Table of Contents for October
2003 · Volume 38 · Number 4
Viral Hepatitis
Immunization with an adjuvant hepatitis B vaccine after
liver transplantation for hepatitis Brelated disease (*Human
Study*)
Ulrich Bienzle, Matthias Günther, Ruth Neuhaus, Pierre Vandepapeliere,
Jens Vollmar, Andreas Lun, Peter Neuhaus
Patients who undergo transplantation for hepatitis B virus (HBV)-related
diseases are treated indefinitely with hepatitis B hyperimmunoglobulin
(HBIG) to prevent endogenous HBV reinfection of the graft. Active
immunization with standard hepatitis B vaccines in these patients
has recently been reported with conflicting results. Two groups
of 10 liver transplant recipients on continuous HBIG substitution
who were hepatitis B surface antigen (HBsAg) positive and HBV
DNA negative before transplantation were immunized in a phase
I study with different concentrations of hepatitis B s antigen
formulated with the new adjuvants 3-deacylated monophosphoryl
lipid A (MPL) and Quillaja saponaria (QS21) (group I/vaccine
A: 20 µg HBsAg, 50 µg MPL, 50 µg QS21; group
II/vaccine B: 100 µg HBsAg, 100 µg MPL, 100 µg
QS21). Participants remained on HBIG prophylaxis and were vaccinated
at weeks 0, 2, 4, 16, and 18. They received 3 additional doses
of vaccine B at bimonthly intervals if they did not reach an
antibody titer against hepatitis B surface antigen (anti-HBs)
greater than 500 IU/L. Sixteen (8 in each group) of 20 patients
(80%) responded (group I: median, 7,293 IU/L; range, 721-45,811
IU/L anti-HBs; group II: median, 44,549 IU/L; range, 900-83,121
IU/L anti-HBs) and discontinued HBIG. They were followed up for
a median of 13.5 months (range, 6-22 months). The vaccine was
well tolerated. In conclusion, most patients immunized with the
new vaccine can stop HBIG immunoprophylaxis for a substantial,
yet to be determined period of time. (HEPATOLOGY 2003;38:811-819.)
Hepatitis C virus core protein activates ERK and p38 MAPK
in cooperation with ethanol in transgenic mice
Takeya Tsutsumi, Tetsuro Suzuki, Kyoji Moriya, Yoshizumi Shintani,
Hajime Fujie, Hideyuki Miyoshi, Yoshiharu Matsuura, Kazuhiko
Koike, Tatsuo Miyamura
In human chronic hepatitis C, alcohol intake is a synergistic
factor for the acceleration of hepatocarcinogenesis. Recently,
we showed a significant increase of reactive oxygen species (ROS)
in hepatitis C virus (HCV) core-transgenic mice fed ethanol-containing
diets. Because previous studies indicated that ROS is closely
associated with mitogen-activated protein kinases (MAPK), we
examined activities of c-Jun N-terminal kinase, p38 MAPK, and
extracellular signal-regulated kinase (ERK) in the liver of core-transgenic
and nontransgenic mice with short-term ethanol feeding. Activity
of ERK and p38 MAPK was increased in core-transgenic mice compared
with nontransgenic mice, whereas neither ERK nor p38 MAPK was
activated in core-transgenic mice with normal diets. In addition,
activity of cyclic-AMP and serum responsive element, downstream
pathways of p38 MAPK and ERK, was also increased. Comparison
of gene expression profiles by cDNA microarray and real-time
PCR revealed that galectin-1, which is associated with cell transformation,
was significantly increased in ethanol-fed core-transgenic mice.
On the other hand, glutathione S-transferase (GST), which plays
a key role in protecting cells from oxidative stress, was decreased.
In conclusion, these results suggest that HCV core protein cooperates
with ethanol for the activation of some MAPK pathways, and leads
to the modulation of several genes, contributing to the pathogenesis
of liver disease of HCV-infected patients with high ethanol consumption.
(HEPATOLOGY 2003;38:820-828.)
Phenotypic and functional characterization of intrahepatic
T lymphocytes during chronic hepatitis C (*Human Study*)
Vincent Leroy, Ines Vigan, Jean-Francois Mosnier, Tania Dufeu-Duchesne,
Martine Pernollet, Jean-Pierre Zarski, Patrice N. Marche, Evelyne
Jouvin-Marche
The pathogenesis of liver cell injury during chronic hepatitis
C (CHC) is poorly understood. The cellular immune response is
thought to play a key role in both inhibition of viral replication
and liver pathology. However, little is currently known about
which lymphocyte populations and which immune effectors contribute
to or control liver damage. We investigated a panel of 15 phenotypic
and functional markers of intrahepatic T-lymphocyte subsets irrespective
of their antigen specificity in 48 hepatitis C virus (HCV)-infected
patients and 8 healthy control subjects. Lymphocyte characteristics
were evaluated from liver biopsy specimens both at gene expression
level by real-time quantitative reverse-transcription polymerase
chain reaction (RT-PCR) and by immunochemistry, in relation with
the degree of liver injury and with intrahepatic HCV-RNA levels.
As compared with controls, we found major changes in T-lymphocyte
subsets in HCV-infected patients, with a significant decrease
of T-cell antigen receptor (TCR) and CD56 gene expression, associated
with a concomitant increase of TCR and CD8 that were correlated
with cytotoxic factors, proinflammatory chemokines, and chemokine
receptors including peforin, interferon gamma (IFN-), tumor necrosis
factor (TNF-), RANTES, and CXCR3. The gene expression of CD8,
a specific marker for conventional TCR+CD8+ lymphocytes, was
correlated by multivariate analysis with both alanine aminotransferase
(ALT) serum levels and histologic activity index. Furthermore,
CD8 staining was observed by immunochemistry in the areas of
lobular and piecemeal necrosis. In contrast, no lymphocyte marker
was correlated with viral load, measured both in serum and in
liver. In conclusion, these results strongly suggest key roles
for CD8+ T cells as effectors of liver damage during chronic
HCV infection and for their inability to control viral replication.
(HEPATOLOGY 2003;38:829-841.)
Inhibition of HBV replication by siRNA in a stable HBV-producing
cell line
Masayoshi Konishi, Catherine H. Wu, George Y. Wu
Potent inhibition of endogenous gene expression by RNA interference
has been achieved by using sequence-specific posttranscriptional
gene silencing through the action of small interfering RNA molecules
(siRNA). In these reports, the natural function of genes could
be deduced through the ensuing loss of function. Based on the
extraordinary effectiveness in silencing endogenous genes, we
wondered whether siRNA could be applied against viral replication
in a hepatitis B virus (HBV) model using HBV-specific siRNA.
To test this idea, HepG2 2.2.15, a human hepatoblastoma cell
line that constitutively produces infectious HBV particles, was
transfected with HBV-specific siRNAs and controls. HBV surface
antigen (HBsAg) secretion into culture media was inhibited by
78%, 67%, and 42% with siRNA against the polyadenylation (PA),
precore (PreC), and surface (S) regions, respectively, compared
with controls as detected by enzyme-linked immunosorbent assay.
After exposure to HBVPA siRNA, Northern blot analysis showed
that HBV pregenomic RNA levels were decreased by 72%, and levels
of HBV RNA containing the polyadenylation signal sequence were
suppressed by 86%, as detected by RNase protection assay. Levels
of HBV core-associated DNA, a replication intermediate, also
decreased by 71%. Immunocytochemistry revealed that 30% to 40%
of the cells transfected with HBVPA siRNA were completely negative
for detectable HBsAg levels. Controls consisting of treatment
with HBV-specific siRNA alone, lipofection reagent alone, or
random double-stranded RNA (dsRNA) lipofection complex failed
to decrease HBV surface antigen, HBV messenger RNA (mRNA), or
core-associated HBV-DNA levels. In conclusion, siRNA inhibits
hepatitis B viral replication in a cell culture system. Future
studies are needed to explore the specific delivery of siRNA
to liver cells in vivo and the applicability of this approach.
(HEPATOLOGY 2003;38:842-850.)
Chronic hepatitis C virus infection established and maintained
in chimpanzees independent of dendritic cell impairment
Christine Rollier, Joost A. R. Drexhage, Babs E. Verstrepen,
Ernst J. Verschoor, Ronald E. Bontrop, Gerrit Koopman, Jonathan
L. Heeney
Chronic hepatitis C virus (HCV) infection in humans is associated
with an impairment of dendritic cells (DC). It has been hypothesized
that impairment of DC function may be a central mechanism facilitating
the establishment of a chronic carrier state. However, the majority
of patients studied with DC impairment to date have been identified
and, thus, inadvertently selected because of clinical manifestations
leading to their diagnosis, which may have been many years following
actual infection. We set out to determine whether impaired DC
function occurred in the earlier asymptomatic phase of infection
and turned to a well-defined cohort of HCV-infected chimpanzees
in which the specific date of infection and the nature of the
inoculum were well characterized. Results revealed that, in contrast
to the observations in human subjects with advanced clinical
hepatitis, there was neither impairment of the allostimulatory
capacity of monocyte-derived DC from HCV chronic carriers nor
impairment of the maturation process. Decreased allostimulatory
capacity was only detected in 2 animals and, interestingly, in
those that possessed the highest viral loads. Nevertheless, HCV
sequences were undetectable in any of the DC derived from HCV-infected
chimpanzees. In conclusion, these findings suggest that the mechanisms
of establishing persistent HCV infection are separate and independent
from those responsible for impaired DC function. Indeed, the
maturation and allostimulatory impairment, as described in patient
studies, are not necessary prerequisites but rather possible
consequences of persistent and active HCV infection associated
with disease progression. (HEPATOLOGY 2003;38:851-858.)
Long-term interleukin 10 therapy in chronic hepatitis C patients
has a proviral and anti-inflammatory effect (*Human Study*)
David R. Nelson, Zhengkun Tu, Consuelo Soldevila-Pico, Manal
Abdelmalek, Haizhen Zhu, Yi Ling Xu, Roniel Cabrera, Chen Liu,
Gary L. Davis
An imbalance in Th1 and Th2 cytokine production is implicated
in disease progression of HCV. Our aim was to determine the effect
of IL-10 administration in patients with HCV-related liver disease.
Thirty patients with advanced fibrosis who had failed antiviral
therapy were enrolled in a 12-month treatment regimen with SQ
IL-10 given daily or thrice weekly. Liver biopsies were performed
before and after therapy. Serum and PBMC were collected for HCV
RNA, ALT, and functional T-cell analysis. IL-10 led to significant
improvement in serum ALT (mean ALT: day 0 = 142 ± 17 vs.
month 12 = 75 ± 10; P < .05). Hepatic inflammation
score decreased by at least 2 in 13 of 28 patients (mean decrease
from 4.6 ± 0.3 to 3.7 ± 0.3, P < .05)
and 11 of 28 showed a reduction in fibrosis score (mean change
from 5.0 ± 0.2 to 4.5 ± 0.3, P < .05).
Serum HCV RNA levels increased by 0.5 log during therapy (mean
HCV RNA day 0: 12.3 ± 3.0 Meq/mL; 12 months: 38 Meq/mL;
P < .05) and returned to baseline at the end of follow-up
(11.0 ± 2.4 Meq/mL). Five patients developed viral loads
of greater than 120 Meq/mL and two of these developed an acute
flare in serum ALT. IL-10 caused a decrease in the number of
HCV-specific CD4+ and CD8+ IFN- secreting T cells and alterations
in PBMC cytokine production towards a Th2 dominant profile. These
changes parallel the improvement in ALT and rise in HCV RNA.
In conclusion, long-term rIL-10 therapy appears to decrease disease
activity, but also leads to increased HCV viral burden via alterations
in immunologic viral surveillance. (HEPATOLOGY 2003;38:859-868.)
Identification of a ribavirin-resistant NS5B mutation of hepatitis
C virus during ribavirin monotherapy (*Human Study*)
Kung-Chia Young, Karen L. Lindsay, Ki-Jeong Lee, Wen-Chun Liu,
Jian-Wen He, Susan L. Milstein, Michael M. C. Lai
Ribavirin (RBV), a guanosine analogue, has been suggested to
exert an antiviral action against hepatitis C virus (HCV) by
causing lethal mutations and suppressing RNA polymerase in
vitro, but the mechanism of its clinical therapeutic effects
is currently unknown. To test the hypothesis that RBV could act
both as an RNA mutagen and inhibit viral RNA synthesis in
vivo, we studied the evolution of the nucleotide sequences
of HCV RNA at the nonstructural (NS) 5B region in patients receiving
RBV, placebo, or interferon alfa (IFN-) monotherapy. The RBV
group showed a slightly more accelerated evolution rate of HCV
RNA quasispecies than either the IFN- or placebo group. RBV caused
preferentially A-to-G and U-to-A mutations. Interestingly, an
NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all
(5 of 5) patients infected with HCV genotype 1a during the RBV
treatment. Subsequently, the parental 415F strain reemerged in
some patients after the treatment was discontinued. The effect
of the amino acid substitution at NS5B415 on HCV RNA replication
was then investigated using an HCV subgenomic replicon in Huh7
cells. We showed that treatment of replicon cells with RBV reduced
the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in
a dose-dependent manner. Thus, NS5B F415Y mutation represents
an RBV-resistant variant. The 3-dimensional modeling and structure
analysis of NS5B protein revealed that the 415th amino acid is
located at the P helix region of the thumb subdomain, which may
interact with the minor groove of the template-primer duplex
in the putative RNA-binding cleft. In conclusion, RBV could work
as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore,
the selection of an RBV-resistant variant with a single amino
acid substitution in NS5B suggested that RBV may directly interact
with HCV RNA polymerase, thus interfering with its enzymatic
activity. (HEPATOLOGY 2003;38:869-878.)
Liver Biology and Pathobiology
p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation
in rat myofibroblasts
Fukiko Furukawa, Koichi Matsuzaki, Shigeo Mori, Yoshiya Tahashi,
Katsunori Yoshida, Yasushi Sugano, Hideo Yamagata, Masanori Matsushita,
Toshihito Seki, Yutaka Inagaki, Mikio Nishizawa, Junichi Fujisawa,
Kyoichi Inoue
Hepatic stellate cells (HSCs) spontaneously transdifferentiate
into myofibroblast (MFB)-phenotype on plastic dishes. This response
recapitulates the features of activation in vivo. Transforming
growth factor (TGF-) plays a prominent role in stimulating liver
fibrogenesis by MFBs. In quiescent HSCs, TGF- signaling involves
TGF- type I receptor (TRI)-mediated phosphorylation of serine
residues within the conserved SSXS motif at the C-terminus of
Smad2 and Smad3. The middle linker regions of Smad2 and Smad3
also are phosphorylated by mitogen-activated protein kinase (MAPK).
This study elucidates the change of Smad3-mediated signals during
the transdifferentiation process. By using antibodies highly
specific to the phosphorylated C-terminal region and the phosphorylated
linker region of Smad3, we found that TGF--dependent Smad3 phosphorylation
at the C-terminal region decreased, but that the phosphorylation
at the linker region increased in the process of transdifferentiation.
TGF- activated the p38 MAPK pathway, further leading to Smad3
phosphorylation at the linker region in the cultured MFBs, irrespective
of Smad2. The phosphorylation promoted hetero-complex formation
and nuclear translocation of Smad3 and Smad4. Once combined with
TRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to
plasminogen activator inhibitor-type I promoter could enhance
the transcription. In addition, Smad3 phosphorylation mediated
by the activated TRI was impaired severely in MFBs during chronic
liver injury, whereas Smad3 phosphorylation at the linker region
was remarkably induced by p38 MAPK pathway. In conclusion, p38
MAPK-dependent Smad3 phosphorylation promoted extracellular matrix
production in MFBs both in vitro and in vivo. (HEPATOLOGY
2003;38:879-889.)
Interferon alfa down-regulates collagen gene transcription
and suppresses experimental hepatic fibrosis in mice
Yutaka Inagaki, Tomoyuki Nemoto, Miwa Kushida, Yin Sheng, Kiyoshi
Higashi, Kazuo Ikeda, Norifumi Kawada, Fumiaki Shirasaki, Kazuhiko
Takehara, Kota Sugiyama, Mitsukiyo Fujii, Hiroshi Yamauchi, Atsuhito
Nakao, Benoit de Crombrugghe, Tetsu Watanabe, Isao Okazaki
The equilibrium between the production and degradation of collagen
is rigorously controlled by a number of growth factors and cytokines.
Interferon alfa (IFN-) is now widely used for the treatment of
chronic hepatitis C, which can improve serum levels of fibrotic
markers and the degree of hepatic fibrosis, not only in patients
who responded to therapy but also in those in whom it is ineffective.
These findings may suggest that IFN- possesses direct antifibrotic
effects in addition to its antiviral activity. However, in contrast
to IFN-, which has been shown to suppress collagen gene transcription,
little is known about the mechanisms responsible for the antifibrotic
effects of IFN-. Here, we report that IFN-, when administered
into transgenic mice harboring the 2(I) collagen gene (COL1A2)
promoter sequence, significantly repressed promoter activation
and prevented the progression of hepatic fibrosis induced by
carbon tetrachloride injection. Transient transfection assays
indicated that IFN- decreased the steady-state levels of COL1A2
messenger RNA (mRNA) and inhibited basal and TGF-/Smad3-stimulated
COL1A2 transcription in activated hepatic stellate cells (HSC).
These inhibitory effects of IFN- on COL1A2 transcription were
exerted through the interaction between phosphorylated Stat1
and p300. Blocking of the IFN- signal by overexpressing the intracellular
domain-deleted IFN receptor increased basal COL1A2 transcription
and abolished the inhibitory effects of IFN-. In conclusion,
our results indicate that IFN- antagonizes the TGF-/Smad3-stimulated
COL1A2 transcription in vitro and suppresses COL1A2 promoter
activation in vivo, providing a molecular basis for antifibrotic
effects of IFN-. (HEPATOLOGY 2003;38:890-899.)
Decreased hepatic nitric oxide production contributes to the
development of rat sinusoidal obstruction syndrome
Laurie D. DeLeve, Xiangdong Wang, Gary C. Kanel, Yoshiya Ito,
Nancy W. Bethea, Margaret K. McCuskey, Zoltan A. Tokes, Jeffrey
Tsai, Robert S. McCuskey
This study examined the role of decreased nitric oxide (NO) in
the microcirculatory obstruction of hepatic sinusoidal obstruction
syndrome (SOS). SOS was induced in rats with monocrotaline. Monocrotaline
caused hepatic vein NO to decrease by 30% at 24 hours and by
70% at 72 hours; this decrease persisted throughout late SOS.
NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO
synthase, exacerbated monocrotaline toxicity, whereas V-PYRRO/NO,
a liver-selective NO donor prodrug, restored NO levels, preserved
sinusoidal endothelial cell (SEC) integrity and sinusoidal perfusion
as assessed by in vivo microscopy and electron microscopy,
and prevented clinical and histologic evidence of SOS. NO production
in vitro by SEC and Kupffer cells, the 2 major liver cell
sources of NO, decreases largely in parallel with loss of cell
viability after exposure to monocrotaline. Increased matrix metalloproteinase
(MMP) activity increases early on in SOS and this increase in
activity has been implicated in initiating SOS. Infusion of V-PYRRO-NO
prevented the monocrotaline-induced increase in MMP-9. In conclusion,
decreased hepatic NO production contributes to the development
of SOS. Infusion of an NO donor preserves SEC integrity and prevents
development of SOS. These findings show that a decrease in NO
contributes to SOS by allowing up-regulation of MMP activity,
loss of sinusoidal integrity, and subsequent disruption of sinusoidal
perfusion. (HEPATOLOGY 2003;38:900-908.)
Heme oxygenase-1 and its reaction product, carbon monoxide,
prevent inflammation-related apoptotic liver damage in mice
Gabriele Sass, Miguel Che Parreira Soares, Kenichiro Yamashita,
Stefan Seyfried, Wolfram-Hubertus Zimmermann, Thomas Eschenhagen,
Elzbieta Kaczmarek, Thomas Ritter, Hans-Dieter Volk, Gisa Tiegs
Heme oxygenase-1 (HO-1), a stress-responsive enzyme that catabolizes
heme into carbon monoxide (CO), biliverdin, and iron, has previously
been shown to protect grafts from ischemia/reperfusion injury
and rejection. Here we investigated the protective potential
of HO-1 in 5 models of immune-mediated liver injury. We found
that up-regulation of endogenous HO-1 by cobalt-protoporphyrin-IX
(CoPP) protected mice from apoptotic liver damage induced by
anti-CD95 antibody (Ab) or d-galactosamine in combination with
either anti-CD3 Ab, lipopolysaccharide (LPS), or tumor necrosis
factor (TNF-). HO-1 induction prevented apoptotic liver injury,
measured by inhibition of caspase 3 activation, although it did
not protect mice from caspase-3-independent necrotic liver damage
caused by concanavalin A (Con A) administration. In addition,
overexpression of HO-1 by adenoviral gene transfer resulted in
protection from apoptotic liver injury, whereas inhibition of
HO-1 enzymatic activity by tin-protoporphyrin-IX (SnPP) abrogated
the protective effect. HO-1-mediated protection seems to target
parenchymal liver cells directly because CoPP treatment protected
isolated primary hepatocytes from anti-CD95-induced apoptosis
in vitro. Furthermore, depletion of Kupffer cells (KCs)
did not interfere with the protective effect in vivo.
Exogenous CO administration or treatment with the CO-releasing
agent methylene chloride mimicked the protective effect of HO-1,
whereas treatment with exogenous biliverdin or overexpression
of ferritin by recombinant adenoviral gene transfer did not.
In conclusion, HO-1 is a potent protective factor for cytokine-
and CD95-mediated apoptotic liver damage. Induction of HO-1 might
be of a therapeutic modality for inflammatory liver diseases.
(HEPATOLOGY 2003;38:909-918.)
Human hepatic stellate cells show features of antigen-presenting
cells and stimulate lymphocyte proliferation
Odette Viñas, Ramón Bataller, Pau Sancho-Bru, Pere
Ginès, Cristina Berenguer, Carlos Enrich, Josep M. Nicolás,
Guadalupe Ercilla, Teresa Gallart, Jordi Vives, Vicente Arroyo,
Juan Rodés
Following cell activation, hepatic stellate cells (HSCs) acquire
proinflammatory and profibrogenic properties. We investigated
whether activated HSCs also display immune properties. Here we
show that cultured human HSCs express membrane proteins involved
in antigen presentation, including members of the HLA family
(HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c),
and factors involved in T-cell activation (CD40 and CD80). Exposure
of HSCs to proinflammatory cytokines markedly up-regulates these
molecules. Importantly, cells freshly isolated from human cirrhotic
livers (in vivo activated HSCs) highly express HLA-II
and CD40, suggesting that HSCs can act as antigen-presenting
cells (APCs) in human fibrogenesis. We also explored whether
human HSCs can efficiently process exogenous antigens. Activated
HSCs internalize low- and high-molecular-weight dextran and transferrin,
indicating that they can perform fluid-phase and receptor-mediated
endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules
because they internalize latex particles as well as bacteria.
Interestingly, both culture-activated and in vivo activated
HSCs express high levels of CD68, a protein involved in antigen
trafficking. Finally, we studied whether HSCs modulate T-lymphocyte
proliferation. In basal conditions, coculture of irradiated HSCs
barely induces allogeneic T-lymphocyte proliferation. However,
cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte
response in an HLA-II-dependent manner. In conclusion, human
activated HSCs express molecules for antigen presentation, internalize
macromolecules, and modulate T-lymphocyte proliferation. These
results suggest that HSCs may play a role in the immune function
of the liver. (HEPATOLOGY 2003;38:919-929.)
Feedback regulation of bile acid synthesis in primary human
hepatocytes: Evidence that CDCA is the strongest inhibitor
Ewa Ellis, Magnus Axelson, Anna Abrahamsson, Gösta Eggertsen,
Anders Thörne, Grzegorz Nowak, Bo-Göran Ericzon, Ingemar
Björkhem, Curt Einarsson
Primary human hepatocytes were used to elucidate the effect of
individual bile acids on bile acid formation in human liver.
Hepatocytes were treated with free as well as glycine-conjugated
bile acids. Bile acid formation and messenger RNA (mRNA) levels
of key enzymes and the nuclear receptor short heterodimer partner
(SHP) were measured after 24 hours. Glycochenodeoxycholic acid
(GCDCA; 100 µmol/L) significantly decreased formation of
cholic acid (CA) to 44% ± 4% of controls and glycodeoxycholic
acid (GDCA) decreased formation of CA to 67% ± 11% of
controls. Glycoursodeoxycholic acid (GUDCA; 100 µmol/L)
had no effect. GDCA or glycocholic acid (GCA) had no significant
effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids
had a similar effect as glycine-conjugated bile acids. Addition
of GCDCA, GDCA, and GCA (100 µmol/L) markedly decreased
cholesterol 7-hydroxylase (CYP7A1) mRNA levels to 2% ±
1%, 2% ± 1%, and 29% ± 11% of controls, respectively,
whereas GUDCA had no effect. Addition of GDCA and GCDCA (100
µmol/L) significantly decreased sterol 12-hydroxylase (CYP8B1)
mRNA levels to 48% ± 5% and 61% ± 4% of controls,
respectively, whereas GCA and GUDCA had no effect. Addition of
GCDCA and GDCA (100 µmol/L) significantly decreased sterol
27-hydroxylase (CYP27A1) mRNA levels to 59% ± 3% and 60%
± 7% of controls, respectively, whereas GUDCA and GCA
had no significant effect. Addition of both GCDCA and GDCA markedly
increased the mRNA levels of SHP to 298% ± 43% and 273%
± 30% of controls, respectively. In conclusion, glycine-conjugated
and free bile acids suppress bile acid synthesis and mRNA levels
of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA
levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree
than CYP7A1. (HEPATOLOGY 2003;38:930-938.)
Influence of newly synthesized cholesterol on bile acid synthesis
during chronic inhibition of bile acid absorption (*Human
Study*)
Marco Bertolotti, Lisa Zambianchi, Lucia Carulli, Maria Sole
Simonini, Marina Del Puppo, Marzia Galli Kienle, Paola Loria,
Adriano Pinetti, Nicola Carulli
The effects of newly synthesized cholesterol availability on
bile acid synthesis are largely unknown, particularly in humans.
The present study was aimed to study the changes induced on bile
acid synthesis by simvastatin, a competitive inhibitor of hydroxymethyl
glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme of
cholesterol synthesis, during pharmacologic interruption of the
enterohepatic circulation. Six patients with primary hypercholesterolemia
were studied in basal conditions, after treatment with the bile
acid binding resin cholestyramine alone (8-16 g/d for 6-8 weeks)
and subsequently in combination with simvastatin (40 mg/d for
6-8 weeks). Cholesterol 7-hydroxylation rate, a measure of total
bile acid synthesis, was assayed in vivo by tritium release
analysis. Serum lathosterol levels were assayed by gas chromatography-mass
spectrometry as a measure of cholesterol synthesis. Serum total
and low-density lipoprotein-cholesterol were reduced significantly
after cholestyramine (by 26% and 30%, respectively) and during
combined treatment (by 47% and 55%). 7-Hydroxylation rates increased
nearly 4-fold with cholestyramine alone; addition of simvastatin
induced a significant decrease of hydroxylation rates (cholestyramine
alone, 1,591 ± 183 mg/d; plus simvastatin, 1,098 ±
232 mg/d; mean ± SEM; P < .05). Hydroxylation
rates significantly correlated with serum lathosterol/cholesterol
ratio (r = 0.79, P < .05). In conclusion, in
conditions of chronic stimulation bile acid synthesis may be
affected by changes in newly synthesized cholesterol availability.
The finding might relate to the degree of substrate saturation
of microsomal cholesterol 7-hydroxylase; alternatively, newly
synthesized cholesterol might induce a stimulatory effect on
cholesterol 7-hydroxylase transcription. (HEPATOLOGY 2003;38:939-946.)
Ontogeny, distribution, and possible functional implications
of an unusual aquaporin, AQP8, in mouse liver
Domenico Ferri, Amelia Mazzone, Giuseppa Esterina Liquori, Grazia
Cassano, Maria Svelto, Giuseppe Calamita
Aquaporins are channel proteins widely expressed in nature and
known to facilitate the rapid movement of water across numerous
cell membranes. A mammalian aquaporin, AQP8, was recently discovered
and found to have a very distinct evolutionary pathway. To understand
the reason for this divergence, here we define the ontogeny and
exact subcellular localization of AQP8 in mouse liver, a representative
organ transporting large volumes of water for secretion of bile.
Northern blotting showed strong AQP8 expression between fetal
day 17 and birth as well as at weaning and thereafter. Interestingly,
this pattern was confirmed by immunohistochemistry and coincided
both temporally and spatially with that of hepatic glycogen accumulation.
As seen by reverse-transcription polymerase chain reaction (RT-PCR)
and immunohistochemistry, fasting was accompanied by remarkable
down-regulation of hepatic AQP8 that paralleled the expected
depletion of glycogen content. The level of hepatic AQP8 returned
to be considerable after refeeding. Immunoelectron microscopy
confirmed AQP8 in hepatocytes where labeling was over smooth
endoplasmic reticulum (SER) membranes adjacent to glycogen granules
and in canalicular membranes, subapical vesicles, and some mitochondria.
In conclusion, in addition to supporting a role for AQP8 in canalicular
water secretion, these findings also suggest an intracellular
involvement of AQP8 in preserving cytoplasmic osmolality during
glycogen metabolism and in maintaining mitochondrial volume.
AQP8 may have evolved separately to feature these intracellular
roles as no other known aquaporin shows this specialization.
(HEPATOLOGY 2003;38:947-957.)
Characterization of premature liver polyploidy in DNA repair
(Ercc1)-deficient mice
Michael D. Chipchase, Mary O'Neill, David W. Melton
ERCC1-XPF is the endonuclease that cuts 5´ of the damage
in nucleotide excision repair (NER). Unlike other NER proteins,
ERCC1-XPF is also involved in recombination and the repair of
DNA interstrand cross-links. Unique among the NER gene knockouts,
Ercc1 null mice are severely runted with high levels of
hepatocyte polyploidy. To understand the link between DNA repair
deficiency and polyploidy we have compared the premature polyploidy
in Ercc1 null liver with the normal development of polyploidy
in aging control mice. Polyploidy was accelerated dramatically
in Ercc1 null hepatocytes, such that ploidy levels were
equivalent in 3-week-old Ercc1 null and 1- to 2-year-old
wild-type mouse liver. Levels of the cyclin-dependent kinase
inhibitor, p21, were increased in the nuclei of Ercc1
null hepatocytes, and this increase was concentrated in, but
not confined to, the polyploid hepatocytes. Much lower levels
of p21 messenger RNA (mRNA) were found in old wild-type liver
with equivalent levels of ploidy. We suggest that the more rapid
accumulation of DNA damage in Ercc1 null liver leads to
an increase in p21 levels, but that there is not a simple direct
link between p21 levels and premature polyploidy. The failure
to observe any link between p21 levels and polyploidy in aged
wild-type liver may be attributable to the much lower levels
of accumulated DNA damage, the much greater timescale involved,
or the existence of a p21-independent mechanism for polyploidy.
In conclusion, the premature polyploidy in Ercc1-deficient
liver differs from the normal aging-related process. (HEPATOLOGY
2003;38:958-966.)
Effects of cell proliferation on the uptake of transferrin-bound
iron by human hepatoma cells
Adrian W. M. Lee, Phillip S. Oates, Deborah Trinder
The effects of cellular proliferation on the uptake of transferrin-bound
iron (Tf-Fe) and expression of transferrin receptor-1 (TfR1)
and transferrin receptor-2 (TfR2) were investigated using a human
hepatoma (HuH7) cell line stably transfected with TfR1 antisense
RNA expression vector to suppress TfR1 expression. At transferrin
(Tf) concentrations of 50 nmol/L and 5 µmol/L, when Tf-Fe
uptake occurs by the TfR1- and TfR1-independent (NTfR1)-mediated
process, respectively, the rate of Fe uptake by proliferating
cells was approximately 250% that of stationary cells. The maximum
rate of Fe uptake by the TfR1- and NTfR1-mediated process by
proliferating cells was increased to 200% and 300% that of stationary
cells, respectively. The maximum binding of Tf by both TfR1-
and NTfR1-mediated processes by proliferating cells was increased
significantly to 160% that of stationary cells. TfR1 and TfR2-
protein levels expressed by proliferating cells was observed
to be approximately 300% and 200% greater than the stationary
cells, respectively. During the proliferating growth phase, expression
of TfR1 messenger RNA (mRNA) increased to 300% whereas TfR2-
mRNA decreased to 50% that of stationary cells. In conclusion,
an increase in Tf-Fe uptake by TfR1-mediated pathway by proliferating
cells was associated with increased TfR1 mRNA and protein expression.
An increase in Tf-Fe uptake by NTfR1-mediated pathway was correlated
with an increase in TfR2- protein expression but not TfR2- mRNA.
In concluion, TfR2- protein is likely to have a role in the mediation
of Tf-Fe uptake by the NTfR1 process by HuH7 hepatoma cell in
proliferating and stationary stages of growth. (HEPATOLOGY 2003;38:967-977.)
Liver Failure and Liver Disease
Sex is a major determinant of CYP3A4 expression in human
liver (*Human Study*)
Renzo Wolbold, Kathrin Klein, Oliver Burk, Andreas K. Nüssler,
Peter Neuhaus, Michel Eichelbaum, Matthias Schwab, Ulrich M.
Zanger
Many drugs that are substrates of CYP3A4, the major human drug-metabolizing
cytochrome P450 (CYP), show higher clearance in women than in
men. Although this effect is believed to be related to drug metabolism,
the underlying cause has not been elucidated. We investigated
CYP3A4 in a large collection (n = 94) of well-characterized surgical
liver samples and found 2-fold higher CYP3A4 levels in female
compared with male samples (P < .0001) and a corresponding
50% increase in the CYP3A-dependent N-dealkylation of
verapamil (P < .01). This expression difference was
not due to preferential induction in women following higher drug
exposure because it was even larger in a subgroup not previously
exposed to drugs. Higher expression in women was also found for
CYP3A4 messenger RNA (mRNA) transcripts, suggesting a pretranslational
mechanism. Expression of the pregnane X receptor (PXR), which
is crucially involved in CYP3A4 induction by xenobiotics, was
strongly correlated to CYP3A4 at the mRNA level in all individuals
as well as in the subgroup not exposed to drugs (r = 0.81;
P < .0001), but no sex-dependent expression of PXR
mRNA was found. The ABC transporter P-glycoprotein, which has
been proposed to be implicated in the mechanism of sex-dependent
drug clearance, was also not differentially expressed. The influence
of drug treatment on expression was examined from patient drug
histories, and strong induction of CYP3A4 by carbamazepine and
St. John's wort was found. In conclusion, sex, in addition to
PXR and drug exposure, is a major factor for CYP3A4 expression
in humans, thus explaining many of the previous observations
of sex-dependent drug clearance. (HEPATOLOGY 2003;38:978-988.)
High frequency of chimerism in transplanted livers (*Human
Study*)
Irene Oi-Lin Ng, Kok-Lung Chan, Wai-Hung Shek, Joyce Man-Fong
Lee, Daniel Yee-Tak Fong, Chung-Mau Lo, Sheung-Tat Fan
Recent studies have shown that primitive stem cells can mobilize
and differentiate into hepatocytes. We investigated the time
and extent in which cells of recipient origins could differentiate
into hepatocytes and other cells in human liver allografts. Microsatellite
analysis, which can assess quantitatively the proportions of
recipient and donor DNA, was performed in posttransplantation
liver biopsy specimens from 17 patients at various times. Combined
fluorescence in situ hybridization (FISH) for Y chromosome
and immunofluorescence for different cell types was also performed
in 10 of these cases with sex mismatch. Organ chimerism in the
transplanted livers was found to be of variable extent, and the
recipients' DNA in the posttransplantation liver biopsy specimens
(excluding portal tracts) amounted up to 50%. The recipient DNA
in the posttransplantation liver biopsy specimens increased after
liver transplantation by as early as 1 week, peaked at around
30 to 40 weeks, and could be shown 63 weeks after transplantation.
Most (64%-75%) of the recipient-derived cells showed macrophage/Kupffer
cell differentiation. Only up to 1.6% of the recipient-derived
cells in the liver grafts showed hepatocytic differentiation
in the liver grafts and made up 0.62% of all hepatocytes of both
donor and recipient origins. These livers had mild or minimal
injury histologically. In conclusion, our results show that most
of the recipient-derived cells in the liver allografts were macrophages/Kupffer
cells and only a small proportion of hepatocytes was recipient
derived. However, with regard to recipient-derived hepatocytes,
our data cannot distinguish between transdifferentiation and
cell fusion. (HEPATOLOGY 2003;38:989-998.)
Defective hepatic mitochondrial respiratory chain in patients
with nonalcoholic steatohepatitis (*Human Study*)
Mercedes Pérez-Carreras, Pilar Del Hoyo, Miguel A. Martín,
Juan C. Rubio, Ana Martín, Gregorio Castellano, Francisco
Colina, Joaquín Arenas, José A. Solis-Herruzo
Mitochondrial dysfunction might play a central role in the pathogenesis
of nonalcoholic steatohepatitis (NASH). The aims of this study
were to evaluate whether free fatty acid (FFA) transport into
the mitochondria or the activity of mitochondria respiratory
chain (MRC) complexes are impaired in NASH. In patients with
NASH and control subjects, we measured free carnitine, short-chain
acylcarnitine (SCAC) and long-chain acylcarnitine (LCAC) esters,
carnitine palmitoyltransferase (CPT) activity, and MRC enzyme
activity in liver tissue as well as serum concentration of tumor
necrosis factor (TNF-), homeostatic metabolic assessment of insulin
resistance (HOMAIR), and body mass index (BMI). In patients with
NASH, the LCAC/free carnitine ratio was significantly increased
and the SCAC/free carnitine ratio was decreased. In patients
with NASH, the activity of the MRC complexes was decreased to
63% ± 20% (complex I), 58.5% ± 16.7% (complex II),
70.6% ± 10.3% (complex III), 62.5% ± 13% (complex
IV), and 42.4% ± 9.1% (adenosine triphosphate synthase)
of the corresponding control values. Activity of these complexes
correlated significantly with serum TNF- and HOMAIR. Serum TNF-
(36.3 ± 23.1 pg/mL), HOMAIR (4.5 ± 2.38), and BMI
(29.9 ± 3.5 kg/m2) values were significantly increased
in patients with NASH. In conclusion, activities of MRC complexes
were decreased in liver tissue of patients with NASH. This dysfunction
correlated with serum TNF-, insulin resistance, and BMI values.
(HEPATOLOGY 2003;38:999-1007.)
Improved nonalcoholic steatohepatitis after 48 weeks of treatment
with the PPAR- ligand rosiglitazone (*Human Study*)
Brent A. Neuschwander-Tetri, Elizabeth M. Brunt, Kent R. Wehmeier,
Dana Oliver, Bruce R. Bacon
Insulin resistance (IR) commonly is associated with nonalcoholic
steatohepatitis (NASH). To establish whether IR causes NASH,
this study was undertaken to determine if improving IR would
improve the histologic features that define NASH. Thirty adults
with prior biopsy evidence of NASH were enrolled to receive rosiglitazone,
4 mg twice daily for 48 weeks. All patients were overweight (body
mass index [BMI] > 25 kg/m2) and 23% were severely obese (BMI
> 35 kg/m2); 50% had impaired glucose tolerance or diabetes.
Liver biopsy specimens were obtained before beginning treatment
and at treatment completion. Twenty-six patients had posttreatment
biopsies; of these, 22 had initial protocol liver biopsies that
met published criteria for NASH on subsequent blinded evaluation.
Within this initial NASH group, the mean global necroinflammatory
score significantly improved with treatment and biopsies of 10
patients (45%) no longer met published criteria for NASH after
treatment. Significant improvement in hepatocellular ballooning
and zone 3 perisinusoidal fibrosis also occurred. Five patients
withdrew early; the 25 patients completing 48 weeks of treatment
had significantly improved insulin sensitivity and mean serum
alanine aminotransferase (ALT) levels (104 initially, 42 U/L
at the end of treatment). Adverse effects led to withdrawal of
3 patients (10%). Weight gain occurred in 67% of patients and
the median weight increase was 7.3%. Within 6 months of completing
treatment, liver enzyme levels had increased to near pretreatment
levels. In conclusion, improving insulin sensitivity with rosiglitazone
resulted in improved histologic markers of NASH, an observation
suggesting that insulin resistance contributes to its development
and that improving insulin sensitivity may be important in treating
this liver disease. (HEPATOLOGY 2003;38:1008-1017.)
Myeloperoxidase-positive inflammatory cells participate in
bile duct damage in primary biliary cirrhosis through nitric
oxide-mediated reactions (*Human Study*)
Chih-Te Wu, Jason P. Eiserich, Aftab A. Ansari, Ross L. Coppel,
Sripriya Balasubramanian, Christopher L. Bowlus, M. Eric Gershwin,
Judy Van De Water
Previous studies have suggested that increased nitric oxide (NO)-mediated
products are found in the livers of subjects with primary biliary
cirrhosis (PBC), but the mechanisms involved remain enigmatic.
We took advantage of immunohistochemistry and several unique
monoclonal antibodies to study inflammatory cells responsible
for the generation of NO, the enzymes responsible for NO production,
the expression of 3-nitrotyrosine, and the presence of CD68+
and/or myeloperoxidase (MPO)+ cells. We examined a total of 113
liver specimens, including 64 with PBC, 19 with primary sclerosing
cholangitis (PSC), 6 with non-A, non-B hepatitis, 6 with alcoholic
liver disease, 4 with cryptogenic cirrhosis, 4 with biliary atresia,
and 10 normal subjects. Twenty-two percent of PBC had elevated
expression of 3-nitrotyrosine in their bile duct epithelial cells
(BECs) (P = .0316). Furthermore, the BECs in PBC also
demonstrated apoptotic changes. MPO-positive inflammatory cells
were also noted adjacent to the basement membrane. In contrast,
the liver of normal subjects showed few apoptotic changes in
the bile ducts, with no evidence of MPO staining in the portal
area. Furthermore, sections from livers of subjects with stage
I or stage II PBC demonstrated significantly increased inflammatory
cell infiltration (P = .0064) and elevated 3-nitrotyrosine
expression in BECs (P = .0246) compared with stage III
and IV. The presence of 3-nitrotyrosine was closely associated
with infiltrating CD68- and/or MPO-positive cells. There was
also a stage-associated difference in the presence of bile duct
infiltrating cells and 3-nitrotyrosine in PBC with an increase
dominant in early stage disease. In conclusion, NO and reactive
oxygen species, collectively determined as 3-nitrotyrosine, are
associated with bile duct destruction in PBC and are particularly
prevalent in early stage disease. (HEPATOLOGY 2003;38:1018-1025.)
T2 hyperintensity along the cortico-spinal tract in cirrhosis
relates to functional abnormalities (*Human Study*)
Juan Cördoba, Nuria Raguer, Montserrat Flavià, Víctor
Vargas, Carlos Jacas, Juli Alonso, Alex Rovira
Magnetic resonance has shown T2 hyperintensity along the cortico-spinal
tract in the brain of cirrhotic patients. This abnormality, which
is reversible after liver transplantation, appears to correspond
to mild edema. Because astrocytic edema present in hepatic encephalopathy
may be responsible for neuronal dysfunction, we studied whether
T2 hyperintensity along the cortico-spinal tract may relate to
functional abnormalities. Twenty patients with cirrhosis underwent
neuropsychologic tests, neurophysiologic study of the cortico-spinal
tract with transcranial magnetic stimulation, and 1H-magnetic
resonance. The study was repeated 6 months after liver transplantation
(n = 15) and was compared with a control group of healthy subjects
(n = 11). Cirrhotic patients exhibited increased T2 signal and
several functional abnormalities along the cortico-spinal tract
(increased central motor conduction time, increased motor cortical
threshold, and decreased motor-evoked potential amplitude). Functional
abnormalities reversed after liver transplantation and were associated
with normalization of T2 cortico-spinal hyperintensity and with
improvement of minimal hepatic encephalopathy. In conclusion,
T2 hyperintensity along the cortico-spinal tract in cirrhosis
relates to functional abnormalities that are reversible after
liver transplantation. These findings suggest that mild cerebral
edema along the cortico-spinal pathway may cause neuronal dysfunction.
These results support the participation of astrocytic edema in
the pathogenesis of hepatic encephalopathy. (HEPATOLOGY 2003;38:1026-1033.)
MRI angiography is superior to helical CT for detection of
HCC prior to liver transplantation: An explant correlation
(*Human Study*)
Marta Burrel, Josep M. Llovet, Carmen Ayuso, Carmela Iglesias,
Margarita Sala, Rosa Miquel, Teresa Caralt, Juan Ramon Ayuso,
Manel Solé, Marcelo Sanchez, Concepció Brú,
Jordi Bruix, Barcelona Clínic Liver Cancer (BCLC) Group
Helical computerized tomography (CT) and magnetic resonance imaging
(MRI) are used for staging of hepatocellular carcinoma (HCC)
prior to curative treatments but underestimate tumor extension
in 30% to 50% of cases when compared with pathologic explants.
This study compares a new technology, MRI angiography (MRA),
with triphasic helical CT in detection of HCC. Fifty cirrhotic
patients, 29 with HCC, undergoing liver transplantation were
analyzed. MRA was performed with a 3-D breath-hold fast spoiled
gradient-echo sequence by using an effective section thickness
of 2 to 2.5 mm. The gold standard was the pathologic examination
(liver cut into 5-mm slices). One hundred twenty-seven lesions
were identified at the explant: 76 HCC, 13 high-grade dysplastic
nodules, 31 macroregenerative nodules, 7 hemangiomas. Diameter
of the main HCC nodules was 29 ± 14 mm and 11 ±
7 mm for the 47 additional nodules. On a per nodule basis, sensitivity
of MRA was superior to CT (58/76 [76%] vs. 43/70 [61%], respectively,
P = .001). Sensitivity of MRA for detection of additional
nodules decreased with size (>20 mm: 6/6 [100%]; 10-20 mm:
16/19 [84%]; <10 mm: 7/22 [32%]) and was superior to CT for
nodules 10 to 20 mm (84% vs. 47%, P = .016). Nonspecific
hypervascular nodules >5 mm at MRA were HCC in two thirds
of the cases. In conclusion, MRA has a high diagnostic accuracy
for HCC 10 mm and is more sensitive than triphasic helical CT
in nodules sized 10 to 20 mm. MRA is the optimal technique for
HCC staging prior to curative therapies. (HEPATOLOGY 2003;38:1034-1042.)
Survival in patients undergoing transjugular intrahepatic
portosystemic shunt: ePTFE-covered stentgrafts versus bare stents
(*Human Study*)
Bernhard Angermayr, Manfred Cejna, Franz Koenig, Franz Karnel,
Franz Hackl, Markus Peck-Radosavljevic, Vienna TIPS Study Group
In patients with liver cirrhosis, implantation of a transjugular
intrahepatic shunt (TIPS) leads to reduction of portal pressure,
but not of mortality compared with other therapies. The high
stenosis rates of conventional bare stents causes high reintervention
rates and costs and may be correlated with poor survival. ePTFE-covered
stentgrafts provide much improved patency rates, but their impact
on survival is unclear. All suitable patients receiving either
bare TIPS (419/466) or undergoing implantation of ePTFE endoprostheses
(89/100) in several centers in Austria up to 2002 were included
in this retrospective analysis. Both patient groups were compared
regarding survival with Kaplan-Meier and Cox regression analysis.
Unmatched and 1:1-matched survival analyses were performed. Patients
undergoing ePTFE stentgraft implantation had significantly higher
survival rates in all analyses. The 3-month, 1-year, and 2-year
survival rates were 93%, 88%, and 76% for the ePTFE-group and
83%, 73%, and 62% for conventional TIPS patients, respectively.
The matched survival analyses validated these findings. The model
of the stent, patient age, and Child-Pugh Class (CPC) were independent
predictors of survival. In conclusion, patients undergoing ePTFE-endoprosthesis
implantation had higher survival rates within 2 years after TIPS-implantation.
This may be the result of improved patency rates after correct
placement (up to the inferior caval vein [ICV]) of the ePTFE
stentgraft. These data should be validated in a prospective series.
(HEPATOLOGY 2003;38:1043-1050.)
Copyright © 2003 by the American Association for the
Study of Liver Diseases. All rights reserved.
Table of Contents for October
2003 · Volume 125 · Number 4
Rapid Communication
Enteric involvement of severe acute respiratory syndrome-associated
coronavirus infection
Wai K. Leung, Ka-fai To, Paul K.S. Chan, Henry L.Y. Chan, Alan
K.L. Wu, Nelson Lee, Kwok Y. Yuen, Joseph J.Y. Sung
Background & Aims: Severe acute respiratory syndrome
(SARS) is a recently emerged infection from a novel coronavirus
(CoV). Apart from fever and respiratory complications, gastrointestinal
symptoms are frequently observed in patients with SARS but the
significance remains undetermined. Herein, we describe the clinical,
pathologic, and virologic features of the intestinal involvement
of this new viral infection. Methods: A retrospective
analysis of the gastrointestinal symptoms and other clinical
parameters of the first 138 patients with confirmed SARS admitted
for a major outbreak in Hong Kong in March 2003 was performed.
Intestinal specimens were obtained by colonoscopy or postmortem
examination to detect the presence of coronavirus by electron
microscopy, virus culture, and reverse-transcription polymerase
chain reaction. Results: Among these 138 patients with
SARS, 28 (20.3%) presented with watery diarrhea and up to 38.4%
of patients had symptoms of diarrhea during the course of illness.
Diarrhea was more frequently observed during the first week of
illness. The mean number of days with diarrhea was 3.7 ±
2.7, and most diarrhea was self-limiting. Intestinal biopsy specimens
obtained by colonoscopy or autopsy showed minimal architectural
disruption but the presence of active viral replication within
both the small and large intestine. Coronavirus was also isolated
by culture from these specimens, and SARS-CoV RNA can be detected
in the stool of patients for more than 10 weeks after symptom
onset. Conclusions: Diarrhea is a common presenting symptom
of SARS. The intestinal tropism of the SARS-CoV has major implications
on clinical presentation and viral transmission.
Clinical-alimentary Tract
Esophagogastric junction opening during relaxation distinguishes
nonhernia reflux patients, hernia patients, and normal subjects
John E. Pandolfino, Guoxiang Shi, Brian Trueworthy, Peter J.
Kahrilas
Background & Aims: Flow across the esophagogastric
junction (EGJ) is strongly related to opening dimensions. This
study aimed to determine whether opening of the relaxed EGJ was
altered in patients with gastroesophageal reflux disease (GERD).
Methods: Seven normal subjects (NL), 9 GERD patients without
hiatus hernia (NHH), and 7 with hiatus hernia (HH) were studied.
Cross-sectional area (CSA) of the relaxed EGJ was measured during
low-pressure distention using a modified barostat technique that
resulted in filling a compliant bag straddling the EGJ with renograffin
to the set pressure. Swallows were imaged fluoroscopically at
distensive pressures of 212 mm Hg. The diameter of the narrowest
point of the EGJ in PA and lateral projections was measured from
digitized images. CSA was determined as a function of intrabag
pressure. Results: The minimal EGJ opening aperture occurred
at the diaphragmatic hiatus in all subjects. At pressures 0 mm
Hg, EGJ opening was observed only in HH patients, making it plausible
for these patients to reflux during deglutitive relaxation. At
pressures >0 mm Hg, there were significant increases in EGJ
CSA both for HH and NHH compared with NL (P < 0.001)
and for HH compared with NHH (P < 0.005). This difference
may explain the diminished air/water discrimination seen during
transient lower esophageal sphincter (LES) relaxation-associated
reflux in GERD patients. Conclusions: Anatomic degradation
of the EGJ distinguishes GERD patients from normal subjects,
and these changes may impact on both the observed mechanisms
of reflux and the constituents of reflux during transient LES
relaxation. Therapy focused on EGJ compliance may benefit GERD
patients.
Randomized, double-blind comparison of 4 mg/kg versus 2 mg/kg
intravenous cyclosporine in severe ulcerative colitis
Gert Van Assche, Geert D'haens, Maja Noman, Séverine Vermeire,
Martin Hiele, Katrien Asnong, Joris Arts, Andre D'hoore, Freddy
Penninckx, Paul Rutgeerts
Background & Aims: Cyclosporine A is highly effective
in severe attacks of ulcerative colitis (UC) but is associated
with important adverse effects that are mainly dose dependent.
Our single center, randomized, double-blind, controlled trial
aimed to evaluate the additional clinical benefit of 4 mg/kg
over 2 mg/kg IV cyclosporine in the acute treatment of severe
UC. Methods: Primary end point was the proportion of patients
with a clinical response. Secondary end points included time
to response, colectomy rate, and adverse effects. Results:
Seventy-three patients were included. Day-8 response rates were
84.2% (32 of 38, 4 mg/kg) and 85.7% (32 of 35, 2 mg/kg) after
a median of 4 days in both groups. Short-term colectomy rates
were 13.1% (4 mg/kg) and 8.6% (2 mg/kg). Mean cyclosporine blood
levels were 237 ± 33 in the 2-mg/kg group and 332 ±
43 ng/mL in the 4-mg/kg group. Active smoking was inversely correlated
with clinical response (odds ratio, 0.06), but concomitant azathioprine
or steroids were not predictive. A trend toward a higher incidence
of hypertension was observed in the 4-mg/kg group (23.7% vs.
8.6%, 2 mg/kg, P < 0.08). Conclusions: High-dose
IV cyclosporine has no additional clinical benefit over low dose
in the treatment of severe UC. Although we did not observe differences
in adverse effects on the short term, the use of 2 mg/kg IV cyclosporine
should provide an improved toxicity profile for medical treatment
of severe UC.
A major non-HLA locus in celiac disease maps to chromosome
19
Martine J. Van Belzen, Jos W.R. Meijer, Lodewijk A. Sandkuijl,
Alfons F.J. Bardoel, Chris J.J. Mulder, Peter L. Pearson, Roderick
H.J. Houwen, Cisca Wijmenga
Background & aims: The pathogenesis of celiac disease
is still unknown despite its well-known association with human
leukocyte antigen (HLA)-DQ2 and DQ8. It is clear that non-HLA
genes contribute to celiac disease development as well, but none
of the previous genome-wide screens in celiac disease have resulted
in identification of these genes. Methods: We, therefore,
performed a 2-stage, genome-wide screen in 101 affected sibpairs
from 82 Dutch families who met strict diagnostic criteria. The
small intestinal biopsy samples, on which the original celiac
disease diagnoses had been based, showed a Marsh III lesion in
all patients on reevaluation by 1 pathologist. For association
analysis of markers in regions linked to celiac disease, 216
independent MIII patients and 216 age- and sex-matched controls
were available. Results: As expected, highly significant
linkage to the HLA-region was detected (multipoint maximum lod
score [MMLS] = 8.14). More importantly, significant linkage was
also present at 19p13.1 (MMLS = 4.31), with the peak at marker
D19S899. Moreover, this marker was also significantly associated
with celiac disease in the case-control study (corrected P
= 0.016). Furthermore, we identified suggestive linkage to 6q2122,
which is ~70 cM downstream from the HLA region (MMLS = 3.10).
Conclusions: Significant linkage of celiac disease to
chromosome region 19p13.1 was detected in our genome-wide screen.
These results were confirmed by the association of D19S899 to
celiac disease in an independent case-control cohort. Furthermore,
we identified a possible second celiac disease locus on chromosome
region 6q2122.
Clinical-liver, Pancreas, and Biliary
Tract
Prognostic significance of the hepatopulmonary syndrome
in patients with cirrhosis
Peter Schenk, Maximilian Schöniger-Hekele, Valentin Fuhrmann,
Christian Madl, Gerd Silberhumer, Christian Müller
Background & aims: The hepatopulmonary syndrome (HPS)
has been defined by chronic liver disease, arterial deoxygenation,
and widespread intrapulmonary vasodilation. Mortality of patients
with HPS is considered to be high, but the effect of HPS on survival
in patients with cirrhosis remains unclear. Methods: A
total of 111 patients with cirrhosis were studied prospectively
by using transthoracic contrast echocardiography for detection
of pulmonary vasodilation, blood gas analysis, and pulmonary
function test. Twenty different clinical characteristics and
survival times were noted. Results: Twenty-seven patients
(24%) had HPS. Their mortality was significantly higher (median
survival, 10.6 months) compared with patients without HPS (40.8
mo, P < 0.05), even after adjusting for liver disease
severity (2.9 vs. 14.7 months in Child-Pugh class C with [n =
15] and without HPS [n = 35, P < 0.05]; 35.3 vs. 44.5
months in Child-Pugh class B with [n = 7] and without HPS [n
= 23, P = NS]), and exclusion of patients who underwent
liver transplantation during follow-up (median survival 4.8 vs.
35.2 months, P = 0.005). Causes of death were mainly nonpulmonary
and liver-related in the 19 patients with and the 35 patients
without HPS who died. In multivariate analysis, HPS was an independent
predictor of survival besides age, Child-Pugh class, and blood
urea nitrogen. Mortality correlates with severity of HPS. Conclusions:
The presence of HPS independently worsens prognosis of patients
with cirrhosis. This should influence patient management and
scoring systems and accelerate the evaluation process for liver
transplantation.
Is obesity a risk factor for cirrhosis-related death or hospitalization?
a population-based cohort study
George N. Ioannou, Noel S. Weiss, Kris V. Kowdley, Jason A. Dominitz
Background & aims: Our aim was to determine whether
increased body mass index (BMI) in the general population is
associated with cirrhosis-related death or hospitalization. Methods:
Participants included 11,465 persons aged 2574 years without
evidence of cirrhosis at entry into the study, or during the
first 5 years of follow-up, who subsequently were followed-up
for a mean of 12.9 years. The BMI was used to categorize participants
into normal-weight (BMI < 25 kg/m2, N = 5752), overweight
(BMI 25 to < 30 kg/m2, N = 3774), and obese categories (BMI
30 kg/m2, N = 1939). Results: Cirrhosis resulted in death
or hospitalization of 89 participants during 150,233 person-years
of follow-up (0.59/1000 person-years). Cirrhosis-related deaths
or hospitalizations were more common in obese persons (0.81/1000
person-years, adjusted hazard ratio 1.69, 95% confidence interval
[CI] 1.03.0) and in overweight persons (0.71/1000 person-years,
adjusted hazard ratio 1.16, 95% CI 0.71.9) compared with
normal-weight persons (0.45/1000 person-years). Among persons
who did not consume alcohol, there was a strong association between
obesity (adjusted hazard ratio 4.1, 95% CI 1.411.4) or being
overweight (adjusted hazard ratio 1.93, 95% CI 0.75.3) and
cirrhosis-related death or hospitalization. In contrast, this
association was weaker among persons who consumed up to 0.3 alcoholic
drinks/day (adjusted hazard ratio 2.48, 95% CI 0.78.4 for
obesity; adjusted hazard ratio 1.31, 95% CI 0.44.2 for overweight)
and no association was identified among those who consumed more
than 0.3 alcoholic drinks/day. Conclusions: Obesity appears
to be a risk factor for cirrhosis-related death or hospitalization
among persons who consume little or no alcohol.
The chemokine CCL21 modulates lymphocyte recruitment and fibrosis
in chronic hepatitis C
Andrea Bonacchi, Ilaria Petrai, Raffaella M.S. Defranco, Elena
Lazzeri, Francesco Annunziato, Eva Efsen, Lorenzo Cosmi, Paola
Romagnani, Stefano Milani, Paola Failli, Giacomo Batignani, Francesco
Liotta, Giacomo Laffi, Massimo Pinzani, Paolo Gentilini, Fabio
Marra
Background & aims: The chemokines CCL19 and CCL21
bind CCR7, which is involved in the organization of secondary
lymphoid tissue and is expressed during chronic tissue inflammation.
We investigated the expression of CCL21 and CCR7 in chronic hepatitis
C. The effects of CCL21 on hepatic stellate cells (HSCs) were
also studied. Methods: Expression of CCL21 was assessed
by in situ hybridization and immunohistochemistry. CCR7 on T
cells was analyzed by flow cytometry. Cultured human HSCs were
studied in their activated phenotype. Results: In patients
with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated.
CCL21 was detected in the portal tracts and around inflammatory
lymphoid follicles, in proximity to T lymphocytes and dendritic
cells, which contributed to expression of this chemokine. Expression
of CCR7 was also increased in patients with primary biliary cirrhosis.
Intrahepatic CD8+ T lymphocytes isolated from patients with chronic
hepatitis C had a significantly higher percentage of positivity
for CCR7 than those from healthy controls, and the expression
of CCR7 was associated with that of CXCR3. Cultured HSCs expressed
functional CCR7, the activation of which stimulated cell migration
and accelerated wound healing in an in vitro model. Exposure
of HSCs to CCL21 triggered several signaling pathways, including
extracellular signal-regulated kinase, Akt, and nuclear factor
B, resulting in induction of proinflammatory genes. Conclusions:
Expression of CCL21 during chronic hepatitis C is implicated
in the recruitment of T lymphocytes and the organization of inflammatory
lymphoid tissue and may promote fibrogenesis in the inflamed
areas via activation of CCR7 on HSCs.
Selection of a secretion-incompetent mutant in the serum of
a patient with severe hepatitis B
Tatyana Kalinina, Anna Riu, Lutz Fischer, Theresa Santantonio,
Hans Will, Martina Sterneck
Background & Aims: A secretion-incompetent, highly
replicating hepatitis B variant was previously found as the dominant
viral population in the serum of a liver transplant recipient
with severe hepatitis B reinfection. The secretion block resulted
from mutations in the S protein, including the Gly145Arg substitution
known to emerge under antibody to hepatitis B surface antigen
immunoglobulin treatment. Here we investigated the mechanisms
that allow selection of a secretion-incompetent virus as the
predominant strain in the serum. Methods: To reproduce
the interaction of viral quasispecies occurring in vivo, cotransfection
experiments were performed with full-length genomes containing
wild-type or mutant sequences. In addition, the relevance of
mutations in the common S part of the surface proteins for the
competence of L and S protein to support viral secretion was
studied. Results: A small amount of wild-type virus or
of a wild-type S protein-expressing variant rescued secretion
of the defective mutant. In the secreted virions, the high-replicating
mutant genome was predominant. Selection of the defective mutant
was further supported by a transdominant negative effect of mutant
S protein on wild-type virion secretion. In contrast, mutant
L protein with the same c-terminal mutations as mutant S protein
efficiently supported virion formation and secretion. Conclusions:
Interaction of the variant with a small amount of wild-type virus
can reverse its secretion-defective phenotype. Mutations in the
common region of S and L protein have different consequences
for the ability of the envelope proteins to support virion assembly
and secretion.
A novel MCP-1 gene polymorphism is associated with hepatic
MCP-1 expression and severity of HCV-related liver disease
Marcus Mühlbauer, Anja K. Bosserhoff, Arndt Hartmann, Wolfgang
E. Thasler, Thomas S. Weiss, Hans Herfarth, Guntram Lock, Jürgen
Schölmerich, Claus Hellerbrand
Background & Aims: Factors influencing the progression
of chronic hepatitis C virus (HCV) infection are poorly understood.
Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine,
and its hepatic expression is up-regulated during chronic HCV
infection mainly in activated hepatic stellate cells (HSC). In
this study, we investigated the correlation of the functional
2518 MCP-1 promoter polymorphism with hepatic MCP-1 expression
and the disease outcome in patients with HCV. Methods:
MCP-1 genotyping was performed in 206 patients and 139 healthy
controls. Hepatic MCP-1 messenger RNA (mRNA) expression was quantified
by real-time PCR in 58 HCV patients. Cytokine-induced MCP-1 secretion
of activated human HSC (n = 13) was determined by enzyme-linked
immunosorbent assay (ELISA). Mobility-shift assays were performed
using probes corresponding to the MCP-1 promoter sequence (2511
to 2528) with or without the A to G mutation at 2518.
Results: Frequency of MCP-1 genotypes did not differ between
HCV patients and controls. However, carriers of the G allele
were significantly more frequent in HCV patients with more advanced
fibrosis and severe inflammation. In accordance, hepatic MCP-1
mRNA levels were significantly higher in patients with more advanced
fibrosis and in patients carrying the G allele. Furthermore,
cytokine-induced MCP-1 secretion of HSC isolated from carriers
of the G allele was significantly higher, and there was binding
activity in nuclear extracts from activated HSC specifically
to the G allele, providing a potential mechanism for the differences
seen. Conclusions: Inheritance of the 2518 MCP-1
G allele, which appears to affect hepatic MCP-1 expression, may
predispose HCV patients to more severe hepatic inflammation and
fibrosis.
Low microvessel density is an unfavorable histoprognostic
factor in pancreatic endocrine tumors
Anne-Marie Marion-Audibert, Cécile Barel, Géraldine
Gouysse, Jérôme Dumortier, Frank Pilleul, Céline
Pourreyron, Valérie Hervieu, Gilles Poncet, Catherine
Lombard-Bohas, Jean-Alain Chayvialle, Christian Partensky, Jean-Yves
Scoazec
Background & Aims: In many malignant tumors, intratumoral
microvascular density (MVD) has been suggested to be a prognostic
parameter. We aimed to provide a quantitative evaluation of intratumoral
microvascular density in a large series of resected endocrine
tumors of the pancreas and to evaluate the potential prognostic
significance of this parameter. Methods: Eighty-two tumors
from 77 patients have been studied. MVD was evaluated by 2 observers
after CD34 immunostaining and correlated with the following parameters:
WHO classification, hormonal profile, tumor size, vascular endothelial
growth factor expression, occurrence of metastasis, duration
of survival. Results: MVD ranged from 5 to 92 vessels/field.
MVD was significantly higher in well-differentiated benign endocrine
tumors than in tumors of uncertain behavior and in carcinomas.
No close correlation was found between MVD and the hormonal profile.
MVD was significantly higher in tumors characterized by the following
histoprognostic parameters: size <2 cm, proliferation index
<2%, no evidence of metastasis. No close correlation was observed
between MVD and VEGF expression. Finally, a MVD <30 vessels/field
was associated with the occurrence of metastasis in tumors <2
cm and/or with a proliferation index <2% and with a significantly
shorter survival after surgery. Conclusions: The quantitative
analysis of microvessel density in pancreatic endocrine tumors
may identify patients who, despite favorable conventional histoprognostic
factors, are at risk of unfavorable evolution.
Basic-alimentary Tract
Characterization of cereal toxicity for celiac disease
patients based on protein homology in grains
L.Willemijn Vader, Dariusz T. Stepniak, Evelien M. Bunnik, Yvonne
M.C. Kooy, Willeke De Haan, Jan Wouter Drijfhout, Peter A. Van
Veelen, Frits Koning
Background & Aims: Celiac disease is caused by T-cell
responses to wheat gluten-derived peptides. The presence of such
peptides in other widely consumed grains, however, has hardly
been studied. Methods: We have performed homology searches
to identify regions with sequence similarity to T-cell stimulatory
gluten peptides in the available gluten sequences: the hordeins
of barley, secalins of rye, and avenins of oats. The identified
peptides were tested for T-cell stimulatory properties. Results:
With 1 exception, no identical matches with T-cell stimulatory
gluten peptides were found in the other grains. However, less
stringent searches identified 11 homologous sequences in hordeins,
secalins, and avenins located in regions similar to those in
the original gluten proteins. Seven of these 11 peptides were
recognized by gluten-specific T-cell lines and/or clones from
patients with celiac disease. Comparison of T-cell stimulatory
sequences with homologous but non-T-cell stimulatory sequences
indicated key amino acids that on substitution either completely
or partially abrogated the T-cell stimulatory activity of the
gluten peptides. Finally, we show that single nucleotide substitutions
in gluten genes will suffice to induce these effects. Conclusions:
These results show that the disease-inducing properties of barley
and rye can in part be explained by T-cell cross-reactivity against
gluten-, secalin-, and hordein-derived peptides. Moreover, the
results provide a first step toward a rational strategy for gluten
detoxification via targeted mutagenesis at the genetic level.
Growth hormone reduces chloride secretion in human colonic
epithelial cells via EGF receptor and extracellular regulated
kinase
Jimmy Y.C. Chow, Katie Carlstrom, Kim E. Barrett
Background & Aims: Growth hormone (GH) has been shown
to alleviate symptoms in patients with Crohn's disease. Chloride
secretion is important in driving intestinal fluid secretion.
We examined whether GH inhibits chloride secretion induced by
carbachol (CCh, a calcium-dependent pathway), and the downstream
effectors responsible. Methods: T84 cells were pretreated
with GH at various concentrations followed by CCh (100 µmol/L).
Chloride secretion was assessed as changes in short circuit current
(Isc) in Ussing chambers. Tyrphostins AG1478 (an epidermal growth
factor receptor [EGFr] inhibitor) and AG490 (a Janus kinase 2
[JAK2] inhibitor), SB203580 (a p38 inhibitor), and PD98059 (a
MEK1 inhibitor) were used. Results: GH inhibited CCh-induced
chloride secretion at up to 10 nmol/L, but higher concentrations
were less effective. GH caused tyrosine phosphorylation of JAK2
and EGFr. AG490 suppressed activation of JAK2 and EGFr in response
to GH. AG1478 prevented GH activation of EGFr and reversed its
inhibitory effect on chloride secretion. GH also induced activation
of both p38 and ERK1/2. AG490 reversed GH-induced tyrosine phosphorylation
of both ERK1/2 and p38, but AG1478 reversed that of ERK1/2 only.
PD98059, but not SB203580, reversed the inhibitory effect of
GH on chloride secretion. Conclusions: GH inhibits CCh-induced
chloride secretion via a JAK2-dependent mechanism involving transactivation
of EGFr and consequent recruitment of ERK1/2. Although activated,
p38 does not contribute to the inhibitory effect of GH on secretion.
These data elucidate mechanisms of GH inhibition of chloride
secretion in intestinal epithelia, which may be relevant to therapeutic
benefits of GH in Crohn's disease or other diarrheal diseases.
Helicobacter pylori strain-selective induction of matrix metalloproteinase-7
in vitro and within gastric mucosa
Howard C. Crawford, Uma S. Krishna, Dawn A. Israel, Lynn M. Matrisian,
M.Kay Washington, Richard M. Peek, Jr
Background & Aims: Helicobacter pylori strains
that possess the cag pathogenicity island (cag+)
augment the risk for distal gastric cancer. Matrix metalloproteinase
(MMP)-7, an epithelial cell-derived MMP that is induced by bacterial
contact, is overexpressed within human gastric adenocarcinoma
specimens and enhances tumor formation in rodents. We determined
whether H. pylori alters MMP-7 expression and investigated
the molecular pathways required for these events. Methods:
MMP-7 was detected in human gastric mucosa by immunohistochemistry
and in H. pylori/AGS gastric epithelial cell coculture
supernatants by Western analysis. AGS cells were cocultured with
wild-type H. pylori, or isogenic cagA, cagE,
or vacA mutants, in the absence or presence of inhibitors
of nuclear factor B activation, p38, or extracellular signal-regulated
kinase (ERK) mitogen-activated protein kinase. Results:
H. pylori cag+ strains increased MMP-7 expression in AGS
cells 57-fold, whereas cag isolates had no effect.
Inactivation of cagE, but not cagA or vacA,
completely attenuated induction of MMP-7, and inhibition of ERK
1/2 decreased MMP-7 production. In vivo, MMP-7 was expressed
in gastric epithelial cells in specimens from 80% of cag+-colonized
persons but in none of the cag or uninfected subjects.
Conclusions: H. pylori cag+ strains enhance levels
of MMP-7 within inflamed mucosa. In vitro, cag+ isolates
selectively induce MMP-7, and this is dependent on activation
of ERK 1/2 by specific components within the cag island.
Differential induction of MMP-7 by H. pylori cag+ isolates
may explain in part the augmentation in gastric cancer risk associated
with these strains.
Postoperative ileus is maintained by intestinal immune infiltrates
that activate inhibitory neural pathways in mice
Wouter J. De Jonge, René M. Van Den Wijngaard, Frans O.
The, Merel-Linde Ter Beek, Roel J. Bennink, Guido N.J. Tytgat,
Ruud M. Buijs, Pieter H. Reitsma, Sander J. Van Deventer, Guy
E. Boeckxstaens
Background & Aims: Postoperative ileus after abdominal
surgery largely contributes to patient morbidity and prolongs
hospitalization. We aimed to study its pathophysiology in a murine
model by determining gastric emptying after manipulation of the
small intestine. Methods: Gastric emptying was determined
at 6, 12, 24, and 48 hours after abdominal surgery by using scintigraphic
imaging. Intestinal or gastric inflammation was assessed by immune-histochemical
staining and measurement of tissue myeloperoxidase activity.
Neuromuscular function of gastric and intestinal muscle strips
was determined in organ baths. Results: Intestinal manipulation
resulted in delayed gastric emptying up to 48 hours after surgery;
gastric half-emptying time 24 hours after surgery increased from
16.0 ± 4.4 minutes after control laparotomy to 35.6 ±
5.4 minutes after intestinal manipulation. The sustained delay
in gastric emptying was associated with the appearance of leukocyte
infiltrates in the muscularis of the manipulated intestine, but
not in untouched stomach or colon. The delay in postoperative
gastric emptying was prevented by inhibition of intestinal leukocyte
recruitment. In addition, postoperative neural blockade with
hexamethonium (1 mg/kg intraperitoneally) or guanethidine (50
mg/kg intraperitoneally) normalized gastric emptying without
affecting small-intestinal transit. The appearance of intestinal
infiltrates after intestinal manipulation was associated with
increased c-fos protein expression in sensory neurons in the
lumbar spinal cord. Conclusions: Sustained postoperative
gastroparesis after intestinal manipulation is mediated by an
inhibitory enterogastric neural pathway that is triggered by
inflammatory infiltrates recruited to the intestinal muscularis.
These findings show new targets to shorten the duration of postoperative
ileus pharmacologically.
cAMP inhibition of murine intestinal Na+/H+
exchange requires CFTR-mediated cell shrinkage of villus epithelium
Lara R. Gawenis, Craig L. Franklin, Janet E. Simpson, Bradley
A. Palmer, Nancy M. Walker, Tarra M. Wiggins, Lane L. Clarke
Background & Aims: Unlike the intestine of normal
subjects, small-intestinal epithelia of cystic fibrosis patients
and cystic fibrosis transmembrane conductance regulator protein-null
(CFTR) mice do not respond to stimulation of intracellular
cyclic adenosine monophosphate with inhibition of electroneutral
NaCl absorption. Because CFTR-mediated anion secretion has been
associated with changes in crypt cell volume, we hypothesized
that CFTR-mediated cell volume reduction in villus epithelium
is required for intracellular cyclic adenosine monophosphate
inhibition of Na+/H+ exchanger (primarily Na+/H+ exchanger 3)
activity in the proximal small intestine. Methods: Transepithelial
22Na flux across the jejuna of CFTR+, CFTR, the basolateral
membrane Na+/K+/2Cl co-transporter protein NKCC1+, and NKCC1
mice were correlated with changes in epithelial cell volume of
the midvillus region. Results: Stimulation of intracellular
cyclic adenosine monophosphate resulted in cessation of Na+/H+
exchanger-mediated Na+ absorption (JmsNHE) in CFTR+ jejunum but
had no effect on JmsNHE across CFTR jejunum. Cell volume
indices indicated an approximately 30% volume reduction of villus
epithelial cells in CFTR+ jejunum but no changes in CFTR
epithelium after intracellular cyclic adenosine monophosphate
stimulation. In contrast, cell shrinkage induced by hypertonic
medium inhibited JmsNHE in both CFTR+ and CFTR mice. Bumetanide
treatment to inhibit Cl secretion by blockade of the Na+/K+/2Cl
co-transporter, NKCC1, of stimulated CFTR+ jejunum prevented
maximal volume reduction of villus epithelium and recovered approximately
40% of JmsNHE. Likewise, JmsNHE and cell volume were unaffected
by intracellular cyclic adenosine monophosphate stimulation in
NKCC1 jejuna. Conclusions: These findings show a
previously unrecognized role of functional CFTR expressed in
villus epithelium: regulation of Na+/H+ exchanger 3-mediated
Na+ absorption by alteration of epithelial cell volume.
Hypoglycemia, defective islet glucagon secretion, but normal
islet mass in mice with a disruption of the gastrin gene
Robin P. Boushey, Amir Abadir, Daisy Flamez, Laurie L. Baggio,
Yazhou Li, Veerle Berger, Bess A. Marshall, Diane Finegood, Timothy
C. Wang, Frans Schuit, Daniel J. Drucker
Background & Aims: Both cholecystokinin (CCK)-A and
CCK-B receptors are expressed in the pancreas, and exogenous
gastrin administration stimulates glucagon secretion from human
islets. Although gastrin action has been linked to islet neogenesis,
transdifferentiation, and beta-cell regeneration, an essential
physiologic role(s) for gastrin in the pancreas has not been
established. Methods: We examined glucose homeostasis,
glucagon gene expression, glucagon secretion, and islet mass
in mice with a targeted gastrin gene disruption. Results:
Gastrin / mice exhibit fasting hypoglycemia and significantly
reduced glycemic excursion following glucose challenge. Insulin
sensitivity was normal and levels of circulating insulin and
insulin messenger RNA transcripts were appropriately reduced
in gastrin / mice. In contrast, levels of circulating
glucagon and pancreatic glucagon messenger RNA transcripts were
not up-regulated in hypoglycemic gastrin / mice. Furthermore,
the glucagon response to epinephrine in isolated perifused islets
was moderately impaired in gastrin / versus gastrin
+/+ islets (40% reduction; P < 0.01, gastrin +/+ vs.
gastrin / mice). Moreover, the glucagon response but
not the epinephrine response to hypoglycemia was significantly
attenuated in gastrin / compared with gastrin +/+ mice
(P < 0.05). Despite gastrin expression in the developing
fetal pancreas, beta-cell area, islet topography, and the islet
proliferative response to experimental injury were normal in
gastrin / mice. Conclusions: These findings
show an essential physiologic role for gastrin in glucose homeostasis;
however, the gastrin gene is not essential for murine islet development
or the adaptive islet proliferative response to beta-cell injury.
Basic-liver, Pancreas, and Biliary
Tract
Loss of Inositol 1,4,5-trisphosphate receptors from bile
duct epithelia is a common event in cholestasis
Kazunori Shibao, Keiji Hirata, Marie E. Robert, Michael H. Nathanson
Background & Aims: Cholestasis is one of the principal
manifestations of liver disease and often results from disorders
involving bile duct epithelia rather than hepatocytes. A range
of disorders affects biliary epithelia, and no unifying pathophysiologic
event in these cells has been identified as the cause of cholestasis.
Here we examined the role of the inositol 1,4,5-trisphosphate
receptor (InsP3R)/Ca2+ release channel in Ca2+ signaling and
ductular secretion in animal models of cholestasis and in patients
with cholestatic disorders. Methods: The expression and
distribution of the InsP3R and related proteins were examined
in rat cholangiocytes before and after bile duct ligation or
treatment with endotoxin. Ca2+ signaling was examined in isolated
bile ducts from these animals, whereas ductular bicarbonate secretion
was examined in isolated perfused livers. Confocal immunofluorescence
was used to examine cholangiocyte InsP3R expression in human
liver biopsy specimens. Results: Expression of the InsP3R
was selectively lost from biliary epithelia after bile duct ligation
or endotoxin treatment. As a result, Ca2+ signaling and Ca2+-mediated
bicarbonate secretion were lost as well, although other components
of the Ca2+ signaling pathway and adenosine 3´,5´-cyclic
monophosphate (cAMP)-mediated bicarbonate secretion both were
preserved. Examination of human liver biopsy specimens showed
that InsP3Rs also were lost from bile duct epithelia in a range
of human cholestatic disorders, although InsP3R expression was
intact in noncholestatic liver disease. Conclusions: InsP3-mediated
Ca2+ signaling in bile duct epithelia appears to be important
for normal bile secretion in the liver, and loss of InsP3Rs may
be a final common pathway for cholestasis.
Extracellular matrix proteins protect pancreatic cancer cells
from death via mitochondrial and nonmitochondrial pathways
Eva C. Vaquero, Mouad Edderkaoui, Kyung J. Nam, Ilya Gukovsky,
Stephen J. Pandol, Anna S. Gukovskaya
Background & Aims: Pancreatic cancer is a very aggressive
malignancy. Normal cells die through apoptosis when detached
from extracellular matrix (ECM), but the role of ECM in cancer
cell survival is poorly understood. Here, we determined the effects
of ECM proteins on death responses and underlying signaling pathways
in human pancreatic cancer cells. Methods: We measured
apoptosis and necrosis, caspase activation, and mitochondrial
dysfunction in MIA PaCa-2 and PANC-1 pancreatic carcinoma cells
both detached and attached to ECM proteins. Results: Detachment
of pancreatic cancer cells from ECM did not induce classic apoptosis,
as it does in normal cells, but induced necrosis and apoptosis
associated with secondary necrosis. It caused a pronounced mitochondrial
depolarization and release of cytochrome c and Smac/DIABLO.
However, as different from normal cells, cytochrome c
release did not result in downstream caspase activation. Executioner
caspases were activated in detached pancreatic cancer cells independent
of cytochrome c. Laminin and fibronectin, but not collagen
I, markedly increased pancreatic cancer cell survival by inhibiting
both mitochondrial dysfunction (leading to inhibition of necrosis)
and caspase activity (leading to decreased apoptotic DNA fragmentation).
Conclusions: ECM proteins greatly protect pancreatic cancer
cells from death by mechanisms different from those operating
in normal cells. The results suggest ECM proteins and their receptors
as potential targets for treatment of pancreatic cancer.
Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally
replicative adenovirus for pancreatic cancer
Masato Yamamoto, Julia Davydova, Minghui Wang, Gene P. Siegal,
Victor Krasnykh, Selwyn M. Vickers, David T. Curiel
Background & aims: Pancreatic cancer is one of the
most aggressive human malignancies. Conditionally replicative
adenoviruses (CRAds) have shown some promise in the treatment
of cancers. However, to date, their application for pancreatic
cancer has met several obstacles: one is lack of a good control
element to regulate replication, and the other is relatively
low adenoviral infectivity. Thus, we constructed infectivity
enhanced cyclooxygenase (COX)-2 promoter-based CRAds to develop
a safe and effective therapeutic modality.Methods: The
CRAds were designed to achieve COX-2 promoter-controlled E1 expression
for regulated replication (COX-2 CRAds). The infectivity-enhanced
CRAds also have an RGD-4C motif in the adenoviral fiber-knob
region. The selectivity and efficacy of these constructs were
analyzed with cell lines in vitro. The in vivo therapeutic effect
and viral replication were analyzed with a xenograft model. Pathology
of the major organs and E1 RNA levels in the liver were also
studied after systemic administration. Results: The COX-2
CRAds showed a selective cytocidal effect in vitro in COX-2-positive
cells and killed most of the pancreatic cancer cells. In vivo,
intratumoral administration of the infectivity-enhanced COX-2
CRAds (109 particles) showed a strong antitumor effect comparable
to wild-type virus, whereas the COX-2 CRAds without infectivity
enhancement showed a limited effect. Viral replication was confirmed
in the xenograft tumors. Systemic administration did not cause
any detectable toxicity; the E1 RNA level in the liver after
COX-2 CRAd administration was minimal. Conclusions: Infectivity-enhanced
COX-2 CRAd is a promising agent for the treatment of pancreatic
cancer.
Case Report
Biliary diversion for progressive familial intrahepatic
cholestasis: Improved liver morphology and bile acid profile
Amethyst C. Kurbegov, Kenneth D.R. Setchell, Joel E. Haas, Gary
W. Mierau, Michael Narkewicz, John D. Bancroft, Frederick Karrer,
Ronald J. Sokol
Background & aims: Progressive familial intrahepatic
cholestasis (PFIC) is characterized by pruritus, intrahepatic
cholestasis, low serum -glutamyltransferase levels, and characteristic
"Byler bile" on electron microscopy. Many patients
require liver transplantation, but partial external biliary diversion
(PEBD) has shown therapeutic promise. However, the effect of
PEBD on liver morphology and bile composition has not been evaluated.
Methods: We reviewed liver biopsy specimens from 3 children
with low -glutamyltransferase PFIC before and after PEBD. Follow-up
liver biopsies were performed 960 months after PEBD. Light
and electron microscopic features were scored blindly. Biliary
bile acid composition was analyzed by gas chromatography-mass
spectrometry before and after PEBD in 1 patient and after PEBD
in 2 patients. Results: Following PEBD, all patients improved
clinically. Preoperative biopsy specimens showed characteristic
features of PFIC, including portal fibrosis, chronic inflammation,
cholestasis, giant cell transformation, and central venous mural
sclerosis. Ultrastructural findings included coarse, granular
canalicular Byler bile, effaced canalicular microvilli, and proliferative
pericanalicular microfilaments. Following diversion, histology
showed almost complete resolution of cholestasis, portal fibrosis,
and inflammation with resolution of ultrastructural abnormalities.
Biliary bile acids before PEBD consisted predominantly of cholic
acid. After PEBD, the proportion of chenodeoxycholic acid increased
significantly in 1 patient and was above the PFIC range in a
second patient. Conclusions: The resolution of hepatic
morphologic abnormalities following PEBD supports PEBD as an
effective therapy for PFIC. The improved biliary bile acid composition
suggests enhanced bile acid secretion after PEBD, perhaps by
induction of alternative canalicular transport proteins.
Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion
injury
Hartmut Jaeschke, John J. Lemasters
Warm and cold hepatic ischemia followed by reperfusion leads
to necrotic cell death (oncosis), which often occurs within minutes
of reperfusion. Recent studies also suggest a large component
of apoptosis after ischemia/reperfusion. Here, we review the
mechanisms underlying adenosine triphosphate depletion-dependent
oncotic necrosis and caspase-dependent apoptosis, with emphasis
on shared features and pathways. Although apoptosis causes internucleosomal
DNA degradation that can be detected by terminal deoxynucleotidyl
transferase-mediated deoxyuridine triphosphate nick-end labeling
and related assays, DNA degradation also occurs after oncotic
necrosis and leads to pervasive terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick-end labeling staining far in excess
of that for apoptosis. Similarly, although apoptosis can occur
in a physiological setting without inflammation, in pathophysiological
settings apoptosis frequently induces inflammation because of
the onset of secondary necrosis and stimulation of cytokine and
chemokine formation. In liver, the mitochondrial permeability
transition represents a shared pathway that leads to both oncotic
necrosis and apoptosis. When the mitochondrial permeability transition
causes severe adenosine triphosphate depletion, plasma membrane
failure and necrosis ensue. If adenosine triphosphate is preserved,
at least in part, cytochrome c release after the mitochondrial
permeability transition activates caspase-dependent apoptosis.
Mitochondrial permeability transition-dependent cell death illustrates
the concept of necrapoptosis, whereby common pathways
lead to both necrosis and apoptosis. In conclusion, oncotic necrosis
and apoptosis can share features and mechanisms, which sometimes
makes discrimination between the 2 forms of cell death difficult.
However, elucidation of critical cell death pathways under clinically
relevant conditions will show potentially important therapeutic
intervention strategies in hepatic ischemia/reperfusion injury.
Copyright © 2001-2003 European Association
for the Study of the Liver. All rights reserved.
Table of Contents Volume
39, Issue 4, October 2003
Biliary Tract and Cholestaris
Gernot Zollner et al.
Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter
regulation by cholic and ursodeoxycholic acid in mouse liver,
kidney and intestine
Background/Aims: Adaptive changes in transporter expression
in liver and kidney provide alternative excretory pathways for
biliary constituents during cholestasis and may thus attenuate
liver injury. Whether adaptive changes in ATP-binding cassette
(ABC) transporter expression are stimulated by bile acids and
their nuclear receptor FXR is unknown. Methods: Hepatic,
renal and intestinal ABC transporter expression was compared
in cholic acid (CA)- and ursodeoxycholic acid (UDCA)-fed wild-type
(FXR+/+) and FXR knock-out mice (FXR/). Expression was assessed
by reverse transcription-polymerase chain reaction, immunoblotting
and immunofluorescence microscopy. Results: CA feeding
stimulated hepatic Mrp2, Mrp3, Bsep and renal Mrp2 as well as
intestinal Mrp2 and Mrp3 expression. Lack of Bsep induction by
CA in FXR/ was associated with disseminated hepatocyte necrosis
which was not prevented by compensatory induction of Mrp2 and
Mrp3. With the exception of Bsep, UDCA stimulated expression
of hepatic, renal and intestinal ABC transporters independent
of FXR without inducing liver toxicity. Conclusions: Toxic
CA and non-toxic UDCA induce adaptive ABC transporter expression,
independent of FXR with the exception of Bsep. Stimulation of
hepatic Mrp3 as well as intestinal and renal Mrp2 by UDCA may
contribute to its therapeutic effects by inducing alternative
excretory routes for bile acids and other cholephiles.
Cell Biology, Metabolism and Transport
Caroline Clair et al.
Hormone receptor gradients supporting directional Ca2+ signals:
direct evidence in rat hepatocytes
Background/Aims: In the liver, InsP3-dependent agonists
such as vasopressin and noradrenaline induce tightly coordinated
sequences of intracellular Ca2+ increases, leading to apparent
unidirectional Ca2+ waves. In previous works, we have postulated
that cell-to-cell differences in hormone receptor density create
a cellular sensitivity gradient that determines which cell initiates
the intercellular Ca2+ wave and the direction of propagation
of the Ca2+ signal. The aim of this study was to test directly
this hypothesis. Methods: Lobular distribution of V1a
vasopressin receptors and 1 adrenergic receptors were observed
by autoradiography in rat liver sections. Cell-to-cell differences
in the number of these receptors were evaluated on hepatocyte
multiplets using specific fluorescent probes. Results:
The relative amount of fluorescence associated with the V1a receptor
differed significantly between cells within multiplets. The `cell-after-cell'
Ca2+ increase induced by vasopressin was correlated with the
number of V1a receptors. These observations may be more general,
as autoradiography revealed similar lobular distributions of
V1a receptors and 1 adrenergic receptors; the amounts of both
were greatest in hepatocytes surrounding central veins. Conclusions:
These data confirm that a fine gradient along liver cell plates
contributes to the molecular basis of the unidirectional hormone-induced
Ca2+ signalling observed in the liver lobule.
Chronic Liver Diseases
Philippe Langlet et al.
Clinicopathological forms and prognostic index in Budd-Chiari
syndrome
Background: A recent study in patients with Budd-Chiari
syndrome showed the value of a prognostic index including age,
Pugh score, ascites and serum creatinine. Surgical portosystemic
shunt did not appear to improve survival. Aims: To validate
these findings in an independent sample; to evaluate a classification
into three forms according to the presence of features of acute
injury, chronic lesions, or both of them (types I, II or III,
respectively); and to assess whether taking into account this
classification would alter our previous conclusions. Methods:
Multivariate Cox model survival analysis, first on 69 new patients;
second, on these 69 and 54 previous patients, all diagnosed since
1985. Results: Previous prognostic index had a significant
prognostic value (P<0.0001) which was further improved
by taking into account type III form (P<0.001). Type
III form was associated with the poorest outcome. No significant
impact of surgical shunting on survival was disclosed. Conclusions:
The prognosis of Budd-Chiari syndrome can be based on age, Pugh
score, ascites, serum creatinine and the presence of features
indicating acute injury superimposed on chronic lesions (type
III form). The idea that surgical shunting has no significant
impact on survival is reinforced by these findings.
Cirrhosis and its Complications
Mauro Bernardi et al.
Systemic and regional hemodynamics in pre-ascitic cirrhosis:
effects of posture
Background/Aims: To clarify the hemodynamic pattern
of pre-ascitic cirrhosis, we compared the impact of posture on
systemic and regional hemodynamics of patients and healthy subjects
without and with plasma volume expansion. Methods: Cardiac
index (CI), peripheral vascular resistance (PVRi), heart rate,
mean arterial pressure, and the mean blood flow velocities of
superior mesenteric (SMAV) and common femoral arteries were evaluated
by duplex-Doppler techniques in 10 patients and 20 healthy controls
after 2 h of standing and 2 h after lying down. Ten healthy controls
received saline infusion (15 ml/kg body weight) when they changed
their posture, and five were also evaluated after plasma volume
expansion in the upright posture. Results: Standing systemic
and regional hemodynamics did not differ between patients and
controls. After saline infusion, standing control subjects showed
greater CI and SMAV than patients. Recumbency caused changes
of CI, PVRi and SMAV greater in patients and controls with plasma
expansion than in controls without expansion, so that supine
patients and controls with expansion were indistinguishable,
showing higher CI and SMAV and lower PVRi than controls without
expansion. Conclusions: Systemic and regional hemodynamics
of patients with pre-ascitic cirrhosis are mainly determined
by blood volume expansion which is compartmentalized within the
splanchnic venous bed during standing and translocates towards
the central and arterial circulatory districts during recumbency.
Delphine Nidegger et al.
Cirrhosis and bleeding: the need for very early management
Background/Aims: Retrospective studies suggest that
the prognosis of patients with cirrhosis and variceal hemorrhage
has improved in more recent decades. In a prospective cohort
study in which the choice of prophylactic therapy was left to
each practitioner, we followed cirrhotic patients with medium/large
varices to determine factors predictive of bleeding and death.
Methods: Three hundred fourteen patients with grades 2
or 3 esophageal varices (Child A and B/C: 218 and 96) were enrolled.
One hundred seventy-three patients had no previous history of
variceal bleeding. Only 245 patients (100% of patients with prior
variceal hemorrhage, 61% of patients without prior hemorrhage)
were receiving some form of prophylactic therapy. The median
follow-up was 18 months. Results: There were 76 bleeding
events and 14 related deaths (18%); nine of these deaths occurred
within 24 h of bleeding onset (two at home, two during hospital
transfer, and five in hospital, a mean of 2.5 h after onset;
six involved Child C patients). Twenty-five deaths were not due
to bleeding but were closely related to cirrhosis. In a Cox model,
the presence of tense ascites (relative risk 3.4, 95% confidence
interval, CI 2.5-5.9) and a prior history of hemorrhage (relative
risk 4.4, 95% CI 2.6-7.5) were independent predictors of variceal
hemorrhage. In patients without a prior history of bleeding,
bleeding risk was higher with more prolonged prothrombin time
and lower when patients were receiving propranolol. Conclusions:
Despite the advent of effective drugs and endoscopic therapy
for variceal bleeding, about a quarter of deaths occur very early
after bleeding onset, confirming the need for rapid specific
management.
Mariona Graupera et al.
Cyclooxygenase-1 inhibition corrects endothelial dysfunction
in cirrhotic rat livers
Background/Aims: Cirrhotic livers exhibit endothelial
dysfunction that contributes to the increased hepatic vascular
resistance. The present study evaluates the role of cyclooxygenase
(COX)-derived prostanoids, implicated in the pathogenesis of
endothelial dysfunction in other settings, in the pathogenesis
of endothelial dysfunction in cirrhotic livers. Methods:
Endothelial dysfunction was evaluated by performing concentration-effect
curves to acetylcholine after precontracting the liver with methoxamine
in groups of control and CCl4-cirrhotic rat livers preincubated
either with vehicle, indomethacin, the COX-1 selective inhibitor,
SC-560, the COX-2 selective inhibitor, SC-236, the thromboxane
A2 receptor antagonist, SQ 29,548 or the nitric oxide (NO) synthase
inhibitor NG-nitro-L-arginine. Thromboxane A2 (TXA2) production
was determined in samples of the perfusate. Results: Cirrhotic
livers exhibited endothelial dysfunction, as shown by the significantly
lower relaxation to acetylcholine than control livers, that was
totally corrected by indomethacin. COX-1 inhibition and TXA2
blockade, but not COX-2 inhibition, also corrected endothelial
dysfunction. Acetylcholine significantly increased TXA2 production
in cirrhotic but not in control livers. Indomethacin and COX-1
inhibition, but not COX-2 or NO inhibition, prevented the increased
production of TXA2. Conclusions: An increased production
of TXA2 is involved in the pathogenesis of endothelial dysfunction
in cirrhotic rat livers. This is mainly mediated by COX-1, but
not by COX-2.
Violina Lozeva, Leena Tuomisto, Juhani Tarhanen and Roger F.
Butterworth
Increased concentrations of histamine and its metabolite,
tele-methylhistamine and down-regulation of histamine H3 receptor
sites in autopsied brain tissue from cirrhotic patients who died
in hepatic coma
Background/Aims: Hepatic encephalopathy (HE) is a
serious neuropsychiatric complication of chronic liver disease.
To determine whether changes in the central histaminergic system
are a feature of human HE, we studied histamine, tele-methylhistamine,
and presynaptic autoregulatory H3 receptors in cerebral cortex
and caudate-putamen obtained at autopsy from six cirrhotic patients
and six appropriately matched controls. Methods: Histamine
was assayed by HPLC; tele-methylhistamine by GC-MS. H3
receptors were studied by in vitro receptor binding using [3H]R--methylhistamine
as ligand. Results: In HE patients, there was a significant
fourfold increase of histamine in caudate-putamen and a significant
increase in all cortical regions studied. tele-Methyhistamine
was also increased and the densities of histamine H3 receptor
sites were significantly decreased in patient material. Conclusions:
These findings are consistent with activation of the histaminergic
system in HE. Given that histamine participates in the regulation
of arousal and circadian rhythmicity, they indicate that induction
of central histamine mechanisms may contribute to the development
of neuropsychiatric symptoms, such as sleep disturbances and
altered circadian rhythms in chronic HE and suggest that pharmacological
manipulation of the histaminergic system could be beneficial
in the treatment of HE in chronic liver failure.
Inflammation and Fibrosis
Gianluca Svegliati-Baroni et al.
Regulation of ERK/JNK/p70S6K in two rat models of liver injury
and fibrosis
Background/Aims: The regulation of three major intracellular
signalling protein kinases was investigated in two models of
liver injury leading to hepatic fibrosis, dimethylnitrosamine
administration (DMN) and bile duct ligation (BDL). Methods:
Extracellular signal-regulated kinases (ERK)1/2, c-Jun terminal
kinase (JNK) and p70S6-kinase (p70S6K) were studied in vivo in
the whole liver, in liver sections and in isolated hepatocytes,
cholangiocytes and hepatic stellate cells (HSC). Results:
In the whole liver, activation of these kinases occurred with
a different kinetic pattern in both models of liver injury. By
immunohistochemistry and Western blot in isolated cells, phosphorylated
kinases were detected in proliferating cells (i.e. hepatocytes
and cholangiocytes after DMN and BDL, respectively), in addition
to stellate-like elements. ERK1/2, JNK and p70S6K activation
was associated with hepatocytes proliferation after DMN, while
JNK activation was not associated with cholangiocytes proliferation
after BDL. In HSC isolated from injured livers, protein kinases
were differentially activated after BDL and DMN. Kinases activation
in HSC in vivo preceded cell proliferation and alpha-smooth muscle
actin appearance, a marker of HSC transformation in myofibroblast-like
cells, and collagen deposition. Conclusions: Our findings
indicate that these kinases are coordinately regulated during
liver regeneration and suggest that their modulation could be
considered as a future therapeutic approach in the management
of liver damage.
Peter Stärkel et al.
Oxidative stress, KLF6 and transforming growth factor- up-regulation
differentiate non-alcoholic steatohepatitis progressing to fibrosis
from uncomplicated steatosis in rats
Background/Aims: Pathogenesis of non-alcoholic steatohepatitis
(NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1),
cytokines, oxidative stress and activation of hepatic stellate
cells seem to play a role in this process. The aim was to determine
the potential implication of these factors in the progression
from uncomplicated steatosis to steatohepatitis with progressive
fibrosis.
Methods: Animals were fed a standard diet, a 5% orotic
acid-diet (OA) developing hepatic steatosis, or the methionine-choline
deficient (MCD) diet inducing steatohepatitis for 2 and 6 weeks.
Lipid peroxidation, CYP 2E1 expression and activity, expression
of UCP-2, interleukin (IL)-6, transforming growth factor (TGF)1,
KLF6 mRNAs, and activation of hepatic stellate cells were examined
by gas chromatography, high-performance liquid chromatography,
Western blotting, quantitative polymerase chain reaction and
immunohistochemistry. Results: Lipid peroxidation increased
in the MCD model whereas only minor changes occurred in the OA
model. KLF6 and TGF1 mRNAs were selectively up-regulated in MCD
animals. Stellate cell activation, inflammation and collagen
deposition only occurred in the MCD group. CYP 2E1 expression
and activity increased in the OA group while both decreased in
MCD animals. UCP-2 and IL-6 mRNA increased in both groups. Conclusions:
In the context of steatosis, lipid peroxidation is associated
with inflammation and stellate cell activation with concomitant
increase in TGF1 production, possibly through up-regulation of
KLF6.
Liver Cell Injury and Liver Failure
P. Aiden McCormick, Darren Treanor, Geraldine McCormack and
Michael Farrell
Early death from paracetamol (acetaminophen) induced fulminant
hepatic failure without cerebral oedema
Background: Paracetamol overdose is a frequent cause
of fulminant hepatic failure. In fatal cases the most frequent
causes of death are cerebral oedema in the early phase or sepsis
and multiorgan failure later. However some patients do not fit
this pattern. Aim: To review cause of death in paracetamol
induced fulminant hepatic failure. Methods: We reviewed
all fatal cases of paracetamol induced fulminant hepatic failure
in our liver unit between 1995 and 2000. Results: Twenty
one patients died without liver transplantation and post mortem
examinations were performed on all. Significant cerebral oedema
was present in 13 patients and absent in eight. The patients
without cerebral oedema were significantly older (55.4±5.3
versus 36.3±3 years: P=0.0034), had a lower arterial
pH on admission (pH 7.0±0.03 versus 7.3±0.05: P=0.0008),
a shorter interval between overdose and death (3.75±0.7
versus 7.6±1.3 days: P=0.043) and a shorter interval
between admission and death (1.9±0.6 versus 5.7±1.0
days: P=0.0097) than patients with cerebral oedema. The
cause of death in the sub-group of patients without cerebral
oedema was predominantly cardiovascular collapse with rapidly
progressive resistant hypotension and/or cardiac arrest. No source
of sepsis was identified in 7/8 patients without cerebral oedema.
Conclusions: In this series the most frequent causes of
death were cerebral oedema or cardiovascular collapse. Patients
without cerebral oedema appear to form a distinct subgroup associated
with early mortality and may require specific management strategies.
Etty Grad-Itach et al.
Liver micro-organs transcribe albumin and clotting factors
and increase survival of 92% hepatectomized rats
Background/Aims: Currently there is no effective non-surgical
therapy for most patients with fulminant or end stage chronic
liver disease. Methods: We have prepared rat liver micro-organs
(LMOs), which preserve the liver micro-architecture and ensure
that no cell is more than 150 µm away from a source of
nutrients and gases. The function of LMOs has been evaluated
in vitro and in a new extra-corporeal liver device termed aLIVE
in which LMOs are exposed to liver-like hemodynamic conditions.Results:
In vitro LMOs maintain normal physiological and biochemical functions
including oxygen consumption, glucose metabolism, conversion
of ammonia to urea, secretion of albumin and de novo transcription
of genes coding for albumin and clotting factors. Inside the
aLIVE bioreactor, LMOs also display sustained oxygen consumption,
glucose metabolism and transcription of albumin and clotting
factors IX and X, when connected both to normal and to 92% hepatectomized
rats. Survival of 92% hepatectomized rats was 40% longer following
a single 4-h treatment with aLIVE, compared to untreated
animals. Conclusions: An extra-corporeal liver device,
aLIVE, which provides key liver functions, has been developed.
When tested in 92% hepatectomized rats, aLIVE improved
the clinical condition and significantly increased survival time
of the treated rats.
Liver Growth and Cancer
Hironobu Watanabe et al.
Clinical significance of serum RCAS1 levels detected by monoclonal
antibody 22-1-1 in patients with cholangiocellular carcinoma
Background/Aims: The tumor-associated antigen, RCAS1,
has been reported to be expressed in various types of cancer,
including cholangiocarcinoma. We measured serum RCAS1 levels
in patients with intrahepatic cholangiocellular carcinoma (CCC)
and other hepatobiliary diseases, and examined the clinical significance
of serum RCAS1 as a tumor marker. Methods: Sera
collected from the patients and healthy volunteers were used
for ELISA for RCAS1. The values of RCAS1 for CCC patients were
compared to those of other tumor marker proteins. Results:
Serum RCAS1 levels exceeded the normal limit in a high percentage
(73.9%) of CCC patients. The positivity rate was higher than
those of CA19-9 and CEA. No correlation was found between the
RCAS1 and CA19-9 concentrations. Serum RCAS1 was positive in
many cases that were negative for CA19-9. Surgical resection
of CCC reduced the RCAS1 level to within the normal range. On
the other hand, serum RCAS1 levels were elevated in very few
cases of benign hepatobiliary disease. Conclusions:
As a tumor marker in CCC, RCAS1 is, at least, of complementary
value to CA19-9 and CEA. Measuring serum RCAS1 contributes to
the diagnostic accuracy, and is useful for estimating tumor progression
or therapeutic effect.
Laura Rossi et al.
Genetic polymorphisms of steroid hormone metabolizing enzymes
and risk of liver cancer in hepatitis C-infected patients
Background/Aims: Genetic polymorphisms of enzymes
involved in hormone metabolism can influence hormonal activities
and risk of hormone-dependent cancers. As progression of chronic
hepatitis C and risk of liver cancer is higher in males than
in females, we evaluated whether the polymorphisms of three enzymes
participating in the pathway of estrogen and androgen biosynthesis
and inactivation, 5-reductase type II (SRD5A2), cytochrome P450c17
(CYP17) and catechol-O-methyltransferase (COMT), might
affect the expression of hepatitis C virus (HCV)-related liver
disease. Methods: The study included 78 healthy subjects
and 387 HCV patients: 100 asymptomatic carriers, 105 hepatitis,
90 cirrhosis and 92 hepatocellular carcinomas (HCC). Variant
positions SRD5A2 V89L and A49T, CYP17 (34)T/C and COMT V108M
were analysed by polymerase chain reaction and restriction fragment
length polymorphism. A cross-sectional study of association was
performed, considering carriers as reference category. Results:
The CYP17 (34)C/C genotype was over-represented in HCC patients
as compared to carriers (22.5 vs. 11.2%, odds ratio (OR): 2.29,
P: 0.05). Females mostly contributed to this association
(OR: 4.95, P: 0.01) and OR values increased in post-menopausal
women (OR: 6.00, P: 0.03). No differences were observed
for SRD5A2 and COMT gene polymorphisms. Conclusions: CYP17
high-activity alleles associated with increased circulating levels
of estrogens and androgens may affect liver cancer risk in HCV-infected
women.
Ignazio Grattagliano et al.
Mitochondrial glutathione content determines the rate of liver
regeneration after partial hepatectomy in eu- and hypothyroid
rats
Background/Aims: Mitochondrial glutathione has been
postulated to affect mitochondrial function and liver regeneration.
Methods: Mitochondrial respiration, total and oxidized
glutathione, and liver regeneration were assessed after partial
hepatectomy in glutathione-depleted and in hypothyroid rats with/without
supplementation of glutathione ester. Results: Mitochondrial,
cytosolic and circulating glutathione levels were lower in glutathione-depleted
rats. Hepatectomy was followed by significant changes of intra-
and extracellular glutathione and of mitochondrial respiration.
In glutathione-deficient rats, the recovery of mitochondrial
function and the liver regeneration were delayed. Administration
of glutathione ester partially corrected the fall of cytosolic
and mitochondrial glutathione following hepatectomy, reduced
mitochondrial oxidative damage, and accelerated the restoration
of mitochondrial respiration and the rate of liver regeneration.
In hypothyroid rats, intracellular glutathione homeostasis and
mitochondrial respiration were impaired already at baseline;
slower regeneration and mitochondrial oxidative alterations were
observed after hepatectomy. Glutathione ester ameliorated the
regenerative response in hypothyroid rats by providing higher
concentrations of cytosolic and mitochondrial glutathione. Conclusions:
Glutathione depletion and hypothyroidism affect the mitochondrial
function during liver regeneration. Liver regenerates more slowly
in glutathione-depleted and in hypothyroid rats. The earlier
restoration of mitochondrial function and the higher rate of
proliferation in glutathione ester treated rats suggest that
the maintenance of intracellular glutathione facilitates liver
regeneration.
Mei Chung Moh, Lay Hoon Lee, Xiaodong Yang and Shali Shen
HEPN1, a novel gene that is frequently down-regulated in hepatocellular
carcinoma, suppresses cell growth and induces apoptosis in HepG2
cells
Background/Aims: Examining genes associated with human
hepatocellular carcinoma (HCC) by subtractive hybridisation,
we identified a novel transcript, designated as HEPN1,
in non-tumorous liver. In this study, we aimed to evaluate HEPN1
gene expression in HCC patients, to characterise and to explore
the functional significance of HEPN1 in vitro. Methods:
One-step reverse transcription-polymerase chain reaction (RT-PCR)
and real-time RT-PCR were employed to determine HEPN1
expression in 23 paired (HCC and the adjacent non-HCC) liver
specimens. Sequence analyses were performed by bioinformatics.
Transfection studies were carried out by expressing HEPN1, V5-fused
HEPN1, and green fluorescent protein-fused HEPN1, individually,
in HepG2 cells. Results: Significant downregulation of
HEPN1 (P<0.0001) was detected in 22/23 of HCC
patients tested. Gene HEPN1 maps to chromosome 11q24.2;
and the predicted gene product, a 10-kDa peptide with 88 amino
acids, has no homology to known proteins. When transfected into
HepG2 cells, HEPN1 reduced cell viability to 37.5±2.5%
(P=0.001), and induced apoptosis with typical morphological
changes as demonstrated by microscopy and Annexin V assay. Conclusions:
Our data show that HEPN1 is frequently silenced in HCC,
and that exogenous HEPN1 exhibits antiproliferative effect
on HepG2 cells, suggesting that silencing of HEPN1 may
be associated with carcinogenesis of hepatocytes.
Transplantation
Caroline Créput et al.
Human leukocyte antigen-G (HLA-G) expression in biliary epithelial
cells is associated with allograft acceptance in liver-kidney
transplantation
Background/Aims: Liver allograft is known to protect
simultaneously transplanted organs from acute rejection. We have
reported that only 6% of combined liver-kidney recipients, versus
32.5% of kidney recipients, develop kidney graft acute rejection.
Release of soluble human leukocyte antigen (HLA) molecules by
the liver has been proposed as a possible tolerogenic mechanism
involved in the better acceptance of double transplants. The
HLA-G molecule is acknowledged to possess tolerogenic properties.
Methods: We investigated the involvement of HLA-G in allogeneic
transplant acceptance by analyzing its expression in kidney and
liver biopsies of 40 combined transplanted patients. Results:
We demonstrate the presence of HLA-G in 14 out of 40 liver and
five out of nine kidney transplants biopsies. HLA-G is expressed
de novo by cells that are otherwise frequently susceptible target
cells of acute rejection, i.e. liver biliary and renal tubular
epithelial cells. We show a significant association between HLA-G
expression in liver biliary epithelial cells and the absence
of liver graft rejection. No acute or chronic rejection of the
kidney graft was observed in patients in whom HLA-G was expressed
in the liver graft. Conclusions: HLA-G expression in the
liver allograft is associated with a lower frequency of hepatic
and renal acute rejection and may be involved in the acceptance
of simultaneously transplanted organs.
Viral Hepatitis
Carolina Boni et al.
Transient restoration of anti-viral T cell responses induced
by lamivudine therapy in chronic hepatitis B
Background/Aims: Lamivudine therapy in patients with
chronic hepatitis B can induce the recovery of antiviral T cell
responses. It is unknown whether the recovery of T cell responsiveness
is long-lasting and persists throughout the treatment and whether
the elevation of viremia which follows therapy withdrawal can
restore a condition of T cell unresponsiveness. Methods:
Frequency and function of circulating hepatitis B virus (HBV)-specific
CD4 and CD8 cells from 12 hepatitis e surface antigen + patients
with chronic hepatitis B were studied longitudinally before,
during and after lamivudine therapy by intracellular cytokine
staining, proliferation and cytotoxicity assays against HBV proteins
and peptides. CD4-mediated responses were analyzed in all patients,
whereas CD8 cells were studied in 6 HLA-A2+ patients. Results:
HBV-specific CD4 and CD8 reactivity showed a bi-phasic behavior
under lamivudine therapy with an early enhancement of T cell
frequency and intensity of responses followed by a persistent
decline starting from the 5th to 6th month of treatment. Conclusions:
Since restoration of HBV-specific T cell reactivity is only transient,
our study indicates that therapeutic stimulation of HBV-specific
T cell responses to complement lamivudine treatment should be
done early after the initiation of lamivudine. Moreover, the
transient nature of the immune reconstitution may represent a
favorable condition for virus reactivation once lamivudine therapy
is withdrawn.
Gerlinde Teuber et al.
Randomized, controlled trial with IFN- combined with ribavirin
with and without amantadine sulphate in non-responders with chronic
hepatitis C
Background/Aims: Efficacy and safety of interferon-
(IFN-)/ribavirin retreatment with or without amantadine sulphate
were evaluated in non-responders with chronic hepatitis C. Methods:
Two hundred twenty five consecutive non-responders to previous
antiviral treatment(s) with IFN- alone or in combination with
ribavirin or amantadine were treated with IFN- 2b 5 MU daily
for 4 weeks, 5 MU tiw for 20 weeks, followed by 3 MU tiw for
additional 24 weeks combined with ribavirin 1000-1200 mg/d. One
hundred fifteen of 225 patients were randomized to receive amantadine
sulphate 100 mg bid for 48 weeks. Treatment was discontinued
in patients with detectable serum hepatitis C virus (HCV)-RNA
at treatment week 24. Results: An overall sustained virologic
response with undectable serum HCV-RNA levels was observed in
49/225 patients (22%). Patients infected with HCV-genotype non-1
(P<0.001), low viremia (P=0.011) and only one
previous antiviral treatment (P=0.032) were more likely
to respond to antiviral retreatment. There was a trend towards
higher sustained virologic response rates in patients receiving
triple retreatment compared with those treated with IFN-/ribavirin
alone (25 versus 18%, P=0.172). Conclusions: The
addition of amantadine was well tolerated and led to an improvement
of sustained virologic responses compared with retreatment with
IFN-/ribavirin alone, in particular in patients with low baseline
viremia.
Soo Hyung Ryu et al.
Long-term additional lamivudine therapy enhances durability
of lamivudine-induced HBeAg loss: a prospective study
Backgrounds/Aims: In the treatment of chronic hepatitis
B (CHB) with lamivudine, adequate duration of the therapy remains
to be determined. In this prospective study, the authors intended
to investigate whether long-term additional administration of
lamivudine might enhance the durability of lamivudine-induced
HBeAg seroconversion. Methods: Eighty-five CHB
patients whzo achieved HBeAg seroconversion by lamivudine received
additional lamivudine therapy for at least 24 months at a dose
of 100 mg per day. Among them, 61 patients whose serum HBeAg
and HBV-DNA (solution hybridization assay) had been negative
persistently for >24 months discontinued lamivudine therapy
and followed-up for >12 months. We calculated the cumulative
reappearance rate of serum HBV-DNA and HBeAg and also evaluated
the predictive factors for post-treatment virologic relapse.
Results: The cumulative reappearance rates of serum
HBV-DNA following cessation of lamivudine therapy at 6 months,
1 year and 2 years were 15%, 21%, and 31%, respectively. The
cumulative reappearance rates of serum HBeAg at 6 months, 1 year
and 2 years were 11%, 13% and 16%, respectively. Old age and
presence of precore mutant were two independent predictive factors
for viral relapse. Conclusions: These results suggested
that long-term additional administration of lamivudine might
enhance the durability of lamivudine-induced HBeAg seroconversion.
Javier P. Gisbert, Luisa García-Buey, José María
Pajares and Ricardo Moreno-Otero
Prevalence of hepatitis C virus infection in porphyria cutanea
tarda: systematic review and meta-analysis
Background/Aims: To conduct a systematic review and
meta-analysis on the prevalence of hepatitis C virus (HCV) infection
in porphyria cutanea tarda (PCT). Methods: Studies evaluating
prevalence of HCV infection in patients with PCT were considered.
Bibliographical searches were conducted in several electronic
databases. Studies comparing HCV prevalence in PCT (cases) and
in a reference group (controls) were included in the meta-analysis,
combining the Odds Ratios (OR) of the individual studies. Results:
Fifty studies including 2,167 patients were identified. Mean
HCV prevalence by serology was 47%, and 50% with polymerase chain
reaction (PCR). HCV prevalence markedly varied depending on the
country and the type of PCT (57% in the sporadic and 26% in the
familial form). Eight case-contol studies were identified. Seven
studies compared HCV prevalence in PCT vs. healthy controls:
40% vs. 0.24%, respectively (OR=275; 95% confidence interval=104-725).
Heterogeneity disappeared when only studies evaluating HCV infection
by PCR were included. Conclusions: HCV prevalence in patients
with PCT is approximately 50%, much higher than that reported
in general population, suggesting a possible etiopathogenic role
of HCV in PCT. The striking geographical variation in this association
suggests that genetic and/or environmental factors may also be
involved in the pathogenesis of this disorder.
Case Report
Igor M. Sauer et al.
Extracorporeal liver support based on primary human liver
cells and albumin dialysis - treatment of a patient with primary
graft non-function
Methods: Following liver transplantation, a 26-year old female
suffered from primary non-function of the transplant. The patient
was subsequently treated with a modular extracorporeal liver
support concept until a suitable organ became available. A bioreactor
was charged with human liver cells, obtained from a discarded
cadaveric graft (470 g, viability: 60%). The bioreactor was integrated
into an extracorporeal circuit with continuous single pass albumin
dialysis and continuous veno-venuous hemodiafiltration for detoxification
and fluid reduction. Results: Over the total system application
time of 79 h, a significant reduction of the plasma levels of
total bilirubin (21.1 mg/dl at start, 10.1 mg/dl at end of therapy)
and ammonia (100 versus 22.7 µmol/l) was achieved. During
treatment the patient's neurological status significantly improved
from coma stage IV to I permitting extubation. Recovery of kidney
function with a urine output of 1325 ml/24 h compared to 45 ml/24
h prior to system application, was noted. Over the treatment
period, an improvement of coagulation status was observed. Adverse
events were absent. Conclusions: This first successful
clinical treatment of a patient with liver failure suggests that
a modular approach combining both primary human liver cell bioreactor
technology and detoxification methods is promising.
Copyright © 2001-2003 European Association
for the Study of the Liver. All rights reserved.
© 2003 BMJ Publishing Group Ltd
The New England Journal of Medicine is owned, published,
and copyrighted © 2003 Massachusetts Medical Society. All
rights reserved.
The Lancet, published, and copyrighted © 2003. All
rights reserved.
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