- Email: [email protected]
PP41 HEPATITIS B VIRUS IN VITRO INFECTION OF HUMAN LIVER PROGENITOR CELLS
Abstracts of XVI National Congress of SIGENP / Digestive and Liver Disease 41S (2009), S199–S239 disease other than WD (36 boys, median age 7.1 years, range 2-16). Among WD children, 35 (81%) were asymptomatic (30 with isolated hypertransaminasemia, 5 picked up by familial screening). Cupruria was evaluated before (basal cupruria [BCu]) and after (penicillamine challenge test cupruria [PCTCu]) penicillamine 500 mg orally 12 hourly × 2. Results: Ceruloplasmin, BCu and PCTCu significantly differed between WD and controls (P<0.0001, P<0.0001 and P<0.03, respectively). ROC analysis for ceruloplasmin at cut-off of ≤20 mg/dl showed sensitivity of 95.4% (95% confidence interval [CI], 84.5-99.4%) and specificity of 84.4% (95% CI, 72.5-92.6%). With a cut-off of ≤10 mg/dl, sensitivity and specificity were 65.9% (95% CI, 50-79.5%) and 96.5% (95% CI, 88-99.6%), respectively. A BCu threshold of ≥100 μg/24h showed sensitivity of 65.8% (95% CI, 49.4-79.9%) and specificity of 98.2% (95% CI, 90.7-99.9%), whereas at ≥40 μg/24h sensitivity was 78% (95% CI, 62.4-89.4%) and specificity 87.9% (95% CI, 76.7-95%). ROC area for PCTCu was not significant (AUC 0.63, P=0.051), and in our cohort this test showed sensitivity of only 15.4% (95% CI, 4.3-34.8%) and specificity of 94.8% (95% CI, 85.6-98.9%). Finally, we assessed the diagnostic accuracy of the Ferenci score [1]: at the proposed score of ≥4, sensitivity was 96.7% (95% CI, 83.3-99.9%) and specificity 93.3% (95% CI, 77.9-99.1%), with a ROC area of 0.95, P<0.0001. Conclusions: Diagnostic accuracy of ceruloplasmin and BCu, considered as single tests, are high in a cohort of predominantly asymptomatic children with WD. We have observed optimal sensitivity and specificity for BCu with a cut-off value of ≥40 μg/24h as recommended by recent guidelines. Instead, we do not recommend to measure PCTCu routinely in asymptomatic children because of its low sensitivity. Our observations confirm that WD diagnosis is multi-step, as pointed out by high accuracy of the diagnostic scoring system.
S219
[1] Ferenci P. et al. Diagnosis and phenotypic classification of Wilson disease. Liver Int 2003;23:139-42.
2007). An HBV-infected human hepatoma cell line (HepAD38) was used for the production of infective virions. AHLPC were incubated with PEG-concentrated virus-rich supernatant from HepAD38 culture, which was initially chosen to standardize infection conditions. A minimum concentration of 100 IU of HBV DNA per cell was used. PEG 6000 was added during incubation to help viral adhesion process. After 72h incubation, cell cultures were washed and cultured in normal culture conditions. Culture medium was changed every 3 days and confluence reached after each 14 days. The whole culture was followed up to 60 days. Total viral DNA, HBsAg and HBeAg were dosed in supernatant at different times post-infection using qPCR and ELISA. Immunoreactivity of HBsAg, HBcAg and fibronectin was investigated by immunofluorescence (IF). All experiments were done in parallel on freshly isolated human hepatocytes, HepAD38 cells and human bone marrow derived mesenchymal stem cells. Results: After incubation of AHLPC with HBV and subsequent extensive washing, both HBsAg and HBeAg were absent from culture supernatant. HBsAg became then detectable from day 3 and remained positive up to day 10 post-infection, while HBeAg was always negative. This was confirmed using IF for both HBsAg and HBcAg. This immunoreactivity was shown to be specific for AHLPC through costaining with fibronectin, a signature of the mesenchymal origin of such cells. Analysis of cell culture supernatant by qPCR revealed the presence of HBV DNA from day 3 up to day 35 post-infection. IF for both HBsAg and HBcAg was negative at that time. Conclusions: Our results clearly demonstrated for the first time that human liver progenitor cells can take up HBV in vitro. Nevertheless the absence of viral DNA in culture supernatant and the negative immunostaining from day 35 post-infection suggest that AHPLC are not permissive for HBV replication. These preliminary data should be supported by the analysis of the involved cellular mechanisms to allow a better understanding of the protective or permissive properties of both the hepatocyte and liver stem cell compartments against hepatitis B virus.
PP41
PP42
HEPATITIS B VIRUS IN VITRO INFECTION OF HUMAN LIVER PROGENITOR CELLS
CONGENITAL HEPATIC SHUNTS: A PEDIATRIC SERIES
References
M. Paganelli a , D.N. Khuu a , M. Najimi a , I. Malla a , B. Kabamba b , P. Goubau b , E.M. Sokal a
G. Capuano a , M. Caropreso a , S. Lenta a , A. Staiano a , N. Di Cosmo a , M. Esposito a , C. Veropalumbo a , D. Pariente b , O. Bernard b , P. Vajro a
a Pediatric
a Dipartimento di Pediatria dell’Università di Napoli “Federico II”, Italy; b Hospital d’Enfants de Bicetre, France
Aim: We investigated the susceptibility of adult liver progenitor cells to infection by hepatitis B virus (HBV) in vitro. Methods: Adult Human Liver Progenitor Cells (AHLPC) were cultured as previously described (Najimi M et al. Cell Transplantation
Background: Isolated congenital hepatic shunts are very rare abnormalities and a small number of cases have hitherto been reported. Complications include portal hypertension (PH) in arterio-portal types, hyperammonemic encephalopathy and hepatic tumours in porto-caval types, failure to thrive (FTT) and pulmonary hypertension in both
Hepatology and Cell Therapy Lab, b Virology Lab, Université Catholique de Louvain, Belgium
Abstract PP42 – Table 1 Patient
Type of Shunt and anatomy
Clinical presentation
Treatment
Outcome
1
Age at onset 6 yrs
Extrahepatic portal-caval fistula
– US hepatic nodules – FTT
– partial external banding
– F.U. 17 yrs - 0 mos – banding complicated by portal thrombosis → PH – new hepatic nodules – FTT – no hyperNH3 – surgical shunt planned
2
7 yrs
Extrahepatic portal-caval fistula/LPV
– Leopard Syndrome – fortuitous diagnosis of US hepatic adenomas – HyperNH3 – FTT
– failure of EV embolization – surgical fistula closure – adenomectomy
– F.U. 9 yrs - 2 mos – good response to surgery – no recurrence of hepatic tumours and of hepatic encephalopathy
3
6 mos
Intrahepatic arterio-portal fistula/LPV
– PH with GI bleeding – FTT
– 2 settings of PC embolizations – propranolol – omeprazole
– F.U 12 yrs - 6 mos – partial response with persistence of PH → new embolization planned – FTT
S219
[1] Ferenci P. et al. Diagnosis and phenotypic classification of Wilson disease. Liver Int 2003;23:139-42.
2007). An HBV-infected human hepatoma cell line (HepAD38) was used for the production of infective virions. AHLPC were incubated with PEG-concentrated virus-rich supernatant from HepAD38 culture, which was initially chosen to standardize infection conditions. A minimum concentration of 100 IU of HBV DNA per cell was used. PEG 6000 was added during incubation to help viral adhesion process. After 72h incubation, cell cultures were washed and cultured in normal culture conditions. Culture medium was changed every 3 days and confluence reached after each 14 days. The whole culture was followed up to 60 days. Total viral DNA, HBsAg and HBeAg were dosed in supernatant at different times post-infection using qPCR and ELISA. Immunoreactivity of HBsAg, HBcAg and fibronectin was investigated by immunofluorescence (IF). All experiments were done in parallel on freshly isolated human hepatocytes, HepAD38 cells and human bone marrow derived mesenchymal stem cells. Results: After incubation of AHLPC with HBV and subsequent extensive washing, both HBsAg and HBeAg were absent from culture supernatant. HBsAg became then detectable from day 3 and remained positive up to day 10 post-infection, while HBeAg was always negative. This was confirmed using IF for both HBsAg and HBcAg. This immunoreactivity was shown to be specific for AHLPC through costaining with fibronectin, a signature of the mesenchymal origin of such cells. Analysis of cell culture supernatant by qPCR revealed the presence of HBV DNA from day 3 up to day 35 post-infection. IF for both HBsAg and HBcAg was negative at that time. Conclusions: Our results clearly demonstrated for the first time that human liver progenitor cells can take up HBV in vitro. Nevertheless the absence of viral DNA in culture supernatant and the negative immunostaining from day 35 post-infection suggest that AHPLC are not permissive for HBV replication. These preliminary data should be supported by the analysis of the involved cellular mechanisms to allow a better understanding of the protective or permissive properties of both the hepatocyte and liver stem cell compartments against hepatitis B virus.
PP41
PP42
HEPATITIS B VIRUS IN VITRO INFECTION OF HUMAN LIVER PROGENITOR CELLS
CONGENITAL HEPATIC SHUNTS: A PEDIATRIC SERIES
References
M. Paganelli a , D.N. Khuu a , M. Najimi a , I. Malla a , B. Kabamba b , P. Goubau b , E.M. Sokal a
G. Capuano a , M. Caropreso a , S. Lenta a , A. Staiano a , N. Di Cosmo a , M. Esposito a , C. Veropalumbo a , D. Pariente b , O. Bernard b , P. Vajro a
a Pediatric
a Dipartimento di Pediatria dell’Università di Napoli “Federico II”, Italy; b Hospital d’Enfants de Bicetre, France
Aim: We investigated the susceptibility of adult liver progenitor cells to infection by hepatitis B virus (HBV) in vitro. Methods: Adult Human Liver Progenitor Cells (AHLPC) were cultured as previously described (Najimi M et al. Cell Transplantation
Background: Isolated congenital hepatic shunts are very rare abnormalities and a small number of cases have hitherto been reported. Complications include portal hypertension (PH) in arterio-portal types, hyperammonemic encephalopathy and hepatic tumours in porto-caval types, failure to thrive (FTT) and pulmonary hypertension in both
Hepatology and Cell Therapy Lab, b Virology Lab, Université Catholique de Louvain, Belgium
Abstract PP42 – Table 1 Patient
Type of Shunt and anatomy
Clinical presentation
Treatment
Outcome
1
Age at onset 6 yrs
Extrahepatic portal-caval fistula
– US hepatic nodules – FTT
– partial external banding
– F.U. 17 yrs - 0 mos – banding complicated by portal thrombosis → PH – new hepatic nodules – FTT – no hyperNH3 – surgical shunt planned
2
7 yrs
Extrahepatic portal-caval fistula/LPV
– Leopard Syndrome – fortuitous diagnosis of US hepatic adenomas – HyperNH3 – FTT
– failure of EV embolization – surgical fistula closure – adenomectomy
– F.U. 9 yrs - 2 mos – good response to surgery – no recurrence of hepatic tumours and of hepatic encephalopathy
3
6 mos
Intrahepatic arterio-portal fistula/LPV
– PH with GI bleeding – FTT
– 2 settings of PC embolizations – propranolol – omeprazole
– F.U 12 yrs - 6 mos – partial response with persistence of PH → new embolization planned – FTT