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Association between Systemic Lupus Erythematosus and Periodontitis

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Title:
Association between Systemic Lupus Erythematosus and Periodontitis
Creator:
STROUT, STEPHEN LEWIS ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Antibodies ( jstor )
Confidence limits ( jstor )
Cytokines ( jstor )
Diseases ( jstor )
DNA ( jstor )
Lupus ( jstor )
Periodontitis ( jstor )
Rheumatoid arthritis ( jstor )
Systemic lupus erythematosus ( jstor )
Trucks ( jstor )

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University of Florida
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University of Florida
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Copyright Stephen Lewis Strout. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
4/30/2005
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73726511 ( OCLC )

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ASSOCIATION BETWEEN SYSTEMIC LUPUS ERYTHEMATOSUS AND PERIODONTITIS By STEPHEN LEWIS STROUT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Stephen Lewis Strout

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This thesis is dedicated to my beautiful a nd supportive wife, Meridith. Thank you for all your love and understanding.

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ACKNOWLEDGMENTS I would like to thank Marlene Sarmiento, RN, BSN of the Autoimmune Center at the University of Florida Shands for her support and organization of the Lupus patients. I would also like to thank Sonali Narain for her much-needed help with organizing the laboratory and all the data. Thanks go to the General Clinical Research Center for collecting all the blood and urine samples. Finally, I would like to thank my committee, (Dr. Carol Stewart, Dr. Westley Reeves, and Dr. Herbert Towle) for their ongoing support and encouragement. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 MATERIALS AND METHODS.................................................................................9 3 RESULTS...................................................................................................................13 4 DISCUSSION.............................................................................................................23 LIST OF REFERENCES...................................................................................................29 BIOGRAPHICAL SKETCH.............................................................................................34 v

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LIST OF TABLES Table page 1 Characteristics of Study Subjects.............................................................................10 2 Complement Factor (C3) Group Analysis...............................................................14 3 Complement Factor (C4) Group Analysis...............................................................15 4 Anti-double-stranded DNA Group Analysis............................................................17 5 Cyclic Citrullinated Peptide Group Analysis...........................................................18 6 Rheumatoid Factor Group Analysis.........................................................................20 7 High-Sensitivity C-Reactive Protein Group Analysis..............................................21 8 Mean Values for High-Sensitivity C-Reactive Protein............................................28 vi

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LIST OF FIGURES Figure page 1 TLR signaling pathways are separated into two groups.............................................3 2 Endotoxin increases vascular permeability and induces cell damage and release of DNA...........................................................................................................................6 3 Number of subjects per group..................................................................................13 4 Complement C3 trend in all four groups.................................................................15 5 Complement C4 trends for all 4 groups...................................................................16 6 Anti-dsDNA trends for all four Groups...................................................................18 7 Anti-Cyclic Citrullinated Peptide (anti-CCP) trends for all four Groups................19 8 Rheumatoid Factor (RF) trends for all four Groups.................................................20 9 High-sensitivity C-reactive protein (HSCRP) trends for all four Groups................22 vii

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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 ASSOCIATION BETWEEN SYSTEMIC LUPUS ERYTHEMATOSUS AND PERIODONTITIS By Stephen Lewis Strout May 2004 Chair: Carol Stewart Cochair: Herbert J. Towle, III Major Department: Periodontics Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting the connective tissues and multiple organs in the body. Periodontitis is a chronic destructive inflammatory disease of the tissues supporting the teeth. Multiple systemic chemicals have been implicated in the initiation, detection, and progression of SLE and periodontitis such as TNF-, C-reactive protein (CRP), C3, C4, Mx-1, -dsDNA, CCP, and Rheumatoid Factor. Our purpose was to measure these chemicals in the serum and urine of subjects with systemic lupus erythematous and periodontitis to determine whether a correlation exists. Forty-nine subjects were divided into four groups. Statistical comparisons were made of each group; and of SLE compared to periodontitis groups. The complement factors C3 and C4 were shown to have an SLE effect (C3; p> 0.003, C4; p> 0.0065) but no periodontitis effect (C3; p> 0.8520, C4; viii

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p> 0.6537). This proved that the SLE subjects did truly bind the complement and that it was thus not available for the test. Values for Rheumatoid Factor showed no effect for SLE (p> 0.0633) nor periodontitis (p> 0.7478). Cyclic Citrullinated Peptide also showed no statistically significant effect for SLE (p> 0.1559) or periodontitis (p> 0.7301). There was no statistically significant association for the Anti-double-stranded DNA values with SLE (p> 0.1841) and periodontitis (p> 0.2211). High-Sensitivity C-Reactive Protein showed no statistically significant association for any of the groups either (SLE p> 0.1048, periodontitis p> 0.4035). However, the mean values for each of the four groups did show a significant trend. Group 1 had the highest mean (11.905) which was expected, since this group had both diseases in it. Groups 2 and 3 had relatively similar means (8.557 and 5.65 respectively) which was expected, since they each represent only one disease. Group 4 had the lowest mean (2.968) which again was expected, since this group had no diseases. ix

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CHAPTER 1 INTRODUCTION Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting the connective tissues and multiple organs in the body (Tan, Cohen et al. 1982). Systemic lupus erthyematous is more common in woman and African Americans apparently, a genetic predisposition exists for the disease (Condemi 1987). The disease is characterized by production of antibodies with specificity for a wide range of self-antigens. The SLE autoantibodies mediate organ damage by directly binding to host tissues and forming immune complexes (Baechler, Batliwalla et al. 2003). Its onset can either be insidious or acute, and is a chronic, remitting and relapsing disease that is characterized principally by damage to the skin, joints, kidney and serous membranes (Leach 1998). The classic description of systemic lupus erythematous includes weight loss, chronic fever, a malar or butterfly rash, effusion, and glomerulonephritis. The skin conditions may be present, including alopecia, vesiculobullous lesions, and discoid plaques on the face and scalp (Burge, Frith et al. 1989). Periodontitis is a chronic destructive inflammatory disease of the teeth-supporting tissues and is initiated by an overgrowth of specific Gram-negative bacteria, such as Porphyromonas gingivalis and Bacteroides forsythus, to name a few (Darveau, Tanner et al. 1997). Specifically the lipopolysaccarhide (LPS) endotoxin found on the outer leaflet of the outer membrane of these Gram-negative bacteria is responsible for most of their destructiveness. Endotoxin was discovered largely through the work of Mary Jane Osborn and Hiroshi Nikaido, and through the work of Andr Boivin (1895-1949), who 1

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2 formulated a suitable extraction procedure that enabled the chemical characterization of endotoxin (Beutler and Rietschel 2003). If left untreated, periodontitis leads to loosened teeth and the potential exfoliation of those teeth. Periodontitis is a multifactorial disease in which many host and environmental factors play a role (Page, Offenbacher et al. 1997). There is no question that the delicate balance between local levels of key cytokines, sequestered in response to periodontopathogenic bacteria and their products, is critical in determining the outcome of a given pathogen’s immune response, such as in SLE and periodontitis (Jiang, Magli et al. 1999). Many clinical and experimental studies have shown that lipopolysaccharide (LPS) and other products of bacteria stimulate host cells resulting in the release of cytokines. Target cells are then stimulated to release other cytokines, inflammatory mediators, and catabolic enzymes (Bickel, Axtelius et al. 2001). Currently, very little information exists on the periodontal status of patients with SLE. One study comparing the gingival indices (GI), plaque indices (PI), and probing depths (PD) showed no evidence for a predisposition to increased periodontal disease in SLE (Mutlu, Richards et al. 1993). However, another study reported an incidence of periodontitis in 94% of the patients with SLE (Rhodus and Johnson 1990). Another study showed that 18 of the 30 patients (60%) with SLE had periodontitis in (Novo, Garcia-MacGregor et al. 1999). Cytokines are small peptides with a wide spectrum of inflammatory, hemopoieic, metabolic, and imunomodulatory properties. They are produced by a multitude of cells, including macrophages, endothelial cells, fibroblasts, and dentritic cells (Arai, Lee et al. 1990). Consequently, cytokines along with their receptors, form a complex network that

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3 is under biologic control with positive and negative feedback by the cytokines themselves. Tumor necrosis factor(TNF-) is a pro-inflammatory cytokine that enhances the microbicidal capacity of macrophages and neutrophils. The TNFalong with IL-12 causes NK cells to release INF-, which further enhances the activity of the macrophages. The TNFwas originally described as an endotoxin-induced, macrophage-derived factor that promoted the hemorrhagic necrosis of solid tumors (Beutler and Cerami 1988). Recently TNFwas implicated in a host of inflammatory, infectious, and malignant disorders. It is one of the strongest of the osteoclastic cytokines produced as a result of inflammation. The TNFcoordinates a lipopolysaccharide-stimulated osteoclastic process via its activation by the NF-B (RANKL) pathway (Figure 1) (Lam, Takeshita et al. 2000; Yamamoto, Takeda et al. 2004). Figure 1 TLR signaling pathways are separated into two groups. A MyD88-dependent pathway that leads to the production of pro-inflammatory cytokines with quick activation of NFB and MAPK, and a MyD88-independent pathway associated with the induction of IFNand IFN-inducible genes, and maturation of dendritic cells with slow activation of NFB and MAPK. (Yamamoto, Takeda et al. 2004)

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4 In bone, TNFinhibits extracellular matrix deposition, stimulates matrix metalloprotease synthesis, and enhances the production of osteoclastogenic cytokines such as RANKL (Horwood, Elliott et al. 1998; Siwik, Chang et al. 2000). Multiple studies have shown that the concentration of TNFin the blood is elevated in both SLE and periodontitis (Kinane, Hodge et al. 1999; Liou 2001; Gorska, Gregorek et al. 2003), which lends itself to the possible connection between these two very distinct diseases. C-reactive protein (CRP) is a well-known acute-phase protein that acts as a proinflammatory cytokine produced by the liver after tissue trauma, injury, or infection. It elicits the synthesis of TNFand IL-1, followed by the production of IL-6 (Baumann and Gauldie 1994). The cytokines TNF-, IL-1, and IL-6 produced in this process then stimulate hepatocytes to synthesize acute phase proteins, one of which is CRP (Baumann and Gauldie 1990). C-reactive protein has been implicated in pathogenesis of both periodontitis and SLE through the acute-phase response. Elevated serum levels of CRP have been shown to be associated with deep periodontal pockets, severe attachment loss, and subgingival microflora in periodontitis (Ebersole, Machen et al. 1997; Tracy, Lemaitre et al. 1997; Slade, Offenbacher et al. 2000; DeNardin 2001; Mercado, Marshall et al. 2001; Noack, Genco et al. 2001). Recent studies have also shown elevated serum CRP levels to be associated with SLE (Liou 2001; Mercado, Marshall et al. 2001). After LPS stimulates the toll-like receptor 4 (TLR4) the sequence then proceeds into the cell (Figure 1). Once inside the cell, the pathway either travels down the myeloid differentiation factor 88 (MyD88) pathway to stimulate the production of the inflammatory cytokines TNF-, IL-6, and IL-12; or it diverges to IRF-3, which eventually leads to the upregulation of the secondary interferon (INF) response gene

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5 myxovirus resistance 1 (Mx-1) via the Stat1 pathway (Yamamoto, Takeda et al. 2004). The Mx-1 production via INFand INFhas been shown (in multiple studies) to be upregulated in SLE subjects (Blanco 2001; Ronnblom 2001; Rozzo 2001; Bennett, Palucka et al. 2003). Although no studies could be found linking Mx-1 to periodontitis, it can be inferred from the data in Figure 1 that the lipopolysaccarhide (LPS) found on the outer membrane of the Gram-negative bacteria associated with periodontitis could (through the TLR4/IRF-3/IFN-/Stat1 cascade) also stimulate an upregulation of Mx-1 production. The detection of autoantibodies against anti-double stranded DNA (-dsDNA), and the complement proteins C3 and C4 has been used routinely in rheumatology to test for the presence of systemic lupus erythematous (SLE) and rheumatoid arthritis (RA) (Horak, Scudla et al. 2001; Tyrrell-Price, Lydyard et al. 2001). During cell death (for example, in an autoantibody response such as in SLE or rheumatoid arthritis) the double-stranded DNA is released into the serum, and binds to the glomerular basement membrane. Anti-double-stranded DNA antibodies are then produced by the activation of B-cells in the glomerular membrane. This in turn leads to local immune complex formation( which if left unregulated, can cause an autoimmune response) (Figure 2) (Cooper 1985; Farries and Atkinson 1991). The complement system (C3/C4) is also part of the innate immune system consisting of the Classic and Alternate pathways. The consequences of complement activation are, opsonization, activation of leucocytes, and lysis of target cells. The classic pathway is generally activated by viruses or Mycoplasma, and is the main protective antibody-directed mechanism.

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6 Figure 2: Endotoxin increases vascular permeability and induces cell damage and release of DNA. The DNA can then become deposited 1) on the collagen of the glomerular basement membrane (GBM) in the kidney. Endotoxin can also induce a polyclonal stimulation of B cells, some of which produce autoantibodies such as anti-DNA and anti-IgG; the latter are known as rheumatoid factors (RF). Anti-DNA antibody can then bind to the deposited DNA forming a local immune complex (2) (Roitt 1996). This system is used by the body to recognize self from non-self and destroy it. In autoimmune diseases, such as SLE and rheumatoid arthritis, the body fails to distinguish self as self and formulates an immune response against itself. Conversely, the Alternate Pathway is activated by viruses and many strains of Gram-positive and Gram-negative bacteria, such as those seen in periodontitis. The alternate pathway is not regulated as well as the classic pathway and lends itself to more cross-over type reactions. The biological activities of the complement system are generally divided into those that are beneficial to the host and those that are harmful. The major beneficial activities are; promotion of the killing of microorganisms, the efficient clearing of immune complexes, and the induction and enhancement of antibody responses. The major harmful activities of the complement system when activated and unregulated are as follows: in large scale systemically, it may cause septicemia, during a myocardial infarction, and by an

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7 autoimmune response to host tissue (as in SLE and rheumatoid arthritis) (Roitt 1996). Increased levels of each of these serum markers are associated with a positive diagnosis of SLE. Testing for all three markers together allows for greater confidence in the diagnosis, almost eliminating any false positive results. Currently, there is no research linking -dsDNA, C3, or C4 with the progression or initiation of periodontal disease. However, knowing that complement is activated in the Alternate Pathway by Gram-negative bacteria, such as those seen in periodontitis, it lends itself to the possible connection between these two diseases. Recently developed assays that detect the antibodies against cyclic citrullinated peptide (anti-CCP antibodies) have been shown to posses a very high specificity of 98% with a sensitivity of 68-80% for systemic lupus erythematous and rheumatoid arthritis (Schellekens, Visser et al. 2000). Citrullination is the conversion of peptidyl-arginine to peptidyl-citruline which induces the appearance of an autoimmune reaction identified by the CCP antibodies (Rantapaa-Dahlqvist, de Jong et al. 2003). CCP tests have been implemented recently in the early detection of Rheumatoid Arthritis (RA). However, Nielen showed that 10 out of 16 of his subjects that tested positive for anti-CCP antibodies developed systemic lupus erythematous (SLE) approximately 0.7-4.5 years later (Nielen, van Schaardenburg et al. 2004). Rheumatoid Factor (RF) is a type of antibody found in the blood of most rheumatoid arthritis patients. Normally antibodies are produced by the immune system to eliminate invading bacteria and viruses. RF is an antibody that can attach to normal body tissue, resulting in damage. A high level of rheumatoid factor can be caused by several

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8 autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematous as well as some severe infections (Roitt 1996). The purpose of the present study was to assess whether a correlation exists in serum and urine between a mixture of subjects with systemic lupus erythematous and periodontitis. Serum and urine was collected to test for specific markers of Systemic Lupus Erythematous and periodontitis (TNF-, HSCRP, Mx-1, -dsDNA, and C3/C4) that have been implicated in the course of these diseases. Also, Rheumatoid Factor (RF) and the Cyclic Citrullinated Peptide (CCP) were measured to test for the presence of rheumatoid arthritis which may have gone undiagnosed previously.

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CHAPTER 2 MATERIALS AND METHODS Fifty subjects were divided into one of four groups. In Group 1 were subjects that were previously diagnosed with SLE and periodontitis (SLE/P). In Group two were subjects who were previously diagnosed with SLE but did not show clinical signs of periodontitis (SLE/X). In Group 3 were subjects who were previously diagnosed with periodontitis but reported they were never diagnosed with SLE (X/P). In Group 4 were subjects who reported they were never diagnosed with SLE nor did they show clinical signs of periodontitis (X/X). Fourteen (14) subjects were in Group 1 (2 males and 12 females; age range 24-58, mean age 40.9 years). Sixteen subjects were in Group 2 (3 males and 13 females; age range 18-54, mean age 30.1 years). All the SLE subjects were followed at the Autoimmune Center, University of Florida SHANDs and were diagnosed according to the classification criteria of the American College of Rheumatology (Tan, Cohen et al. 1982). Eight subjects were in Group 3 (1 male and 7 females; age range 39-58, mean age 48.9 years). Twelve subjects were in Group 4 (3 males and 9 females; age range 21-57, mean age 38.9 years). The Group 3 subjects were recruited from the Periodontics Clinic at the University of Florida College of Dentistry and the Group 4 subjects from the University of Florida College of Dentistry. An Informed consent was obtained from all participants in the study, and the study was approved by the University of Florida Health Center Institutional Review Board for the use and disclosure of protected health information. 9

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10 To participate in the study the subjects had to satisfy the following inclusion criteria, which was determined by a screening questionnaire: diagnosis of systemic lupus erythematous based on the 1982 American Academy of Rheumatology criteria with recent revisions for anti-phospholipid antibodies, presence of at least 16 teeth, in the age range of 18-65 years old, the clinical presence of at least 6 periodontal sites probing 4.0 mm being represented on each side and each arch of the mouth, and bleeding on gentle probing at the aforementioned periodontitis sites. The subjects could not participate in the study if they possessed any of the following exclusion criteria: the presence of drug induced systemic lupus erythematous, previous periodontal treatment within the past 6 months, a positive history of systemic infections within the last 6 months (such as Hepatitis, Pancreitis, Diabetes, etc.), currently pregnant, the presence of any other severe concurrent systemic diseases that may impact on the periodontal condition (such as uncontrolled diabetes). Table 1 further explains the characteristics of the study participants in all 4 groups in the study. Table 1: Characteristics of Study Subjects Characteristic SLE Subjects With Periodontitis (SLE/P: n=13) SLE Subjects Without Periodontitis (SLE/X: n=16) Non-SLE Subjects With Periodontitis (X/P: n=8) Non-SLE Subjects Without Periodontitis (X/X: n=12) Mean Age in years(Std Dev) 40.9 (10.578) 30.1 (12.316) 48.9 (6.325) 38.9 (10.933) Female/Male 12/1 13/3 7/1 9/3 Race: AA 8 6 2 0 C 4 6 6 10 H 1 2 0 1 A 0 2 0 1 AA = African American. C = Caucasian. H = Hispanic. A = Asian. The initial visit for all the subjects involved periodontal probing with a standard 15.0 mm UNC periodontal probe, the identification of the presence of bleeding on gentle

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11 probing, identification of at least 16 teeth in the mouth, and a review of medical and dental history. Subjects also had a urine sample and approximately 60 mL of blood collected. The blood and urine samples were collected by the General Clinical Research Center (GCRC) at the University of Florida SHANDs in Gainesville, Florida. Anti dsDNA antibodies (-dsDNA antibodies) were measured by the enzyme-linked immunosorbent assay (ELISA) method. Testing was done by coating 96-well Nunc Immobilizer amino polystyrene plates with calf thymus double stranded DNA (Sigma, D-4522) using Reacti-Bind DNA coating solution (Pierce, 17250) as the binding method. Plates were then incubated for 12 hours at +4C and then subsequently washed with TBS-Tween20 and blocked with 0.5% BSA NET/NP40 0.05% NaN3 . This was followed by a 12 hour incubation period. Plates were then incubated for another 2 hours with diluted serum samples (1:500) and serially diluted standard. After incubation with the primary antibody, the plates were then washed and a diluted enzyme labeled second antibody (1:1000) (Goat anti-human IgG AP, Southern Biotech, 2040-04) was added into the wells and was incubated for another 1.5 hours. After washing, the plates were then developed with a phosphatase substrate (Sigma, S-0942). Absorbance was read at the wavelength of 405nm and the results were analyzed by SOFTmax Pro 4.3 LS. Using results from serial standard dilution SOFTmax Pro 4.3 LS calculated a standard curve that was used to convert absorbance into units. Each of the samples was tested in triplicates and the final result was the mean value of the 3 tests which was then converted into units. Cyclic citrullinated peptide (CCP) antibodies were measured by the enzyme-linked immunosorbent assay (ELISA) method. A standardized reagent kit was purchased from

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12 INOVA Diagnostics based out of California and used for the test. The ELISA was performed according to the manufacturer’s specifications and the measurements were recorded. High-sensitivity C-reactive protein (HSCRP), rheumatoid factor (RF), Complement factor 3 (C3), and Complement factor 4 (C4) were all tested simultaneously with a Nephelometer. All tests were performed according to the manufacturers specifications and measurements recorded as Units.

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CHAPTER 3 RESULTS For the study four groups of fifty (50) subjects were divided based on their SLE and periodontitis. In Group one were subjects that were previously diagnosed with SLE and periodontitis, SLE/P (n=14). In Group two were subjects who were previously diagnosed with SLE but did not show clinical signs of periodontitis, SLE/X (n=16). In Group 3 were subjects who were previously diagnosed with periodontitis but reported they were never diagnosed with SLE, X/P (n=8). In Group 4 were subjects who reported they were never diagnosed with SLE nor showed clinical signs of periodontitis, X/X (n=10) (Figure 3). Subjects Per Group05101520Group 1 (SLE/P)Group 2 (SLE/X)Group 3 (X/P)Group 4 (X/X)Number Figure 3: Number of subjects per group. The mean age and standard deviation of the whole study population was 37.5 years. The percentage of female subjects in the study was 93.75%. The percentage of male 13

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14 subjects was 6.25%. The mean age, percent female/male, and race is calculated in Table 1. Complement protein C3 has a normal range of 90-180 Mg/dl. A total of 48 subjects had C3 levels recorded (Table 2). Table 2: Complement Factor (C3) Group Analysis. Test Group N Mean (Norm 90-180 Mg/dl) Standard Deviation Lower Confidence Limit Upper Confidence Limit C3 SLE/PERIO 12 81.62 35.28 59.20 104.03 SLE/NONPERIO 16 100.50 23.38 88.04 112.96 NONSLE/PERIO 8 131.50 16.27 117.90 145.10 NONSLE/NONPERIO 12 109.76 21.51 96.09 123.43 Twelve subjects in Group 1 (SLE/P) had C3 levels tested. Five out of the twelve (5/12) subjects had C3 levels below normal levels ranging from 25.1 to 66.3 Mg/dL (Figure3). There were 7/12 subjects that had normal levels, ranging from 95.4-130 Mg/dL with 6/7 of them within 13 Mg/dL of each other. Group 2 (SLE/X) had 16 subjects who had C3 levels recorded. Five out of the sixteen (5/16) recorded levels below normal, ranging from 42.5-83.1 Mg/dL. The remaining 11/16 subjects all had normal levels ranging from 96.7-131 Mg/dL. Group 3 (X/P) had 8 subjects with C3 values. All eight of the subjects (100%) had normal values, ranging from 106-151 Mg/dL. The range for Group 3 started higher than Group 1 and Group 2, and also had a higher upper limit than both groups. Group 4 (X/X) had 12 subjects with C3 values recorded. Two of the 12 subjects were below normal levels with a range of 74.2-87.4 Mg/dL. The remaining 10/12 subjects all fell into the normal range (93.1 to 142 Mg/dl). Groups 1 and 2 both showed a positive SLE effect (p> 0.0003) over the non-SLE groups. There was no periodontitis effect that could be shown (p> 0.8520). When it was

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15 evaluated for any statistical interactions between the groups only a very slight interaction was found (p> 0.0107), but was not found to be statistically significant. Complement (C3)050100150200SLE/PSLE/XX/PX/XGroupMd/dl <90 = Below Normal 90-180 = Normal Figure 4: Complement C3 trend in all four groups. Complement protein C4 had a normal range of 10-40 Mg/dL (Table 3). A total of 47 subjects had C4 values recorded (Figure 4). Twelve subjects in Group 1 had C4 values recorded. Table 3: Complement Factor (C4) Group Analysis. TEST GROUP N MEAN (Norm 10-40 Mg/dl) STANDARD DEVIATION LOWER CONFIDENCE LIMIT UPPER CONFIDENCE LIMIT C4 SLE/PERIO 12 14.58 9.30 8.67 20.49 SLE/NONPERIO 15 17.41 8.06 12.95 21.87 NONSLE/PERIO 8 24.98 6.26 19.74 30.21 NONSLE/NONPERIO 12 20.08 5.75 16.43 23.74 Five out of the twelve (5/12) had values below normal, ranging from 4.12 to 8.54 Mg/dL. The remaining 7/12 subjects fell into the normal range, with values ranging from 13.8 to 36.5 Mg/dL. Group 2 had a total of 15 subjects with C4 values, with 3/15 falling into the below normal range (4.83 to 8.15 Mg/dL). The remaining 12/15 subjects were

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16 all in the normal range from13.5 to 32.3 Mg/dL. The values for Groups 1 and 2 were matched together relatively close, from the low to the high values. In Group 3 there were 8 subjects with C4 values and all 8/8 were in the normal range (from 17 to 33 Mg/dL). Group 4 had 12 subjects with C4 values, one out of the twelve (1/12) was below the normal range (9.3 Mg/dL). The remaining 11/12 ranged from 13.3 to 26.7 Mg/dL. This test had a positive SLE effect (p> 0.0065) and a negative periodontitis effect (p> 0.6537) similar to that seen in the C3 test. When evaluated for any interactions between the groups, statistically there was only a very slight interaction (p> 0.0979) but not statistically significant. Complement (C4)010203040SLE/PSLE/XX/PX/XGroupMg/dl <10 Below Normal 10-40 Normal Figure 5: Complement C4 trends for all 4 groups. Anti-double-stranded DNA was considered negative if the value was less than 0.552 Units, it “may be” positive if the value was between 0.552-1.124 Units, and was positive if the value was greater than 1.124 Units. A total of forty-nine subjects had -dsDNA values recorded (Figure 5). Group 1 had a total of 12 subjects with -dsDNA values recorded. One out of the twelve subjects was negative with a value of 0.536 Units. There were 6/12 that may be positive for -dsDNA with a range of 0.566 to 0.696 Units. Group 1 had a total of 5/12 subjects that were positive for -dsDNA with a range

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17 of 1.25 to 974.51 Units (Table 4). There were two patients that recorded extremely high values for -dsDNA (735.208 and 974.51 Units). Figure 2 was adjusted for these Table 4: Anti-double-stranded DNA Group Analysis. TEST GROUP N MEAN STANDARD DEVIATION LOWER CONFIDENCE LIMIT UPPER CONFIDENCE LIMIT dsDNA SLE/PERIO 13 132.63 324.24 -63.31 328.56 SLE/NON-PERIO 16 5.56 18.90 -4.51 15.63 NON-SLE/PERIO 8 0.21 0.04 0.17 0.24 NON-SLE/NONPERIO 12 4.39 14.58 -4.87 13.66 severely high recordings, any value that was above 2.0 Units was recorded as 2.1 Units to allow for easier interpretation of the trends. Since the figure only represents the trends associated with -dsDNA, it is irrelevant what the upper limit is numerically. Group 2 had a total of 17 subjects with -dsDNA values recorded. There were 7/17 that were negative (range 0.211 to 0.445 Units), and 2/17 that may be positive (range 0.801 to 1.042 Units), and 8/17 subjects that were positive for -dsDNA (range 1.14 to 76.411 Units). Group 3 had a total of 8 subjects with -dsDNA values recorded. All 8/8 subjects in Group 3 were negative for the -dsDNA antibodies, with values ranging from 0.154 to 0.261 Units. Group 4 had a total of 12 subjects with -dsDNA values. There were 11/12 subjects that were negative, with a range of 0.105 to 0.427 Units. A single subject (1/12) was positive with a value of 50.678 Units. For the -dsDNA group statistically there was shown to be no SLE effect (p> 0.1841), nor a periodontitis effect (p> 0.2211). When evaluated for any interactions between the groups, none were found (p> 0.1918).

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18 Anti-Double-Stranded DNA (anti-dsDNA)0 0.5 1 1.5 2 2.5 SLE/PSLE/XX/PX/XGroupUnits <0.552 NEG (-) 0.552-1.124 Maybe (+) >1.124 Positive (+) Figure 6: Anti-dsDNA trends for all four Groups. Cyclic Citrullinated Peptide (CCP) was considered negative if it is less than 20 Units, it has a low positive result if it is between 20-40 Units, and is considered high positive if it is above 40 Units. A total of fo rty-nine subjects had CCP values calculated (Figure 6). Group 1 had a total of 13 subjects with CCP values (Table 5). There was 10/13 subjects that were negativ e (range 6.57 to 14.13 Units). Table 5: Cyclic Citrullina ted Peptide Group Analysis. TEST GROUP N MEAN STANDARD DEVIATION LOWER CONFIDENCE LIMIT UPPER CONFIDENCE LIMIT CCP SLE/PERIO 13 14.17 10.11 8.06 20.28 SLE/NONPERIO 16 13.17 13.11 6.18 20.16 NONSLE/PERIO 8 10.11 2.75 7.81 12.41 NON-SLE/NONPERIO 12 9.17 3.96 6.66 11.69 A total of 2/13 subjects were consider ed low positive with values of 21.98 and 22.12 Units. A single subject (1/13) was reco rded as a high positive with 42.89 Units. Group 2 had a total of 16 subjects with CCP va lues recorded. The group was divided into 14/16 with a negative score (ranging fr om 5.28 to 14.84 Units) and 2/16 with a high

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19 positive score with values of 45.75 and 46.39 Units. Group 3 and Group 4 had eight and twelve subjects respectively that had CCP values recorded. The range for the Groups was 5.71 to 14.84 Units for Group 3, and 5.57 to 16.7 Units for Group 4. The values for Groups 3 and 4 showed similarities in thei r trends, with the values almost matching between the two groups. No statistically significan t interaction between any of the groups was found for the CCP test (p> 0.9913). Neither SLE (p> 0.1559) , nor periodontitis (p> 0.7301) had a statistical effect on any of the groups studied. Cyclic Citrullinated Peptide (CCP)0 10 20 30 40 50 SLE/PSLE/XX/PX/XGroupUnits <20 NEG (-) 20-40 LOW (+) >40 HIGH (+) Figure 7: Anti-Cyclic Citrullinated Pep tide (anti-CCP) trends for all four Groups. Rheumatoid Factor (RF) was recorded as being within the normal range when the values were between 0-15 IU/mL. There was a total of 48 subjects that had their Rheumatoid Factor (RF) values recorded (F igure 7). Group 1 had a total of 12 subjects with RF values. There were 10/12 subject s that fell into the normal range (10-13.9 IU/mL) with 9/10 having a value of 10 IU/m L. There were 2/12 that had above normal values (21 and 31.3 IU/mL) (Table 6).

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20 Table 6: Rheumatoid Factor Group Analysis. TEST GROUP N MEAN (Norm 0-10 IU/mL) STANDARD DEVIATION LOWER CONFIDENCE LIMIT UPPER CONFIDENCE LIMIT RF SLE/PERIO 12 15.12 13.19 6.74 23.50 SLE/NONPERIO 16 13.91 6.84 10.26 17.55 NONSLE/PERIO 8 10.28 0.78 9.62 10.93 NONSLE/NONPERIO 12 10.00 0.00 . . In Group 2 a total of 16 subjects had RF valu es recorded. Thirteen out of sixteen (13/16) had normal values (ranging from 10 to 13.9 IU/mL), with 12/ 13 having a value of 10 IU/mL. Group 3 and Group 4 had eight and twelve subjects re spectively, with all 20/20 within the normal range. Group 3 had 7/8 with a 10 IU/mL score and 1/8 with a score of 12.2 IU/mL. All twelve subjects in Group 4 had a value of 10 IU/mL. Statistically, neither SLE (p> 0.0633) nor periodo ntitis (p> 0.7478) were shown to have a significant effect on the results. In addition, there was not statistical in teraction (p> 0.8395) found between any of the groups studied. Rheumatoid Factor (RF)0 10 20 30 40 SLE/PSLE/XX/PX/XGroupI.U./ml 0-15 = NORM >15 HIGH Figure 8: Rheumatoid Factor (RF) trends for all four Groups.

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21 High-sensitivity C-reactive protein (HSCRP) is considered normal when the value falls in the range 0-3 Mg/L. A total of fort y-seven subjects had HSCRP values recorded (Figure 8). Group 1 had a total of 12 subjects with HSCRP values with 5/12 having normal values (range 0.798 to 2.11 Mg/L), and the remaining 7/12 having above normal values (ranging from 9.22 to 31.6 Mg/L) (Table 7). Group 2 had a total of 15 Table 7: High-Sensitivity C-Reactiv e Protein Group Analysis. TEST GROUP N MEAN (Norm 0-3 Mg/L) STANDARD DEVIATION LOWER CONFIDENCE LIMIT UPPER CONFIDENCE LIMIT HSCRP SLE/PERIO 12 11.90 11.19 4.79 19.02 SLE/NONPERIO 15 8.56 18.07 -1.45 18.56 NONSLE/PERIO 8 5.65 3.27 2.91 8.39 NONSLE/NONPERIO 12 2.97 3.08 1.01 4.93 subjects with their HSCRP values recorded. Normal values were found in 8/15 subjects (range 0.401 to 2.91 Mg/L) and higher than normal values in the remaining 7/15 subjects (range 4.88 to 71.2 Mg/L). Group 3 had a tota l of 8 subjects with HSCRP, a normal value was found for 2/8 subjects (range 2.15 to 2.31 Mg/L) and a higher than normal value was found for the remaining 6/8 subjec ts (range 3.57 to 10.7 Mg/L). In Group 4 there were a total of 12 subjects with HSCRP values. A total of 8/12 had normal values (ranging from 0.222 to 2.38 Mg/L). And the rema ining 4/12 subjects showed higher than normal values (range 3.02 to 9.7 Mg/L). In both Group 3 and in Group 4 the higher than normal values were significantly lower than the higher than normal values for Group 1 and Group 2. Statistically there was no interaction found betw een any of the groups studi ed (p> 0.9263). Neither was

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22 there a statistically pos itive effect found for SLE (p> 0.1048) nor for periodontitis (p> 0.4035) in regards to High-Sensit ivity C-Reactive Protein values. High-Sensitivity C-Reactive Protein (HSCRP)0 10 20 30 40GroupMg/L 0-3 = NORM >3 = HIGH Figure 9: High-sensitivity C-reactive prot ein (HSCRP) trends for all four Groups.

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CHAPTER 4 DISCUSSION A vast array of system diseases have been implicated to have an association with periodontitis, such as diabetes, atherogenesis, cardiovascular diseases (CVD), and myocardial infarction (MI) (Ross 1999; Slade, Offenbacher et al. 2000). At this time there is only a very limited amount of information on the association of periodontitis with SLE. One study comparing the gingival indices (GI), plaque indices (PI), and probing depths (PD) showed no evidence for a predisposition to increased periodontal disease in SLE (Mutlu, Richards et al. 1993). However, another study reported an incidence of periodontitis in 94% of the patients with SLE (Rhodus and Johnson 1990), and another case showed 18 of the 30 patients (60%) had periodontitis in their SLE group (Novo, Garcia-MacGregor et al. 1999). It was the aim of this study to discover an association between systemic lupus erythematous and periodontitis. Several serum and urine markers were measured in a cross-sectional study design in four different groups of patients with and without systemic lupus erythematous and periodontitis. Each of the four groups was tested against each other to find an association. The complement factors (C3 & C4) were measured together using a Nephelometer. The normal range for C3 is from 90-180 Mg/dL and the normal range for C4 is from 10-40 Mg/dL. A decrease in the values for the SLE subjects was expected due to the already proven decrease found in SLE patients. This decrease is due to complement 23

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24 being in a bound state when SLE is active, therefore less “free” complement is in serum to be identified by the test antibody. An increase in the periodontitis patients was expected due to the fact that Gram-negative bacteria have been shown to activate the alternate pathway of complement. The results (Figures 4 & 5) showed that none of the patients in any of the groups had higher than normal values for either C3 or C4. Both of the SLE groups had a wide range of values with a small percentage of the subjects below the normal range as expected. It would be expected that Group 1 and Group2 have the lowest values for complement and Groups 3 and 4 have the highest. This was shown statistically with a positive SLE effect (p> 0.0003) and a negative periodontitis effect for C3 (p> 0.8520). As well as in the C4 test with a positive SLE effect (p> 0.0065) and a negative periodontitis effect (p> 0.6537). The positive SLE effect showed that the subjects that were positive for SLE were the only subjects that showed a decrease in their C3 and C4 values. The negative periodontitis effect could be explained by the intermittent nature of periodontitis. Patients with periodontitis can go through years of inactivity before any further loss of attachment is seen. Theoretically, the subjects in this study could have all possessed a stable periodontium therefore no complement would need to be active. Anti-double-stranded DNA antibodies that were concentrated below 0.552 Units were considered as being a negative result. When the antibodies were concentrated between 0.552 and 1.124 Units it was interpreted that the subject may be positive. A positive result was when the antibodies were concentrated above 1.124 Units.

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25 It has been shown that an increase in -dsDNA antibodies is seen in subjects with SLE (Horak, Scudla et al. 2001; Tyrrell-Price, Lydyard et al. 2001), although no studies to date have shown an association between -dsDNA and periodontitis. As expected 11/12 (91.67%) of the subjects in Group 1 were either positive or may be positive for -dsDNA antibodies, with only one subject with a negative score (Figure 6, Table 4). Groups 3 and 4 both had all negative results, except for one subject in Group 4. The single subject of Group 4, which had a positive result (50.678 Units), most likely has one or more of the following; SLE, RA, or some other inflammatory response that they were not previously diagnosed with. These trends do not lend themselves to the possibility of an association between SLE and periodontitis but only show that the patients who reported they had SLE did in fact have SLE. Statistically, there was no association found between any of the groups (Interaction p> 0.1918) nor was there an SLE effect (p> 0.1841) nor a periodontitis effect (p> 0.2211). A positive -dsDNA effect, in the SLE groups, was expected due to the already proven relationship between them in the literature. Cyclic Citrulinated Peptide (CCP) antibodies have been shown in multiple studies to have a high positive correlation with rheumatoid arthritis and only a weak association with SLE (Schellekens, de Jong et al. 1998; Schellekens, Visser et al. 2000; Bizzaro, Mazzanti et al. 2001; Avcin, Cimaz et al. 2002; Rantapaa-Dahlqvist, de Jong et al. 2003; Nielen, van Schaardenburg et al. 2004). In the present study, CCP was measured to test the idea that periodontitis may be the “rheumatoid arthritis of the mouth”. The majority of the subjects in the study, 44 out of the 49 (89.8%), reported a negative result for CCP (Figure 7, Table 5), with all of the subjects in Group 3 and 4 being negative as expected.

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26 Although CCP did not show a correlation between SLE and periodontitis, it did prove that none of the non-SLE patients had rheumatoid arthritis. On the other hand, five of the Group 1 and 2 subjects were either low positive or high positive for CCP antibodies. This lends itself to the possibility that these five subjects may be positive for rheumatoid arthritis, but the clinical signs are not yet evident (Rantapaa-Dahlqvist, de Jong et al. 2003; Nielen, van Schaardenburg et al. 2004; van Gaalen, Linn-Rasker et al. 2004). Rheumatoid Factor (RF) is a type of antibody found in the blood of most rheumatoid arthritis patients. A high level of rheumatoid factor can be caused by several autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematous as well as some severe infections (Roitt 1996). When RF is used concomitantly with CCP the specificity of a positive test for RA increases to a 95% confidence (van Gaalen, Linn-Rasker et al. 2004). A normal value for RF is in the range of 0-15 I.U./mL. Groups 3 and 4 had 100% (20 out of 20) of the subjects in the normal range for RF. Groups 1 and 2 had 23 out of 28 (82.1%) of the subjects in the normal range with 5 out of 28 (17.9%) having a high value for RF. This result, combined with the low positive and high positive result for the CCP antibodies, again points to the possibility that these subjects may be positive for RA but the clinical signs of the disease are not yet evident. The results did not, however, give evidence to the adage that “periodontitis is the rheumatoid arthritis of the mouth”. C-reactive protein (CRP) is a well known acute-phase protein which acts as a pro-inflammatory cytokine. C-reactive protein has been implicated in the pathogenesis of both periodontitis and SLE through the acute phase response. Elevated serum levels of

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27 CRP have been shown to be associated with deep periodontal pockets, severe attachment loss, and subgingival microflora in periodontitis (Ebersole, Machen et al. 1997; Tracy, Lemaitre et al. 1997; Slade, Offenbacher et al. 2000; DeNardin 2001; Mercado, Marshall et al. 2001; Noack, Genco et al. 2001). Recent studies, also show elevated serum CRP levels to be associated with SLE (Liou 2001; Mercado, Marshall et al. 2001). High-sensitivity C-reactive protein (HSCRP) was considered normal in this study when the values were in the range of 0-3 Mg/L. As seen in Figure 9, Groups 1 and 2 have higher values for HSCRP than either Groups 3 or 4. Both groups, however, have subjects within the normal range. A correlation can be inferred from the data, that subjects with SLE have higher values for HSCRP than the non-SLE subjects. No statistically significant association between the two diseases can be made with these results (p> 0.9263). Neither SLE (p> 0.1048) nor periodontitis (p> 0.4035) were shown to have a positive effect on the values of HSCRP in any of the groups studied. An interesting finding can be seen in Table 8, related to the mean values of the four groups. Group 1 had the highest mean value for HSCRP with Groups 2 and 3 having relatively similar mean values and Group 4 having a significantly lower mean value. Since C-reactive protein has been shown to be elevated in both SLE and periodontitis patients, these means are as expected. Group 2 would be elevated from the influence of SLE and Group 3 elevated from the influence of the periodontitis. Since Group 4 had neither SLE nor periodontitis, this group should have the lowest mean value. This study corroborates the results found in multiple other studies (Ebersole, Machen et al. 1997; Tracy, Lemaitre et al. 1997; Slade, Offenbacher et al. 2000; DeNardin 2001; Liou 2001; Mercado, Marshall et al. 2001; Noack, Genco et al.

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28 2001) that C-reactive protein levels are elevated in both Systemic Lupus Erythematosus and in periodontitis. Table 8: Mean Values for High-Sensitivity C-Reactive Protein. Group N Mean Std. Dev. SLE/P 12 11.905 11.195 SLE/X 15 8.557 18.071 X/SLE 8 5.65 3.272 X/X 12 2.968 3.081 Also interesting was the relative difficultly in finding Systemic Lupus Erythematosus subjects above age 35 who did not have any clinical signs of periodontitis. This is reflected in the 10 year difference between the mean ages in Groups 1 and 2. Periodontitis is known to be a disease primarily of the older population, and contributes to this discrepancy. If a larger sample size could have been taken, it would be interesting to study the prevalence of SLE subjects who showed signs of periodontitis.

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LIST OF REFERENCES Arai, K. I.; Lee, F.; Miyajima, A.; Miyatake, S.; Arai, N.; Yokota, T. (1990). "Cytokines: coordinators of immune and inflammatory responses." Annu Rev Biochem 59: 783-836. Avcin, T.; Cimaz, R.; Falcini, F.; Zulian, F.; Martini, G.; Simonini, G.; Porenta-Besic, V.; Cecchini, G.; Borghi, M. O.; Meroni, P. L. (2002). "Prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies in juvenile idiopathic arthritis." Ann Rheum Dis 61(7): 608-11. Baechler, E. C.; Batliwalla, F. M.; Karypis, G.; Gaffney, P. M.; Ortmann, W. A.; Espe, K. J.; Shark, K. B.; Grande, W. J.; Hughes, K. M.; Kapur, V.; Gregersen, P. K.; Behrens, T. W. (2003). "Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus." Proc Natl Acad Sci U S A 100(5): 2610-5. Baumann, H. and J. Gauldie (1990). "Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation." Mol Biol Med 7(2): 147-59. Baumann, H. and J. Gauldie (1994). "The acute phase response." Immunol Today 15(2): 74-80. Bennett, L.; Palucka, A. K.; Arce, E.; Cantrell, V.; Borvak, J.; Banchereau, J.; Pascual, V. (2003). "Interferon and granulopoiesis signatures in systemic lupus erythematosus blood." J Exp Med 197(6): 711-23. Beutler, B. and A. Cerami (1988). "Tumor necrosis, cachexia, shock, and inflammation: a common mediator." Annu Rev Biochem 57: 505-18. Beutler, B. and E. T. Rietschel (2003). "Innate immune sensing and its roots: the story of endotoxin." Nat Rev Immunol 3(2): 169-76. Bickel, M.; Axtelius, B.; Solioz, C.; Attstrom, R. (2001). "Cytokine gene expression in chronic periodontitis." J Clin Periodontol 28(9): 840-7. Bizzaro, N.; Mazzanti, G.; Tonutti, E.; Villalta, D.; Tozzoli, R. (2001). "Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis." Clin Chem 47(6): 1089-93. 29

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30 Blanco, P., AK Palucka, M. Gill, V. Pascual, J. Banchereau (2001). "Induction of dendritic cell differentiation by IFN-alpha insystemic lupus erythematosus." Science 294: 1540-1543. Burge, S. M.; Frith, P. A.; Juniper, R. P.; Wojnarowska, F. (1989). "Mucosal involvement in systemic and chronic cutaneous lupus erythematosus." Br J Dermatol 121(6): 727-41. Condemi, J. J. (1987). "The autoimmune diseases." Jama 258(20): 2920-9. Cooper, N. R. (1985). "The classical complement pathway: activation and regulation of the first complement component." Adv Immunol 37: 151-216. Darveau, R. P.; Tanner, A.; Page, R. C. (1997). "The microbial challenge in periodontitis." Periodontol 2000 14: 12-32. DeNardin, E. (2001). "The role of inflammatory and immunological mediators in periodontitis and cardiovascular disease." Ann Periodontol 6(1): 30-40. Ebersole, J. L.; Machen, R. L.; Steffen, M. J.; Willmann, D. E. (1997). "Systemic acute-phase reactants, C-reactive protein and haptoglobin, in adult periodontitis." Clin Exp Immunol 107(2): 347-52. Farries, T. C. and J. P. Atkinson (1991). "Evolution of the complement system." Immunol Today 12(9): 295-300. Gorska, R.; Gregorek, H.; Kowalski, J.; Laskus-Perendyk, A.; Syczewska, M.; Madalinski, K (2003). "Relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum samples from patients with chronic periodontitis." J Clin Periodontol 30(12): 1046-52. Horak, P.; Scudla, V.; Hermanovo, Z.; Pospisil, Z.; Faltynek, L.; Budikova, M.; Kusa, L. (2001). "Clinical utility of selected disease activity markers in patients with systemic lupus erythematosus." Clin Rheumatol 20(5): 337-44. Horwood, N. J.; Elliott, J.; Martin, T. J.; Gillespie, M. T. (1998). "Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells." Endocrinology 139(11): 4743-6. Jiang, Y.; Magli, L.; Russo, M. (1999). "Bacterium-dependent induction of cytokines in mononuclear cells and their pathologic consequences in vivo." Infect Immun 67(5): 2125-30. Kinane, D. F.; Hodge, P.; Eskdale, J.; Ellis, R.; Gallagher, G. (1999). "Analysis of genetic polymorphisms at the interleukin-10 and tumour necrosis factor loci in early-onset periodontitis." J Periodontal Res 34(7): 379-86.

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31 Lam, J.; Takeshita, S.; Barker, J. E.; Kanagawa, O.; Ross, F. P.; Teitelbaum, S. L. (2000). "TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand." J Clin Invest 106(12): 1481-8. Leach, M. (1998). "Signs and symptoms of systemic lupus erythematosus." Nursing Times 94(13): 50-52. Liou, L. B. (2001). "Serum and in vitro production of IL-1 receptor antagonist correlate with C-reactive protein levels in newly diagnosed, untreated lupus patients." Clin Exp Rheumatol 19(5): 515-23. Mercado, F. B.; Marshall, R. I.; Klestov, A. C.; Bartold, P. M. (2001). "Relationship between rheumatoid arthritis and periodontitis." J Periodontol 72(6): 779-87. Mutlu, S.; Richards, A.; Maddison, P.; Scully, C. (1993). "Gingival and periodontal health in systemic lupus erythematosus." Community Dent Oral Epidemiol 21(3): 158-61. Nielen, M. M.; van Schaardenburg, D.; Reesink, H. W.; van de Stadt, R. J.; van der Horst-Bruinsma, I. E.; de Koning, M. H.; Habibuw, M. R.; Vandenbroucke, J. P.; Dijkmans, B. A. (2004). "Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors." Arthritis Rheum 50(2): 380-6. Noack, B.; Genco, R. J.; Trevisan, M.; Grossi, S.; Zambon, J. J.; De Nardin, E. (2001). "Periodontal infections contribute to elevated systemic C-reactive protein level." J Periodontol 72(9): 1221-7. Novo, E.; Garcia-MacGregor, E.; Viera, N.; Chaparro, N.; Crozzoli, Y. (1999). "Periodontitis and anti-neutrophil cytoplasmic antibodies in systemic lupus erythematosus and rheumatoid arthritis: a comparative study." J Periodontol 70(2): 185-8. Page, R. C.; Offenbacher, S.; Schroeder, H. E.; Seymour, G. J.; Kornman, K. S. (1997). "Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions." Periodontol 2000 14: 216-48. Rantapaa-Dahlqvist, S.; de Jong, B. A.; Berglin, E.; Hallmans, G.; Wadell, G.; Stenlund, H.; Sundin, U.; van Venrooij, W. J. (2003). "Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis." Arthritis Rheum 48(10): 2741-9. Rhodus, N. L. and D. K. Johnson (1990). "The prevalence of oral manifestations of systemic lupus erythematosus." Quintessence Int 21(6): 461-5. Roitt, I. (1996). "Immunology." 28.14.

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32 Ronnblom, L., and G.V. Alm (2001). "A pivotal role fr the natural interferon alpha-producing cells (plasmacytoid dendritic cells) in the pathogenesis of lupus." J. Exp. Med. 194: F59-F63. Ross, R. (1999). "Atherosclerosis--an inflammatory disease." N Engl J Med 340(2): 115-26. Rozzo, S. J. (2001). "Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus." Immunity 15: 435-443. Schellekens, G. A.; de Jong, B. A.; van den Hoogen, F. H.; van de Putte, L. B.; van Venrooij, W. J. (1998). "Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies." J Clin Invest 101(1): 273-81. Schellekens, G. A.; Visser, H.; de Jong, B. A.; van den Hoogen, F. H.; Hazes, J. M.; Breedveld, F. C.; van Venrooij, W. J. (2000). "The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide." Arthritis Rheum 43(1): 155-63. Siwik, D. A.; Chang, D. L.; Colucci, W. S. (2000). "Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metalloproteinase activity in cardiac fibroblasts in vitro." Circ Res 86(12): 1259-65. Slade, G. D.; Offenbacher, S.; Beck, J. D.; Heiss, G.; Pankow, J. S. (2000). "Acute-phase inflammatory response to periodontal disease in the US population." J Dent Res 79(1): 49-57. Tan, E. M.; Cohen, A. S.; Fries, J. F.; Masi, A. T.; McShane, D. J.; Rothfield, N. F.; Schaller, J. G.; Talal, N.; Winchester, R. J. (1982). "The 1982 revised criteria for the classification of systemic lupus erythematosus." Arthritis Rheum 25(11): 1271-7. Tracy, R. P.; Lemaitre, R. N.; Psaty, B. M.; Ives, D. G.; Evans, R. W.; Cushman, M.; Meilahn, E. N.; Kuller, L. H. (1997). "Relationship of C-reactive protein to risk of cardiovascular disease in the elderly. Results from the Cardiovascular Health Study and the Rural Health Promotion Project." Arterioscler Thromb Vasc Biol 17(6): 1121-7. Tyrrell-Price, J.; Lydyard, P. M.; Isenberg, D. A. (2001). "The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus." Clin Exp Immunol 124(1): 118-25.

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33 van Gaalen, F. A.; Linn-Rasker, S. P.; van Venrooij, W. J.; de Jong, B. A.; Breedveld, F. C.; Verweij, C. L.; Toes, R. E.; Huizinga, T. W. (2004). "Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study." Arthritis Rheum 50(3): 709-15. Yamamoto, M.; Takeda, K.; Akira, S. (2004). "TIR domain-containing adaptors define the specificity of TLR signaling." Mol Immunol 40(12): 861-8.

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BIOGRAPHICAL SKETCH Stephen L. Strout graduated from Apopka High School in Apopka, Florida in 1993, with Honors. He was accepted to the University of Florida where he pursued his bachelor’s degree in microbiology with a minor in chemistry and a minor in business administration. He was a member of the Pre-Professional Service Organization, and Habitat for Humanity; played multiple intramural sports; and volunteered at Shands, and the VA hospital. After graduation in 1997 (with his bachelor’s degree), he attended the University of Florida College of Dentistry. While in dental school, Stephen was part of the American Student Dental Organization, Psi Omega dental fraternity, and participated in a service trip to the Dominican Republic providing dental care to the indigent population there. While in dental school, Stephen was accepted into the University of Florida College of Dentistry Periodontology resident program. While a resident, Stephen became a member of the American Academy of Periodontology, the American Dental Association, the Southern Academy of Periodontology, the Florida Association of Periodontists, and the Academy of Osseointegration. He is currently the chief resident in the periodontic department and has participated in multiple off-site continuing education seminars. After graduation from his Periodontics residency, he will practice in Daytona Beach, accompanied by his wife Meridith. 34