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  • 1. Periodontal Disease and Rheumatoid Arthritis: From Epidemiologic Associations toShared Disease MechanismsBy: Clifton O. Bingham III, MDDivision of Rheumatology, Department of MedicineJohns Hopkins UniversityBaltimore, MD  There are no disclosures relevant to this talk  There will be no discussion of off-label uses of medications, or investigational agents in this presentationOutline  A Primer on Periodontal Disease  Associations of Periodontal Disease and Rheumatoid Arthritis  Citrullination as a shared mechanism of diseaseBackgroundThere have been multiple reports that have described an association between periodontal diseaseand rheumatoid arthritis. Its been recognized that periodontal disease is associated with othersystemic conditions, including diabetes, atherosclerotic cardiovascular disease, low birth weightinfants and preterm labor. Intervention by treating periodontal disease can actually improvediabetes control, can improve the weight of infants, and can decrease preterm labor. There is aninterventional study that has been ongoing looking at the effect in atherosclerotic cardiovasculardisease. This implies that there may be relationships between oral health and systemic diseases.Periodontal DiseasePeriodontitis  Inflammatory disease  Consequence of an infectious trigger  Initially involves gingival soft tissue  Characterized by destruction of surrounding connective tissue matrixStages of Periodontitis  Gingivitis, Chronic inflammation  Colonization by periopathogenic “red complex” organisms  Loss of connective tissue attachments to teeth  Bone resorption  Tooth lossBiofilm Development in PD
  • 2. This is a biofilm on the tooth of a patient with periodontitis - there are multiple bacteria that are piled on top of each other. Certain bacteria come along and colonize and then there are bacteria that grow on top of them, due to the specific adhesion molecules between the two. Then additional bacteria that come along with additional properties that build up on top of those.Colonization in Periodontal DiseaseBelow is an image of the late colonizers that really describe patients with periodontal disease.
  • 3. Porphyromonas gingivalisPatients with periodontitis have specific flora in their mouths, of which the most important isPorphyromonas gingivalis. Others that are also of importance and track along with it, areTreponema denticola, certain species of Eubacterium, Providencia intermedia andactinomycetemcomitans organism here as well. When these come along the process goes frominflammation to one that has the potential for destruction.Porphyromonas gingivalis is a gram negative anaerobic bacteria, and its one of the terminal “redcomplex” organisms and a late colonizers. A property that is unique to P. gingivalis is itsexpression of LPS. Most of the other bacteria that are underneath there dont have LPS—assuch, it can activate toll-like receptors. Also, P. gingivalis has many proteolytic enzymes, manyof which are involved in arginine, lysine and cysteine metabolism, including a peptidyl argininedeiminase. There are also many enzymes that are directly secreted by this bacteria that causecollagen degradation as well as other exotoxins and gingipains which make many proteinssusceptible to further cleavage. P. gingivalis has DNA for enolase, which has overlappingsequence with human alpha enolase, which is susceptible to citrullination. Antibodies to P.gingivalis are a marker for periodontal disease—one of the ways that people can be identified forhaving exposure to this organism is by looking at serum antibody responses against it.Periodontal Disease Epidemiology  Leading cause of tooth loss in the US  Estimates of Prevalence of Periodontal Disease  NHANES III 35% of adults > 35 years  30% of these are moderate to severe (13% total)  Substantial proportion of severe PD is progressive  Risk factors  Cigarette smoking, medications, systemic diseases  Twin studies ≈50% heritability  Genetic Associations reported  DR4, TNF, IL1β, IL6, TLR, CD14 polymorphismsHealthy/Gingivitis/ Moderate Periodontitis/Severe Periodontitis
  • 4. The progression of gum disease: 1. Healthy gums 2. Gingivitis 1. Some hyperemia of the gingiva 2. A little bit of swelling 3. Some colonization 4. Excessive plaque accumulation—toothbrushing and good oral hygiene will help 3. Moderate periodontitis 1. Gum inflammation 2. Recession of the gum line—parts of the tooth that werent visible now exposed 3. Regression of the gingiva 4. Severe periodontitis 1. Triangles and black spaces between teeth 2. Significant loss of the gingiva 3. Beginnings of loss of bone 4. Much plaque accumulationThis is a progressive disease and a progressive cycle.Radiographic Changes in Periodontal Disease
  • 5. There are many things that are similar between periodontal disease and rheumatoid arthritis, andone of those is the effect on bone. A normal bitewing x-ray with healthy gums has a normalbone level at the bottom of the tooth.The bone level in a patient with periodontal disease is regressed significantly and there arespaces underneath the roots of the teeth.Pathogenesis of Periodontal Disease  Bacteria necessary but not sufficient  Bacterial eradication does not necessarily lead to resolution  Submicrobicidal tetracyclines improve PD (MMP Activity)  Antibody response to bacteria predicts progressive disease and bone loss (e.g. IgG Anti- P. gingivalis)  T-cell component (HLA, oligoclonal expansions)  Cytokine-mediated effector damage (osteoclast, RANKL)  Genetic Associations (DR4, IL1a promoter, etc)Tooth DiagramThis diagram shows what happens in periodontal disease. The accumulation of bacteria andplaque and biofilm formation that develops in the pocket between the tooth and the gingiva.
  • 6. These bacteria begin to release substances such as LPS, activating neutrophils, macrophages, andrecruiting them into the site. There are T-cells that proliferate and there are plasma cells in thegingiva as well. These proliferate and rheumatoid factors have also been described in patientswith periodontal disease. There are also the antibodies that develop again to the bacteria. Therelease of multiple cytokines are detectable and are expressed in the gingival tissue as well asreleased into the gingival crevicular fluid. There is also activation of fibroblasts that ends upleading to further connective tissue matrix degradation and osteoclast activation that leads to thedegradation of bone. Many of the players that we are seen in rheumatoid arthritis arerecapitulated in the periodontal microenvironment.Previous Studies of RA and PDNHANES III (dePablo P, et al)  Subjects ≥60 years with musculoskeletal and single quadrant dental exams  RA more likely edentulous (OR=2.27) and have PD (OR=1.82) compared with non-RA subjects  Adjusted for age, sex, race/ethnicity, and smokingSmall case-control studies have reported similar results (Mercado FB, et al; Pischon N, et al)  OR 2.3-8.0 for PD in RA patents vs controlsMarotte H, et al  High association-- periodontal bone loss and wrist X-ray damage (x2=11.82)  RA shared epitope associated with both periodontal bone loss and wrist damage (OR=2.2)Recent RA-PD Case Control Study (Pischon N et al)  57 RA subjects, 52 healthy controls (age and gender-matched)  Measurements at 4 sites per tooth, PI, GI, PoD, CAL=CEJ-pocket bottom  Periodontally Diseased = mean CAL>4  In stepwise logistic regression with RA status, age, gender, education, alcohol and BMI, only RA and age remained predictors of periodontal disease  Adjusted OR of 8.05 in RA patients for periodontitis compared with controls (2.93-22.09)  When adjusted for PI and GI, OR drops to 6.09 (1.72-21.58)  RA patients higher GI, BOP, CAL 4.37 +/- 1.3mm vs 3.4 +/- 0.89 mm , PoD 3.71 +/- 0.73mm vs 3.16 +/- 0.58  Limitations: No information of RA disease characteristics, nonstandard definition of diseaseRA, PD, and CVD (Abou-Raya S et al)  100 RA patients with CVD, 50 RA patients without CVD, 50 controls without RA  84% of RA patients with CAD, had PD compared to 60% of RA patients without CAD and to 10% of control subjects. BOP, PoD >4mm, AL >4mm, missing teeth, calculus significantly higher in RA patients with CAD as compared to RA patients without CAD and to controls  The severity of PD significantly correlated to RA disease duration, DAS28 score, TNF-α level, ESR, hs-CRP, fibrinogen and HDL.
  • 7. This study gets at this issue of relationships between rheumatoid arthritis, periodontal diseaseand cardiovascular disease. Periodontal disease has been described as a risk factor foratherosclerosis and intervention studies have been ongoing trying to look at this relationshipbetter. There was an additive effect of RA plus cardiovascular disease and the association withperiodontal disease, periodontal disease further increasing the cardiovascular risk.TNF Inhibition Improves Experimental PeriodontitisThere have been several studies that have shown that cytokines that are involved in rheumatoidarthritis are also important in periodontal disease. Several studies have looked at models ofexperimental periodontitis and shown decreased inflammatory cell infiltration and bone loss withTNF inhibition, including a study that looked at P55 receptor knockout mice that had less boneloss and less severe periodontitis with an induced model of bacteria.TNF in Patients with Periodontal DiseaseTNF has been described as increased in patients with periodontal disease and higher in patientswho are smokers than are non-smokers. A TNF polymorphism has been reported and confirmedrecently in Japanese patients with severe periodontal disease. A very recent study looked at 19patients with rheumatoid arthritis versus 30 healthy controls and found that the serum TNF levelwas the only independent variable to predict probing death and attachment loss of bleeding onprobing. Its a small study with not many controls but it suggests an association with TNF withthe extent of periodontal disease.That was followed, however, with a study in the Journal of Periodontology that showed thatinfliximab treatment did not improve gingivitis:  40 RA subjects  20 IFX treated (mean 22.2 infusions)  20 with no infliximab  No difference in baseline RA disease activity  Perio status of IFX patients every 6 weeks  9 subjects from non inflix group with PD (mean probing depth > 4mm) treated with IFX and followed 9 months.  Exams on only 6 teeth, max values for each tooth and mean calculated  PD and AL not affected by IFX Rx in Group I vs 2  Patients with pre/post IFX did have decrease in AL (6.27 +/- 0.97mm vs 5.22 +/- 1.05mm, p<0.05), but PD constant  Gingivitis and bleeding index actually increased in 9 patients.  Limitations: 6 teeth examined, No control for confounders, Pre-post only 9 subjectsShared Mechanisms of Disease in PD and RAMediatorsIL-1TNFIL-6IL-8
  • 8. MMPNitric OxideLipoxinsPGE2CellsT cellsB cellsPlasma cellsMacrophagesNeutrophilsFibroblastsOsteoclastsAngiogenesisIn the general paradigm of RA vs. PD, mediators that are seen in many cell types can be lined up.Histopathologically, this is a patient with periodontal disease, with an intense neutrophilic, andwith mixed cellular infiltrate and even some small lymphoid follicles that appear to bedeveloping in the gingiva, which is very similar to what we might see in a rheumatoid joint.These relationships that have been described epidemiologically raise the question: Whichcomes first, the RA or the periodontal disease? The traditional paradigm is that RA precededthe development of periodontal disease but thats far from conclusive—its time to begin thinkingin the other direction.Prior Studies of RA and PDThe problems with many of these other studies that have been case-control and otherwise is thatthey have only looked at patients with established longstanding rheumatoid arthritis. There hasbeen very poor characterization of RA disease activity. The influence of medications and othercomorbid medical conditions has been unclear and uncontrolled. There has been poorcharacterization of any type of functional ability for the RA patients, especially hand function.Also, there has been no control for Sjögrens, and they have been poorly controlled for smoking.Old vs. New HypothesesThe old hypothesis: RA is Risk Factor for Periodontal Disease. Patients with RA had poororal hygiene because they couldnt grip their toothbrush and brush well, or they had RA and hadTMJ joints that were affected and couldn’t open their mouth enough to be able to brush theirteeth. Or they were immunosuppressed or received NSAIDs that could be risk factors for thedevelopment of periodontal disease, or they had secondary Sjögrens. None of these were provenin any way, however.The new hypothesis: Periodontal Disease is a Risk Factor for RA. There are similar geneticsusceptibilities, in terms of HLA-DR4 by two studies and some of these other polymorphismsthat have been described. There are similar lesions in the inflammatory focus and we there areother bacterial infections that can directly cause inflammatory arthritis, either by molecularmimicry or by other mechanisms, including rheumatic fever, Lyme disease and reactive arthritis.
  • 9. Question #1 - Do patients with Early RA have increased periodontal disease?Periodontal Assessment in RA Study (PARAS, R03 DE018094)  Inclusion  18-75 yrs  Definite or probable RA  RA diagnosis within 12 months  At least 20 teeth  Exclusion  Other inflammatory arthritis  Systemic or oral topical antibiotics within 30 days  Phenytoin, cyclosporine, coumadin, or other medications associated with gingival hyperplasia  Contraindications to probing (e.g. coagulopathy)  Valvular heart disease, prosthesis, or other conditions requiring ABX prophylaxis for dental procedures (unless willing to receive standard prophylaxis)RA Characteristics Explored RA Severity and RA Disease Activity RA Treatment Other Extra-articular Factors • RA duration • DAS28 • DMARDs • Demographic • RF • Swollen joints • Prednisone • Smoking • Anti-CCP • Tender joints • NSAIDs • Alcohol • Nodules • MD global • Aspirin use • Damage • Pt Global • Brushing • HAQ • Flossing • Ro/La • CRP • Diabetes • Schirmer test • Functional Class • Concom Meds • Medical HxDental Characteristics Explored Dental History Periodontal Questions Sjogrens Questions Dental Exam • Dental History • Bleeding Gums • Oral Dryness • Overall state of • Tooth loss • Swollen Gums • Ocular Dryness dentition • Hygiene • Halitosis • Examine all teeth • Brushing • Pockets • 6 sites per tooth • Flossing • Gaps between teeth • Multiple measures • Smoking • Unstimulated • Alcohol salivary flow and saliva collectionPeriodontal ExaminationThe periodontal examination being performed is an overall oral assessment. All teeth except thewisdom teeth are examined at six sites on every tooth for the following indices: plaque,gingivitis, pocket depth, distance from the edge of the gum down to where the bottom of thepocket is, the amount of recession or attachment loss, bleeding on probing, tooth mobility andfurcation (sticking the probe in and seeing if it can get underneath a root).E. Examples of Healthy/Gingivitis/Moderate Periodontitis/Severe Periodontitis
  • 10. Definitions  Periodontally Healthy  At least 24 teeth  No attachment loss > 3mm  Periodontally Diseased  At least 20 teeth  At least 4 sites with pocket depths greater than 3mm and  At least 4 sites with attachment loss greater than 3mm
  • 11.  Periodontal Progression  More than 2mm loss of attachment from baseline to 6- month measurement at more than 4 sitesOral Health Assessment in RA PatientsAssessment of the first 26 patients that were evaluated, breaking down patients between thosewith early rheumatoid arthritis and those with established rheumatoid arthritis showed that thedifference in disease duration is shorter disease in the patients with early RA. The CRPs were abit lower in the group of patients with early RA probably because there is a little bit more steroiduse in these patients. The tender joint counts were higher in the early rheumatoid arthritispatients. The DAS values were not statistically significantly different, but the numbers ofpatients remained somewhat low with this preliminary data.PD is Equally Present And Severe Early And Late In The Course of RAFor the periodontal parameters, the numbers of teeth were similar. With regards to the numberof patients who met our definition for periodontally diseased: 65.4 percent overall, 54.5 percentin the early and 73.3 percent in the established. Based on the NHANES III definition, theexpected value in the population would be about 35 percent.Conclusions  Periodontal disease is both prevalent and often severe in patients with RA  Characteristics of periodontal disease are similar in patients with early and established diseaseQuestion #2: Do patients with RA and periodontal disease have more severe RA?Hypotheses  Periodontal disease and oral symptoms will be common in RA patients  RA disease activity and severity will be associated with periodontal diseaseObjectives  To estimate the prevalence of periodontal disease in patients with RA using self-report oral health questionnaires  To explore the associations of RA disease characteristics with periodontal and oral health measures in RA patientsMethodsThe method used was to administer questionnaires asking about periodontal disease to patients toget data concerning the relationships with rheumatoid arthritis. The available population ofpatients with the ESCAPE cohort were administered questionnaires regarding periodontaldisease and oral health. (ESCAPE RA is the Evaluation of Subclinical Atherosclerosis andPredictors of Events in RA.) 183 patients from the ESCAPE cohort took the questionnaire. RAdisease characteristics and their oral health parameters were analyzed, and a validation study inthe 33 patients that evaluated with oral examinations and questionnaires was also performed.ESCAPE patients are aged 45 to 84 and are recruited from the Johns Hopkins Arthritis Centerand community rheumatologists. Patients with prior physician-diagnosed cardiovascular eventsor procedures are excluded.Methods: Analysis  Univariate associations for RA disease characteristics with periodontal disease characteristics  Poisson regression of periodontal symptoms with RA characteristics with robust variance estimation
  • 12. Prevalence ratios (PR) calculated in simple and multivariable models  adjustments for socio-demographic, lifestyle, oral hygiene, and co-morbid disease variables  Association of periodontal and oral/ocular dryness symptoms with log transformed DAS28 score modeled using multivariable linear regression in zero order and complex models  Simpler multivariable models constructed by comparing Akaike’s Information Criteria (AIC) values for complex vs. simpler nested models  Shapiro-Wilk test to examine normality across the extent of independent variables in multivariable modeling  Adjusted coefficient of determination (R2) calculated for each model to represent the proportion of the variability in outcome explained by aggregate of modeled covariatesCharacteristics Explored RA Severity and RA Disease Activity RA Treatment Other Extra-articular Factors • RA duration • DAS28 • DMARDs • Smoking • RF • Swollen joints • Prednisone • Alcohol • Anti-CCP • Tender joints • NSAIDs • Aspirin use • Shared epitope • MD global • Brushing • Sharp score • HAQ • Flossing • Nodules • CRP • Diabetes •Ro/La • Schirmer testRA Disease Characteristics (n=183) Age, years 61.5 +/- 8.3 Female (percentage) 60 RA Duration yrs, median (IQR) 10.8 (5.5-18.8) RF-positive (percentage) 64 CCP-positive (percentage) 69 HLA-DRB1 Shared epitope (percentage) 71 Nodules (percentage) 20 Swollen 28 Joint Count, median (IQR) 3 (1-5) Tender 28 Joint Count, median (IQR) 3 (1-6) CRP, mg/L, median (IQR) 2.75 (0.95-7.24) DAS28 CRP, median (IQR) 3.12 (2.43-3.79) HAQ, units, median (IQR) 0.75 (0.13-0.15)RA patients have longstanding disease, which is relatively well-controlled.Oral Health Characteristics Current smoking (percentage) 11.5
  • 13. Any periodontal symptoms (percentage) 78.7 Receding gums (percentage) 36.5 Bleeding gums with brushing (percentage) 67.2 Bleeding gums without brushing (percentage) 9.3 Swollen gums (percentage) 18.7 Bleeding OR swollen gums (percentage) 52.5 Bleeding AND swollen gums (percentage) 16.4 Oral dryness (percentage) 35.5 Ocular dryness (percentage) 29.0 Schirmer positive (percentage) 43.0 Ro or La positive (percentage) 7.3Self-reported oral symptoms are common in RA patients.Univariate Associations Between RA Characteristics and Gum Bleeding/SwellingUnivariate associations between the RA characteristics and whether or not patients had gumbleeding or gum swelling were examined.. Nodules, disease activity, swollen joint count, tenderjoint count, HAQ and physician global were all statistically significantly associated with patientsself-report of periodontal symptoms. In bivariate analysis the only RA characteristics thatremained significantly associated with self-reported periodontal symptoms, swollen gums, orbleeding gums were DAS28 and rheumatoid nodules.Associations of RA Disease Activity (DAS28) with Periodontal SymptomsThe only characteristics that remained associated after controls, however, were rheumatoidnodules in DAS28. Looking further into the relationship between DAS28, with any periodontalsymptoms, with swollen gums and with bleeding gums and breaking out DAS28 levels bypatients with high disease activity, moderate disease activity and low disease activity, there iswhat looks like a dose-response relationship between the presence of periodontal symptoms anddisease activity with RA. This data was adjusted for gender, race, age, education, smoking,alcohol, brushing, flossing and aspirin use. All of the other variables (diabetes, family history ofbleeding disorder, RA duration, HAQ, RF, anti-CCP, Ro, La, nodules, Sharp score, cumulativeprednisone dose, and HLA-DRB1 “Shared Epitope”) were also examined and none of them weresignificant.DAS28 Scores According to Combined Gum Bleeding, Swelling, and Oral Dryness
  • 14. It appears that patient report of oral dryness further increases the disease activity for rheumatoidarthritis. These are patients reporting whether or not they had these periodontal symptoms ofbleeding, swelling of their gums and now reporting whether or not they had dryness in theirmouth. There appeared to be an additive effect on top of the effect from the bleeding andswelling in terms of relationship with RA disease activity.Periodontal Symptoms Account for 22% of variability in DAS28  Despite adjusting for confounders, gum bleeding and swelling remained significantly associated with higher log DAS28 scores.  Proportion of the variability in log DAS28 score explained by the full model was 33% (i.e. adjusted R2=0.326), reduced to 25% when periodontal symptoms were excluded from the model.  Difference in explainable variability in log DAS28 attributed to the independent association of gum bleeding and swelling (22% of the total explainable variability) was highly significant (p<0.001).Validation of Periodontal Self-Report  26 RA subjects had comprehensive full mouth oral examinations with questionnaires administered  The odds of having PD* in those reporting periodontal symptoms was 2.3 times higher than those without symptoms  Performance of questionnaires  Specificity: 0.75  Sensitivity: 0.40  Positive predictive value: 0.595  High specificity of individual questions for bleeding/swelling with bleeding on probing and gingivitis  Questionnaires, while not detecting all cases, provide some indication of a true association* - PD defined as Pocket depth ≥3mm at ≥4 sites, and Attachment loss ≥3mm at ≥4 sites
  • 15. Self Reported Gum Bleeding with ExaminationThere is a statistically significant relationship between these.Correlation of Gingival Bleeding with PeriodontitisLooking at the correlation between gingival bleeding and periodontitis there relationship that ishighly statistically significantly different. Even though periodontal examinations were notperformed there is some evidence that the questionnaires that were administered do show arelationship with the ultimate question of periodontal disease being there or not.Limitations of Study  Questionnaires and self-report  Patients with longstanding RA  Relatively well-controlled RA diseaseConclusions of this Study  In this large cohort of established RA patients, a high prevalence of self-reported periodontal symptoms was observed, consistent with our earlier report based on oral examination  Questionnaires had a moderately high positive predictive value to detect periodontal disease  Periodontal symptoms were associated with increasing RA disease activity  Even after adjusting for multiple pertinent confounders  Oral dryness was an additive factor associated with RA disease activityAdditional Questions, and Citrullination  Do patients with RA have laboratory evidence of exposure to oral pathogens?  Do patients with periodontal disease have antibodies associated with RA?  Can careful attention to oral health improve outcomes for patients with RA?  Does periodontal disease contribute to increased cardiac risk in RA patients?P. gingivalis is a unique organism in the mouth in that it has an endogenous peptidylargininedeiminase (PAD) enzyme. Citrullination is one of the critical first steps in the development ofrheumatoid arthritis where markers are detected as antibodies against citrullinated peptides, adisease specific marker that also correlates with disease and with damage.Citrullination is the conversion of a charged arginine residue to an uncharged citrulline residue,which leads to secondary and tertiary conformational changes in proteins, potentially formingneoepitopes. In rheumatoid arthritis numerous putative targets of citrullination have beenidentified, including fibrinogen, vimentin, enolase and even PAD4. (There are five PAD4s inhumans.) PAD2, PAD4 and PAD6 are expressed in RA synovium and PAD4 polymorphismshave been described in some RA patients. Recently it has been learned that PAD2 is upregulatedwith cigarette smoking and also that P. gingivalis has an endogenous PAD.P. gingivalis has an endogenous PAD because one of the things that happens through thisconversion of arginine to citrulline is the release of ammonia. In the mouth, one of the NAimmune defenses is acidification of the environment of the mouth, which is typicallyinhospitable to bacteria. In the microenvironment at the base of the tooth it would be veryadvantageous for a bacteria to be able to neutralize acid. A bacteria that is able to citrullinateproteins that are readily available, therefore generating ammonia, can provide for itself a more
  • 16. hospitable environment for it to evade host defense. The process of citrullination, at leastthrough human PADs, can also inactivate certain chemokines that could lead to neutrophilrecruitment into the area. Thus, two potential mechanisms for host defense evasion from thebacteria through a PAD.Roles of Periodontitis and PADs in CitrullinationNow, throw periodontitis into the mix. For example, smoking is potentially a trigger for turningon human PAD, such as PAD2, activating and releasing IL1 and TNF, which may also act tostimulate human PADs and periodontitis acting through LPS and toll-like receptors, which couldpotentially have an additional impact on human PAD regulation. P. gingivalis specifically hasits own peptidylarginine deiminase and also has gingipains, which make many of these proteinspotentially more susceptible to cleavage by other proteases. There is a whole panel of differentputative citrullinated substrates that have been implicated for rheumatoid arthritis as well.It has also been shown that human enolase, alpha enolase, is a putative autoantigen for patientswith rheumatoid arthritis. The site that becomes citrullinated in this human enolase has sharedsequence hemology with bacterial enolase that is made by P. gingivalis. The response to andgeneration of autoantigens against these enolase products are very highly MHC restricted, notjust within the shared epitope but with specific ileals. This indicates that there may be a veryfine specificity between what becomes citrullinated and what becomes neoantigenic in specificindividuals. This is possibly where, based on this hypothesis, that periodontitis could come intoplay.
  • 17. Citrullinated Proteins are Detected in Saliva from Patients with Systemic AutoimmuneDiseasesThe question then becomes do we see any evidence for citrullination in patients with periodontaldisease and in patients with rheumatoid arthritis in the mouth?Logistic Regression for Periodontal Disease with RA Parameters (n=26) Definition Female Gender Current Smoking Anti-CCP DAS28Perio R03 Definition 1.10 2.39 11.25 1.83CDC 2007 Moderate 6.40 3.25 2.70 2.00CDC 2007 Severe 3.40 3.16 1.40 0.80  In logistic regression, cigarette smoking, anti-CCP status, RA disease activity (DAS28), and female gender were associated with increased odds for PD using various definitions  Anti-CCP with PD and smoking with severe and moderate PD reached or approached significanceThis is saliva just collected from patients with RA, psoriatic arthritis and Sjögrens syndrome runout on a blot and then probed with the AMC citrullination detection kit. It is a positive control ofneutrophils stimulated with calcium and ionomycin. Saliva is from patients with variousautoimmune conditions showing citrullinated substrates across the board. Citrullinated proteinsare detected in the saliva, in some more than others, the identity of which are not clear. Ifprotease inhibitors arent put in immediately after the saliva is obtained, much of it will be lost.The procedure for saliva collection has been modified to include a cocktail of protease inhibitorsto try to stabilize what being studied so it can be evaluated further in the future.IgG Anti-CCP Antibodies are Associated with Antibody responses to P. gingivalis  Random sample of RA database patient serum  76 patients equally distributed between CCP-positive and CCP-negative  Serum batch shipped, without reference to CCP state, for IgG antibody against cell surface antigens for P gingivalis using a previously established ELISA.  Results: Odds Ratio of 2.67 (CI 1.04-6.8) for anti-CCP with anti-P gingivalis antibodies.  These results are consistent with a recent report by Mikuls who showed arelationship with IgM anti-CCP and IgG2 anti-CCP (but not IgA or total IgG) with anti-P gingivalis.  Taken together these studies show consistent associations between periodontal bacteria and antibody responses against citrullinated peptides.PAD-4 is Detected in Periodontal Tissue  PAD4 expression was examined using immunohistochemistry of gingival tissue from patients with periodontal disease undergoing surgery.  Preliminary Results: Nuclear and cytoplasmic expression of PAD4 was seen in periodontitis predominantly in nonkeratinized junctional and pocket epithelium adjacent to the gingival sulcus; the level of expression was somewhat less than seen in other inflammatory tissues such as RA synovium.
  • 18. Summary  Moderate to severe periodontal disease is common in RA patients at disease initiation and with established disease  RA patients with more periodontal symptoms have higher levels of RA disease activity  In RA patients, PD is a strong predictor of anti-CCP antibodies  Antibodies indicating exposure to an oral bacteria with the ability to citrullinate, P. gingivalis, are associated with anti-CCP antibodies.Further Studies  Comprehensive oral assessments of RA patients and matched controls are underway to further confirm these self-report findings  Evaluations of the mechanistic underpinnings of disease interrelationships are also in progress  Whether aggressive management of periodontal disease in RA patients will result in improved RA clinical outcomes remains to be determinedClinical Studies  Continued longitudinal evaluation of early RA patients with sequential periodontal exams  Oral examinations of ESCAPE patients for validation of questionnaire data and additional associations  Effects of RA medications on PD parameters  Further evaluation of Sjogren’s and relationships with PD and RA  Analysis of additional questionnaire data from BRASS cohort  Gingival tissue collection of patients undergoing periodontal surgery  Interventional studies to determine the effect of aggressive treatment of periodontal disease on RA outcomesCurrent RA Patient Recruitment for Oral Health Assessment  Screened: 97  Enrolled: 56  First Exam completed: 38  Second Exam completed: 10Treatment of PD Improves RA Disease Activity (Ortiz P, et al)A very recent treatment study of PD in RA patients just came out which took 40 patients withRA, all of whom had generalized severe periodontitis. (It does speak again to the prevalence ofsevere periodontal disease in RA patients if they could identify 40 patients over a year and a halfto do the study.) Twenty of the patients were on DMARDS and twenty were on DMARDS plusTNF. In each group the patients were randomized either to get treatment for their periodontaldisease or to wait six weeks, and then the periodontal exam was performed again. There was areduction in SED rate in DAS28, in periodontal parameters with the periodontal treatment oversix weeks. Interestingly, there was a reduction from baseline TNF levels in 40% of the patientswho received the periodontal treatment versus 20% of those who had not been treated, even inthe patients receiving TNF antagonists. This is very intriguing data—its a small study, but it
  • 19. does hint at the possibility of doing an interventional study looking at RA with periodontaldisease.Triangulation of InflammationMechanistic Studies  Bacterial PAD  Cloning, sequencing, expression  Functional analysis  Calcium and pH dependence  Substrate specificity  Comparison with human PADs  Gingival tissue, gingival crevicular fluid, and salivary expression  Citrullinated proteins, PADs, relation to inflammation  Role of citrullination in periodontal disease and Sjogren’s  PD +/- RA, Healthy (no-PD, non-RA)  Antibodies to bacterial flora vs colonization patterns  Examination of RA, PsA, non-arthritis controls  Relation to anti-CCP and RA parameters  Relationship of bacterial colonization, PD, and SE status
  • 20. Conclusions  Periodontal disease is prevalent and often severe in patients with RA  Significant evidence suggests that citrullination may link periodontal disease with RA  Additional studies are needed to better understand these mechanismsClinical Implications  Oral health parameters should be more closely monitored in patients with RA and autoimmune diseases  Interventions to improve oral pathology may have direct and indirect systemic benefits

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