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Local and Systemic Inflammatory Response in Type II Diabetic Patients with Periodontal Disease

Permanent Link: http://ufdc.ufl.edu/UFE0042989/00001

Material Information

Title: Local and Systemic Inflammatory Response in Type II Diabetic Patients with Periodontal Disease
Physical Description: 1 online resource (52 p.)
Language: english
Creator: MESIA,RUBEN FERNANDO
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: CREVICULAR -- CYTOKINES -- DIABETES -- LPS -- PERIODONTAL
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: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science LOCAL AND SYSTEMIC INFLAMMATORY RESPONSE IN TYPE II DIABETIC PATIENTS WITH PERIODONTAL DISEASE By Ruben Fernando Mesia May 2011 Chair: Luciana Shaddox Major: Dental Science Numerous studies have shown a correlation between chronic inflammatory periodontal disease and diabetes, in which both of them influence the progression and response to treatment of the other. However, the mechanisms behind the association of these two diseases are not yet elucidated. The objective of this study was to quantify local and systemic inflammatory responses in Type II diabetic patients as compared to normoglycemic subjects with periodontal disease. Gingival crevicular fluid (GCF) and blood samples were collected from 20 patients with chronic periodontal disease (10 Type II diabetes-DBT; 10 non-diabetic-NDBT). GCF samples were collected from a diseased site (pocket depth-PD?5mm, attachment loss ?AL?2mm and bleeding on probing-BOP) and a healthy site (no AL, no BoP). Blood samples were stimulated with ultra-pure TLR-2 and TLR-4 agonists for 24h. Twenty-two cyto/chemokines were quantified in GCF and culture supernatants using Luminex (Milliplex?). Results were compared between groups using t-test. DBT patients showed higher systemic unstimulated levels of IL-8, TNF?, IL-10, MIP1? and MIP1?; higher stimulated levels of IL-6, IL-8, IL-10, MIP1? and MIP1?; and lower stimulated and unstimulated levels of GM-CSF when compared to NDBT (p<0.05). DBT patients also showed higher levels of MIP1? in GCF from healthy sites (p<0.001) than NDBT, while NDBT showed higher levels of IL-7 in GCF from diseased sites (p<0.05). Among patients with chronic periodontitis, those with diabetes seem to have a higher systemic inflammatory response than those without diabetes, as measured by pro-inflammatory cytokine release. Lower levels of certain cytokines associated with wound healing in diabetes (DBT) patients upon stimulation with toll like receptors (TLRs) could also indicate an impaired immune response in this patient population. Longitudinal studies to assess the relationship between degree of inflammation, magnitude of diabetes control and extent/severity of periodontal disease are justified to better understand the role of inflammatory response and periodontal disease control.
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 RUBEN FERNANDO MESIA.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Shaddox, Luciana.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042989:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042989/00001

Material Information

Title: Local and Systemic Inflammatory Response in Type II Diabetic Patients with Periodontal Disease
Physical Description: 1 online resource (52 p.)
Language: english
Creator: MESIA,RUBEN FERNANDO
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: CREVICULAR -- CYTOKINES -- DIABETES -- LPS -- PERIODONTAL
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: Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science LOCAL AND SYSTEMIC INFLAMMATORY RESPONSE IN TYPE II DIABETIC PATIENTS WITH PERIODONTAL DISEASE By Ruben Fernando Mesia May 2011 Chair: Luciana Shaddox Major: Dental Science Numerous studies have shown a correlation between chronic inflammatory periodontal disease and diabetes, in which both of them influence the progression and response to treatment of the other. However, the mechanisms behind the association of these two diseases are not yet elucidated. The objective of this study was to quantify local and systemic inflammatory responses in Type II diabetic patients as compared to normoglycemic subjects with periodontal disease. Gingival crevicular fluid (GCF) and blood samples were collected from 20 patients with chronic periodontal disease (10 Type II diabetes-DBT; 10 non-diabetic-NDBT). GCF samples were collected from a diseased site (pocket depth-PD?5mm, attachment loss ?AL?2mm and bleeding on probing-BOP) and a healthy site (no AL, no BoP). Blood samples were stimulated with ultra-pure TLR-2 and TLR-4 agonists for 24h. Twenty-two cyto/chemokines were quantified in GCF and culture supernatants using Luminex (Milliplex?). Results were compared between groups using t-test. DBT patients showed higher systemic unstimulated levels of IL-8, TNF?, IL-10, MIP1? and MIP1?; higher stimulated levels of IL-6, IL-8, IL-10, MIP1? and MIP1?; and lower stimulated and unstimulated levels of GM-CSF when compared to NDBT (p<0.05). DBT patients also showed higher levels of MIP1? in GCF from healthy sites (p<0.001) than NDBT, while NDBT showed higher levels of IL-7 in GCF from diseased sites (p<0.05). Among patients with chronic periodontitis, those with diabetes seem to have a higher systemic inflammatory response than those without diabetes, as measured by pro-inflammatory cytokine release. Lower levels of certain cytokines associated with wound healing in diabetes (DBT) patients upon stimulation with toll like receptors (TLRs) could also indicate an impaired immune response in this patient population. Longitudinal studies to assess the relationship between degree of inflammation, magnitude of diabetes control and extent/severity of periodontal disease are justified to better understand the role of inflammatory response and periodontal disease control.
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 RUBEN FERNANDO MESIA.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Shaddox, Luciana.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042989:00001


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1 LOCAL AND SYSTEMIC I NFLAMMA TORY RESPONSE IN TYPE II DIABETIC PATIENTS WITH PERIOD ONTAL DISEASE By RUBEN FERNANDO MESIA 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 2011

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2 2011 Ruben Fernando Mesia

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3

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4 ACKNOWLEDGMENTS I thank my research ment or Dr. Luciana Shaddox as well as Dr. Shannon Wallet, for dedicating themselves and inspiring me with their passion for the search of deep knowledge. I also would like to thank to the entire faculty at the University of Florida, for their tireless commitme nt in providing an outstanding education in the field of periodontics and helping me to develop solid clinical skills.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 BACKGROUND ................................ ................................ ................................ ...... 13 Diabetes ................................ ................................ ................................ .................. 13 Periodontal Disease ................................ ................................ ................................ 14 Diabetes and Periodontal Disease ................................ ................................ .......... 16 Local and Systemic Response in Periodontal Disease and Diabetes ..................... 18 3 MATERIALS AND METHODS ................................ ................................ ................ 23 Patient Selection ................................ ................................ ................................ ..... 23 Periodontal Examination ................................ ................................ ......................... 24 Collection of GCF (Local Response) ................................ ................................ ...... 24 Co llection of Blood Samples (Systemic Response): ................................ ............... 25 La boratory Procedures ................................ ................................ ........................... 25 Luminex Analysis ................................ ................................ ................................ .... 26 Statis tical Analysis ................................ ................................ ................................ .. 26 4 RESULTS ................................ ................................ ................................ ............... 31 5 DISCUSSION ................................ ................................ ................................ ......... 42 LIST OF REF ERENCES ................................ ................................ ............................... 47 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 52

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6 LIST OF TABLES Table page 4 1 Clinical results between diabetics and non diabetics ................................ .......... 33

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7 LIST OF FIGURES Figure page 3 1 Periodontal clinical evaluation. ................................ ................................ ........... 27 3 2 Gingival fl uid collection: ................................ ................................ ...................... 28 3 3 Laboratory procedures:. ................................ ................................ ..................... 29 3 4 Luminex ................................ ................................ ................................ .............. 30 4 1 Systemic markers (Unstimulate): IL 8 and TNF alpha.. ................................ ..... 34 4 2 Systemic markers (Unstimulate): IL 10.. ................................ ............................ 35 4 3 Systemic marke rs ( U nstimulate): MIP1 alpha and MIP1 beta.. .......................... 36 4 4 Systemic markers (Stimulate): IL 6 and IL 8.. ................................ ..................... 37 4 5 Systemic markers (Stimu late): IL 10.. ................................ ................................ 38 4 6 Systemic markers (Stimulate): MIP1 alpha and MIP1 beta.. ............................... 39 4 7 Systemic markers (Unstimulate and Stimulat e): GM CSF ................................ 40 4 8 Local markers 7. ................................ ................................ .......... 41

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8 LIST OF ABBREVIATION S AL Attachment loss BOP Bleeding on probing CAL Clinical attachment levels CRP C reactive protein DBT Diabetes or diabetic FMPE Full mouth periodontal examination GCF Gingival cr evicular fluid GM CSF Granulocyte macrophage colony stimulating factor HbA1c Hemoglobin A1c IDDM Insulin dependent diabetes mellitus IL Interleukins LPS Lipopolysaccharides MIP1 Macrophage inflammatory protein 1 NDBT Non diabetes or Non diabetic PD Pocket s depths PMN Polymor phonuclear neutrophilic leukocytes PMPE Partial mouth periodontal examination TLR Toll like receptor Tumor necrosis factor alpha

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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 Degree of Master of Science LOCAL AND SYS TEMIC INFLAMMATORY R ESPONSE IN TYPE II DIABETIC PATIENTS WITH PERIOD ONTAL DISEASE By Ruben Fernando Mesia May 2011 Chair: Luciana Shaddox Major: Dental Science Numerous studies have shown a correlation between chronic inflammatory periodontal disease and diabetes, in which both of them influence the progression and response to treatment of the other. However, the mechanisms behind the association of these two diseases are not yet elucidated. The objective of this study was to quantify local and systemic inflamma tory responses in T ype II diabetic patients as compared to normoglycemic subjects with periodontal disease. Gingival crevicular fluid (GCF) and blood samples were collected from 20 patients with chronic perio dontal disease (10 T ype II diabetes DBT; 10 non diabetic NDBT). GC F samples were collected from a diseas e d site (pocket depth attachment loss and bleeding on probing BO P ) and a healthy sit e (no AL, no BoP). Blood samples were stimulated with ultra pure TLR 2 and TLR 4 agonists for 24h. Twenty two cyto/chemokines were quantified in GCF and cult ure supernatants using Luminex ( Milliplex ) Results were compared between groups using t test. DBT patients showed higher systemic unstimulated levels of IL 10, 6, IL 8, IL nd

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10 lower stimulated and unstimulated levels of GM CSF when compared to NDBT (p<0.001) than NDBT, while NDBT showed higher levels of IL 7 in GCF from diseased sites (p<0.05) Among patients with chronic periodontitis, those with diabetes seem to have a higher systemic inflammatory response than those without diabetes, as measured by pro inflammatory cytokine release. Lower levels of certain cytokines associated with wound he aling in diabetes ( DBT ) patients upon stimulation with toll like receptors ( TLRs ) could also indicate an impaired immune response in this patient population. Longitudinal studies to assess the relationship between degree of inflammation, magnitude of diabe tes control and extent/se verity of periodontal disease are justified to bet ter understand the role of inflammatory response and periodontal disease control.

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11 CHAPTER 1 INTRODUCTION Diabetes type II is a heterogeneous disease that arises from and intera ction among environmental factors such as obesity, sedentary lifestyle, high calorie food intake, and genetic susceptibility that result in increased insulin resistance and the clinical manifestation of disease ( Lopez, Valenzuela et al. 2009 ) Periodontitis is a common disease; the clinical sings include a chronic, tissue destructive inflammation, which degrades the attachment around teeth, resulting in tooth loss. T he interpretation of epidemiological data of periodontal disease is difficult, due to inconsistencies in the methodology used. It is not possible, therefore, to accurately assess if the prevalence of periodontal diseases shows a worldwide decline ( Papapanou 199 6 ) most severe form of the disease is present in approximately 4.8% of t he population, whereas the 17.5 % exhibit moderate to mild signs of disease ( Papapanou 1996 ; Eke, Thornton Evans et al. 2010 ) Several studi es have reported association between periodontal disease and diabetes ( Shlossman, Knowler et al. 1990 ; Emrich, S hlossman et al. 1991 ; Taylor, Burt et al. 1998 ) ; one possible mechanism for the reported association between periodontitis and diabetes could be the release of bacteria, bacterial products or pro inflamm atory cytokines from the chronic periodontal lesion into the blood stream. This might lead to a systemic inflammatory response in diabetic patients and may lead to a worsening of diabetic control.

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12 New data support the concept that in diabetes associated pe riodontitis, the altered host inflammatory response plays a critical role. Elevated circulating levels of inter leukins, tumor necrosis factor alpha can worsen insulin resistant in individual with diabetes and thereby impair the glycemic control ( Taylor, Burt et al. 1996 ) Thus, periodontal disease may have a significant impact on the metabolic state in diabetes ( Mealey and Oates 2006 ) The extent of the local and systemic inflammation mediators associate with diabetes remains unclear. The aim of this study w as to quantify local and systemic inflammatory responses in Type II diabetic patients as compared to normoglycemic subjects with periodontal disease The levels of inflammatory mediators such as cytokine s and chemokines were evaluated locally (GCF gingiv al crevicular fluid) and systemically (serum levels) in both groups. We hypothesized that a mong patients with chronic periodontitis, those with diabetes have a higher systemic inflammatory response than those without diabetes, as measured by pro inflammato ry cytokine release upon s timulation with toll like receptors ( TLR s) agonists.

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13 CHAPTER 2 BACKGROUND Diabetes Diabetes is a syndrome in which chronic hyperglycemia leads to long term damage to various organs including the heart, eyes, kidneys, nerves an d vascular system ( Mealey and Oates 2006 ) Diabetes mellitus affects over 21 million Americans, including >9% of the adult population ( Harris, Hadden et al. 1987 ; Mokdad, Bowman et al. 2001 ) The current cla ssification of diabetes is based upon the pathophysiol ogy of each form of the disease. Type I diabetes is a cellular mediated auto immune cells of the pancreas resulting in life long dependence on exogenous insulin Type II diabetes results from insulin resistance in which the use of endogenously produc ed insulin is altered at the tar get cells ( Mealey and Oates 2006 ) Type II diabetes is the most prevalent type, affecting 85 to 95% of all diseased patients. While Type II diabetic patients have some altered insulin production, the patients retain the capacity to produce insulin. However, stresses from additional illnesses, such as infection can exa cerbate the insulin resistance such that it results in ketoacidosis, a life threatening condition ( DeFro nzo and Ferrannini 1991 ) Therefore appropriate treatment of these secondary illnesses is imperative to the diabetic long term health. Elevated systemic proinflammatory mediators in subjects with diabetes have a tremendous impact. Adipose tiss ue has been found to release pro in flam m atory cytokines, this cytokines are involved in an Insulin regulation. Insulin resistance are strongly linked to the actions of the proinflammatory cytokines IL 6 and TNF ( Crook 2004 ) IL 6 stimulates increased production of IL 6 f r o m

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14 r exacerbate insulin resistance ( Natali, Toschi et al. 2006 ) Traditionally, diabetics have been considered more susceptible to severe periodontal disease due to metabolic and host response that re sults in impaired wound healing. People with diabetes often have a shift in m onocyte/macrophage phenotype, which results in the overproduction of these same inflammatory cytokines in response to periodontal pathogens ( Salvi, Yalda et al. 1997 ) Diabetic patients who also have periodontitis may present with an even greater systemic inflammatory condition with elevated serum levels of IL 6, TNF aggravate the glycemic response. This could confirm the hypothesis that severe periodontitis in persons with non insulin dependent diabetic increases th e risk of poor glycemic control ( Taylor, Burt et al. 1996 ) Pe riodontal D isease Periodontitis is a multifactorial polymicrobial infection initiated by the presence of Gram negative bacteria, which accumulates in the gingiva crevice region. Bacterial plaque accumulation on the tooth surface leads to inflammation of th e marginal tissues which may lead to destruction of periodontal ligament (clinical attachment loss) and the adjacent supporting bone, causing tooth loss The prevalence of periodontal disease varies among the adult population depending on severity and ext ent. The most severe form of the disease is present in approximately 10 15% of the population, whereas 35% exhibit moderate to mild sig n s of disease (Papapanou 1996). Recently, the National Health and Nutrition Examination Survey (NHANES) reviewed the dat a for the assessment of national prevalence of periodontitis in the US. Historically, because of time, labor and cost constraints variations of a partial mouth periodontal examination

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15 (PMPE) have been the clinical protocol of choice for large scale studie s However, because periodontitis is not evenly distributed in the mouth PMPE protocols do unde restimate prevalence. T he accuracy of previous NHANES periodontal examination protocols was questioned it was found that both PMPE protocols used in previous studies (NHANES III and 2001 04) substantially underestimate the true prevalence of periodontitis by 50% or more. The total true prevalence of pe (4.8% severe and 17.5% moderate) vs. 8.9% found in the NHANES III protocol ( Eke, Thornton Evans et al. 2010 ) Periodontal destruction is considered as a result of the response of a suscept ible host to bacterial challenge. Periodontal disease progression by tissue destruction i s host mediated by locally produced pro inflammatory cytokines in response to the bac terial flora and its products. Lipopolysaccharides (LPSs) of Gram negative microorganisms are recognized by host receptors such as toll like receptors (TRLs) T oll like receptors (TLRs) are germ line encoded pattern recognition receptors expressed on cell s of the innate immune system t hat recognize structural compo nents co nserved among classes of micro organisms, also called pathogen associated molecular patterns. Inflammation in the gingival sulcus is initiated and maintained by the host defense against ba cteria migrating from dental plaque. Toll like receptor (TLR) 2 and TLR4 are constitutively expressed in periodontal tissue and the TLRs can recognize a variety of bacterial components. Cell walls of Gram positive and Gram negative bacteria are the ligands for TLR2, and lipopolysaccharide (LPS), an outer membrane of

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16 Gram negative bacteria, is the ligand for TLR4 ( Yoshioka, Yoshimura et al. 2008 ) The interactions between the LPS and the toll like receptors stimulate the production of cytokines. Cytokines are soluble proteins, secreted by cells involved in both the innate and adaptive host response. Interleukins are important members of the cytokine group and are p rimarily involved in communication between leukocytes and others cell such us epithelial cells, endothelial cells, and fibroblasts engaged in the inflammatory process. There is c lear evidence that the cell wall components of gram bacteria in the periodonti tis such as Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum and Tannerella forsythensis stimula te, via TLR2 and TLR4, the pro duction of proinflammatory cytokines from the host, such as interle ukin 1 and tumo r necrosis factor, which induce alveolar bone resorption and the pr oduc tion of matrix metalloproteases ( Kikkert, Laine et al. 2007 ) It is possible that the production of local cytokine and/or low level asymptomatic bacteremia affects the plasma concentrations of biomarkers ( Loos, Craandijk et al. 2000 ) Elevate levels of proinflammatory cytokines, such as TNF alpha, IL 6 and IL 8 can stimulate a numbers of events that occur during periodontal disease, including the induction of adhesion molecules, amplification of inflammatory responses, and stimulation of matrix metalloproteinases and bone resorption. Anti inflammatory cytokine, such as IL 10, plays a major roll in the suppression of inflammatory responses by inhibiting the antigen presenting capac ity of macrophages and T helper 1 cell differentiation ( Yamaguchi, Yoshimura et al. 2009 ) Diabetes and P eriodontal D isease Research has shown that periodontal disease is closely associated with diabetes

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17 mellitus ( Rylander, Ramberg et al. 1987 ; Shlossman, Knowler et al 1990 ; Emrich, Shlossman et al. 1991 ; Novaes, Pereira et al. 1991 ; Seppala, Seppala et al. 1993 ; Nishimura, Takahashi et al. 1998 ; Taylor, Burt et al. 1998 ) Indeed a meta analysis using 3,500 diabetic adults concluded that the m ajority of studies dem onstrate more severe periodontal disease in diabetic patients than in adults without diabetes, confirming a significant association between periodontal disease and diabetes ( Papapanou 1996 ) Further studies have shown that compared to non diabetic individuals type II diabetics were 2.8 1 times more likely to have clinical attachment loss and 3.4 3 times more likely to have radiographic bone loss than normoglycemic controls ( Emrich, Shlossman et al. 1991 ) Also, a 2 year longitudinal study demonstrated that diabetic subjects had a significantly increased risk for alveolar bone loss compared to non diabetic individuals with an odds ratio of 4.2. Among these patients, poorly controlled diabetics had an odds ratio of 11.4 compared to 2.2 of well controlled diabetics ( Taylor, Burt e t al. 1998 ) The strength of evidence on the relationship between diabetes and periodontal disease have led some to suggest that periodontal ( Loe 1993 ) Yet, the relationship between metabolic control of diabetes and pe riodontal disease is still unclear. Just as diabetes contributes to increased incidence and severity of periodontal disease, periodontal disease can have a significant impact on the metabolic state in diabetes ( Mealey and Oates 2006 ) For instance, in a 2 year longitudinal trial, diabetic subjects with severe periodontitis at baseline had a six fold increased risk of worsening of glycemic control compared to diabetic subjects without periodontitis ( Taylor, Burt et

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18 al. 1998 ) An additional study reported that 82% o f diabetic patients with severe periodontitis experienced the onset of one or more diabetic complications such as major cardiovascular, cerebrovascular or peripheral vascular events compared to only 21% of diabetic subjects without periodontitis ( Thorstensson, Kuylenstierna et al. 1996 ) These and other studies support the notion that the presence of periodontal disease in diabetic patients may increase i nsulin resistance and contribute to a worsening of the diabetic state and diabetic complications ( Williams and Mahan 1960 ; Sammalkorpi 1989 ; Yki Jarvinen, Sammalkorpi et al. 1989 ; Miller, Manwell et al. 1992 ; Grossi, Skrepcinski et al. 1996 ; Taylor, Burt et al. 1996 ; Thorstensson, Kuylenstierna et al. 1996 ; Grossi, Skrepcinski et al. 1997 ) although the mechanism of how this occurs is still unclear. L ocal and S ystemic R esponse in P eriodontal D isease and D iabetes Gingival crevicular fluid (GCF), can be collected from the gingival sulcus surrounding the teeth, and exists as either a serum transudate or inflammatory exudate. The GCF contains substances from the host as well as from microorganism in the supra or subgingival plaque. A vast number of studies began to identify enzymes and host response in the crevicular fluid since Genco and Slots ( 1984 ) demonstrate d the importance of the host response to the progression of periodontal disease, in different stages a) colonization, b) invasion c) destruct ion and d) healing and placed into perspective the various host responses as they may affect each of these four stages. Traditional methods to collect GCF use micropipettes. The most applicable method is the use of precut methylcellulose filter paper stri p s placed in the sulcus. The fluid is adsorbed and later analyzed. T his is noninvasive, can be time consuming and very technique sensitive method Before collection of the GCF, plaque needs to be removed from the tooth, since plaque, saliva and blood can i nfluence the volume of fluid

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19 collected. T he volume of GCF present at a given site may be directly related to inflammation and ulceration of the gingival crevicular epithelium. GCF can be analyzed to determine whether specific markers of systemic disease ca n be identified in the oral cavity. It has been shown that a g reater volume of GCF is present in severe inflamed sites than in less inflamed sites. However, no studies have demonstrated that an increase of this volume is related to t he risk for periodontal disease ( Lamster and Ahlo 2007 ) In looking specifically at diabetes influence on local inflamm atory markers, Engebretson et al. ( 2004 ) found that there was a correlation between poor glycemic control and increased levels of IL 1 beta levels in GCF, emp hasizing a plausible explanation for the increased incidence and severity of periodontal disease in patients with diabetes. Evidence exists suggesting that proinflammatory cytokines in particular IL 1 beta, may play an important role in the etiology of per iodontal disease. IL 1 beta is biologically active in low concentrations and is a potent bone resorptive cytokine. Engebretson et al. ( 2002 ) compared GCF IL 1 beta expression in patie nts with different degrees of periodontal disease; baseline clinical parameters were significant associated with total IL 1 beta concentration in GCF and patients with severe periodontitis demonstrate increased of levels of IL 1 beta than those with mild/m oderate disease. Interleukin 6 (IL 6) is a major mediator of the host response to tissue injury, infection and bone resorption. Kurtis et al. ( 1999 ) measured IL 6 levels in GCF from twenty four patients with type II diabetes with periodontitis twenty four adult periodontitis, and twenty four healthy controls. GCF sampling was performed on the vestibular aspects of the maxillary incisors and canine tee th. Higher GCF IL 6 levels

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20 were observed in the diabetic with periodontitis and adult with chronic periodontitis alone patients versus the healthy control (p<0.05) These findings suggested that GCF IL 6 levels were significantly higher in the area of infl ammation and periodontal destruction locally. Interleukin 8 (IL 8) is a potent chemokine with a distinct target for recruitment and activation of human granulocytes and mediation of inflammatory process. Monocytes/macrophages, lymphocytes, fibroblasts, en dothelial cells and epithelial cells can secrete IL 8 and plays an important role in regulation of neutrophil function. Under normal conditions respond well to neutrophil infiltration, under inappropriate release the ils and uncontrolled release of IL 8 can cause tissue damage by hyperactivity of neutrophils ( Jin, Leung et al. 2002 ) Regarding systemic markers, Manouchehr Pour et a l. ( 1981 ) demonstrated that diabetic patients with severe periodontitis have significant impairment of PMN chemotaxis. In contrast, diabetic patients with mild periodontal disease as well as non diabetic subjects with either severe or mild periodontitis did not show such impairment. In addition, p eriodontitis and diabetes share a common pathogenesis involving an increased inflammatory response at the local and systemic level. Elevated serum levels of proinflammatory cytokines is often seen i n patients with periodontal disease while patients with diabetes have hyper inflammatory immune cells that can exacerbate the elevated production of proinflammatory cytokines ( Dag, Firat et al. 2009 ) S alvi et al. ( 1997 ; 1998 ) studied 39 Insulin dependent diabetes mellitus (IDDM) patients and 64 systemically health y individuals. The patients were divided into groups A (gingivitis and mild periodontitis) and group B (moderate and severe periodontitis), 17

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21 patients from the systemic healthy individuals were used as a control. These authors showed that d iabetic patient s had significant higher GCF levels of IL 1 beta as compared to non diabetic s Within the diabetic population the GCF level s of this inflammatory mediator were higher in group B than group A. Furthermore, diabetes as a group showed a higher systemic respon se of IL 1 beta upon stimulation by Escherichia coli and Porphyromonas gingivalis lipo polysaccharide (LPS) as compar ed to non diabetic patients with adult periodontitis. They suggested that diabetes type II is a significant risk factor for more severe peri odontal disease because they found that diabetic patients have exaggerated inflammatory responses when compared to non diabetic controls. Tumor necrosis factor (TNF alpha) has been reported to play a key role in the pathogenesis of type II diabetes. TNF is associated with insulin resistance; the etiology of highest circulation levels in diabetics is not fully understood. Adipose tissue may be a major source of TNF alpha secreting cells in ty pe II diabetes but not al l the studies have shown TNF to be as sociated with obesity. There are at least three main sources for increased circulating TNF alpha levels in patients with diabetes, including the release of TNF from adipose tissues, from the stimulation of monocyte macrophage cells/ leukocytes by advance d glycation end products, and from periodontal inflammation ( Dag, Firat et al. 2009 ; Chen, Wei et al. 2010 ) Recent evi dence suggests that inflammation influence the circulation TNF rather than obesity. Forty six patients with periodontitis and type II diabetes were evaluated in a cross sectional study the results demonstrated that chronic periodontitis is associated with plasma TNF s ubjects with type II diabetes sup po rting the hypothesis that

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22 periodontal infection and inflammation may contribute to insulin resistance ( Engebretson, Chertog et al. 2007 ) Correa et al. ( 2010 ) investigated the effect of periodontal therapy in diabetic s the sample was composed of 23 type II diabetes subjects, and higher levels of TNF alpha, IL 4, IL 6, IL 8 and IL 10 were found at the baseline. T he main finding of this prospective study was that the satisfactory clinical response to non surgical periodontal therapy was followed by a reduction of circulation TNF diabetes. The oth er circulating cytokines investigate d were also reduced but did reach sta tistical significance The literature suggests a two way relationship between periodontal diseases and diabetes, although the mechanisms in which this relationship occurs is still not completely understood. It seems that diabetics are more susceptible to periodontal diseases due to both a hyper inflammatory component leading to more tissue natural heali ng/regenerative capabilities during the disease course. On the other hand, periodontitis may aggravate the diabetic host inflammatory component both locally and systemically, leading to worsening of diabetes status/control. The objective of the present in vestigation is to better understand the contribution of diabetes to the local and systemic inflammatory response in individuals with periodontitis

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23 CHAPTER 3 MATERIALS AND METHODS Patient S election Two volunteer populations with periodontal disease were recruited for this study: Ten type II diabetic DBT patients and ten non diabetic NDBT subjects. Diabetic patients were recruited from the Endocrinology clinic, at the Shands Medical Plaza. Non Diabetic patients were recruited from the Graduate Periodonto logy clinic at the University of Florida college of Dentistry. All subjects signed an informed consent in order to participate in the study according to the UF institutional Review Board approval (protocol #70 2007 ). These patients were included according to the following criteria: Inclusion criteria for a ll patients (diabetics and non diabetics) Subjects aged 40 75 years old; Presence of at least 20 teeth; Diagnosis of chronic periodontal disease defin ed by the presence of at least 4 sites with probing d epth of 5mm or more and attachment loss of 3 mm or more with bleeding on probing Exclusion criteria for a ll patients (diabetics and non diabetics) Subjects diagnosed with any forms of aggressive or necrotizing periodontal disease; any systemic diseases o r conditions that could influence the course of either periodontal disease and/or glucose control; Subjects under any medications that could influence the characteristics or response to treatment of either diabetes and/or periodontal disease; Pregnant or l actating women. Inclusion criteria for d iabetic patients Diagnosed with type II diabetes (HbA1c control; Have not altered their medication to treat diabetes in the past 3 months.

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24 Exclusion criteria for d iabetic patients Type I diabetic patients, d iabetic complications such as macrovascular diseases, kidney or liver failure, alteration of diabetes medications three months prior to study enrollment Periodontal E xamination All patients received a periodontal evaluation. All c lini cal parameters were recorded at six sites per tooth : probing depth s, gingival marginal position clinical attachment levels, plaqu e index and bleeding on probing. All parameters were measured and recorded by two calibrated examiners (LS & RM) with a period ontal electronic probe (Florida Probe, Gainesville, Fl) (Figure 3 1) Subject disease status was classified according to the 1999 Classification of Periodontal Disease established by the Ame rican Academy of Periodontology ( Armitage 1999 ) T he two examiners calibration and reproducibility were ensured in calibration sessions at the beginning of the study, obtaining duplicates measurements of pockets depths and gingival margin positions on diff erent patients from the study. Calibration intra and inter examiner was obtained once >80% of agreement (measurements within 1mm) was obtained between duplicate measurements of pocket depth and gingival margin position. Col lection of GCF (Local R esponse) GCF samples were collected from 2 selected periodontal sites, one disease site ( probing depths 5mm or greater, with bleeding on probing ), and one healthy site ( pocket depth < 3mm and no bleeding on probing ) to serve as a con trol. Prior to GCF fluid sampling, supragingival plaque was removed using a sterile curette. These surfaces were gently air dried and isolated with cotton rolls. GCF fluid was collected using PerioPaper GCF collection strips (Oraflow Inc, Plainview, NY) by inserting the strip

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25 roughly 1 2 mm into the gingival sulcus for ~10 sec each. The volume of GCF obtained was quantified with a Periotron 8000 (Oraflow Inc, Plainview, NY). The strips were placed into a sterile polypropylene eppendorf tube and then the sam ples were stored at 8 0 C until assayed (Figure 3 2). Co llection of B lood S amples ( S ystemic R esponse): One vacutainer tube (4mL ) of venous blood samples was collected ; the plasma was used to measure systemic levels (stimulated and non stimulated) of cytoki nes and chemokines. The blood samples were obtaine d from each subject by a certified phlebotomist under the direct supervision of the examiners. The skin was wiped with 70% isopropyl alcohol and venipuncture was performed with an intravenous 22 gauge and v acuum tube (BD Biosciences, MD, USA) La boratory P rocedures Two ml of peripheral blood from the two groups (diabetics and non diabetics ) were diluted in 6 ml of RPMI 1640 (Fisher Scientific, PA) one ml of this diluted solution was placed into three wells o f a 24 well plate. Five microliter of ultra pure TLR 2 and TRL 4 agonist alone was added to separate wells. One well was not stimulated to serve as a control. Samples were incubated at 37 C in CO 2 for 24 hours (Figure 3 3). After 24 hours the blood from ea ch well was transferred into the corresponding eppendorf tube The labele d tubes containing the blood were placed into a small centrifuge and run for 5 min utes After centrifuged, the supernatant from each of those tubes where placed into t he corresponding eppendorf tube Those tubes were stored in 80 C until assayed.

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26 Luminex A nalysis F luorescence detection kits ( Milliplex 22 plex cyto/chemokine detection kits, Millipore, St. Charles, MO ) wer e used to detect and quantify 22 cyto/chemokines (IL1 L2, IL4 IL5, IL6, IL7, IL8, IL10, IL12(p4 0), IL12(p70), IL13, IL15, Eotaxin GM CSF, IFN IP10, MCP1, MIP1 TNF VEGF). Briefly, 50 of eluates and 25 of a cyto/chemokine capture bead cocktail were placed in a 96 well filter plate and incubat ed overnight at 4 C. Wells were washed 3 times with assay buffer, after which 25 of a biotin labeled anti cytokine cocktail and 75 of assay buffer were plated and incubated for 1.5 hours After which 25 of SAV PE was added and incubated for 30 min ut e s. Twenty five of stop reagent was added for 5 min utes followed by 3 washes with assay buffer and beads resuspended in 125 of sheath fluid. All incubations were performed at room temperature in the dark while gently shaking. Data were acquired usi ng instrumentation ( Luminex100 TM Millipore, St. Charles, MO ) and analyzed using a software ( Milliplex Analyst, Viagene Tech, Carlisle, MA ) standard curves and five parameter logistics. Some samples were diluted 1:100 in assay buffer prior to running t he above described assay. Statis tical A nalysis R esults were analyzed for each Cytokine / chemokine between diabetes and non diabetes group using t test, with a significance level of =0.05.

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27 Figure 3 1. Periodontal clinical evaluation: A ll patients received a periodontal evaluation. Clinical parameters at six sites per tooth were taken (probing depths, gingival marginal positioning, attachment levels, plaque index and bleeding on probing). A) Florida probe before reading, B) Florida probe in the periodontal (Photo courtesy to Ruben Mesia)

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28 Figure 3 2. Gingival fluid collection: P erioPaper GCF strips were use to collect GCF from 2 selected periodontal sites, 1 disease and 1 healthy site. The volume obtained was quantified with a Periotron 8000. The samples were stored at A) PerioPaper GCF strips in the gingival pocket, B) PerioPaper GCF strips quantify with periotron 8000. C) PerioPaper GCF strips ready to be assayed (Photo courtesy to Ruben Mesia)

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29 Figure 3 3. Laboratory procedures: 1 mL of blood was stimulated with 1 TRL 2 and TRL 4 agonists. One well was not stimulated to serve as a control. Samples were incubated at 37 C in CO2 for 24 hours. A) Blood samples, B) Stimulation with TRL 2, C) A fter 24 hours of incubation, each well was centrifuged and storage at 80 C until assayed (Photo courtesy to Ruben Mesia)

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30 Figure 3 4. Luminex was used to analyze local (GCF) and systemic inflammatory (blood samples) markers (Photo courtesy to Ruben Mesia)

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31 CHAPTER 4 RESULTS The distribution of clinical results of these two populations is illustrated in T able 4 1. The ages of subjects in non diabetics ( N DBT ) and diabetics ( DBT ) range from 44 to 64 and 51 to 69 years old with mean sta ndard deviation of 53.3 6.4 and 61.3 5.4, respectively. The ratios of female/male gender in groups N DBT and DBT were 5/5 (1.0) and 4/6 (0.66) respectively. Among all subjects, t he mean percent age of site s per subject with bleeding on probing ( BOP ) was 45.7 22.4 % and 25. 4 13.8 % for N DBT and DBT respectively. The mean percentages of plaque index were 57.3 14.5 % and 20.6 18.1 % for N DBT and DBT respectively (p<0.05) The mean percentage of pockets depths greater than 4 mm were 41.8 13.4% and 11 .9 8.4 % for N DBT and DBT respectively (p<0.05) The Periodontal probing depths was 5.22 0.5 and 4.50 0.3 for N DBT and DBT respectively (p<0.05) M ean clinical attachment levels ( CAL ) in N DBT and DBT were 5.64 0. 9 and 4.47 1.2 respectively (p< 0.05) and the m ean HbA1c value for diabetic subjects was 8.13 1.23 The Analysis of systemic markers showed higher unstimulated levels of IL 8, in the serum of diabetics ( DBT ) patients (p<0.05, F igure s 4 1, 4 2, 4 3). After Lipopolysaccharides ( LPS ) stim ulation with toll like receptors two and toll like receptors four ( TRL 2 and TLR 4 ) DBT patients showed higher stim ulated levels of IL 6, IL 8, IL (p<0.05, Figure s 4 4, 4 5, 4 6). The DBT population also showed lower stimulated and unstimulated levels of G M CSF tha n NDBT (p<0.05, Figure 4 7 ). Regarding the local inflammatory response (local marker s) DBT patients also gingival crevicular fluid ( GCF ) from healthy sites

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32 (p<0.001) than NDBT, while NDBT showed higher levels of IL 7 in GCF from diseased sites (p<0.05) (Figure 4 8 ).

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33 Table 4 1. Clinical results between d iabetics and non diabetics. All results shown by mean standard deviation Non Diabetic Diabetic % BOP 45.7 22.4 25.4 13.8 % PLAQUE 57.3 14.5 20.6 18.1 %PD>4 mm 41.8 13.4 11.9 8.4 Mean PD 5.22 0.5 4.50 0.3 Mean CAL 5.64 0. 9 4.47 1.2 % Female 50 40 Mean age 53.3 6.4 61.3 5.4 Mean HbA1c -8.13 1.23 Denotes significant differences between groups (p<0.05).

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34 Figure 4 1. Systemic markers (Unstimulate): Diabetes patients present higher inflammatory markers in serum in IL 8 and TNF alpha. Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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35 Figure 4 2. Systemic markers (Unstimulate): Diabetes patients present higher inflammatory markers in s erum in IL 10. Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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36 Figure 4 3. Systemic markers ( U nstimulate): Diabetes patients present higher inflammatory markers in serum in MIP1 alp ha and MIP1 beta. Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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37 Figure 4 4. Systemic markers (Stimulate): Diabetes patients present higher inflammatory markers upon LPS stimulati on in IL 6 and IL 8. Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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38 Figure 4 5 Systemic markers (Stimulate): Diabetes patients present higher inflammatory markers upon LPS stimulatio n in IL 10 Denotes significant differences between diabetic and non diabetic groups (p<0.05 ) (Photo courtesy to Ruben Mesia)

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39 Figure 4 6. Systemic markers (Stimulate): Diabetes patients present higher inflammatory markers upon LPS stimulation in MI P1 alpha and MIP1 beta. Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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40 Figure 4 7. Systemic markers (Unstimulate and Stimulate): Diabetes patients present lower stimulated and unsti mulated levels of G M CSF than NDBT Denotes significant differences between diabetic and non diabetic groups (p<0.05) (Photo courtesy to Ruben Mesia)

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41 Figure 4 8. Local markers and IL 7 : Diabetes patients also showed higher levels GCF from healthy sites than NDBT, while NDBT showed higher levels of IL 7 in GCF from diseased s ites Bars indicate differences between groups or sites, (p<0.05), *** (p<0.001) (Photo courtesy to Ruben Mesia)

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42 CHAPTER 5 DISCUSSION The influence of d iabetes on periodontal disease has been discussed in the literature, and there is substantial evidence indicating that diabetes is a risk factor for periodontal disease ( Emrich, Shlossman et al. 1991 ; Taylor, Burt et al. 1998 ) T he present study has reported, among patients with chronic gingival disease, those with diabetes seem to have more severe systemic inflammat ory processes than those without diabetes, as measured by cytokine /chemokine expression. Tho se high levels of cytokines were previously highly correlated with active progressive periodontal lesions in non diabetic healthy individuals ( Tonetti, Imboden et al. 1994 ; Loos, Craandijk et al. 2000 ; Kikkert, Laine et al. 20 07 ) However, the inflammatory response with regard to IL 6, IL in diabetes patients was exaggerate we also reported l ower levels of certain cytokines such as GM CSF could indicate an impaired immune response in this patient population. An interesting finding in this study was the observation of higher inflammatory response in the diabetic group even before lipopolysaccharides ( LPS ) stimulation Those exaggerate responses may be a phenotypic marker for susceptibility in diabetics. In agreement with previous studies, we found elevate d levels of TNF in serum in subject with diabetes with less severity of periodontal disease. T his finding tend to support recent research by Chen et al. ( 2010 ) in which they faile d to find a correlation between the severity of periodontal disease and serum TNF levels. One possible reason may be that the contribution of periodontal infection to circulating TNF was so minor that it was overwhelmed by others sources of TNF in pa tients with T ype 2 diabetes. Other studies found no significant differences in serum levels of TNF at the

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43 baseline between DBT and NDBT ( Dag, Firat et al. 2009 ; Kardesler, Buduneli et al. 2010 ) P revious investigations have demonstrated that chronic periodontitis severity is significantly associated with plasma TN F in Ty pe II diabetes ( Salvi, Beck et al. 1998 ; Crook 2004 ; Correa, Goncalves et al. 2010 ) However, Engebretson et al. ( 2007 ) measured plasma levels of T NF in subjects with type II diab etes and chronic periodontitis and found that TNF showed a significant positive correlation with attachment loss but not with probing depth, r a ising the question whether TNF levels influence diabetes severity, or indeed wh ether circulating TNF influences periodontitis severity Salvi et al. ( 1998 ) also found a significant upregulated monocytic secretion of TNF alpha (4.6 fold) when c o mpared to non diabetic controls, which is in agreement with the present findings. According to the present findings, IL 6 levels of the groups with diabetes were higher than the NDBT after LPS stimulation Loos et al. ( Loos, Craandijk et al. 2000 ) reported that IL 6 can be detected in plasma of >50% of patients with severe periodontitis Some studies show that elevated level s of IL 6 is found in diabetes T ype II ( Correa, Goncalves et al. 2010 ; Kardesler, Buduneli et al. 2010 ) We found higher levels of IL 6 in diabetics ( DB T ) than non diabetics ( NDBT ) even though DBT did not have the same degree of periodontal severity. Ross et al. ( 2010 ) found that periodontal IL 6 expression increase s (periodontitis and diabetes suggest that diabetes further aggravates systemic inflammation. The present study

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44 findings may suggest tha t diabetes may play a more aggravating role to the systemic inflammatory response than the severity of periodontitis. Interestingly, IL 8 was found to be elevated in DBT patients before and after stimulation in the present study. IL 8 is known to be involv ed in the recruitment of polymorphonuclear neutrophilic leukocytes ( PMNs ) and is highly expressed in the junctional epithelium adjacent to infected periodontal defects, where PMNs infiltrate. Therefore, IL 8 may be involved in the initial stages of periodo ntal breakdown ( Tonetti, Imboden et al. 1994 ) The elevated levels of this cytokine in DBT patients in the present investigation may suggest that these patients ar e more susceptible to periodontal breakdown initiation than NDBT patients. In addition, the fact that the DBT patients in the present study had a more localized and milder form of periodontitis indicates that this disease status could be in its initial dev elopment which further explain s the over expression of this cyt okine Similarly, MIP1 has been correlated to high levels of IL 8 in early mucositis lesions ( Petkovic, Matic et al. 2010 ) w hich corroborates with the initial periodontal breakdown role of IL 8 previously mentioned in these patients. DBT patients also expressed high systemic levels of IL 10 before and after stimulation with LPS. Since this cytokine is involved with inhibition o f cytokine synthesis due to its inhibitory effect on macrophage monocytes ( Fiorentino, Zlotnik et al. 1991 ) high levels of this cytokine in this population could then be detrimental to the immune response activation and maintenance. On the other hand, DBT patients showed lower levels of GM CSF systemically when compared to NDBT patients. Abnormal maturation and other defects of dendritic

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45 cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self tolerance can be restored and maintained by semimature DCs induced by GM CS F ( Gaudreau, Guindi et al. 2007 ) Thus, a lower level of GM CSF in poorly controlled diabetic patients is expected. While previous studies ( Salvi, Beck et al. 1998 ; Engebretson, Grbic et al. 2002 ; Engebretson, Hey Hadavi et al. 2004 ) found higher levels of IL 1 in DBT patients our present findings did not show significant differences between IL 1 locally and systemically between DBT and NDBT. The significant differences between periodontal parameters in our groups may have contribute d to this fin ding One li mitation of th e present study was the significant difference in periodontal parameters between these two populations. The reason behind that difference was possible selection bias, since diabetic patients were recruited from a medical facility while non di abetic patients were recruited from the graduate periodontal program, where periodontal disease status is expected to be more severe. This difference in periodontal severity between the two groups could have influenced the levels of certain cytokines, main ly locally, in the GCF. However, interestingly, this difference did not influence the higher systemic inflammatory response in DBT patients, which lead us to believe that non controlled diabetes may have a stronger influence in systemic inflammation than periodontal disease alone. Other limitation was the absence of a healthy control group. The inclusion of a healthy control group would enable us to evaluate the role of periodontal disease alone in local and systemic inflammatory response. However, our obj ective in the present study was to evaluate the added role of diabetes into periodontal disease. Lastly, it is clear that a larger study population would

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46 also be needed to draw any final conclusions regarding the biomarkers in diabetic patients with differ ent levels of periodontitis and diabetic control In summary, s ignificantly higher systemic pro inflammatory response in DBT patients could explain the increased susceptibility to periodontal disease in th ese patients in general Conversely, this increas e in mediators could be a reflection of the negative effect periodontal disease has on diabetic metabolic control. In addition, the lower levels of certain defensive cytokines in DBT patients upon stimulation with TLRs could indicate an impaired immune re sponse in this patient population. Further studies to assess the relationship between degree of inflammation, magnitude of diabetes control and extent/se verity of periodontal disease are justified

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47 LIST OF REFERENCES Armitage, G. C. (1999) Development of a classification system for periodontal diseases and conditions. Ann Periodontol 4, 1 6. Chen, L., Wei, B., Li, J., Liu, F., Xuan, D., Xie, B. & Zhang, J. (2010) Association of periodontal parameters with metabolic level and sys temic inflammatory markers in patients with type 2 diabetes. J Periodontol 81, 364 371. Correa, F. O., Goncalves, D., Figueredo, C. M., Bastos, A. S., Gustafsson, A. & Orrico, S. R. (2010) Effect of periodontal treatment on metabolic control, systemic inf lammation and cytokines in patients with type 2 diabetes. J Clin Periodontol 37, 53 58. Crook, M. (2004) Type 2 diabetes mellitus: a disease of the innate immune system? An update. Diabet Med 21, 203 207. Dag, A., Firat, E. T., Arikan, S., Kadiroglu, A. K. & Kaplan, A. (2009) The effect of periodontal therapy on serum TNF alpha and HbA1c levels in type 2 diabetic patients. Aust Dent J 54, 17 22. DeFronzo, R. A. & Ferrannini, E. (1991) Insulin resistance. A multifaceted syndrome responsible for NIDDM, obe sity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14, 173 194. Eke, P. I., Thornton Evans, G. O., Wei, L., Borgnakke, W. S. & Dye, B. A. (2010) Accuracy of NHANES periodontal examination protocols. J Dent Res 89, 1208 1213. Emrich, L. J., Shlossman, M. & Genco, R. J. (1991) Periodontal disease in non insulin dependent diabetes mellitus. J Periodontol 62, 123 131. Engebretson, S., Chertog, R., Nichols, A., Hey Hadavi, J., Celenti, R. & Grbic, J. (2007) Plasma lev els of tumour necrosis factor alpha in patients with chronic periodontitis and type 2 diabetes. J Clin Periodontol 34, 18 24. Engebretson, S. P., Grbic, J. T., Singer, R. & Lamster, I. B. (2002) GCF IL 1beta profiles in periodontal disease. J Clin Period ontol 29, 48 53. Engebretson, S. P., Hey Hadavi, J., Ehrhardt, F. J., Hsu, D., Celenti, R. S., Grbic, J. T. & Lamster, I. B. (2004) Gingival crevicular fluid levels of interleukin 1beta and glycemic control in patients with chronic periodontitis and type 2 diabetes. J Periodontol 75, 1203 1208. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M. & O'Garra, A. (1991) IL 10 inhibits cytokine production by activated macrophages. J Immunol 147, 3815 3822.

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48 Gaudreau, S., Guindi, C., Menard, M., Besin, G., Dupuis, G. & Amrani, A. (2007) Granulocyte macrophage colony stimulating factor prevents diabetes development in NOD mice by inducing tolerogenic dendritic cells that sustain the suppressive function of CD4+CD25+ regulatory T cells. J Immunol 179, 3638 3647. Genco, R. J. & Slots, J. (1984) Host responses in periodontal diseases. J Dent Res 63, 441 451. Grossi, S. G., Skrepcinski, F. B., DeCaro, T., Robertson, D. C., Ho, A. W., Dunford, R. G. & Genco, R. J. (1997) Treatment of periodontal disease in di abetics reduces glycated hemoglobin. J Periodontol 68, 713 719. Grossi, S. G., Skrepcinski, F. B., DeCaro, T., Zambon, J. J., Cummins, D. & Genco, R. J. (1996) Response to periodontal therapy in diabetics and smokers. J Periodontol 67, 1094 1102. Harris, M. I., Hadden, W. C., Knowler, W. C. & Bennett, P. H. (1987) Prevalence of diabetes and impaired glucose tolerance and plasma glucose levels in U.S. population aged 20 74 yr. Diabetes 36, 523 534. Jin, L. J., Leung, W. K., Corbet, E. F. & Soder, B. (2002 ) Relationship of changes in interleukin 8 levels and granulocyte elastase activity in gingival crevicular fluid to subgingival periodontopathogens following non surgical periodontal therapy in subjects with chronic periodontitis. J Clin Periodontol 29, 60 4 614. Kardesler, L., Buduneli, N., Cetinkalp, S. & Kinane, D. F. (2010) Adipokines and inflammatory mediators after initial periodontal treatment in patients with type 2 diabetes and chronic periodontitis. J Periodontol 81, 24 33. Kikkert, R., Laine, M. L., Aarden, L. A. & van Winkelhoff, A. J. (2007) Activation of toll like receptors 2 and 4 by gram negative periodontal bacteria. Oral Microbiol Immunol 22, 145 151. Kurtis, B., Develioglu, H., Taner, I. L., Balos, K. & Tekin, I. O. (1999) IL 6 levels in gingival crevicular fluid (GCF) from patients with non insulin dependent diabetes mellitus (NIDDM), adult periodontitis and healthy subjects. J Oral Sci 41, 163 167. Lamster, I. B. & Ahlo, J. K. (2007) Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Ann N Y Acad Sci 1098, 216 229. Loe, H. (1993) Periodontal disease. The sixth complication of diabetes mellitus. Diabetes Care 16, 329 334.

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49 Loos, B. G., Craandijk, J., Hoek, F. J., Wertheim van Dillen, P. M. & van der Velden, U. (2000) Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J Periodontol 71, 1528 1534. Lopez, N. J., Valenzuela, C. Y. & Jara, L. (2009) Interleukin 1 gene cluster poly morphisms associated with periodontal disease in type 2 diabetes. J Periodontol 80, 1590 1598. Manouchehr Pour, M., Spagnuolo, P. J., Rodman, H. M. & Bissada, N. F. (1981) Comparison of neutrophil chemotactic response in diabetic patients with mild and s evere periodontal disease. J Periodontol 52, 410 415. Mealey, B. L. & Oates, T. W. (2006) Diabetes mellitus and periodontal diseases. J Periodontol 77, 1289 1303. Miller, L. S., Manwell, M. A., Newbold, D., Reding, M. E., Rasheed, A., Blodgett, J. & Kor nman, K. S. (1992) The relationship between reduction in periodontal inflammation and diabetes control: a report of 9 cases. J Periodontol 63, 843 848. Mokdad, A. H., Bowman, B. A., Ford, E. S., Vinicor, F., Marks, J. S. & Koplan, J. P. (2001) The continu ing epidemics of obesity and diabetes in the United States. JAMA 286, 1195 1200. Natali, A., Toschi, E., Baldeweg, S., Ciociaro, D., Favilla, S., Sacca, L. & Ferrannini, E. (2006) Clustering of insulin resistance with vascular dysfunction and low grade in flammation in type 2 diabetes. Diabetes 55, 1133 1140. Nishimura, F., Takahashi, K., Kurihara, M., Takashiba, S. & Murayama, Y. (1998) Periodontal disease as a complication of diabetes mellitus. Ann Periodontol 3, 20 29. Novaes, A. B., Jr., Pereira, A. L ., de Moraes, N. & Novaes, A. B. (1991) Manifestations of insulin dependent diabetes mellitus in the periodontium of young Brazilian patients. J Periodontol 62, 116 122. Papapanou, P. N. (1996) Periodontal diseases: epidemiology. Ann Periodontol 1, 1 36. Petkovic, A. B., Matic, S. M., Stamatovic, N. V., Vojvodic, D. V., Todorovic, T. M., Lazic, Z. R. & Kozomara, R. J. (2010) Proinflammatory cytokines (IL 1beta and TNF alpha) and chemokines (IL 8 and MIP 1alpha) as markers of peri implant tissue condition. Int J Oral Maxillofac Surg 39, 478 485. Ross, J. H., Hardy, D. C., Schuyler, C. A., Slate, E. H., Mize, T. W. & Huang, Y. (2010) Expression of periodontal interleukin 6 protein is increased across patients with

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50 neither periodontal disease nor diabetes, p atients with periodontal disease alone and patients with both diseases. J Periodontal Res 45, 688 694. Rylander, H., Ramberg, P., Blohme, G. & Lindhe, J. (1987) Prevalence of periodontal disease in young diabetics. J Clin Periodontol 14, 38 43. Salvi, G. E., Beck, J. D. & Offenbacher, S. (1998) PGE2, IL 1 beta, and TNF alpha responses in diabetics as modifiers of periodontal disease expression. Ann Periodontol 3, 40 50. Salvi, G. E., Yalda, B., Collins, J. G., Jones, B. H., Smith, F. W., Arnold, R. R. & Offenbacher, S. (1997) Inflammatory mediator response as a potential risk marker for periodontal diseases in insulin dependent diabetes mellitus patients. J Periodontol 68, 127 135. Sammalkorpi, K. (1989) Glucose intolerance in acute infections. J Intern Med 225, 15 19. Seppala, B., Seppala, M. & Ainamo, J. (1993) A longitudinal study on insulin dependent diabetes mellitus and periodontal disease. J Clin Periodontol 20, 161 165. Shlossman, M., Knowler, W. C., Pettitt, D. J. & Genco, R. J. (1990) Type 2 d iabetes mellitus and periodontal disease. J Am Dent Assoc 121, 532 536. Taylor, G. W., Burt, B. A., Becker, M. P., Genco, R. J., Shlossman, M., Knowler, W. C. & Pettitt, D. J. (1996) Severe periodontitis and risk for poor glycemic control in patients with non insulin dependent diabetes mellitus. J Periodontol 67, 1085 1093. Taylor, G. W., Burt, B. A., Becker, M. P., Genco, R. J., Shlossman, M., Knowler, W. C. & Pettitt, D. J. (1998) Non insulin dependent diabetes mellitus and alveolar bone loss progressio n over 2 years. J Periodontol 69, 76 83. Thorstensson, H., Kuylenstierna, J. & Hugoson, A. (1996) Medical status and complications in relation to periodontal disease experience in insulin dependent diabetics. J Clin Periodontol 23, 194 202. Tonetti, M. S ., Imboden, M. A., Gerber, L., Lang, N. P., Laissue, J. & Mueller, C. (1994) Localized expression of mRNA for phagocyte specific chemotactic cytokines in human periodontal infections. Infect Immun 62, 4005 4014. Williams, R. C., Jr. & Mahan, C. J. (1960) Periodontal disease and diabetes in young adults. J Am Med Assoc 172, 776 778. Yamaguchi, R., Yoshimura, A., Yoshioka, H., Kaneko, T. & Hara, Y. (2009) Ability of supragingival plaque to induce toll like receptor 4 mediated stimulation is

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51 associated with cytokine production by peripheral blood mononuclear cells. J Periodontol 80, 512 520. Yki Jarvinen, H., Sammalkorpi, K., Koivisto, V. A. & Nikkila, E. A. (1989) Severity, duration, and mechanisms of insulin resistance during acute infections. J Clin Endoc rinol Metab 69, 317 323. Yoshioka, H., Yoshimura, A., Kaneko, T., Golenbock, D. T. & Hara, Y. (2008) Analysis of the activity to induce toll like receptor (TLR)2 and TLR4 mediated stimulation of supragingival plaque. J Periodontol 79, 920 928.

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52 BI OGRAPHICAL SKETCH Ruben Fernando Mesia was born in Yurimaguas, Peru. He has got his dental degree in Cayetano Heredia Peruvian University in Lima, Peru. He completed further training in advanced education in g ener al d entistry (AEGD) at the University of Co nnecticut School of Dental Medicine. Currently, Ruben Mesia is completing his post doctoral residency in p eriodontics at the University of Florida College of Dentistry Upon graduation in the s pring of 2011, Ruben plans to practice clinical p eriodontics i n Tampa, FL.