| ObjectiveHepatitis C is a global epidemical viral infectious disease. There are 170 million people infected with hepatitis C virus (HCV) and the number of patients with hepatitis C emerging each year is about 35,000 in the world. According to epidemical data, 3.2% of Chinese general population was anti-HCV positive. Chronic hepatitis C is considered the main cause of cirrhosis and liver cancer. There is no effective vaccine to prevent hepatitis C up to date as the high variability of hepatitis C virus genotypes and multiple influencing factors. Therefore, effective treatment for hepatitis C has become more important. Currently, pegylated interferon and ribavirin combination therapy has become the standard clinical treatment, but some clinical trials showed that its sustained virological response (SVR) rate was only 48-88%. There were still a large number of patients with viral clearance failure, among which the genotype 2 or 3 HCV infection was 18%-24% and the genotype 1 HCV infection was 54%-58%. Difficult-to-treat hepatitis C (DT-CHC) was defined as a patient with chronic hepatitis C with one of following criteria:HCV genotype 1, non-response or relapse, high viral load (viral load≥2×106copies/ml), cirrhosis, insulin resistance or non-insulin dependent diabetes and low viral clearance rate. In Chinese patients with HCV infection, the majority is DT-CHC for the major genotype 1b HCV infection. How to raise the SVR of hepatitis C has become a focus around the world. Up to now, the features of clinic and pathology have not been identified and the impact of the immune condition in liver on SVR has not been illuminated, which are significance for the diagnosis and treatment of DT-CHC. In this study, we compared the DT-CHC with the common hepatitis C (C-CHC), based on a large number of patients with chronic hepatitis C who underwent liver biopsy and clinical laboratory investigation, to determine the clinical, pathological and epidemiological features, and to provide a reliable basis for evaluation of therapeutic effection and individualized anti-viral treatment. Meanwhile, in order to explore the immune regulatory mechanisms of the DT-CHC, we investigated the lymphocytic condition of intrahepatic Th1, Th2,Th17 and Treg by detecting a panel of cytokines.MethodsA total of 222 subjects who had undergone liver biopsies were consecutively enrolled in this study from the inpatients in 302 Hospital of PLA between January 1996 and December 2009, and were divided into two groups: the difficult-to-treat hepatitis C group (117 cases) and the common chronic hepatitis C group (105 cases), according to the criteria of DT-CHC. The liver function and blood biochemical parameters were detected by automatic biochemical analyzer. Serum anti-HCV and HCV RNA levels were determined by using enhanced chemiluminescence immunization and fluorescence quantitative PCR (RT-PCR). HCV antigens and HCV RNA in liver tissues were detected by immunohistochemistry staining and in situ hybridization staining respectively. The liver biopsies from the two groups were evaluated by a single experienced histopathologist who was blinded to clinical datas. A novel pathological assessment system was used to evaluate the histopathological changes in liver. Briefly, grade of inflammatory activity was scored 0-18; stage of fibrosis was scored 0-6; degree of hepatic steatosis was divided into mild, moderate and severe; and other items such as lymphatic follicle, duct injury and plasm cells infiltration were showed none or have. Clinical, pathological and epidemiological data were analyzed statistically. Lymphocyte subsets of peripheral blood, collected on the same day of performed liver biopsy or within a week, were tested by using flow cytometry (FCM). A panel of cytokines related to Th1, Th2, Th17 and Treg immunocytes in liver tissues were detected by immunohistochemical staining. The immunostaining positive cells in each slide were calculated by computer image analysis system. All statistical analysis was performed with SPSS version 11.5 software package for Windows.Results1. Features of clinical epidemiologyThe mean age of patients in the common group and in the difficult-to-treat group was 35.11±11.47 years (ranged from 16 years to 57 years) and 34.17±14.11 years (ranged from 16 years to 63 years) repectively. There were no significant differences between the two groups in age (P=0.556) and gender (x2=0.165, P=0.685), but in the population of 16-19 years, the number of male patients in the difficult-to-treat group was significantly more than that in the common group (x2=5.182, P=0.046). The male proportion of the lower 40 years old patients was significantly higher than the over 40 years old (x2=12.306, P=0.001) in the difficult-to-treat group. In this study, patients with genotype 2a HCV infection were all come from the southern areas of China. 89.77% (79 cases) of genotype 1b hepatitis C patients was from the northern areas of China. The main ways of HCV infection, including blood transfusion or blood product application, were not different between the two groups (x2=6.042, P=0.110).2. Virological examination in serum and liver tissuesAll the recruited patients with chronic hepatitis C were serum anti-HCV positive. In the difficult-to-treat group, the patients with serum HCV RNA positive were 114 cases (97.44%), and which were 71 cases (67.62%) in the common group. In situ hybridization results showed that expression of HCV RNA in liver tissues were higher in the difficult-to-treat group than that in the common group.3. Features of Clinic and pathologyThe serum levels of ALT and AST in the difficult-to-treat group were 77.63±77.01 and 55.02±45.05 respectively, which increased significantly compared with the common group (P=0.026, 0.037, respectively). The portal inflammation, steatosis in difficult-to-treat group were more serious than those in the common group (P=0.036, 0.012, respectively). The biliary duct injury, folliculus lymphacyte aggregation in the protal tracts was more frenquent than those in the common group (P=0.011, 0.006, respectively). The perisinusoidal fibrosis (P=0.038) and plasma cell infiltration (x2=6.936,P=0.008) were more easily observed in the difficult-to-treat group. Interestingly, the degree of liver fibrosis was positively correlated with steatosis, serum AST, HCV RNA level repectively in difficult-to-treat group. Meanwhile, closely positive correlation between ALT, AST and inflammatory activity, steatosis, biliary duct injury and lymphatic folliculus were found in the difficult-to-treat group.4. Changes of lymphocyte subsets in peripheral blood and liver tissuesThe frequencies of CD3, CD4 posisitive cells in peripheral blood were higher in the difficult-to-treat group than those in the common group (P=0.036, 0.024, respectively), while there were no significant difference between the two groups in the frequencies of CD8, B, NK posisitive cells; Meanwhile, the amounts of CD3, CD4, CD8 positive cells in liver tissues from the difficult-to-treat group were significantly higher than those from the common group (P=0.032, 0.004, 0.033, respectively), but there were no differences in the amouts of CD20, CD56 positive cells in liver tissues between the two groups.5. Cytokine analysis in liver tissueThe amounts of IL-4, IL-10, IL-6, IL-17, TNF-αpositive cells in liver tissues in difficult-to-treat group were significantly higher than those in the common group (P=0.031, 0.028, 0.028, 0.036, 0.023, respectively), but the amounts of IL-2, IFN-γ, TGF-β, Foxp3 positive cells in liver tissues were significantly lower than those in the common group (P=0.036, 0.021, 0.004, 0.036, respectively). The amounts of IL-6, IL-17 positive cells in liver tissues from difficult-to-treat group were positively correlate with plasma cells infiltration. Meanwhile, positive correlations were also found between the amount of TGF-βpositive cells and the degree of liver fibrosis in the difficult-to-treat group.Conclusions:1. DT-CHC had its pathological features compared with C-CHC. portal inflammation, steatosis were more serious and biliary duct injury, lymphatic folliculus, plasma cells infiltration and perisinusoidal fibrosis were observed more easily in the liver tissue in DT-CHC, which may be the pathological basis of elevated serum transaminase and low SVR. The histological scoring system, building in this study, could present the pathological features of CHC in detail, and could be used perfectly in evaluation of histological changes of hepatitis C and drug verification .2. Clinical data showed that the liver functions of patients with DT-CHC were more serious than those with C-CHC, which could provide reference for deciding treatment opportunity and evaluation of liver injury. In addition, the age of male patients who developed to DT-CHC was younger than the female, and the male patients proportion of lower 40 years old was higher than the female, suggesting that gender and age may be the influence factors of SVR. Patients with DT-CHC caused by genotype 1b HCV possessed a large proportion in the northern areas of China. 3. Th1/Th2 cytokine profile imbalance was more serious in DT-CHC compared with C-CHC. The imbalance of Th1/Th2 cytokine profile may be one important reason for poor antiviral efficiency.4. Cytokines associated with Th17 significantly increased in DT-CHC, and the amount of IL-17 positive cells was closely correlated with plasma cells infiltration in liver tissue, suggesting that Th17 probably affected the antiviral response of CHC and may be associated with high incidence of autoimmune reaction in CHC. |
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