Patients
Twenty-eight patients with CD were enrolled. These patients were treated with UST at the Shiga University of Medical Science Hospital. The demographic characteristics of the patients are described in Table 1. Clinical disease activity was evaluated using the Crohn’s disease activity index (CDAI) score [23].
UST was introduced by a one-time intravenous infusion according to the patient’s body weight (260mg for patients <55kg, 390mg for patients between 55kg and 85kg, and 520mg for patients >85kg). The patients then received a UST subcutaneous injection (90mg/body) every 8 weeks. Blood was collected before the next injection (trough concentration). There was an average of 6.5 UST injections at the time of endoscopy.
Ethics
The study protocol was approved by the institutional review boards of the Shiga University of Medical Science (permission No. R2017-136). All patients gave their written informed consent prior to their inclusion in this study. The registration number of the University Hospital Medical Information Network Center (UMIN) was 000033552.
Endoscopic examination
Trans-anal approach using the single-balloon enteroscope Olympus SIF-Q260 (Olympus, Tokyo Japan) was performed in all patients except those with a stoma. For a patient with a stoma, the colonoscope Olympus PCF-Q260 (Olympus) was used.
The trans-oral approach was applied if jejunal lesions were suspected by other diagnostic modalities, i.e., small bowel follow-through, computed tomography, and/or magnetic resonance enteroclysis.
Evaluation of small bowel lesions
Endoscopic activity of CD was assessed using the modified Rutgeerts scoring system as described in our previous report [24] (Table 2)_ENREF_25. The original Rutgeerts score [25] was developed for evaluation of anastomosis lesions after ileocolic resection, but we adapted the scoring system for entire endoscopic lesions. The score of the most serious lesion was adopted. Endoscopic remission was defined as a score of 0 (no lesions or scar) or 1 (≤ 5 aphthous lesions). At least two well-trained endoscopists calculated the disease scores in patients.
Measurement of serum UST concentrations
Serum UST levels were determined by an immunoassay developed in our laboratory [26]. Briefly, an avidin ELISA plate® (blocking-less type; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) was coated with biotinylated-IL-12 p40 (100 ml of 0.5 μg/mL) by incubation for 2 h. After extensive washing, a further blocking was performed with Block Ace® (DS Pharma Biomedical, Co., Ltd., Suita, Japan). After washing, samples (100 μL of 100-fold diluted serum) were incubated overnight at 4 °C. Finally, the reacted UST was detected by horseradish peroxidase -labeled F(ab')2 fragments of chicken anti-human IgG (x 20,000 diluted; Thermo Fisher Scientific Co., Ltd., Waltham, MA). 3,3’,5,5’-Tetramethylbenzidine (Nacalai Tesque, Kyoto, Japan) was used for color development.
Measurement of serum AUA concentrations
Serum levels of anti-UST antibodies (AUAs) were measured using a drug-tolerant assay developed in our laboratory [26].
Statistical Analyses
The Chi-square or Mann-Whitney U test was used to evaluate the difference between two independent groups. The Spearman’s rank correlation coefficient was used to evaluate associations between parameters. The cut-off values of UST concentration associated with normal C-reactive protein (CRP), serum albumin and endoscopic remission were determined using receiver operating characteristic (ROC) curve analysis. All statistical testing was performed at the 0.05 significance level.