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RESEARCH REPORT |
1 Department of Periodontology, Eastman Dental Institute and Hospital, University College London, 256 Gray’s Inn Road, London WC1X 8LD, UK;
2 Eastman Clinical Investigation Center, Eastman Dental Institute and Hospital, University College London; and
3 Microbiology Unit, Eastman Dental Hospital, University College London Hospitals, NHS Trust;
* corresponding author, t.maurizio{at}eastman.ucl.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: periodontitis • inflammation • atherosclerosis • infection • C-reactive protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Support for the hypothesis that periodontitis-driven inflammatory responses are of significance for otherwise healthy individuals is at least three-fold: (i) Periodontitis has been associated with increased odds of cardiovascular events (Genco et al., 2002; Joshipura et al., 2003), of delivering pre-term low-birthweight babies (Offenbacher et al., 1996), and of having sub-optimal control of type II diabetes (Grossi and Genco, 1998); (ii) the strength of association between periodontitis or other chronic infections and cardiovascular events seems to be of similar magnitude (Danesh, 1999); and (iii) experimental pre-clinical models have indicated that chronic infection with periodontal pathogens leads to thickening of the carotid intima (Li et al., 2002) and to fetal growth restriction (Collins et al., 1994).
Independently of the underlying mechanism(s), systemic inflammation seems to be central for explaining the nature of the link between chronic infections and atherosclerosis (Ridker et al., 1997; Danesh, 1999; Ross, 1999; Libby et al., 2002; Pearson et al., 2003). Within this context, CRP represents an emerging and reliable marker of the acute phase response to infectious burdens and/or inflammation. As a consequence of its kinetics, it best describes the inflammatory status of the individual (de Maat and Kluft, 2001). CRP hepatic production is usually elicited by an inflammatory stimulus and mediated through a complex network of cytokines (mainly IL-6) (Ablij and Meinders, 2002). CRP has also assumed a significant role as a predictor for future coronary events in healthy populations (Blake and Ridker, 2002).
The aim of this pilot intervention trial was to assess whether the degree of individual response to periodontal treatment was associated with changes in serological markers of systemic inflammation, i.e., CRP and IL-6, in otherwise healthy individuals.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A baseline visit was conducted by a blind calibrated examiner who collected a complete medical history, standard clinical periodontal parameters (O’Leary et al., 1972; Tonetti et al., 2002) and blood samples. Thereafter, patients underwent a standard phase of non-surgical periodontal treatment that was performed by a periodontist. All other necessary dental treatments (extractions of hopeless teeth, restorative treatments) were carried out before completion of the periodontal treatment, consisting of oral hygiene instructions and subgingival scaling and root planing with the subjects under local anesthesia, with the use of a piezoelectric instrument equipped with fine subgingival tips (Electro Medical Systems, Nyon, Switzerland). The therapeutic phase was completed within 1–3 mos of the baseline visit. Patients were then re-examined at 2 and 6 mos after the completion of the treatment.
Serum CRP and IL-6 Analysis
Serum samples were collected at different time points (baseline, 2 and 6 mos), were processed, and were stored at -70°C until analysis in a standardized fashion to limit intra-individual variations. CRP levels were assessed by an automated immunoturbidimetric high-sensitivity assay (Cobas Integra, Roche, Mannheim, Germany; detection limit of 0.25 mg/L); IL-6 was measured with a high-sensitivity sandwich ELISA (Quantikine HS, R&D System, Minneapolis, MN, USA; mean detection limit, 0.04 ng/L).
Microbiological Analysis
Samples of subgingival periodontal plaque were collected at baseline from the 4 deepest pockets, one in each quadrant. DNA was isolated by the use of a commercial DNA extraction kit (Puregene, Minneapolis, MN, USA) and subsequently used as a template to carry out amplification of the bacterial 16S rRNA genes of three different periodontal pathogens by the use of multiplex-polymerase chain-reaction (Tran and Rudney, 1999). The use of specific primers produced three different-sized DNA products for Tannerella forsythensis (Tf), Porphyromonas gingivalis (Pg), and Actinobacillus actinomycetemcomitans (Aa). PCR products were then visualized on an agarose gel.
DNA Genotyping
For genotyping, DNA was extracted from subjects’ leukocytes in peripheral venous blood samples collected at the baseline visit by means of a commercial kit (Nucleon® BACC2 kit, Nucleon Bioscience, Coatbridge, UK). Sequences of the oligonucleotide primers used for PCR amplification, the sizes of the predicted PCR products, and the PCR amplification program used were as previously described (Kornman et al., 1997; Fishman et al., 1998). Briefly, the following primer sets were used: for IL-1A (-889), 5'-AAG CTT GTT CTA CCA CCT GAA CTA GGC-3' and 5'-TTA CAT ATG AGC CTT CCA TG-3'; for IL-1B (-511), 5'-TGG CAT TGA TCT GGT TCA TC-3' and 5'-GTT TAG GAA ATC TTC CCA CTT-3'; for IL-1B (+3954), 5'-CTC AGG TGT CCT CGA AGA AAT CAA A-3' and 5'-GCT TTT TTG CTG TGA GTC CCG-3'; and finally, for IL-6 (-174 G/C), 5'-TGA CTT CAG CTT TAC TCT TGT-3', 5'-CTG ATT GGA ACC CTT ATT AAG-3'. Alleles were identified following digestion with restriction endonucleases on a 3% agarose gel containing 0.5 mg/mL ethidium bromide in 1 TBE buffer at 100 V for 2 hrs. The bands were visualized with the use of a UV transilluminator.
Statistical Analysis
Since no previous estimates of the variance of periodontal-treatment-associated changes in the primary outcome variables (CRP and IL-6) were available, the sample size was based not on a formal calculation but rather on logistic and empiric considerations. All data were entered into a computer file, proofed for entry errors, and analyzed with a statistical package (SAS version 8•1, Cary, NC). Changes in serum concentrations of CRP and IL-6 following periodontal therapy were used as the primary outcome variables. Variables not normally distributed were logarithmically transformed before being used in parametric analyses. Continuous normally distributed variables were reported as mean ± standard deviation (SD), whereas median and interquartile ranges (IQR) were used to describe data not normally distributed. Between visits, differences for each inflammatory marker were assessed with a paired Wilcoxon rank-sum test. We assessed the association between changes in CRP and IL-6 concentrations between 0 and 6 mos and the individual response to periodontal therapy by constructing a generalized linear model (using the SAS PROC GLM and REPEATED statement) that included the following confounding factors: age, gender, body mass index, cigarette smoking, and cytokine polymorphisms. The alpha value was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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On average, 58 ± 20.7% of sites in a patient presented with detectable plaque (O’Leary et al., 1972). Microbial analysis showed the presence of Tf in 76.3% of cases, Pg in 72.8%, and Aa in 43.8%. Participants presented with high levels of gingival inflammation (full-mouth bleeding scores of 63.5 ± 16.4%, N = 94) and severe widespread periodontitis (average of 77 ± 23 periodontal lesions per subject, with an average clinical attachment level loss of 4.93 ± 1.13 mm, N = 94). Significant reductions in full-mouth bleeding (45.5 ± 16.7%, P < 0.0001 t test, N = 94) and in number of periodontal pockets greater than 4 mm (57 ± 24, P < 0.0001 t test, N = 94) were achieved 6 mos after treatment.
The baseline median level of CRP was 1.9 mg/L (3.6 IQR, N = 94, Fig. 1
), whereas the level of IL-6 was 1.8 ng/L (1.5 IQR, N = 94, Fig. 2). No significant differences in concentrations were found among different groups according to age, gender, and smoking status. Differences were statistically significant between baseline and 2 and 6 mos for serum IL-6 (P = 0.021, P = 0.006, respectively, Wilcoxon test, N = 94) and between baseline and 6 mos for CRP (P < 0.0001, Wilcoxon test, N = 94). Improvements in serum IL-6 and CRP were observed in comparison of baseline and six-month values in 73% and 62% of subjects, respectively. The median change in CRP concentrations between baseline and 6 mos was 0.5 mg/L, with a 95% distribution-free confidence interval (95% CI) of 0.4 to 0.7 mg/L. Corresponding values for IL-6 were 0.2 ng/L (95% CI 0.1 to 0.4 ng/L). Since response to periodontal treatment was not homogeneous across the population, subjects were dichotomized as planned a priori according to the level of clinical response to periodontal therapy (based on the median number of residual periodontal lesions < 30 and gingival bleeding < 30% as the cut-off). Among subjects with the better periodontal response, 79.2% of patients displayed a decrease in serum CRP.
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). Serum CRP concentrations were also significantly associated with carriage of specific polymorphic functional alleles in the IL-6 (-174G/C) and IL-1A (-889) promoter regions. A similar analysis with Log IL-6 as the dependent variable indicated that serum IL-6 concentrations were associated with the outcome of periodontal treatment as well as with the age of the subjects and being homozygous for allele 2 of a specific functional polymorphism in the promoter region of the IL-1B gene (- 511, see Table).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The improvements in CRP were obtained in individuals whose baseline values were in the upper quartiles of normality (median of 1.9 mg/L, IQR 3.6 mg/L). Analysis of these data confirms previous observations that otherwise healthy subjects suffering from chronic periodontitis display a moderate increase in systemic inflammation (Loos et al., 2000). Analyses of recent data have indicated that CRP levels in the upper quartiles of normality are good predictors of future coronary events in healthy populations (Ridker et al., 1997; Blake and Ridker, 2002). Several lines of evidence support the causative role of chronic infections and/or the associated inflammatory responses in atherosclerosis: (i) increased odds ratios; (ii) chronological consistency between infection and atherosclerosis; (iii) data from experimental models; and, in the end, (iv) emerging results of intervention trials. Data supporting the role of periodontal infections have been limited to the first three lines of evidence. In the majority of studies, subjects with severe generalized periodontitis displayed increased odds ratios of cardiovascular events. Reports from large prospective studies have associated periodontitis and tooth loss, with an increased risk for carotid atherosclerosis (1-mm thickening of the intima) after correction for possible confounding factors (Beck et al., 2001; Desvarieux et al., 2003). In cell culture studies, Pg, one of the most important periodontal pathogens, has shown the ability to invade endothelial cells (Dorn et al., 2000). Periodontal pathogens have been identified in carotid atheromatous plaques of patients undergoing endarterectomy (Haraszthy et al., 2000). Furthermore, Pg chronic inoculations in ApoE +/- mice increased lipid profiles, enhanced atheroma formation, and produced calcifications of the aortal atherosclerotic plaques (Li et al., 2002). The current study expands this evidence to include the results of an initial intervention trial focusing on inflammatory responses. The positive results obtained warrant confirmation in a randomized controlled clinical trial and expansion of these findings to include, in the first instance, the evaluation of the effects of periodontal treatment on parameters of arterial function.
Periodontitis is an infection caused by Gram-negative bacteria that are organized in a biofilm in a sub-gingival location between the diseased root surface of the tooth and the junctional epithelium. As such, it is relatively insensitive to the effects of systemic antibiotics, and its treatment requires, in the first instance, the removal of the biofilm by mechanical professional instrumentation. Following successful treatment, bacterial load is significantly reduced, while antibody titers and avidity to the specific pathogens are improved. As a result of these changes, local inflammation significantly decreases, and there is a significant improvement of the clinical parameters (probing pocket depths, bleeding on probing, etc.). In this study, decreases in serum CRP were significantly associated with the half of the population that responded better to periodontal treatment in terms of decreases in the infection burden and the associated periodontal inflammation as assessed by clinical parameters. These findings add further strengths to the observed results by providing an internal comparison: The lack of significant CRP decreases in the half of the population with the worse clinical periodontal outcomes indicates that factors other than periodontal treatment are unlikely to account fully for the observed decreases in serum CRP.
In agreement with previous observations, analysis of the data also indicated that, besides the significance of the outcome of periodontal therapy, CRP serum levels were associated with a variety of patient-specific attributes and exposures, notably cigarette smoking, body mass index, age of the subject, and carriage of specific polymorphisms in the IL-1A and IL-6 promoter regions. In terms of inflammatory cytokines, being homozygous for allele 2 for IL-1A or IL-6 was associated with significantly higher serum CRP concentrations. These findings are in agreement with the hypothesis that the periodontal burden (infection plus associated inflammatory responses) acts on an individual susceptibility profile that amplifies the systemic inflammatory responses and, in turn, may play a role in systemic inflammatory diseases such as atherosclerosis. On the other hand, given the relatively small sample size of this pilot and the high number of tested covariates, these analyses will require confirmation in further, larger-scale investigations.
In summary, periodontitis seems to contribute to systemic inflammation. The potential significance of the reported findings relates to the magnitude of the observed decreases in CRP, the high prevalence of periodontitis in the population, and the fact that periodontitis can be treated. Results of this investigation should be taken into account in the design and implementation of a definitive trial.
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ACKNOWLEDGMENTS |
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Received May 15, 2003; Last revision September 26, 2003; Accepted November 17, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Beck JD, Elter JR, Heiss G, Couper D, Mauriello SM, Offenbacher S (2001). Relationship of periodontal disease to carotid artery intima-media wall thickness: the atherosclerosis risk in communities (ARIC) study. Arterioscler Thromb Vasc Biol 21:1816–1822.
Blake GJ, Ridker PM (2002). Inflammatory bio-markers and cardiovascular risk prediction. J Intern Med 252:283–294.[ISI][Medline]
Collins JG, Windley HW 3rd, Arnold RR, Offenbacher S (1994). Effects of a Porphyromonas gingivalis infection on inflammatory mediator response and pregnancy outcome in hamsters. Infect Immun 62:4356–4361.
Danesh J (1999). Coronary heart disease, Helicobacter pylori, dental disease, Chlamydia pneumoniae, and cytomegalovirus: meta-analyses of prospective studies. Am Heart J 138(5 Pt 2):S434–S437.[ISI][Medline]
de Maat MP, Kluft C (2001). Determinants of C-reactive protein concentration in blood. Ital Heart J 2:189–195.[Medline]
Desvarieux M, Demmer RT, Rundek T, Boden-Albala B, Jacobs DR Jr, Papapanou PN, et al. (2003). Relationship between periodontal disease, tooth loss, and carotid artery plaque: the Oral Infections and Vascular Disease Epidemiology Study (INVEST). Stroke 34:2120–2125.
Dorn BR, Burks JN, Seifert KN, Progulske-Fox A (2000). Invasion of endothelial and epithelial cells by strains of Porphyromonas gingivalis. FEMS Microbiol Lett 187:139–144.[ISI][Medline]
Ebersole JL, Machen RL, Steffen MJ, Willmann DE (1997). Systemic acute-phase reactants, C-reactive protein and haptoglobin, in adult periodontitis. Clin Exp Immunol 107:347–352.[ISI][Medline]
Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al. (1998). The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102:1369–1376.[ISI][Medline]
Genco R, Offenbacher S, Beck J (2002). Periodontal disease and cardiovascular disease: epidemiology and possible mechanisms. J Am Dent Assoc 133(Suppl):14S–22S.
Grossi SG, Genco RJ (1998). Periodontal disease and diabetes mellitus: a two-way relationship. Ann Periodontol 3:51–61.[Medline]
Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ (2000). Identification of periodontal pathogens in atheromatous plaques. J Periodontol 71:1554–1560.[ISI][Medline]
Hutter JW, van der Venden U, Varoufaki A, Huffels RA, Hoek FJ, Loos BG (2001). Lower numbers of erythrocytes and lower levels of hemoglobin in periodontitis patients compared to control subjects. J Clin Periodontol 28:930–936.[ISI][Medline]
Joshipura KJ, Hung HC, Rimm EB, Willett WC, Ascherio A (2003). Periodontal disease, tooth loss, and incidence of ischemic stroke. Stroke 34:47–52.
Kornman KS, Crane A, Wang HY, di Giovine FS, Newman MG, Pirk FW, et al. (1997). The interleukin-1 genotype as a severity factor in adult periodontal disease. J Clin Periodontol 24:72–77.[ISI][Medline]
Kweider M, Lowe GD, Murray GD, Kinane DF, McGowan DA (1993). Dental disease, fibrinogen and white cell count; links with myocardial infarction? Scott Med J 38:73–74.[ISI][Medline]
Li L, Messas E, Batista EL Jr, Levine RA, Amar S (2002). Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 105:861–867.
Libby P, Ridker PM, Maseri A (2002). Inflammation and atherosclerosis. Circulation 105:1135–1143.
Loos BG, Craandijk J, Hoek FJ, Wertheim-van Dillen PM, van der Velden U (2000). Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J Periodontol 71:1528–1534.[ISI][Medline]
Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ, De Nardin E (2001). Periodontal infections contribute to elevated systemic C-reactive protein level. J Periodontol 72:1221–1227.[ISI][Medline]
O’Leary TJ, Drake RB, Naylor JE (1972). The plaque control record. J Periodontol 43:38.[ISI][Medline]
Offenbacher S, Katz V, Fertik G, Collins J, Boyd D, Maynor G, et al. (1996). Periodontal infection as a possible risk factor for preterm low birth weight. J Periodontol 67(10 Suppl):1103–1113.[ISI][Medline]
Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO 3rd, Criqui M, et al. (2003). Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 107:499–511.
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH (1997). Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 336:973–979.
Ross R (1999). Atherosclerosis—an inflammatory disease. N Engl J Med 340:115–126.
Slade GD, Offenbacher S, Beck JD, Heiss G, Pankow JS (2000). Acute-phase inflammatory response to periodontal disease in the US population. J Dent Res 79:49–57.
Slade GD, Ghezzi EM, Heiss G, Beck JD, Riche E, Offenbacher S (2003). Relationship between periodontal disease and C-reactive protein among adults in the Atherosclerosis Risk in Communities study. Arch Intern Med 163:1172–1179.
Tonetti MS, Lang NP, Cortellini P, Suvan JE, Adriaens P, Dubravec D, et al. (2002). Enamel matrix proteins in the regenerative therapy of deep intrabony defects. J Clin Periodontol 29:317–325.[ISI][Medline]
Tran SD, Rudney JD (1999). Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis. J Clin Microbiol 37:3504–3508.
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P < 0.0001 Wilcoxon rank-sum paired test compared with baseline.
P = 0.021 and 
P = 0.006 Wilcoxon rank-sum paired test compared with baseline.