Johns Hopkins Arthritis Center Grand RoundsPeriodontal Disease and Rheumatoid Arthritis: From Epidemiologic Associations to Shared DiseaseMechanismsClifton O. Bingham III, MDJune 8, 2009Slide 1 (“Periodontal Disease and Rheumatoid Arthritis: From Epidemiologic Associations to Shared Disease Mechanisms”)Slide 2 (“Disclosures”)Slide 3 (“Absolutely Complete Disclosure”)…inhibitors of these proteins and cells. So thats my full disclosure, which Anthony encouraged all ofus to do. So hopefully that gives you some better insight in terms of my conflicts of interest. So…butif we do everything that Ed Miller wants us to do according to the rules of the law, thats what weneed to do now.Slide 4 (“Outline”)So the outline today. Im going to be giving you sort of a primer on oral health and periodontaldisease. We are going to talk about associations between periodontal disease and rheumatoid arthritis.And finally, we are going to discuss citrullination as a shared mechanism of disease.Slide 5 (“Background”)In terms of background, there have been multiple reports, and Ill go through some of these with you,that have described an association between periodontal disease and rheumatoid arthritis. And its beenrecognized that periodontal disease is associated with other systemic conditions, including diabetes,atherosclerotic cardiovascular disease, low birth weight infants and preterm labor. And in fact,intervention by treating periodontal disease can actually improve diabetes control, can improve theweight of infants and decrease preterm labor. And there is an interventional study that has beenongoing looking at the effect in atherosclerotic cardiovascular disease. So it implies that there may berelationships between oral health and systemic diseases.Slide 6 (“Periodontal Disease”)Now periodontal disease is something that we need to learn about to understand its relevance to thistalk – or to this topic. Periodontitis is an inflammatory disease affecting the gums and structures.While it initiates from an infectious trigger and involves gingival inflammation, which many of us –or most of us – have to some extent, the characterization of periodontal disease is when it goes beyondinflammation and becomes destructive, where there is separation of the tooth from its surroundingstructure, destruction of the collagen, destruction of the matrix that begins to make the tooth loose.The stages begin with gingivitis, or chronic inflammation, and again this if you look in your mouth,most of us will have some evidence of gingival inflammation. But its beyond this when there iscolonization of what are called the red complex organisms that we really look at the process ofperiodontitis beginning. And then again, this sequence that occurs with loss of connective tissueattachments, bone resorption and ultimately tooth loss.
Slide 7 (“Biofilm Development in PD”)This is what a biofilm looks like on the tooth of a patient with periodontitis, and you see that there aremultiple bacteria that are sort of piled on top of each other. And this little cartoon sort of outlineswhat happens in the stages of biofilm development in the mouth. There are certain bacteria that comealong and colonize, but then there are bacteria that set up shop on top of this, the specific adhesionmolecules between the two. And then there are further bacteria that come along with additionalproperties that build up on top of that.Slide 8 (“Colonization in Periodontal Disease”)And these are the late colonizers that really describe patients with periodontal disease. So we all havemultiple organisms in our mouths. And we know that the mouth is very dirty and human bites are badthings that we have to treat with antibiotics because of all of the anaerobes.Slide 9 (“Porphyromonas gingivalis”)But patients with periodontitis have specific flora in their mouths, one of the most important of whichis this bug called Porphyromonas gingivalis. Others that are also of importance, and they track alongwith it, are treponema denticola, certain species of Eubacterium, Providencia intermedia andactinomycetemcomitans organism here as well. But its when these come along that the process goesfrom inflammation to one that has the potential for destruction.Porphyromonas gingivalis is a gram negative anaerobic bacteria, and again its one of these terminalcomplex organisms – these late colonizers. Several properties that are unique about P. gingivalisinclude its expression of LPS. Most of the other bugs that are down underneath there dont have LPs.Therefore, it can activate toll-like receptors. Also, Porphyromonas gingivalis has an awful lot ofproteolytic enzymes. Many are involved in arginine, lysine and cysteine metabolism, and one of thosewhich well talk about more is a peptidyl arginine deiminase. There are also many enzymes that aredirectly secreted by this bacteria that cause collagen degradation and other exotoxins and gingipainswhich make many proteins susceptible to further cleavage.Recently it has been described that P. gingivalis has DNA for enolase, which has overlappingsequence with human alpha enolase, which is susceptible to citrullination. And finally, antibodies toP. gingivalis are a marker for periodontal disease. And one of the ways that people can be identifiedfor having exposure to this organism by looking at serum antibody responses against it.Slide 10 (“Periodontal Disease Epidemiology”)The epidemiology of periodontal disease is something as well we need to learn about. Periodontaldisease is the leading cause of tooth loss in the United States. Now the estimates of the prevalence ofperiodontal disease depend on the definition that you use, but using the NHANES III survey, about35% of adults over age 35 have some degree of periodontal disease; 30% of these were classified asbeing moderate to severe; and a substantial proportion of these severe patients go on to progressivedisease. So if we look at the expected prevalence using NHANES definition, the expected prevalenceof moderate to severe disease would be about 13% of the general population. Now the risk factors forthe development of periodontal disease are cigarette smoking, certain medications, systemic diseasesand what we have learned is that twin studies have shown us that about 50% of periodontal disease is
explained by some heritability, and many genetic associations have been reported, none of them wellvalidated, but some of the ones that I will put up here that have at least one report include HLA-DR4polymorphisms and TNF, and the TNF promoter that has been described for rheumatoid arthritis aswell as in the IL1 promoter.QUESTION: What is the magnitude of the effect of cigarette smoking?Cigarette smoking is the major risk factor, so – for periodontal disease. It is the one that is absolutelyvery strongly associated. Its unusual, and Melanie, you can weigh in for me as well, that people whoare not smokers will develop it although people do, as you will see from some of our data.QUESTION: [Inaudible]I dont know in terms of relative risk for cigarette smoking, but it is the major, major risk.Other polymorphisms that have been described are in the toll-like receptor and CD14.Slide 11 (“Photos: Healthy/Gingivitis/ Moderate Periodontitis/Severe Periodontitis”)So the progression of gum disease is as follows. These are nice, healthy gums. These are the gumsthat most of us or many of us would have that have gingivitis. And what you can see is somehyperemia of the gingiva. You might also be able to appreciate a little bit of swelling here that is notpresent here. And this is a mild condition where you have colonization. You may have excessiveplaque accumulation, and this is the type of thing that toothbrushing will help calm down, and goodoral hygiene. But this is the progression to periodontitis. I hope you can appreciate some of thedifferences. Now you not only have the gum inflammation, but what you begin to see is recession ofthe gum line, and you begin to see parts of the tooth that werent visible before. So you have hadregression of the gingiva. You begin to expose parts of the tooth that werent there. You begin to getthese triangles and black spaces between teeth. And then this it the later stages of the disease whereyou see significant loss of the gingiva. You begin to see loss of the bone, and Ill show you what thislooks like radiographically in just a second. And here obviously a lot of plaque accumulation as well.So this is a progressive disease and a progressive cycle. And what were talking about is this type oflesion when we speak of periodontal disease.Slide 12 (“Radiographic Changes in Periodontal Disease”)Now there are many things that are similar between periodontal disease and rheumatoid arthritis, andone of those is the effect on bone. This is a normal bitewing x-ray that you might get when you go tothe dentist with nice, healthy gums and heres a bone level demonstrated here around the bottom of thetooth. Here is that normal bone level again in a patient with periodontal disease. You see that thebone level has regressed significantly. And you begin to see even these spaces underneath the roots ofthe teeth, and Ill describe to you furcation later, which is putting a probe into the mouth and seeing ifit will slip underneath this place in the tooth. In fact, thats something that I will show you some dataon that we see very frequently in the RA patient population.Slide 13 (“Pathogenesis of Periodontal Disease”)
So what do we know about the pathogenesis of this? Well, bacteria are necessary, but they are notsufficient. Eradication of the bacteria does not necessarily lead to resolution of the condition. Andsubmicrobicidal concentrations of tetracyclines improve periodontal disease, explained for in part bymatrix metalloproteinase activity. Antibody responses to certain bacteria predict progressive diseaseand bone loss, one of which is anti-P. gingivalis. There is a T-cell component of the disease that isseen, again, some suggestions of HLA associations, oligoclonal expansions of T-cells seen in thegingiva. There is cytokine-mediated effector damage driven by osteoclasts, RANK ligand and otherpro-inflammatory cytokines, including TNF and IL1. And again, these genetic associations.Slide 14 (“Cartoon: Tooth”)So if we look at a diagram – I think a diagram sort of will tell us a lot about what happens in disease –weve gotten used to seeing what this looks like in rheumatoid arthritis. I tried to set up a similar typeof diagram for periodontal disease. Now Ive shown you the accumulation of bacteria and plaque andbiofilm formation that develops in this pocket. These bacteria begin to release substances such asLPS, activating neutrophils, macrophages, recruiting them into the site. But there are also T-cells thatproliferate. There are plasma cells in the gingiva as well. These proliferate, and rheumatoid factorshave also been described in patients with periodontal disease. There are also the antibodies thatdevelop again to the bacteria. The release of multiple cytokines that are detectable both expressed inthe gingival tissue as well as released into whats called the gingival crevicular fluid, which sits righthere. Activation of fibroblasts that ends up leading to further connective tissue matrix degradationand osteoclast activation that leads to the degradation of bone. So many of the players that we areaccustomed to in rheumatoid arthritis we see recapitulated in the periodontal microenvironment.Slide 15 (“Previous Studies of RA and PD”)So what about studies that have been done looking at RA and periodontal disease? One of the largeststudies that was recently reported looked again back to the NHANES III study. It looked at subjectswho were greater than 60 years of age, who had musculoskeletal disease – they looked at patientswho were 60 years of age. Musculoskeletal examinations were performed, and there was a singlequadrant dental examination performed on these patients as well. That means one-fourth of the teethwere looked at in patients to define oral health. From this study it was found that RA patients weremore likely to be edentulous – an odds ratio of 2.27 – and have periodontal disease, with an odds ratioof 1.82 compared with non-RA subjects. This level of risk increase is similar to that seen for otherconditions such as diabetes, preterm labor and low birth weight. So again, similar odds ratios to thoseconditions. And these data were adjusted for age, sex, race, ethnicity and smoking. But there reallywere no details whatsoever regarding the rheumatoid arthritis in this study.Several other small case-control studies have reported similar results, and the odds ratios have rangedfrom 2.3 up to 8. And another study that I think is of interest is a study by a French group that lookedat the association between bone loss on periodontal x-rays compared to bone loss on wrist radiographsof patients with rheumatoid arthritis and found that the RA-shared epitope is associated both withperiodontal bone loss as well as with wrist damage. So sort of guilt by association from severaldifferent studies.Slide 16 (“Recent RA-PD Case Control Study”)
Here is one of the recent case-control studies that has a little bit more information and betterexaminations than many of the others. It is in 57 RA subjects and 52 healthy controls. They didmeasurements at four sites in each tooth looking at plaque index, gingival index, pocket depth, clinicalattachment loss, and they defined patients who are periodontally diseased as having attachment lossgreater than 4 mm. Now in this study that did step-wise logistic regression, and they found out thatthe odds were 8.05 for patients with RA for periodontitis compared with controls. When this wasadjusted for the plaque and gingival index the odds ratio dropped, but it was still markedly significant.And it was also found that RA patients had higher indices of all of the different markers ofperiodontitis, including bleeding on probing, attachment loss, gingivitis and other measures ofperiodontal disease.QUESTION: The definition of periodontitis there was….?No, this was a standard – a single definition that was a mean attachment loss of 4 mm. This is not astandard definition, and thats one of the limitations of this study that is significant. There was noinformation on RA disease characteristics, and it was a non-standard definition of periodontal disease.So there are several definitions that Ill go through with you that have been established, but no studieshave really fulfilled all of the obligations of having good information on RA disease activity onperiodontal disease and using standard definitions of activity for both.QUESTIONS: In the NHANES data on the prior slide, how is RA defined?Its self-report by the participant.Slide 17 (“RA, PD, and CVD”)And heres another study that I just put up here because its a little bit larger. But it begins to get atthis issue of relationships between rheumatoid arthritis, periodontal disease and cardiovasculardisease. As I said, periodontal disease has been described as a risk factor for atherosclerosis andintervention studies have been ongoing trying to look at this relationship a little bit better.This is an Egyptian group who has been working on oral health for a while. They took 100 RApatients who had cardiovascular disease – Im sorry – 100 RA patients, 50 of whom hadcardiovascular disease, 50 of whom did not have cardiovascular disease and 50 controls without RA.Eighty-four percent of the RA patients with cardiovascular disease had periodontal disease comparedto 60% of the patients without cardiovascular disease and 10% of the control subjects. So theconclusion of the group was that there was an additive effect of RA plus cardiovascular disease andthe association with periodontal disease, periodontal disease further increasing the cardiovascular risk.Now, it was not a terribly well-done study, but I think there was some interesting information thatcame out of it. They did have some information on RA disease activity in the study, and they foundthat the severity of periodontal disease was significantly correlated to RA disease duration, to theDAS28, to TNF levels and inflammatory markers. And they also found that again these periodontalindices were higher in RA patients compared to controls, and they were higher in RA patients withcardiovascular disease than those without cardiovascular disease.Slide 18 (“TNF inhibition Improves Experimental Periodontitis”)
There have been several studies that have told us 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 with TNFinhibition, and there was a very nice study that looked at P55 receptor knockout mice who had – thathad less bone loss and less severe periodontitis with an induced model of bacteria.Slide 19 (“TNF in Patients with Periodontal Disease”)And TNF has been described increased in patients with periodontal disease, higher in patients who aresmokers than are non-smokers. A TNF polymorphism has been reported and confirmed recently inJapanese patients with severe periodontal disease. And in a very recent study that came out at the endof last year, it looked at 19 patients with rheumatoid arthritis versus 30 healthy controls and found thatthe serum TNF level was the only independent variable to predict probing death, attachment loss ofbleeding on probing. Again, a small study, not many controls, but again, since the suggestion of anassociation with TNF with the extent of periodontal disease.Slide 20 (“Infliximab Treatment Does Not Improve Gingivitis”)That was followed, however, with a study at the end of last year that showed that infliximab treatmentdid not improve gingivitis and this was a study from the Journal of Periodontology. It has a lot ofweaknesses but some interesting data that came out of it. They took 40 RA subjects, 20 of whom hadbeen treated with infliximab, 20 who had not been treated with infliximab. They looked at theperiodontal status of these infliximab-treated patients every six weeks. Nine patients who had notbeen treated with infliximab began being treated with infliximab, and they looked at those patientsover the next nine months. Now, there are many weaknesses in the study. In the whole mouth theyonly evaluated six index teeth. They recorded the maximum values for many parameters andcalculated a mean to give a periodontal index. What they found, though, is that periodontal diseasedid not appear to be affected by infliximab in this group versus this group. And in these nine patientsthey had pre and post data with infliximab. There was a decrease in attachment loss, but theperiodontal disease remained constant. In the gingivitis and bleeding index in contrast to what youmight expect with TNF inhibition, actually were increased in these nine patients. So this is, youknow, a study that would argue against there being a role of TNF significantly with periodontaldisease, but I think there are many limitations to this study that make it far from conclusive in terms ofthis activity.Slide 21 (“Shared Mechanisms of Disease in PD and RA”)So turning back to the general paradigm of RA versus periodontal disease, we can really line up manyof the mediators that we have seen in many of the cell types. And if you look histopathologically, thisis a patient actually with periodontal disease here with an intense neutrophilic and mixed cellularinfiltrate and even some small lymphoid follicles that appear to be developing in the gingiva, verysimilar to what we might see in a rheumatoid joint.Slide 22 (“Rheumatoid Arthritis ? Periodontal Disease”)Now, these relationships that have been described epidemiologically raise the question of whichcomes first, the RA or the periodontal disease. And the traditional paradigm was that RA preceded
the development of periodontal disease. But I think that its far from conclusive, and I think its timefor us to potentially begin thinking in the other direction.Slide 23 (“Prior Studies of RA and PD”)So problems with many of these other studies that have been case-control and otherwise – they haveonly looked at patients with established longstanding rheumatoid arthritis. There has been very poorcharacterization of RA disease activity. The influence of medications and other comorbid medicalconditions has been unclear and uncontrolled. There has been poor characterization of any type offunctional ability for the RA patients, especially hand function. There has been no control forSjögrens, and they have been poorly controlled for smoking.Slide 24 (“Old Hypothesis: RA is Risk Factor for Periodontal Disease”)Again, this was the old hypothesis that RA came first. Patients with RA had poor oral hygienebecause they couldnt grip their toothbrush and brush well or patients with RA had TMJ joints thatwere affected and couldn’t open their mouth enough to be able to brush their teeth. Or patients wereimmunosuppressed or received NSAIDs that could be risk factors for the development of periodontaldisease or they had secondary Sjögrens. But none of these were proven in any way.Slide 25 (“New Hypothesis: Periodontal Disease is a Risk Factor for Rheumatoid Arthritis”)But I would like to take us back to what weve learned from the pathophysiology of the disease andsay that perhaps periodontal disease is what comes first, before the rheumatoid arthritis. And we haveseen that there are similar genetic susceptibilities, at least in terms of HLA-DR4 by two studies andsome of these other polymorphisms that have been described. We see similar lesions in theinflammatory focus. And we also know that other bacterial infections can directly causeinflammatory arthritis, either by molecular mimicry or by other mechanisms, including rheumaticfever, lyme disease and reactive arthritis.Slide 26 (“Questions”)So as I started looking at this, and my entry into this area actually came from clinical concerns andclinical observations while I was in New York at Bellevue where we saw a lot of patients who hadvery low socioeconomic status, many patients very, very poor oral health, and it was at the time whenTNF antagonists were just coming to the market. I had extreme concerns about whether or not thesedrugs would be safe in patients who had significant periodontal disease. I was worried about the riskpotentially of these patients developing endocarditis or other abnormalities. But because of the extentof the arthritis in the patients, we had to really bite the bullet and treat their arthritis. And in fact wedidnt see patients that were coming down with endocarditis or getting these overwhelming dentalabscesses. And in fact, if anything, it appeared that some of the gingival inflammation wasimproving. So I started working with dental colleagues at NYU School of Dentistry, then I moveddown here to Johns Hopkins and began collaborations with the group over at University of Maryland.And as I thought through this issue of periodontal disease and RA, these are the types of questions thatstarted to come up. I told you that most of the studies that have been done were looking at patientswith established rheumatoid arthritis. They were cross-sectional evaluations of patients which reallytell you nothing about cause and effect. So it seemed to me that one of the most important things tostudy was a group of patients with incident rheumatoid arthritis to find out if periodontal disease was
present at the beginning of the disease and then what happened to patients over time as theirrheumatoid arthritis progressed. I also wondered if patients with rheumatoid arthritis and periodontaldisease would have more severe rheumatoid arthritis and if patients with RA have laboratory evidenceof exposure to oral pathogens or did patients with periodontal disease have antibodies that areassociated with RA. And sort of the ultimate clinical question is can careful attention to oral healthimprove the outcomes for patients with RA and potentially does periodontal disease contributeincreased cardiovascular risk to patients with rheumatoid arthritis.Slide 27 (“Do Patients with Early RA have Increased Periodontal Disease?”)So if we go to the first question – this was do patients with early have increased periodontal disease.Slide 28 (“Periodontal Assessment in RA Study (PARAS, R03 DE018094)”)This takes me to an R03 that was funded that we called the periodontal assessment in RA study thatcontinues to be ongoing. We looked at patients who are ages 18 to 75 who had definite or probableRA. RA was diagnosed within 12 months. One of our inclusion criteria was that patients have at least20 teeth in their mouths that we could score, and this became a problem for us in terms of patientrecruitment. Exclusions were other forms of inflammatory arthritis, other topical or systemicantibiotics, medications that may be associated with gingival hyperplasia, contraindications to probingsuch as coagulopathy, valvular heart disease, prosthesis and other conditions requiring antibioticprophylaxis for dental procedures. We didnt exclude these patients unless they were unwilling toreceive standard prophylaxis. So the patients could come into the study. We gave them antibioticsbefore we did any of the probing; even though the probing that were doing should probably not causebacteremia, we always wanted to be on the safe side.Slide 29 (“RA Characteristics Explored”)The RA characteristics that we explored included RA severity and extraarticular factors that havebeen looked at with the ESCAPE cohort, RA duration, rheumatoid factor, CCP status, nodules,damage. We also looked at Ro/La in this population with Antony and Livia and the group upstairs.We looked at Schirmers test in patients. We measured measure of RA disease activity. We looked attreatment, and we also evaluated comorbidities, alcohol, smoking, aspirin use and other habits.Slide 30 (“Dental Characteristics Explored”)We also explored multiple measures related to dental health, including a dental history of tooth loss,oral hygiene, brushing, flossing habits, cigarette smoking and alcohol use and we designed severalperiodontal questions that we gave to patients looking at bleeding gums, swollen gums, halitosis,pockets and gaps between teeth.We asked standard six-question Sjogrens questionnaires for oral dryness and ocular dryness and weconducted a dental examination looking at all of the teeth, six sites on every tooth with multiplemeasures, and we have begun to evaluate unstimulated salivary flow and collected saliva frompatients as well.Slide 31 (“Periodontal Examination”)
The periodontal examination that were performing is an overall oral assessment. We are examiningall teeth except the wisdom teeth. Were measuring at six sites on every tooth 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 and thisissue of furcation where you stick the probe in and see if it can get underneath a root.Slide 32 (“Periodontal Examination”)This is what the probe looks like in the teeth. Its non-painful, and people really dont complain toomuch. But you go around six sites on every tooth. One, two, three on the front and one, two, three onthe back. And then the probe had gradations in millimeters, and you can measure how deep the probegoes down into the pocket and get these different measurements. The exam itself takes about an hourto an hour and a half for patients to conduct. So it is an intensive experience for them. We have beendoing this on a standard examination table, which in patients with RA can be quite uncomfortable, butnonetheless we have managed to do it. But it has presented an additional challenge to the study. Soagain you might have had this done by your own dentist, going around doing a periodontalexamination on you. But this is the probe going into the pocket and showing you how with moderateperiodontitis there is this attachment loss that begins where this probe goes deeper into a pocket andyou begin to get exposed roots and exposed parts of the teeth that form.Slide 33 (“Cartoons: Healthy/Gingivitis/ Moderate Periodontitis/Severe Periodontitis”)Slide 34 (“Definitions”)The definition that we used for the initial study was one that had been used for other studies ofperiodontal disease. There was no real well-established definition at the time we started with this forperiodontal disease, but we looked at patients and called them diseased if they had at least 20 teethand had at least 4 sites with pocket depths greater than 3 mm and 4 sites with attachment loss greaterthan 3 mm. And this definition approaches that of the NHANES III definition that was used inlooking at periodontal disease. And we also looked at patients sequentially every six months forperiodontal progression, and we set up a definition for this as well.Slide 35 (“Oral Health Assessment in RA Patients”)This is information on the first 26 patients that we have evaluated breaking down patients betweenthose with early rheumatoid arthritis, and we also looked at a group of patients with establishedrheumatoid arthritis. And you can see that the difference in disease duration is shorter disease in thepatients with early RA. The CRPs were a little bit lower in the group of patients with early RA,probably because there is a little bit more steroid use in these patients that we were just seeing. Thetender joint counts were higher in the early rheumatoid arthritis patients. The Das values were notstatistically significantly different, but the numbers of patients remained somewhat slow – low – withthis preliminary data.Slide 36 (“PD is Equally Present And Severe Early And Late In The Course of RA”)We looked at the periodontal parameters. The numbers of teeth were similar. The number of patientswho met our definition for periodontally diseased, 65.4% overall, 54.5% in the early and 73.3% in the
established. And I remind you that based on the NHANES III definition, the expected value in thepopulation would be about 35%.Slide 37 (“Conclusions”)Looking at a more stringent definition defined by the CDC in 2007 for moderate disease and severedisease, our numbers have increased to 38% with moderate disease and 26.9% with severe disease.And again, the expected levels for these would be somewhere around 10% and less than 5% in thegeneral population. What you notice from here is that the parameters related to periodontal diseaseare all very similar between the two groups with no statistically significant difference except forbleeding on probing and number of sites. And I bring you back to this issue of furcation. Over 80%of patients we are able to probe down and get to the level of a root of a tooth, indicating prettysignificant periodontal disease in most patients that participated in the study. And there was really noscreening for patients. We asked patients as they came through the clinic, would you be willing toparticipate. Probably there is a bias because I actually look at peoples teeth and think about this. Butits – nonetheless, I think that these data strongly suggest that this phenomenon takes place in earlydisease as well as with established disease. So I think the conclusions from the first part of the studyare that periodontal disease is both prevalent and often severe in patients with RA. And thecharacteristics of the periodontal disease are also similar between patients in early and establisheddisease.Slide 38 (“Do Patients with RA and Periodontal Disease have More Severe RA?”)So that brings us to question number two: Do patients with rheumatoid arthritis and periodontaldisease have more severe RA? That is, does periodontal disease influence the level of rheumatoidarthritis?Slide 39 (“Hypotheses/Objectives”)Our hypothesis was that periodontal disease and oral symptoms would be common in RA patients,and we already – we had shown this in terms of the previous work. But what we wanted to do was toanswer this question as quickly and easily as we could with a larger group of patients.Slide 40 (“Methods”)So we elected to move forward administering questionnaires, asking about periodontal disease topatients to try to get some idea concerning the relationships with rheumatoid arthritis. We had anavailable population of patients with the ESCAPE cohort, and we administered questionnairesregarding periodontal disease and oral health to these patients at their second visit.Slide 41 (“Escape RA”)So we took patients from the ESCAPE cohort, 183 patients took our questionnaires. We got all of theinformation that I showed you before that we were looking at in the patients who we were also doingexaminations on. We analyzed RA disease characteristics and their oral health parameters, and we dida validation study in the 33 patients that we had evaluated with oral examinations and also given thesame questionnaires to.
So ESCAPE weve heard about from Joan and John multiple times in the past, but just to remind you,these are patients aged 45 to 84. It was a cohort to evaluate the prevalence and progression ofsubclinical cardiovascular disease in men and women with RA.Slide 42 (“Methods: Analysis”)We looked at univariate associations for RA disease characteristics with periodontal characteristics.We calculated prevalence ratios and generated simple and multivariable models that were adjusted formultiple confounders between periodontal symptoms and oral symptoms.Slide 43 (“Characteristics Explored”)The characteristics explored were, again, were the same as what we had done with our early RAcohort.Slide 44 (“RA Disease Characteristics (n=183)”)And here were the characteristics of the ESCAPE group. Disease duration of 10.8 years, relativelylow levels of disease activity with low swollen and low tender joint counts. HAQ scores also in thelow range.Slide 45 (“Oral Health Characteristics”)But the oral health characterization from patients with self-reported questionnaires – periodontalsymptoms – 78.7% of patients. Specifically, bleeding of gums with brushing – 67% of patients. Self-reported swelling of the gums in 18.7%. Bleeding or swollen gums in 52.5%. Oral dryness beingreported by about 35.5%. Schirmer test positive in about 43% of these patients. Ro/La positive onlyin 7%.Slide 46 (“Univariate Associations Between RA Characteristics and Gum Bleeding/Swelling”)And we looked at univariate associations between the RA characteristics and whether or not patientshad gum bleeding or gum swelling. Here are the patients with no gum symptoms. Here are thosewith – who reported frequent gum bleeding or swelling. And here are those that reported bothfrequent gum bleeding and gum swelling. And we looked and found that nodules, disease activity,swollen joint count, tender joint count, HAQ and physician global were all statistically significantlyassociated with patients self-report of periodontal symptoms.Slide 47 (“Associations of RA Disease Activity (DAS28) with Periodontal Symptoms”)The only characteristics that remained associated after controls, though, were rheumatoid nodules inDAS28. When we looked further into the relationship between dAS28, here again with anyperiodontal symptoms, with swollen gums and with bleeding gums and we broke out DAS28 levels bypatients with high disease activity, moderate disease activity and low disease activity, we found whatlooked like a dose-response relationship between the presence of periodontal symptoms and diseaseactivity with RA. We adjusted for gender, race, age, education, smoking, alcohol, brushing, flossingand aspirin use. We also looked at all of the other variables that are listed here, and none of themwere significant.
Slide 48 (“DAS28 Scores According to Combined Gum Bleeding, Swelling, and Oral Dryness”)When we looked further into this relationship and threw oral dryness into the equation, it appearedthat patient report of oral dryness further increased the disease activity for rheumatoid arthritis. Sothese are patients now reporting whether or not they had these periodontal symptoms of bleeding,swelling of their gums and now reporting whether or not they had dryness in their mouth, and thereappeared to be an additive effect on top of the effect from the bleeding and swelling in terms ofrelationship with RA disease activity.Slide 49 (“Periodontal Symptoms Account for 22% of variability in DAS28”)In further modeling, John has shown that the periodontal symptoms account for about 22% of thevariability thats associated with the DAS28, which is a pretty high level of association. And this waswith multiple controls.Im not going to go into the data for that. Im going to move on to some additional data that we havefrom the study. The data that I presented before that last one I presented is an oral presentation atACR.Slide 50 (“Validation of Periodontal Self-Report”)Now these were self-reported data, and you would raise questions about how good is self-report inrelating to the actual examination of patients. We didnt do the exams on the patients from theESCAPE cohort, but we had been doing examinations on other patients with rheumatoid arthritis whocompleted the same questionnaires. We did a validation study initially based on 26 patients and thenincreased up to 33 patients. And we found that the odds of patients having periodontal disease inthose reporting periodontal symptoms was 2.3 times higher than those without symptoms. Our simplequestionnaire gave a specificity of 0.75 and a positive predictive value of about 0.6. So thequestionnaires, although they did not detect all of the cases, they provided some indication of a trueassociation with periodontal disease.Slide 51 (“Self Reported Gum Bleeding with Examination”)If we look at self-reported gum bleeding with the examination for bleeding on probing, we found thatthere was a significantly – statistically significant relationship between these. So patients can report –if the patient says their gums bleed, we stick a probe into their mouth, they bleed – they do a prettygood job.Slide 52 (“Correlation of Gingival Bleeding with Periodontitis”)And then we look at the correlation between gingival bleeding and periodontitis. We see a very nicerelationship that is highly statistically significantly different. So even though in this group of 183patients we havent done periodontal examinations, we do have some evidence that the questionnairesthat we have administered do have a relationship with the ultimate question of periodontal diseasebeing there or not.Slide 53 (“Limitations of Study”)
So the limitations of this part of the study, obviously, are that these are questionnaires and self-reports.The patients that I showed you had longstanding rheumatoid arthritis, and their disease activity wasrelatively well-controlled.Slide 54 (“Conclusions of this Study”)So the conclusions from this study – in a large cohort of patients with established RA there was a highprevalence of self-reported periodontal symptoms consistent with our earlier report based on oralexamination. The questionnaires had a moderately high positive predictive value to detect periodontaldisease. And periodontal symptoms were associated with increasing RA disease activity, even afteradjustment for multiple confounders. And oral dryness appeared to be an additive factor associatedwith RA disease activity.Slide 55 (“Untitled: Questions”)So now we have early disease looking at actual examinations compared to later disease. We have alarge cohort where weve tried to evaluate these questions related to periodontal disease and RAdisease activity. And then the next questions that I told you about were trying to understand themechanism between these two disease processes. The question is, do patients with RA havelaboratory evidence of exposure to oral pathogens and do patients with periodontal disease haveantibodies associated with RA?Slide 56 (“Citrullination”)And this comes back to the issue of citrullination. I told you that P. gingivalis was a unique organismin the mouth and that it has an endogenous peptidylarginine deiminase enzyme. And as we know,citrullination is one of the critical first steps in the development of rheumatoid arthritis where we havemarkers detected as antibodies against citrullinated peptides, a disease specific marker that alsocorrelates with disease – with damage.Slide 57 (“Citrullination”)Citrullination is the conversion of a charged arginine residue to an uncharged citrulline residue, whichis thought to lead to secondary and tertiary conformational changes in proteins, potentially formingneoepitopes. In rheumatoid arthritis numerous putative targets of citrullination have been identified,including fibrinogen, vimentin, enolase and even PAD4. And we know that there are five PAD4s inthe human. We know that PAD2, PAD4 and PAD6 are expressed in RA synovium. We know thatPAD4 polymorphisms have been described in some RA patients. We have also recently learned thatPAD2 is upregulated with cigarette smoking. And Ive told you that P. gingivalis also has anendogenous PAD. So why in the world would a bacteria had a peptidylarginine deiminase as one ofthe enzymes that it has? Well, its felt that the reason that P. gingivalis has this is that one of thethings that happens through this conversion of arginine to citrulline is the release of ammonia. And Iremind you that in the mouth one of the NA immune defenses that we have is acidification of theenvironment of the mouth, which is typically inhospitable to bacteria. Now around the base of a toothyou think about the microenvironment there, it would be very advantageous for a bacteria to be able toneutralize acid. So if you can imagine a bacteria that is able to citrullinate proteins that are readilyavailable, therefore generating ammonia, it may provide a more hospitable environment for it to evade
host defense. So very interestingly, we have also learned that the process of citrullination at leastthrough human PADs can also inactivate certain chemokines that could lead to neutrophil recruitmentinto the area. So again, two potential mechanisms for host defense evasion from the bacteria througha PAD.QUESTION: [Inaudible]In the space around the tooth, the environment is normally acidic.COMMENT: Okay, so my instinct is if saliva is alkaline and loss of saliva gives you rampant dentalcares, that alkaline is good. Ive always had that sense that alkaline is good, so if alkaline is good,then maybe be the ammonia from the [inaudible] isnt necessarily due to pH change but due tosomething else. I think the mechanism I would leave somewhat open as to [inaudible].Yes. Again, this is one of the hypothesized mechanisms. I think that there are many other things thatdont – that are a little bit atypical as well between Sjögrens and periodontal disease. Most of thestudies that have actually been done looking at Sjögrens and periodontal disease at least with primarySjögrens, not secondary Sjögrens, have shown that there is not really a relationship between the two,although there have been some suggestions in the RA population with secondary Sjögrens that theremay be an effect. So – and Im not quite sure how it all goes together. But at least from the dentalliterature, this has been the hypothesized mechanism for this issue of changing pH and making a morehospitable microenvironment.Slide 58 (“Citrullinated Peptides in the Pathogenesis of RA”)So when we put this all together in terms of the pathogenesis of rheumatoid arthritis, we have thesePADs that are here. We have these human PAD mutations. We have innate immune responses andother things that can activate human PADs. The process of citrullination releases autoantigens thatturn on T cells, autoantibody formation and other cells in the microenvironment.Slide 59 (“Roles of Periodontitis and PADs in Citrullination”)And then we throw in periodontitis into the mix. We think about smoking, which could be a triggerfor periodontitis, potentially a trigger for turning on human PAD, such as PAD2. Periodontitisactivating and releasing IL1 and TNF, which may also act to stimulate human PADs and periodontitisacting through LPS and toll-like receptors, which could potentially have an additional impact onhuman PAD regulation. P. gingivalis specifically having its own peptidylarginine deiminase. AndP. gingivalis also having gingipains, which make many of these proteins potentially more susceptibleto cleavage by other proteases. And then we have a whole panel of different putative citrullinatedsubstrates that have been implicated for rheumatoid arthritis.I point out here this bacterial enolase again. The group from London has shown that human enolase,alpha enolase, is a putative autoantigen for patients with rheumatoid arthritis. They found that the sitethat becomes citrullinated in this human enolase has shared sequence hemology with bacterial enolasethat is made by P. gingivalis. They have also found that the response to and generation ofautoantigens against these enolase products are very highly MHC restricted, not just within the sharedepitope but with specific ileals. So it indicates that there may be a very fine specificity between whatbecomes citrullinated and what becomes neoantigenic in specific individuals. I think this is where I
would say based on this entire hypothesis, that periodontitis could play in. We really dont know if itsthis mechanism, if its this mechanism or if its some other mechanism between the two.Slide 60 (“Citrullinated Proteins are Detected in Saliva from Patients with Systemic Autoimmune Diseases”)The question becomes do we see any evidence for citrullination in patients with periodontal diseaseand in patients with rheumatoid arthritis in the mouth.Slide 61 (“Logistic Regression for Periodontal Disease with RA Parameters (n=26)”)This is a blot from Felipe. I appreciate his letting me use it for the grant in terms of preliminary data.This is saliva just collected from patients with RA, psoriatic arthritis and Sjögrens syndrome run outon a blot and then probed with the AMC citrullination detection kit. And what you see is here is apositive control of neutrophils stimulated with calcium and ionomycin. And here is saliva frompatients with various autoimmune conditions showing citrullinated substrates across the board. Socertainly in saliva we detect citrullinated proteins, in some more than others, the identity of which arenot clear. Ill also mention that we have recently done some additional experiments withGinny VanIkes group that have shown that this method of just taking saliva and running it out on agel, if you dont put in protease inhibitors immediately after the saliva is obtained, you will lose a lotof what used to be there. So we now have modified our procedure for saliva collection to include acocktail of protease inhibitors to try to stabilize what were looking at so we can evaluate it further inthe future.Slide 62 (“IgG Anti-CCP Antibodies are Associated with Antibody responses to P. gingivalis”)Now, going back to the data that we had from our small group of patients in whom we had periodontalexaminations and serum collected, we looked at the relationship between periodontal disease andvarious RA parameters. Now here are the three different definitions that we use for looking atperiodontal disease. Our definition that we established – which is about the NHANES III definition,the CDC definition for moderate disease and the CDC definition for severe disease. Again, this is asmall number of patients that this data is taken from. But what we see is that anti-CCP antibodieswith our definition of any periodontal disease are extremely high. Fortunately, this approachedsignificance, but with the small numbers it still was not statistically significant. But this level of effectwas even stronger than that attributed by cigarette smoking, which is the major risk factor known tobe associated with periodontal disease. So we really thought that this was something that was highlysuggestive of a relationship between anti-CCP antibodies in the presence of periodontal disease.QUESTION: [Inaudible]Yeah, in the ESCAPE cohort the relationship didnt pan out, but again, based on symptoms.QUESTION: [Inaudible]The shared epitope and anti-CCP antibodies had no effect on the relationships. It was independent.So we also looked at a random sample of RA patient serum to find out if we had evidence of exposureto periodontal bacteria. So we divided these patients between patients who were CCP positive and
CCP negative. There were an equal number in each group. Serum was batch-shipped up to NYU,where an antibody assay is available to look at antibodies against P. gingivalis using an ELISA testthat was there. We found an odds of 2.67 for having anti-CCP antibodies along with anti-P. gingivalisantibodies. Now, recently Ted Mickels in the group from BRASS with Jim ODell showed arelationship as well between the presence of IgM and IgG2 subclass anti-CCP antibodies withantibodies against P. gingivalis. But this relationship didnt hold out with total IgG antibodies againstP. gingivalis as we had detected here. But at least from our preliminary data as well as from thisgroup there seems to be a consistent association that shows that antibody responses againstperiodontal bacteria and antibodies against citrullinated peptides appear to go together in patients withrheumatoid arthritis.QUESTION: [Inaudible]Im sorry?QUESTION: [Inaudible]In terms of the….QUESTION: [Inaudible]And I think thats one of the limitations of the work that weve done at least in our original study onour patients, is we didnt look – we used whatever was available from the patients medical records forCCPs, which were from various assays and from various labs because we didnt have funding to lookat CCPs ourselves within the study. But I think it does get into this issue of some of how you aretesting for these, but I did find it interesting that the level of association that they describe and thelevel that we saw were quite similar.Slide 63 (“PAD-4 is Detected in Periodontal Tissue”)We have also begun to look at periodontal tissue itself in collaboration with our colleagues over at theUniversity of Maryland. Some of the very preliminary data that we have we have stained to see ifPAD4 is expressed and upregulated in periodontal tissue. And I thank Livia for her work on thisbasically looking at staining for PAD4, here visualized by brown staining in the periodontium. Andone of the things that Livia said is that PAD4 is not as abundantly expressed as it would be in thesynovium or other places where it has been upregulated. So the question becomes are we dealing witha reason for citrullination in periodontal disease, is it due to endogenous upregulation of the PADs aswe might hypothesize in rheumatoid arthritis or might it be due to one of these exogenous causes,such as the PAD obtained from the bacteria. I think we dont know. But it does imply that at leastcitrullination is taking place in the microenvironment of the periodontium, and we would expect to seecitrullinated proteins that are present as well, and those studies are ongoing.Slide 64 (“Summary”)QUESTION: [Inaudible]This was our preliminary data that was used for the grant, and thats one of my specific aims. So wealso submitted an R21 that will be going in for reconsideration and one of the specific aims
specifically is to look with multiple methods in addition to immunohistochemistry at the range ofPAD expression in the tissue and the range of citrullinated proteins.QUESTION: [Inaudible]Thats a good question, and thats another future specific aim for another grant that would look at awell-characterized periodontal cohort of patients because nobody has looked at anti-CCP antibodies inoverall patients with periodontal disease. Is that what youre –QUESTION: [Inaudible]Well, my reasoning would be is because we dont all have the appropriate HLA-DR4 shared epitopethat would then make this something that becomes a true neoantigen.Slide 65 (“Further Studies”)In terms of further studies, comprehensive oral assessments of RA patients and match controls areunderway to confirm the self-report findings that I showed you before. The grant that was funded bythe Arthritis Foundation will allow us to go into the ESCAPE cohort now and perform periodontalexaminations on all of those patients. Obviously evaluation of the mechanistic underpinnings of thedisease relationships is also in progress. And I think this is the question that nobody knows – whetheraggressive management of periodontal disease will result in improved RA outcomes.Slide 66 (“Clinical Studies”)The clinical studies that are ongoing include the continued longitudinal evaluation of the early RApatients with sequential exams to look at progression of periodontal disease, oral exams of theESCAPE patients, determining the effect of RA related medications on PD parameters, furtherevaluation of some of these relationships between Sjögrens and periodontal disease. I have also giventhe questionnaire to Nancy Shaddock, who has administered it to over 1,000 RA patients in theBRASS cohort of RA patients cross-sectionally, so well be able to have a confirmation cohort for ourdata from the ESCAPE set. We have gingival tissue collection ongoing for patients undergoingperiodontal surgery, hopefully identifying patients with RA that have severe enough periodontaldisease that would be willing to have periodontal surgery done over at University of Maryland, wherewe can obtain their tissue. And ultimately, one would like to design interventional studies todetermine the effect of treatment.Slide 67 (“Current RA Patient Recruitment for Oral Health Assessment”)So far with the study weve screened 97 patients. We have enrolled 56, and weve completedexaminations on 38 and a second exam on 10. And this will be ongoing now with funding.Slide 68 (“Treatment of PD Improves RA Disease Activity”)I put up one recent study that came out actually just last week of a treatment study of periodontaldisease in rheumatoid arthritis patients. Its a very strange study. It took 40 patients with RA, all ofwhom had generalized severe periodontitis. So how they found these patients, I dont – you know,they obviously must have a pretty large RA population. But I think it does speak again to the
prevalence of severe periodontal disease in RA patients if they could identify 40 patients over a yearand a half to do the study. Twenty of the patients were on DMARDS. Twenty were on DMARDSplus TNF. And then in each group they were randomized either to get treatment for their periodontaldisease or to wait six weeks, and the looked at the periodontal exam again after six weeks. There wasa reduction in sed rate in DAS28, in periodontal parameters with the periodontal treatment over sixweeks. An interestingly, there was a reduction in baseline – in that should not be baseline TNF levels– but a reduction from baseline TNF levels in 40% of the patients who received the periodontaltreatment versus 20% of those who had not been treated, even in the patients receiving TNFantagonists. I think this is very intriguing data. Again, its a small study, but it does hint at thepossibility of doing an interventional study looking at RA with periodontal disease.Slide 69 (“Triangulation of Inflammation”)Now I finally come back to this diagram that looks at this what I call now a triad of inflammationwhere we have rheumatoid arthritis and periodontal disease that we have been working with where wehave the opportunity to work with the RA cardiovascular disease link that Joan is working with andbegin to put these three diseases together potentially for a future grant application.Slide 70 (“Mechanistic Studies”)The mechanistic studies from this REF grant – were going to be looking at bacterial PAD expressionand trying to understand how this differs from human PADs. For an R21 were looking at gingivaltissue, crevicular fluid and salivary expression of PADs and citrullinated proteins. And looking atantibodies to bacterial flora versus the colonization patterns which give a little bit better idea ofexactly whats taking place in the mouth of an individual.Slide 71 (“Untitled”)So we go back to my original diagram, and now I think it can become a little bit more complicated,when we throw periodontal disease into the model. So we have cigarette smoking turning on PAD2.Cigarette smoking is a risk factor for periodontal disease. Periodontal disease through P. gingivalispotentially leading to upregulation of PADs and citrullination. Periodontal disease leading to aninflammatory disease state with TNF upregulation that may drive the upregulation of PADs.Periodontal disease itself activating innate immune responses and causing localized inflammation andimmunologic activation.Slide 72 (“Conclusions”)And finally a system as we have with RA with the disease acting back upon itself with periodontaldisease potentially acting back on itself as well. So potentially a way to put these two diseaseprocesses together.Slide 73 (“Clinical Implications”)The clinical implications of this, I think, are number one that oral health parameters needs to bemonitored much more closely in patients with RA and with autoimmune disease. I think well find alot if we start looking for it. And now we might have the opportunity to do that, not only for RA but
also for other diseases. And interventions to improve oral pathology may have direct and indirectsystemic benefits.Slide 74 (“Past (and Future?) Arthritis Treatment”)I mentioned that until the 1930s one of the ways that rheumatism was treated was by pulling the teeth.And you wonder if people might have been onto something back then beyond the relationship withrheumatic fever.Slide 75 (“Happy Teeth Make Happy Joints”)So I put out for you maybe a future plug "Happy Teeth Make Happy Joints." And the control of yourarthritis pain – brush more, hurt less.Slide 76 (“Acknowledgements”)So Id like to acknowledge all the many people who have helped me up to this point, includingeveryone in the Arthritis Center, all the research coordinators, Antony, Livia, Felipe, Erika, mycolleagues at University of Maryland, at NYU, including the late John Ship, who was really one of thegreat researches in Sjögrens syndrome who died last year, John Gunsolley – or Jack Gunsolley – whois now at Virginia Commonwealth, and then the multiple funding sources that have come into play forthis work up to this point. Thank you.Slide 77 (“Progression to Periodontitis”)A couple of minutes of questions.QUESTION: [Inaudible.]It hasnt been – it hasnt been looked at well. But the associations when it has been looked at havecertainly been stronger for RA than other diseases. I think we now have the opportunity as we get aninfrastructure set up to do this obviously to expand beyond RA and look into other diseases, at leastother autoimmune diseases, and obviously another inflammatory disease control would be ideal. Oneof the sets – the groups of patients that I wanted to use is sort of the ideal control that was in myoriginal grant application that the NIH pooh-poohed a long time again and the second iteration of myR03 was to have a control group of patients with psoriatic arthritis who have inflammatory arthritiswho should not have anti-CCP antibodies to see if the relationship holds or doesnt hold in that groupof patients as would be expected.QUESTION: [Inaudible.]I think some of the things that you can see again with aggressive periodontal treatment – you are notgoing to get bone growing back. Thats never going to happen. But you can see an impact on some ofthe indices of gingival inflammation, for instance, that will go down, bleeding on probing that will godown again. So youre treating the inflammatory component. You can also, you know, some studieshave shown improvement in some of the different pieces of periodontal disease with treatment. Butits more an issue of, I think, prevention of further progression because really with periodontal disease
once it enters to a certain state it does become really sort of an inevitable type of progressive responsedownward until the tooth is lost.QUESTION: [Inaudible.]Right. So you may be able to, for instance, monitor radiographically over shorter periods of time andlook for alveolar bone loss or not lost as one of the potential ways, as we do with Sharp scores in RA.It is one of the things that we have talked about and thought about that might be, you know, again sortof a way to judge a potential intervention.