| Infection with the hepatitis C virus (HCV) and/or hepatitis B virus (HBV) is characterized by a broad spectrum of possible outcomes. Infection is self-limited in a very minority, while the majority of infections develop persistent infection. Among those individuals with persistent HCV and/or HBV infection, the majority develops chronic hepatitis, some progress to fibrosis and even primary hepatocellular carcinoma. The variation may be attributable to viral and host genetic background. However it has been reported that the natural outcome in HCV or HBV infection varies among individuals though infected by the same viral. So there is strong evidence in HCV/HBV infection that host genetic factors play a major role in determining the outcome of infection.The immunity responses to HCV and HBV infection vary among individuals to a far extent. The single nucleotide polymorphism (SNP) in some cytokines gene greatly influences the clinical outcome of individuals with HCV and HBV infection. Helper T cell (Th) plays a key role in mediated immunity. Th1 cell is produced by the origin of Th in the stimulation of interferon-gamma and Th1 secrete cytokines such as interleukin 2, interferon-gamma, and so on. Th1 cytokines participate in cell-mediated immunity for controlling such intracellular pathogens as viruses, cytotoxic T lymphocyte (CTL) proliferation and activation, and natural killer cells (NK) activation. Th2 cell is produced by the origin of Th with the stimulation of interleukin 4. The major lymphokines secreted by Th2 cell are interleukin 4, interleukin 10 and so on. Th2 cytokines are essential for antibody-mediated immunity by proliferating and activating the B lymphocell. The cytokines from Th1 cells inhibit Th2 cells, and vice versa. The imbalance of Th1 and Th2 heavily disturbed the balance between the cell-mediated immunity and antibody-mediated immunity, which may be the key to the clinical outcome of chronic HCV and/or HBV infection.There is compelling evidence that cytokines play a significant role in chronic HCV and HBV disease pathogenesis. However, contradictory reports exist as to the exact effect of the cytokines promoter polymorphisms on the natural outcome of HCV and HBV infection. In order to explore the possible association of the cytokines gene polymorphism with the susceptibility of chronic HCV/HBV infection and the outcome of these infections, the IFN-γ+874, IL-2-330, IL-4-589, IL-10-1082, IL-10-592 gene polymorphisms were studied in subjects of mild hepatitis, moderate and severe hepatitis, and cirrhosis infected with HCV, HBV, HCV co-infected with HBV. These results would explore the immune genetic mechanisms of the clinical outcome of HCV and HBV infection.Part 1 Result comparison of anti-HCV detected by ELISA and HCV RNA detected by RT-n PCR in chronic hepatitis C virus infectionObjective: In order to provide the basis for the clinical test and the blood station screening the health donator, the results of anti-HCV tested by enzyme-linked-immuno-absorbed assay (ELISA) and HCV RNA tested by Reverse-Transcript–nested-Polymerase-Chain-Reaction (RT-n PCR) were compared in the chronic HCV infection.Methods: Venous blood samples of 133 chronic HCV infections, which were infected with HCV nearly in 1990 through plasma donator and diagnosed in 1993 in a rural area of Zhao County of Hebei Province, and 52 health controls were collected in May 2005. The antibody to HCV was tested by ELISA method and HCV RNA was tested by RT-n PCR method.Results:①In 185 cases, the rate of both anti-HCV and HCV RNA positive was 49.73% (92/185). The rate of anti-HCV negative but HCV RNA positive was 9.73% (18/185). The rate of anti-HCV positive but HCV RNA negative was 11.89% (22/185). The rate of both anti-HCV and HCV RNA negative was 28.65% (53/185). The result-agreement rate of ELISA and RT-n PCR testing methods was 78.38% ((92+53)/185). Kappa value equals 0.548 (u=8.65, P<0.01). The disagreement rate was not obviously different between ELISA and RT-n PCR testing methods (χ2 paired =0.40, P>0.05), which illustrated high agreement between the ELISA and RT-nPCR methods.②In the diagnosis of chronic HCV infection, the sensitivity of anti-HCV tested by ELISA was 82.71%, the specificity was 92.31%, and the omitting rate was 17.29%. The sensitivity of HCV RNA tested by RT-n PCR was 81.20%, and the specificity was 96.15%, and the omitting rate was 18.80%. The sensitivity was not obviously different between ELISA and RT-n PCR (χ2=0.10,P>0.05).③The sensitivity tested by ELISA parallel with RT-n PCR was 96.75%, which was obviously higher than that of single ELISA (82.71%) (χ2=9.62,P=0.00) and ELISA series with RT-n PCR(χ2=29.39, P=0.00).Conclusion: The false negative rate was nearly 17% when anti-HCV was used to detect HCV infection. The positive testing rate of HCV infection was increased remarkably when ELISA and RT-n PCR were used simultaneously. Part 2 The study of Th1 cytokine SNP in chronic HCV and/or HBV infectionObjective: To explore the possible association of the polymorphisms in interferon-gamma gene intron 1 at position +874 (IFN-γ+874), interleukin-2 gene at position–330 (IL-2-330) with the susceptibility of HCV and/or HBV infection and the outcome of these infections.Methods: IFN-γ+874 gene and IL-2-330 gene SNP were detected in 277 subjects including 79 chronic HCV co-HBV infection, 55 individuals only with HCV infection, 69 individuals only with HBV infection and 74 healthy controls, by sequence specific primers-PCR (SSP-PCR) and by restricted fragment long polymorphism-PCR (RFLP-PCR). Hepatocellular injury as suggested by alanine aminotransferase (ALT) was detected by BeckmanLX-20. The presence or absence of viral particles in serum was determined by RT-nPCR. The possible association of the polymorphism of IFN-γ+874, IL-2-330 with the susceptibility of HCV and/or HBV infection and the outcome of these infections were analyzed. Results:①IFN-γ+874 AA genotype frequency in individuals with chronic HCV, HBV, HCV co-HBV infection was significant higher than that in controls(χ2=16.15,P=0.01). Odds Ratio (OR) and 95% Confidence Interval (95% CI) of IFN-γ+874 AA genotype in chronic infection with HCV, HBV, HCV co- HBV were 3.22 (1.43~7.25), 2.70 (1.24~5.92) and 4.02 (1.88~8.55) compared with TA genotype. No significant differences were found among the HCV, HBV, HCV and HBV infections (P>0.05). There were no significant association of IFN-γ+874 A/T allele frequency with HCV/HBV infections (χ2=4.87, P=0.18); Chronic infection with HCV, HBV, HCV co-HBV was associated with higher IL-2-330 TT genotype frequency (χ2=14.24, P=0.03; OR (95%CI) = 3.46 (1.17~10.20), 7.14 (2.13~23.81), 2.93 (1.15~7.46)) and T allele frequency (χ2=12.33, P=0.01; OR (95%CI)=1.82 (1.09~3.03), 1.73 (1.10~ 2.73), 2.26 (1.39~3.69)). No significant differences were found among the HCV and / or HBV infections (P>0.05).②The outcome of mild chronic hepatitis (CH), moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with IFN-γ+874 AA genotype (χ2=14.78,P=0.02, OR=3.09 (1.51~6.33), 3.85 (1.70~8.70), 3.14 (1.08~9.17)); No significant relationships were found between IFN-γ+874 A/T allele frequency and the clinical outcome of HBV/HCV infection (χ2=5.68, P=0.13); The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with higher IL-2-330 TT genotype frequency (χ2=13.46, P=0.04; OR=3.42 (1.45~8.13), 3.29 (1.10~9.80), 11.11 (1.32~90.91)) and T allele frequency (χ2=11.69, p=0.01; OR=1.83 (1.20~2.80), 1.70 (1.02~2.82), 2.75 (1.32~5.63)), and the IL-2 (-330) GG genotype frequency showed negative correlation with the clinical progression (γ=-0.92, p<0.05).③There were no significant associations of IFN-γ+874 genotype / allele frequency with HCV duplication (χ2=2.36,P=0.31;χ2=1.92,P=0.17). No significant relationship was found between IL-2-330 genotype/allele frequency and HCV duplication (χ2=0.83,P=0.66;χ2=0.20,P=0.66).④There were no significant associations of IFN-γ+874 genotype/allele, IL-2-330 genotype/allele frequency with abnormal ALT (χ2=0.23,P=0.89;χ2=0.12, P=0.74;χ2=1.35,P=0.51;χ2=0.24, P=0.62).Conclusion: These results suggest that polymorphisms in the IFN-γ+874, IL-2-330 appear to have some influence on the chronic HCV and/or HBV infection, and on the outcome of HCV and/or HBV infections. IFN-γ+874AA, IL-2-330 TT genotype and T allele are possible risk to the chronic HCV and/or HBV infection and to the outcome of HCV and/or HBV infection, however IFN-γ+874 TA, IL-2-330 GG genotype and G allele is possible protective to them.Part 3 The study of Th2 cytokine SNP in chronic HCV and/or HBV infectionObjective: To explore the possible associations of the polymorphism of interleukin-4 (IL-4) gene at position–589, interleukin-10 (IL-10) gene at position -1082, -592 with the susceptibility of hepatitis C and/or hepatitis B virus infection and the outcome of these infections.Methods: IL-4-589, IL-10-1082, IL-10-592 gene SNP were detected in 277 subjects including 79 HCV co-HBV infection, 55 individuals only with HCV infection, 69 individuals only with HBV infection and 74 health control, by SSP-PCR and RFLP-PCR. Hepato-cellular injury as suggested by ALT was detected by BeckmanLX-20. The presence or absence of viral particles in serum was determined by RT-nPCR. The possible associations of the polymorphisms of IL-4-589, IL-10-1082 and IL-10-592 with the susceptibility of HCV and/or HBV infection and the outcome of these infections were analyzed.Results:①Chronic infection with HCV, HBV, HCV co-HBV was not associated with IL-4-589 genotype and allele (χ2=4.41,P=0.62;χ2=0.26,P=0.97); Chronic infection with HCV, HBV, HCV and HBV was associated with higher IL-10-1082 AA genotype frequency (χ2=13.05, P=0.04; OR=2.34 (1.07~5.10), 2.29 (1.10~4.78), 2.56 (1.25~5.21)). However no significant differences were found among the HCV, HBV, HCV and HBV infection (P>0.05). There were no significant association of IL-10-1082 allele frequency among HCV, HBV and HCV co-HBV infections (χ2=4.65,P=0.20). There were no significant association of IL-10-592 genotype and allele frequency with chronic HBV and/or HCV infection(χ2=2.83,P=0.83;χ2=1.10,P=0.78);②The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with IL-10-1082 AA genotype frequency (χ2=11.40, p=0.07; OR=2.03 (1.03~3.98), 2.70 (1.24~5.88), 4.26 (1.59~11.36)), and the IL-10-1082 AA genotype frequency showed positive correlation with the clinical outcome (γ=-0.99, p<0.01), however IL-10-1082 AG genotype frequency showed negative correlation with the clinical outcome (γ=-0.99, p<0.01). There were no significant associations of IL-10-1082 allele frequency, IL-10-592 genotype and allele frequency with HCV/HBV infection and clinical outcome (χ2=5.85, P=0.12;χ2=2.50, P=0.87;χ2=0.92, P=0.82); The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was not associated with IL-4-589 genotype and allele frequency (χ2=4.58, P=0.60;χ2=2.46, P=0.48).③There were obviously associations of IL-10-1082 higher AA genotype frequency and A allele frequency with HCV replication (χ2=12.27, P=0.00;χ2=5.36, P=0.02). The risk of subjects with AA genotype or A allele was 3.36 times (1.67~6.76), 1.67 times (1.08~2.57) than that with AG genotype and G allele; There were no significant associations of IL-10-592, IL-4-589 genotype/allele frequency with HCV replication (χ2=2.10, P=0.35;χ2=1.88, P=0.17;χ2=1.53, P=0.47;χ2=1.24, P=0.27).④There were no significant associations of IL-10-1082 genotype and allele frequency and IL-10-592 allele frequency with abnormal ALT (χ2=3.25, P=0.20;χ2=1.79, P=0.18;χ2=2.30, P=0.13); Abnormal ALT level was associated with higher IL-10-592 AC genotype (χ2=6.32, P=0.04; OR=2.83(1.26~6.37)); Abnormal serum ALT level was associated with IL-4-589 CC and CT genotype (χ2=12.46, P=0.00; OR=8.25 (1.74~ 39.22), 3.43 (1.47~8.00)) and the C allele (χ2=9.97, P=0.00, OR=2.31 (1.36~3.93)).Conclusion: These results suggest that polymorphism in the IL-10-1082 appears to have some influence on the chronic HCV and/or HBV infection, and on the outcome of HCV and/or HBV infections and HCV replication. IL-10-1082 AA genotype is possible risk to the chronic HCV, HBV and HCV co-HBV infection and the clinical outcome, however AG genotype is possible protective to them. IL-10-592 AC genotype frequency has influence on the liver damage, and IL-4-589 CT, CC genotype, and C allele are possible risk to the liver inflammatory injury, and TT genotype and T allele is possible protective to it.Part 4 The interaction of Th1 and Th2 cytokine SNP in the clinical outcome infected with chronic HCV and/or HBVObjective: To analyze the interaction of the Th1 and Th2 cytokine SNP in HCV/HBV infection and in the outcome of these infections. Methods: The interaction of the Th1 and Th2 cytokine SNP in HCV and/ or HBV infection and the outcome of these infections was analyzed by multifactor dimensionality reduction (MDR). The OR was used to evaluate the risk of clinical outcome during HCV/HBV infection. The synergy index (S), Attributable proportion of interaction (API), Relative excess risk of interaction (RERI) and 95% CI were used to evaluate the interaction degree.Results:①The interaction of IFN-γ+874 AA and IL-2-330 TT genotype was found in the clinical outcome of chronic HCV/HBV infection by MDR (P=0.00). When individuals carried with IFN-γ+874 AA genotype and IL-2-330 TT genotype, the OR and 95% CI for the HCV/HBV to develop to mild CH, moderate/severe CH and cirrhosis was 10.1(2.8~36.0), 10.6(2.9~38.8) and 13.6(4.9~66.8) respectively, and the OR in individuals with IFN-γ+874 AA and IL-2-330 TT was 6.8 times, 4.1 times and 6.6 times compared with individuals of only IFN-γ+874 AA, and was 8.4 times, 8.1 times and 6.6 times comparied with individuals of only. The S of chronic HCV/HBV infection developing to mild CH, moderate/severe CH and cirrhosis in IFN-γ+874 AA and IL-2-330 TT genotype was 13.04, 5.01 and 7.22 respectively. RERI and 95% CI were 8.43(2.61~27.23), 7.65(2.50~23.42) and 10.83(2.53~46.32). API was 83.22%, 72.44% and 79.81% respectively.②Interaction of IL-10-592 gene and IL-4-589 gene was found in the hepato-cellular injury of chronic HCV and / or HBV infection by MDR (P=0.00). When only with IL-10-592 AC, there was no significant effect on the hepato-cellular injury of chronic HCV/HBV infection (OR=1.0). However together with IL-4-589 CC or CT and IL-10-592 AC, the OR was 5.5 (2.6~11.5). It was found to have significant interaction to the hepato-cellular injury, which the S was 7.1, RERI was 3.7(2.11~6.49), and API was 67.6%.③The interaction of IFN-γ+874 gene, IL-2-330 gene and IL-10-1082 gene was found in the clinical outcome of chronic HCV/HBV infection by MDR (P=0.01). When one SNP were analyzed, the IL-10-1082 AA genotype had the highest risk (OR=2.7, 4.3, 7.1), IFN-γ+874 AA genotype had the middle risk (OR=2.0, 3.9, 2.4), IL-2-330 TT had the lowest risk (OR=1.2, 1.3, 2.8) to the outcome of HCV/HBV infection. The IL-10-1082 AA genotype was positive correlation with the clinical progression (γ=0.99, P<0.05). When two SNP were analyzed at a time, IFN-γ+874 AA genotype and IL-2-330 TT genotype had a high risk for the HCV/HBV to develop to mild CH, moderate/severe CH and cirrhosis, OR=7.5(2.0~28.4), 9.6(2.6~35.6) and 10.7(1.4~ 80.2)), which were not equal to the OR=10.1(2.8~36.0), 10.6(2.9~38.8) and 13.6(4.9~66.8) of IFN-γ+874 AA genotype and IL-2-330 TT genotype was due to with or without considering the interaction of IL-10-1082 AA genotype. The risk of both IFN-γ+874 AA genotype and IL-2-330 TT genotype in mild CH, moderate/severe CH and cirrhosis was 3.8 times, 2.5 times and 4.5 times comparied with only IFN-γ+874 AA genotype, and was 6.3 times, 7.4 times and 3.8 times comparied with only IL-2-330 TT genotype. The S was 5.3, 2.7 and 3.0 respectively , the RERI was 5.25(1.1~24.5), 5.41(1.8~16.2) and 6.5(1.3~31.5), the API was 70.28%, 56.35%, 60.54%. When together with IFN-γ+874 AA genotype and IL-10-1082 AA genotype, the OR in mild CH, moderate/severe CH and cirrhosis was 1.6(1.0~2.5), 4.0(1.6~9.9) and 5.3(1.5~ 19.1) respectively, which was lower than that with only IL-10-1082 AA genotype. The S was 0.22, 0.49, 0.58 respectively, the RERI was -2.1(-1.1~-4.1), -3.2(-1.5~ -6.8), -3.15 (-1.3~ -7.6) respectively, the API was -130.00%, -79.50%, -59.10% respectively. When together with IL-2-330 TT genotype and IL-10-1082 AA genotype, the OR in mild CH, moderate/severe CH and cirrhosis was 3.0(1.3~7.2), 4.5(1.6~12.3), 8.5(2.0~35.7), which was similar to the OR of only with IL-10-1082 AA genotype. S was 1.06, 0.98, 0.95 respectively, RERI was 0.11, -0.07, -0.42, API was 3.68%, -0.02%, -0.42%. When three SNPs were analyzed at a time, OR in mild CH, moderate/severe CH and cirrhosis was 9.6(2.9~31.8), 12.8(2.0~80.2) and 32.0(5.1~200.9). The OR was the 1.3 times, 1.3 times and 3.0 times compared with IFN-γ+874 AA and IL-2-330 TT. S was 1.1, 1.0 and 2.0, API was 4.2%, -0.8% and 47.5%, RERI was 0.4(0.2~0.7), -0.1(0.0~0.5) and 15.2(3.6~64.3) respectively.Conclusion: These results suggest that subjects with IFN-γ+874 AA genotype and IL-2-330 TT genotype had higher risk for the clinical outcome of chronic HCV/HBV infection, the subjects with IL-10-592 AC genotype and IL-4-589 CC or CT genotype had a higher risk for the hepato-cellular injury of chronic HCV/HBV infection. IFN-γ+874 AA and IL-2-330 TT together with IL-10-1082 AA had no significant interaction for HCV/HBV infection to the mild CH, moderate and severe CH, however had positive interaction for HCV/HBV infection develop to cirrhosis. |
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