Our study is aimed at analysing and comparing distinctive intracellular cytokines in patients with autoimmune thyroiditis associated or not with selected non-endocrine autoimmune diseases. A total GDC-0449 in vivo of 78 Caucasian patients agreed to participate in this study. The inclusion criteria were a definite diagnosis of HT associated or not with the most representative non-endocrine autoimmune diseases (chronic atrophic gastritis, CD, generalized vitiligo and Sjögren’s syndrome). Exclusion criteria
were: (a) the presence of anti-thyrotrophin (TSH)-receptor antibodies or ultrasonographic evidence of thyroid atrophy; (b) clinical history of hyperthyroidism; (c) evidence of infectious diseases in the last 3 months; (d) treatment with drugs known to interfere with the immune system, namely cytokines, interferon, corticosteroids, non-steroidal anti-inflammatory
drugs (NSAIDs), amiodarone, lithium; (e) pregnancy and lactation over the previous 6 months; and (f) presence of acute or chronic systemic diseases other than those included above. Ten patients were subsequently excluded because they took drugs for concomitant diseases, became pregnant or because they had simultaneous infectious diseases. Of the remaining 68 (55 female, 13 male; mean age = 40 ± 16 years), 33 met the criteria for isolated chronic lymphocytic thyroiditis (28 females, five males; mean age = 34 ± 13 years). The remaining 35 patients (27 females, eight males; mean age = 47 ± 16 years), besides chronic lymphocytic thyroiditis, also had chronic atrophic gastritis (n = 18; seven patients also with pernicious anaemia), AZD2014 purchase CD (n = 7), generalized vitiligo (n = 6) and Sjögren’s syndrome (n = 4). The study was conducted with written informed consent and as part of the diagnostic work-up of the patients involved, according to the local ethical rules and the guidelines in the
Declaration of Helsinki. RPMI-1640 supplemented with 25 mm Hepes buffer, 2 mm glutamine, 100 U/ml Sclareol penicillin, heat-inactivated fetal calf serum (FCS) and phosphate-buffered saline (PBS) Dulbecco’s medium without calcium and magnesium and sodium bicarbonate were purchased from Gibco (Grand Island, NY, USA). Fycoll Hypaque (Lymphoprep) density 1·077 ± 0·001 g/ml, osmolality 280 ± 15 mOsm, was from Axis-Shield (Oslo, Norway). Phorbol-12-myristate-13-acetate (PMA), ionomycin, monensin and digitonin were purchased from Sigma (St Louis, MO, USA). Paraformaldehyde (PFA) was from Merck (Darmstadt, Germany). Monoclonal antibodies (anti-CD4, anti-CD8, anti-CD2, anti-IL-2, anti-IL-4, anti-IFN-γ fluorescein isothiocyanate (FITC)-conjugate and anti-CD8 phycoerythrin (PE)-conjugate) and isotype-matched antibodies were purchased from IL-Coulter (Hialeah, FL, USA). Blood samples were sampled from all patients at the same time of day and processed immediately.