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Role of Oral Epithelial Cell Toll-Like Receptors in the Onset and Progression of Periodontal Disease

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Title:
Role of Oral Epithelial Cell Toll-Like Receptors in the Onset and Progression of Periodontal Disease
Creator:
Rasmussen, Richard Allyn III
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
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1 online resource (45 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Dental Sciences
Dentistry
Committee Chair:
WALLET,SHANNON MARGARET
Committee Co-Chair:
KOUTOUZIS,THEOFILOS
Committee Members:
LAKSHMYYA,KESAVALU NAIDU
YILMAZ,OZLEM
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Bones ( jstor )
Cells ( jstor )
Connective tissues ( jstor )
Diseases ( jstor )
Epithelial cells ( jstor )
Epithelium ( jstor )
Inflammation ( jstor )
Mice ( jstor )
Periodontal diseases ( jstor )
Teeth ( jstor )
Dentistry -- Dissertations, Academic -- UF
epithelial-cells -- inflammation -- myd88 -- periodontitis -- toll-like
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Dental Sciences thesis, M.S.

Notes

Abstract:
The oral cavity is a gateway for a wide array of antigenic challenges where alterations in oral mucosal homeostasis can lead to inflammatory diseases such as periodontitis. Epithelial barrier function and mucosal immunity depend on interactions between commensal microbiota and pathogens respectively with toll-like receptors (TLRs) on epithelial cells. Here we wanted to determine if TLR induced oral epithelial cell responses contribute to periodontal disease. Myeloid differentiation factor 88 (MyD88) is a required signaling adaptor protein for most TLRs and is expressed in oral epithelium and resident immune cell populations. In mice, expression of dominant- negative MyD88 in intestinal epithelial cells causes spontaneous intestinal inflammation. In contrast, ubiquitous MyD88 deficiency prevents induced intestinal inflammation. These data support the hypothesis that MyD88-dependent signaling plays distinct roles in epithelial cells versus resident innate immune cells. The purpose of this research is to evaluate the role(s) of TLR oral epithelial cell specific responses in the progression of periodontal disease. A well described poly-microbial model of periodontal disease induction and inducible epithelial cell specific MyD88 knockout mice (B6K5Cre.MyD88plox) were utilized. Following knockdown of MyD88 in the oral epithelium, mice were infected with Porphorymonas gingivalis and Aggregatibacter actinomycetemcomitans by oral lavage 4 times per week, every other week for 12 weeks, after which maxillae and mandibles were subjected to bone morphometric and histological analysis. An increase in total bone loss and soft tissue changes were observed in the epithelial cell specific MyD88 deficient mice compared to infected controls. A difference in the level of local inflammation was also observed. These results indicate that oral epithelial cell MyD88-dependent TLR signaling is responsible for tolerogenic immune tuning which counteracts otherwise inflammatory responses and that oral epithelial cells can be a target for periodontal therapies aimed at controlling inflammation. ( en )
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.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: WALLET,SHANNON MARGARET.
Local:
Co-adviser: KOUTOUZIS,THEOFILOS.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31
Statement of Responsibility:
by Richard Allyn III Rasmussen.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2015
Classification:
LD1780 2014 ( lcc )

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1 ROLE OF ORAL EPITHELIAL CELL TOLL LIKE RECEPTORS IN THE ONSET AND PROGRESSION OF PERIODONTAL DISEASE By RICHARD ALLYN RASMUSSEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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2 2014 Richard Allyn Rasmussen

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3 To my father, Richard Allyn Rasmussen, Jr., the person who has most inspired me in my life

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4 ACKNOWLEDGMENTS First and foremost, I would like to thank my father. Without his constant support, I would not be the man I am today. I can only hope that one day I can become half the man he is. I would also like to thank my family, which has supported me throughout my professional endeavors without hesitation. Additionally, I would like to thank my fiance, whom has tirelessly supported my goals and encouraged me throughout this journey.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 8 CHAPTER 1 INTRODUCTION .................................................................................................... 11 2 BACKGROUND ...................................................................................................... 15 The Periodontium in Health .................................................................................... 15 Periodontal Tissue Destruction ............................................................................... 16 Tolllike Receptors and Periodontal Disease .......................................................... 19 Role of Epithelial Cells in Health and Disease ........................................................ 20 Murine Models of Periodontal Disease ................................................................... 21 3 MATERIALS AND METHODS ................................................................................ 24 Mouse Models ........................................................................................................ 24 Induction of Periodontal Disease ............................................................................ 24 Immunohistochemistry ............................................................................................ 25 Histochemical Analysis of Periodontal Tissue ......................................................... 25 Bone Morphometric Analysis of Bone Loss ............................................................ 26 ......................................................... 26 Statistics ................................................................................................................. 27 4 RESULTS ............................................................................................................... 29 Epithelial Cell Specific Knockdown of MyD88 ......................................................... 29 Oral Epithelial Cell MyD88 / Results in Exacerbated Soft Tissue Changes ........... 29 Oral Epithelial Cell MyD88 / Results in Exacerbated Bone Loss ........................... 30 Oral Epithelial Cell MyD88 / Results in Exacerbated ProInflammatory Cytokine Production ........................................................................................................... 30 5 DISCUSSION ......................................................................................................... 35 REFERENCES .............................................................................................................. 40 BIOGRAPHICAL SKETCH ............................................................................................ 45

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6 LIST OF TABLES Table page 3 1 Experimental groups ............................................................................................. 28

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7 LIST OF FIGURES Figure page 1 1 Experimental design. ............................................................................................. 14 4 1 Murine oral epithelial cell knockdown of MyD88. .................................................. 31 4 2 S oft tissue changes in the presence and absence of epithelial cell MyD88 expression. ......................................................................................................... 32 4 3 B one loss in the presence and absence of epithelial cell MyD88 expression. ...... 33 4 4 Mucosal TNF expression in the presence and absence of epithelial cell MyD88 expression. ............................................................................................. 34

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8 LIST OF ABBREVIATIONS CEJ cement enamel junction I/I induced infected I/U i nduced uninfected K5 keratin 5 MMPS matrix metalloproteinases MYD88 myeloid differentiation factor 88 PAMP pathogen associated molecular pattern PDL periodontal ligament PerioGard 0.12% chlorhexidine gluconate PR progesterone receptor TLR tolllike receptors U/I uninduced infected

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science ROLE OF ORAL EPITHELIAL CELL TOLL LIKE RECEPTORS IN THE ONSET AND PROGRESSION OF PERIODONTAL DISEASE By Richard Allyn Rasmussen May 2014 The oral cavity is a gateway for a wide array of antigenic challenges where alterations in oral mucosal homeostasis can lead to inflammatory diseases such as periodontitis. Epithelial barrier function and mucosal immunity depend on interactions between com mensal microbiota and pathogens respectively with tolllike receptors ( TLRs ) on epithelial cells. Here we wanted to determine if TLR induced oral epithelial cell responses contribute to periodontal disease. Myeloid differentiation factor 88 (MyD88) is a re quired signaling adaptor protein for most TLRs and is expressed in oral epithelium and resident immune cell populations. In mice, expression of dominant negative MyD88 in intestinal epithelial cells causes spontaneous intestinal inflammation. In contrast, ubiquitous MyD88 deficiency prevents induced intestinal inflammation. These data support the hypothesis that MyD88dependent signaling plays distinct roles in epithelial cells versus resident innate immune cells The purpose of this research is to evaluat e the role(s) of TLR oral epithelial cell specific responses in the progression of periodontal disease. A well described poly microbial model of periodontal disease induction and inducible epithelial cell specific MyD88 k nockout mice (B6K5Cre.MyD88plox) we re utilized. Following knockdown of MyD88 in the oral epithelium, mice were infected with Porphorymonas gingivalis and Aggregatibacter

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10 actinomycetemcomitans by oral lavage 4 times per week, every other week for 12 weeks, after which maxillae and mandibles were subjected to bone morphometric and histological analysis. An increase in total bone loss and soft tissue changes were observed in the epithelial cell specific MyD88 deficient mice compared to infected controls. A difference in the level of local infla mmation was also observed. These results indicate that oral epithelial cell MyD88dependent TLR signaling is responsible for tolerogenic immune tuning which counteracts otherwise inflammatory responses and that oral epithelial cells can be a target for periodontal therapies aimed at controlling inflammation.

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11 CHAPTER 1 INTRODUCTION A key requirement of the mucosal immune system is a mechanism(s) to limit or regulate immune responses to commensal microbes1. Dysfunc tion of mucosal innate systems impairs mucosal barrier function leading to localized and systemic inflammation1. The oral cavity is a gateway for a wide array of antigenic challenges where alterations in oral mucosal homeostasis, can lead to inflammatory diseases such as gingivitis and periodontitis1. To this end, the aim of my research was to understand the contribution of oral mucosal homeostasis in the localized inflammatory response of periodontal disease. Specifically, to determine if hyper active TLR induced oral epithelial cell responses contribute to the exacerbated periodontal disease. Thus I hypothesized, that TLR induced proinflammatory hyper reactivity of oral epithelial cells compromises barrier integrity allowing for hyper activation of the mucosal innate immunity resulting in increased soft and hard tissue destruction observed in periodontal disease. I ut ilize d mice that allow for the distinction between the roles of epithelial cell specific TLR responsiveness. Specifically, I utilize d the C57Bl/6 Keratin CrePR mice ( B6K5Cre)2. Keratin CrePR mice express a Cre rec ombinase progesterone receptor (PR) fusion protein whose expression is restricted by a Keratin 5 (K5) promoter. The CrePR is inducible by the progesterone antagonist RU486 but not by endogenous progesterone2. Upon induction with RU486, C rePR translocates into the nucleus and excises DNA sequences flanked by Lox P sites. I cross ed these mice with the B6.CgMyd88tm1Defr/J [B6MyD88plox]. These mice possess loxP sites on either side of exon 3 of the MyD88 gene. When these mutant mice are bred to B6K5Cre mice, offspring

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12 ( B6K5Cre.MyD88plox) will have MyD88 deleted in epithelial cells upon the administration of RU486. To test my hypothesis the following experimental design was implemented where I utilize d the mous e models described along with a well described poly microbial model of periodontal disease induction (Fig. 1 1). C 5 7Bl/6 and B6K5Cre.MyD88plox mice w ere treated daily with RU486 (allows for the knockdown of MYD88) for one week prior to receiving a polymicrobial infection consisting of P. gingivalis strain 381 and A actinomycetemcomitans strain 29522. 2.5x109 of each bacterium were mixed with 2% low viscosity carboxy methyl cellulose. Following a 3day antibiotic washout using 0.12% chlorhexidine gluconate ( PerioGard), mice were orally lavaged on 4 consecutive days per week for 12 weeks. Three weeks following the last infection protocol animals were sacrificed and the following analysis performed. Local inflammation: The maxillae and mandibles of the animals were removed and fixed in 10% buffered formalin, decalcified with Immunocal and all will be embedded and sectioned for histological analysis. After which 5 micron sections were stained with hematoxylin and eosin for inflammatory scoring. 10 images per animal were captured at 20x magnification and inflammation scored using PMN/mononuclear cell infiltration [0, no inflammatory cells; 1, minimal inflammation (scattered inflammatory cells close to the junctional epit helium); and 2, moderate inflammation (numerous inflammatory cells in the gingival connective tissue). Bone loss: The maxilla and mandible of the animals were removed, defleshed and immersed in 3% hydrogen peroxide. After which the cementoenamel junction (CEJ) was identified. To quantify horizontal bone loss, color digital images were

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13 captured under a 10x stereo dissecting microscope. Using morphometry image analysis software, the area of the horizontal bone resorption in mm2 was determined and expressed as the mean of measurements obtained for each of the four quadrants (left and right maxillae and mandibles). Soluble Mediators of Soft Tissue : The maxillae and manibles of the animals were removed and placed in protein lysis buffer with protease inhibit ors After which the evaluated using ELISA Epithelial cell specific knockdown of MyD88 was successful and resulting in exacerbated soft tissue destruction and an increa se in total bone loss along with elevated level s of local TNF These results indicate that oral epithelial cell MyD88dependent TLR signaling is responsible for tolerogenic immune tuning which counteracts otherwise inflammatory responses and that oral epithelial cells can be a target for periodontal therapies aimed at controlling inflammation.

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14 Figure 1 1 Experimental design. C57Bl/6K5Cre.MyD88plox mice were either left uninduced and uninfected (U/U), induced and left uninfected (I/U), left uninduced and infected (U/I), or induced and infected (I/I) (n=4/group). Induction = oral administration of RU486 for two days. All mice were lavaged with 0.12% chlorhexidine gluconate for three days. Infection consisted of an oral lavage with Porphorymonas gingivalis strain 831 and Aggregatibacter actinomycetemcomitans strain 29522 4 times per week, every other week for 12 weeks. After which the maxillae and mandibles were harvested for h istolo gy, b one m orphometric a nalysis, and ELISA.

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15 CHAPTER 2 BACKGROUND The Periodontium in Health The periodontium, or the supporting structures surrounding teeth, consists of several tissues. These tissues serve not only to support the teeth, but, to provide protection against bacterial infiltration and disease. These tissues consist of bone, cementum, connective tissue, and epithelium3. In health, these separate tissues comprise a very strong support structure for the teeth that provide adequate protection against bacterial plaque3. The normal, healthy structure of the periodontium has been studied extensively. T he epithelium attaches to the tooth anywhere from 0.671mm apical to the cementoenamel junction (CEJ) and is approximately 1mm in dimension. The connective tissue typically comprised of collagen, ground substance and fibroblasts, attaches to the tooth from the point of epithelial attachment and comprises an additional millimete r4. The connective tissue attachment approximates the bone and from the bone to the apex of the tooth root, the periodontal ligamen t (PDL) and bone are continuous. There is minor variation among people, as well as tooth type with respect to the above dimensions. The sum of these measurements is termed the biologic width4. In health, these measurements do not stray far from one millimeter each for the sulcus, epithelial attachment and connective tissue attachment. The epithelium serves to provide protection to bacterial invasion of the PDL and bone, as well as mechanical protection. In disease, through repeated insult, the epithelial attachment may become weakened and eventually detached4. The lack of attachment of the epithelial barrier allows a bacterial plaque biof ilm to form

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16 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 plaque that serves as a constant irritant to the connective tissue attachment. Given more time, plaque further accumulates apically and the disease progresses5. The main function of bone in the peridontium is to support teeth in the process of mastication6. Forces in the human masticatory system sometimes reach 600750N in Americans and Europeans6. T issues in the system, such as the PDL, tend to distribute the load through a slig htly movable system6 (10) While the PDL does provide some of the support, the majority of forces are borne by the mandible and the maxilla6. The main functional capacity of the PDL is to stabilize the tooth within its bony socket, as well as to provide somatosensory information and nutrition6. The destruction of these tissues results in loss of attachment, increas ed probing depth and ultimately, loss of the tooth due to lack of proper bony support. Periodontal Tissue Destruction Diagnosis of periodontal disease is currently accomplished according to collection of clinical data namely probing depth, recession, ble eding 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 millimeter, to which the probe will reach. Typicall y, this is in the two to three millimeter range in health and does not extend beyond the epithelial attachment. In disease, the periodontal probe passes the epithelium and extends into connective tissue7. Recession indicates t issue loss from the cementoenamel junction (CEJ) to the gingival margin. While t he presence of bleeding on probing does not necessarily

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17 indicate disease, it can indicate the possibility that inflammation is present at a particular site8. Involvement of furcation is when there exists enough bone loss as to expose the area between roots to probing, where by mo bility indicates the lateral movement of a tooth. Lack of mobility is typically considered a sign of better prognosis and health9. The sum of probing depth and recession, from the l evel of the CEJ is termed attachment loss. The higher the degree of attachment loss, the more support has been lost from the tooth. Periodontal disease is an inflammatory disease classified according to the type of tissue involved as well as the location, duration and severity of the disease. The current classification system, established in 1999, is widely used to diagnose periodontal di sease10. In addition to nonmicrobially induced forms of the disease, such as viral infections, fungal infections, etc. the disease origins includes those of the bacterial nature (plaqueinduced)10. 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 occurred10. Many bacterial species have been implicated in the process of periodontal destruction. In 1983, Haffajee and Socransky identified several bacterial species associated with periodontal diseases11. In addition to identifying some of the present bacteria via DNA checkerboard hybridization, they attempted to associate certain species with more severe forms of the disease11. Several groups were described, with the red and orange complexes being associated more closely with disease t han the other groups12. Two species of bacteria that have been readily identified with periodontal disease, both chronic and aggressive, respectively, are Porphorymonas

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18 gingivalis and Aggregatibacter Actinomycetemcomita ns12. Each of these species is associated with the Red complex and Green complexes, respectively12. Both of these species have been s hown to persist in the periodontium despite effort of nonsurgical therapies. Aggregatibacter Actinomycetemcomitans has been shown to be able to invade gingival tissues13. This invasion was found to be a multi step process that i nvolves: entry, escape from vacuole, rapid multiplication, and inter/intra cellular spread13. Additionally, Porphorymonas gingivali s has been demonstrated to invade within epithelial cells and replicate within them13. The ability of these bacterial species to invade and persist within the periodontal soft tissues likely contributes to the chronicity of periodontal diseases and could possible explain why nonsurgical therapy alone cannot completely eliminate pathogens13. The immune response mounted by the body to the bacterial infiltration releases a host of inflammatory mediators that are able to destroy the periodontal tissues14. Soon after the junctional epithelium has been violated, access to the PDL and underlying connective tissues is possible14. Bacterial toxins, such as lipopolysaccharides, induce the production of inflammatory mediators, such as proteases, cytokines and prostaglandins to assist in resistance of the bacterial invasion14. This response, intended to neutralize bacteria, also has the untoward effect of destroying the periodontal tissues along with the bacteria. The resultant tissue destruction is the basis for the loss of tissue seen in periodontitis6. Extension of the inflammation is made possible by the proliferous blood supply within the periodontium. Vessels extending into the connective tissue, periosteum and bone allow migration of bacteria, bacterial toxins, and proinflammatory mediators

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19 througho ut the tissues6. The resultant immune response via proinflammatory cytokines, such as IL1 atrix metalloproteinases (MMPs) leads to the resorption of bone and other connective tissues6. This inflammatory process is capable of destroying supporting periodontal tissues over a long period of time, as with c hronic periodontitis, or within a short time frame, as with aggressive periodontitis. Toll like Receptors and Periodontal Disease As mentioned abouve, periodontitis is characterized by an active increase in the immune system, both innate, as well as adapt i ve as a result of bacterial insult. The early periodontal lesion can be histologically typified by t he acute invasion of PMNs Later in this progression, the established lesion c onsists of an intense plasma cell invasion15. The advanced lesion, however, is the first stage of the lesion 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 involvement of the innate immune response16. Integral in this response are toll like receptors (TLRs)16. Tolllike receptors are intracellular and extracellular proteins that form the first response to bacterial and viral infiltration16. They often initiate a response based upon bacterial or viral products that are present, such as lipopolysaccharides, peptidoglycan, flagellin, foreign DNA and RNA, as well as bacterial byproducts16. TLRs are homologous to the IL1 str ucture, but, contain a number of leucinerich repeats in their structure16. The leucinerich repeat areas correspond to certain P athogenassociated molecular patterns (PAMPs) whereby TLRs dimerize following recognize of these different bacterial products16. Lipopolysaccarides and other components of bacter ial products function as PAMPs and thus trigger a signaling cascade from TLRs on specific

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20 cells within the innate immune system and architecture of the periodontium, including monocytes, neutrophils, dendritic cells, epithelial and endothelial cells. Specifically, it has been show n that both TLR2 and TLR4 are upregulated in the presence of bacterial insult within the periodontium, although which cell ty pes are involved is unclear17, 18. Triggering of TLR2 and TLR4, among other toll like receptors, result in the upregulation of many soluble mediators such as cytokines, ch emokines and growth factors19. These soluble mediators initiate multiple cell signaling pathways resulting in the activation of T cells and B cells, the hallmark of the established periodontal lesion and part of the adaptive immune system19. Chronic stimulation of toll like receptors, as is found in plaqueinduced periodontitis, releases a continuous stream of pro inflammatory mediators, one of the mechanisms responsible for the tissue destruction seen in periodontitis16. Role of Epithelial Cells in Health and Disease The oral epithelial cells involved in protection of the periodontal apparatus (cementum, periodontal ligament, and alveolar bone) can be broken into several parts: the masticatory gingival epithelium, the crevicular gingival epithelium, and the junctional epithelium. Gargiulo et al. established the dimensions and relationships of the dentogingival junction in humans. It was determined that the average mean measurements for sulcus depth was 0.69 mm; the epithelial attachment, 0.97 mm; and the connective tissue attachment, 1.07 mm5. The connective tissue attachment length was the most consistent and the epithelial attachment length was the most variable20. Histologically, hemidesmosomes, form the attachment of epithelial cells to basement membrane and to the tooth surface and are a key component in defense against

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21 bacterial invasion and sub sequent inflammation21. Several specializ ed cells exist within the epithelium, including melanocytes, Merkel cells, and Langerhans cells. Langerhans cells are dendritic cells that play a role in the immune surveillance system. Their numbers increase in inflamed tissue (2 to 10x) with cells migrat ing from the underlying connective tissue in response to antigenic challenge22. It is postulated that the role of Langerhans cells is in the uptake and presentation of antigens to T cells, possible constituting one of the first lines of defense against surface penetration of the host by forcing antigens22. It has more recently been determined that oral epithelial cells are also a first line of defense to potential infectious pathogens within the oral cavity23. These key cells serve multiple immune roles along the mucosal surface functioning as: 1) a physical barrier to microbes; 2) a source of antimicrobial peptides which i nhibit microbial growth; 3) inflammatory immune cells which produce cytokines to trigger antimicrobial immunity; and 4) tolerogenic immune cells which can produce immunoregulatory cytokines or remain quiescent in response to nonpathogenic commensal microo rganisms23. Murine Models of Periodontal Disease There are four main mouse models that are used to study the pathogenesis of periodontitis and to assess therapeutic modalities against the disease: the Baker mouse model, chemically induced mouse model, m urine incisor abscess model, and the murine back abscess model24. The B aker mouse model of periodonti tis has been used to measure alveolar bone resorption caused by oral bacterial inoculums as an outcome for the clinical presentation of periodontitis in humans25. To assess the virulence of periodontal

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22 pathogens, specific pathogenfree mice are orally infected with strains of A. actinomycetemocomitans and/or P. gingivalis Prior to infection, mice are given antibiotics (sulfamethoxazole and trimethoprim) in their water for about 10 days to suppress the normal oral microflora. Mice are then treated by oral gavage five times at 2 day intervals with one type or an admixture of bacteria resuspended in carboxymethylcellulose to establish the infection15, 26, 27. The chemically induced mouse model is an alternative method for inducing inflammation of oral tissues is by using trinitrobenzene sulfonic acid (TNBS) or dextran sulphate sodium (DSS)28, 29. Oral administrat ion of TNBS resulted in a localized action on periodontal tissues with alveolar bone loss observed in both maxilla and mandibles with progression in a timedependent manner. In the murine incisor abscess model, r odent incisors do not have roots and are co ntinually erupting. To induce a gum pocket abscess model, outbred ICR mice (3 6 weeks old) were injected for 3 days into the gums of lower incisors with F. nucleatum that naturally does not colonize mice which induses a short term infection resulting in sw elling at the site of injection30. Histological examination using H&E staining will demonstrate granuloma formation within the inflamed gum. This model needs repeated injections of the bacteria and has a limited use in studying gum pocket abscess to mimic chronic halitosis caused by microbial infection30. The murine back abscess model has also been used to investigate the interactions of both oral microbial species and host responses to various oral pathogens as monomicrobial infections leading to soft tissue destruction3133. Although, the lesions are not located in the oral cavity, this model has some value for examining bacterially

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23 induced infections/coinfections that result in soft tissue destruction33. In the studies presented here a modified Baker model was utilized whereby a polymicrobial model of periodontal disease induction and inducible epithelial cell specific MyD88 knockout mice were utilized. Following knockdown of MyD88 in the oral epithelium, mice were incfected with P.g. and A.a. by oral lavage 4 times per week, every other week for 12 weeks, after which m axillae and mandibles were subjected to bone morphometric and histological analysis.

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24 CHAPTER 3 MATERIALS AND METHODS Mouse Models C57Bl/6 Keratin CrePR mice ( B6K5Cre) Keratin CrePR mice express a Cre rec ombinase progesterone receptor (PR) fusion protein whose expression is restricted by a Keratin 5 (K5) promoter. The CrePR is inducible by the progesterone antagonist RU486 (Sigma Aldrich, St. Louis, MO) but not by endogenous progesterone. Upon induction with RU486, CrePR translocates into the nucleus and excises DNA sequences flanked by Lox P sites. T hese mice were crossed with the B6.Cg Myd88tm1Defr/J ( B6MyD88plox) These mice possess loxP sites on either side of exon 3 of the MyD88 gene. When these mutant mice are bred to B6K5Cre mi ce, offspring ( B6K5Cre.MyD88plox) will have MyD88 deleted in epithelial cells upon the administration of RU486. Four experimental groups were utilized in this experimental design (Table 31). C57Bl/6K5Cre.MyD88plox mice were either : 1) left uninduced and uninfected (U/U) (MyD88 sufficient) 2) induced and left uninfected (I/U) (MyD88 deficient) 3) left uninduced and infected (U/I) (MyD88 sufficient) or 4 ) induced and infected (I/I) (MyD88 deficient) (n=4/group) Induction of Periodontal Disease C57Bl/6K5Cre.MyD88plox m ice were either induced or not Induction consisted of oral administration of 500ug of RU486 solubilized in 25ul of sesame oil (Publix, Gainesville, FL) for three consecutive days. After which all mice were lavaged with 0.12% chlorhexidine gluconate (3M, St. Paul, MN) for three days. Infection consisted of an oral lavage with 2.5x109 P gingivalis strain 831 and 2.5x109 A actinomycetemcomitans

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25 strain 29522 (ATCC Manassas, VA ) resuspended in 2% low viscosity carboxy methyl cellulose (Sigma Aldrich) on 4 consecutive days every other week for 12 weeks. After which the maxillae and mandibles were harvested for histology, bone m orphometric a nalysis, and ELISA Immunohistochemistry Buccal tissues from C57Bl/6K5Cre.MyD88plox 7 days following mock or RU486 induction of cre recombinase expression were removed and fixed in 10% buffered formalin (Sigma Aldrich) and sent to Histology Tech Services, Inc (Gainesville, Fl), where paraffin embedded and 5uc sections were mounted to microscope slides. Mounted sections were deparafinzed in xylene (25min), followed by stepwise rehydration in 100% (5min) 95% (5min), and 70%(5min) ethanol. Rehydrated sections were then rinsed in distilled water (5min) followed by equilibration in PBS (5min). Sections were probed with 1:500 dilution of goat anti murine MyD88 (Cell Signaling Technology, Boston, MA) overnight at 4C in a humidified chamber in the dark. Probed sections were rinsed 3x in PBS (5min), followed by a 2hr incubation with 1:5000 dilution of PE conjugated goat anti mouse Ig (red) ( Cell Signaling Technology ) and counterstained with DAPI (blue) (Cell Signaling Technology) and coversliped. I mages were captured on a spinning disc confocal microscope at 40x magnification. Histochemical Analysis of Periodont al Tissue The maxillae and mandibles of animals were removed and fixed in 10% buffered formalin, decalcified, embedded and sectioned for histological analysis. After which 5micron sections were stained with hematoxylin and eosin (Sigma Aldrich) Images were captured at 20x magnification and inflammation scored using PMN/mononuclear cell infiltration (0, no inflammatory cells; 1, minimal inflammation (scattered inflammatory

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26 cells close to the junctional epithelium); 2, moderate inflammation (numerous infl ammatory cells in the gingival connective tissue; and 3, severe inflammation) was to be performed. In addition, changes in epithelial cell attachment, elongation and eruption were qualitatively assessed. Bone Morphometric Analysis of Bone Loss The maxilla and mandible of mice were removed, defleshed, immersed in 3% hydrogen peroxide (Fisher Scientific) and stained with methylene blue (Sigma Aldrich) to delineate the cementoenamel junction (CEJ). To quantify bone loss, color digital images were captured under a 10x stereo dissecting microscope (Leica, Buffalo Grove, IL) Using morphometry image analysis software, the total area of bone resorption in mm2 was determined (lingual and buccal surfaces for each of the four quadrants (left and right maxillae and mandibles). Specifically, t he maxilla and mandible were imaged under 2X magnification perpendicular to the alveolar bone. Scale bar was calibrated using a predetermined measure at 2X magnification. The distance between the CEJs of murine posteri or teeth and alveolar c rest were outlined and measured. Right and left maxillary bone loss was measured on both the lingual and buccal surfaces in mice from all experimental groups. Data are presented as corrected total surface area. The average total surface a rea from induced uninfected (I/U ) mice was subtracted from total surface area in uninduced infected (U/I) and induced infected (I/I) mice. Tissues from t he maxilla and mandible were homogenized using a bead bea ting technique in a protein lysis buffer containing protease inhibitors. After which ELISA was used to determine the concentration of TNF according to the manufacturers instructions (BD Optiplex San Jose, CA ). Specifically, supernatant s from tissue

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27 homogenates were incubated in microtiter polystyrene plates (Costar, Corning, NY, USA) coated with anti for 2 hours at room temperature. After washing, alkaline phosphataseconjugated anti was added and incubated for additional 2 hours at room temperature. 1mg/ml of p nitrophenylphosphate ( Sigma Aldrich ) was added to the washed plates for 30 minutes and the reaction was terminated by using 3M NaOH (Sigma Aldrich) The concentration was quantified using a linear standard curve, where the colorimetric reaction was quantified using a spectrophotometer at 450nm with a reference of 655nm. A standard curve and a best fit linear trend line were used to determine pg/mL concentrations. pg/ml of w as normalized to total protein content as foll ows ( raw pg/ml x total protein content corrective ratio) where the corrective ratio = (lowest protein content) / ( protein content for sample of interest) Statistics Statistical analyses were performed using Prism Software (GraphPad Software, Inc; La Joll a, CA) where oneway ANOVA with Bonferronis multiple comparisons test was used to determine significance when comparing groups of three or more while Students t test was used when comparing two groups. P value <0.05 was considered significant.

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28 Table 3 1 Experimental groups Abbreviation Description Induction Infection MyD88 Status U/U uninduced/uninfected No No Sufficient I/U induced/uninfected Yes No Deficient U/I uninduced/infected No Yes Sufficient I/I induced/infected Yes Yes Deficient

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29 CHAPTER 4 RESULTS The purpose of this research was to evaluate the role(s) of TLR oral epithelial cell specific responses in the progression of periodontal disease. We hypothesized that oral epithelial cell MyD88 dependent TLR signaling is responsible for tolerogenic immune tuning which counteracts otherwise inflammatory responses. Epithelial Cell Specific Knockdown of MyD88 In order to determine if RU486 induced the knockdown of MyD88 in the oral epithelial cells of mice following three days of adminis tration, the buccal tissues of C57Bl/6K5Cre.MyD88plox mice were harvested seven days following the last administration and probed for MyD88 using immunohistochemistry (Fig. 41B) As a control the buccal tissues of sham treated C57Bl/6K5Cre.MyD88plox mice were also evaluated (Fig. 41A). A lack of MyD88 expression, as measured by a lack of immunofluorescence, was noted in the oral epithelial cells of RU486 treated but not sham treated C57Bl/6K5Cre.MyD88plox mice (Fig. 4 1) This means that in C57Bl/6K5Cre.MyD88plox mice RU486 is effective in knocking down MyD88 specifically in oral epithelial cells. Oral Epithelial Cell MyD88 / Results in Exacerbated Soft Tissue Changes In order to evaluate the soft tissue changes in the presence and absence of epit helial cell MyD88 expression in mice, mandibles were fixed in formaldehyde followed by decalcification, sectioning (5uM sections) and H/E staining. Sections were then analyzed histologically for loss of attachment and/or epithelialization. Following oral i nfection, a loss of oral epithelial cell MyD88 expression resulted in exacerbated soft tissue changes including loss of attachment and/or extension of long junctional

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30 epithelium (Fig. 4 2) This indicates that MyD88 expression is necessary for protection against normal flora of the mouth. Oral Epithelial Cell MyD88 / Results in Exacerbated Bone Loss In order to evaluate bone loss in the presence and absence of epithelial cell MyD88 expression, maxillae were imaged under 2X magnification perpendicular to t he alveolar bone. Right and left maxillary bone loss was measured on both the lingual and buccal surfaces in mice from all experimental groups. The average total surface area from induced uninfected (I/U) mice was subtracted from the total surface area in uninduced infected (U/I) and induced infected (I/I) mice. An increase in total bone loss was observed in the B6K5Cre.MyD88plox mice compared to infected controls (Fig. 4 3) This means that following oral infection, a loss of oral epithelial cell MyD88 expression results in exacerbated maxillary bone loss. Oral Epithelial Cell MyD88 / Results in Exacerbated Pro Inflammatory Cytokine Production In order to assess mucosal TNF expression in the presence and absence of epithelial cell MyD88 expression, mandibles were subjected to bead beating to homogenize the soft tissue into a buffer containing protease inhibitors. After which ELISA was used to determine the concentration of T NF Concentrations were normalized to total protein. Following infection, higher levels of was observed in the B6K5Cre.MyD88plox mice compared to infected controls (Fig. 4 4). In addition, higher levels of were observed in uninfected B6K5Cre.M yD88plox mice compared to uninfected controls (Fig. 44). This means that a loss of oral epithelial cell MyD88 expression result s in increased local TNF production even in the absence of infection

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31 Figure 4 2 Murine o ral e pithelial cell k nockdown of MyD88. Buccal tissues from C57Bl/6K5Cre.MyD88plox mice 7 days following (A) sham or (B) Ru486 induction of cre recombinase expression were probed with anit MyD88 (red) and a nuclear stain (blue). Note lack of MyD88 expression in the oral epithelial cells following induction of cre expression (B).

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32 Figure 4 2 S oft tissue changes in the presence and absence of epithelial cell MyD88 expression. Mandibles were fixed in formaldehyde followed by decalcification, sectioning (5uM sections) and H/E staining. (A, D) U/U uninduced uninfected (MyD88 sufficient) ; (B,E ) U/I uninduced infected (MyD88 sufficient) ; (C,F) I/I induced infected (MyD88 deficient) Brackets indicated loss of attachment and/or epithelialization.

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33 Figure 4 3 B one loss in the presence and absence of epithelial cell MyD88 expression. (A D) Maxillae were imaged under 2X magnification perpendicular to the alveolar bone. Scale bar was calibrated using a predetermined measure at 2X magnification. The distance between the CEJs of murine posterior teeth and alveolar crest were outlined and measured. Right and left maxillar y bone loss was measured on both the lingual and buccal surfaces in mice from all experimental groups. (A B) induced ininfected (I/I) (MyD88 deficient) ; (C D) uninduced infected U/I (MyD88 sufficient) (E) Data are presented as corrected total surface area. The average total surface area from induced uninfected (I/U ) mice was subtracted from total surface area in uninduced infected (U/I) and induced infected (I/I) mice. *p value = 0.0116 Students T test.

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34 Figure 4 4 Mucosal TNF expression in the presence and absence of epithelial cell MyD88 expression. Mandibles were subjected to bead beating to homogenize the soft tissue into a buffer containing protease inhibitors. After which ELISA was used to determine the concentration of TNF Concentrations were normalized to total protein. U/U = uninduced uninfected (MyD88 sufficient); I/U= induced uninfected (MyD88 deficient); U/I = uninduced infected (MyD88 sufficient) ; I/I = induced infected (MyD88 deficient). *p value <0.05. Oneway AN OVA with Bonferronis correction.

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35 CHAPTER 5 DISCUSSION TLRs respond to various PAMPs of infectious and resident bacteria, viruses and fungi TLRs are predominantly expressed on cells of the innate immune system where they induce a wide variety of innate and adaptive immune responses34. Research has established that TLRs are also expressed in periodontal tissues on nonhematopoietic immune cells such as epithelial cells35 TLR responses of oral epithelial cells are the first line of defense to potential infectious pathogens within the oral cavity. However recent research has indicated that under steady state conditions activation of TLRs by commensal bacteria is critical for the maintenance of oral health36 Thus, TLR induced signaling in oral epithelial cells results in divergent cellular outcomes which can be a two edgedsword. O ver production of proinflammatory cytokines by oral epithelial cells either due to chronic stimulation or inappr opriate TLR responsiveness can lead to exacerbated soft and hard tissue destruction. I t has recently been demonstrate that while the repertoire of TLRs expressed by oral epithelial cells and traditional immune cells such as macrophages are similar, the re lative levels and ratios are significantly different37. This differential expression of TLRs resulted in an attenuated TLR response profile similar but not identical to macrophages37. can be induced in response to multip le bacterial PAMPS through their engagement with TLRs on oral epithelial cells37. IL8 has significant pleiotropic activities, where IL8 causes an increase in 1) the release of enzymes from granules, 2) the metabolism of reactive oxygen species (ROS), and the 3) chemotaxis and retainment of immune cells all of which can amplify the inflammatory cascade38. Importantly, IL8 can also induce osteoclastogenesis and

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36 osteoclast activation39. Thus over expression of IL8 as seen periodontal disease can result in the exacerbation of both soft and hard tissue destruction observed in periodontal disease. On the other hand, epithelial cells have been shown to express and in response to some bacterial PAMPs preventing the the proliferation of most adaptive immune cells40 of bone matrix proteins in osteoblasts as well as most major extracellular matrix proteins40. n prevents the control of inflammation and progression of wound healing necessary for the control of periodontal disease. Indeed here we demonstrate that a lack of MyD88dependent TLR signaling within the oral epithelium results in exacerbated soft and har d tissue destruction in a well established murine model of periodontal disease. These data suggest the TLR signaling within the oral epithelium is not only imperative for homeostasis, but may be a good target for periodontal therapies, especially in refractory patients. MyD88 is a constitutive intermediate signaling molecule for most TLR signaling pathways. Here we have demonstrated that loss of MyD88 and thus TLR signaling within the OEC results in enhanced soft tissue inflammation and hard tissue destruction in a mouse model of periodontal disease, suggesting that TLR signaling within the oral epithelium i s important for maintenance of homeostasis. It also suggests that alterations in OEC TLR signaling could result in exacerbated periodontal disease such as we have observed under the conditions of diabetes mellitus

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37 An association between diabetes mellitus and destructive periodontal diseases has been reported in the literature since 196041. A well described bi directional relationship between diabetes mellitus and periodontal diseases has been established whereby diabetes is a major risk factor for periodontitis and the severity of periodontitis influences glycemic control42. In addition, it has been suggested that controlling periodontal inflammation has the potential to reduce the systemic inflammation responsible for secondary complications of diabetes42. Specifically, in type 1 diabetes (T1D), a toll like receptor (TLR) hyper inflammatory monocytic phenotype has been implicated as a mechanism of exacerbated tissue destruction observed. In addition, our laborat ory h as demonstrated that t he aberrant TLR activation of OEC in T1D which is concomitant with an absence of TLR induced regulatory or homeostatic responsiveness. Thus this dysregulation of OEC TLR responsiveness has the potential to contribute to excessive soft and hard tissue destruction observed under the conditions of T1D Thus, the data presented in this thesis and our previously published data will now direct the investigation into novel accessible targets for personalized intervention in periodontal patients with T1D. Periodontal diseases are a class of pathologies wherein oral microbes induce harmful immune responses in a susceptible host. Therefore, an agent which can both reduce microbial burden and lessen pathogenesis of localized inflammation would h ave beneficial effects in periodontal disease. 2,4,4trichloro 2 hydroxydiphenyl ether ( triclosan ) is currently used in oral care products due to broad spectrum anti microbial and anti inflammatory properties. A novel anti microbial mechanism by which tr iclosan improves plaque control and an additional anti inflammatory property which could have

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38 beneficial effects in periodontal disease resolution has recently been reported43. Because plaque is the main cause of periodontal diseases, its control has long been the cornerstone in the management of periodontal disease44. Over the past decade, supplement al treatments, including triclosan/copolymer toothpaste, have been developed which claim a therapeutic effect beyond conventional periodontal therapy44. Results from scientific and clinical studies together with two systematic reviews and a meta analysis provide strong evidence that twice daily use of triclosan significantly improves clinical plaque control and slows periodontal disease progression4552. For many years it has been recognized that triclosan has anti bacterial activity, however in the early 1990s it was suggested that the anti inflammatory properties of triclosan provide an additional benefit in the management of the inflammatory periodontal disease44. Finally, data from our lab suggests that triclosan promotes accelerated LPS tolerance through regulation of the TLR induced signaling cascade thus preventing over activation of oral epithelial cells43. Importantly we have demonstrated that triclosans anti inflammatory property is also effective in abrogating the TLR induced OEC hyper activity. Future directi ons of the work presented include evaluating the efficacy of triclosans effect on MyD88/ induced exac erbation of periodontal disease and its associated mechanisms. Although triclosan is used in a multitude of household products, the pleiotropic actions of triclosan including controlling microbial growth/colonization, inflammation as well as hormonal and endocrine function are still being uncovered 5355 Because triclosan has many beneficial properties, it is important that the mechanism(s) of action continue to be investigated to better understand the role

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39 triclosan plays in controlling cellular and biological processes. This would allow for co ntinued identification of targets for intervention in periodontal patients with excessive inflammation associated with their periodontal disease.

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40 REFERENCES 1. Rodrigues, P.H. et al. Porphyromonas gingivalis strain specific interac tions with human coronary artery endothelial cells: a comparative study. PloS one 7 e52606 (2012). 2. Malkoski, S.P., Cleaver, T.G., Lu, S.L., Lighthall, J.G. & Wang, X.J. Keratin promoter based gene manipulation in the murine conducting airway. Internati onal journal of biological sciences 6 68 79 (2010). 3. Nanci, A. & Bosshardt, D.D. Structure of periodontal tissues in health and disease. Periodontology 2000 40 11 28 (2006). 4. Gargiulo, A., Krajewski, J. & Gargiulo, M. Defining biologic width in crown lengthening. CDS review 88, 20 23 (1995). 5. Oshrain, H.I., Salkind, A. & Mandel, I.D. An histologic comparison of supra and subgingival plaque and calculus. Journal of periodontology 42, 31 33 (1971). 6. Kornman, K.S., Page, R.C. & Tonetti, M.S. The host response to the microbial challenge in periodontitis: assembling the players. Periodontology 2000 14, 33 53 (1997). 7. Anderson, G.B., Caffesse, R.G., Nasjleti, C.E. & Smith, B.A. Correlation of periodontal probe penetration and degree of inflammation. American journal of dentistry 4 177 183 (1991). 8. Lang, N.P., Adler, R., Joss, A. & Nyman, S. Absence of bleeding on probing. An indicator of periodontal stability. Journal of clinical periodontology 17 714 721 (1990). 9. McGuire, M.K. & Nunn, M.E. Prognosis versus actual outcome. II. The effectiveness of clinical parameters in developing an accurate prognosis. Journal of periodontology 67 658 665 (1996). 10. Armitage, G.C. Development of a classification system for periodontal diseases and conditions. Annals of periodontology / the American Academy of Periodontology 4 1 6 (1999). 11. Haffajee, A.D., Socransky, S.S. & Goodson, J.M. Clinical parameters as predi ctors of destructive periodontal disease activity. Journal of clinical periodontology 10 257 265 (1983).

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41 12. Socransky, S.S., Haffajee, A.D., Cugini, M.A., Smith, C. & Kent, R.L., Jr. Microbial complexes in subgingival plaque. Journal of clinical periodontology 25 134144 (1998). 13. Saglie, F.R., Carranza, F.A., Jr., Newman, M.G., Cheng, L. & Lewin, K.J. Identification of tissue invading bacteria in human periodontal disease. Journal of periodontal research 17 452 455 (1982). 14. Snyderman, R. Immunological mechanisms of periodontal tissue destruction. Journal of the American Dental Association 87 10201026 (1973). 15. Pierce, D.L. et al. Host adhesive activities and virulence of novel fimbrial proteins of Porphyromonas gingivalis. Infection and immuni ty 77, 32943301 (2009). 16. Mahanonda, R. & Pichyangkul, S. Toll like receptors and their role in periodontal health and disease. Periodontology 2000 43 41 55 (2007). 17. Mochizuki, S. et al. Gamma interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. Journal of periodontal research 39, 333343 (2004). 18. Hirschfeld, M. et al. Signaling by toll like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infection and immunity 69, 14771482 (2001). 19. Abreu, M.T. Immunologic regulation of toll like receptors in gut epithelium. Current opinion i n gastroenterology 19, 559 564 (2003). 20. Gargiulo, A.W., Wentz, F.M. & Orban, B. Mitotic activity of human oral epithelium exposed to 30 per cent hydrogen peroxide. Oral surgery, oral medicine, and oral pathology 14, 474 492 (1961). 21. Thilander, H. Epi thelial changes in gingivitis. An electron microscopy study. Journal of periodontal research 3 303312 (1968). 22. DiFranco, C.F. et al. Identification of Langerhans cells in human gingival epithelium. Journal of periodontology 56 48 54 (1985). 23. McCo rmick, T.S. & Weinberg, A. Epithelial cell derived antimicrobial peptides are multifunctional agents that bridge innate and adaptive immunity. Periodontology 2000 54 195 206 (2010). 24. Oz, H.S. & Puleo, D.A. Animal models for periodontal disease. Journal of biomedicine & biotechnology 2011, 754857 (2011).

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42 25. Baker, P.J., Evans, R.T. & Roopenian, D.C. Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Archives of oral bi ology 39 10351040 (1994). 26. Novak, E.A., Shao, H., Daep, C.A. & Demuth, D.R. Autoinducer 2 and QseC control biofilm formation and in vivo virulence of Aggregatibacter actinomycetemcomitans. Infection and immunity 78, 29192926 (2010). 27. Polak, D. et al. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response. Journal of clinical periodontology 36 406 410 (2009). 28. Oz, H.S., Chen, T. & Ebersole, J.L. A model for ch ronic mucosal inflammation in IBD and periodontitis. Digestive diseases and sciences 55 21942202 (2010). 29. Oz, H.S. & Ebersole, J.L. A novel murine model for chronic inflammatory alveolar bone loss. Journal of periodontal research 45 94 99 (2010). 30. Liu, P.F., Haake, S.K., Gallo, R.L. & Huang, C.M. A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis. Vaccine 27, 15891595 (2009). 31. Kesavalu, L., Ebersole, J.L., Machen, R.L. & Holt, S.C. Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components. Infection and immunity 60, 14551464 (1992). 32. Kesavalu, L., Holt, S.C. & Ebersole, J.L. Trypsinlike protease activity of Porphyromonas gingivalis as a potential virulence factor in a murine lesion model. Microbial pathogenesis 20, 1 10 (1996). 33. Kesavalu, L., Holt, S.C. & Ebersole, J.L. Porphyromonas gingivalis virulence in a murine lesion model: effects of immune alterations. Microbial pathogenesis 23, 317326 (1997). 34. Hans, M. & Hans, V.M. Toll like receptors and their dual role in periodontitis: a review. Journal of oral science 53, 263271 (2011). 35. Kusumoto, Y. et al. Human gingival epithelial cells produce chemotactic factors interleukin8 and monocyte chemoattractant protein1 after stimulation with Porphyromonas gingivalis via toll like receptor 2. Journal of periodontology 75 370379 (2004). 36. Hatakeyama, J. et al. Contrasting responses of human gingival and periodontal ligament fibr oblasts to bacterial cell surface components through the CD14/Toll like receptor system. Oral microbiology and immunology 18, 14 23 (2003).

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43 37. Neiva, K.G., Calderon, N.L., Alonso, T.R., Panagakos, F. & Wallet, S.M. Type 1 Diabetes associated TLR Responsiveness of Oral Epithelial Cells. Journal of dental research (2013). 38. Rosenkilde, M.M. & Schwartz, T.W. The chemokine system -a major regulator of angiogenesis in health and disease. APMIS : acta pat hologica, microbiologica, et immunologica Scandinavica 112 481 495 (2004). 39. Bendre, M.S. et al. Interleukin8 stimulation of osteoclastogenesis and bone resorption is a mechanism for the increased osteolysis of metastatic bone disease. Bone 33, 28 37 (2003). 40. Letterio, J.J. & Roberts, A.B. Regulation of immune responses by TGF beta. Annual review of immunology 16, 137 161 (1998). 41. Williams, R.C., Jr. & Mahan, C.J. Periodontal disease and diabetes in young adults. Journal of the American Medical A ssociation 172, 776 778 (1960). 42. Preshaw, P.M. et al. Periodontitis and diabetes: a twoway relationship. Diabetologia 55, 21 31 (2012). 43. Wallet, M.A., et al. Triclosan alters antimicrobial and inflammatory responses of epithelial cells. Oral diseas es 19, 296 302 (2013). 44. Blinkhorn, A. et al. Is there a role for triclosan/copolymer toothpaste in the management of periodontal disease? British dental journal 207 117 125 (2009). 45. Rosling, B. et al. Effect of triclosan on the subgingival microbiota of periodontitis susceptible subjects. Journal of clinical periodontology 24 881887 (1997). 46. Rosling, B. et al. The use of a triclosan/copolymer dentifrice may retard the progression of periodontitis Journal of clinical periodontology 24 873 880 (1997). 47. Furuichi, Y., Rosling, B., Volpe, A.R. & Lindhe, J. The effect of a triclosan/copolymer dentifrice on healing after nonsurgical treatment of recurrent periodontitis. Journal of clinical periodontology 26, 63 66 (1999). 48. Ellwood, R.P., Worthington, H.V., Blinkhorn, A.S., Volpe, A.R. & Davies, R.M. Effect of a triclosan/copolymer dentifrice on the incidence of periodontal attachment loss in adolescents. Journal of clinical periodontology 25, 363367 (1998).

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44 49. Cullinan, M.P. et al. Acquisition and loss of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia over a 5year period: effect of a triclosan/copolymer dentifrice. Journal of clinical periodontology 30 532 541 (2003). 50. Cullinan, M.P. et al. The effect of a triclosancontaining dentifrice on the progression of periodontal disease in an adult population. Journal of clinical periodontology 30 414 419 (2003). 51. Papas, A. et al. Comparative effica cy of stabilized stannous fluoride/sodium hexametaphosphate dentifrice and sodium fluoride/triclosan/copolymer dentifrice for the prevention of periodontitis in xerostomic patients: a 2year randomized clinical trial. Journal of periodontology 78 1505151 4 (2007). 52. Kerdvongbundit, V. & Wikesjo, U.M. Effect of triclosan on healing following nonsurgical periodontal therapy in smokers. Journal of clinical periodontology 30, 10241030 (2003). 53. James, M.O., Li, W., Summerlot, D.P., Rowland Faux, L. & Woo d, C.E. Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep placenta. Environment international 36 942 949 (2010). 54. Zorrilla, L.M. et al. The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicol Sci 107 56 64 (2009). 55. Gee, R.H., Charles, A., Taylor, N. & Darbre, P.D. Oestrogenic and androgenic activity of triclosan in breast cancer cells. J Appl Toxicol 28, 78 91 (2008).

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45 BIOGRAPHICAL SKETCH Dr. Richard A. Rasmussen III studied Human Nut rition at the University of Florida, where he graduated Cum Laude with research distinction in the spring of 2007. After which, he attended dental school at Nova Southeastern University College of Dental Medicine where he received his Doctor of Dental Medi cine degree and was elected into the Omicron Kappa Upsilon National Dental Honor Society in the summer of 2011. Currently, Richard Rasmussen is completing his post doctoral residency in Periodontics at the University of Florida. Upon graduation in the summ er of 2014, Richard will be returning to Tampa, Florida to practice clinical Periodontics with his f ather, Richard Rasmussen Jr., at Implant and Periodontal Therapy.


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