*4.2. Study Protocol*

All patients were treated weekly for 6 months with 15 mg MTX (orally) plus 5 mg folic acid. The MTX dose was adjusted by increments of 5 to 25–30 mg weekly until the disease response criteria were met. Patients were advised to take 5 mg of Prednisone (orally) daily and a non-teroidal anti-inflammatory drug at fixed doses during the study. All patients were monitored monthly for clinical and analytical tolerance to MTX treatment and at 3 and 6 months to assess the clinical response and to undertake immunological studies. Disease activity was determined by the DAS28 score according to EULAR criteria and using a validated Spanish version of the Health Assessment Questionnaire (HAQ) [45]. The clinical response of the patients to MTX was defined according to EULAR criteria for RA [46], classifying patients as responders or nonresponders. The responder group included those patients with an actual DAS28 < 3.2 score, plus a DAS28 score decrease by at least 1.2 with respect to the initial value, after 6 months of MTX treatment.

Three peripheral blood samples were obtained from each patient by antecubital venipuncture at baseline (before starting MTX treatment) and at 3 and 6 months after starting MTX treatment.
