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Innate Immune Receptor Expression in Periodontally Diseased and Healthy Tissues

Permanent Link: http://ufdc.ufl.edu/UFE0024420/00001

Material Information

Title: Innate Immune Receptor Expression in Periodontally Diseased and Healthy Tissues
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Waite, Matthew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: advanced, periodontal, periodontitis, rage, tlr2, tlr4
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The innate immune system, of which toll-like receptors (TLRs) and receptor for advanced glycated end products (RAGE) are an integral part, has been implicated in the pathogenesis of periodontal disease. Recent work has shown expression of RAGE to be up regulated in gingival tissues from patients suffering from chronic periodontitis and diabetes, although the reason for the increased expression has not been elucidated. However, RAGE has been implicated in a number of other pro-inflammatory processes and has been implicated in the disease progression of coronary artery disease, hypertension, arthritis and Alzheimer's disease. Additionally, toll-like receptors two (TLR-2) and four (TLR-4) are up regulated in chronic periodontitis as a result of bacterial infiltration by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. It was the purpose of this investigation to determine whether there was an association among the up-regulation of RAGE, TLR-2, TLR-4 in patients diagnosed with chronic periodontitis. To determine the possible associations, gingival tissue samples were collected from patients during periodontal surgeries, after which, RNA isolation and real time polymerase chain reaction (qPCR) were used to quantify expression of RAGE, TLR-2 and TLR-4. Our results demonstrate that RAGE and TLR-4 expression were up regulated in chronic periodontitis patients, whereas TLR-2 expression was down regulated when compared to expression in healthy gingival tissues, with a correlation coefficient of r=0.9964, indicating that in a given participant when RAGE expression was increased, so was TLR-4 expression while TLR-2 expression was decreased.
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 Matthew Waite.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Katz, Joseph.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024420:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024420/00001

Material Information

Title: Innate Immune Receptor Expression in Periodontally Diseased and Healthy Tissues
Physical Description: 1 online resource (48 p.)
Language: english
Creator: Waite, Matthew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: advanced, periodontal, periodontitis, rage, tlr2, tlr4
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The innate immune system, of which toll-like receptors (TLRs) and receptor for advanced glycated end products (RAGE) are an integral part, has been implicated in the pathogenesis of periodontal disease. Recent work has shown expression of RAGE to be up regulated in gingival tissues from patients suffering from chronic periodontitis and diabetes, although the reason for the increased expression has not been elucidated. However, RAGE has been implicated in a number of other pro-inflammatory processes and has been implicated in the disease progression of coronary artery disease, hypertension, arthritis and Alzheimer's disease. Additionally, toll-like receptors two (TLR-2) and four (TLR-4) are up regulated in chronic periodontitis as a result of bacterial infiltration by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. It was the purpose of this investigation to determine whether there was an association among the up-regulation of RAGE, TLR-2, TLR-4 in patients diagnosed with chronic periodontitis. To determine the possible associations, gingival tissue samples were collected from patients during periodontal surgeries, after which, RNA isolation and real time polymerase chain reaction (qPCR) were used to quantify expression of RAGE, TLR-2 and TLR-4. Our results demonstrate that RAGE and TLR-4 expression were up regulated in chronic periodontitis patients, whereas TLR-2 expression was down regulated when compared to expression in healthy gingival tissues, with a correlation coefficient of r=0.9964, indicating that in a given participant when RAGE expression was increased, so was TLR-4 expression while TLR-2 expression was decreased.
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 Matthew Waite.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Katz, Joseph.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024420:00001


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1 INNATE IMMUNE RECEPTOR EXPRESSI ON IN PERIODONTALLY DISEASED AND HEALTHY TISSUES By MATTHEW THOMAS WAITE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Matthew Thomas Waite

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3 To my wife, who has tirelessly suppor ted my goals throughout this journey.

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4 ACKNOWLEDGMENTS I would like to thank m y family, who ha ve supported me throughout my professional endeavors, without hesitation. Additionally, I would like to extend my gratitude to the faculty members at the University of Florida Departme nt of Periodontology for their contribution to my education and their continuing commitment to our profession.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF FIGURES .........................................................................................................................7ABSTRACT ...................................................................................................................... ...............8CHAPTER 1 INTRODUCTION .................................................................................................................. 102 BACKGROUND ....................................................................................................................13The Periodontium in Health .................................................................................................... 13Periodontal Tissue Destruction ............................................................................................... 14Risk Factors for Periodontal Disease ......................................................................................16Treatment for Periodontal Disease .........................................................................................17Toll-Like Receptors and Periodontal Disease ........................................................................ 18Advanced Glycation Endproducts and Periodontal Disease ..................................................193 MATERIALS AND METHODS ...........................................................................................22Participant Population ........................................................................................................ .....22Surgical Procedure ..................................................................................................................22Tissue Preparation and Storage .............................................................................................. 23RNA Harvesting ................................................................................................................ .....23Reverse Transcription (RT) .................................................................................................... 23Real-time Polymerase Chain Reaction (qPCR) ...................................................................... 24Hematoxylin and Eosin Staining ............................................................................................ 24Immunohistology ............................................................................................................... .....254 RESULTS ....................................................................................................................... ........26Expression of Receptor for AGE in Periodontal Tissues ....................................................... 26Expression of Toll-Like Recepto r 2 in Periodontal Tissues ................................................... 27Expression of Toll-Like Recepto r 4 in Periodontal Tissues ................................................... 28Comparison of Expression Patterns for RAGE, TLR-2 and TLR-4 ....................................... 28Analysis of Cell Types of the Pe riodontal Tissue Expressing RAGE ....................................295 DISCUSSION .................................................................................................................... .....40LIST OF REFERENCES ...............................................................................................................44BIOGRAPHICAL SKETCH .........................................................................................................48

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6 LIST OF TABLES Table page 4-1 Demographic data of participant population ......................................................................30

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7 LIST OF FIGURES Figure page 2-1 Periodontal Health. Diagram of a tooth in health (left) and disease (right) ...................... 214-1 Quantitative gene expressi on in periodontal tissues .......................................................... 314-2 RAGE expression in healt hy and diseased subjects .......................................................... 324-3 TLR-2 and TLR-4 expression comparison for healthy and diseased subjects ................... 334-4 Gene expression in all subjects, excluding smokers and diabetics. ...................................344-5 Correlation Data among RAGE, TLR-2 and TLR-4 ......................................................... 354-6 Histological examination of healthy periodontal tissue .................................................... 364-7 Histological examination of healthy periodontal tissue .................................................... 374-8 Immunohistology staining for RAGE in healthy periodontal tissues. ...............................384-9 Immunohistology staining for RAGE in diseased periodontal tissues ..............................39

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INNATE IMMUNE RECEPTOR EXPRESSI ON IN PERIODONTALLY DISEASED AND HEALTHY TISSUES By Matthew Thomas Waite May 2009 Chair: Joseph Katz Major: Dental Sciences The innate immune system, of which toll-like receptors (TLRs) and receptor for advanced glycated end products (RAGE) are an integral part, has been implicated in the pathogenesis of periodontal disease. Recent work has shown expr ession of RAGE to be up regulated in gingival tissues from patients suffering from chronic pe riodontitis and diabetes although the reason for the increased expression has not been elucidated. However, RAGE has been implicated in a number of other pro-inflammatory processes and has been implicated in the disease progression of coronary artery disease, hypertension, arthritis and Alzheimers disease. Additionally, tolllike receptors two (TLR-2) and four (TLR-4) are up regulated in chronic periodontitis as a result of bacterial infiltration by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis It was the purpose of this investigation to determine wh ether there was an association among the up-regulation of RAGE, TLR-2, TLR-4 in patients diagnosed with chronic periodontitis. To determine the possible associations, gi ngival tissue samples were collected from patients during periodontal surgeries, after which, RNA isolation and real time polymerase chain reaction (qPCR) were used to quantify expres sion of RAGE, TLR-2 and TLR-4. Our results demonstrate that RAGE and TLR-4 expression were up regulated in chronic periodontitis

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9 patients, whereas TLR-2 expression was down re gulated when compared to expression in healthy gingival tissues, with a correlation coefficient of r= 0.9964, indicating that in a given participant when RAGE expression was incr eased, so was TLR-4 expression while TLR-2 expression was decreased.

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10 CHAPTER 1 INTRODUCTION Periodontal disease is one of the m ost prevalent diseases in the United States, with nearly one quarter of the population being affected with some form of the disease. World estimates, according to the World Health Organization put mo re severe forms of the disease within the range of ten to 15% worldwide.1 While periodontal disease its elf is not necessarily lifethreatening, there is an incr easing body of evidence supporting the notion that periodontal disease may significantly contribute to systemic diseases that carry a higher morbidity and mortality, such as diabetes, cardiovascular disease and cerebrovascular disease.2, 3 Although specific links have not yet been elucidated, ma ny of these diseases share the common link of chronic inflammation, increased le vels of inflammatory products and markers, as well as higher levels of Advanced Glycation Endproducts, or AGEs.4 The immunological response begins with th e innate immune system, which is highly conserved throughout nature. The innate respons e is targeted specifically toward common molecular identifiers of pathogens, known as pathogen associated molecular proteins, or PAMPs. PAMPs sequences identified incl ude lipopolysaccharides (LPS), peptidoglycans, bacterial DNA, as well as lipoproteins.5 On the front line of the innate immune response are immunologic receptors initially identified in mice in 1985 Toll-like receptors, or TLRs. TLRs recognize these conserved patterns and trigger a cas cade within the innate immune response.5 Many organisms known to contribute to peri odontal disease also express many of these PAMPS. For instance, th e LPS from the bacteria A. actinomycetemcomitans and P. gingivalis bind to TLR-2 and TLR-4 respectively.5 Several studies have indicated an increase in the expression of these innate immune recep tors under inflammatory conditions.

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11 AGEs form via a non-enzymatic reaction from reducing sugars, such as glucose with amino groups in proteins, lipids and nucleic acids, by way of a series of reactions, Schiff bases and Amadori products are produced.4 They can be formed exogenously, such as in tobacco and cooked foods, or endogenously due to spontaneous r eactions especially in those persons with high circulating blood glucose levels, such as diabetics.6 AGEs are insoluble and are capable of permanently modifying proteins after which th ey bind to the endogenous receptors for known as RAGE resulting in a pro-in flammatory response. The inflammatory nature of periodontitis has been well established. Periodontitis has been associated directly with pro-inflammatory mediat ors and cytokines, such as interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor (TNF), mainly through the induction of the innate immune response using TLR ligation.7 In addition, studies indi cate a relationship between RAGE signaling and those induced by TLR ligat ion. Therefore, determining a relationship between chronic bacterial infection, such as in ch ronic periodontitis, and an association with an increase in the expression of RAGE, TLR-2, and TLR-4 wher e a clear relationship between periodontal disease and these receptors has not been esta blished is of interest. In order to elucidate a possible relationsh ip among RAGE expre ssion and TLR-2 and TLR4 expression, periodontally healthy and patient s diagnosed with chronic periodontitis were recruited to donate gingival tissu es from surgical procedures. He re, the level of gene expression for RAGE, TLR-2 and TLR-4 was analyzed using real-time polymerase chain reaction (qPCR). In addition, histological sections were used to determine the cell types harvested in these tissues as well as the expression patte rn of RAGE in these tissues. These data demonstrate that periodontally di seased tissue expressed a higher amount of RAGE and TLR-4 and a lower amount of TLR-2 when compared to periodontally healthy

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12 tissues. We were able to determine that epit helial, endothelial, fibrobl ast, etc. cells were included in the cell types analyzed for gene expression. In addition, mononuclear cells were found to be present in the diseas ed tissues as well. Here, RAGE protein expression was also qualitatively up regulated on most cells types, not just the immune infiltrate. These data indicate that increases in gene expressi on was not solely due to increase s in immune cell infiltrate Hypothesis: The state of inflammation or periodontal health status has an effect on the expression level of innate immune receptors.

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13 CHAPTER 2 BACKGROUND The Periodontium in Health The periodontium or the supporting struct ures surrounding teeth, consists of several tissues. These tissues serve not only to suppor t the teeth, but, to provi de protection against bacterial infiltration and disease. These tissues consist of bone, cementum, connective tissue, and epithelium.7 In health, these separate tissues comp rise a very strong support structure for the teeth that provide adequate prot ection against bacterial plaque. The normal, healthy structure of the healthy periodontium has been studied extensively. Dimensi onally, the epithelium attaches to the tooth anywhere from 0.67-1mm apical to the cementoenamel junction (CEJ) and is approximately 1mm in dimension. The connect ive tissue, typically comprised of collagen, ground substance and fibroblasts, attaches to th e tooth from that point on and comprises an additional millimeter.8 The connective tissue attachment approximates the bone and from the bone to the apex of the tooth root, the periodonta l ligament (PDL) and bone are continuous. (Figure 2-1) There is minor variation among people, as well as t ooth type with respect to the above dimensions. The sum of these measur ements is termed the biologic width.8 In health, these measurements do not stray far from one millimeter each for the sulcus, epithelial attachment and connective tissue attachment. Th e epithelium serves to provide protection from bacterial invasion into the PDL a nd bone, as well as mechanical protection. In disease, through repeated insult, the epithelial attachment ma y become weakened and eventually detached.8 The lack of attachment of the epithelial barrier allows a bacterial plaque biofilm to form subgingivally, making removal of the plaque very difficult for the patient. With time, this plaque is able to harden into calculus a mineralized form of the bacterial pl aque that serves as a

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14 constant irritant to the connective tissue at tachment. Given more time, plaque accumulates further apically and the disease progresses.9 The main function of bone in the peridontiu m is to support teeth in the process of mastication. Forces in the human masticator y system sometimes reach 600-750N in Americans and Europeans. The separate tissues in the system such as the PDL, tend to distribute the load through a slightly movable system. While the PDL does provide some of the support, the majority of forces are borne by the mandible and the maxilla. The main functional capacity of the PDL is to stabilize the tooth within its bony socket, as well as to provide somatosensory information and nutrition.10 The destruction of these tissues results in loss of attachment, increased probing depth and ultimately, loss of the tooth due to lack of proper bony support. Periodontal Tissue Destruction Diagnosis of periodontal diseas e is currently acco mplished according to collection of clin ical data namely probing depth, reces sion, bleeding upon probing, furcation involvement and mobility of teeth. Probing depth gives us an indication as to the current status of the PDL attachment around a tooth by determining the depth, to the nearest millimet er, to which the probe will reach. Typically, this is in the two to three millimeter range in health and does not extend beyond the epithelial attachment. In disease, the periodontal probe pa sses the epithelium and extends into connective tissue.11 Recession indicates tissue loss from the cemento-enamel junction (CEJ) to the gingival margin. Bleedi ng upon probing indicates the possibility that inflammation is present at a part icular site. The presence of bleeding does not necessarily indicate disease, however.12 A tooth can be described as furcation involved when there exists enough bone loss as to expose the ar ea between roots to probing. M obility indicates the lateral movement of a tooth. Lack of mobility is t ypically considered a sign of better prognosis and health.16 The sum of probing depth and recession, from the level of the CEJ is termed

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15 attachment loss. The higher the degree of attach ment loss, the more support has been lost from the tooth. Periodontal disease is an inflam matory disease classified acco rding to the type of tissue involved, the location, duration and severity of the disease. Th e current classification system, established in 1999, is widely used to diagnose periodontal disease.13 In addition to nonmicrobially induced forms of the disease, such as viral infections, fungal infections, etc. the system includes bacterial origin s (plaque-induced) of th e disease. For periodontal diseases of bacterial origin, diagnoses range from chronic to aggressive, localized to generalized, and slight to severe, dependent upon the amount of attachment loss that has occurred.13 Tissue destruction (or attachment loss) in peri odontitis is the result of several factors. Many bacterial species have been implicated in the process of periodontal destruction. In 1998, Haffajee and Socransky identified several bacteria l species associated with periodontal diseases. In addition to identifying some of the presen t bacteria via DNA checkerboard hybridization, they attempted to associate certain species with more severe forms of the disease. Several groups were described, with the red and orange complexes being associated more closely with disease than the other groups.14 Two species of bacteria that have been readily identified with periodontal disease, both chronic and aggressive, respectively, are Porphorymonas gingivalis and Aggregatibacter Actinomycetemcomitans Each of these species is associated with the Red complex and Green complexes, respectively.14 The immune response mounted by the body to the bacterial infiltration releases a host of inflammatory mediators that are able to destroy the periodontal ti ssues. Soon after the junctional epithelium has been violated, access to the PD L and underlying connective tissues is possible.15 Bacterial toxins, such as lipopolysaccharides, induce the prod uction of inflammatory mediators,

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16 such as proteases, cytokines and prostaglandins to assist in resistance of the bacterial invasion. This response, intended to neut ralize bacteria, also has the unto ward effect of destroying the periodontal tissues along with the b acteria. The resultant tissue de struction is the basis for the loss of tissue seen in periodontitis.17 Extension of the inflammation is made possibl e by the proliferous blood supply within the periodontium. Vessels extendi ng into the connectiv e tissue, periosteum and bone allow migration of bacteria, bacterial toxins, and pro-inflammatory mediators through out the tissues. The resultant immune response via pro-infl ammatory cytokines, such as IL-1, TNFand matrix metalloproteinases (MMPs) leads to the reso rption of bone and other connective tissues.17 This inflammatory process is capable of destroyi ng supporting periodontal ti ssues over a long period of time, as with chronic periodontitis, or with in a short time frame, as with aggressive periodontitis. Risk Factors for Periodontal Disease As with any disease, the risk for devel oping periodontal disease can be m odified by outside, controllable factors. These modifiable traits are often called risk factors.18 Within a population of patients, use of speci fic tools to identify which patie nts are at risk for a certain disease is often of great value in prevention or modification of the severity of a disease. Several factors have been associated with increased risk of developing periodontal disease, including socioeconomic status and age. High on the list, however, are tobacco smoking, as well as diabetes.19 The negative effects of smoking on oral he alth have been well-documented. Tobacco smoke, in addition to being an exogenous source of AGEs, retards healing, vascularization, and can impair collagen growth.20 The effects of diabetes have also been well-documented, with

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17 impaired healing and host immune response sp ecifically contributing to the morbidity of periodontal diseases.20 Treatment for Periodontal Disease Although both bacteria and inflamm atory pr ocesses contribute to the initiation and progression of periodontal diseases, the majority of treatments to date are aimed at the removal of bacterial complexes. Indeed, the first line in treatment for pe riodontal disease is non-surgical treatment, which typically takes the form of s caling and root planing. Scaling is defined as removal of supra and subgingival bacterial deposits (plaque and calculus). By root planing, the outermost layer of cementum from a tooth root is removed and planed to an even consistency, thereby providing for smooth, deposit-free root surfaces. The removal of bacterial deposits, combined with root surface modification, allows reattachment of periodontal tissues, typically in the form of a long junc tional epithelium (LJE).21 Outcome measures for periodontal health include reduction of probing de pths, gain of attachment leve ls, and evidence of bleeding upon probing. After traditional, non-surgical periodontal treatment is perf ormed and the results do not attain health, surgical treatment of periodontal disease becomes the ne xt line of treatment. With surgical treatment, the gingival tissues are refl ected, oftentimes removing a small (1-3mm) collar of diseased tissues, granulomatous or tissues that are undergoing an intense inflammatory response and replace lost bone and other conn ective tissues are removed and discarded, and scaling and root planing is performed with better visual ization of bacterial deposits.22 The enhanced visibility of involved teeth allows for more complete removal of deposits and recontouring of tooth roots. Typically, the remaining bone ar ound involved teeth is recontoured in order to achieve positive ar chitecture of the supporting bone.22 The goal of these treatments

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18 results in the removal of bacteria, allowing for d ecreased insults to the immune system. This, in turn, leads to a decreased activation of the immu ne response and the result ant tissue destruction. Toll-Like Receptors and Periodontal Disease Periodontitis is characterized by an active increase in the i mmune system both innate, as well as adapted, as a result of bacterial in sult. The early periodontal lesion can be histologically typified by the acute invasion of lym phocytes, which includes monocytes, B and T cells. Later in this progression, the established lesion consists of an intense neutrophil invasion. The advanced lesion, however, is the firs t stage of the le sion that can be described as having resultant bone loss and supporting tissue destruction. Throughout these stages of the periodontal lesion, a common thread is the involvemen t of the innate immune response. Integral in this response are toll-like receptors (TLRs). Toll-like receptors are intracellular and extracellu lar proteins that form the first response to bacterial and viral infiltration. They often initiate a respons e based upon bacterial or viral products that are present, such as lipopolysacch arides, peptidoglycan, flagellin, foreign DNA and RNA, as well as bacterial byproduc ts. TLRs are homologous to the IL-1 strucuture, but, contain a number of leucine-rich repeats in their structure. The leucin e-rich repeat areas correspond to certain PAMPs and TLRs are capable of dimerizi ng to recognize different bacterial products.5 Specifically, it has been shown that both TLR-2 and TLR-4 are up regulated in the presence of bacterial insult within the periodontium, although which cell types are involved is unclear.24, 25 Lipopolysaccarides and other components of bacterial products function as PAMPs, trigger a signaling cascade from TLRs on specifi c cells within the innate immune system and architecture of the periodontium, including monocytes, neutrophils, dendritic cells, epithelial and endothelial cells. Triggering of TLR-2 and TLR4, among other toll-like r eceptors, result in the upregulation of many soluble mediators such as cytokines, chemokine s and growth factors.26

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19 These soluble mediators initiate multiple cell si gnaling pathways result in the activation of T cells and B cells, the hallmark of the estab lished periodontal lesion a nd part of the adaptive immune system. Chronic stimulation of tolllike receptors, as is found in plaque-induced periodontitis, releases a c ontinuous stream of pro-inflammatory mediators,5 one of the mechanisms responsible for the tissu e destruction seen in periodontitis. Advanced Glycation Endproducts and Periodontal Disease AGEs for m via a non-enzymatic reaction from reducing sugars, such as glucose with amino groups in proteins, lipids and nucleic acids. By way of a series of reactions, Schiff bases and Amadori products are produced, forming AGEs.4 This process has been described as early as the 1900s and is commonly referred to as the Maillard reaction. They can be formed exogenously, such as in tobacco and cooked foods, or endogenously due to spontaneous reactions especially in thos e persons with high circulating blood glucose levels, such as diabetics.6 AGEs are insoluble and are capable of pe rmanently modifying proteins, and bind to an endogenous receptor known as RAGE. The receptor for AGE is a cell surface r eceptor that is capable of inducing a proinflammatory response. RAGE expression oc curs within a wide variety of cell types, including epithelial, en dothelial, smooth muscle, lymphocytes, monocytes, as well as neurons.4 Activation of RAGE triggers cellular activati on of proinflammatory mediators, such as interleukin 1 (IL-1), IL-6 and tu mor necrosis factor alpha (TNF). Once bound, RAGE stimulates further expression of RAGE via a positive feedback mechanism and generates oxidative stress, synthesis of proinflamma tory cytokines, as well as chemotaxis. RAGE also has the unique capability of bi nding many types of ligands. Some of the ligands, in addition to AGEs, that have been shown to bind RAGE are S100/calgranulins, High

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20 Mobility Group Box-1 (HMGB1) and amyloid beta peptide (Abeta).38 S100 has been shown to act as a pro-inflammatory cyt okine, triggering a cascade of re lease of IL-1, IL-6, and TNF, as well.31 MHGB1 is a proinflammatory protein that has been implicat ed in a growing number of transformational processes, includi ng breast cancer and adenocarcinoma.32 AGEs have been implicated in the pathogenesis of tissue destruction as a result of diseases such as diabetes and cardiovascular disease, as well as the natural aging process. Macular degeneration, atherosclerosis, diabetic neuropath y, as well as periodontitis all have a link to increased expression of RAGE, as well as higher levels of circulating AGEs.28 Signaling pathways pursuant to RAGE ligati on results in higher levels of in flammatory mediators, as well, such as C-reactive protein, and an increased degree of overall systemic inflammation.29 RAGE expression has been previously been a ssociated with risk factors for periodontal disease, such as diabetes.6 Lalla et al. also reported increas ed formation and deposition of AGEs in the gingival of diabetic mice inoculated with P. gingivalis Additionally, a study using the same murine model found that periodontal dise ase was arrested by administration of soluble RAGE (sRAGE), most likely through the inhibition of the RAGE-AGE interaction.6 Finally, Katz et al. has shown human subjects with diab etes and periodontal di sease to have a higher expression of RAGE in gingival tissues compared to controls. These evidence together point to the possible relationship of RAGE expression and pe riodontal disease status. 33

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21 Figure 2-1. Periodontal Hea lth. Diagram of a tooth in health (l eft) and disease (right) (A) Note the level of the free gingival margin. Apical migration of the free gingival margin is termed recession. (B) Note the height of th e alveolar process. Bone levels are reduced in disease. (C) Note periodontal probe extending deep within the pocket on the diseased site. In disease the period ontal probe extends beyond the epithelial attachment and into the connective tissue. (D) Note junctional epithelium near CEJ in health and long junctional epit helium in disease. (E) Note the presence of plaque and calculus and its access to periodontal disease. Adapted from: dental-arts.net/ images/Periodontology.jpg

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22 CHAPTER 3 MATERIALS AND METHODS A prospective observational study w as conducted to determine if periodo ntally healthy and diseased tissues had a difference in expression of RAGE, TLR-2 and/or TLR-4. Quantification of gene expression for these three genes was completed utilizing real -time polymerase chain reaction (qPCR). In order to determine which cell types were contributing to the expression patterns observed, gingival samples were al so analyzed using hematoxylin and eosin immunohistochemistry on parrafin embedded sections. Participant Population Fifty patients were recruited from the Univ ersity of Florida College of Dentistry, Department of Periodontology. All patients c onsented to the study, following Institutional Review Board approval. Incl usion criteria were as follows: diagnosis including chronic periodontitis or health, an age range between 18 and 70 years of age, necessity for surgical treatment (periodontal surgery, dental implant surg ery, etc.). Patients were excluded if they had a history of severe or chronic systemic diseas e, were pregnant or lactating or were taking medications known to affect the gingiva. Surgical Procedure Tissue harv esting was completed in conjunction with multiple types of oral surgical procedures performed by different surgeons. Th e majority of diseased specimens were removed as waste tissue during surgery to treat moderate to advanced ch ronic periodontitis. The healthy specimens were removed as waste tissue during procedures not related to periodontitis, such as dental implant placement, uncove ry, clinical crown le ngthening, or distal wedge surgeries. Patients were seen for regular follow-up seven to fourteen days post surgery.

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23 Tissue Preparation and Storage Following each surgical proce dure, equal portions of the sa mpled tissue were placed in Trizol, RNA later, transfer Media and/or form ali n. After which, the specimens were stored at 80C or room temperature (RT) until RNA ha rvesting or histology respectively could be performed. Formalin-fixed tissues were sent for embedment in paraffin, sectioning and slide preparation. RNA Harvesting Sa mples frozen in Trizol were allowed to thaw under a fume hood, after which 200 L of chloroform was then added to each tube, afte r which each tube was vigorously shaken by hand for 15 seconds. The tubes were left to incubate for 15 minutes at room temperature and then centrifuged for 15 minutes at 4 C at 12,000g. 500 L of isopropynol was then added to the resulting supernatant in a new tube and left to incubate at -80 C overnight. After which, samples were centrifuged at 4 C for 10 minutes at 12,000g. The supernatant was then decanted and 1000ul of 75% EtOH was added to each tube, vort exed for 15 seconds, and centrifuged at 4 C for 5 minutes at 7500g. The supernatant was again decanted and allowed the tubes were allowed to air dry for 15 minutes. Finally, 20 L of RNase/DNase free H20 was added to each tube, which were then incubated at 65 C for 1 hour. Resultant RNA was stored at -20 C until Reverse Transcription (RT) could be performed. Reverse Transcription (RT) cDNA was r everse transcribed using normalized RNA and a standard reverse transcription master mix containing 5X buffer (Invitrog en), 1mM DTTs (Invitrogen), 2.5mM dNTPs (Invitrogen), Superscript III reverse transcriptase (Invitrogen), oligonucleotides (Invitrogen), and RNase, DNase free water in a final volume of 25 L using the thermocycler conditions: 40 C for

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24 40 minutes, 70 for 15 minutes hold at 4 C. All cDNA was stored at 4 C until qPCR could be performed. Real-time Polymerase Ch ain Reaction (qPCR) 10ul of cDNA were placed into indi vidual wells of a 96-well polypropylene RNase/DNase-free plate (USA Scientific) along with a standard qPCR master m ix including qPCR primers specific for either GAPDH: 5' TC C ACC ACC CTG TTG CTG TA 3' (reverse), 5' ACC ACA GTC CAT GCC ATC AC 3' (forward ) (Integrated DNA Technologies, Coralville, IA); hRAGE: 5' GGA CTT CAC AGG TCA GGG TT A C 3' (reverse), 5' GAC TCT TAG CTG GCA CTT GGA T 3' (forward) (Integrated DNA Technologies, Coralville, IA); TLR-2 RT2 qPCR primers, (SABiosciences, Frederick, MD) and TLR-4 RT2 qPCR primers (SABiosciences). Standard curves that were ge nerated from serial dilutions of each gene were used to measure mRNA transcript copy number. Each gene was detected in independent qPCR reactions. Data are expressed as a copy numb er normalized GAPDH content. The normalized mRNA copy number for a gene was determined by: [raw transcript copy number derived from standard curve] [GAPDH corrective ratio]. The GAPDH corrective ratio was calculated as [lowest GAPDH copy number within sample set] /[GAPDH copy number for cell of interest]. Hematoxylin and Eosin Staining Tissue specim ens in formalin were sent for paraffin embedment, sectioning into 5 m slices and placement on slides. Upon receipt, the slides we re then soaked in successive baths for five minutes at a time: xylene, xylen e, 100% ethanol, 95% ethanol, 70% ethanol, deionized water. Following this, the sections were soaked for fi ve minutes in Harris Hematoxylin, rinsed in deionized water. The slides were then soaked in bluing solution for one minute, (1.5mL NH4OH in 98.5mL of 70% ethanol), followed by rinsing in de ionized water. The slides were soaked in

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25 an Eosin bath for five minutes, after which were rehydrated in ethanol and xylene baths: 70% ethanol, 95% ethanol, 100% etha nol and xylene for five minutes each. Cover slips were then placed on the slides using Permount and dried under the fume hood for one hour. Sections were visualized using a Zeiss microscope and fields captured using Image Pro-Software (BioRad). Immunohistology Tissue specim ens in formalin were sent for paraffin embedment, sectioning into 5 m slices and placement on slides. Upon receipt, the slides were soaked in successive baths for five minutes at a time: xylene, xylene, 100% ethano l, 95% ethanol, 70% ethanol, deionized water PBS. The slides were quenched by incuba ting in 0.5% H2O2 for 30 minutes, followed by rinsing for five minutes in PBS. Slides were probed with goat anti-hRAGE (1:500) and allowed to incubate in a humidified chamber overnight at 4C. After a rinse in PBS for five minutes, slides were probed with anti-goa t HRP (1:1000) and allowed to incubated in a humidifier for one hour at room temperature. The slides were then developed with 33-daminobenzidine (DAB) substrate in a humidified chamber for 45 minutes Slides were counterstained by washing in water for five minutes, dipping three times in hemotoxylin, followed by two successive five minute baths in distilled water. Slides were th en rehydrated in ethanol and xylene baths: 70% ethanol, 95% ethanol, 100% etha nol and xylene for five minutes each. Cover slips were then placed on the slides using Permount and dried under the fume hood for one hour. Sections were visualized using a Zeiss microscope and fields captured using Image Pro-Software (BioRad).

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26 CHAPTER 4 RESULTS Currently, a total of 47 subjects have participated in this st udy. Thirty nine of which donated tissue samples consisting of epithelium and connective tissue (Fig. 2-2, 2-3). Thirty four (19 diseased, 15 healthy) of these samples were analyzed via PCR for gene expression of RAGE, TLR-2, as well as TLR-4. RAGE was chosen, as it is a marker allowing for measurement of exposure to exogenous and endogenous AGEs. TLR-2 and TLR-4 were chosen, as they represent innate immune respons e to lipopolysaccharide of partic ular bacterial species involved in periodontitis P. gingivalis and A. actinomycetemcomitans respectively. Patients that participated had a diagnosis chronic periodontitis (moderate or severe; localized or generalized) necessi tating surgical intervention, or were considered periodontally healthy, undergoing an alternate pr ocedure related to dental impla ntology or removal of healthy tissues for clinical crown lengthening or tissue wedge removal. Patients were considered having periodontitis if they met the disease classifications set forth by the American Academy of Periodontology. (13) The demographics of the participant populat ion are summarized in Table 1. Briefly, among the 19 subjects studied in the diseas ed population, there were 7 smokers and 12 nonsmokers. In addition, 3 of these periodontally diseas ed participants were diabetic and 1 of these participants was a diabetic smoker. Males comp rised 6 of the subjects, with 13 being females. With respect to the healthy populations 15 subjec ts, 2 were smokers, with no diabetics present in the periodontally healthy group. Five males and 10 females made up the healthy population. Expression of Receptor for AGE in Periodontal Tissues RAGE expr ession occurs in many different cell types, including epithelial, endothelial, as well as gingival fibroblasts. (4) Expression is a result of insult by endogenous, as well as

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27 exogenous sources. The binding of RAGE to its ligand, AGE, triggers an immune response that involves release of pro-inflammato ry mediators and cytokine rel ease resulting in tissue damage. (28) It has been hypothesized that diseased pe riodontal tissues express RAGE to a higher degree than periodontal tissues th at are not diseased. To determine the validity of this hypothesis, qPCR was performed on 19 diseased and 15 healthy tis sue samples harvested during periodontal surgeries. RAGE-specific primers were used to determ ine expression and GAPDHspecific primers were used as a normalization c ontrol. After the data was normalized for cDNA content, it was determined that there was a significantly higher ( p value < 0 .0004, MannWhitney) expression of RAGE in periodontally diseased tissues versus normal, healthy tissue samples (Fig. 4-1, 4-2). These analysis included tissues from diabetic participants and well as those from smokers. Both of these conditions have been shown to increase the expression of RAGE, therefore, we repeated the analysis in the absence of these participant samples. Here the difference in RAGE expression remained significant. ( p value < 0.0004, Mann-Whitney Test) (Fig. 4-4) Expression of Toll-Like Recep tor 2 in Periodontal Tissues Toll-like receptors, upon binding to ligands signal the transc ription of many proinflammatory cytokines. Again, TLRS are expres sed in many of the cells associated with the periodontium and periodontal lesions. TLRs have also been described to be up regulated in response to bacterial stimuli as seen in periodont al disease. In additi on the LPS of periodontal pathogen P. gingivalis has been demonstrated to induce an immune response through ligation of TLR-2. Therefore, in order to elucidate as to whether periodontal disease classification correlated positively with TLR-2 expression qPCR was performed on tissue samples utilizing TLR-2-specific primers, normalized to GAPDH. After normalization, it was determined that

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28 diseased tissues expressed TLR -2 to a lesser degree than healthy periodontal tissues ( p value < 0.0007, Mann Whitney Test) (Fig 4-1, 4-3). Agai n, after adjusting the analysis without smokers and diabetic patients the decreased expression of TLR-2 remained significant (p value < 0.0007, Mann Whitney Test) (4-4). Expression of Toll-Like Recep tor 4 in Periodontal Tissues Induction of expression of TLR-4 occurs in a similar manner to that of TLR-2, being expressed by multiple cell types within the perio dontium, such as epithelial cells, fibroblasts, cementum, osteoblasts and oste oclasts. (5) In addition, th e LPS of multiple periodontal pathogens such as A. actinomycetemcomitans and Fusobacterium nucleatum ligate TLR-4 inducing a pro-inflammatory response (5) Similar to TLR-2 expression experiments, our intent was to establish the relationship between periodontal health status and TLR-4 expression in periodontal tissues. Just as with here we used TLR-4 specific primers and qPCR with pe riodontally healthy and diseased tissues again normalizing to GAPDH. In these experiments, TLR-4 was shown to be up regulated in periodontally diseased tissues ( p value < 0.0001 via the Mann Whitney Test.) (Fig 4-1, 4-3) Adjustment to the data analysis by removing thos e with a smoking and/or diabetes status did not alter the significance of the up regulation of TLR-4 observed in periodontally dise ased tissues (44). Comparison of Expression Patterns for RAGE, TL R-2 and TLR-4 While the above experiments demonstrated th at both RAGE and TLR-4 were up regulated in periodontally diseased tissues, it was not clear whether those patients with high levels of RAGE also had high levels of TLR-4. At the sa me time the inverse relationship of TLR-2 and TLR-4 expression by a given participant was not addressed. To answer these questions, we performed regression analysis to determine if there was a correlation between the expression of

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29 RAGE, TLR-4 and TLR-2 within a given individual Here we determined if RAGE expression was high in diseased tissues, TLR -2 expression was consistently low in the same individual (r=0.7201) (Fig 4-5). Additionally, if RAGE expr ession was high, along with decreased TLR-2 expression TLR-4 expression was al so relatively high (r=0.9964) (Fig 4-5). Therefore we can state that both increased RAGE and TLR-4 expressi on directly correlate w ith periodontal disease status. The opposite held true for TLR-2 expres sion, with high expression levels having an inverse relationship with periodon tal disease status (Fig 4-5). Analysis of Cell Types of the Pe rio dontal Tissue Expressing RAGE Utilizing histological analysis, we were able to determine that epithe lial, endothelial, and fibroblasts, among other cells were included in the cell types analyzed for gene expression. In addition, mononuclear cells were found to be pr esent in the diseased tissues as well. Hemotoxylin and Eosin stained sect ions (figures 4-6, 4-7) were pr epared in order to determine typical cellular contents of samples taken. These cell types were identified in healthy, as well as diseased tissues. Additionally, RAGE expression was qualitatively determ ined by utilization of immunohistochemistry. Analysis of expression in healthy and diseas ed tissues revealed relatively little expression in the healthy segmen t of tissues. (Figure 4-8) The diseased tissues, however, demonstrated strong e xpression in areas of many cell types, including epithelial, endothelial, fibroblast and macropha ge cells. (Figure 4-9) RAGE protein expression was also qualitatively up regulated on most cells types, not just the immune infiltrate.

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30 Table 4-1. Demographic data of participant population. Table indicates sex, smoking status, diabetes status and number of participants that were diabetics in addition to smokers. Healthy (15) Diseased (19) Males/Females 5/10 6/13 Smokers/Non-Smokers 2/13 7/12 Diabetics/Non-Diabetics 0/153/16 Smoker+Diabetic 01 Age Range 18-5725-70

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31 RAGE TLR2 TLR4 RAGE TLR2 TLR4 10-1 100 101 102 103 104 105 106 107 108 109 1010 Healthy Diseased Figure 4-1. Quantitative gene expression in peri odontal tissues. Periodontal tissues from 19 diseased (black) and 15 healt hy (white) participants were subjected to qPCR. Gene expression for RAGE (squares), TLR-2 (circles) and TLR-4 (triangles) was normalized to GAPDH and copy number quantita ted using amplicon specific standard curves. *p value < 0.0004 RAGE healthy vs. diseased, # p value < 0.0007 TLR-2 healthy vs. diseased, ^p value <0.000 1 TLR-4 healthy vs. diseased.

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32 Figure 4-2. RAGE expression in healthy and diseased subjects. Differences between expression in healthy and disease subj ects were significant (p<.05)

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33 10-1 100 101 102 103 104 105 106 107 108 109 1010 1011 healthy (n=16) diseased (n=19) TLR 2 TLR 4 healthy (n=16) diseased (n=19) Figure 4-3. TLR-2 and TLR-4 expression compar ison for healthy and diseased subjects. Differences between TLR-2 expression in healthy and diseased subjects was significant (p<.05). Differences between TLR -4 expression in healthy and diseased subjects was significant (p<.05).

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34 Figure 4-4. Gene expression in all subjects, excluding smokers and diabetics. Exclusion of smokers and diabetics yielded similar re sults, with differences among RAGE, TLR-2 and TLR-4 staying significant (p<.05) after exclusion.

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35 10-2 10-1 100 101 102 103 104 105 106 107 108 109 1010 100 101 102 103 104 105 106 107 108 109 1010r value = 0.9964 p value < 0.0001 copy number of RAGE 10-2 10-1 100 101 102 103 104 105 106 107 108 109 1010 10-1 100 101 102 103 104 105 106 107 108 109 1010r value = -0.7201 p value < 0.0001 copy number of RAGE 100 101 102 103 104 105 106 107 108 109 1010 10-1 100 101 102 103 104 105 106 107 108 109 1010r value = -0.5716 p value = 0.0004 copy number of TLR4 Figure 4-5. Correlation Data among RAGE, TLR-2 and TLR4. RAGE-TLR-4 correlation r=0.9964, p<.0001. RAGE-TLR-2 correlation r=-0.7201, p<.0001. TLR-2-TLR-4 correlation r=-0.5716, p=.0004.

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36 Figure 4-6. Histological examination of health y periodontal tissue. Re presentative section of healthy tissue which was s ectioned and stained with hemotoxylin and eosin for histological examination of ce ll types present in sample (n=5 ). Note the presence of epithelial cells (A), fibroblast s (B) endothelial cells (C) In addition, note the lack of inflammatory infiltrate and hyper-vascularity.

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37 Figure 4-7. Histological examination of healthy periodontal tissue.. Representative section of diseased tissue which was sectioned and stained with hemotoxylin and eosin for histological examination of ce ll types present in sample (n=5 ). Note the presence of epithelial cells (A), fibrobla sts (B), endothelial cells (C) macrophages (dark-staining cells). In addition, note the inflammato ry infiltrate (D and Circle) invading connective tissue as well as th e high degree of vascularity.(E).

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38 Figure 4-8. Immunohistol ogy staining for RAGE in healthy pe riodontal tissues. Darker brown staining areas indicate RAGE expression. Expr ession is seen in epithelial cells (A), fibroblasts (B) and endothelial cells (C). Note distinct lack of heavy macrophage infiltration, as well as normal vascularity.

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39 Figure 4-9. Immunohistology staining for RAGE in diseased pe riodontal tissues. Darker brown staining areas indicate RAGE expression. Expr ession is seen in epithelial cells (A), fibroblasts (B) endothelial cells (C ) and macrophages (D and circle).

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40 CHAPTER 5 DISCUSSION Periodontal disease, as well as m any other degenerative diseas es, is characterized by tissue damage as a result of an inflammatory response. The goal of periodontal therapy is reduction of the inflammatory response by removal of offe nding pathogens and their products, which results in improved tissue health and increased support for the affected dentition.40 It was the intention of this investigation to quantify the expression of innate immune receptors commonly found to be up regulated as a result of periodontal disease, as well as expression of RAGE, which has been shown to contribute to a host of inflammatory-based diseases.4 Utilizing qPCR, expression of innate immune receptors TLR-2 and TLR-4 and RAGE were quantified in peridontally diseased and periodontally healt hy tissues. It was demonstrated that both tissue types expressed varying amounts of all three receptors studied (Fig. 4-1, 4-4). However, there were significant differences betw een expression for diseased and healthy tissues (Fig. 4-1, 4-4). RAGE, which is capable of binding several di fferent ligands, (AGEs, S100, HMGB1) was shown to be up regulated in periodontally diseased versus healthy tissues (Fig. 4-1, 4-2, 4-4). While it has been demonstrated th at RAGE expression is increased in diseased gingival samples in the past, those studies involved pa tients with diabetes and/or smoking.33 To our knowledge, this is the first demonstration of increased RAGE expression without confounding variables already shown to increase R AGE expression (Fig. 4-4). Interestingly, TLR-2 did not follow the expression pattern that has been traditionally elucidated5 (Fig. 4-3). In contrast to the expected increased expression in diseased tissues, a down-regulation was demonstrated in this population of patients (Fig. 4-3). Reasons for this difference are unknown at this time. However, recen t work in our lab (Amir et al.) has shown an

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41 anti-inflammatory response by gingival epithelia l cells (GEC) when stimulated with LPS from P. gingivalis. This response resulted in th e release of anti-inflammatory cytokines IL-4 and IL-10. This type of response does not lead to the clearance of pathogen and ther efore may be the result of a bodily response to maintain homeostatic inte grity in the face of dead bacterial components some of which maybe from commensal organism s consistently stimulating the epithelial component of the innate immune system. Therefor e, in the face of an in tense bacterial infection such as periodontal disease the immune syst em down-regulate those receptors, which, upon ligation result in anti-inflammato ry responses, allowing for a prope r immune response to occur. It would be interesting to determine if there is a relationship with state of disease progression and TLR-2 expression. One would hypothesize that once the bacterial insult has been controlled by treatment and the pro-inflammatory response is no longer needed, that TLR-2 expression levels may rise to again maintain homeostasis between commensal organisms and the innate immune response. Unlike TLR-2 expression, there was a signi ficant and definitive increase in TLR-4 expression in diseased tissues as compared to periodontally healthy tissues (Fig. 4-1, 4-3, 4-4). The up regulation of TLR-4 in diseased periodontal tissues seems to be the prototypical response of the innate immune system25 Here, TLR-4 ligation by PAMPs, which are highly conserved structures in many pathogenic bact erial species results in a robus t pro-inflammatory response in order to immediately destroy an d/or control the offending agent .. It has been hypothesized by many that TLR-4 and RAGE interact to augmen t and/or enhance inflammatory responses under certain conditions. Indeed recent research, has demonstrated a corr elation between TLR-4 ligation and RAGE ligand interactions. Here, low density lipoprotein (LDL) AGEs were used to stimulate human coronary artery endothelial cells, as well as human and mouse macrophages.41

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42 When TLR-4 loss of function was induced or TLR-4 binding was blocked by a competitive inhibitor, in mouse macrophages, cyt okine synthesis was markedly reduced.41 These results indicated that there may be an activation of TLR -4 signaling pathways by stimulation with AGE. In addition, HMGB1 a RAGE ligand and pro-infla mmatory cytokine. implicated in cancers, mostly due to its pro-angiogenic factor, seems to potentiates the pro-inflammatory response through activation of NFkB, using a similar si gnaling pathway as TLR-2 and TLR-4, again resulting in a positive feedback mechanism that signals for other inflammatory mediators.42 These data taken together coupled with the data presented here i ndicate there may be implications for RAGE and TLR interactions be yond the cardiovascular system and cancer, such as the dentogingival complex. We were able to determine that epithelial, e ndothelial, fibroblast, etc. cells were included in the cell types analyzed for gene expression. Hematoxylin and Eosin stained sections were analyzed for the above cell types. It was determ ined that representative sections of healthy and diseased tissues contained the above cell types, regardless of di sease status. However, there were heavy immune cell infiltrates present within the diseased tissues. (Figures 4-6, 4-7) Immunohistology was also completed for patient samples. Healthy and diseased tissues were shown to express RAGE, although, diseased sa mples demonstrated a much higher intensity in certain locales. RAGE prot ein expression was also qualitativ ely up regulated on most cells types, not just the immune infilt rate. There appeared to be expression from all identifiable tissue types, however, the expression was not homogene ous per tissue type. The healthy tissues showed a more homogeneous expression profile, indicating normal, homeostatic expression, without distinct expression in any one area or tissue.

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43 Correlation analysis was also completed with th e data. It was confirmed that patients who exhibited an increased expression of RAGE al so had increased expression of TLR-4 and a decreased expression of TLR-2. What remains to be seen, however, is the interaction among AGE levels, the innate and adapted immune responses and chronic disease states. With increasing elucidation of the mechanisms at a cellular level by which the interactions occur, the question continues to remain as to whether there are implicati ons for the body as a whole, and to what extent. AGE levels have documented effects on disease process such as cardiovascular disease, sequalae from diabetes and periodontal disease. The grow ing perception seems to point toward an interrelationship among all of these diseases and that there is an oral-systemic connection. In essence, that the presence of periodontal disease may have a causal effect for chronic inflammatory disease due to its contribution to the regulation of TLR a nd RAGE expression. At this point, the evidence is strong for association, but, not causality. As expected, the receptor for AGE seems to be a likely participant in this relationship. RAGE has the capability to bind to several li gands (AGE, Calgranulin/S100, HMGB1), which is unique in terms of immune receptors. Additionall y, RAGE is expressed in a variety of tissues, and systems, including endothelial, fibroblasts, m acrophages, etc. Due to the ability for RAGE to bind to many ligands and its ubiquitous nature throughout tissues, coupled with the potential of the innate immune response, RAGE up regulation may hold the key to understanding many of the mechanisms chronic inflammatory diseases. While quite a bit is known about RAGE expression and its association with inflammatory diseases, such as periodontal dis ease as described here, further re search is certainly needed to elucidate the mechanisms of how it contri butes to disease init iation and progression.

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44 LIST OF REFERENCES 1. Petersen PE, Ogawa H. Strengthening the prevention of periodontal disease: the W HO approach. J Periodontol. 2005 Dec;76(12):2187-93. Review. 2. Beck JD, Garcia R, Heiss G, Vokonas PS, Offenbacher S. Periodontal disease and cardiovascular disease. J Periodontol 1996;67:1123-1137. 3. Grossi SG, Genco RJ. Periodontal disease and diabetes mellitus: a two-way relationship. Ann Periodontol 1998; 3:51-61. 4. Singh R, Barden A, Mori T, Beilin L. Advanced glycation en d-products: a review. Diabetologia. 2001 Feb;44(2):129-46. Review. Erratum in: Diabetologia 2002 Feb;45(2):293. 5. Mahanonda R, Pichyangkul S. Toll-like receptors and their role in periodontal health and disease. Periodontology 2000. 2007;43:41-55 6. Lalla E, Lamster IB, Schmidt AM. Enhanced interaction of adva nced glycation end products with their cellular receptor RAGE: implications for the pathogenesis of accelerated periodontal disease in diabetes Ann Periodontol. 1998 Jul;3(1):13-9. Review. 7. Nanci A, Bosshardt DD. Structure of periodontal tissues in health and disease. Periodontology 2000. 2006;40;11-28 8. Gargiulo A, Krajewski J, Gargiulo M. Defining biologic width in crown lengthening. CDS Rev. 1995 Jun;88(5):20-3. Review. 9. Oshrain HI, Salkind A, Mandel ID. An histologic com parison of supra and subgingival plaque and calculus. J Pe riodontol. 1971 Jan;42(1):31-3 10. Pileicikiene G, Surna A. The human ma sticatory system from a biomechanical perspective: a review. Stomatologija, Baltic Dental and Maxillofacial Journal, 2004, Vol. 6., N. 3. 11. Andersen GB, Caffasse JJ, Nasjleti C, Smith BA. (1991) Correlation of periodontal probe penetration and the degree of inflammation. American Dental Journal. 4, 177-183 12. Lang NP, Adler R, Joss A, Nyman S. Absen ce of bleeding on probing. An indicator of periodontal stability. J Clin Pe riodontol. 1990 Nov;17(10):714-21. 13. Armitage, GC. Development of a classifi cation system for periodontal diseases and conditions. Annals of Periodontology. 1999; 4:1-6 14. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. Journal of C linical Periodontology. 1998;25(2):134-44

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45 15. Weinmann J. Progress of gingival inflammation into the supporting st ructure of the teeth. J Periodontol 1941;12:71. 16. McGuire MK, Nunn ME. Prognosis versus actual outcome, part II: the effectiveness of clinical parameters in developing an accu rate prognosis. J Periodontol 1996;67(7):658 17. Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge. Periodontology 2000. 1997;14:33-53 18. Salvi GE, Lawrence HP, Offenbacher S, Beck JD. Influence of risk factors on the pathogenesis of periodontitis Periodontol 2000 1997;14: 173. 19. Grossi, SG; Zambon, JJ, Ho AW. Assessment of risk for periodontal disease. I. Risk indicators, for attachment loss. Journal of Periodontology. l994;65:260. 20. Ah MK, Johnson GK, Kaldahl WB, Patil KD, Kalkwarf KL. The effect of smoking on the response to periodontal therapy. J Clin Periodontol. 1994 F eb;21(2):91-7. 21. Caton JG, Zander HA. The attachment between tooth and gin gival tissues after periodic root planing and soft tissue curetta ge. J Periodontol. 1979 Sep;50(9):462-6. 22. Prichard, J. Advanced Periodontal Disease: Surgical and Prosthetic Management. 2nd ed. s.l.: 265, 1965 23. Page RC, Schroeder HE. Pathogenesis of inflammatory pe riodontal disease. A summ ary of current work. Lab Invest 1976 Mar;34(3):235-49. Review. 24. Mochizuki S, Kobayashi M, Suzuki T, Oikawa A, Koseki T, Nishihara T, Hasegawa K. Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival broblasts. J Periodont al Res 2004: 39: 333. 25. Hirschfeld M, Weis JJ, Toshchakov V, Sal kowski CA, Cody MJ, Ward DC, Qureshi N, Michalek SM, Vogel SN. Signaling by toll-l ike receptor 2 and 4 agonists results in differential gene expression in murine m acrophages. Infect I mmun 2001: 69: 1477. 26. Abreu MT. Immunologic regulation of toll-like receptors in gut epithelium. Curr Opin Gastroenterol 2003: 19: 559 564. 27. Mitsuhashi T, Vlassara H, Founds HW, Li YM. 1997. Standardising the immunological measurement of advanced glycation end pr oducts using normal human serum. J Immunol Methods 207: 79-88 28. Bucala R, Cerami A. 1992. Advanced glycat ion: chemistry, biology and implications for diabetes and aging. Adv Pharmacol 23: 1-34

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46 29. Li JT, Hou FF, Guo ZJ, Shan YX, Zhang X, Liu ZQ. Advanced glycation end products upregulate C-reactive protein synthesis by human hepatocytes through stim ulation of monocyte IL-6 and IL-1 beta producti on.. Scand J Immunol. 2007 Nov;66(5):555-62. 30. Hsieh HL, Schfer BW, Sasaki N, Heizmann CW. Expression analysis of S100 proteins and RAGE i n human tumors using tissue microarrays. Biochem Biophys Res Commun. 2003 Jul 25;307(2):375-81. 31. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Nagashima M, Lundh ER, Vijay S, Nitecki D. RAGE is a cellular binding s ite for amphoterin: mediation of neurite outgrowth and co-expression of RAGE and amphoterin in the developing nervous system. J Biol Chem. 1995;270:25752 32. Ellerman JE, Brown CK, de Vera M, Zeh HJ, Billiar T, Rubartelli A, Lotze MT. Masquerader: high mobility group box-1 and cancer. Clin Cancer Res. 2007 May 15;13(10):2836-48. Review. 33. Katz J, Bhattacharyya I, Farkhondeh-Ki sh F, Perez FM, Caudle RM, Heft M W. Expression of the receptor of advanced glyca tion end products in gingival tissues of type 2 diabetes patients with chronic pe riodontal disease: a study utilizing immunohistochemistry and RT-PCR. Journa l of Clinical Periodontology. 2005. 32:40-44 34. Katz J, Caudle RM, Bhattacharyya I, Stew art CM, Cohen DM. Receptor for advanced glycation end product (RAGE) upregulation in human gingival fibroblasts incubated with nornicotine. Journal of Periodontology. 2005;76:1171-1174 35. Socransky SS. New concepts of destructive periodontal disease. Journal of Clinical Periodontology. 1984;11:21-32 36. Lalla E, Lamster IB, Feit M, Huang L, Spesso t A, Qu W, Kislinger T, Lu Y, Stern DM, Schmidt AM. Blockade of RAGE suppresse s periodontitis-associated bone loss in diabetic mice. The Journal of C linical Investigation. 2000;105:1117-1124 37. Kirkwood KL, Cirelli JA, Rogers JE, Giannob ile WV. Novel host response therapeutic approaches to treat periodontal di seases. Periodontology 2000. 2007; 43:294-315 38. Schmidt AM, Yan SD, Yan SF, Stern DM. The biology of the receptor for advanced glycation end products and its ligands. Biochimica et Biophysica Acta 1498. 2000; 99111 39. Lin L. RAGE on the toll road? The Chinese Society of Immunology. 2006. 351-358 40. Meador HL, Lane JJ, Suddick RP. The long-term effectiveness of periodontal therapy in a clinical practice. J Peri odontol. 1985 May;56(5):253-8. 41. Hodgkinson CP, Laxton RC, Patel K, Ye S. Adva nced glycation end-product of low density lipoprotein activates the toll-like 4 receptor pathwa y implications for diabetic atherosclerosis. Arterioscler Thromb Vasc Bi ol. 2008 Dec;28(12):2275-81.

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48 BIOGRAPHICAL SKETCH Dr. Matthew Waite studied International Relations at the University of South Florida, where he graduated in the spring of 1997. After which, he attended dental school at Louisiana State University where he received his Doctor of Dental Surgery degree in the summer of 2006. Currently, Matthew Waite is co mpleted his post-doctoral residency in periodontics at the University of Florida. Upon graduation in the spring of 2009, Matthew returned to Tampa, Florida to practice clinical periodontics.