Serum Antibodies to Oral Anaerobic Bacteria in Patients With Rheumatoid Arthritis

Mesut Ogrendik, MD; Siranus Kokino, MD; Ferda Ozdemir, MD; Philip S. Bird, BSc, MSc, PhD; Stephen Hamlet, BSc, MSc

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In This Article

Materials and Methods

This study was conducted from August 2001 to August 2002 in Turkey and Australia. The study was conducted in accordance with the principles of Good Clinical Practice, according to the Declaration of Helsinki. Before this study, all patients gave written informed consent.

Thirty patients (5 men, 25 women) who fulfilled the American College of Rheumatology criteria for RA were included.[10] The mean age of RA patients was 49 years with a range of 19-69. The mean disease duration was 4.9 ± 1.3 years. Patients were ineligible to participate in the study if they met any of the following exclusion criteria: Sjögren's syndrome, other infectious disease, metabolic disease, periodontal disease, gingivitis. treatment with antibiotics, and using tobacco.

Rheumatoid factor (RF) was measured by agglutination assay (latex) test in the 30 patients with RA. For each patient, the disease activity score (DAS28) was also calculated from the number of tender and swollen joints (both by 28-joint-count), erythrocyte sedimentation rate (ESR), and patient's general health assessment by visual analog scale.

The presence of extra-articular manifestations, such as Sjögren's syndrome, rheumatoid nodules, rheumatoid vasculitis, pleuritis, nephropathy, anemia, Raynaud's syndrome, or Felty's syndrome, was recorded and vasculitis was diagnosed when one of the following symptoms was present: polyneuropathy/mononeuritis multiplex, cutaneous vasculitis, digital gangrene, and visceral infarction, not attributable to any disease.

The control group consisted of blood serum samples obtained from 20 (5 men, 15 women) healthy donors. The mean age of healthy donors was 47 years with a range of 23-69. There was no clinical evidence of periodontitis and gingivitis in any of the controls. The serum samples were sent to the University of Queensland School of Dentistry (Oral Biology and Pathology Laboratory) in Brisbane, Australia, for the bacteria antibody determination and quantification.

Bacteria were revived from liquid nitrogen stocks and P gingivalis , Actinobacillus actinomycetemcomitans , and P intermedia were grown on a Trypticase Soy Agar (TSA). This agar was prepared from at Trypticase soy broth base (30 g/L; BBL, Becton Dickinson, Cockeyville, Maryland) with the addition of agar (10 g/L), yeast extract (5 g/L), L-cysteine hydrochloride (.5 g/L), sodium formate (2 g/L), sodium fumerate (3 g/L), menadione (1 mg/L), haemin (5 mg/L), L-cysteine HCl (.5 g/L), and 5% defibrinated horse blood. P melaninogenica was grown on Wilken Chalgrens sheep blood agar plates. Tannerella forsythensis was grown on TSA with a disk containing 300 mcg of N -acetylmuramic acid (NAM) and co-cultured with streak S aureus . Plates were incubated at 37°C in atmosphere of 80% N2, 10% CO2, and 10% H2 in anaerobic jars for 4-5 days. Purity was monitored by Gram stain, colonial morphology on blood agar plates, and identification confirmed using API-ZYM. The strains were expanded in 550-mL batch cultures of brain heart infusion broth (Difco Laboratories, Detroit, Michigan), supplemented with yeast extract (5 g/L), haemin (5 mg/L), and menadione (1 mg/L), L-cysteine HCl (1 g/L), and for the cultivation of B forsythus , NAM (15 mg/L) was added to the broth medium. Bacteria were incubated at 37°C in an atmosphere 80% N2, 10% CO2, 10% H2 for 48-72 hours. P melaninogenica was harvested from Wilken Chalgrens sheep blood agar plates. Purity was assessed by Gram stain and colonial morphology on agar plates. Bacteria were harvested at late log phase by centrifugation (10,000 x g for 20 minutes) and washed twice in .15 M sodium chloride. Bacteria were revived from liquid nitrogen stocks and grown on a TSA su . B forsythus was prepared from a trypticase soy broth base (30 g/L; BBL, Becton Dickinson, Cockeyville) with the addition of agar (10 g/L), yeast extract (5 g/L), L-cysteine hydrochloride (0.5 g/L), menadione (1 mg/L), haemin (5 mg/L), and 5% defibrinated horse blood. B forsythus was grown on TSA with a disk containing 300 mcg of NAM and co-cultured with streak of S aureus . Plates were incubated for 7-10 days at 37°C in an atmosphere of 80% N2, 10% CO2, and 10% H2 in anaerobic jars. Purity was monitored by Gram stain, colonial morphology on blood agar plates, and identification confirmed using API-ZYM. The bacteria was expanded in a 500-mL batch culture of brain heart infusion broth (Difco Laboratories, Detroit), supplemented with yeast extract (5 g/L), haemin (5 mg/L), menadione (1 mg/L), and L-cysteine HCL (1 g/L), and NAM (15 mg/L). Broth cultures were incubated at 37°C in an atmosphere of 80% N2, 10% CO2, and 10% H2 for 48-72 hours. Purity was assessed by Gram stain and colonial morphology on TSA. Bacteria were harvested at late log phase by centrifugation (10,000 x g for min) and washed twice in .15 M sodium chloride.

Pathogen-specific immunoglobulin (Ig)G was quantitated with an ELISA. Microtiter plate wells (Nunc, Maxisorp) were coated with 100-mcL aliquots of bacteria in .1 M carbonate buffer (pH 9.6) at a concentration of 1 mcg/mL. To allow quantification of antibody titer, 100-mcL aliquots of known concentrations of IgG prepared from purified human IgG (Zymed) was also transferred to the plate. Negative control wells for each serum sample were prepared by adding carbonate buffer only to the appropriate wells. After overnight incubation at 4°C, the plates were washed (x 3) in PBS-Tween and blocked with 1% BSA for 1 hour at room temperature. One hundred-microliter aliquots of diluted serum were then added to the appropriate wells, and the plates incubated at room temperature for 2 hours. After a further wash (x 3) in PBS-T, plates were incubated for 1 hour at room temperature with diluted rabbit antihuman IgG-specific horseradish peroxidase-labeled monoclonal antibody (Dako). After a further wash (x 3) in PBS-T, color development was achieved by adding 150 mcL of 2.5 mM tolidine (Eastman Kodak, Rochester, New York) in 100 mM phosphate citrate buffer pH 3.5 containing .025 mM ethylenediaminetetraacetic acid and activated with 3% H2O2. The resulting reaction was stopped after 10 minutes by adding 50 mcL of 1 M HCl. Plates were then read in a Bio-Rad microplate reader, model 3550 (Bio-Rad Laboratories, Regents Park NSW, Australia ) at 450 nm and 655 nm.

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