studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1031A
We have shown previously that viral quasispecies evolution
and diversity may play a pathogenic role in development of
HBeAg seroconversion. However, the role of viral quasispecies
in pathophysiology of reactivation in HBeAg-negative chronic
hepatitis B is uncertain. Methods: Twenty-two HBeAg-negative
chronic hepatitis B patients who had serial serum samples
collected before and after HBV reactivation were analysed.
Full length HBV genomes were amplified from serum extracted
DNA and were cloned into pCR®2.1-TOPO vector. At least
20 positive clones were randomly selected and sequenced
using Big Dye Terminator. The samples were analyzed for point
mutation in precore stop codon, core promoter- Enhancer II, S1
promoter, S2 promoter and Enhancer I- X promoter regions.
Ten inactive HBV carriers were also analysed as controls. Fulllength
HBV genome collected before and after reactivation
were transfected into Huh7 cells for functional analysis of their
replication fitness. Results: In subjects with clear clinical reactivation
profile, clonal selection was demonstrated (9 out of
10 subjects) by DNA phylogenetic analysis (p=0.005). The
genetic diversity and viral evolution rates were not statistically
different from the inactive controls except except at the precore/core
ORF (p=0.035, Mann-Whitney U test). 80% of reactivation
patients had precore stop codon G to A substitution at
nucleotide position 1896. In addition, other mutations in the
promoter/enhancer regions were identified with more than fifty
percent difference between time point 1 and time point 2 (and
not found in control subjects). HBV clones isolated during HBV
reactivation showed higher HBV DNA and HBsAg production
rates than the clones isolated during inactive phase of the same
patient in transfected Huh7 cell line. Conclusions: Significant
clonal selection occurred during HBeAg-negative reactivation.
Precore stop codon substitution G1896A occurs significantly
in majority of the patients. The nucleotide variability in the precore
promoter, and possibly other promoter/enhancer regions
may play an important role in the progression of HBV disease,
although more functional analyses of individual mutations are
required.
Disclosures:
Seng Gee Lim - Advisory Committees or Review Panels: Bristol-Myers Squibb,
Novartis Pharmaceuticals, Merck Sharp and Dohme Pharmaceuticals, Gilead
Pharmaceuticals, Roche Pharmaceuticals; Speaking and Teaching: Bristol-Myers
Squibb, Novartis Pharmaceuticals, Merck Sharp and Dohme Pharmaceuticals,
Gilead Pharmaceuticals
The following authors have nothing to disclose: Guan Huei Lee, Hui Heng Chong
1687
Hepatic macrophage IL-1 beta expression and bile and
sterol transporter suppression precede cholestasis in a
parenteral nutrition mouse model
Karim C. El Kasmi, Aimee Anderson, Michael W. Devereaux,
Ronald J. Sokol; Pediatrics, UC Denver, Aurora, CO
Background: Parenteral Nutrition Associated Cholestasis
(PNAC) is a serious complication of long term PN infusion in
children and adults with intestinal failure. We have previously
reported in a PNAC mouse model that activation of hepatic
macrophages and up-regulation of pro-inflammatory cytokines
(IL-1 beta) was associated with suppression of hepatic bile
(Abcb11/BSEP, Abcc2/MRP2) and sterol (ABCG5/8/sterolin)
transporters. IL-1R-/- mice were protected from PNAC, with
normalization of hepatic transporter expression, supporting
a causal role for IL-1 beta. The objective of this study was
to determine the role of IL-1 beta at early stages of PNAC
by examining the temporal relationship at early time points
between IL-1 beta, transporter expression, and cholestasis.
Methods and Results: PNAC model: wild type C57/B6 mice
were exposed to dextran sulfate sodium (DSS) in drinking
water (to induce intestinal injury) for 4 days followed by infusion
of soy lipid emulsion-based PN through a central venous
catheter for 3 or 7 days (DSS-PN mice); appropriate controls
were conducted. Cholestasis (increased serum bile acids and
bilirubin) and hepatocyte injury (increased AST and ALT)
were absent in DSS/PN3d mice but developed in all DSS/
PN7d mice. IL-1 beta transcription was significantly induced
in liver homogenate and in isolated hepatic mononuclear cells
(hMNCs) at 3 days in DSS/PN mice and persisted at 7 days.
Concomitantly, transcription of Abcb11, Abcc2, and Abcg5/8
was suppressed at both of these time points in DSS/PN mice,
indicating that macrophage activation and transporter suppression
preceded the onset of biochemical cholestasis. To further
determine if IL-1 beta was responsible for the transcriptional
suppression, wild type mice were injected i.p. with recombinant
IL-1 beta (200ng/mouse) and sampled after 4 hrs, and
HuH7 and HepG2 cells (human hepatocyte cell lines) were
incubated with IL-1b (10-50ng/ml) for 4 hrs, and gene expression
measured. IL-1 beta treatment significantly suppressed
expression of Abcb11, Abcc2, and Abcg5/8 in the mice and
in both hepatocyte cell lines. Incubation of these cell lines with
LPS (100-1000ng/ml) had no effect on these transporters. Conclusions:
Activation of hepatic macrophages and generation of
IL-1 beta, as well as suppression of bile and sterol transporters,
occurred after only 3 days of PN infusion and preceded onset
of cholestasis. Furthermore, IL-1 beta was sufficient to suppress
these transporters in cultured hepatocytes and in vivo. These
data support IL-1 beta as a critical early mediator in the pathogenesis
of PNAC and suggest IL1-beta signaling as a potential
therapeutic target in this disorder.
Disclosures:
Ronald J. Sokol - Advisory Committees or Review Panels: Yasoo Health, Inc.; Consulting:
Roche, Ikaria, Otsuka American Pharmaceuticals, Alnylam, Retrophin;
Grant/Research Support: Mead Johnson Nutritionals, Lumena, FFF Enterprises
The following authors have nothing to disclose: Karim C. El Kasmi, Aimee Anderson,
Michael W. Devereaux
1688
Molecular mechanism of IL-1 beta mediated transcriptional
suppression of the sterol transporter Abcg5/8 in
a parenteral nutrition mouse model
Karim C. El Kasmi, Aimee Anderson, Padade M. Vue, Michael
W. Devereaux, Natarajan Balasubramaniyan, Frederick J. Suchy,
Ronald J. Sokol; Pediatrics, UC Denver, Aurora, CO
Background: Parenteral nutrition associated cholestasis (PNAC)
is the leading indication for combined intestine/liver transplant.
A possible role of intravenous soy lipid emulsions in PNAC
pathogenesis has led to concern about the hepatic toxicity of
phytosterols contained in soy lipid emulsions. We have demonstrated
in a novel PNAC mouse model that soy lipid emulsions
in PN are associated with induction of hepatic macrophage
IL1b expression concomitant with accumulation of hepatic phytosterols
and transcriptional suppression of hepatocyte Abcg5/
g8, coding for the canalicular transporter of sterols, as well
as BSEP and MRP2. The aim of this study was to elucidate the
role of IL1b signaling in suppression of ABCG5/8 transcription
during PNAC. Methods and Results: Wild type C57/B6 mice
were exposed to dextran sulfate sodium (DSS) (to induce intestinal
injury) for 4d followed by infusion of phytosterol-containing
(soy lipid) PN solution through a central venous catheter for 14
d (DSS-PN mice) and developed cholestasis (increased serum
bile acids and bilirubin) and hepatocyte injury (increased AST
and ALT). DSS-PN mice had hepatic IL1b induction and markedly
reduced hepatic mRNA for Abcb11, Abcc2, Abcg5/8
compared to control mice. To further elucidate the role of IL1b
in this transcriptional suppression, mice with genetic deletion