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Periodontal and Serum Protein Profiles in Patients With Rheumatoid Arthritis Treated With Tumor Necrosis Factor Inhibitor Adalimumab , Tetsuo Kobayashi* †, DDS, PhD, Tomoko Yokoyama*, DDS, PhD, Satoshi Ito‡, MD, PhD, Daisuke Kobayashi‡,||, MD, PhD, Akira Yamagata§, PhD, Moe Okada*, DDS, PhD, Ken Oofusa§, PhD, Ichiei Narita||, MD, PhD, Akira Murasawa‡, MD, PhD, Kiyoshi Nakazono‡, MD, PhD, and Hiromasa Yoshie*, DDS, PhD

* Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. † General Dentistry and Clinical Education Unit, Niigata University Medical and Dental Hospital, Niigata, Japan. ‡ Niigata Rheumatic Center, Shibata, Japan. §

ProPhoenix Division, Towa Environment Science Co., Ltd. Osaka, Japan.

||

Division of Clinical Nephrology and Rheumatology, Department of Homeostatic Regulation Developments, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Background: Tumor necrosis factor-alpha (TNF-α) inhibitor has been implicated to have an effect on periodontal condition in patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody adalimumab (ADA) on periodontal condition in patients with RA, and to compare serum protein profiles before and after ADA therapy. Methods: The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were determined, and identified with mass spectrometry and protein databases. Results: The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P = 0.0004), and serum levels of TNF-α (P = 0.0003) and interleukin-6 (P < 0.0001) after ADA medication, although plaque levels were comparable. Among a total of 495 protein spots obtained, nine spots were significantly decreased in abundance at reassessment, which corresponds to complement factor H, phospholipase D, serum amyloid A, complement component 4, and alpha-1-acid glycoprotein (P < 0.01). Conclusions: These results suggest an beneficial effect of ADA therapy on periodontal condition in patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

KEYWORDS: Arthritis, rheumatoid; immunotherapy; periodontitis; proteomics; serum; tumor necrosis factor-alpha

Rheumatoid arthritis (RA) and periodontitis have been suggested to share many clinical and pathological features.1,2 Both diseases represent chronic inflammatory diseases characterized by accumulation and persistence of inflammatory infiltrates in the local lesions. Patients with RA exhibiting synovitis and destruction of the joint were more likely to have periodontitis,3-5 whereas patients with moderate-to-severe periodontitis had a higher prevalence of RA than those without periodontitis.5-7 These observations imply that certain features of the inflammatory responses are common to both diseases, which might be underpinned by biological pathways.2

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A similar profile of cytokines has been involved in the pathogenesis of RA and periodontitis.1,2 In particular, tumor necrosis factor-alpha (TNF-α) has been suggested as one of the most potent cytokines associated with these two inflammatory diseases.8,9 It is well recognized that TNF-α plays a central role in inflammatory reactions including alveolar bone resorption and the loss of connective tissue attachment in periodonttis.9 It has also been documented that TNF antagonists inhibited loss of tissue attachment and bone in a model of experimental periodontitis.10,11 It was further reported that TNF-α blockade led to improvement of clinical arthritis condition in a mouse model of collageninduced arthritis.12 These findings suggest that constitutive overproduction of TNF-α may play a pathological role in RA and periodontitis. Recently, TNF inhibitors have been evaluated for their efficacy on periodontal condition in patients with RA.13-17 It has been shown that treatment with infliximab (IFX: a chimeric mouse/human anti-TNF-α monoclonal antibody) proved beneficial in suppressing periodontal diseases in patients with RA.13,14,16 Other studies also demonstrated an improvement of clinical periodontal condition in the patients who were treated with IFX, etanercept (ETN: a recombinant fusion protein linked to human type II TNF receptor-Fc portion), or adalimumab (ADA: a fully humanized anti-TNF-α monoclonal antibody).15,17 However, there has been no study that evaluated the effect of ADA therapy alone on periodontal condition. These three TNF inhibitors, IFX, ETN, and ADA were shown to be comparative in effectiveness for the treatment of RA,18 but their difference in biological effects has been suggested.19-21 ETN is able to bind TNF-α and TNF-β, whereas IFX and ADA can bind only TNF-α. It has been reported that both IFX and ADA exerted complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), and outside-to-inside signals through transmembrane TNFα, while ETN had only ADCC activity.19 Therefore, it is necessary to perform a comparative proteomic study that allows global assessment of changes in serum protein/peptide abundance before and after anti-TNF therapy, which might provide the molecular mechanisms by which TNF inhibitors proved effective in the treatment of RA and periodontitis. There has been a few studies showing a difference in serum protein profile before and after treatment with IFX and ETN.20,21 However, there is no information to date on the effects of treatment with ADA alone on serum protein profile in patients with RA. Therefore, the aim of the present study is to assess the effects of ADA therapy on periodontal condition in patients with RA, as determined by gingival index (GI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL). Additionally, serum protein profiles were compared before and after ADA therapy with two-dimensional gel electrophoresis and mass spectrometry of the proteins.

MATERIALS AND METHODS Participants A total of 20 Japanese patients with RA (19 females and 1 male; mean ± standard error [SE] 51.7 ± 2.2 years) followed at the Niigata Rheumatic Center, Shibata, Japan, were enrolled in the present study between July 2011 and February 2014. The study was approved by the Institutional Review Board of the Niigata University Faculty of Dentistry (No. 23-R2-11-05, on July 12, 2011) and Niigata Rheumatic Center (No. 2, on June 1, 2011). Signed informed consent was obtained from all participants before inclusion. All patients were confirmed to fulfill the 1987 revised classification criteria of American Rheumatism Association,22 as well as the 2010 RA classification criteria of American College of Rheumatology and European League Against Rheumatism.23 In brief, RA was defined on the confirmed presence of synovitis in at least 1 joint, absence of an alternative diagnosis that better explains the synovitis, and achievement of a total score of 6 or greater (of a possible 10) 2

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from the individual scores in 4 domains: number and site of involved joints (score range 0-5), serologic abnormality (score range 0-3) elevated acute-phase response (score range 0-1), and symptom duration (2 levels; range 0-1). All participants were found to take corticosteroids, diseasemodifying antirheumatic drugs (DMARDs), or non-steroidal anti-inflammatory drugs (NSAIDs), and to fulfill the following two criteria: 1) the absence of diabetes mellitus and pregnancy, 2) having neither any periodontal therapy nor mouth rinse usage within the preceding 3 months. Clinical Assessments Clinical periodontal assessments were performed by two trained and calibrated examiners (TK and MO) who were masked from the periodontal and rheumatologic data. The calibration was performed before the study with 5 volunteer subjects in Niigata University Faculty of Dentistry. Reproducibility of the clinical measurements was calculated by means of the κ index, and a value of 0.857 was obtained for CAL with a difference of ± 1 mm. All patients were evaluated clinically at the Niigata Rheumatic Center in the following measurements: number of teeth present, GI,24 PD, CAL, supragingival plaque accumulation, and BOP. The presence or absence of supragingival plaque and BOP were recorded at four and six sites around each tooth, respectively. Measurements of PD and CAL were conducted with a Williams probe at six sites around each tooth, recorded to the nearest millimeter, and every observation close to 0.5 mm was rounded to the lower whole number. The averaged score for whole-mouth PD, CAL, and the number of sites with supragingival plaque and BOP divided by the total number of sites per mouth and multiplied by 100 were calculated for each subject. The disease activity of RA was determined with Disease Activity Score including 28 joints using C-reactive protein (DAS28-CRP),25 which is calculated with a formula that takes into account the number of tender and swollen joints (TJC and SJC), the patient’s general assessment of their condition scored on a visual analog scale (VAS), and CRP. DAS28-CRP constitutes four categories: remission (DAS28-CRP < 2.3), low (2.3 ≤ DAS28-CRP < 2.7), moderate (2.7 ≤ DAS28-CRP < 4.1), and high disease activity (4.1 ≤ DAS28-CRP), which underestimates disease activity compared with DAS28 using erythrocyte sedimentation rate (DAS28-ESR) in Japan.25 Smoking status of the participants was classified as current-smokers, former-smokers, or never-smokers, according to information provided on a standard questionnaire. Demographic and disease-related characteristics of 20 participants at baseline are summarized in Table 1. Adalimumab Medication After the clinical and laboratory analyses at baseline, all patients with RA received ADA at a dosage of 40 mg subcutaneously every two weeks, and were then subjected to the same analyses (reassessment) at 3 months later. Neither periodontal treatment such as professional scaling and prophylaxis nor tooth-brushing instruction was performed during and before the study period. In addition, all participants were instructed not to change their oral hygiene regimens throughout the study period. Measurements of Serum RF, Anti-CCP Antibodies, CRP, MMP-3, IL-6, TNF-α Levels Peripheral venous blood samples were obtained by venipuncture from all participants. Serum was isolated from the blood by centrifugation at 1,500 x g for 20 minutes, and stored at -70°C until used. Serum concentrations of rheumatoid factor (RF) and high-sensitive CRP were determined with a latex particle-enhanced and a simple nephelometric method.¶ Serum levels of anti-cyclic citrullinated peptide (CCP) antibodies, and those of matrix metalloproteinase-3 (MMP-3), interleukin-6 (IL-6), and TNF-α were determined by sensitive enzyme-linked immunosorbent assay (ELISA) with the commercially available kits ,** , according to the manufacturer’s 3

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instructions. The microtiter plates were read at a wavelength of 450 nm for anti-CCP antibodies and †† MMP-3, or 490 nm for IL-6 and TNF-α, with an automated microplate reader. The lower limits of detection for these measurements were as follows: RF, 1.25 IU/ml; anti-CCP antibodies, 0.4 U/ml; CRP, 0.004 mg/dl; MMP-3, 0.009 ng/ml; IL-6, 0.016 pg/ml; TNF-α, 0.038 pg/ml. Positivity of RF and anti-CCP antibodies was defined as showing more than15 IU/ml and 4.5 U/ml, respectively. Levels of measurements below the lower limit of detection were recorded as being not determined, and were deleted from the statistical analyses. Two-Dimensional (2-D) Gel Electrophoresis Serum protein profiles were analyzed as previously described.26 In brief, 100 μL of sera from 6 donors who were randomly selected were mixed together. Six high-abundance proteins (albumin, IgG, IgA, antitrypsin, haptoglobin, and transferrin) were deleted from the mixed sera with affinity ‡‡ column and optimized buffer reagent kit, according to the manufacturer’s instructions, to avoid the masking effect of well-characterized high-abundance proteins. The protein concentration of each sample was measured by a commercially available kit. §§ After the samples were concentrated and desalted with a filter device,|||| the first-dimensional separation of proteins was carried out on immobilized pH gradient (IPG) strips,¶¶ in accordance with published procedures.27,28 Briefly, the protein samples (200 μg of total protein) were applied overnight to IPG strips by in-gel rehydration. The rehydrated gels were then gently dried with sheets of tissue paper to remove excess of fluid, and were subjected to an isoelectric focusing electrophoresis unit, according to the manufacturer’s instructions. This was followed by second-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 9–18% acrylamide gradient gels using an electrophoresis chamber. The proteins separated on the 2-D acrylamide gels were stained with fluorescent dye SYPRO *** ††† ‡‡‡ Ruby, and were detected with an imaging system. An image analysis software was used to identify matched and unmatched protein spots on gel images of sera before and after ADA therapy, showing that more than 99 % of the spots were coincident in each pair of three replicates, when the densitometric volume (related to the quantity of protein) of the spots was > 200. The higher- and lower-abundance of the matched protein spot was identified when difference in the spot volume was significantly different before and after ADA therapy. Mass Spectrometric Analysis The protein spots with significant high- and low-abundance were cut for in-gel digestion and subsequently were subjected to mass spectrometric analysis, as described by Gerashchenko et al.28 The fluorescent stained 2-D gels were further stained with silver to visualize spots. Briefly, the selected matched protein spots were excised from the dried silver stained 2-D gels,29 and rehydrated in 100 mM ammonium bicarbonate for 20 min. The gel spots were then destained in solution of 15 mM potassium ferricyanide and 50 mM thiosulfate for 20 min, rinsed twice in deionized water, and finally dehydrated in 100% acetonitrile (ACN) until they turned opaque white. The spots were then dried in a vacuum centrifuge, followed by overnight rehydration in digestion solution consisting of 100 mM ammonium bicarbonate, 10% ACN, 0.01 μg/μL modified sequence-grade trypsin§§§ at 37oC. The digestion of spots was terminated by treatment with 5% trifluoroacetic acid (TFA) for 20 min. Peptides were extracted from the spots three times with 5% TFA in 50% ACN for 20 min, and were pooled and dried in a vacuum centrifuge. This was followed by their collection/concentration with pipette tips||||||, according to the manufacturer’s protocol. The peptide mass spectra were measured using a mass spectrometer.¶¶¶ Identification of protein was performed with a search engine using the National Center for Biotechnology Information (NCBI) database.

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Statistical Analyses ****

The sample size calculation test with the statistical software suggested that a minimum of 12 patients were needed to detect a significant difference in % sites with BOP, with 80% power and 5% of level, after the cytokine-targeted therapy.30 Differences between the baseline and reassessment in the value of clinical and serum parameters and the spot volume of matched serum proteins were evaluated by Wilcoxon signed rank tests and paired t-tests, respectively, after evaluating the normality of distribution by Kolmogorov-Smirnov tests. Statistical significance was accepted at 1% (P < 0.01).

RESULTS Changes in Rheumatologic and Serum Characteristics No adverse events were observed in gingival, oral mucosal, and rheumatologic condition of all participants during the study period. As shown in Table 2, ADA therapy led to a significant decrease in DAS28-CRP (P = 0.0004), VAS (P = 0.001), and frequencies of the patients with high activity (P = 0.0008). Likewise, serum levels of anti-CCP antibodies (P = 0.0005), RF (P = 0.0002), CRP (P = 0.0003), MMP-3 (P = 0.007), IL-6 (P < 0.0001), and TNF-α (P = 0.0003) were significantly decreased after ADA therapy (Table 2). Smoking status and RA medication were unchanged in the distribution and dose from the baseline to reassessment (data not shown). Changes in Periodontal Characteristics The patients showed a significant decrease in GI (P = 0.002), % sites with BOP (P = 0.003), and PD (P = 0.002) after ADA therapy (Table 3). Other periodontal parameters including % sites with plaque and CAL proved comparable between the baseline and reassessment (P > 0.01) (Table 3). Changes in Serum Protein Profiles A total of 495 protein spots were obtained in the 2-D electrophoretograms of sera from all participants both at the baseline and reassessment. The representative 2-D electrophoretograms of sera from the patients before and after ADA therapy were shown in Figure 1. Most of the protein spots were distributed between pI 4 and 7, and between 10 and 225 kDa. More than 99% of the spots were coincident in each pair (baseline and reassessment) of three replicates (data not shown). As shown in Figure 2, the 2-D gel image analysis revealed that 9 spots were significantly different in abundance before and after ADA therapy (P < 0.01). All 9 spots were significantly lower in abundance at reassessment than those at baseline, which corresponds to 5 proteins: complement factor H (CFH), phospholipase D (PLD), serum amyloid A (SAA), complement component 4 (C4), and alpha-1-acid glycoprotein (AGP) (P < 0.01) (Table 4).

DISCUSSION The results of periodontal assessments demonstrated a significant improvement of periodontal condition, as determined by GI, BOP, and PD, in patients with RA after ADA therapy. These results are consistent with the findings of other studies13-17, suggesting a therapeutic efficacy of TNFinhibition on periodontal condition. The data also showed the comparable levels of supragingival plaque, and the same status of smoking and RA medication between the baseline and reassessment. It has been documented that periodontits occurs as a result of excessive host inflammatory responses such as TNF-α overproduction to microbial challenge.31 Therefore, it is suggested that the improvement of periodontal condition might be independent of the confounding environmental factors related to periodontal disease. To the best of the authors' knowledge, this is the first study to 5

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assess ADA therapy on periodontal condition in patients with RA. However, it cannot be concluded from these findings alone whether ADA has a beneficial effect on periodontal condition. Similar analyses have now been undertaken in the test and control individuals with RA before, and 6 months after ADA therapy. The results of RA assessments indicated a significant improvement of rheumatologic condition, as indicated by a decrease in DAS28-CRP and VAS score, in the patients after ADA medication. The data also showed that serum levels of anti-CCP antibodies, RF, CRP, MMP-3, IL-6, and TNF-α were significantly decreased at reassessment than those at baseline. Moreover, the frequencies of corticosteroids, DMARDs, and NSAIDs medication were unchanged throughout the study period, although these drugs have been shown to affect systemic inflammatory markers such as TNF-α.32-34 These results imply that ADA therapy may not only decrease RA activities, but also reduce systemic inflammation. These observations might be supported by the results of other studies,18,35-38 showing that the clinical efficacy of ADA therapy was associated with decreased serum levels of RF, antiCCP antibody, and inflammatory mediators including CRP, Pro-MMP3, chemerin, IL-6, and TNFα. However, it does not rule out the possibility that ADA plays an inhibitory effect on local inflammation as well, although the antibody levels have not been studied in the periodontium. Serum proteomic analysis has been shown as a valuable strategy for identification of novel candidate protein biomarkers in relation to RA, or new molecular targets for RA therapy.20,21 Serum contains not only dynamic concentration range of protein components, but also several groups of high-abundance proteins that interfere with the detection of low-abundance potential biomarker proteins.39,40 Therefore, a serum proteomic approach was employed with a high throughput method for efficient depletion of high-abundance proteins in the present study. The proteomic results indicated that 5 low-abundant proteins (SAA, AGP, C4, CFH, and PLD) were identified in sera from the patients after ADA therapy, which were classified as an acute-phase protein except for PLD. These results are different from the findings of other proteomic studies,20,21 which identified serum proteins before and after treatments with IFX and ETN. The present study is the first proteomic analysis of sera from patients with RA before and after ADA therapy. However, it would be further necessary to confirm these proteomic results with ELISA and western blot. Furthermore, the clinical and proteomic results indicated an association between these 5 serum proteins and the improvement of periodontal inflammatory conditions. These results imply the direct or indirect impact of changes in serum proteins within the periodontal tissues. However, the limitation of the present study is the lack of determination of serum protein levels in gingival crevicular fluid, which would be necessary to clarify whether the serum proteins can be responsible for the clinical improvement directly or indirectly. SAA is an acute-phase 12.5 kDa apolipoprotein associated with serum high-density lipoprotein, and represents the soluble precursors of inflammation-related amyloid A protein. AGP is an acutephase glycoprotein with complex N-linked oligosaccharide chains, which is known to increase several-fold in sera from individuals with inflammation. It has been reported that serum levels of SAA and AGP were increased in patients with RA and periodontitis than those in control individuals.41-43 Additionally, serum levels of these proteins were decreased following TNF-targeted and periodontal therapies, respectively.,41,42,44 Moreover, it has been shown that serum SAA levels were correlated with RA activity and MMP-3 levels and had the ability to induce TNF-α.41 In particular, reactive amyloid A (AA) amyloidosis has been shown as a serious systemic complication of RA, which is associated with elevated SAA levels.45 It is thus implicated that ADA therapy may also have a possible beneficial effect on prevention of AA amyloidosis in patients with RA. C4 and CFH constitute the complement system, and play an important role in immunity. C4 cleavage is necessary for the generation of complement component 3 convertase in the classical/lectin pathway, whereas CFH is an abundant 150 kDa single chain serum glycoprotein 6

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involved in the alternative pathway.46 Activation of these pathways leads to induction and progression of inflammatory reactions, which may contribute to the pathogenesis of RA and periodontitis.26,46 The proteomic results showed that C4 and CFH were low-abundant proteins in the patients treated with ADA, which is supported by the findings of other study showing that anti-TNFα treatment reduced complement activation in patients with RA.47 PLD is an enzyme that hydrolyzes the phosphodiester bound in phophatidylcholine, yielding choline and phosphatidic acid. It has been proposed by an in vitro study that PLD may play a role in osteoclastogenesis by targeting osteoclast precursors.48 However, the clinical relevance of PLD to RA and periodontitis has not been evaluated. Caution is necessary when the proteomic results are interpreted, because the detection of protein spots on the 2-D gels is influenced by the employed techniques. The results showed 9 serum protein spots were identified with different abundance before and after ADA therapy, which were distributed on the gels between pI 4 and 7, and between 10 and 225 kDa. However, it is technically difficult to detect the spots appeared in the lower or higher range of pH and molecular weight, which were not visible on the gel. Another caution relates to the reliability of the proteomic findings, which may be due to the complexity of procedures. For improvement of the reliability, the sample pooling was used to reduce nonspecific spot volume in the 2-D electrophoresis.49 The 2D gel image analysis revealed that more than 99% of the spots were coincident in each pair (baseline and reassessment) of three replicates, reflecting very low levels of nonspecific spot volume in the present study.

CONCLUSION The results of the present study suggest an beneficial effect of ADA therapy on periodontal condition in patients with RA, which might be related to differences in serum protein profiles before and after treatment with ADA. ACKNOWLEDGMENTS This work was supported by Grant-in Aid for Scientific Research A (No. 25253104) and C (No. 22592309) and Grant-in Aid for Challenging Exploratory Research (No. 24659922) from the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan. Dr. Oofusa is a division manager and Dr. Yamagata is a chief researcher at ProPhoenix Division, Towa Environment Science Co., Ltd. Osaka, Japan. The authors declare no conflict of interest related to this study.

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9. Graves DT, Cochran D. The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J Periodontol 2003;74:391-401. 10. Assuma R, Oates T, Cochran D, Amar S, Graves DT. IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J Immunol 1998;160: 403-409. 11. Delima AJ, Oates T, Assuma R, et al. Soluble antagonists to interleukin-1 (IL-1) and tumor necrosis factor (TNF) inhibits loss of tissue attachment in experimental periodontitis. J Clin Periodontol 2001;28:233-240. 12. Williams RO, Feldmann M, Maini RN. Cartilage destruction and bone erosion in arthritis: the role of tumor necrosis factor α. Ann Rheum Dis 2000;59(Suppl I):i75-i80. 13. Pers J-O, Saraux A, Pierre R, Youinou P. Anti-TNF-α immunotherapy is associated with increased gingival inflammation without clinical attachment loss in subjects with rheumatoid arthritis. J Periodontol 2008;79:16451651. 14. Mayer Y, Balbir-Gurman A, Machtei EE. Anti-tumor necrosis factor-alpha therapy and periodontal parameters in patients with rheumatoid arthritis. J Periodontol 2009;80:1414-1420. 15. Ortiz P, Bissada NF, Palomo L, et al. Periodontal therapy reduces the severity of active rheumatoid arthritis in patients treated with or without tumor necrosis factor inhibitors. J Periodontol 2009;80:535-540. 16. Mayer Y, Elimelech R, Balbir-Gurman A, Braun-Moscovici Y, Machtei EE. Periodontal condition of patients with autoimmune diseases and the effect of anti-tumor necrosis factor-α therapy. J Periodontol 2013;84:136-142. 17. Üstün K, Erciyas K, Kısacık B, et al. Host modulation in rheumatoid arthritis patients with TNF blockers significantly decreases biochemical parameters in periodontitis. Inflammation 2013;36:1171-1177. 18. Greenberg JD, Reed G, Decktor D, et al. A comparative effectiveness study of adalimumab, etanercept and infliximab in biologically naive and switched rheumatoid arthritis patients: results from the US CORRONA registry. Ann Rheum Dis 2012;71:1134-1142. 19. Mitoma H, Horiuchi T, Tsukamoto H, et al. Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor α-expressing cells. comparison among infliximab, etanercept, and adalimumab. Arthritis Rheum 2008;58:1248-1257. 20. Sekigawa I, Yanagida M, Iwabuchi K, et al. Protein biomarker analysis by mass spectrometry in patients with rheumatoid arthritis receiving anti-tumor necrosis factor-α antibody therapy. Clin Exp Rheumatol 2008;26:261-267. 21. Yanagida M, Jung G, Tanaka Y, et al. Serum proteome analysis in patients with rheumatoid arthritis receiving therapy with etanercept, a chimeric tumor necrosis factor-alpha receptor. Int J Rheum Dis 2012;15:486-495. 22. Arnett FC, Edworthy SM, Bloch DA, et al. The American rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-324. 23. Aletaha D, Neogi T, Silman AJ, et al. 2010 Rheumatoid arthritis classification criteria. An American college of rheumatology/European league against rheumatism collaborative initiative. Arthritis Rheum 2010;62:2569-2581. 24. Löe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity; Acta Odontol Scand 1963;21:533-551. 25. Inoue E, Yamanaka H, Hara M, Tomatsu T, Kamatani N. Comparison of disease activity score (DAS) 28-erythrocyte sedimentation rate and DAS28-C-reactive protein threshold values. Ann Rheum Dis 2007;66:407-409. 26. Yokoyama T, Kobayashi T, Ito S, et al. Comparative analysis of serum proteins in relation to rheumatoid arthritis and chronic periodontitis. J Periodontol 2014;85:103-112. 27. Yamagata A, Kristensen DB, Takeda Y, et al. Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2002;2: 1267-1276. 28. Gerashchenko BI, Yamagata A, Oofusa K, Yoshizato K, de Toledo SM, Howell RW. Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation. Proteomics 2007;7:20002008. 29. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20: 601-605. 30. Kobayashi T, Okada M, Ito S, et al. Assessment of interleukin-6 receptor inhibition therapy on periodontal condition in patients with rheumatoud arthritis and chronic periodonttis. J Periodontol 2014;85: 57-67.

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31. Graves DT, Li J, Cochran DL. Inflammation and uncoupling as mechanisms of periodontal bone loss. J Dent Res 2011;90:143-153. 32. Debets JM, Ruers TJ, van der Linden MP, van der Linden CJ, Buurman WA. Inhibitory effect of corticosteroids on the secretion of tumor necrosis factor (TNF) by monocytes is dependent on the stimulus inducing THF synthesis. Clin Exp Immunol 1989;78:224-229. 33. Gerards AH, de Lathouder S, de Groot ER, Dijkmans BAC, Aarden LA. Inhibition of cytokine production by methotrexate. Studies in healthy volunteers and patients with rheumatoid arthritis. Rheumatology 2003;42:11891196. 34. Renvert S, Lindahl C, Roos-Jansåker A-M, Lessem J. Short-term effects of an anti-inflammatory treatment on clinical parameters and serum levels of C-reactive protein and proinflammatory cytokines in subjects with periodontitis. J Periodontol 2009;80:892-900. 35. Weinblatt ME, Keystone EC, Furst DE, et al. Adalimumab, a fully human anti-tumor necrosis factor α monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate. the ARMADA trial. Arthritis Rheum 2003;48:35-45. 36. Atzeni F, Sarzi-Puttini P, Acqua DD, et al. Adalimumab clinical efficacy is associated with rheumatoid factor and anti-cyclic citrullinated peptide antibody titer reduction: a one-year prospective study. Arthritis Res Ther 2006;8:R3. 37. Potter C, Hyrich KL, Tracey A, et al. Association of rheumatoid factor and anti-cyclic citrullinated peptide positivity, but not carriage of shared epitope or PTPN22 susceptibility variants, with anti-tumor necrosis factor response in rheumatoid arthritis. Ann Rheum Dis 2009;68:69-74. 38. Herenius MMJ C, Oliveria ASF, Wijbrandts CA, Gerlag DM, Tak PP, Lebre MC. Anti-TNF therapy reduces serum levels of chemerin in rheumatoid arthritis: a new mechanism by which anti-TNF might reduce inflammation. PLoS One 2013;8:e57802. 39. Cho SY, Lee E-Y, Lee JS, et al. Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimentional map. Proteomics 2005;5:3386-3396. 40. Bandow JE. Comparison of protein enrichment strategies for proteome analysis of plasma. Proteomics 2010;10:1416-1425. 41. Connolly M, Mullan RH, McCormick J, et al. Acute-phase serum amyloid A regulates tumor necrosis factor α and matrix turnover and predicts disease progression in patients with inflammatory arthritis before and after biologic therapy. Arthritis Rheum 2012;64:1035-1045. 42. Vuletic S, Taylor BA, Tofler GH, et al. SAA and PLTP activity in plasma of periodontal patients before and after full-mouth tooth extraction. Oral Dis 2008;14:514-519. 43. Olewicz-Gawilik A, Korczowska-Lacka I, Lącki JK, Klama K, Hrycaj P. Fucosylation of serum α1-acid glycoprotein in rheumatoid arthritis patients treated with. Clin Rheumatol 2007;26:1679-1684. 44. Pinho MdeN, Oliveira RDR, Novaes ABJr, Voltarelli JC. Relationship between periodontitis and rheumatoid arthritis and the effect of non-surgical periodontal treatment. Braz Dent J 2009;20:355-364. 45. Kuroda T, Tanabe N, Harada T, et al. Long-term mortality outcome in patients with reactive amyloidosis associated with rheumatoid arthritis. Clin Rheumatol 2006;25:498-505. 46. Markiewski MM, Lambris JD. The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol 2007;171:715-727. 47. Familian A, Voskuyl AE, van Mierlo GJ, et al. Infliximab treatment reduces complement activation in patients with rheumatoid arthritis. Ann Rheum Dis 2005;64:1003-1008. 48. Park MK, Her Y-M, Cho ML, et al. IL-15 promotes osteoclastogenesis via the PLD pathway in rheumatoid arthritis. Immunol Lett 2011;139:42-51. 49. Weinkauf M, Hiddemann W, Dreyling M. Sample pooling in 2-D gel electrophoresis: a new approach to reduce nonspecific expression background. Electrophoresis 2006;27:4555-4558.

Correspondence: Tetsuo Kobayashi, DDS, Ph D. Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. Phone: (+81) 25-227-2870, Fax: (+81) 25-2270808, E-mail: [email protected] 9

Journal of Periodontology; Copyright 2014

DOI: 10.1902/jop.2014.140194

Submitted April 04, 2014; accepted for publication May 02, 2014. Figure 1. Representative protein profiles in sera from patients with RA before and after ADA therapy. The vertical axis represents the molecular weight of proteins (kDa). The horizontal axis represents the range of isometric point (pI) values of proteins between 4 and 7. The circles indicate 9 matched protein spots with significant difference in abundance before and after ADA therapy, as assessed by paired t-test (P < 0.01). Figure 2. Nine protein spots with significant difference in abundance before and after ADA therapy. The regions of the matched spots that are marked with the circles are shown enlarged. These proteins were cut for in-gel digestion and were subjected to mass spectrometric analysis. The protein assignments are presented in Table 4. Table 1. Demographic and disease-related characteristics of 20 patients with RA at baseline of ADA therapy

Characteristics Age (years; mean ± SE) Female (n [%])

51.7 ± 2.2 19 (95.0)

Smoking status Current smoker (n [%])

0 (0.0)

Former smoker (n [%])

3 (15.0)

Never smoker (n [%])

17 (85.0)

Number of teeth present (mean ± SE)

26.6 ± 0.5

Duration of RA (months; mean ± 108.6 ± 24.4 SE) RA medication Corticosteroids (n [%])

13 (65.0)

DMARDs (n [%])

19 (95.0)

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Journal of Periodontology; Copyright 2014

DOI: 10.1902/jop.2014.140194

Table 2. Rheumatologic and serum characteristics of 20 patients with RA at baseline and reassessment of ADA therapy

Characteristics

Baseline

Reassessment

P value

DAS28-CRP (mean ± SE)

3.7 ± 0.2

2.4 ± 0.2

0.0004*

DAS28-CRP category

0.0008*

Remission (n [%])

2 (10.0)

10 (50.0)

Low activity (n [%])

1 (5.0)

2 (10.0)

10 (50.0)

7 (35.0)

7 (35.0)

1 (5.0)

TJC (mean ± SE)

4.7 ± 1.2

1.8 ± 0.4

0.01

SJC (mean ± SE)

3.4 ± 0.5

1.6 ± 0.4

0.02

45.0 ± 3.9

20.8 ± 3.7

0.001*

105.2 ± 23.4

90.7 ± 21.7

0.0005*

Anti-CCP antibody positive (n [%])

16 (80.0)

16 (80.0)

Serum RF levels (IU/ml; mean ± SE)

140.4 ± 39.5

75.4 ± 22.9

0.0002*

19 (95.0)

15 (75.0)

0.04

1.38 ± 0.42

0.33 ± 0.21

0.0003*

212.5 ± 32.0

114.0 ± 27.0

0.007*

Serum IL-6 levels (pg/ml; mean ± SE)

6.3 ± 0.9

1.6 ± 0.4

< 0.0001*

Serum TNF-a levels (pg/ml; mean ± SE)

2.4 ± 0.6

1.6 ± 0.4

0.0003*

Moderate activity (n [%]) High activity (n [%])

VAS (mm; mean ± SE) Serum anti-CCP titer (U/ml; mean ± SE)

RF positive (n [%]) Serum CRP levels (mg/dl; mean ± SE) Serum MMP-3 levels (ng/ml; mean ± SE)

ND

* Significantly different between the baseline and reassessment, as assessed by Wilcoxon signed rank test (P < 0.01). ND = not determined due to the statistical difficulties. Table 3. Periodontal characteristics of 20 patients with RA at baseline and reassessment of ADA therapy

Characteristics GI (mean ± SE) % sites with plaque (mean ± SE) % sites with bleeding on probing (mean ±SE) PD (mm; mean ± SE) % sites with PD < 4 mm (mean ±SE)

Baseline 0.89 ± 0.06 32.7 ± 3.7 10.8 ± 1.8 2.60 ± 0.06

Reassessment 0.83 ± 0.05 29.8 ± 3.0 4.4 ± 0.7 2.51 ± 0.05

P value 0.002* 0.12 0.003* 0.002*

90.2 ± 2.0

93.3 ± 1.6

0.03

9.6 ± 2.0

6.6 ± 1.5

0.04

0.2 ± 0.1 2.62 ± 0.06

0.1 ± 0.1 2.58 ± 0.06

0.59 0.42

90.0 ± 2.1

91.4 ± 1.9

0.27

9.9 ± 2.1

8.5 ± 1.9

0.24

0.3 ± 0.2

0.2 ± 0.1

0.59

% sites with PD 4-6 mm (mean ±SE) % sites with PD > 6 mm (mean ±SE) CAL (mm; mean ± SE) % sites with CAL < 4 mm (mean ±SE) % sites with CAL 4-6 mm (mean ±SE) % sites with CAL > 6 mm (mean ±SE)

* Significantly different between the baseline and reassessment, as assessed by Wilcoxon signed rank test (P < 0.01).

11

Journal of Periodontology; Copyright 2014

DOI: 10.1902/jop.2014.140194

Table 4. Identification of protein spots with a significant difference in abundance before and after ADA therapy . Mean spot volume NCBInr Molecular No. of Spot MOWSE ratio Accession mass peptides Protein name No. After/before number score matched (Da) 4 Complement factor H 0.7 gi|183763 38777 47 2 6 Phospholipase D 0.5 gi|388765 92943 47 2 38 Serum amyloid A 0.3 gi|36310 10161 39 1 40 Serum amyloid A 0.2 gi|36310 10161 52 1 41 Serum amyloid A 0.3 gi|36310 10161 52 1 47 Serum amyloid A 0.1 gi|36310 10161 50 1 53 Serum amyloid A 0.4 gi|36310 10161 52 1 131 Complement component 4 0.7 gi|34782950 23679 101 4 201 Alpha-1-acid glycoprotein 0.4 gi|112877 23725 66 2 NCBInr = NCBI non-redundant; MOWSE = molecular weight search. The proteins correspond to 9 spots with significant differences in abundance before and after ADA therapy by paired t-test (P < 0.01). Probability-based MOWSE scores showed significantly matches, with ions scores > 34 for the spot no. 47, > 35 for the spot nos. 40, 41, and 53, > 36 for the spot no. 201, > 37 for the spot nos. 4 and 6, and > 38 for the spot nos. 38 and 131 (P < 0.01). This work was supported by Grant-in Aid for Scientific Research A (No. 25253104) and C (No. 22592309) and Grant-in Aid for Challenging Exploratory Research (No. 24659922) from the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan. The authors declare no conflict of interest related to this study. ¶

SRL, Tokyo, Japan. Mesacup-2 test CCP, Medical & Biological Laboratories, Nagoya, Japan.

**

Quantikine ELISA, R&D Systems, Minneapolis, MN.

††

Bio-Rad Japan Laboratories, Tokyo, Japan.

‡‡

Multiple Affinity Removal System, Agilent Technologies, Tokyo, Japan.

§§

Bio-Rad, Hercules, CA.

||||

Ultrafree-0.5 Centrifugal Filter Device, Millipore, Billerica, MA.

¶¶

Immobiline DryStrip pH 4-7, GE Healthcare Life Sciences, Chalfont St. Giles, UK. CoolPhoreStar IPG-IEF Type P electrophoresis unit, Anatech, Tokyo, Japan.

*** ††† ‡‡‡ §§§ ||||||

Sypro Ruby, Life Technologies Japan, Tokyo, Japan. Molecular Imager FX Systems, Bio-Rad Japan Laboratories, Tokyo, Japan. ImageMaster 2D Platinum, GE Healthcare Japan Cooperation, Tokyo, Japan. Promega, Madison, WI.

Zip-Tip, Millipore, Bedford, MA.

¶¶¶

Ultraflex TOF/TOF, Bruker Daltonics, Billerica, MA. Mascot, Matrix Science, London, UK.

****

IBM SPSS SamplePower version 3.0, SPSS Inc., an IMB Company, Chicago, IL.

12

Journal of Periodontology; Copyright 2014

DOI: 10.1902/jop.2014.140194

13

Journal of Periodontology; Copyright 2014

DOI: 10.1902/jop.2014.140194

14

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the ...
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