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Different effects of type I-interferons on the HBV-enhancer I-, enhancer II- and core promoter-regulated transcription
Viral hepatitis." basic aspects
I P/C05/17 I
[ P/C05/19 I
ICAM-1 POLYMORPHISM: NEW RISK FACTOR FOR HEPATOCELLULAR CARCINOMA IN PATIENTS WITH CHRONIC HEPATITIS C? P.K. Constantini, M. Egg, I.W. Graziadei, W. Vogel Dp. of Hepatology, Innsbruck University, Austria. Recent studies have shown an association between genetic polymorphism and the outcome of chronic Hepatitis C infection (I-ICV), which is known to be a class II mediated type of inflammation. Intercellular adhesion molecules like ICAM-1 influence the cell to cell interaction and thus the activation of this pathway. The aim of this study was to investigate a possible association between ICAM-1 polymorphism at codon 241 (exon 4) and clinical progression of chronic HCV-infection. Patients and Methods: We investigated a group of 93 patients with chronic HCV infection, all of them were antibody and RNA positive and had cirrhosis, 20 of them developed hepatoeellular carcinoma (HCC). ICAM polymorphism 241 was performed by using PCR and sequence specific primers. The genotype distribution of the population with HCC (HCV+, HCC+) was compared with those 73, who had no evidence of carcinoma (HCV+, HCC-). Results: The 241 A,G 8enotype was found at reduced frequency in patients of the HCV+ and HCC+ group. Only 5% (1/20) in the HCV+, HCC+ group versus 22% (16/73) of the HCV+ but HCC- population were found to have the 241 A,G genotype (p< 0.05). None (0/20) of the HCV+, HCC+ patients and only one (1/73) of the HCV+, HCC- group were found to be 241 A,A homozyote. Discussion and Conclusion: Our investigation indicates a significant association between ICAM-1 241 polymorphism and the risk of HCC occurring in HCV patients. If this novel observation indicates a causative role for ICAM-1 polymorphism in the development of HCC in HCV patients, or if it is due to linkage disequilibrium needs further investigations.
DECREASE IN CD3-CD8 AND Yy9/V62 TCR+ T CELL COUNT AND LOW PERFORIN EXPRESSION IS ASSOCIATED WITH AN IMPAIRMENT OF NATURAL KILLER CELL ACTIVITY IN HEPATITIS C VIRUS (HCV) INFECTION. THE ENHANCING EFFECTS OF INTERFERONa 2B TREATMENT G. P/tr, A. P~,r, D. Rukavina l, M. Horfinvi, J. Szekeres-Bartho, G. Hegedtis, M. Pail.l, GY. M6zsik University Medical School, P~cs, Hungary. IUniversity of Rijeka, Rijeka, Croatia. Aim: As chronic HCV infection is associated with an impaired NK cytotoxicity, our aim was to elucidate the molecular background of this phenomenon. We studied the phenotype changes in peripheral blood lymphocytes, distribution of perforin-bearing lymphocytes and NK cytotoxicity in chronic HCV infection and followed-up the effect of IFN therapy. Patients: A total of 52 HCV patients have been studied, out of them 12 have been on IFN treatment, 8 were HCV carriers with normal serum ALT. Methods: Three-color fluorescence-activated cytometric analysis was used to measure the percentage of CD3*/CD8, CD3*CD4, 78 TCR+, CD16, CD56, CD19, CD28 and perforin-positive cells, the percentage of Vy9/V82 TCR+ T cells were detected by immunocytochemistry. Results: Patients with active hepatitis C showed a significant decrease in the CD3CD8 (a natural killer subtype) and perforin-positive cells as well as in V79/V82 TCR+ lymphocytes as compared to normal control. IFN raised the percentage of CD28*CD8 (cytotoxic T cell) from 50% to 67% and perforinpositive lymphocytes from 17% to 29%, and it was accompanied with an 141% increase in NK cytotoxicity. Conclusion: Our findings suggest that in chronic HCV infection CD3"CD8, V79/V82 TCR+ and perforin-positive cells are responsible for the impaired NK cell activity, which can be enhanced by IFN treatment.
I P/C05/18 I
I P/C05/20 I
BENEFICIAL EFFECT OF INTERFERON-a 2b ON FREE RADICAL GENERATION AND LIPID PEROXIDATION DURING TUMOR PROMOTION G. Koci6, R. Koci6, D. Pavlovic, T. Jevtovic, D. Sokolovic, B. Kocic Inst. of Biochem. and Clin. for Endocrinol. Fac. Med Nis, Yugoslavia. A substantial amount of evidence implicated that active oxygen species may play., a role in the tumor promotion process of multistage carcinogenesis, xanthine oxidase, as a rate-limitmg enzyme in purine degradation has a critical importance as a generator of free oxygen radicals. Persistent hepatitis B or C seems to be associated with the accelerated progression towards hepatocellular carcinoma and it was already documented that INFct-2b is able to down regulate the transcription of genes whose expression is initiated upon the activation of tumor promoters-activators of protein kinase C. The aim of this study was to explore whether 1NF-ct2b can be effective in preventing effect of protein kinase C activating agent-phorbol myristate acetate (PMA) on lipid peroxidation and xanthine oxidase activation. Hepatocytes were isolated under sterile conditions, by using collagenase (Img/ml of RPMI 1640 medium) and collagenase was inbibited by using 10%FCS. Hepatocytes were maintained in RPMI 1640 medium (with 5%FCS) for 24h divided in four groups: exposed to PMA (25ng/ml), exposed to iNF-ct2b(0.06U/ml), preincubated for lh wit~ PMA (25ng/ml) and then exposed simultaneously to INF-2b(0.06U/ml) and control ones. The activity of xanthine oxidase increased after PMA treatment (3.424-0.28 vs control 2.97+0.53 U/g prot. p<0.05) and correlates with its ability to produce free radicals, since the lipid peroxidation(MDA) level was also increased (9.574-0.71 vs control 8.544-0.62 nM MDA/ml p<0.05). The si=nultaneous incubation with INFc~-2b appears to be sufficient to decrease xanthine oxidase activity (3.214-0.31 p<0.05) and decreased lipid peroxide level less than control value (7.744-0.89 p<0.001). Given alone INFot-2b was very effective in decreasing xanthine oxidase activity bellow normal value (2.6+0.30 p<0.05) and also lipid peroxide level (5.48+0.10 P<0.001). In accord with this view it can be proposed that observed effect of lNFct-2b can be one of the pathways how INFct-2b can antagonize cellular response to positive effectors of tumor promotion.
DIFFERENT EFFECTS OF TYPE I-INTERFERONS ON THE HBVENHANCER I-, ENHANCER H- AND CORE PROMOTER-REGULATED TRANSCRIPTION E. Schulte-Frohlinde, I. Burkard, T. Freilinger, M. Classen, G. Foster l, B. Seidler Department of Medicine II, Technical University of Munich, Munich, Germany. 1The Liver Unit, St. Mary's Hospital, London, UK.
Background:Type I -Interferons (IFNs) exert a direct antiviral effect on Hepatitis B Virus (HBV)- replication in vitro and in vivo. At least 9 IFNc-subtypes can be isolated from human blood leucocytes. A characterization of the effect of different type I-IFNs on the HBVenhancer (enh)l, enhU and core promoter-regulated transcription has not been performed yet. Methods: HuH7-cells were transiently transfected with a reporter-plasmid, which encodes the firefly luciferase(luc.)--gene under control of the HBV-enhl,enhll and core promoter of HBVadw2. A deleted plasmid encoding the renilla luc.-gene served as a control. The transfected cells were incubated with 0.1 and 0.5 ng/ml of IFN~I, 2a, 2b, 5, 8, 10, 14, 17, 21, 131a, con1. The specific inhibition was determined by the ratio of firefiy/renilla-luc.activities. Results: A dosedependent inhibition of luc. activities could be observed for each IFN tested. Specific inhibition at a dose of 0.1 ng/ml ranged from 14 + 2.5 % for IFN~zl to 42 + 4.5% for IFNc~17. At equimolar concentrations inhibition was most pronounced for IFN¢8, IFNI~la and consensus IFN. Conclusion: The effect of different type I-IFNs on HBVenhl. enhll and core promoter-regulated transcription varies markedly. However, the type I-IFNs commonly used for antiviral treatment of chronic hepatitis B do not represent the most efficient ones in the assay applied.
93
I P/C05/17 I
[ P/C05/19 I
ICAM-1 POLYMORPHISM: NEW RISK FACTOR FOR HEPATOCELLULAR CARCINOMA IN PATIENTS WITH CHRONIC HEPATITIS C? P.K. Constantini, M. Egg, I.W. Graziadei, W. Vogel Dp. of Hepatology, Innsbruck University, Austria. Recent studies have shown an association between genetic polymorphism and the outcome of chronic Hepatitis C infection (I-ICV), which is known to be a class II mediated type of inflammation. Intercellular adhesion molecules like ICAM-1 influence the cell to cell interaction and thus the activation of this pathway. The aim of this study was to investigate a possible association between ICAM-1 polymorphism at codon 241 (exon 4) and clinical progression of chronic HCV-infection. Patients and Methods: We investigated a group of 93 patients with chronic HCV infection, all of them were antibody and RNA positive and had cirrhosis, 20 of them developed hepatoeellular carcinoma (HCC). ICAM polymorphism 241 was performed by using PCR and sequence specific primers. The genotype distribution of the population with HCC (HCV+, HCC+) was compared with those 73, who had no evidence of carcinoma (HCV+, HCC-). Results: The 241 A,G 8enotype was found at reduced frequency in patients of the HCV+ and HCC+ group. Only 5% (1/20) in the HCV+, HCC+ group versus 22% (16/73) of the HCV+ but HCC- population were found to have the 241 A,G genotype (p< 0.05). None (0/20) of the HCV+, HCC+ patients and only one (1/73) of the HCV+, HCC- group were found to be 241 A,A homozyote. Discussion and Conclusion: Our investigation indicates a significant association between ICAM-1 241 polymorphism and the risk of HCC occurring in HCV patients. If this novel observation indicates a causative role for ICAM-1 polymorphism in the development of HCC in HCV patients, or if it is due to linkage disequilibrium needs further investigations.
DECREASE IN CD3-CD8 AND Yy9/V62 TCR+ T CELL COUNT AND LOW PERFORIN EXPRESSION IS ASSOCIATED WITH AN IMPAIRMENT OF NATURAL KILLER CELL ACTIVITY IN HEPATITIS C VIRUS (HCV) INFECTION. THE ENHANCING EFFECTS OF INTERFERONa 2B TREATMENT G. P/tr, A. P~,r, D. Rukavina l, M. Horfinvi, J. Szekeres-Bartho, G. Hegedtis, M. Pail.l, GY. M6zsik University Medical School, P~cs, Hungary. IUniversity of Rijeka, Rijeka, Croatia. Aim: As chronic HCV infection is associated with an impaired NK cytotoxicity, our aim was to elucidate the molecular background of this phenomenon. We studied the phenotype changes in peripheral blood lymphocytes, distribution of perforin-bearing lymphocytes and NK cytotoxicity in chronic HCV infection and followed-up the effect of IFN therapy. Patients: A total of 52 HCV patients have been studied, out of them 12 have been on IFN treatment, 8 were HCV carriers with normal serum ALT. Methods: Three-color fluorescence-activated cytometric analysis was used to measure the percentage of CD3*/CD8, CD3*CD4, 78 TCR+, CD16, CD56, CD19, CD28 and perforin-positive cells, the percentage of Vy9/V82 TCR+ T cells were detected by immunocytochemistry. Results: Patients with active hepatitis C showed a significant decrease in the CD3CD8 (a natural killer subtype) and perforin-positive cells as well as in V79/V82 TCR+ lymphocytes as compared to normal control. IFN raised the percentage of CD28*CD8 (cytotoxic T cell) from 50% to 67% and perforinpositive lymphocytes from 17% to 29%, and it was accompanied with an 141% increase in NK cytotoxicity. Conclusion: Our findings suggest that in chronic HCV infection CD3"CD8, V79/V82 TCR+ and perforin-positive cells are responsible for the impaired NK cell activity, which can be enhanced by IFN treatment.
I P/C05/18 I
I P/C05/20 I
BENEFICIAL EFFECT OF INTERFERON-a 2b ON FREE RADICAL GENERATION AND LIPID PEROXIDATION DURING TUMOR PROMOTION G. Koci6, R. Koci6, D. Pavlovic, T. Jevtovic, D. Sokolovic, B. Kocic Inst. of Biochem. and Clin. for Endocrinol. Fac. Med Nis, Yugoslavia. A substantial amount of evidence implicated that active oxygen species may play., a role in the tumor promotion process of multistage carcinogenesis, xanthine oxidase, as a rate-limitmg enzyme in purine degradation has a critical importance as a generator of free oxygen radicals. Persistent hepatitis B or C seems to be associated with the accelerated progression towards hepatocellular carcinoma and it was already documented that INFct-2b is able to down regulate the transcription of genes whose expression is initiated upon the activation of tumor promoters-activators of protein kinase C. The aim of this study was to explore whether 1NF-ct2b can be effective in preventing effect of protein kinase C activating agent-phorbol myristate acetate (PMA) on lipid peroxidation and xanthine oxidase activation. Hepatocytes were isolated under sterile conditions, by using collagenase (Img/ml of RPMI 1640 medium) and collagenase was inbibited by using 10%FCS. Hepatocytes were maintained in RPMI 1640 medium (with 5%FCS) for 24h divided in four groups: exposed to PMA (25ng/ml), exposed to iNF-ct2b(0.06U/ml), preincubated for lh wit~ PMA (25ng/ml) and then exposed simultaneously to INF-2b(0.06U/ml) and control ones. The activity of xanthine oxidase increased after PMA treatment (3.424-0.28 vs control 2.97+0.53 U/g prot. p<0.05) and correlates with its ability to produce free radicals, since the lipid peroxidation(MDA) level was also increased (9.574-0.71 vs control 8.544-0.62 nM MDA/ml p<0.05). The si=nultaneous incubation with INFc~-2b appears to be sufficient to decrease xanthine oxidase activity (3.214-0.31 p<0.05) and decreased lipid peroxide level less than control value (7.744-0.89 p<0.001). Given alone INFot-2b was very effective in decreasing xanthine oxidase activity bellow normal value (2.6+0.30 p<0.05) and also lipid peroxide level (5.48+0.10 P<0.001). In accord with this view it can be proposed that observed effect of lNFct-2b can be one of the pathways how INFct-2b can antagonize cellular response to positive effectors of tumor promotion.
DIFFERENT EFFECTS OF TYPE I-INTERFERONS ON THE HBVENHANCER I-, ENHANCER H- AND CORE PROMOTER-REGULATED TRANSCRIPTION E. Schulte-Frohlinde, I. Burkard, T. Freilinger, M. Classen, G. Foster l, B. Seidler Department of Medicine II, Technical University of Munich, Munich, Germany. 1The Liver Unit, St. Mary's Hospital, London, UK.
Background:Type I -Interferons (IFNs) exert a direct antiviral effect on Hepatitis B Virus (HBV)- replication in vitro and in vivo. At least 9 IFNc-subtypes can be isolated from human blood leucocytes. A characterization of the effect of different type I-IFNs on the HBVenhancer (enh)l, enhU and core promoter-regulated transcription has not been performed yet. Methods: HuH7-cells were transiently transfected with a reporter-plasmid, which encodes the firefly luciferase(luc.)--gene under control of the HBV-enhl,enhll and core promoter of HBVadw2. A deleted plasmid encoding the renilla luc.-gene served as a control. The transfected cells were incubated with 0.1 and 0.5 ng/ml of IFN~I, 2a, 2b, 5, 8, 10, 14, 17, 21, 131a, con1. The specific inhibition was determined by the ratio of firefiy/renilla-luc.activities. Results: A dosedependent inhibition of luc. activities could be observed for each IFN tested. Specific inhibition at a dose of 0.1 ng/ml ranged from 14 + 2.5 % for IFN~zl to 42 + 4.5% for IFNc~17. At equimolar concentrations inhibition was most pronounced for IFN¢8, IFNI~la and consensus IFN. Conclusion: The effect of different type I-IFNs on HBVenhl. enhll and core promoter-regulated transcription varies markedly. However, the type I-IFNs commonly used for antiviral treatment of chronic hepatitis B do not represent the most efficient ones in the assay applied.
93