| Section oneCLINICAL VALUES OF SERUM IL-33 AND ST2 TESTS FOR PATIENTS WITH ACUTE-ON-CHRONIC HEPATITIS B LIVER FAILUREBackgroundHepatitis B virus (HBV) infection is a global public health problem across the world. An estimated 350 million people worldwide are chronically infected with HBV. The carriers of HBV are at high risk of developing rapid deterioration of liver function and progressing into acute-on-chronic hepatitis B liver failure (ACHBLF). ACHBLF is a life threatening condition and usually associates with high mortality. In Asia, ACHBLF accounts for more than 70% of acute-on-chronic liver failure (ACLF) because of the high prevalence of HBV and almost 120,000 patients die of ACHBLF each year. Until now, the pathogenesis of ACHBLF is still not fully understood. Accumulating evidences suggest that several pro-inflammatory cytokines may play pivotal roles in ACHBLF and contribute to the ACHBLF progression.Interleukin-33 (IL-33) is one of the newly described members in IL-1 cytokine family. It is also known as nuclear factor of high endothelial venules (NF-HEV) and DVS 27. As a ligand for the interleukin-1 receptor-like 1 (ST2), IL-33 plays an important role in T helper 2 (Th2)-associated immune responses through activating the MyD88 and NF-KB-related signal pathway. ST2 has transmembrane form (ST2L) and soluble form (sST2). ST2L functions as a mediator of IL-33 bioactivities, while sST2 acts as a decoy receptor for IL-33.In previous studies, serum IL-33 and sST2 were found to be overexpressed in liver damage during acute or chronic hepatitis. Therefore, there is a possibility that serum IL-33 and sST2 may also be overexpressed in ACHBLF and associated with its disease severity or prognosis.Then, we investigated the association of serum IL-33 and sST2 with clinical and laboratory parameters in ACHBLF and determined their predictive value for prognosis.ObjectiveTo study the expressions and significances of IL-33 and sST2 in different liver diseases. Acute and chronic liver failure represents for acute inflammatory reaction. We detect the serum IL-33 and sST2 levels in patients with ACHBLF, patients with CHB and HCs. We evaluate the roles of the serum IL-33 and sST2 levels in patients with ACHBLF. We want to know the relationships between the expression of the serum IL-33 and sST2 levels and the prognosis of patients with ACHBLF patients, and their relationships with clinical indexes and clinical significances.Materials and methods60 patients with ACHBLF,58 patients with CHB and 30 HCs were enrolled from September 2011 and June 2013 in the Department of Hepatology, Qilu Hospital of Shandong University. The diagnosis of the patients is in compliance with consensus recommendations of Asian-Pacific Association for the Study of the Liver (APASL).The care provided to patients with ACHBLF was in accordance with the APASL consensus recommendations, which routinely included antiviral therapy, bed rest, energy supplements and vitamins, intravenous infusion albumin, maintenance water, electrolyte and acid-base equilibrium, and prevention and treatment complications, etc. In this study, no patient with ACHBLF received liver transplantation. The serum IL-33 and sST2 levels of the participants were determined by ELISA using human IL-33 and ST2 ELISA Kits (ImmunoWay, USA) according to the manufacturer’s instructions. Hepatitis B e antigen (HBeAg), HBV DNA load and the serum biochemical markers included alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin (ALB).Hemostasis markers included prothrombin time activity (PTA) were measured using standard methodologies in Department of Laboratory Medicine, of Shandong University.Model for end-stage liver diseases (MELD) score was employed to estimate the severity of ACHBLF. It was calculated according to the formula:r= 9.57×loge[creatinine(mg/dl)] +3.78×loge[bilirubin(mg/dl)]+11.2×loge(INR)+6.43x (aetiology:0 if cholestatic or alcoholic,1 otherwise).The start date of the follow-up was the date of the diagnosis of ACHBLF. All the patients with ACHBLF were followed up for at least 3 months and the outcome (death or survival) of each patient was recorded.Quantitative variables were expressed as the median (centile25; centile75). Categorical variables were expressed as number (percentage). The data were analyzed using SPSS 16.0 statistical software. Student’s t-est, Kruskal-Wallis test and One-Way ANOVA test were used to compare the quantitative variables. Chi2 test was used to compare the categorical variables. The correlation between variables was evaluated using the Spearman rank correlation test. The area under the ROC curve (AUC) was used to assess the diagnostic value of IL-33, sST2 and MELD score in predicting 3-month mortality for patients with ACHBLF. Diagnostic accuracy was assessed by sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Survival curves were drawn using the Kaplan-Meier method and the statistical significance was determined using the log-rank test. To test the diagnostic value when both serum sST2 and MELD score were measured, we estimated function of the combined marker by binary logistic regression. All statistical analyses were two sided, and P-value< 0.05 was considered to be statistically significant.Results1. There was no significant difference between ACHBLF and CHB patients with respect to HBeAg (P=0.92; x2 test) and Log10[HBV DNA] (P=0.71; student’s t-test). No significant difference was found among ACHBLF, CHB and HC groups for sex (P=0.60; x2 test) and age (P=0.33; One-Way ANOVA test). However, significantly different ALT (P<0.01; Kruskal-Wallis test), AST (P<0.01; Kruskal-Wallis test), TBIL (P<0.01; Kruskal-Wallis test), ALB (P<0.01; One-Way ANOVA test), PTA (P<0.01; Kruskal-Wallis test), IL-33 (P<0.01; Kruskal-Wallis test) and sST2 levels (P<0.01; One-Way ANOVA test) were found among the three groups.2. IL-33 and sST2 expression in different participants groupThe serum IL-33 level in patients with ACHBLF (313.10±419.97 pg/ml) was significantly higher than that with CHB (97.25± 174.67 pg/ml, P<0.01) and HCs (28.39±6.53pg/ml, P<0.01). The serum IL-33 level in patients with CHB was also significantly higher than that with HCs (P<0.01).The serum sST2 level was 1545.87±1135.70 pg/ml in patients with ACHBLF, which was significantly higher compared with that in patients with CHB (152.55±93.28 pg/ml, P<0.01) and HCs (149.27±104.90 pg/ml, P<0.01). However, no significant difference was found between serum sST2 level in patients with CHB and HCs(P> 0.05).3. Correlations between serum IL-33, sST2 level and liver function parametersWe analyzed all participants’ data and examined the correlation between serum IL-33 and sST2 level. We found that the serum IL-33 was significantly positively correlated with serum sST2 level (Spearman’s r=0.43, P<0.01).Then, we examined the correlation between serum IL-33, sST2 level and the liver function parameters in patients with ACHBLF. The serum IL-33 was significantly associated with ALT (Spearman’s r=0.26, P=0.04). No correlation could be observed between serum IL-33 and age (Spearman’s r=-0.23,P=0.07), AST (Spearman’s r=0.23, P=0.08), TBIL (Spearman’s r=0.05, P=0.70), ALB (Spearman’s r=0.19,P=0.15), PTA (Spearman’s r=-0.21, P=0.11), Log10 [HBVDNA] (Spearman’s r=0.17, P=0.20) and MELD score (Spearman’s r=-0.03, P=0.80). There were significant correlation between serum sST2 and TBIL (Spearman’s r=0.59, P<0.01), Log10[HBVDNA] (Spearman’s r=-0.47,P< 0.01) and MELD score (Spearman’s r=0.28, P=0.03). No correlation could be observed between serum sST2 and age (Spearman’s r=0.08, P= 0.55), ALT (Spearman’s r=-0.08,P=0.52), AST (Spearman’s r=-0.12, P=0.38), ALB (Spearman’sr=0.01, P=0.93), PTA (Spearman’s r=-0.08, P=0.52).4. The predictive value and accuracy of serum IL-33 and sST2 for prognosis of ACHBLFThe three-month mortality of patients with ACHBLF was 43.33%(26/60). The three-month mortality was significantly associated with TBIL, PTA, serum sST2 level and MELD score (P< 0.05, respectively). No significant association could be observed between sex, age, HBeAg, Log10[HBVDNA], ALT, AST, ALB and serum IL-33 level (P> 0.05, respectively) and three-month mortality.The predictive values of serum IL-33 level, sST2 level and MELD in predicting 3-month mortality for patients with ACHBLF were assessed by AUC. The serum sST2 level had an AUC of 0.81 (SE 0.06,95% CI 0.68-0.90), while MELD score had an AUC of 0.77 (SE 0.06,95% CI 0.64-0.87). No significant difference (P=0.64) could be observed between them. The AUC of serum IL-33 level was 0.53 (SE 0.08, 95% CI 0.40-0.66), which was significantly lower than that of serum sST2 level and MELD score (P<0.05, respectively).Different cut-off points of serum sST2 level were chosen to predict 3-month mortality for patients with ACHBLF.A low cut-off point was chosen based on the ROC analysis to obtain sensitivity of at least 90%. A moderate cut-off point was chosen to provide the optimum Youden index. A high cut-off point was also chosen to provide a specificity of at least 85%. The low cut-off point (serum sST2>695 pg/ml) showed sensitivity of 92%, specificity of 47%, PPV of 57% and NPV of 89%. It successfully identified 24 of 26 death group patients. Meanwhile, only 2 of 18 patients with sST2<695 pg/ml died after followed up for 3 months. The moderate cut-off point (serum sST2>1507 pg/ml) showed sensitivity of 77%, specificity of 76%, PPV of 71% and NPV of 81%. At this cut-off point, serum sST2 provided an optimum Youden index of 0.53. The high cut-off point (serum sST2>1811 pg/ml) showed sensitivity of 58%, specificity of 85%, PPV of 75% and NPV of 73%. It correctly identified 29 of 34 survival group patients. Meanwhile,15 of 20 patients with serum sST2>1811 pg/ml died after followed up for 3 months.The mean survival time (from hospital admission) of patients with ACHBLF was 67.55 (SE 3.80,95% CI 60.09-75.01) days. Compared with patients with ACHBLF who had serum sST2<1507 pg/ml, survival in those who had serum sST2>1507 pg/ml were statistically significantly poorer (P<0.01, log-rank test). The mean survival time (from hospital admission) for patients with serum sST2>1507 pg/ml were 51.00 (SE 5.69,95% CI 39.86—62.15) days. The mean survival time for patients with serum sST2<1507 pg/ml were 82.03 (SE 3.48,95% CI 75.22—88.84) days.To assess whether the combined use of serum sST2 and MELD score was better than either of these two biomarkers alone, a new variable predicted probability (P) for 3-month mortality of ACHBLF was created on the basis of an equation obtained by binary logistic regression.Variables (sST2 plus MELD)=log(p/1-p)=0.01×sST1+0.238×MELD-6.59) ROC analysis was used to compare the diagnostic value of sST2 plus MELD score and either test alone for 3-month mortality of ACHBLF. The sST2 plus MELD score had an AUC of 0.86 (SE 0.05,95% CI 0.74-0.93), which was significantly higher than that of MELD score (P<0.05).ConclusionsIn conclusion, our study revealed that serum IL-33 and sST2 levels were elevated significantly in patients with ACHBLF.It also showed that serum sST2 was significantly associated with the 3-month mortality of ACHBLF. Therefore, serum IL-33 and sST2 levels might potentially serve as prognostic markers for ACHBLF in further clinical work. This study provided evidence for further evaluation and treatment of ACHBLF.Section twoCLINICAL VALUES OF SERUM IL-33 AND ST2 TESTS FOR PATIENTS WITH CHRONIC HEPATITIS BBackgroundInterleukin-33 (IL-33), a new member of the IL-1 family, induced the production of pro-inflammatory and T helper-2(Th2)-associated cytokines. ST2 was to weaken Th2 inflammatory responses as its receptor. Hepatitis B virus (HBV) infection is still a major health problem worldwide. There are approximately 350 million people infected chronically with HBV globally, and they are at great risk of developing liver cirrhosis and hepatocellular carcinoma. About 5% of adulthood and over 90% of infants and young children who are infected with HBV will evolve chronicity. Many patients with chronic hepatitis B (CHB) ultimately progress to liver cirrhosis and hepatocellular carcinoma. Previous studies showed that the interaction of HBV, hepatocytes and the host immune system determines the persistence of HBV infection and chronic inflammation. The immune system were known to be suppressed by the viral infection and related hepatocyte injuries. Although experimental evidence suggested that antigen-specific Thl immunity and pro-inflammatory cytokines played an important role in the HBV related liver injury and clearance of viruses, serum IL-33 Levels was associated with liver damage of patients with CHB, serum level of IL-33 and soluble ST2 elevated in liver failure, which could be a sign of immune hyperactivation, and/or a mechanism to down-regulate inflammation. It is widely accepted that the adaptive immune responses play major roles in the clearance of HBV infection. However, the role of innate immunity during HBV infection appears not to be well understood, which can be attributed to the fact that the recruitment of patients in the very early, asymptomatic phase of HBV infection is very difficult. The natural course of chronic HBV infection was perceived as consisting of 4 phases: immune tolerance, immune clearance (HBeAg-positive chronic hepatitis B, EAPCHB), inactive carrier state, and reactivation (HBeAg-negative chronic hepatitis B, EANCHB).Although IL-33 and ST2 serum levels were up-regulated in liver failure and CHB, the relevance of serum levels of both molecules and patients with hepatitis B virus infection needed further research. And serum alanine aminotransferase (ALT) activity is an important marker for liver damage in patients with CHB.We therefore sought to investigate the serum measurements of IL-33 and ST2 in patients with chronic hepatitis B virus infection and study the relationship of IL-33 and ST2 with ALT and HBeAg. We examined the measurements of serum IL-33, ST2 and ALT in patients with CHB, chronic hepatitis B virus carriers in immuno-tolerant phase (CHBVIT) and healthy controls (HCs) so that we could determine whether IL-33 and ST2 had relevance with chronic HBV infection in our study.ObjectiveInterleukin-33 (IL-33) and ST2 have been demonstrated to be associated with liver damage. However, their potential value in hepatitis B virus (HBV) infection remains unknown. This study was designed to investigate the clinical correlation of serum IL-33 and ST2 levels and the course of chronic HBV infection.MethodsA total of 120 patients with chronic hepatitis B (CHB),20 chronic hepatitis B virus carriers in immuno-tolerant phase (CHBVIT) and 28 healthy controls (HCs) were enrolled in this study. All patients with CHB were divided into four groups according to their serum ALT levels. The serum levels of IL-33 and ST2 of all participants were determined by enzyme-linked immunosorbent assay (ELISA), and compared between each two out of those six groups.ResultsNo significant differences were found in serum levels of IL-33 and ST2 between the CHB ALT1-2ULN group and the HC, or CHBVIT (P>0.05, respectively). Other than that, there were significant differences when serum levels of IL-33 and ST2 were compared between any other two out of those six groups (P<0.05, respectively). The overall correlation analysis indicated that changes of serum IL-33 and ST2 levels were positively associated with ALT levels in patients with chronic HBV infection (P <0.05, respectively), as well as all study participants (P<0.05, respectively). No significant differences were found when the serum levels of ALT, IL-33 and ST2 were compared between patients with HBeAg-positive CHB and HBeAg-negative CHB (P>0.05, respectively).ConclusionsOur study revealed that the serum IL-33 and ST2 levels changed in the different courses of chronic hepatitis B virus infection. The serum levels of IL-33 and ST2 elevated as serum ALT levels increased in patients with CHB. They might indicate liver damage for patients with CHB as ALT. They were associated with the natural course of chronic henatitis B virus infection... |
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