IL-1β, IL-6 and IL-17 were studied in supernatants from biopsy cultures. To evaluate the effects of IL-17 on epithelium, the expression of the apoptotic markers BAX and selleck bcl-2 was studied in IL-17 stimulated CaCo-2 cells.

In this study we collected distal duodenal biopsy samples taken with the capsule method from patients in Finland and Sweden for different immunological analyses (see Table 1). The intestinal biopsy specimens were cut into 7-µm sections and stored at −80°C prior to immunohistochemical staining. The lamina propria lymphocytes on frozen sections were stained with the avidin–biotin immunoperoxidase system according to Vectastain ABC Elite kit instructions (Vector Laboratories, Burlingame, CA, USA). After acetone fixation the slides were blocked in normal serum for 30 min. The slides were incubated for 1 h with the following primary antibodies: mouse monoclonal antibodies for FoxP3 (clone 236 A/E7; Abcam, Cambridge, UK), click here CD4 (clone RPA-T4; BD Pharmingen, San Jose,

CA, USA) or rabbit polyclonal antibody for IL-17 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Methanol–hydrogen peroxidase was used to quench endogenous peroxidase activity. The slides were incubated with a biotinylated antibody for 30 min and thereafter in avidin–biotin complex (ABC) reagent for 30 min; 9-amino-ethyl-cardatzole was used as a chromogen. Harris haematoxylin was used to counterstain the slides. Between

the staining protocol steps the slides were washed in phosphate-buffered saline (PBS). Negative controls were performed by omission of the primary antibodies. The slides were evaluated blinded to the clinical data. The number of positively stained cells in the lamina propria was counted systematically under a Leica DM4000B light eltoprazine microscope through a calibrated eyepiece graticule: positive cells in approximately 30 fields were counted using an objective of × 100 and an eyepiece at × 10. The cell densities were expressed as the mean number of positive cells/mm2 which were used in the statistical analysis. Remaining biopsies from Finnish subjects were dissected from matrix of optimal cutting temperature (OCT) compound (Miles Laboratories, Elkhart, IN, USA) and homogenized using a pestle (Starlab, Ahrensburg, Germany) in a 0·5 ml Eppendorf tube containing 250 µl of lysis buffer (Sigma, St Louis, MO, USA). Total RNA was isolated with a GenElute mammalian total RNA miniprep kit (Sigma). RNA concentration and purity was measured by a spectrophotometer (ND-1000; NanoDrop Technologies Inc., Wilmington, DE, USA). Reverse transcription was performed with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) with additional treatment of 200 ng of total RNA with DNAse I (0·01 U/ul) (Roche Diagnostics, Mannheim, Germany) to eliminate genomic DNA.