A total of 70 patients with ulcerative colitis were enrolled in this study. All patients attended the gastroenterology outpatient clinic at the Hospital of the Kyoto Prefectural University of Medicine and were diagnosed with clinical remission. The characteristics of the enrolled patients are shown in Table 1.
Table 1
Patient characteristics and background.
Total number
|
70
|
Sex (female / male)
|
32 / 38
|
Age (years)
|
45.0 ± 16.0
|
Disease duration (month)
|
111.2 ± 104.0
|
Smoking history (%)
|
7 (10.0)
|
Disease location (%)
|
|
|
Extensive
|
54 (77.1)
|
Left-sided
|
11 (15.7)
|
Rectum
|
5 (7.1)
|
Current medication (%)
|
|
5-Aminosalicylates
|
62 (88.6)
|
Prednisolone
|
1 (1.4)
|
Azathioprine
|
13 (18.6)
|
Biologics
|
IFX
|
3 (4.3)
|
ADA
|
5 (7.1)
|
GLM
|
2 (2.9)
|
Diagnostic evaluation
Three endoscopists who were blinded to the patient details of the endoscopic images and clinical data evaluated all endoscopic images. Two were expert endoscopists (experts A and B) who had previously performed >8000 conventional colonoscopies, while the other was a non-expert (non-expert C) who had previously performed <1000 conventional colonoscopies. This analysis was performed based on the previous report [16].
Assessment of disease activity
Clinical disease activity was determined using the Lichtiger Colitis Activity Index (LCAI) [17]. All patients enrolled in this study were in clinical remission, defined as a score of 4 or below on LCAI. The relapse of ulcerative colitis was defined as aggravation of clinical symptoms of ulcerative colitis, and is characterized by an aggravation of endoscopic findings.
Sample collection
All participants underwent total colonoscopy, and endoscopic activity was determined using the Mayo endoscopic subscore (MES). Rectal biopsy was performed to analyze cytokine mRNA expression and histological activity, which is the same diagnostic area as the total colonic mucosal diagnosis evaluated by MES. All specimens were collected during the remission phase. Based on whether the patient later relapsed or not, the samples were grouped into “relapse” and “remission”, respectively.
Histopathological assessment
Inflammation in the biopsy specimen was evaluated according to the Geboes score [18] by an expert pathologist. Biopsies were collected from the same sites that were subjected to MES, and active histological inflammation was defined as a Geboes score ≥ 2B.1.
mRNA analysis
The mRNA expression of human colonic mucosal cytokines was determined by real-time polymerase chain reaction (RT-PCR) using biopsy samples from patients with UC. A total of 70 samples from all patients were analyzed. Total RNA was isolated from human biopsy samples by the acid guanidinium phenol-chloroform method, using an ISOGEN kit (Nippon Gene, Tokyo, Japan). The concentration of RNA was determined by the ratio of absorbance at 260 and 280 nm (A260/280). The isolated RNA was stored at -80°C until RT-PCR was performed. Extracted RNA (1 mg) was reverse-transcribed into cDNA at 42°C for 40 min, using 100 U/mL of reverse transcriptase (Takara Biomedicals, Shiga, Japan) and 0.1 mM of oligo(dT)-adapter primer (Takara Biomedicals) in a 50-μL reaction mixture. Real-time PCR for human and mouse Serpin B1 was carried out with the 7300 Real-time PCR DNA-binding dye SYBR green I for the detection of PCR products. The reaction mixture (RT-PCR kit, Code RRO43A; Takara Biochemicals) contained 12.5 mL Premix Ex Taq, 2.5 mL SYBR green I, custom-synthesized primers, ROX reference dye, and cDNA (equivalent to 20 ng total RNA) in a final reaction volume of 25 mL. The PCR settings were as follows: the initial denaturation for 15 s at 95°C was followed by 40 cycles of amplification for 3 s at 95°C and 31 s at 60°C, with subsequent melting curve analysis increasing the temperature from 60°C to 95°C. A total of 22 cytokine mRNAs (IFNg, IL-12, IL-17A, IL-17F, IL-23) were quantified from biopsy specimens, and the sequences of primers are shown in Supplementary figure 1. GAPDH was used as an internal control. This analysis was performed based on the previous report [19].
Statistical analysis
Continuous data were described as mean ± standard deviation (SD), if normally distributed, or median and interquartile range [IQR] (25%, 75%), if not normally distributed. Kappa values of <0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80, and >0.80 indicate poor, fair, moderate, good, and excellent agreement, respectively. Values of p < 0.05 were considered statistically significant. The analysis of variance (ANOVA) was performed to assess the trend of the mean, stratified according to the normally distributed continuous variables; the trend test was based on liner contrast. If a non-parametric method was required, the Jonckheere–Terpstra trend test was performed. Values of p < 0.05 were considered statistically significant. All analyses were performed with SPSS version 22.0 (IBM Japan, Ltd, Japan). The non-relapse rate was plotted using the Kaplan–Meier method and compared using the log-rank test. Thirty months was defined as the time interval between endoscopic diagnosis and relapse (censored observation). This analysis was performed based on the previous report [16].