Patients and samples
CD patients undergoing intestinal resection for a chronically active disease poorly responsive to medical treatment were prospectively enrolled. Four specimens were taken from inflamed, but not ulcerated, ileal mucosa of these patients. Surgical samples were collected in completed medium 30-60 minutes after the intestinal resection and immediately processed. After surgery, all the patients underwent ileocolonoscopy at 6 or 12 months depending on the clinical activity or presence of risk factors for severe disease (i.e. smoking habit, young age onset, rectal disease). Endoscopic recurrence was graded according to the Rutgeerts’s score (0: no lesions; 1: less than 5 aphthous lesions; 2: more than 5 aphthous lesions with normal mucosa between the lesions, or skip areas of larger lesions, or lesions confined to the ileocolonic anastomotic lining; 3: diffuse aphthous ileitis with diffusely inflamed mucosa; and 4: diffuse ileal inflammation with larger ulcers, nodules, or narrowing. Hyperaemia and oedema alone were not considered as signs of recurrence). (6) During ileocolonoscopy, 4 adjacent ileal biopsy samples were systematically taken about 10 cm above the anastomosis from the most inflamed, but not ulcerated, ileal mucosa or from macroscopically unaffected mucosa. Four biopsies were also taken in the terminal ileum, 10-25 cm above the ileo-cecal valve, of normal controls (CTR) who underwent ileocolonoscopy for irritable bowel syndrome: no endoscopic lesion was found in these patients and the ileal mucosa was histologically normal.
Each patient who took part in the study gave informed consent and the study was approved by the local Ethics Committee.
Immunohistochemistry
All reagents were from Sigma-Aldrich (Milan, Italy) unless specified. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of CD patients and CTR. The sections were deparaffinized and dehydrated through xylene and ethanol and the antigen retrieval was performed in citrate buffer (pH 6.0) for 20 minutes in microwave. Immunohistochemical staining was performed using a rabbit anti-SMAD7 (orb11386, Biorbyt Ltd), at room temperature for 1 hour. Immunoreactive cells were visualized using MACH4 Universal HRP-Polymer kit (Bio- care Medical, Concord, CA, USA) with 3,3’-Diaminobenzidine (DAB) (Dako North America, Carpinteria, CA, USA) as a chromogen system, according to the manufacturer’s instructions, and lightly counterstained with hematoxylin. Isotype control IgG-stained sections were prepared under identical immunohistochemical conditions as described above, replacing the primary antibody with a purified rabbit normal IgG control antibody (R&D Systems, Minneapolis, MN, USA). Both Smad7-positive epithelial cells and Smad7-positive lamina propria cells were identified by high power magnification of the immunohistochemical images and the positive cells were manually counted in 5 high power fields from each slide. Sections were analyzed by LEICA DMI4000 B microscope expressed as number of cells for high power field (hpf).
Lamina propria mononuclear cell isolation
Lamina propria mononuclear cells (LPMC) were isolated from ileal biopsy samples and intestinal resection specimens of CD patients and CTR as described elsewhere. (21) LPMC were suspended in RPMI 1640 medium, supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (P) (100 U/ml), and streptomycin (S) (100 µg/ml) (Life Technologies-GibcoCRL, Milan, Italy) at concentration of 1 million per ml and used to assess cytokine expression by flow cytometry.
Flow-cytometry analysis
LPMC were seeded in 96-well U-bottom culture dishes and stimulated with phorbol myristate acetate (PMA) (10 ng/mL), ionomycin (1 µg/mL), and brefeldinA (10 µg/mL;eBioscience, San Diego, CA). After 5h, cells were stained with anti-CD3-PerCP (1:50, final dilution, BD Biosciences, San Jose, CA) and fixed with 1% formaldehyde for 20’. Subsequently cells were permeabilized with 0.5% saponin in 1% BSA FACS buffer and stained with the following Abs: anti-interferon (IFN)-g-PE (1:50, final dilution; BD Biosciences), anti–interleukin (IL)-17A–APC (1:50, final dilution, eBioscience). Appropriate isotype-matched controls from BD Biosciences were included in all of the experiments. Cells were analysed using a FACS Calibur cytometer and Cell-QuestPro software.
Statistical analysis.
Since this was a pilot study, no calculation of the simple size was made. Statistical differences were assessed with the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA). Nonparametric data were analyzed using the Mann–Whitney U‐test for comparison between two groups or Kruskal-Wallis test for multiple comparison. Significance of correlation was determined using the Spearman non-parametric correlation.
A p value of less than 0.05 was considered statistically significant