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Hypervariable region 1 quasispecies in chronic hepatitis C patients: influence of genotype
110
Poster Sessions
dot blot hybridization. We have found that both RC and ccc DNA species are present in sera samples from HBV infected patients, and that both DNA forms were resistant to digestion with DNase-I. By employing CsCl and sucrose gradients, as well as Immunoprecipitation techniques, we show that similar to the RC-DNA, HBV ccc-DNA is associated with the viral envelope and core proteins. These results suggest that the ccc DNA, found in the serum of HBV infected patients, is packed within the HBV Dane particles. We could not find any correlation between the clinical condition of the patients or HBV titers and the presence of HBV ccc-DNA in the sera.
0 91
DIRECT DETECTION OF HCV BY SOLID PHASE HYBRIDIZATION WITH FLUORESCENT PROBE
E.N. Ilina, V.M. Govorun, E.A. Klimova, E.A. Gagikulieva ‘, N.D. Yuschuk, E.V. Generozov. Institute of Physical-Chemical Medicine, Moscow; I M. Pirogovskaia, Russia Hepatitis C virus (HCV) account for approximately 20% of cases of acute hepatitis and 70% of chronic hepatitis in the world. Difficulties in clinical diagnostics as well as often asymptomatic exposure to virus and high probability of disease transformation in a chronic infection demand improvement of diagnostics and monitoring methods. Up to day in modem diagnostic practice the most common method of virus DNA detection is gel-electrophoresis of RT-PCR amplification products from patient serum. We developed the detection system based on solid phase direct hybridization with FITC-labeled probes. Usage of highly sensitive fluorometer Fluoroskan Ascent (“Labsystems”) allow us to detect the complex of amplicone and probe directly after hybridization step. Intensity of the signal in this case reflect the concentration of amplicone in a probe. Amplification parameters were selected in a way to allow the detection of the samples with different concentration. This method was clinically tested on a group of HCV positive patients.
0 92
SIGNIFICANCE OF CHANGING IN GENETIC HETEROGENEITY OF HCV GENOME IN ACUTE HEPATITIS C
E.N. Ilina, V.M. Govorun, E.A. Klimova, E.A. Gagikulieva, N.D. Yuschuk. Institute of physico-chemical medicine, Russian ministry of health; The Moscow State Medico-stomathologi Universaty, Russia Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of sub-types have been reported. More over in infected individuals HCV exists as a variable complex population of related genetic variants known as quasispecies. Some researchers suggest that the high level of genomic heterogeneity was mostly associated with nonresponse to interferon-alpha, relapes and cirrhosis. Presented this work we assume that measurements in acute phase of illness the divergention of individual population of HCV using HVRl loci as a marker could help in hepatitis C development prediction. The aim of present investigation was the estimation of the clinical significance of the genomic heterogeneity and diversity of individual HCV population on patients with acute hepatitis C. The hypervariable region (HVRl) of the HCV was amplified by reverse transcription and PCR. The PCR products were analyzed by SSCP. Our data suggest that there is no correlation between genome heterogeneity degree, titer of HCV RNA and serum alanine aminotransferase (ALT) level. The observed changes in HCV genomic heterogeneity and fluctuation of serum viremia level may be used as indirect markers of viral replication in hepatocites.
I
100
ROLE OF REACTIVE OXYGEN SPECIES INDUCED BY IRON LOAD IN CHRONIC HEPATlTlS C
Carmen Fierbinteanu Braticevici, D. Andronescu. Medical Clinic Iv Department of Gastroenterology, University Hospital, Bucharest, Romania Iron is potentially toxic to tissues and increased formation of reactive oxygen species which explain lipid peroxidation and development of hepatic lesions. Aim: to evaluate the relationship between iron load and liver inflamatory degenerative processes in chronic viral hepatitic C. Methods: 100 subjects (aged 3345) were included in this study and divided as follows: 50 patients affected by chronic hepatitis C virus and 50 healthy controls matched for sex and age. Iron load was evaluated by serum iron, ferritin and trensferin saturation. Blood lipid peroxidation assesed by the level of malonyl-dialdehyde (MDA) and serum total gluthation (GSH) were analysed in both groups. In hepatitis C virus patients liver biopsy specimens were obtained for hepatic iron determination and for histologic examination; a semiquantitative score for portal inflamation necrosis and fibrosis was applied to liver biopsy. Results: Serum indices of iron load were higher in hepatitis C virus patients than in controls and were higher in cirrhotic than in chronic hepatitic cases. The level of MDA was correlated with higher iron status and GSH decrease was reverse proportionale with the severity of hepatic lesions. Conclusions: The increase of iron load stimulates inflammatory and degenerative hepatic processes adding oxygen free radical injury to damage of viral infection.
0 105
HYPERVARIABLE REGION 1 QUASISPECIES IN CHRONIC HEPATITIS C PATIENTS: INFLUENCE OF GENOTYPE
Tarik Asselah, Michelle Martinot-Peignoux, Dominique Cazals-Hatem, Nathalie Boyer, Dominique Valla, Claude Degott, Patrick Marcellin. INSERM 17481,Hopital Beaujon, Clichy, France All studies on HCV heterogeneity focused on patients infected with genotype lb infection and abnormal serum ALT levels. Our aim was to study the hypervariable region 1 (HVRI) heterogeneity in patients with chronic hepatitis C infected with genotype lb or 3 and normal or abnormal serum ALT levels. Patients and Methods: HVRl quasispecies were assessed in 67 patients with chronic hepatitis C, including 35 patients with persistently normal serum ALT levels and 32 patients with abnormal serum ALT levels. A PCR-SSCP was performed with specific primers for genotype lb and 3a located in the HVRl. The characteristics studied were gender, age, source and duration of infection, serum ALT levels, HCV genotype, serum HCV RNA levels, histologic lesions. Results: Among the 67 patients, 40 were infected with genotype lb and 27 with genotype 3. The mean band observed was 3.05 f 1.36 (range: l-7). In univariate analysis, low heterogeneity was significantly associated with persistently normal serum ALT levels (p < O.OOl), genotype lb (p = 0.07), and milder histologic lesions (activity, p = 0.02; fibrosis, p = 0.04). In multivariate analysis, low heterogeneity was significantly and independently associated with persistently normal serum ALT levels (p = 0.0005) and genotype lb (p = 0.03). Conclusion: In our study with a multivariate analysis we show that a low viral heterogeneity is significantly and independently associated with normal serum ALT levels and genotype lb.
Poster Sessions
dot blot hybridization. We have found that both RC and ccc DNA species are present in sera samples from HBV infected patients, and that both DNA forms were resistant to digestion with DNase-I. By employing CsCl and sucrose gradients, as well as Immunoprecipitation techniques, we show that similar to the RC-DNA, HBV ccc-DNA is associated with the viral envelope and core proteins. These results suggest that the ccc DNA, found in the serum of HBV infected patients, is packed within the HBV Dane particles. We could not find any correlation between the clinical condition of the patients or HBV titers and the presence of HBV ccc-DNA in the sera.
0 91
DIRECT DETECTION OF HCV BY SOLID PHASE HYBRIDIZATION WITH FLUORESCENT PROBE
E.N. Ilina, V.M. Govorun, E.A. Klimova, E.A. Gagikulieva ‘, N.D. Yuschuk, E.V. Generozov. Institute of Physical-Chemical Medicine, Moscow; I M. Pirogovskaia, Russia Hepatitis C virus (HCV) account for approximately 20% of cases of acute hepatitis and 70% of chronic hepatitis in the world. Difficulties in clinical diagnostics as well as often asymptomatic exposure to virus and high probability of disease transformation in a chronic infection demand improvement of diagnostics and monitoring methods. Up to day in modem diagnostic practice the most common method of virus DNA detection is gel-electrophoresis of RT-PCR amplification products from patient serum. We developed the detection system based on solid phase direct hybridization with FITC-labeled probes. Usage of highly sensitive fluorometer Fluoroskan Ascent (“Labsystems”) allow us to detect the complex of amplicone and probe directly after hybridization step. Intensity of the signal in this case reflect the concentration of amplicone in a probe. Amplification parameters were selected in a way to allow the detection of the samples with different concentration. This method was clinically tested on a group of HCV positive patients.
0 92
SIGNIFICANCE OF CHANGING IN GENETIC HETEROGENEITY OF HCV GENOME IN ACUTE HEPATITIS C
E.N. Ilina, V.M. Govorun, E.A. Klimova, E.A. Gagikulieva, N.D. Yuschuk. Institute of physico-chemical medicine, Russian ministry of health; The Moscow State Medico-stomathologi Universaty, Russia Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of sub-types have been reported. More over in infected individuals HCV exists as a variable complex population of related genetic variants known as quasispecies. Some researchers suggest that the high level of genomic heterogeneity was mostly associated with nonresponse to interferon-alpha, relapes and cirrhosis. Presented this work we assume that measurements in acute phase of illness the divergention of individual population of HCV using HVRl loci as a marker could help in hepatitis C development prediction. The aim of present investigation was the estimation of the clinical significance of the genomic heterogeneity and diversity of individual HCV population on patients with acute hepatitis C. The hypervariable region (HVRl) of the HCV was amplified by reverse transcription and PCR. The PCR products were analyzed by SSCP. Our data suggest that there is no correlation between genome heterogeneity degree, titer of HCV RNA and serum alanine aminotransferase (ALT) level. The observed changes in HCV genomic heterogeneity and fluctuation of serum viremia level may be used as indirect markers of viral replication in hepatocites.
I
100
ROLE OF REACTIVE OXYGEN SPECIES INDUCED BY IRON LOAD IN CHRONIC HEPATlTlS C
Carmen Fierbinteanu Braticevici, D. Andronescu. Medical Clinic Iv Department of Gastroenterology, University Hospital, Bucharest, Romania Iron is potentially toxic to tissues and increased formation of reactive oxygen species which explain lipid peroxidation and development of hepatic lesions. Aim: to evaluate the relationship between iron load and liver inflamatory degenerative processes in chronic viral hepatitic C. Methods: 100 subjects (aged 3345) were included in this study and divided as follows: 50 patients affected by chronic hepatitis C virus and 50 healthy controls matched for sex and age. Iron load was evaluated by serum iron, ferritin and trensferin saturation. Blood lipid peroxidation assesed by the level of malonyl-dialdehyde (MDA) and serum total gluthation (GSH) were analysed in both groups. In hepatitis C virus patients liver biopsy specimens were obtained for hepatic iron determination and for histologic examination; a semiquantitative score for portal inflamation necrosis and fibrosis was applied to liver biopsy. Results: Serum indices of iron load were higher in hepatitis C virus patients than in controls and were higher in cirrhotic than in chronic hepatitic cases. The level of MDA was correlated with higher iron status and GSH decrease was reverse proportionale with the severity of hepatic lesions. Conclusions: The increase of iron load stimulates inflammatory and degenerative hepatic processes adding oxygen free radical injury to damage of viral infection.
0 105
HYPERVARIABLE REGION 1 QUASISPECIES IN CHRONIC HEPATITIS C PATIENTS: INFLUENCE OF GENOTYPE
Tarik Asselah, Michelle Martinot-Peignoux, Dominique Cazals-Hatem, Nathalie Boyer, Dominique Valla, Claude Degott, Patrick Marcellin. INSERM 17481,Hopital Beaujon, Clichy, France All studies on HCV heterogeneity focused on patients infected with genotype lb infection and abnormal serum ALT levels. Our aim was to study the hypervariable region 1 (HVRI) heterogeneity in patients with chronic hepatitis C infected with genotype lb or 3 and normal or abnormal serum ALT levels. Patients and Methods: HVRl quasispecies were assessed in 67 patients with chronic hepatitis C, including 35 patients with persistently normal serum ALT levels and 32 patients with abnormal serum ALT levels. A PCR-SSCP was performed with specific primers for genotype lb and 3a located in the HVRl. The characteristics studied were gender, age, source and duration of infection, serum ALT levels, HCV genotype, serum HCV RNA levels, histologic lesions. Results: Among the 67 patients, 40 were infected with genotype lb and 27 with genotype 3. The mean band observed was 3.05 f 1.36 (range: l-7). In univariate analysis, low heterogeneity was significantly associated with persistently normal serum ALT levels (p < O.OOl), genotype lb (p = 0.07), and milder histologic lesions (activity, p = 0.02; fibrosis, p = 0.04). In multivariate analysis, low heterogeneity was significantly and independently associated with persistently normal serum ALT levels (p = 0.0005) and genotype lb (p = 0.03). Conclusion: In our study with a multivariate analysis we show that a low viral heterogeneity is significantly and independently associated with normal serum ALT levels and genotype lb.