Cell Immunity Response to Vaccination Against Influenza in Patients With Rheumatoid Arthritis

Sixty patients with RA and 30 healthy , aged matched controls will participate in the study.

After signing informed consent, all subjects will be vaccinated with the inactivated split virion vaccine which will be recommended by the WHO next fall.

Patients will be evaluated at weel 0 and 6 weeks later. Clinical evaluation will be based on the Disease Activity Score 28 (DAS 28) which includes number of swollen and tender joints, visual global evaluation of the physician , ESR and CRP Blood with be collected on the day of vaccination and 6 weeks later.

Preparation of peripheral blood mononuclear cells Blood samples will be obtained prior to and 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) will be isolated by centrifugation of heparinized blood on a Lymphoprep gradient (Nycomed, Oslo, Norway). Cells will be washed twice in tissue culture medium RPMI (RPMI1640-Hepes with Glutamax-I, GIBCO BRL, NY, USA) and resuspended to a concentration of 2 106/ml in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (FBS) (GIBCO BRL, NY, USA).

ELISPOT assay The 96-well PVDF (polyvinylidene difluoride, Millipore Corp., MA, USA) plates will be prewet with 70% EtOH, then coated with 100 l/well of anti-human IFN- monoclonal antibody (mAb) 1-D1K , diluted to 15 g/ml in sterile filtered phosphate-buffered saline (PBS) . The plates will be kept overnight at 4°C. A total of 100 000 PBMC plus 5 l/well (1 g/ml) peptides will be added to each well in duplicate for each condition and incubated for 24 h at 37°C in 5% CO2. After incubation the plates will be washed with PBS then 100 l of biotinylated mAb (7-B6-1-biotin, Mabtech AB), diluted in filtered PBS with 0.5% FBS to the concentration 1 g/ml were added to each well. The plates will be incubated 2 h at room temperature, washed with PBS and then incubated with 100 l/well of streptavidine-alkaline phosphatase (Mabtech AB) diluted 1-1000 in PBS with 0.5% FBS for 1 h at room temperature. After washing, cells will be developed by adding 100 l/well of colorimetric substrate (BCIP/NBT-plus, Mabtech AB) and then counted in an ELISPOT reader. Each spot was produced by a single IFN- secreting T-cell-spot-forming cell (SFC). All tests were performed in duplicate and the mean values were calculated. A response will be considered to be positive when the number of spots in the wells with peptide-stimulated cells, after subtraction of the background (wells without peptide stimulation), will be at least two-fold greater than the number of background spots14 and more than 20/106 SFC.