Laboratory procedures
Serum was investigated for C-reactive protein (CRP) concentration by enzyme-linked immunosorbent assay (ELISA) (DuoSet; R&D Systems, Abingdon, UK). IgM-RF (in international units per milliliter) was measured by using an in-house validated ELISA (cutoff point for positivity: 25 U/mL) [
37]. Total IgG anti-cyclic citrullinated protein (anti-CCP) antibodies (ACPAs) (in units per milliliter) were determined by using the Phadia analyzer (Phadia Laboratory Systems, Phadia AB, Uppsala, Sweden) with an upper detection limit of 340 U/mL (cutoff point for positivity: 10 U/mL). Antibody levels to five synthetic native peptides representing the best-established auto-antigens in RA [
38] (that is, enolase-1 fibrinogen-1, fibrinogen-2, fibrinogen-3, and vimentin-1 and their citrullinated forms) were measured by using the same ELISA as described by van de Stadt and colleagues [
39].
IgA, IgG, and IgM antibodies to P. gingivalis were determined by using an in-house developed ELISA, in which a pooled lysate of four randomly selected clinical isolates of P. gingivalis from patients with periodontitis was used as an antigen. These monocultures were suspended in ice-cold PBS with protease inhibitors (Complete Mini EDTA-free Protease Inhibitor Cocktail Tablets; Roche Diagnostics, Basel, Switzerland). After centrifuging and discarding of the supernatant, pellets were resuspended in an ice-cold lysis buffer containing non-denaturing detergent (Noninet P-40; Sigma-Aldrich, St. Louis, MO, USA) and sonicated for 15 minutes (Bioruptor Standard sonication device; Diagenode s.a., Liège, Belgium). Protein concentration was determined by using the BCA Protein Assay Kit (Pierce Protein Biology Products, Thermo Fisher Scientific, Rockford, IL, USA). Microtiter plates (Costar 96-Well; Corning, Amsterdam, The Netherlands) were coated overnight with 1 μg/mL of antigen. Standard curves were made from protein standard (N protein-standard SL; Siemens Healthcare Diagnostics, Den Haag, The Netherlands) diluted twofold (highest standard curve values were for 300, 25, and 250 ng/mL for IgA, IgG, and IgM, respectively). For the standard curves, the following capture antibodies were used: monoclonal anti-human IgA (1:4,000, clone GA-112; Sigma-Aldrich, Zwijndrecht, The Netherlands), monoclonal anti-human IgM (1:10,000, clone MB-11, μ-chain-specific; Sigma-Aldrich), and goat anti-human IgG (1:5,000, F(ab')2 fragment-specific; Jackson ImmunoResearch Europe Ltd., Newmarket, UK), respectively. Serum samples were incubated in fourfold serial dilutions (1:100, 1:400, 1:1,600, and 1:6,400). Detection of anti-P. gingivalis antibodies was carried out with horseradish peroxidase-labeled goat anti-human IgA, mouse anti-human IgG (Fc fragment-specific, clone JDC-10), and mouse anti-human IgM (μ-chain-specific, clone SA-DA4; all from SouthernBiotech, Birmingham, AL, USA) followed by color reaction with tetramythylbenzidin and hydrogen peroxide. Absorbance was read at 450 nm in an EMax microplate reader and corrected for background binding, and concentration of antibodies was calculated by SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, USA) according to the IgA, IgG, or IgM standard curves included on each ELISA plate and expressed in nanograms per milliliter. As an internal control, two serum samples with a repeatedly high and a low titer were tested at each plate. The variation coefficients were 22% for IgA, 20% for IgG, and 25% for IgM anti-P. gingivalis. Interference of IgM-RF was investigated by spiking samples with sera with known high titers of RF. Adding RF had no measurable effect on anti-P. gingivalis titers.
In paired samples of serum and GCF of patients with RA, the presence of IgG-ACPAs was assessed by using the anti-CCP2 ELISA (Euro Diagnostica, Nijmegen, The Netherlands). Because the HLA-DRB1 shared epitope (SE) is a known genetic risk factor for RA and a possible genetic risk factor for periodontitis [
40], HLA-DRB1-SE-containing alleles were genotyped from genomic DNA in the patients with RA. The presence of an RA SE was analyzed by a polymerase chain reaction-based sequence-specific oligonucleotide probe hybridization (SSOP) approach by using a commercial kit (Hologic Gen-Probe Incorporated, San Diego, CA, USA) and Luminex xMAP technology (Luminex Corporation, Austin, TX, USA) in accordance with the instructions of the manufacturer.
Microbiology
Microbiological samples were processed by using standard anaerobic culture techniques [
41,
42]. Paper point samples were vortexed for 2 minutes, and appropriate 10-fold serial dilutions (100 μL) in PBS were plated on blood agar plates (Oxoid no. 2; Oxoid Limited, Basingstoke, UK), which were supplemented with horse blood (5% vol/vol), hemin (5 mg/L), and menadione (1 mg/L) and incubated in 80% N
2, 10% H
2, and 10% CO
2 at 37°C for up to 14 days. Besides the presence and proportions of
P. gingivalis, those of other established periodontal pathogens, including
Prevotella intermedia,
Fusobacterium nucleatum,
Parvimonas micra,
Tannerella forsythia, and
Campylobacter rectus, were assessed [
43]. Identification was based on microscopic morphology, Gram reaction, and the production of a set of metabolic enzymes (API/ID 32; BioMérieux, La Balme Les Grottes, France). Additional tests for identification included detection of a trypsin-like activity based on the degradation of benzoyl-DL-arginine-2-naphthylamide (Sigma-Aldrich) [
44]. Finally, the total number of colony-forming units per sample was determined.