Patients and specimens
From 2004 to 2011, this study included 183 patients who underwent IPAA in our institution. One hundred fifteen patients excluded for the following reasons: unable to obtain the frozen tissue samples of ileal mucosa because of emergency surgery (subtotal colectomy) (n=65), poor quality of samples(n=12) and lost to follow-up(n=38). In total, frozen tissue samples of ileal mucosa were obtained from 68 patients who underwent IPAA in our institution. Samples were obtained from the terminal ileum of resected specimens at time of total colectomy or subtotal colectomy. Patients with obvious backwash ileitis were excluded. Samples were soaked in RNA later and stored at −80°C until RNA extraction. The protocol for this research project has been approved by a suitably constituted Ethics Committee of the institution and it conforms to the provisions of the Declaration of Helsinki. All informed consent was obtained from the subjects and guardians.
Surgical procedure and diagnosis of pouchitis
All patients underwent IPAA and routinely performed endoscopy each year after stoma closure. In addition, patients with suspected pouchitis were assessed by endoscopic assessments. Patients with pouchitis were diagnosed by a modified Pouchitis Disease Activity Index (mPDAI) score ≥519. Patients with CD of pouch were diagnosed with stenosing, fistulising and with no granulomas during follow-up. Patients with CD of the pouch and Secondary pouchitis (pouch ischemia, anastomotic stricture, pelvic sepsis, Cytomegalovirus infection, Clostridium difficile infection, and regular use of nonsteroidal anti-inflammatory drugs) were excluded. The onset of pouchitis was defined as the time from stoma closure to clinical and endoscopic diagnosis at the first episode. Acute antibiotic-responsive pouchitis (ADP) was defined as episodes of symptomatic pouch inflammation occurring fewer than 4 times per year with symptomatic response to a 2-week course of a single antibiotic such as ciprofloxacin. Chronic antibiotic-refractory pouchitis (CARP) included patients with greater than 4 episodes of symptomatic pouch inflammation per year, individuals who require continuous antibiotic therapy to maintain symptom remission, or patients whose symptoms were refractory to antibiotic therapy 20.
RNA extraction and cDNA synthesis
Ileal mucosa was homogenized with a Mixer Mill MM 300 homogenizer (Qiagen, Chatsworth, CA, USA). Total RNA was isolated using a RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Then, purity and concentration of RNA was estimated with Nano-Drop. Pure RNA was obtained with a A260/A280 ratio of 2.0–2.2. Degraded RNA could be detected with different ratio. cDNA was synthesized from 5.0 mg total RNA with random hexamer primers and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
qRT-PCR analysis was performed using the TaqMan universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). We investigated the mRNA expression of 5 cytokines in the terminal ileum focusing on Th2 responses (IL-5, IL-6, IL-13, IL-17A and IL-23), and IFN-γ. The relative abundance of target transcripts was measured using TaqMan probes (Applied Biosystems, Foster City, CA, USA) for IFN-γ (Assay ID: Hs00989291_m1), IL-5 (Assay ID: Hs01548712_g1), IL-13 (Assay ID: Hs00174379_m1), IL-17A (Assay ID: Hs001743883_m1), and IL-23A (Assay ID: Hs00900828_g1). Glyceraldehyde-3-phosphate dehydrogenase: GAPDH (Assay ID, Hs02758991_g1; Applied Biosystems) was measured as an internal housekeeping gene. cDNA was amplified and quantified using Applied Biosystems StepOne Plus Real-Time PCR System and analyzed by Software version 2.2.2 (Applied Biosystems).
Quantification of the relative expression levels of Th cytokines
Relative gene expression was determined using the standard curve method. Standard curves and line equations were generated using five-fold serially diluted solutions of cDNA generated by the reverse transcription of qPCR Human Reference Total RNA (Clontech, Mountain View, CA, USA). All standard curves were linear in the analyzed range with an acceptable correlation coefficient (R2). Target gene expression was calculated from the standard curve followed by the quantitative normalization of cDNA in each sample using GAPDH as an internal control. Assays were performed in duplicate for each sample and the mean value was used for analysis.
Statistical analysis
Results were expressed as median values (interquartile range). The cutoff value of each continuous variable was determined by the median value. Comparisons were performed using non-parametric Wilcoxon signed-rank test for continuous variables. Cumulative incidence of pouchitis was evaluated by the Kaplan-Meier method. Differences between two groups were determined by the log-rank test. Cox proportional hazard regression analysis was used to evaluate the independent influence of factors on the onset of pouchitis. All statistical analyses were carried out using JMP 10 for Windows software (SAS Institute, Cary, NC, USA). Two-sided p-values <0.05 were considered statistically significant.