In the current study, we performed two independent sample collections. The Study 1 aimed to investigate the infiltration of CD4⁺ and CD8⁺ T cells in the hepatic tissue of BA patients and its prognostic value for early postoperative cholangitis. Based on the findings of the study, we subsequently designed Study 2 to further investigate the subtypes of inflammatory cells and if the autophagy is involved in the hepatic tissue of BA patients. Due to the small size of paraffin specimens used in Study 1 (about 2 cm × 1 cm), some paraffin-embedded tissue blocks were insufficient for the Study 2 after serially sectioned into 5–8 sections in the Study 1. Therefore, some new specimens were supplemented in the Study 2. A total of 31 BA patients receiving surgical treatment at our center from January 2007 to January 2009 were included in Study 2. There were 11 patients overlapping between Study 1 and Study 2. The inclusion criteria were: 1) patients received exploratory laparotomy at our center, and had a confirmed diagnosis of type III BA by intraoperative extrahepatic cholangiography or methylene blue injection. The patients then underwent Kasai operation; 2) intraoperative wedge biopsy specimens were collected from the anterior right lobe of the liver.

Diagnostic criteria of early postoperative cholangitis were within one month after surgery, patients with: 1) high fever or remittent, bile discharge reduced or completely stopped; 2) abdominal distension, vomiting and reduced liver function; 3) worsening jaundice in a short period, elevated levels of serum bilirubin and alanine aminotransferase; 4) pale or clay-colored stools, dark yellow-colored urine; 5) level of white blood cells elevated significantly, especially the neutrophils.

BA Patients were treated with hilar jejunum anastomosis surgical treatment. Postoperative adjuvant treatments for all patients included: 1) fasting and intravenous nutrition support for 3 or 7 days after the Kasai operation; 2) intravenous dexamethasone (0.4 mg/kg) for one week at the 2nd week postoperation; 3) for anti-infective treatment, intravenous or oral third-generation cephalosporins at least 1 month postoperation. For patients with repeated fever, the fourth-generation cephalosporins would be used: 4) intravenous infusion of Yinzhihuang injection (proprietary Chinese medicine) and polyene phosphatidylcholine for liver protection; 5) after discharge, long-term oral administration of ursodeoxycholic acid, Yinzhihuang decoction, glucurolactone to protect gallbladder and liver.

Paraffin-embedded tissue blocks were pre-cooled at −20°C for 30 minutes, and were then serially sectioned in 3 µm–4 µm thick sections, transferred to slides. Sections were incubated at 40°C overnight, then de-paraffinized in dimethylbenzene and rehydrated in graded ethanol solutions. For H & E staining, slides were stained in hematoxylin solution for 5 minutes, and immersed in 0.5% hydrochloric alcohol, 0.5% ammonia and 0.5% eosin 3 minutes, slides were extensively washed between each immersion step. The slides were then dehydrated in graded ethanol solutions and dimethylbenzene, followed by sealed with neutral resin. For immunohistochemical (IHC) staining, slides were treated with 10 mM sodium citrate buffer (pH 6.0) at 96°C for 10 minutes for antigen retrieval. The slides were then incubated with blocking solution (5%–10% goat serum) at room temperature for 20 minutes, and subsequently primary antibodies (20 × dilution) at 37°C for 1 hour and secondary antibodies at 37°C for 30 minutes. After another PBS wash, the peroxidase reaction was developed using a DAB detection kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) according to manufacturer's protocol. The slides were stained with hematoxylin for 2 minutes, differentiated in 0.1% hydrochloric acid alcohol for 5 seconds, treated with bluing in running water for 10 minutes, dehydrated by gradient ethanol treatments. The sections were then sealed with neutral resin. The slides were visualized and photographed using a phase contrast microscope (CX41; Olympus, Tokyo, Japan). Primary antibodies (mouse anti-human) specific for CD4 (Novocastra, London, UK), CD8 (Novocastra), CD68 (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), CD45PRO (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), PCNA (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd)., Beclin1 (Abcam, Cambridge, MA, USA), TGF-β1 (Santa Cruz, Dallas, TX, USA) and secondary antibody goat anti-mouse IgG/HRP (DAKO, Carpenteria, CA, USA) were all used in the IHC analyses.

Each IHC slide was examined by two pathologists (JZ and MS) blinded to the subject's clinical history. Slide of lymph node specimen from children without biliary disease was used as a positive control. A negative control slide was prepared by substituting saline buffer for the primary antibodies. Five representative high power fields were selected for each slide, and the total number of cells should be over 1,000. Semi-quantitative analysis was performed for the results based on the proportion of positive cells in all the cells and the intensity of antibody staining in positive cells.22 The staining intensity was scored as 0 (no staining), 1 (light yellow), 2 (yellow), 3 (brown) or 4 (dark brown). The proportion of positive cells was scored as 0 (< 5%), 1 (5% to 25%), 2 (26% to 50%), 3 (51% to 75%) or 4 (> 75%). The scoring results from two pathologists were averaged, and the average score from staining intensity and proportion of positive cells were summed up as the total score. Total scores were ranked into 4 grades: rank 1 (0–2 points), rank 2 (3–4 points), 3 (5–6 points), 4 (7–8 points).

Continuous data were presented as the mean ± standard deviation (SD) and tested by Mann-Whitney U test. Categorical data were demonstrated by number (N) and percentage in cross-tables and tested by χ² test. Rank data including CD4, CD8, CD68, CD45RO, and Beclin1 were analyzed as both continuous and categorical variables. Univariate and multivariate logistic regression were utilized to investigate the independent variables which were associated with early cholangitis. Significant independent variables would be further analyzed by receiver operating characteristic (ROC) curve to observe their area under ROC curve (AUROC) and potential capacity in diagnosis. A rough guide for assessing the utility of a biomarker based on its AUC is as follows: 0.9–1.0 = excellent; 0.8–0.9 = good; 0.7–0.8 = fair; 0.6–0.7 = poor; 0.5–0.6 = fail.23 All statistical analyses were performed using IBM SPSS Version 20 (SPSS Statistics V20; IBM Corporation, Somers, NY, USA). The significance level was set at P < 0.05, two-tailed.

This study was approved by the Institutional Review Board of the First Affiliated Hospital of Sun Yat-Sen University on January 9, 2009, and written informed consent was obtained from all patients. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

A total of 25 and 31 BA patients receiving Kasai operation were included in Study 1 and Study 2, respectively. In both studies, patients were divided into non-cholangitis and cholangitis groups depending on whether they had early postoperative cholangitis or not. At one month postoperation, there were 14 and 16 patients diagnosed with early postoperative cholangitis according to the clinical manifestations and laboratory tests in the Study 1 and Study 2, respectively. The demographic and clinical baseline characteristics of the patients were summarized in Table 1. There was no significant difference in the demographic and clinical baseline characteristics, including pathological diagnosis, jaundice duration, levels of total bilirubin, direct bilirubin, indirect bilirubin, white blood cell red blood cell, neutrophil and hemoglobin between non-cholangitis and cholangitis groups in both studies (all P > 0.05, Table 1), indicating the two groups are comparable.

Tissue samples from Study 1 were used to investigate the infiltration degree of CD4⁺ and CD8⁺ T cells. IHC analysis showed that varying degrees of infiltration of CD4⁺ (Supplementary Fig. 1A) and CD8⁺ T (Supplementary Fig. 1B) cells were observed in the hepatic tissue of all the BA patients. The infiltrated CD4⁺ and CD8⁺ T cells mainly distributed in the portal area and some cells infiltrated to the liver sinusoid. To compare degrees of infiltration between the two groups, the IHC data was quantitated. As shown in Table 2, there is no significant difference in CD4⁺ T infiltration between non-cholangitis and cholangitis groups (both P > 0.05), while the level or rank of CD8⁺ T infiltration was significantly higher in the non-cholangitis group than in cholangitis group (both P < 0.01). This data suggested that patients with early postoperative cholangitis had a lower infiltration degree of CD8⁺ T cells.

In Study 2, we compared the infiltration degrees of CD68⁺ cells and CD45RO⁺ T cells and the expression levels of Beclin. IHC data showed that CD68⁺ cells mainly distributed among the hepatocytes, especially within the liver sinusoid, and around the central venous, but less in the portal area (Supplementary Fig. 2). In contrast, CD45RO⁺ T cells were more abundant in the portal area, particularly in areas with other infiltrated inflammatory cells, but less among the hepatocytes (Supplementary Fig. 3). Beclin1 expressed in the cytoplasm and cell membrane (Supplementary Fig. 4). The IHC data was quantitated to compare levels of these markers between two groups. As shown in Table 3, the cholangitis group had a higher level or rank of CD45RO⁺ T cells infiltration and a significantly lower level or rank of Beclin1 protein as compared with the non-cholangitis group. No significant difference was found in the level of CD68⁺ cells between the two groups. This data suggested that patients with early postoperative cholangitis had a higher level of CD45RO⁺ T infiltration and a lower level of Beclin1 protein.

Since differential levels of Beclin1 and infiltration of immune cells were observed between non-cholangitis and cholangitis groups, we attempted to identify the influence factors associated with early postoperative cholangitis. In Study 1, logistic regression showed that CD8⁺ cells level was a significant influence factor both in the univariate and multivariate analyses (both P < 0.05). The estimated odds ratio (OR) of CD8⁺ T cells after adjusting gender and age was 0.140 (95% confidence interval [CI], 0.027–0.721). Even including CD4⁺ cells level in the multivariate model, CD8⁺ T cells still reached significant (OR, 0.112; 95% CI, 0.022–0.577; P = 0.009). These results indicated that the low infiltration of CD8⁺ T cells was a risk factor for early postoperative cholangitis.

In Study 2, CD45RO⁺ T cells and Beclin1 were found steady significant in both univariate and multivariate results (all P < 0.05). In the multivariate model with adjustment for gender and age, the estimated ORs of CD45RO⁺ T cells and Beclin1 were 3.88 (95% CI, 1.37–11.03) and 0.088 (95% CI, 0.018–0.452). These data indicated that high infiltration of CD45RO⁺ T cells and low Beclin1 were risks factors for early postoperative cholangitis.

To further evaluate the potential prognostic value of the independent influence factors for early postoperative cholangitis (CD8⁺, CD45RO⁺ T cells, and Beclin1), ROC curve analysis was utilized. As shown in Fig. 1, ROC analysis showed that all the three factors reached statistical significance (all P < 0.05) and have a good AUROC. The AUROC values were 0.857, 0.738 and 0.900 for CD8⁺, CD45RO⁺ cells and Beclin1, respectively. These data suggested that CD8⁺, CD45RO⁺ cells and Beclin1 possessed the prognostic value for early postoperative cholangitis in BA patients undergoing Kasai operation.