Mucosal biopsy samples of ICD patients and normal controls were placed on transwell (transwell permeable support, Costar, Corning incorporated, New York, USA) in 24-well plates containing RPMI medium supplemented with 1% P/S and 50 μg/ml gentamycin in the absence or presence of PT (1 mg/ml). In addition, mucosal biopsy samples of ICD patients were placed on transwell in 24-well plates containing RPMI medium supplemented with 1% P/S and 50 μg/ml gentamycin in the presence of IFN-γ or IL-21 (50 ng/ml final concentration, Peprotech, London, UK). In parallel, samples were cultured with or without cytokines in the presence or absence of the JAK/STAT inhibitor AG-490 (100 μM final concentration, Inalco, Milan, Italy). Mucosal biopsy samples of ACD patients were cultured as above in the presence of antibodies neutralizing IFN-γ or IL-21 (both used at 10 μg/ml final concentration, R&D Systems, Minneapolis, MN, USA). In further experiments, mucosal biopsy samples of ACD patients were cultured in the presence or absence of rapamycin (50 or 100 ng/ml final concentration).
The cultures were performed in an organ culture chamber at 37 °C in a 5% CO2/95% O2 atmosphere and after 24 h the samples were used for protein extraction.
The cultures were performed in an organ culture chamber at 37 °C in a 5% CO2/95% O2 atmosphere and after 24 h the samples were used for protein extraction.