraction Viability (in percent) A 67.4 (16.2) B 80.9 (8.1) C 80.8 (8.0) D 82.1 (8.8) E 76.0 (10.5) F 46.4 (10.0) Control 82.9 (6.5) The data are presented as mean viability in percent along with the standard deviation of four experiments in parentheses. The asterisk, *, denotes a significant difference compared to the control viability.

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43 CHAPTER 4 CYTOTOXICITY OF CRANBERRY FRACTIONS AND LIPOPOLYSACCHARIDE O N DIFFERENTIATED HL 60 CELLS Introduction The next part of the hypothesis was to determine the cranberry fractions effects on particular protein levels in differentiated cells Before approaching this task, it was necessary to determine the highest dose of each fraction that differentiated cells can tolerate The methods and results are discussed in this chapter. Materials and Methods HL 60 Cell Differentiation HL 60 cells were transferred to a separate flask at a concentration of approximately 6 x 105 cells/mL to 8 x 105 cells/mL and incubated for 96 hours in 1 M alltrans retinoic acid ( ATRA ) (Sigma) dissolved in 95% ethanol at 37C, 5% CO2Cytotoxicity and humidity as previously repor ted by Freeman et al. [41 ]. After the incubation, cells were considered differentiated and ready for experiments. To verify that differentiation occurred, the respiratory burst assay previously mentioned in Chapter 3 was run using the ATRA treated cells and the same design as discussed in that chapter. ATRA treated cells were compared to vehicletreated control cells. Five independent experiments were conducted. A Mann Whitney rank sum t test was used to test for significant differences between the med ians due to a failed normality test Differentiated HL60 cells were plated at 5 x 105 cells/mL in a clear, tissue culture treated 12well plate and incubated for 24 hours at 37C, 5% CO2, and humidity in 250 ng/mL lipopolysaccharide ( LPS ) (Si gma) and 50 g/mL, 20 g/mL, 5 g/mL, or 0.1 g/mL of one of the fractions which were dissolved in dimethyl sufloxide ( DMSO )

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44 and Iscoves Modified Dulbeccos medium ( IMDM) media. Cont rol cells were incubated in only vehicle, excluding DMSO, with no LPS or cranberry fractions present. A second set of cells was incubated with only 250 ng/mL of LPS and no cranberry fractions but included 0.1% DMSO, the highest amount of DMSO found in the cells incubated with cranberry fractions At the e nd of the 24 hour i ncubation, cell counts and viability were assessed using the trypan blue dye exclusion method. Four independent experiments were conducted. A one way analysis of variance (ANOVA) was used to test for significant differences among the means of the data. T o test for which means differed, a Student NewmanKeuls post hoc all pairwise analysis was used. Results HL 60 Cell Differentiation The superoxide aniondriven reduction of cytochrome C in the ATRA treated cells was approximately 20 times that of the reduc tion of cytochrome C seen in the control cells as shown in Figure 41 There was a significant difference between the untreated ce lls and the ATRA treated cells ( P <0.001). Cytotoxicity The ANOVA showed that the differences betw een the data, shown in figur e 42, was due to more than just chance (P value = 0.025). However, there were no significant differences found by the pairwise multiple comparison between any of the fractions or the controls. Only one concentration of one fraction, the 50 g/mL fraction E treated cells, approached significance (P value = 0.051) versus the negative LPS, negative DMSO control. The trend was for the cells under this treatment to have a

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45 slightly higher viability compared to cells that were incubated with no DMSO and no LPS. However, this was not statistically significant. Discussion HL 60 Cell Differentiation It is evident that 1 M ATRA induces considerable superoxide anion production in HL 60 cells after a 96hour incubation and stimulation with phorbol myristic acid ( PM A ) This is classic behavior indicative of mature immune cells. It is also an established fact that prolonged exposure to ATRA induces HL60 cell differ entiation toward the neutrophil like cells [46,53 ] It was determined that the previously described procedure for differentiation was effective and was used in further experiments where differentiated cells were needed. Cytotoxicity There was no significant difference in the viabilities of any fractions tested versus the controls. It appeared that the am ount of DMSO used to dissolve the cranberry fraction, the concentration of LPS used to stimulate the differentiated cells, or the concentration of any fraction studied had no effect on the viability of the differentiated HL 60 cells. Due to this finding, the concentration of F raction A, E, and F used in further experiments was 50 g/mL. The concentrations of LPS and DMSO used in the cytotoxicity experiment was also the same as those used in the inhibitor of kappa B alpha ( ) interleukin1 receptor associated kinase 4 ( IRAK4) and tumor necrosis factor alpha ( ) studies.

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46 Figure 41. Rate of cytochrome c reduction in ATRA treated cells versus untreated cells. The asterisk, *, denotes a significant difference betw een the ATRA treated cells and the control cells. Figure 42. Viability of differentiated HL60 cells treated with various concentrations of six cranberry fractions and LPS.

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47 CHAPTER 5 TOTAL AND PHOSPHORYL ATED INHIBITOR OF KA PPA B ALPHA AND INTERLEUKI N 1 RECEPTOR ASSOCIATED KINASE 4 PROTEIN L EVELS IN HL 60 CELLS AFTER INCUBATI ON WITH CRANBERRY FRACTIONS AND LIPOPOLYSACCHARIDE Introduction Inhibitor of kappa B ( ) is an important regulatory protein in the nuclear factor kappa B ( NF ) pathway. It binds NF prevents the increased transcription of NF inducible inflammatory genes. The first step in the separati on of NF phosphorylated, it is marked for ubiquitination and subsequent degra dation. The NF pathway can be stimulated by various pathogen components and through a variety of mechanisms. The toll like receptor ( TLR ) pathway is one that eventually results in the cleavage of N F Interleukin1 receptor associated kinase 4 ( IRAK 4 ) is one of many downstream signaling proteins in the TLR pathway Any changes in its expression have been associated with changes in NF [ 5463 ]. Materials and Methods Differentiated HL60 cells were treated with 50 g/mL of either F raction A, E, or F dissolved in dimethyl sulfoxide ( DMSO ) and media and 250 ng/mL of lipopolysaccharide (LPS) dissolved in media Control cells were treated with a DMSO and media control solution and 250 ng/mL of LPS or a vehicleonly replacement In past research the greatest difference between total and ( ) has been seen 10 to 15 minutes after treatment [ 57,64 ] The 2 hour incubation time chosen for IRAK4 was also based on the procedures of previous research [65] Fractions were incubated with cells for 10 minutes before

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48 for IRAK4 ana lysis. Immediately after addition of the fractions, 250 ng/mL of LPS was also added to the cells before incubation began. LPS was also added to cells that had only been treated with a vehicle control. This was the LPS control. A second control, the neg ative control, had only vehicle added in place of a cranberry fraction and LPS. After incubation, between 2 x 107 and 3 x 107For detection, blots were blocked overnight in 5% nonfat dry milk blocking buffer at 4C. The primary antibodies were diluted 1:1000 and incubated with blots for 1 hour at room temperature. cells were collected, washed twice with phosphate buffered saline and resuspended in an aqueous buffer containing protease inhibit ors including 1 mM phenylmethylsulfonyl fluoride (Fisher), 10 g/mL pepstatin A (Sigma) 20 g/mL leupeptin (Sigma) 10 g/mL aprotinin (Sigma) 2 mM ethylenediaminet etraacetic acid (Fisher), and 5 mM dl dithiothreitol (Sigma) Phosphatase inhibitors, 25 mM sodium fluoride (Fisher), 1 mM sodium orthovanadate (Santa Cruz Biotechnology, Santa Cruz, CA) and 10 glycerophosphate (Sigma) and sonicated for 610 second bursts. After sonication, Triton X 100 (Sigma) was added at a final concentration of 1 mM Samples were, then, centrifuged at 4500 rpm to pellet cellular debris. Supernatant was removed and used for Western blotting. Protein content was determined by the Bradford method. Between 40 and 75 g of protein were loaded into each well of the gel. Samples were separate using a 12% sodium dodecyl sulfatepolyacrylamide gel and transferred to a nitrocellulose membrane (Bio Rad, Hercules, CA) for one hour at 95 V with a 10% methanol (Fisher), 25 mM Tris (Sigma) 192 mM glycine (Bio Rad) transfer buffer.

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49 Signaling Tech nology (Danvers, MA) and g lyceraldehyde3 phosphate dehydrogenase antibody was purchased from Imgenex (San Diego, CA). The secondary anti rabbit antibody (GE Healthcare, Buckinghamshire, UK) was also incubated with the blot under these conditions. Detect ion solutions were mixed 1:1 and incubated with the blot for one minute and detection occurred immediately after. Films (Fisher) were exposed to blots between 1 and 20 minutes depending on the amount of protein on the blot Proteins of interest were detected first and, then, the blots were stripped and reprobed for the loading control proteins. IRAK4s loading control was glyceraldehyde3 phosphate dehydrogenase tubulin. Films were scanned into the SigmaGel software for analysis. Protei n bands were compared based on pixel intensities and a ratio of the proteinof interest to the loading control was obtained for each fraction and controls. To account for differences between gels in the amount of protein loaded into the wells of a gel, the ratio of proteinof interest to loading control for the LPS control for each blot was set to one arbitrarily. The negative control and fractiontreated samples were then nor malized to the LPS control. and IRAK4 protein, while 3 protein levels. A one way analysis of variance (ANOVA) was used to test for significant differences among the means of the data. To test for which means differed, a Student NewmanKeuls post hoc all pairwise analysis was used. For the IRAK4 and Wallis one way ANOVA was used to test for significant differences among the ranks of the data due to failed normality tests. To test for which

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50 medians differed, a Dunns post hoc multiple comparison versus the LPS control analysis was used. Results Fraction A and F treated cells had a significant increase ( P protein levels compared to the LPS control cells as shown in Figure 51A protein levels illustrated in Figure 51B, were significantly lower than the LPS control in F raction A treated cells ( P = 0.039) F raction F treated cells ( P = 0.043), and in the negative control cells ( P = 0.032). Figure 51C shows that IRAK4 protein levels were significantly lower in the F raction A treated cells ( P <0.05) compared to the LPS control cells. Discussion NF of inflamm atory genes for growth factors, stress proteins adhesion molecules and cytokines and their receptors [ 66 in the cytosol of cells until The inhibitor of kinase (IKK) phosphorylates which signals it for ubiquitination. Once unbound, NF nucleus. Many types of pathogens activate NF triggers for NF this pathway ends up activati ng NF While an inflammatory response is necessary to mount an attack on invading pathogens, inflammation due to autoimmune disease or ex cessive or chronic inflammation can have serious negative effects on ones health. For th is reason, anything, including plant polyphenols, that can dampen an exaggerated immune response has been of great interest to researchers.

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51 According to the protein data, F ractions A and F have shown the most promise across the board. After a 10minute in cubation with LPS and F raction s A or F the neutrophil like cells had a lower amount of p compared to the LPS control Also, the cells treated with F raction A and LPS had lower IRAK4 protein levels in comparison to the LPS controls Taken together this data paints a picture of the ability of the p roanthocyanidinrich cranberry fractions to diminish the inflammatory effect of LPS in neutrophils. It appears that the proantho cyanidinrich fractions thus it can continue to keep NF of the cell. The lower levels of IRAK4, a signaling protein in the TLR4 pathway, suggest a mechanism by which the proanthoc yanidinrich fractions have their effect. For these experiments, it is important to note the possibility of the incubation time havi ng an effect on the results. The incubation times were carefully chosen based on published research in order to find times that allow for a large change from baseline. A time course over 12 or 24 hours would be helpful in verifying the changes in protein levels and would give a clearer picture of exactly when these changes occur

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52 A Figure 5fractions. Cells were treated with 50 g/mL of F raction A, E, or F or with a vehicle control. LPS was added to all samples containing fractions and the LPS control. A negative control contained no cranberry fractions and no LPS. Protein expression is in arbitrary units with the LPS control protein expression set to one. A) Total protein levels. Asterisks, *, above the bars indicate statistical significance in comparison to the LSP control.

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53 B C Figure 51. Continued

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54 CHAPTER 6 TUMOR NECROSIS FACTO R ALPHA SECRETION IN CREASES WITH I NCUBATION IN CRANBERRY FRACTIO NS Introduction One of the hallmarks of nuclear factor kappa B ( NF ) activation is the production and release of inflammatory cytokines. Tumor necrosis factor alpha ( ) is one of these cytokines. An enzyme linked immunosorbent assay ( ELISA ) assay, described in like HL 60 cells after stimulation with LPS and treatment with cranberry polyphenols. Materials and Methods HL 60 cells were incubated with 50 g/mL of cranberry fraction A, E, or F and 250 ng/mL of lipopolysaccharide (LPS) for 24 hours at 37C, 5% CO2 San Diego, CA in the supernatant. The ELISA procedure provided by the eBioscence kit was followed, but a brief description of the procedure can be found below. and in a humidified environment. Cells were collected after incubation and centrifuged at 600 times gravity for 10 minutes. The resultant supernatant was used for further analysis of TNF Plates were coated wit h capture antibody overnight at 4C. Wells were washed 5 times for one minute per wash using wash buffer. Then, wells were blocked with assay diluents for 1 hour at room temperature. The plate was washed again as was plated in the following concentrations: 500 pg/mL, 350 pg/mL, 250 pg/mL, 125 pg/mL, 50 pg/mL, 10 pg/mL, and 4 pg/mL. Samples were plated in triplicate. The plate was incubated overnight at 4C and, then, washed. Detection antibody at a 1/250 dilut ion was added to each well and incubated

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55 for 1 hour at room temperature. The plate was washed again. The avidinhorseradish peroxidase enzyme was diluted 1 to 250 and added to the plate for a 30minute incubation at room temperature. Wells were washed 7 times for one minute per wash and substrate solution was added to each well. The plate was incubated for 15 minutes at room temperature. A 2 normal sulfuric acid stop solution was added to each well and the plate was read at 450 nm and 570 nm on a Spect raMax 340PC 384. The second wavelength was subtracted from the first Four independent experiments were conducted. A one way analysis of variance ( ANOVA) was used to test for significant differences among the means. To test for which means differed, a Student NewmanKeuls post hoc all pairwise analysis was used. Results shown in Figure 61, were approximately 10 times higher than cells treated with LPS alone. Fraction A and F raction F approximately 600 pg/mL, while F raction E treated cells had slightly less at about 400 level in the supernatant of unstimulated differentiated HL60 cells was not detectable, while the LPS stimulated cells had a n average concentration of approximately 60 pg/mL. The differences between all fractiontreated cells and the controls all have P values less than 0.001. The difference between F raction A treated cells and F raction E treate d cells has a P value of 0.005, while the difference between F raction F treated cells and F raction E treated cells has a P value of 0.002.

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56 Discussion what was expected. The experiments involving inhibitor of kappa B alpha ( ) and interleukin1 receptor associated kinase 4 ( IRAK4 ) protein levels suggested that signalin g through tolllike receptor 4 ( TLR4 ) and NF unstimulated cells or at least reduced in comparison to the LPS control. However, the cranberry polyph Despite the large body of research that shows plant polyphenols have anti inflammatory capabilities there are several studies that have found seemingly opposing data [1 2 67,68 ] Wang et al stud i ed 23 polyphenols, including phenolic acids, isoflavones, flavonols, and anthocyanins, and berry extracts in relation to their effects monocyte/macrophage cell line.[12 ] Cells were incubated with 16 to 500 M concentrations of various polyphenols or 16 to 500 g/mL of berry extracts for 1 hour and then exposed to LPS and IFN nitric oxide production. At higher concentrations daidzin, genistin, cyanidin, malvidin, pelargonidin, peonidin crude blackberry extract, and blueberry concentrate all increased At most or all concentrations kaempferol, myricetin, delphinidin, cyanidin 3 glucoside, crude Saskatoon berry extract, blackberry concentrate, and black curr ant concentrate. Park et al. [ 67] used the same cell line to study monomeric, dimeric, and trimeric flavonoids and pycnogenol, a maritime pine bark extract. The monomeric and dimeric

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57 flavonoids at 100 g/mL However, a trimeric flavonoid and pycnogenol at 100 g/mL interferongamma ( IFN ) stimulated cells, monomeric flavonoids, like catechin, and dimeric flavonoids reduced secretion at 100 g/mL The trimeric flavonoid and the IFN s e experiments also showed that NF dependent gene expression nearly doubled when c ells were incubated with a trimeric flavonoid or pycnogenol and IFN the unstimulated control and the dimeric flavonoids had slightly reduced expression in comparison to the IFN trol. A third study showed that quercetin and resveratrol at a concentration of 0.2 mM and 0.1 mM, respectively, did not inhibit LPS stimulated p50/65, an NF activation in RAW 264.7 cells [ 68 ]. Quercetin did inhibit the activation of the p50/50 cells treated with LPS. It is important to note the purity of the treatment in addition to the effects on immune cells. A quercetin or genistin treatement is expect ed to be relatively pure, while a crude extract of blueberry is likely to be just that. It is possible that compounds other than polyphenols in these crude extracts may be having an effect on the cells. Chemicals within these extracts may inhibit the act ivity of polyphenols or may synergistically enhance their activity. While the preponderance of data on polyphenols from cranberry and other plant sources suggests they are generally anti inflammatory. The nearly 10fold increase in

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58 an inflammatory cytokine, secretion in polyphenol treated neutrophils found in my study seems to suggest otherwise. The research discussed above present s evidence to corroborate these findings It is interesting to note that the data reported by Wang et al. [12] and Park et al. [67] was the result of using higher concentrations of polyphenols compared to my study. These two studies used up to 500 g/mL and 100 g/mL, respectively, while my study used concentrations of 50 g/mL. The Wang et al. research [1 2] which found a few production and several that enhance it is a fitting illustration of the dichotomy of polyphenols effects on inflammatory responses in cell culture.

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59 Figure 61. TNF secretion after 24hour incubation in cranberry fractions and LPS. The thick bars represent the mean of four experiments and the tails represent the standard deviation. Bars with the same letter above are not significantly different.

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60 CHAPTER 7 CONCLUSION This project has shown that cranb erry fractions enriched in presscake proanthocyanidins, F raction A, and juice proanthocyanidins, F raction F, have the highest nitrogen radical quenching ability of all of the fractions tested in the 2,2 d iphenyl 1 picrylhydrazyl ( DPPH ) assay. The juice polyphenol rich fraction F raction E, and F raction F are best at quenching the peroxyl radical from the oxygen radical absorbance capacity assay (ORAC) Differentiated HL60 cells tolerated all fractions up to 50 g/mL. Lipopolysaccharide ( LPS ) stimulated differentiated cells showed reduced phosphorylated inhibitor of kappa B ( ) protein levels when treated with F raction A and F raction F. The protein levels of total inhibitor of kappa B alpha ( ) were significantly higher in cells t reated with these two fractions. Furthermore, i nterleukin1 receptor associated kinase 4 ( IRAK4 ) protein levels were decreased in cells treated with F raction A and LPS. The decreases in this protein in F raction F treated cells are suggestive, but not significant. These data indicates that the pro anthocyanidinrich fractions from cranberry pr esscake and juice attenuate LPSinduced nuclear factor kappa B ( NF ) activation by preventing the phosphorylat They also suggests that at least one way it accomplishes this is through interrupting the tolllike receptor 4 ( TLR4 ) pathway, as shown by the reduced IRA K4 protein levels in F racti on A treated cells and the downward trend of IRAK4 protein levels in F raction F treated cells However, the large increases in secreted tumor necrosis factor alpha ( ) seen in fractiontreated cells point to the activation of another pathway by the cranberry

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61 fractions. This is stimulation above that seen by LPS alone. Taken together, this shows that the cranberry polyphenols studied have promising immune modulating abilities. They can prevent mass activation of inflammatory genes, but still allow for targeted inflammatory cytokine secretion in response to a pathogen. This may make them better targets for further research into the treatment of severe or chronic inflammation, like sepsis or autoimmune disease. My study tested polyphenol rich cranberry f ractions at a concentration of 50 g/mL in an in vitro system. This concentration is approximately 10 to 1000 times higher than previously measured plasma levels of various flavonoids [69]. It is difficult to measure plasma polyphenols that originate from the diet because gut bacteria metabolize them. This modification likely changes their activity in the body and increases the number of metabolites to measure in the blood. However, it is important to note that gut associated lymphoid tissues sample int estinal contents for pathogens and likely come in contact with concentrations of polyphenols in excess of 50 g/mL. This may have implications in the way these immune cells react to gastrointestinal (GI) disorders, like inflammatory bowel disease or the m anner in which an immune response is mounted to pathogens detected in the GI tract. Furthermore, my study is important as a jumping off point for further research of these cranberry fractions. It reveals the activities of these fractions as antioxidants and in an in vitro model. These experiments have revealed a new avenue for research in the field of cranberry polyphenols. However, they have raised as many questions as they may have answered. More study should be conducted to elucidate the manner in which these polyphenols affect the TLR4 pathway. Also, what other inflammatory genes do

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62 these fractions turn on? It is possible that they encourage the transcription and translation of anti inflammatory genes as well. While in vitro experiments are a help ful way to start off in a new area of research, animal and human studies allow for stronger data. The immune modulating effects of cranberry polyphenols should be tested in an animal model to verify their validity

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70 BIOGRAPHICAL SKETCH Catherine Elizabeth Muller was born in Melbourne, Florida. Catherine grew up in Brevard County Florida and attended West Shore Junior Senior High School in Melbourne, 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 2007. In the spring of 2008, Catherine enrolled as a master s student in the Food Science and Human Nut rition Department at UF. While working toward her degree, she studied under Dr. Susan S. Percival. In her free time, Catherine enjoys running and the art of stained glass. After graduation, she intends on pursuing a doctor of philosophy degree in food science.