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In vitro infection of peripheral blood mononuclear cells by a defective hepatitis B virus
Poster Sessions
112
infusions, day 0, and on days 112 and 394 after infusions were started. In CH1439,59%, 50%. 79%, and 89% of the clones represented the master sequence in four samples, respectively. A second major variant, found in three samples before infusions (12%, 38%, 16% of clones) disappeared by day 112 (end of infusions). In CH1483, the master sequence was depleted from 79% to 45% of clones in samples obtained after the infusions ended. Aminor variant (57%) present during 5-year follow-up period emerged as a predominant sequence (55%) after the infusions. A year later, the master sequence reemerged as a single (100%) species. In CH1556, the master sequence present at about 90% frequency over the 2-year follow-up period was reduced to 67% after the HCIg infusions and six new minor variants emerged. Molecular analysis of HVRl in chronically infected chimpanzees revealed that the immune pressure induced by the passive transfer of HCIg markedly altered the quasispecies pattern of HCV in serum.
I 222
INHIBITION OF CLASS I MHC PRESENTATION ON HEPATOCYTES AND LYMPHOID CELLS IN CHRONIC VIRAL HEPATITIS IN WOODCHUCKS
T.I. Michalak, PD. Hodgson, N.D. Churchill. Molecular virology and Hepatology Research, Faculty of Medicine, Memorial University of Newfoundland, Canada
Woodchuck hepatitis virus (WI-IV), as hepatitis B virus (HBV), casuse acute liver inflammation that can progress to chronic hepatitis and HCC. WI-IV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnaviral hepatitis is characterized by a profound difference in the expression of class I MHC molecules on infected hepatocytes and, notably, lymphoid cells. While acute infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on hepatocytes is a uniform hallmark of chronic WI-IV infection. This inhibition occurs despite augmented expression of hepatic genes for class I MHC heavy chain, beta-2microglobulin, and TAP1 and TAP2. Further, the class I antigen inhibition is not related to the histological severity of liver injury, the level of intrahepatic gamma interferon induction or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained demonstrate that the defective presentation of class I MHC is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHCdependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.
I
239
IN VITRO INFECTION OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY A DEFECTIVE HEPATITIS B VIRUS
M. Cabrerizo, J. Bartolome, V. Carreho. Fundacidn Estudio Hepatitis Virales and Instituo de Hepatologia, Hospital Pardo de Aravaca, Madrid, Spain
We identified a defective HBV strain which contains a 183 deletion in the Sgene promoter in sera from HBsAg negative patients with serum HBV-DNA. To analyze the infectivity of this mutant, peripheral blood mononuclear cells (PBMCs) from a healthy donor were incubated with serum samples from 2 HBsAg negative patients with serum HBV-DNA (with both the wild-type and the deletion mutant), from a HBsAg chronic carrier (only wild-type HBV) and from a healthy donor. After I week, HBV-DNA was detected by PCR in all supematants and PBMCs incubated with the HBV-DNA positive inocula but not with the negative
control. DNaseI and trypsin pretreatment confirmed the intracellular location of HBV in the cells. HBV-RNA and covalently closed circular HBV-DNA were also detected in PBMCs, indicating that the viral DNA infecting these cells was transcriptionally active. Deletion mutant and wild-type HBV were detected in supematant of the PBMCs infected with the 2 HBsAg negative HBV-DNA sera, while only wild-type HBV was detected in the supematant of the cells incubated with the HBsAg inoculum. In conclusion, this HBV deletion mutant can infect and replicate in PBMCs cultured in vitro, and release viral particles to the supematant.
I 249
SERUM FERRITIN CONCENTRATIONS AND HEPATOCELLULAR MITOCHONDRIAL ALTERATIONS IN PATIENTS WITH CHRONIC HEPATITIS C RELATED TO THE HEPATITIS C VIRUS GENOTYPE
G. Barbarini, G. Barbaro, A. Asti, G. Belloni, A. Pellicelli, G. Di Lorenzo, B. Grisorio. Infectious and Tropical Diseases, IRCCS S: Matteo-University of Pavia, Italy
Aim of the Study and Methods: The study considered 130 CHC patients and 30 control cases. In these patients we correlated the levels of serum ferritin and concentrations of hepatic, plasmatic and lymphocytic GSH and erytbrocyte malonyldialdehyde. Moreover, we correlated the ultrastructural findings of liver biopsy specimens with thelipoperoxidation markers and the contents of mt. DNA in a selected cohort of patients in relation to HCV genotype. Results: Patients with genotype I showed higher levels of serum ferritin compared to patients with genotype 2 and 3 and to controls with a significant reduction of H-GSH, P-GSH, L-GSH concentrations and PBMC cytotoxic activity. Ultrastructural alterations of mithocondria were documented in 93% of genotype 1, 60% of genotype 2 and 24% of genotype 3 patients. A significant depletion of mt. DNA with a reduction of mt. DNA/nDNA ratio which correlated significantly with lipoperoxidation markers was documented in patients with genotype 1 compared to patients with genotype 2 and 3 and controls. Conclusions: The increased production of free radicals with a reduced PBMC cytotoxic activity may represent a factor underlying the resistence to interferon therapy and may influence the evolution of liver disease by enhancement of the cytopathic effect of HCV. In genotype I patients the observed phosphorylation of the mithocondria and the depletion of mt. DNA may represent the expression of a greater impairment of oxidative phosphorilation which may lead to a more rapid process of hepatocellular apoptosis.
I
312
EVIDENCE OF l-lV REPLICATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS INFECTED IN VITRO
J.M. Lopez-Alcorocho, L.F. Mariscal, I. Castillo, J. Bartolome, V. Carrefio. Fundacion Estudio Hepatitis Wales and Instituto de Hepatologia, Hospital Pardo de Aravaca, Madrid, Spain
Although TTV DNA has been detected in peripheral blood mononuclear cells (PBMCs), no direct evidence concerning the replication of TI’V in these cells has been reported. Thus, PBMCs from 2 healthy TTV DNA negative donors were incubated with a TTV DNA positive serum and cultured for 3 weeks. DNA from cells and supematants was extracted and electrophoresed on agarose gels. Gels were cut into slides at 150 or 500 bp intervals, DNA was extracted from each slide and TTV DNA was amplified by PCR. TTV DNA from cells migrated at 1.5 to 3.0 Kb (sensitive to Sl nuclease but resistant to the restriction endonuclease NdeI) and at 3.5 to 6.0 Kb (resistant to Sl nuclease but sensitive to NdeI). TTV DNA extracted from the supematants migrated at 1.5 to 3.0 Kb, being sensitive to Sl nuclease but resistant to NdeI. ‘ITV DNA forms from 1.5 to 3.0 Kb may represent circular single-stranded full-lenght genome, while the 3.5 to 6.0 Kb forms are consistent with circular
112
infusions, day 0, and on days 112 and 394 after infusions were started. In CH1439,59%, 50%. 79%, and 89% of the clones represented the master sequence in four samples, respectively. A second major variant, found in three samples before infusions (12%, 38%, 16% of clones) disappeared by day 112 (end of infusions). In CH1483, the master sequence was depleted from 79% to 45% of clones in samples obtained after the infusions ended. Aminor variant (57%) present during 5-year follow-up period emerged as a predominant sequence (55%) after the infusions. A year later, the master sequence reemerged as a single (100%) species. In CH1556, the master sequence present at about 90% frequency over the 2-year follow-up period was reduced to 67% after the HCIg infusions and six new minor variants emerged. Molecular analysis of HVRl in chronically infected chimpanzees revealed that the immune pressure induced by the passive transfer of HCIg markedly altered the quasispecies pattern of HCV in serum.
I 222
INHIBITION OF CLASS I MHC PRESENTATION ON HEPATOCYTES AND LYMPHOID CELLS IN CHRONIC VIRAL HEPATITIS IN WOODCHUCKS
T.I. Michalak, PD. Hodgson, N.D. Churchill. Molecular virology and Hepatology Research, Faculty of Medicine, Memorial University of Newfoundland, Canada
Woodchuck hepatitis virus (WI-IV), as hepatitis B virus (HBV), casuse acute liver inflammation that can progress to chronic hepatitis and HCC. WI-IV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnaviral hepatitis is characterized by a profound difference in the expression of class I MHC molecules on infected hepatocytes and, notably, lymphoid cells. While acute infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on hepatocytes is a uniform hallmark of chronic WI-IV infection. This inhibition occurs despite augmented expression of hepatic genes for class I MHC heavy chain, beta-2microglobulin, and TAP1 and TAP2. Further, the class I antigen inhibition is not related to the histological severity of liver injury, the level of intrahepatic gamma interferon induction or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained demonstrate that the defective presentation of class I MHC is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHCdependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.
I
239
IN VITRO INFECTION OF PERIPHERAL BLOOD MONONUCLEAR CELLS BY A DEFECTIVE HEPATITIS B VIRUS
M. Cabrerizo, J. Bartolome, V. Carreho. Fundacidn Estudio Hepatitis Virales and Instituo de Hepatologia, Hospital Pardo de Aravaca, Madrid, Spain
We identified a defective HBV strain which contains a 183 deletion in the Sgene promoter in sera from HBsAg negative patients with serum HBV-DNA. To analyze the infectivity of this mutant, peripheral blood mononuclear cells (PBMCs) from a healthy donor were incubated with serum samples from 2 HBsAg negative patients with serum HBV-DNA (with both the wild-type and the deletion mutant), from a HBsAg chronic carrier (only wild-type HBV) and from a healthy donor. After I week, HBV-DNA was detected by PCR in all supematants and PBMCs incubated with the HBV-DNA positive inocula but not with the negative
control. DNaseI and trypsin pretreatment confirmed the intracellular location of HBV in the cells. HBV-RNA and covalently closed circular HBV-DNA were also detected in PBMCs, indicating that the viral DNA infecting these cells was transcriptionally active. Deletion mutant and wild-type HBV were detected in supematant of the PBMCs infected with the 2 HBsAg negative HBV-DNA sera, while only wild-type HBV was detected in the supematant of the cells incubated with the HBsAg inoculum. In conclusion, this HBV deletion mutant can infect and replicate in PBMCs cultured in vitro, and release viral particles to the supematant.
I 249
SERUM FERRITIN CONCENTRATIONS AND HEPATOCELLULAR MITOCHONDRIAL ALTERATIONS IN PATIENTS WITH CHRONIC HEPATITIS C RELATED TO THE HEPATITIS C VIRUS GENOTYPE
G. Barbarini, G. Barbaro, A. Asti, G. Belloni, A. Pellicelli, G. Di Lorenzo, B. Grisorio. Infectious and Tropical Diseases, IRCCS S: Matteo-University of Pavia, Italy
Aim of the Study and Methods: The study considered 130 CHC patients and 30 control cases. In these patients we correlated the levels of serum ferritin and concentrations of hepatic, plasmatic and lymphocytic GSH and erytbrocyte malonyldialdehyde. Moreover, we correlated the ultrastructural findings of liver biopsy specimens with thelipoperoxidation markers and the contents of mt. DNA in a selected cohort of patients in relation to HCV genotype. Results: Patients with genotype I showed higher levels of serum ferritin compared to patients with genotype 2 and 3 and to controls with a significant reduction of H-GSH, P-GSH, L-GSH concentrations and PBMC cytotoxic activity. Ultrastructural alterations of mithocondria were documented in 93% of genotype 1, 60% of genotype 2 and 24% of genotype 3 patients. A significant depletion of mt. DNA with a reduction of mt. DNA/nDNA ratio which correlated significantly with lipoperoxidation markers was documented in patients with genotype 1 compared to patients with genotype 2 and 3 and controls. Conclusions: The increased production of free radicals with a reduced PBMC cytotoxic activity may represent a factor underlying the resistence to interferon therapy and may influence the evolution of liver disease by enhancement of the cytopathic effect of HCV. In genotype I patients the observed phosphorylation of the mithocondria and the depletion of mt. DNA may represent the expression of a greater impairment of oxidative phosphorilation which may lead to a more rapid process of hepatocellular apoptosis.
I
312
EVIDENCE OF l-lV REPLICATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS INFECTED IN VITRO
J.M. Lopez-Alcorocho, L.F. Mariscal, I. Castillo, J. Bartolome, V. Carrefio. Fundacion Estudio Hepatitis Wales and Instituto de Hepatologia, Hospital Pardo de Aravaca, Madrid, Spain
Although TTV DNA has been detected in peripheral blood mononuclear cells (PBMCs), no direct evidence concerning the replication of TI’V in these cells has been reported. Thus, PBMCs from 2 healthy TTV DNA negative donors were incubated with a TTV DNA positive serum and cultured for 3 weeks. DNA from cells and supematants was extracted and electrophoresed on agarose gels. Gels were cut into slides at 150 or 500 bp intervals, DNA was extracted from each slide and TTV DNA was amplified by PCR. TTV DNA from cells migrated at 1.5 to 3.0 Kb (sensitive to Sl nuclease but resistant to the restriction endonuclease NdeI) and at 3.5 to 6.0 Kb (resistant to Sl nuclease but sensitive to NdeI). TTV DNA extracted from the supematants migrated at 1.5 to 3.0 Kb, being sensitive to Sl nuclease but resistant to NdeI. ‘ITV DNA forms from 1.5 to 3.0 Kb may represent circular single-stranded full-lenght genome, while the 3.5 to 6.0 Kb forms are consistent with circular