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Liver HCV RNA is correlated to Fas mRNA and to apoptosis in liver transplanted patients infected by hepatitis C
Viral hepatitis: basic aspects
TYPE I INTERFERON GENE EXPRESSION IN CHRONIC HEPATITIS C VIRUS INFECTION Y. Castelruiz. E. Larrea. A. Alberdi. P. Bova. M.P. Civeira. J. Prieto
Dpt. of Internal Medicine, University of Navarra, Pamplona, Spain. Type I interferons (IFN-a and IFN-8) are a family of cytokines produced in response to viral infection. Their main function is to make the cell resistant to viral replication. Hepatitis C virus (HCV) has a strong tendency to establish chrome infection, suggesting deficient activation of the endogenous IPN system in the infected patients. The aim of this study was to determine IFN-a and IPN-8 gene expression in peripheral blood mononuclear cells (PBMC) and in the liver of patients with chronic hepatitis C. Methods: We used semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR) to measure IFN-a and IPN-8 mRNA levels in PBMC and in liver of 15 HCV-RNA positive patients and in 15 controls. 8-actin gene expression was used as internal control. We also determined EN-a mRNA levels in PBMC after in vitro Sendai virus stimulation (16 HAU/mL, 6 hours, 37 ‘C, 5% CO,) from 34 HCV-RNA (+) patients and 20 controls. None of the patients had been treated at the time of the study. &g&: The expression of IFN-a and IPN-B in PBMC was significantly higher in HCV-RNA (+) patients compared to controls (3.2f0.48 vs. 1.141t0.26; p=O.OOl and 1.66kO.2 vs. 0.88*0.16; p=O.O08, IF%a and IFN-8 respectively). &N-l3 mRNA levels in liver were, also, significantly higher in HCV-RNA (f) patients than in controls (1.73f0.23 vs. 0.941tO.12; p=O.OOZ), however we found a significant decrease in EN-a gene expression in the liver of the patients as compared to those found in normal livers (0.12*0.03 vs. 0.37iO.09; p=O.OOS). When we stimulated PBMC with Sendai virus, IFN-a mRNA levels in these cells rose in both groups reaching similar values. Conclusions: Patients with chronic hepatitis C show increased expression of IPN-8 in both, the liver and PBMC. In contrast, in these patients IPN-a mRNA levels are increased in PBMC but reduced in liver tissue, suggesting a selective blockade of this form of interferon in infected hepatocytes which could contribute to chronicity of the infection.
1 P/CO5/014
97
EPITOPE MAPPING OF THE ‘A’ DETERMINANT REGION OF HBV VARIANTS REVEAL MARKED CHANGES: IMPLICATIONS FOR DESIGN OF ASSAYS FOR DIAGNOSIS OF HBV INFECTION. S. Tonekaboni. J. Waters, S. Jeffers. P. Karaviannis. H.C.Thomas Department of Medicine A, Imperial College School of Medicine, Mary’s Hospital, London, UK.
St
Hepatitis B surface antigen (HBsAg) induces a protective immune response to hepatitis B virus (HBV), a response which can be induced following recovery from infection and after vaccination. The antibody is directed against a hydrophilic region of HBsAg known as the ‘a’ antigenic determinant, which lies between amino-acids (aa) 110-160. In recent years, it has become apparent that HBsAg and anti-HBs can co-exist in the serum of some patients, particularly in those who developed anti-HBs after immunisation. A variant virus with an aa change at residue 145 from gly to arg, first described in a vaccinated child born to a carrier mother, has an altered antigenicity and immunogenicity. Other variants have mutations affecting positions 141 (lys to glu), 142 (pro to ser), 146 (asn to asp) or insertions of two or three aa between positions 122 and 124. Sequences encoding the entire HBsAg region from the serum of patients with atypical serological profiles (HBsAg -ve, anti-HBe +ve, HBVDNA+ve, anti-HBs +ve), were amplified by PCR and cloned into the insertion vector pHILD2 The latter was used to generate recombinant Picnia pastoris. The recombinant variant proteins expressed by the yeast, were released by mechanical disruption of the cells and precipitated by ammonium sulphate. Following dialysis the proteins were employed in a range of immunoassays to test their ability to bind to both polyclonal and monoclonal antibodies recognising the native protein. The binding studies showed marked changes in the epitopes displayed by the variants, These observations have important implications for design of HBsAg assays for blood screening and diagnostic practice
]
LIVER HCV RNA IS CORRELATED TO FAS mRNA AND TO APOPTOSIS IN LIVER TRANSPLANTED PATIENTS INFECTED BY HEPATITIS C. C Fhav. C Brenot, V Di M&no. D Samuel. F Saurini, MA Petit. M Revnbs. H Bismuth. Centre Hepato-Biliaire. HBpital Paul Brousse. Villejuif. France. Previous studies suggested that fas-mediated apoptosis is involved during infection by Hepatitis C Virus. We studied liver apoptosis, transcription of Fas and replication of HCV during hepatitis C after liver transplantation (LT). Samples. Sixty-one paraftinembeddedliver graft biopses harvested in 28 HCV-RNA positive patients (pts) transplanted for HCV-related cirrhosis showing after LT either normal histology (n=13), lobular hepatitis (LH) (n=16), chronic hepatitis (CH) (n=27) or rejection (n=15). Methods. DNA fragmentation was determined and semi-quantified by In Situ End-Labelling (ISEL) method (Apoptag, Oncor,) on deparaflined 3 pm-thick sections and semi-quantified (from 0 to 3). Liver Fas mRNA and liver HCV RNA were quantified according competitive PCR and titration respectively and normalized to the quantitation of an internal standard (ribosomal 28s RNA). Liver tissues were scored through Metavir Score in case of CH. Results. Lobular apoptosis (LA) demonstrated by ISEL was significantly more pronouncedin pts with LH (0.94f0.8 vs 0.42 ti.65; ~~0.01) and portal apoptosis (PA) was more frequent in case of CH (1.4fl vs 0.8*85; p=O.O2). Both LA andPA were significantly more elevatedin case of rejection. Liver HCV RNA and titration of Fas mRNA were significantly elevated in case of LH or of CH. An extremely strong positive correlation was observed between the levels of liver Fas mRNA and of liver HCV RNA (p
PHYLOGENETIC ANALYSIS OF HEPATITIS G VIRUS (HGV) S-NON CODING REGION AND DEVELOPMENT OF A RAPID PCR-BASED TYPING ASSAY. 0 M.U. Departments of Pathology and ‘Infectious Diseases, University and IRCCS S. Matteo, §IGBE, CNR, Pavia; *A.S.A.E.V., Bonate Sotto, Italy; #R&D Boebrlnger Mannheim, Pen&erg, Germany. Sequence analysis of viral isolates is a powerful epidemiologic and phylogenetic tool, which can shed light on the patterns of regional and pandemic spread of infections. We studied 80 worldwide isolates of HGV for sequence variation in subgenomic fragments of the 5’ non coding (SNCR), E2 and NS3 regions using 3 methods of phylogenetic inference and statistical comparison of pairwise evolutionary distances, according to a critical framework elaborated for HCV analysis. In agreement with previous studies, phylogenetic informativeness was restricted to SNCR, whereas no significant segregation was observed for variants from other regions. Reproducible phylogenetic relationships were obtained supporting the existence of at least 4 main HGV types. Three types (l-3) were as previously described (AS Muerhoff et al, J Viral 1997, DB Smith et al, J Gen Viral 1997) and segregated according to geographical origins and major ethnic groups. South African isolates were phylogenetically distinct from other African isolates (type 1) and showed and intra!mter-type distribution of variation compatible with the segregation into a new HGV type (type 4). Asian isolates (Japan, Indonesia, Taiwan) were essentially type 3. European (Italy, Germany, Eastern European countries) and Midlle Eastern (Egypt, Saudi Arabia) isolates were mainly type 2, a notable exception being type 1 isolates which were restricted to young intravenous drug users. This suggests a recent change in the epidemiology of HGV infection as observed for HCV. Comparing a large database of SNCR sequences, we identified signature motifs specific for the different HGV types and developed and validated a rapid PCR-based typing assay which makes use of 5 (1, 2a, 2b, 3 and 4) type-specific primers and gel sizing of PCR products. Using this assay, we typed over 150 Italian isolates from patients with cryptogenic and HCVrelated liver disease of different severity. No differences in type distribution were observed according to the epidemiologic and pathologic variables analyzed with the exception of a history of drug use in type 1 patients.
TYPE I INTERFERON GENE EXPRESSION IN CHRONIC HEPATITIS C VIRUS INFECTION Y. Castelruiz. E. Larrea. A. Alberdi. P. Bova. M.P. Civeira. J. Prieto
Dpt. of Internal Medicine, University of Navarra, Pamplona, Spain. Type I interferons (IFN-a and IFN-8) are a family of cytokines produced in response to viral infection. Their main function is to make the cell resistant to viral replication. Hepatitis C virus (HCV) has a strong tendency to establish chrome infection, suggesting deficient activation of the endogenous IPN system in the infected patients. The aim of this study was to determine IFN-a and IPN-8 gene expression in peripheral blood mononuclear cells (PBMC) and in the liver of patients with chronic hepatitis C. Methods: We used semiquantitative reverse transcriptasepolymerase chain reaction (RT-PCR) to measure IFN-a and IPN-8 mRNA levels in PBMC and in liver of 15 HCV-RNA positive patients and in 15 controls. 8-actin gene expression was used as internal control. We also determined EN-a mRNA levels in PBMC after in vitro Sendai virus stimulation (16 HAU/mL, 6 hours, 37 ‘C, 5% CO,) from 34 HCV-RNA (+) patients and 20 controls. None of the patients had been treated at the time of the study. &g&: The expression of IFN-a and IPN-B in PBMC was significantly higher in HCV-RNA (+) patients compared to controls (3.2f0.48 vs. 1.141t0.26; p=O.OOl and 1.66kO.2 vs. 0.88*0.16; p=O.O08, IF%a and IFN-8 respectively). &N-l3 mRNA levels in liver were, also, significantly higher in HCV-RNA (f) patients than in controls (1.73f0.23 vs. 0.941tO.12; p=O.OOZ), however we found a significant decrease in EN-a gene expression in the liver of the patients as compared to those found in normal livers (0.12*0.03 vs. 0.37iO.09; p=O.OOS). When we stimulated PBMC with Sendai virus, IFN-a mRNA levels in these cells rose in both groups reaching similar values. Conclusions: Patients with chronic hepatitis C show increased expression of IPN-8 in both, the liver and PBMC. In contrast, in these patients IPN-a mRNA levels are increased in PBMC but reduced in liver tissue, suggesting a selective blockade of this form of interferon in infected hepatocytes which could contribute to chronicity of the infection.
1 P/CO5/014
97
EPITOPE MAPPING OF THE ‘A’ DETERMINANT REGION OF HBV VARIANTS REVEAL MARKED CHANGES: IMPLICATIONS FOR DESIGN OF ASSAYS FOR DIAGNOSIS OF HBV INFECTION. S. Tonekaboni. J. Waters, S. Jeffers. P. Karaviannis. H.C.Thomas Department of Medicine A, Imperial College School of Medicine, Mary’s Hospital, London, UK.
St
Hepatitis B surface antigen (HBsAg) induces a protective immune response to hepatitis B virus (HBV), a response which can be induced following recovery from infection and after vaccination. The antibody is directed against a hydrophilic region of HBsAg known as the ‘a’ antigenic determinant, which lies between amino-acids (aa) 110-160. In recent years, it has become apparent that HBsAg and anti-HBs can co-exist in the serum of some patients, particularly in those who developed anti-HBs after immunisation. A variant virus with an aa change at residue 145 from gly to arg, first described in a vaccinated child born to a carrier mother, has an altered antigenicity and immunogenicity. Other variants have mutations affecting positions 141 (lys to glu), 142 (pro to ser), 146 (asn to asp) or insertions of two or three aa between positions 122 and 124. Sequences encoding the entire HBsAg region from the serum of patients with atypical serological profiles (HBsAg -ve, anti-HBe +ve, HBVDNA+ve, anti-HBs +ve), were amplified by PCR and cloned into the insertion vector pHILD2 The latter was used to generate recombinant Picnia pastoris. The recombinant variant proteins expressed by the yeast, were released by mechanical disruption of the cells and precipitated by ammonium sulphate. Following dialysis the proteins were employed in a range of immunoassays to test their ability to bind to both polyclonal and monoclonal antibodies recognising the native protein. The binding studies showed marked changes in the epitopes displayed by the variants, These observations have important implications for design of HBsAg assays for blood screening and diagnostic practice
]
LIVER HCV RNA IS CORRELATED TO FAS mRNA AND TO APOPTOSIS IN LIVER TRANSPLANTED PATIENTS INFECTED BY HEPATITIS C. C Fhav. C Brenot, V Di M&no. D Samuel. F Saurini, MA Petit. M Revnbs. H Bismuth. Centre Hepato-Biliaire. HBpital Paul Brousse. Villejuif. France. Previous studies suggested that fas-mediated apoptosis is involved during infection by Hepatitis C Virus. We studied liver apoptosis, transcription of Fas and replication of HCV during hepatitis C after liver transplantation (LT). Samples. Sixty-one paraftinembeddedliver graft biopses harvested in 28 HCV-RNA positive patients (pts) transplanted for HCV-related cirrhosis showing after LT either normal histology (n=13), lobular hepatitis (LH) (n=16), chronic hepatitis (CH) (n=27) or rejection (n=15). Methods. DNA fragmentation was determined and semi-quantified by In Situ End-Labelling (ISEL) method (Apoptag, Oncor,) on deparaflined 3 pm-thick sections and semi-quantified (from 0 to 3). Liver Fas mRNA and liver HCV RNA were quantified according competitive PCR and titration respectively and normalized to the quantitation of an internal standard (ribosomal 28s RNA). Liver tissues were scored through Metavir Score in case of CH. Results. Lobular apoptosis (LA) demonstrated by ISEL was significantly more pronouncedin pts with LH (0.94f0.8 vs 0.42 ti.65; ~~0.01) and portal apoptosis (PA) was more frequent in case of CH (1.4fl vs 0.8*85; p=O.O2). Both LA andPA were significantly more elevatedin case of rejection. Liver HCV RNA and titration of Fas mRNA were significantly elevated in case of LH or of CH. An extremely strong positive correlation was observed between the levels of liver Fas mRNA and of liver HCV RNA (p
PHYLOGENETIC ANALYSIS OF HEPATITIS G VIRUS (HGV) S-NON CODING REGION AND DEVELOPMENT OF A RAPID PCR-BASED TYPING ASSAY. 0 M.U. Departments of Pathology and ‘Infectious Diseases, University and IRCCS S. Matteo, §IGBE, CNR, Pavia; *A.S.A.E.V., Bonate Sotto, Italy; #R&D Boebrlnger Mannheim, Pen&erg, Germany. Sequence analysis of viral isolates is a powerful epidemiologic and phylogenetic tool, which can shed light on the patterns of regional and pandemic spread of infections. We studied 80 worldwide isolates of HGV for sequence variation in subgenomic fragments of the 5’ non coding (SNCR), E2 and NS3 regions using 3 methods of phylogenetic inference and statistical comparison of pairwise evolutionary distances, according to a critical framework elaborated for HCV analysis. In agreement with previous studies, phylogenetic informativeness was restricted to SNCR, whereas no significant segregation was observed for variants from other regions. Reproducible phylogenetic relationships were obtained supporting the existence of at least 4 main HGV types. Three types (l-3) were as previously described (AS Muerhoff et al, J Viral 1997, DB Smith et al, J Gen Viral 1997) and segregated according to geographical origins and major ethnic groups. South African isolates were phylogenetically distinct from other African isolates (type 1) and showed and intra!mter-type distribution of variation compatible with the segregation into a new HGV type (type 4). Asian isolates (Japan, Indonesia, Taiwan) were essentially type 3. European (Italy, Germany, Eastern European countries) and Midlle Eastern (Egypt, Saudi Arabia) isolates were mainly type 2, a notable exception being type 1 isolates which were restricted to young intravenous drug users. This suggests a recent change in the epidemiology of HGV infection as observed for HCV. Comparing a large database of SNCR sequences, we identified signature motifs specific for the different HGV types and developed and validated a rapid PCR-based typing assay which makes use of 5 (1, 2a, 2b, 3 and 4) type-specific primers and gel sizing of PCR products. Using this assay, we typed over 150 Italian isolates from patients with cryptogenic and HCVrelated liver disease of different severity. No differences in type distribution were observed according to the epidemiologic and pathologic variables analyzed with the exception of a history of drug use in type 1 patients.