Cellular Immunity after Chronic Hepatitis B Jan. 2006 to Aug. 2010. This study was conducted in our hospital in Wuxi, China. Diagnoses of CHB and chronic severe hepatitis were made per the diagnostic criteria in the Program for Prevention and Treatment of Viral Hepatitis (2). The requirements for enrollment included positivity for HBV DNA (HBV DNA > 103 copies/mL), ala- nine aminotransferase (ALT) > 2× upper limit of normal value, and age over 18 years. Exclusion criteria included patients with hepatitis A, C, D, or E infection; a medical history of autoimmune disease; alcohol abuse; medi- cation with hepatotoxic agents; and use of antiviral agents, such as nucleoside analogs and interferons, and immune-enhancing or immunosuppressive agents, such as glucocorticoids. The 255 CHB patients were divided into two groups. Group A consisted of 49 cases whose disease progressed to chronic severe hepatitis B; 35 were male (71.45%), the average age was 35.68 ± 9.63 years, and the average course of illness was 4.27 ± 2.40 years. Group B contained 206 cases in whom the disease did not progress to chron- ic severe hepatitis B; 147 were male (71.36%), the average age was 35.49 ± 10.09 years, and the average course of ill- ness was 4.23 ± 2.32 years. There was no significant differ- Xibing G et al. 811 3.4. HBV Markers and HBV DNA HBV markers (HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc) were measured by 1235 time-resolved fluores- cence method; the reagents were purchased from Xinbo, Shanghai. HBV DNA was assayed by real-time fluores- cence quantitative PCR; the reagents were purchased from Kehua, Shanghai. 3.5. Identification of HLA-A2 Allotypes Fresh blood samples (100 μL) that were treated with heparin sodium were loaded into test and control tubes. After adding 10 μL HLA-A2-PE and the same type control, the tubes were incubated at room temperature for 30 min in the dark and analyzed on a Beckman-Coulter 3XL after hemolysis. Reagents were purchased from Proim- mune, UK. 3.6. HBV-Specific CTL Test HBV-specific CD8+ T cells were measured by HLA-peptide tetramer flow cytometry, the principle of which is as fol- lows (3): The tetramer is labeled with phycoerythin (PE), and CD8+ T cells from peripheral blood that are specific Table 1. Comparison of ALT, Total Bilirubin (TBIL), Albumin (ALB), HBV DNA and HBeAg Positive Rate between Group A and Group B at Baseline (mean ± SD) ALT a, u/L TBIL a, μmol/L HBVDNA a, log10 Copies/mL HBeAg a Positive, No. (%) ALB a, g/L 41.95 ± 3.31 Group A, (n = 49) 426.35 ± 182.31 38.25 ± 22.17 5.58 ± 0.76 Group B, (n = 206) 423.61 ± 190.91 37.88 ± 21.01 42.03 ± 1.42 5.59 ± 0.81 t value 0.115 0.146 0.273 0.113 29 (59.18) 132 (64.08) χ2 = 0.409 P value < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 a Abbreviations: ALB, albumin; ALT, alanine aminotransferase; HBeAg; hepatitis B e antigen ; HBVDNA, hepatitis B virus DNA; TBIL, total bilirubin ence in sex, age, or course of illness between the groups (P > 0.05). There was no significant difference in ALT, HBV DNA, or HbeAg-positive rate between groups (P >0.05) on the second day of admission (Table 1). The two groups were comparable in every aspect. Thirty healthy blood donors were enrolled as controls; 22 were male (73.33%), and the average age was 35.18 ± 7.01 years. 3.2. Treatment Methods Both groups were treated with 200-mg silybin meglu- mine tablets (Jiangsu Zhongxing Pharmaceutical) orally three times daily to protect the liver. For patients whose disease deteriorated, antiviral treatments were given af- ter the laboratory tests when indicated. For those whose disease did not progress into severe hepatitis by Day 20 after admission, antiviral therapy was given when the therapy was indicated. 3.3. Prothrombin Time (Activity) Prothrombin time was measured by magnetic bead method (reagents and ACL-200 were purchased from Coulter, USA). for an epitope identify and bind short peptides in the antigen channel through the T cell receptor (TCR); these CD8+ T cells are detected by FITC-labeled anti-CD8, and HBV antigen-specific CD8+ T cells are detected by dual- C T I 8F 28 D D C C TET-PE Figure 1. Flow Cytometry of HBV Specific CTL Hepat Mon. 2011;11(10):810-815