Table of Contents for November
2003 · Volume 38 · Number 5
Liver Biology and Pathobiology
Establishment, characterization, and long-term maintenance
of cultures of human fetal hepatocytes (*Human Study*)
Catherine A. Lázaro, Emma J. Croager, Claudia Mitchell,
Jean S. Campbell, Changpu Yu, Jessica Foraker, Jonathan A. Rhim,
George C. T. Yeoh, Nelson Fausto
Cultured human hepatocytes have broad research and clinical applications;
however, the difficulties in culturing rodent and human hepatocytes
are well known. These problems include the rapid loss of the
hepatocytic phenotype in primary culture and the limited replicating
capacity of the cultured cells. We describe the establishment
of serum-free primary cultures of human fetal hepatocytes (HFHs)
that retain hepatocytic morphology and gene expression patterns
for several months and maintain sufficient proliferative activity
to permit subculturing for at least 2 passages. Initially, HFH
cultures contained 2 main cell types that morphologically resembled
large and small hepatocytes. The fetal hepatocytes expressed
-fetoprotein (AFP), cytokeratin (CK) 19, albumin, and other hepatic
proteins. Treatment of the cultures with oncostatin M (OSM) increased
cell size and enhanced cell differentiation and formation of
bile canaliculi, probably through an effect on hepatocyte nuclear
factor (HNF) 4. Approximately 1 month after plating, multiple
clusters of very small cells became apparent in the cultures.
These cells had very few organelles and are referred to as blast-like
cells. Flow cytometric analysis of these cells showed that they
express oval cell/stem cell markers such as CD90 (Thy-1), CD34,
and OV-6 but do not stain with antibodies to 2-microglobulin.
HFH cultures maintained for 9 to 12 months produced grossly visible
organoids containing ductular structures that stained for CK18,
CK19, and AFP. In conclusion, HFH cultures, which might contain
a population of hepatic stem cells, constitute an excellent tool
for a variety of studies with human hepatocytes, including the
mechanisms of viral infection. (HEPATOLOGY 2003;38:1095-1106.)
Adenovirus-mediated overexpression of follistatin enlarges
intact liver of adult rats
Kazuaki Takabe, Lili Wang, Angela M. O. Leal, Leigh A. MacConell,
Ezra Wiater, Tomoaki Tomiya, Akihiko Ohno, Inder M. Verma, Wylie
Vale
Under normal physiologic conditions, liver size is under strict
regulatory control. Activin, a member of the transforming growth
factor (TGF-) superfamily, is expressed in the intact adult liver
and is an inhibitor of hepatocyte growth. However, the exact
role played by endogenous activin in maintaining the size of
a normal adult liver has yet to be completely examined in
vivo. Here, we report the development of an adenoviral vector
(AdexCAFS288) that expressed human follistatin-288, which binds
to activin and neutralizes its biologic activities. AdexCAGFP,
a control virus, expressed green fluorescent protein. AdexCAFS288
effectively expressed follistatin-288, as measured both in HepG2
cell lysate and conditioned medium and blocked activin signaling
and its biologic functions in vitro. Intraperitoneal injection
of AdexCAFS288 in vivo resulted in significant liver growth
(146% of control) in intact liver of adult male rats 12 days
following treatment without significant dysfunctions. The increase
in liver size was attributed to increased hepatocyte proliferation,
as monitored by the mitotic index. Furthermore, there was a significant
correlation between serum follistatin levels and liver weight.
In conclusion, our results suggest that activin plays a critical
role in maintaining optimal liver size and implicates the endogenous
activin system as a therapeutic target in the treatment of liver
disease. (HEPATOLOGY 2003;38:1107-1115.)
Role of V 14 NKT cells in the development of impaired liver
regeneration in vivo
Hiroyasu Ito, Kazuki Ando, Toshinori Nakayama, Masaru Taniguchi,
Takayuki Ezaki, Kuniaki Saito, Masao Takemura, Kenji Sekikawa,
Michio Imawari, Mitsuru Seishima, Hisataka Moriwaki
Although we have previously demonstrated that IL-12 stimulation
increases the number of hepatic natural killer (NK) T (NKT) cells
and enhances liver injury during the early phase of liver regeneration,
the role of NKT cells has remained unknown. We therefore evaluated
the influence of NKT cells activated by IL-12 or by -galactosylceramide
(-GalCer) on murine liver regeneration using V 14 NKT knockout
(J 281/) mice. Levels of serum alanine aminotransferase
(sALT) 24 hours after partial hepatectomy were enhanced in J
281+/+ but not in J 281/ mice by both procedures. Hepatic
NKT cells expressed considerably more interferon (IFN) and tumor
necrosis factor (TNF-) messenger RNA (mRNA) after stimulation
with both factors in J 281+/+ mice. Either anti-IFN- or TNF-
antibody inhibited the enhancement of liver injury. Furthermore,
recombinant TNF- injection similarly caused injury in hepatectomized
livers of both J 281+/+ and J 281/ mice; indeed, adoptively
transferred TNF-+/+ NKT cells enhanced liver injury after hepatectomy
in TNF- knockout mice. TNF receptor expressions on hepatocytes
increased and peaked 24 hours after partial hepatectomy. In conclusion,
simultaneous TNF- synthesis and high levels of TNF receptor expression
on hepatocytes cause severe liver damage by activated NKT cells
during liver regeneration. (HEPATOLOGY 2003;38:1116-1124.)
Gap junction-mediated intercellular communication in a long-term
primary mouse hepatocyte culture system
Stephanie A. Stoehr, Harriet C. Isom
Gap junction-mediated intercellular communication (GJIC) is critical
for maintaining integral cellular processes including differentiation
and growth control. The disruption of GJIC has been correlated
with aberrant function in many cell types, including hepatocytes
in vivo; therefore it is imperative that cellular model
systems support intercellular communication to simulate normal
cellular functions. Functional GJIC has been shown in long-term
primary rat hepatocyte cultures, which have been implemented
widely to study various aspects of hepatocellular function; however,
the onset of transgenic technology in murine species has necessitated
the development of a primary mouse hepatocyte system. In this
report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing
long-term primary mouse hepatocyte culture system. The cells
retain morphologic and biochemical characteristics of differentiated
hepatocytes through day 30 post plating, including liver-specific
gene expression. We further show that connexin32 and connexin26
expression and gap junction plaque formation increase over time
in culture concomitant with an increase in GJIC between adjoining
primary mouse hepatocytes. In conclusion, the findings described
in this study make it possible to maintain differentiated primary
mouse hepatocytes that also show GJIC in long-term culture for
30 days. In addition, this system has the potential to be extended
to study primary mouse hepatocytes isolated from genetically
engineered mice. (HEPATOLOGY 2003;38:1125-1135.)
Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide
dismutase
Irina G. Kessova, Ye-Shih Ho, Swan Thung, Arthur I. Cederbaum
Because alcoholic liver disease has been linked to oxidative
stress, we investigated the effect of a compromised antioxidant
defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency,
on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1+/+)
and Sod1 knockout (Sod1/) mice were fed
dextrose or ethanol (10% of total calories) liquid diets for
3 weeks. Histologic evaluation of liver specimens of Sod1/
mice fed ethanol showed the development of liver injury ranging
from mild to extensive centrilobular necrosis and inflammation.
Sod1+/+ mice fed ethanol showed mild steatosis; both Sod1+/+
and Sod1/ mice fed the dextrose diet had normal
histology. Alanine transaminase levels were significantly elevated
only in Sod1/ mice fed ethanol. Cytochrome P450
2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in
Sod1+/+ and Sod1/ mice. Ethanol consumption
increased levels of protein carbonyls and lipid peroxidation
aldehydic products in the liver of Sod1/ mice.
Hepatic adenosine triphosphate (ATP) content was reduced dramatically
in Sod1/ mice fed ethanol in association with
a decrease in the mitochondrial reduced glutathione (GSH) level
and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine
(3NT) residues in liver sections of the Sod1 knockout
mice treated with ethanol showed a significant increase of 3NT
staining in the centrilobular areas. In conclusion, a rather
moderate ethanol consumption promoted oxidative stress in Sod1/
mice, with increased formation of peroxynitrite, protein carbonyls,
and lipid peroxidation and decreased mitochondrial GSH and MnSOD.
We speculate that the increased oxidative stress causes mitochondrial
damage and reduction of ATP content, leading to alcoholic liver
injury. This model may be useful in further mechanistic studies
on alcohol-induced liver injury. (HEPATOLOGY 2003;38:1136-1145.)
Adenovirus-mediated expression of Cu/Zn- or Mn-superoxide
dismutase protects against CYP2E1-dependent toxicity
María José Pérez, Arthur I. Cederbaum
CYP2E1 induction by ethanol is one mechanism by which ethanol
creates oxidative stress in the liver. The superoxide dismutases
(SODs) are an important antioxidant enzyme defense system against
reactive oxygen species (ROS). To investigate the protective
role of SOD against CYP2E1-dependent toxicity, a transfected
HepG2 cell line overexpressing CYP2E1 (E47 cells) was infected
with adenoviral vectors containing Cu/Zn-SOD complementary DNA
(cDNA) (Ad.SOD1) and Mn-SOD cDNA (Ad.SOD2). Forty-eight hours
after infection, intracellular levels and activity of Cu/Zn-SOD
and Mn-SOD were increased about 2- and 3-fold, respectively.
Localization of the overexpressed Cu/Zn-SOD in the cytosol and
Mn-SOD in the mitochondria was confirmed by assaying the levels
and activity of SOD in the corresponding isolated fractions.
Arachidonic acid (AA) plus iron-induced cell death was partially
prevented in both Ad.SOD1- and Ad.SOD2-infected E47 cells. Overexpression
of Cu/Zn-SOD and Mn-SOD also partially protected E47 cells from
the increase in reactive oxygen production and lipid peroxidation
and the loss of mitochondrial membrane potential induced by AA
and iron. Infection with Cu/Zn-SOD and Mn-SOD also protected
the E47 cells against AA toxicity or buthionine sulfoximine (BSO)-dependent
toxicity. CYP2E1 levels and catalytic activity were not altered
by overexpression of Cu/Zn-SOD or Mn-SOD. Cu/Zn-SOD in the cytosol
and Mn-SOD in mitochondria each are capable of protecting HepG2
cells expressing CYP2E1 against cytotoxicity induced by pro-oxidants.
In conclusion, these enzymes may be useful in the prevention
or improvement of liver injury produced by agents known to be
metabolized by CYP2E1 to reactive intermediates and to cause
oxidative stress. (HEPATOLOGY 2003;38:1146-1158.)
Oxidative stress-mediated down-regulation of rat hydroxyacid
oxidase 1, a liver-specific peroxisomal enzyme
Stefania Recalcati, Lorenza Tacchini, Alessandra Alberghini,
Dario Conte, Gaetano Cairo
Hydroxyacid oxidase 1 (Hao1) is a liver-specific peroxisomal
enzyme that oxidizes glycolate to glyoxylate with concomitant
production of H2O2. In Hao1 messenger RNA (mRNA), an iron-responsive
element (IRE) homologous to the sequence recognized by iron regulatory
proteins (IRP), key regulators of iron homeostasis, is present,
but the involvement of iron in Hao1 regulation remains unclear.
In this study, we found a reduction of Hao1 mRNA content in livers
of rats with chronic dietary iron overload, which showed decreased
IRP activity and higher ferritin expression as expected, but
also induction of heme oxygenase (HO-1), a marker of oxidative
damage, and lipid peroxidation. Hao1 mRNA levels were not altered
significantly in livers of rats administered doses of iron sufficient
to induce ferritin expression and to repress IRP activity, but
not to activate HO-1 and to promote lipid peroxidation, as well
as in the liver of iron-deficient rats. These observations were
not consistent with a post-transcriptional down-regulation of
Hao1 by iron through the IRE/IRP pathway and suggested an effect
of reactive oxygen species (ROS). Indeed, a marked decrease of
Hao1 mRNA was observed in the liver of rats subjected to oxidative
stress induced by either glutathione depletion or postischemic
reperfusion. Nuclear run-on analysis showed an effect of ROS
at the transcriptional level. In conclusion, down-regulation
of Hao1 expression during oxidative stress may provide a mechanism
to prevent excessive H2O2 formation in liver peroxisomes and
may represent the prototype of a poorly recognized but potentially
relevant response to oxidative injury involving down-regulation
of ROS-producing enzymes. (HEPATOLOGY 2003;38:1159-1166.)
Membrane cholesterol but not putative receptors mediates anandamide-induced
hepatocyte apoptosis
Kamal Krishna Biswas, Krishna Pada Sarker, Kazuhiro Abeyama,
Ko-ichi Kawahara, Satoshi Iino, Yasuharu Otsubo, Kazuhiko Saigo,
Hiroyuki Izumi, Teruto Hashiguchi, Munekazu Yamakuchi, Kazuyo
Yamaji, Ryujin Endo, Kazuyuki Suzuki, Hitoshi Imaizumi, Ikuro
Maruyama
The endogenous cannabinoid anandamide, a lipid mediator, induces
various physiologic events such as vascular relaxation, inhibition
of gap-junctions formation, tumor proliferation, neurologic analgesia,
and apoptosis. Although increased concentration of anandamide
in plasma has been implicated in pathophysiologic states including
endotoxin-induced hypotension, the effects of anandamide on hepatocytes
still remain unclear. In this study, we present evidence that
plasma anandamide concentration is highly increased in severe
hepatitis and cirrhosis patients. In addition, concentrations
of anandamide within the pathophysiologic range potently induced
apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes,
suggesting a possible link between increased anandamide level
and hepatocyte damage. Anandamide-induced cell death was preceded
by G0/G1 cell-cycle arrest, activation of proapoptotic signaling
(i.e., p38 MAPK and JNK), and inhibition of antiapoptotic
signaling (i.e., PKB/Akt) pathways. Moreover, anandamide
increased susceptibility to oxidative stress-induced hepatocyte
damage. In this context, methyl--cyclodextrin (MCD), a membrane
cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor,
or N-acetyl cysteine, an antioxidant, potently inhibited the
anandamide-induced proapoptotic events and cell death, whereas
putative cannabinoid receptor antagonists did not exhibit an
inhibitory effect on anandamide-induced cell death. Furthermore,
binding assay using polymyxin beads revealed that anandamide
could interact with cholesterol. In conclusion, our data suggest
that cholesterol present in the cell membrane determines the
fate of hepatocytes exposed to anandamide, possibly functioning
as an anandamide receptor. (HEPATOLOGY 2003;38:1167-1177.)
Inhibition of proteasome function leads to NF-B-independent
IL-8 expression in human hepatocytes
Swati Joshi-Barve, Shirish S. Barve, Waseem Butt, Jon Klein,
Craig J. McClain
Breakdown of cellular proteins is a highly regulated process,
and the ubiquitin-proteasome pathway is the major proteolytic
system in the cell. It regulates the levels of numerous proteins
that control gene expression and cell division, as well as responses
to stress and inflammation. Recent studies have reported abnormalities
in proteasome function in alcoholic liver disease (ALD). Moreover,
a direct relation has been reported between impaired proteasome
function and oxidative stress in experimental models of ALD.
Neutrophil infiltration is a hallmark of ALD, and activated neutrophils
are thought to play a role in the pathology of ALD. As a potent
neutrophil chemoattractant and activator, interleukin 8 (IL-8)
likely plays a key mechanistic role in many forms of liver injury.
In this study, we evaluated the effects of inhibition of proteasome
function on expression and release of IL-8 by human fetal hepatocytes
and hepatoma cells. Our data demonstrate that inhibition of proteasome
function in hepatocytes leads to apoptotic cell death. Decreased
hepatocyte survival coincides with enhanced expression of IL-8,
both at the protein and the messenger RNA (mRNA) levels. This
increase in IL-8 is independent of nuclear factor B (NF-B) activation
and is associated with an increase in c-Jun N-terminal kinase
(JNK) and activator protein-1 (AP-1) activity. In conclusion,
hepatocytes dying because of inhibition of proteasome function
produce massive quantities of the proinflammatory chemokine IL-8,
possibly resulting in neutrophil infiltration, increased inflammation,
and liver injury. (HEPATOLOGY 2003;38:1178-1187.)
Kupffer cell engulfment of apoptotic bodies stimulates death
ligand and cytokine expression
Ali Canbay, Ariel E. Feldstein, Hajime Higuchi, Nate Werneburg,
Annette Grambihler, Steve F. Bronk, Gregory J. Gores
Hepatocyte apoptosis by death receptors, hepatic inflammation,
and fibrosis are prominent features of liver diseases. However,
the link between these processes remains unclear. Our aim was
to ascertain whether engulfment of apoptotic bodies by Kupffer
cells promotes hepatic inflammation and fibrosis. Isolated murine
Kupffer cells efficiently engulfed apoptotic bodies generated
from UV-treated mouse hepatocytes. Engulfment of the apoptotic
bodies, but not latex beads, stimulated Kupffer cell generation
of death ligands, including Fas ligand, and tumor necrosis factor
(TNF-). Both apoptotic body phagocytosis and death ligand generation
were attenuated by gadolinium chloride, a Kupffer cell toxicant.
Kupffer cells isolated from 3-day bile duct-ligated (BDL) mice
were phenotypically similar to apoptotic body-"fed"
Kupffer cells with enhanced death ligand expression; inhibition
of hepatocyte apoptosis with a caspase inhibitor prevented this
Kupffer cell activation. Consistent with a role for Kupffer cells
in liver inflammation and fibrosis, gadolinium chloride attenuated
neutrophil infiltration and markers for stellate cell activation.
In conclusion, these findings support a model of cholestatic
liver injury where Kupffer cell engulfment of apoptotic bodies
promotes inflammation and fibrogenesis. (HEPATOLOGY 2003;38:1188-1198.)
Intracellular accumulation of pIgA-R and regulators of transcytotic
trafficking in cholestatic rat hepatocytes
Janet M. Larkin, Hope Coleman, Angelica Espinosa, Amy Levenson,
Mee Soon Park, Bonnie Woo, Alice Zervoudakis, Vu Tinh
Bile duct ligation (BDL) impairs basolateral-to-apical transcytosis
in hepatocytes, causing accumulation of transcytotic carriers
for the polymeric IgA receptor (pIgA-R) and redistribution of
secretory component (SC) from bile to blood. To gain insight
into the mechanisms regulating transcytosis and the pathophysiology
of cholestasis, we investigated nascent protein trafficking in
control and BDL livers using cell fractionation in the context
of in vivo pulse-chase experiments and immunoblot analysis.
Control and cholestatic hepatocytes trafficked [35S]-labeled
serum proteins and the pIgA-R along the secretory pathway with
identical kinetics. However, BDL impaired transcytosis, causing
(1) accumulation of the pIgA-R, rab3D, rab11a, and other candidate
regulators of apical-directed secretion in a crude vesicle carrier
fraction (CVCF) enriched in transcytotic carriers; (2) slow delivery
of [35S]-labeled SC to bile; and (3) paracellular reflux of SC
from bile to blood. In conclusion, these data indicate that the
secretory and transcytotic pathways remain polarized in cholestatic
hepatocytes and suggest that the pIgA-R traffics through postendosomal
rab3D-, rab11a-, and syntaxin 2-associated compartments, implicating
these proteins in the regulation of transcytosis. (HEPATOLOGY
2003;38:1199-1209.)
Liver Failure and Liver Disease
Systemic, renal, and hepatic hemodynamic derangement in
cirrhotic patients with spontaneous bacterial peritonitis (*Human
Study*)
Luis Ruiz-del-Arbol, Jesús Urman, Javier Fernández,
Mónica González, Miguel Navasa, Alberto Monescillo,
Agustín Albillos, Wladimiro Jiménez, Vicente Arroyo
Spontaneous bacterial peritonitis (SBP) is frequently associated
with renal failure. This study assessed if systemic and hepatic
hemodynamics are also affected by this condition. Standard laboratory
tests, tumor necrosis factor (TNF-) in plasma and ascitic fluid,
plasma renin activity (PRA) and norepinephrine (NE), and systemic
and hepatic hemodynamics were determined in 23 patients with
SBP at diagnosis and after resolution of infection. Eight patients
developed renal failure during treatment. At diagnosis of infection,
patients developing renal failure showed significantly higher
values of TNF-, blood urea nitrogen (BUN), PRA and NE, peripheral
vascular resistance, and hepatic venous pressure gradient (HVPG)
and lower cardiac output than patients not developing renal failure.
During treatment, a significant reduction in cardiac output and
arterial pressure and increase in PRA and NE, HVPG, and Child-Pugh
score were observed in the first group but not in the second.
Peripheral vascular resistance remained unmodified in both groups.
Changes in PRA and NE correlated inversely with changes in arterial
pressure and directly with changes in BUN, Child-Pugh score,
and HVPG. Five patients in the renal failure group developed
encephalopathy, and 6 died. In the group without renal failure,
none of the patients developed encephalopathy or expired. In
conclusion, patients with SBP frequently develop a rapidly progressive
impairment in systemic hemodynamics, leading to severe renal
and hepatic failure, aggravation of portal hypertension, encephalopathy,
and death. This occurs despite rapid resolution of infection
and is associated with an extremely poor prognosis. (HEPATOLOGY
2003;38:1210-1218.)
Quantitative T1 mapping of hepatic encephalopathy using
magnetic resonance imaging (*Human Study*)
Nadim Joni Shah, Heiko Neeb, Maxim Zaitsev, Sven Steinhoff, Gerald
Kircheis, Katrin Amunts, Dieter Häussinger, Karl Zilles
Changes are shown in the spin-lattice (T1) relaxation time caused
by the putative deposition of manganese in various brain regions
of hepatic encephalopathy (HE) patients using a novel and fast
magnetic resonance imaging (MRI) method for quantitative relaxation
time mapping. A new method, T1 mapping with partial inversion
recovery (TAPIR), was used to obtain a series of T1-weighted
images to produce T1 maps. Imaging of 15 control subjects and
11 patients was performed on a 1.5T MRI scanner. The measurement
time per patient with this technique, including adjustments,
was ~5 minutes. Regions of interest in the globus pallidus, the
caudate nucleus, the posterior and anterior limbs of the internal
capsule, the putamen, the frontal and occipital white matter,
the white matter of the corona radiata, the occipital visual
and frontal cortices, and the thalamus were interactively defined
in the left hemisphere and analyzed with respect to their T1
values. T1 changes in the brains of HE patients can be determined
quantitatively with TAPIR in short, clinically relevant measurement
times. Significant correlations between the change in T1 and
HE severity have been shown in the globus pallidus, the caudate
nucleus, and the posterior limb of the internal capsule. No significant
correlation of T1 with grade of HE was found in the putamen,
frontal white matter, white matter of the corona radiata, white
matter in the occipital lobe, the anterior limb of the internal
capsule, visual cortex, thalamus, or frontal cortex. In conclusion,
these measurements show that T1 mapping is feasible in short,
clinically relevant acquisition times. (HEPATOLOGY 2003;38:1219-1226.)
Validity of the 13C-caffeine breath test as a noninvasive,
quantitative test of liver function (*Human Study*)
Gordon Jung-Hyuk Park, Peter Harry Katelaris, D. Brian Jones,
Francis Seow, David George Le Couteur, Meng Chong Ngu
The properties of caffeine render it an ideal substrate for a
quantitative test of liver function. The aim of this study was
to determine whether the caffeine breath test (CBT) using orally
administered 13C-caffeine correlates reliably with plasma caffeine
clearance and reflects varying degrees of liver dysfunction.
The CBT was performed in 25 healthy controls; 20 subjects with
noncirrhotic, chronic hepatitis B or C; and 20 subjects with
cirrhosis. Plasma caffeine clearance was assayed simultaneously
with the CBT in a cohort of these subjects. Over a broad range
of caffeine clearances, the CBT exhibited a highly significant
correlation with plasma clearance (r = 0.85, P
< .001). Cirrhotic patients were characterized by significantly
reduced CBT values (1.15 ± 0.75 mg1) compared with
controls (2.23 ± 0.76; P = .001) and hepatitic
patients (1.83 ± 1.05; P = .04). There was a significant
inverse relationship between the CBT and Child-Pugh score (r
= .74, P = .002). The intraclass correlation coefficient
between repeated CBTs in 20 subjects with normal and cirrhotic
livers was 0.89. Although smoking was associated with an 86%
to 141% increase in CBT in all groups, the CBT was able to distinguish
control, hepatitic, and cirrhotic smokers (5.36 ± 0.82,
3.63 ± 1.21, and 2.14 ± 1.14, respectively, P
= .001). Multivariate analysis revealed that only smoking (P
< .001) and disease state (P = .001) were significant
predictors of the CBT. In conclusion, the 13C-CBT represents
a valid indicator of plasma caffeine clearance and correlates
reproducibly with hepatic dysfunction. (HEPATOLOGY 2003;38:1227-1236.)
Adjuvant intra-arterial injection of iodine-131labeled
lipiodol after resection of hepatocellular carcinoma (*Human
Study*)
Eveline Boucher, Stéphane Corbinais, Yan Rolland, Patrick
Bourguet, Dominique Guyader, Karim Boudjema, Bernard Meunier,
Jean-Luc Raoul
The high rate of recurrence after surgical resection of hepatocellular
carcinoma (HCC) is a major therapeutic challenge. Postoperative
injection of 131-iodine-labeled lipiodol (131I-Lip) into the
hepatic artery has been proposed as adjuvant treatment (Lau et
al.). We analyzed 2 retrospective series of matched patients
treated in our unit before and after addition of 131I-Lip adjuvant
therapy to our standard surgical strategy. Thirty-eight patients
who had undergone surgical resection of HCC after January 1999
were given adjuvant intra-arterial injection of 131I-Lip after
surgery. These patients were matched with 38 other patients who
had undergone surgical resection only between January 1997 and
January 1999. The frequency of recurrences, disease-free rates,
and overall survival rates were compared. The 2 groups were similar
for clinical, biologic, or histologic parameters studied and
Cancer Liver Italian Program scores. There were 15 recurrences
in the group without adjuvant treatment and 9 in the group with
131I-Lip adjuvant treatment. The 1- , 2-, and 3-year disease-free
survival rates (±95% confidence interval) were different
(P < .02): 94.7% ± 3.6%, 83.7% ± 6.1%,
and 68.4% ± 9.7%, respectively, in the 131I-Lip group
versus 73.7% ± 7.1%, 54.3% ± 8.2%, and 41.5% ±
10.5% in the surgery group. The 1- , 2-, and 3-year survival
rates (±95% confidence interval) also were different (P
< .02): 94.7% ± 3.6%, 91.7% ± 4.6%, and 91.7%
± 4.6%, respectively, in the 131I-Lip group versus 94.7%
± 3.6%, 71.3% ± 7.8%, and 49.9% ± 10% in
the surgery group. In conclusion, this retrospective analysis
supports the promising contribution of postoperative injection
of 131I-Lip after resection of HCC. A randomized study including
more patients would be necessary to confirm its contribution
to therapeutic management. (HEPATOLOGY 2003;38:1237-1241.)
*PTK2 and EIF3S3 genes may be amplification targets
at 8q23-q24 and are associated with large hepatocellular carcinomas
(*Human Study*)
Hiroyuki Okamoto, Kohichiroh Yasui, Chen Zhao, Shigeki Arii,
Johji Inazawa
We investigated 39 primary hepatocellular carcinomas (HCCs) for
aberrations in DNA copy number, using comparative genomic hybridization
(CGH). Gain of DNA at 8q was common in these tumors; high-level
gains, indicative of gene amplification, occurred most frequently
at 8q23-q24. Gains of 8q correlated with large (>5 cm) tumor
size. To identify targets of the amplification events involving
8q, we determined expression levels of 14 candidate genes within
that region in a total of 41 HCCs by means of real-time quantitative
reverse-transcription polymerase chain reactions (RT-PCR). Significant
correlation was found between elevated levels of expression and
increases in copy number for PTK2 (located at 8q24.3)
and EIF3S3 (at 8q23.3), but for none of the other candidates,
which included MYC (8q24.1). Southern blot analyses confirmed
that PTK2 and EIF3S3 were amplified, respectively,
in 5 (19%) and 7 (26%) of the 27 tumors examined in accordance
with expression patterns, an indication that expression of PTK2
and EIF3S3 was probably up-regulated by the amplification
mechanism. When we analyzed potential relationships between elevated
expression of PTK2 and EIF3S3 and clinicopathologic
parameters, high expression of the 2 transcripts was significantly
associated with large (>5 cm) tumor size and with hepatitis
B virus (HBV) infection. In conclusion, PTK2 and EIF3S3,
which, respectively, encode focal adhesion kinase and the p40
subunit of the eukaryotic initiation factor 3, were probable
targets within the amplification at 8q23-q24 and may be involved
in progression of HCC. (HEPATOLOGY 2003;38:1242-1249.)
*Patients with primary biliary cirrhosis react against a ubiquitous
xenobiotic-metabolizing bacterium (*Human Study*)
Carlo Selmi, David L. Balkwill, Pietro Invernizzi, Aftab A. Ansari,
Ross L. Coppel, Mauro Podda, Patrick S. Leung, Thomas P. Kenny,
Judy Van De Water, Michael H. Nantz, Mark J. Kurth, M. Eric Gershwin
Infectious and environmental agents have been proposed as immunologic
triggers for primary biliary cirrhosis (PBC). Recently, a ubiquitous
organism that metabolizes organic compounds and estrogens, Novosphingobium
aromaticivorans, has been defined. Importantly, 2 bacterial
proteins have homology with the E2 component of the pyruvate
dehydrogenase complex (PDC-E2). Sera from 97 patients with PBC,
46 first-degree relatives, 10 spouses, and 195 controls were
studied for reactivity against N. aromaticivorans and
Escherichia coli. The reactivity was defined by absorption,
affinity purification, and using monoclonal antibodies to PDC-E2.
Stool samples from 20 patients with PBC and 34 controls were
analyzed by polymerase chain reaction (PCR) for the presence
of N. aromaticivorans. Sera from 100% of anti-PDC-E2 positive
(77/77), 33% of anti-BCOADC E2 positive (1/3), and 12% of antimitochondrial
antibody (AMA) negative patients with PBC (2/17) reacted with
titers up to 106 against two known lipoylated bacterial
proteins (47 and 50 kd) from N. aromaticivorans, including
patients with early disease. This titer was approximately 100-
to 1,000-fold higher than against E. coli and verified
by absorption, use of affinity-purified sera, and monoclonal
antibody reagents. Moreover, 78 of 80 AMA-positive and 5 of 17
AMA-negative patients with PBC had antibodies against 3 other
N. aromaticivorans proteins. In contrast, 0 of 195 control
sera reacted against N. aromaticivorans. Approximately
25% of patients and controls had N. aromaticivorans in
their fecal specimens. In conclusion, based on protein homology,
capacity to metabolize xenobiotics as well as modulate estrogens,
its presence in feces, and specific immunologic response, we
propose that N. aromaticivorans is a candidate for the
induction of PBC. (HEPATOLOGY 2003;38:1250-1257.)
Viral Hepatitis
Hepatitis B virus X protein regulates transactivation activity
and protein stability of the cancer-amplified transcription coactivator
ASC-2
Hee Jeong Kong, Min Jung Park, SunHwa Hong, Hyun Jung Yu, Young
Chul Lee, Young Hyun Choi, JaeHun Cheong
Hepatitis B virus X protein (HBx) is a transcriptional coactivator
that plays a significant role in the regulation of genes involved
in inflammation and cell survival. A recently identified cellular
coactivator, activating signal cointegrator 2 (ASC-2), is enriched
in liver cancer cells and associates with many transcription
factors that are active in hepatocytes. The tissue colocalization
of these 2 proteins, in view of their similar regulatory functions,
led us to examine whether HBx and ASC-2 cooperate in transcriptional
activation of gene expression. Glutathione S-transferase
(GST) pull-down assays and mammalian 2-hybrid analysis show that
the transactivation domain of HBx interacts with the C-terminal
domain of ASC-2. In fact, these 2 proteins associated in a ternary
complex that included the transcriptional activator retinoid
X receptor (RXR). Mechanistically, on expression of HBx, the
half-life of the ASC-2 coactivator is observed to increase in
concordance with the observed increase in ASC-2-dependent coactivation
of transcription. In conclusion, these results show that HBx
stabilizes the cellular coactivator ASC-2 through direct protein-protein
interaction, affecting the regulation of genes actively transcribed
in liver cancer cells. (HEPATOLOGY 2003;38:1258-1266.)
Determinants for sustained HBeAg response to lamivudine therapy
(*Human Study*)
Rong-Nan Chien, Chau-Ting Yeh, Sun-Lung Tsai, Chia-Ming Chu,
Yun-Fan Liaw
There are inconsistent data on the durability of hepatitis B
e antigen (HBeAg) seroconversion after lamivudine is discontinued.
The aim of this study was to examine the determinants for sustained
HBeAg response to lamivudine therapy. Both host and viral factors
as well as the drug factor were compared between 43 patients
with sustained HBeAg response and 39 patients whose response
was not sustained. All of them received a mean period of 16 months
(range, 3-55 months) lamivudine therapy and had achieved complete
response (HBeAg seroconversion plus HBV DNA seroclearance by
hybrid capture assay and normal alanine aminotransferase [ALT])
and were followed-up for a mean period of 44 months (range, 12-88
months). Stepwise logistic regression model was used to estimate
the sustained response on the presence of the following variables:
age; gender; pretherapy ALT; total bilirubin and HBV DNA levels;
time to HBeAg seroconversion; additional lamivudine treatment
after HBeAg seroconversion; total duration of treatment; hepatitis
activity index scores; periportal, intralobular, and portal inflammation
and fibrosis scores; scores excluding fibrosis; status of precore
mutation; basal core promoter mutation; and genotype. The results
showed that genotype (OR, 5.922; 95% CI, 1.611-21.768; P
= .007), age (OR, 0.943; 95% CI, 0.891-0.997; P = .040),
and additional treatment (OR, 1.097; 95% CI, 1.028-1.171; P
= .005) were independent factors to sustained HBeAg response.
Further categorical analysis disclosed that patients with genotype
B, age 36 years, and additional lamivudine treatment over 8 months
have higher sustained response. In conclusion, HBV genotype,
age, and additional treatment are the major determinants for
the sustained HBeAg response to lamivudine therapy. (HEPATOLOGY
2003;38:1267-1273.)
Deficiency in virion secretion and decreased stability of
the hepatitis B virus immune escape mutant G145R
Tatyana Kalinina, Alicja Iwanski, Hans Will, Martina Sterneck
Hepatitis B virus with a G145R mutation in the small surface
protein is considered the quintessential immune escape mutant
because it frequently is found in vaccinated individuals with
breakthrough infections and liver transplant recipients under
anti-hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis.
Nowadays the prevalence of the variant progressively increases.
However, because spread of a virus depends not only on immune
pressure but also on the viral phenotype, we investigated the
biologic properties of the G145R variant. The G145R mutation
was introduced into wild-type (Wt) virus genome by in vitro
mutagenesis. After transfection into human hepatoma cells, the
DNA, RNA, and protein synthesis and viral secretion ability of
the mutant were studied. Furthermore, cotransfection studies
were performed with the G145R variant and a Wt virus S-protein
expressing construct and vice versa. Production and stability
of viral messenger RNAs (mRNAs), DNA, and proteins were not affected
by the G145R mutation. In contrast, secretion of mutant virions
was reduced significantly. Only 20% of virions were found in
the medium of G145R variant-transfected cells compared with Wt
virus. Furthermore, mutant virions were more sensitive to detergent
treatment suggesting a diminished stability. In cotransfection
studies, Wt virus S-protein rescued secretion of mutant virions,
whereas mutant S-protein had a transdominant negative effect
on secretion of Wt virus. Both mechanisms may support persistence
of the defective mutant in a mixed population with Wt virus.
In conclusion, the significant defect of the G145R mutant for
secretion of infectious virions and the diminished stability
of mutant virions may limit global spread of the mutant. (HEPATOLOGY
2003;38:1274-1281.)
Cyclosporin A suppresses replication of hepatitis C virus
genome in cultured hepatocytes
Koichi Watashi, Makoto Hijikata, Masahiro Hosaka, Masashi Yamaji,
Kunitada Shimotohno
Persistent infection of hepatitis C virus (HCV) is a major cause
of liver diseases such as chronic hepatitis, liver cirrhosis,
and hepatocellular carcinoma. Searching for a substance with
anti-HCV potential, we examined the effects of a variety of compounds
on HCV replication using a HCV subgenomic replicon cell culture
system. Consequently, the immunosuppressant cyclosporin A (CsA)
was found to have a suppressive effect on the HCV replicon RNA
level and HCV protein expression in these cells. CsA also inhibited
multiplication of the HCV genome in a cultured human hepatocyte
cell line infected with HCV using HCV-positive plasma. This anti-HCV
activity of CsA appeared to be independent of its immunosuppressive
function. In conclusion, our results suggest that CsA may represent
a new approach for the development of anti-HCV therapy. (HEPATOLOGY
2003;38:1282-1288.)
A pilot study of therapeutic vaccination with envelope
protein E1 in 35 patients with chronic hepatitis C (*Human Study*)
Frederik Nevens, Tania Roskams, Hans Van Vlierberghe, Yves Horsmans,
Dirk Sprengers, Ann Elewaut, Valeer Desmet, Geert Leroux-Roels,
Emmanuel Quinaux, Erik Depla, Stephanie Dincq, Christine Vander
Stichele, Geert Maertens, Frank Hulstaert
New treatments are needed for chronic hepatitis C patients in
whom viral clearance cannot be achieved. Thirty-five chronic
hepatitis C patients (genotype 1) were randomized to receive
20 µg of recombinant HCV E1 (E1) (n = 26) or placebo (n
= 9) intramuscularly at weeks 0, 4, 8, 12, and 24. Thirty-four
then received open-label E1 vaccine at weeks 50, 53, 56, 59,
62, and 65. Twenty-four patients (12 men, 12 women; mean age,
52 y; 18 interferon-based treatment failures; mean baseline alanine
aminotransferase [ALT] level, 118 IU/L) underwent a biopsy before
and after 2 courses of E1, 17 months later. Liver histology was
scored by 2 blinded pathologists according to the Ishak and Metavir
systems. Postinjection reactions were similar to placebo (alum
only). Nine of 24 patients (38%) had improvement of 2 points
or more, 10 (41%) remained stable, and 5 (21%) showed worsening
in total Ishak score. Nine patients (38%) improved both on Ishak
and Metavir fibrosis scores. Plasma HCV-RNA levels remained unchanged,
whereas ALT levels showed a trend toward a decrease during treatment.
All but 3 patients developed a significant de novo E1-specific
T-cell response. The increase in anti-E1 antibody levels correlated
with the decrease in total Ishak score and with the relative
decreases in both Ishak fibrosis score and ALT level (all P
.01). In conclusion, E1 therapeutic vaccination is well tolerated
and the observed effects warrant further study. (HEPATOLOGY 2003;38:1289-1296.)
Characterization of HCV-specific Patr class II restricted
CD4+ T cell responses in an acutely infected chimpanzee
David J. Woollard, Arash Grakoui, Naglaa H. Shoukry, Krishna
K. Murthy, Katherine J. Campbell, Christopher M. Walker
Resolution of hepatitis C virus (HCV) infection is associated
with strong and sustained virus-specific CD4+ T cell responses.
In this study, we investigated the evolution of functional T
cell responses during acute infection of a chimpanzee and the
longevity of these lymphocytes in blood and liver after resolution
of infection. Viremia increased through the first 3 weeks of
infection and then remained stable until the onset of T cell
responses at weeks 6 and 8 postinfection. CD4+ T cells targeting
nonstructural HCV proteins were detected in proliferation assays
by week 6 postinfection, but they failed to produce interferon
(IFN-). HCV-specific CD4+ and CD8+ T cells with the ability to
produce IFN- appeared at week 8 when a rapid 10-fold reduction
in plasma viremia was first observed. This cytokine response
persisted through to week 24 when infection apparently resolved.
T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell
epitope were derived from liver and their Patr major histocompatibility
complex (MHC) restriction elements were identified. In retrospective
studies performed on cryopreserved peripheral blood mononuclear
cells (PBMCs) collected at various timepoints after infection,
the onset of an IFN- response measured against the class II restricted
epitopes correlated with viral clearance. In conclusion, the
characterization of the HCV epitopes and MHC class II restriction
elements described here will facilitate a detailed comparison
of CD4+ T cell function in animals with resolved and persistent
infections. (HEPATOLOGY 2003;38:1297-1306.)
Copyright © 2003 by the American Association for the
Study of Liver Diseases. All rights reserved.
Table of Contents for November
2003 · Volume 125 · Number 5
Rapid Communication
Defects in cholangiocyte fibrocystin expression and ciliary
structure in the PCK rat
Tatyana V. Masyuk, Bing Q. Huang, Christopher J. Ward, Anatoliy
I. Masyuk, David Yuan, Patrick L. Splinter, Rachaneekorn Punyashthiti,
Eric L. Ritman, Vicente E. Torres, Peter C. Harris, Nicholas
F. Larusso
Background & Aims: Recent studies have showed that
proteins associated with polycystic kidney disease (PKD) are
expressed in cilia, linking this organelle and cyst formation
in the kidney, but involvement of cilia in PKD-related biliary
cystogenesis has not been shown. We investigated: (1) the expression
of fibrocystin (a product of PKHD1, the autosomal-recessive
PKD [ARPKD] gene) in cholangiocyte cilia; (2) biliary cyst formation
in an orthologous rat model, PCK; and (3) the effect of Pkhd1
mutation on ciliary structure. Methods: Biliary cystogenesis
was assessed by microcomputed tomography. Fibrocystin expression
in cholangiocytes of isolated intrahepatic bile ducts (IBDUs)
and liver cysts was analyzed by confocal and immunoelectron microscopy,
and ciliary structure and length by scanning and transmission
electron microscopy. Small interfering RNAs (siRNA) were used
to examine the effect of fibrocystin loss on ciliary structure.
Results: The biliary tree in the PCK rat was distorted
markedly, showing multiple bile duct dilatation and focal budding.
In normal IBDUs, each cholangiocyte had a single cilium that
expressed fibrocystin. In contrast, cilia in the PCK rat were
abnormal with bulbous extensions and diminished length, and were
devoid of fibrocystin. In cholangiocytes of normal IBDUs, specific
siRNA reduced Pkhd1 messenger RNA by 80%, the length of
cilia by 41%, and fibrocystin ciliary expression to an undetectable
level. Conclusions: Our results indicate that fibrocystin
is expressed in cholangiocyte cilia and that disruption of Pkhd1
by a germ line mutation in the PCK rat or by siRNA in IBDUs results
in abnormalities in ciliary morphology and possibly biliary cystogenesis.
Clinical-alimentary Tract
Progression of flat
low-grade dysplasia to advanced neoplasia in patients with ulcerative
colitis
Thomas Ullman, Victoria Croog, Noam Harpaz, David Sachar, Steven
Itzkowitz
Background & Aims: Long-standing ulcerative colitis
has long been recognized as a risk factor for colorectal cancer,
but there is still no universal consensus on the optimal management
of ulcerative colitis patients with low-grade dysplasia in flat
mucosa. Some authorities favor prompt colectomy, whereas others
recommend continued surveillance. The purpose of our study was
to determine the frequency with which flat low-grade dysplasia
in ulcerative colitis progresses to advanced neoplasia (high-grade
dysplasia or colorectal cancer) and whether specific variables
could predict such progression. Methods: We reviewed the
medical histories, colonoscopic findings, and surgical and pathology
reports of 46 patients with ulcerative colitis diagnosed with
flat low-grade dysplasia on a surveillance colonoscopy. The rates
of neoplastic progression, as well as the frequency of advanced
neoplasia, were tabulated. We correlated progression with several
clinical and colonoscopic variables: the number of biopsy samples
positive for flat low-grade dysplasia, the duration and anatomic
extent of disease, patient age, and medication use. Results:
Among these 46 patients, there were 7 cases of colorectal cancer,
5 of which were stage II or higher. Unexpected advanced neoplasia
occurred in 4 of 17 (23.5%) patients who underwent colectomy
for flat low-grade dysplasia. On an actuarial basis, the rate
of neoplastic progression was 53% at 5 years. No clinical features
predicted progression to advanced neoplasia. Cancers, including
2 at advanced stages, developed despite frequent follow-up surveillance
examinations. Conclusions: A finding of flat low-grade
dysplasia during ulcerative colitis surveillance is a strong
predictor of progression to advanced neoplasia. Early colectomy
should be recommended for such patients.
Randomized controlled trial of biofeedback for fecal incontinence
Christine Norton, Sonya Chelvanayagam, Jenifer Wilson-Barnett,
Sally Redfern, Michael A. Kamm
Background & Aims: Behavioral treatment (biofeedback)
has been reported to improve fecal incontinence but has not been
compared with standard care. Methods: A total of 171 patients
with fecal incontinence were randomized to 1 of 4 groups: (1)
standard care (advice); (2) advice plus instruction on sphincter
exercises; (3) hospital-based computer-assisted sphincter pressure
biofeedback; and (4) hospital biofeedback plus the use of a home
electromyelogram biofeedback device. Outcome measures included
diary, symptom questionnaire, continence score, patient's rating
of change, quality of life (short-form 36 and disease specific),
psychologic status (Hospital Anxiety and Depression scale), and
anal manometry. Results: Biofeedback yielded no greater
benefit than standard care with advice (53% improved in group
3 vs. 54% in group 1). There was no difference between the groups
on any of the following measures: episodes of incontinence decreased
from a median of 2 to 0 per week (P < 0.001). Continence
score (worst = 20) decreased from a median of 11 to 8 (P
< 0.001). Disease-specific quality of life, short-form 36
(vitality, social functioning, and mental health), and Hospital
Anxiety and Depression scale all significantly improved. Patients
improved resting, squeeze, and sustained squeeze pressures (all
P < 0.002). These improvements were largely maintained
1 year after finishing treatment. Conclusions: Conservative
therapy for fecal incontinence improves continence, quality of
life, psychologic well-being, and anal sphincter function. Benefit
is maintained in the medium term. Neither pelvic floor exercises
nor biofeedback was superior to standard care supplemented by
advice and education.
The genomic damage estimated by arbitrarily primed PCR DNA
fingerprinting is useful for the prognosis of gastric cancer
Koichi Suzuki, Sumiko Ohnami, Chikako Tanabe, Hiroki Sasaki,
Jun Yasuda, Hitoshi Katai, Kimio Yoshimura, Masaaki Terada, Manuel
Perucho, Teruhiko Yoshida
Background & Aims: Genomic instability and the accompanying
alteration of cancer genes play a major role in tumorigenesis.
We evaluated the prognostic significance in gastric cancer of
the degree of accumulation of relative genomic damage, assessed
by arbitrarily primed polymerase chain reaction DNA fingerprinting.
Methods: Genomic damage was assessed by comparative analysis
of paired normal and tumor tissue DNA fingerprints. The total
number of alterations, scored as decreases and increases of band
intensity with 2 arbitrary primers, were used as an estimation
of the genomic damage fraction in 74 primary gastric cancers.
Increases in DNA copy number were also analyzed by array comparative
genomic hybridization in a subset of 30 cases. Results:
The number of altered bands varied among the tumors from none
or a few to more than one third of the approximately 40 fingerprint
bands. The relative values of genomic damage were consistent
with the quantitative chromosomal alterations observed by array
comparative genomic hybridization. When the tumors were stratified
into 2 groups-above or below the cutoff of 0.22 for average genomic
damage fraction-genomic damage fraction was a valuable prognostic
indicator regardless of microsatellite instability status. Multivariate
Cox analysis showed that the genomic damage fraction was a prognostic
indicator, as well as a stage indicator (P = 0.0189).
Survival was significantly diminished in tumors with a genomic
damage fraction >0.22 (P = 0.0009). Moreover, in the
46 curative cases, genomic damage fraction was the only independent
factor for predicting survival (P = 0.0061). Conclusions:
Our results indicate that the degree of genomic damage estimated
by arbitrarily primed polymerase chain reaction fingerprinting
is a useful prognostic indicator for gastric cancer.
Reduced migration of fibroblasts in inflammatory bowel disease:
Role of inflammatory mediators and focal adhesion kinase
Sandra Nicole Leeb, Daniela Vogl, Manuela Gunckel, Stephan Kiessling,
Werner Falk, Michael Göke, Jürgen Schölmerich,
Cornelia Maria Gelbmann, Gerhard Rogler
Background & Aims: Crohn's disease (CD) and ulcerative
colitis (UC) are associated with chronic tissue damage and continuous
tissue repair. A central, but not well-characterized, event during
this process is the migration of activated fibroblasts to the
wound. Methods: Human colonic lamina propria fibroblasts
(CLPF) were isolated from patients with CD and UC and from healthy
controls and were characterized by immunocytochemistry. Migration
assays of CLPF were performed in the modified 48-well Boyden
chamber. Focal adhesion kinase (FAK) and FAK autophosphorylation
in migrating CLPF were determined by Western blotting. FAK mRNA
expression was investigated by Northern blotting. Results:
The migration of CD-CLPF and UC-CLPF was significantly reduced
when compared with control-CLPF. This was correlated with a decrease
in FAK phosphorylation, whereas, in migrating control-CLPF, an
increase was found. Similarly, the presence of the inflammatory
mediators interferon (IFN)- (50 ng/mL) or tumor necrosis factor
(TNF) (30 ng/mL) in conditioned medium significantly reduced
the migration of control-CLPF to 41% ± 4% or 30% ±
7%, respectively. Preincubation of control-CLPF with TNF (20
ng/mL) and IFN- (10 ng/mL) for 3 days reduced their migratory
response to 10% of control (P < 0.001), which also
was correlated with a decrease in FAK phosphorylation. Culture
of IFN-/TNF-treated CLPF for a further 7 days without cytokines
did not restore the migratory potential and FAK phosphorylation,
indicating a persistent functional change. Conclusions:
CD- and UC-CLPF have a reduced migratory potential compared with
normal CLPF. That may be caused by contact with IFN- and TNF.
This loss of migratory potential was correlated with diminished
FAK phosphorylation.
Clinical-liver Pancreas and Biliary
Tract
Successful photodynamic therapy for nonresectable cholangiocarcinoma:
A randomized prospective study
Marianne E.J. Ortner, Karel Caca, Frieder Berr, Jochen Liebetruth,
Ulrich Mansmann, Dominik Huster, Winfried Voderholzer, Guido
Schachschal, Joachim Mössner, Herbert Lochs
Background & Aims: In nonrandomized trials, photodynamic
therapy (PDT) had a promising effect on nonresectable cholangiocarcinoma
(NCC). This prospective, open-label, randomized, multicenter
study with a group sequential design compared PDT in addition
to stenting (group A) with stenting alone (group B) in patients
with NCC. Methods: In patients with histologically confirmed
cholangiocarcinoma, endoscopic or percutaneous double stenting
was performed. Patients fulfilling inclusion criteria were randomized
to group A (stenting and subsequent PDT) and group B (stenting
alone). For PDT, Photofrin 2 mg/kg body wt was injected intravenously
2 days before intraluminal photoactivation (wavelength, 630 nm;
light dose, 180 J/cm2). Further treatments were performed in
cases of residual tumor in the bile duct. The primary outcome
parameter was survival time. Secondary outcome parameters were
cholestasis and quality of life. Results: PDT resulted
in prolongation of survival (group A: n = 20, median 493 days;
group B: n = 19, median 98 days; P < 0.0001). It also
improved biliary drainage and quality of life. Conclusions:
PDT, given in addition to best supportive care, improves survival
in patients with NCC. The study was terminated prematurely because
PDT proved to be so superior to simple stenting treatment that
further randomization was deemed unethical.
Incidence, clinical spectrum, and outcomes of primary sclerosing
cholangitis in a united states community
Kiran Bambha, W. Ray Kim, Jayant Talwalkar, Heidi Torgerson,
Joanne T. Benson, Terry M. Therneau, Edward V. Loftus, Jr, Barbara
P. Yawn, E. Rolland Dickson, L. Joseph Melton, III
Background & Aims: The epidemiology of primary sclerosing
cholangitis (PSC) in the United States is unknown. We report
the incidence, clinical spectrum, and outcomes of PSC in Olmsted
County, Minnesota. Methods: Using the Rochester Epidemiology
Project, a medical records linkage system in Olmsted County,
Minnesota, we identified county residents with PSC, and the diagnosis
was confirmed according to clinical, biochemical, radiographic,
and histologic criteria. Results: Twenty-two patients
met diagnostic criteria for PSC in 19762000. The age-adjusted
(to 2000 U.S. whites) incidence of PSC in men was 1.25 per 100,000
person-years (95% CI, 0.70 to 2.06) compared with 0.54 per 100,000
person-years (95% CI, 0.22 to 1.12) in women. The prevalence
of PSC in 2000 was 20.9 per 100,000 men (95% CI, 9.5 to 32.4)
and only 6.3 per 100,000 women (95% CI, 0.1 to 12.5). Seventy-three
percent of cases had inflammatory bowel disease, the majority
with ulcerative colitis. Survival among PSC patients was significantly
less than expected for the Minnesota white population of similar
age and gender (P < 0.001). Conclusions: These
data represent the first population-based estimates of the incidence
and prevalence of PSC in the United States. The incidence and
prevalence of PSC were approximately one third of those previously
described for primary biliary cirrhosis in the same population.
Our data suggest that the prevalence of PSC in the United States,
with its attendant medical burdens, is significantly greater
than previously estimated.
Sequence variation upstream of precore translation initiation
codon reduces hepatitis B virus e antigen production
Sang Hoon Ahn, Anna Kramvis, Shigenobu Kawai, Hans Christian
Spangenberg, Jisu Li, Gerald Kimbi, Michael Kew, Jack Wands,
Shuping Tong
Background & Aims: Most South African hepatitis B
virus strains harbor point mutations immediately upstream of
the precore AUG codon. The aim of this study was to determine
their effect on hepatitis B e antigen expression. Methods:
The hepatitis B virus DNA sequence around the precore region
was determined from sera of 45 black South Africans. The South
African mutations were introduced into hepatitis B virus dimers
of the same genotype, and hepatitis B e antigen was quantified
from culture medium of transfected HepG2 or Huh7 cells. Results:
The South African sequence changes were easily detectable in
the acute, hepatitis B e antigen-positive phase of infection,
suggesting that they were stable traits and were not selected
by immune pressure. Triple mutations at the 5, 3, and
2 positions of the AUG codon severely impaired hepatitis
B e antigen expression (P < 0.001). The frequent double
mutation at the 5 and 2 positions moderately reduced
hepatitis B e antigen levels (P < 0.001) to an extent
comparable to that of the common core promoter mutations (1762T1764A).
The presence of both South African and core promoter mutations
diminished hepatitis B e antigen expression in an additive manner.
It is interesting to note that the triple South African mutations
enabled core protein translation from precore messenger RNA,
which could rescue the replication defect of a hepatitis B virus
genome with an ablated core gene. Conclusions: We have
identified a novel class of hepatitis B e antigen variants with
reduced hepatitis B e antigen translation by a ribosomal leaky
scanning mechanism. Reduction in hepatitis B e antigen expression
may contribute to accelerated seroconversion from hepatitis B
e antigen to its antibody in black South Africans infected with
hepatitis B virus very early in life.
Distinct costimulation dependent and independent autoreactive
T-cell clones in primary biliary cirrhosis
Takashi Kamihira, Shinji Shimoda, Kenichi Harada, Akira Kawano,
Mizuki Handa, Eishi Baba, Koichi Tsuneyama, Minoru Nakamura,
Hiromi Ishibashi, Yasuni Nakanuma, M. Eric Gershwin, Mine Harada
Background & Aims: Previous work has suggested that
CD4+ CD28 or costimulation-independent T cells are increased
in autoimmune diseases. In this study, we compared frequency
and qualitative characteristics of autoreactive costimulation-independent
or CD4+ CD28 T cells in primary biliary cirrhosis (PBC)
by taking advantage of the well-defined immunodominant autoepitope
of the E2 component of pyruvate dehydrogenase (PDC-E2). Methods:
We determined the frequency of costimulation-independent autoreactive
T cells that respond to PDC-E2 163176 and the frequency
of CD4+ CD28 T cells. Finally, we determined the role of
biliary epithelial cells (BEC) as both an antigen-presenting
cell or, alternatively, as a target cell for T-cell-mediated
cytotoxicity. Results: The precursor frequency of costimulation-independent
CD4+ T cells that respond to PDC-E2 163176 and the frequency
of CD4+ CD28 T cells were dramatically elevated in PBC.
Furthermore, 2 types of T-cell clones that respond to PDC-E2
163176 emerged from this study. One type was costimulation
dependent and the other costimulation independent. Both types
of clones lyse BEC in a similar effector target (E/T) ratio distribution.
However, BEC did not help the proliferation of any T-cell clones.
Furthermore, costimulation-independent T-cell clones do not become
anergic by BEC. Conclusions: In PBC, costimulation-independent
autoreactive T cells, which do not become anergic, increase and
maintain the autoimmune response. In controls, although autoantigens
are expressed on BEC and autoantigen-reactive T cells exist around
BEC, autoantigen-reactive T cells are costimulation dependent
and will become anergic and maintain peripheral tolerance.
Basic-alimentary Tract
Bacterial colonization leads to the colonic secretion of
RELM/FIZZ2, a novel goblet cell-specific protein
Weimian He, Mei-Lun Wang, Han-Qing Jiang, Claire M. Steppan,
Marcus E. Shin, M. Christine Thurnheer, John J. Cebra, Mitchell
A. Lazar, Gary D. Wu
Background & Aims: Goblet cells are highly polarized
exocrine cells found throughout the small and large intestine
that have a characteristic morphology due to the accumulation
of apical secretory granules. These granules contain proteins
that play important physiologic roles in cellular protection,
barrier function, and proliferation. A limited number of intestinal
goblet cell-specific proteins have been identified. In this study,
we investigate the expression and regulation of RELM, a novel
colon-specific gene. Methods: The regulation of RELM messenger
RNA expression was determined in LS174T, Caco-2, and HT-29 cell
lines in response to stimulation with interleukin 13 and lipopolysaccharide.
Quantitative reverse-transcription polymerase chain reaction,
immunoblots, and immunohistochemistry were used to examine the
expression of RELM in BALB/c and C.B17.SCID mice housed in conventional,
germ-free, and gnotobiotic environments. Results: Messenger
RNA for RELM is restricted to the undifferentiated, proliferating
colonic epithelium. Immunohistochemistry shows that this protein
is expressed in goblet cells located primarily in the distal
half of the colon and cecum with lower levels detectable in the
proximal colon. High levels of RELM can be detected in the stool
of mice and humans, where it exists as a homodimer under nonreducing
conditions. Interestingly, the secretion of RELM is dramatically
reduced in germ-free mice. Furthermore, introduction of germ-free
mice into a conventional environment results in enhanced expression
and robust secretion of RELM within 48 hours. Conclusions:
These studies define a new goblet cell-specific protein and provide
the first evidence that colon-specific gene expression can be
regulated by colonization with normal enteric bacteria.
Purinergic mechanisms contribute to mechanosensory transduction
in the rat colorectum
Gregory Wynn, Weifang Rong, Zhenghua Xiang, Geoffrey Burnstock
Background & Aims: Adenosine 5´-triphosphate
plays a role in peripheral sensory mechanisms and, in particular,
mechanosensory transduction in the urinary system. P2X3 receptors
are selectively expressed on small-diameter sensory neurons in
the dorsal root ganglia; sensory neurons from dorsal root ganglia
L1 and S1 supply the colorectum. This study investigated whether
purinergic signaling contributes to mechanosensory transduction
in the rat colorectum. Methods: A novel in vitro rat colorectal
preparation was used to elucidate whether adenosine 5´-triphosphate
is released from the mucosa in response to distention and to
evaluate whether it contributes to sensory nerve discharge during
distention. Results: P2X3 receptor immunostaining was
present on subpopulations of neurons in L1 and S1 dorsal root
ganglia, which supply the rat colorectum. Distention of the colorectum
led to pressure-dependent increases in adenosine 5´-triphosphate
release from colorectal epithelial cells and also evoked pelvic
nerve excitation, which was mimicked by application of adenosine
5´-triphosphate and ,-methylene adenosine 5´-triphosphate.
The sensory nerve discharges evoked by distention were potentiated
by ,-methylene adenosine 5´-triphosphate and ARL-67156,
an adenosine triphosphatase inhibitor, and were attenuated by
the selective P2X1, P2X3, and P2X2/3 antagonist 2´,3´-O-trinitrophenyl-adenosine
5´-triphosphate and by the nonselective P2 antagonists
pyridoxyl 5-phosphate 6-azophenyl-2´,4´-disulfonic
acid and suramin. Adenosine, after ectoenzymatic breakdown of
adenosine 5´-triphosphate, seems to be involved in the
longer-lasting distention-evoked sensory discharge. Single-fiber
analysis showed that high-threshold fibers were particularly
affected by ,-methylene adenosine 5´-triphosphate, suggesting
a correlation between purinergic activation and nociceptive stimuli.
Conclusions: Adenosine 5´-triphosphate contributes
to mechanosensory transduction in the rat colorectum, and this
is probably associated with pain.
Gastric inflammation triggers hypersensitivity to acid in
awake rats
Kenneth Lamb, Yu-Ming Kang, Gerald F. Gebhart, Klaus Bielefeldt
Background & Aims: Changes in visceral sensation contribute
to the development of dyspepsia. Nonhuman models have previously
focused on responses to mechanical stimulation. We studied the
response to acid stimulation in the normal and inflamed stomach
in rats. Methods: A balloon and gastrostomy catheter were
implanted into the stomach. Electromyographic responses to gastric
balloon distention or acid administration through the gastrostomy
were recorded from the acromiotrapezius muscle. To characterize
chemonociceptive pathways, 0.75 mL HCl (0.050.3 N) or saline
were given intragastrically in controls and animals after vagotomy,
splanchnic nerve resection, or chemical denervation with capsaicin.
The effect of inflammation was examined after induction of mild
diffuse gastritis using iodoacetamide or creating gastric ulcers
by injecting 60% acetic acid for 45 seconds into a clamped area
of the stomach. Results: Visceromotor electromyographic
responses increased within 2 minutes after HCl administration
(0.15 and 0.3 mol/L) but not saline or lower acid concentrations.
Vagotomy and pretreatment with capsaicin but not splanchnic nerve
resection abolished this response. Prior acid administration
did not acutely sensitize animals to subsequent gastric distention.
Gastritis and gastric ulcers enhanced the visceromotor responses
to intragastric acid. Conclusions: In awake rats, visceromotor
responses to intragastric acid are quantifiable, reliable, and
reproducible. Aversive responses to acute noxious chemical stimuli
primarily require vagal but not spinal sensory pathways. Injury-induced
sensitization to intragastric acid administration is consistent
with a potential role of chemical stimulation in triggering dyspeptic
symptoms.
Intratracheal IL-13 induces eosinophilic esophagitis by an
IL-5, eotaxin-1, and STAT6-dependent mechanism
Anil Mishra, Marc E. Rothenberg
Background & Aims: Eosinophil infiltration into the
esophagus occurs in a wide range of diseases; however, the underlying
pathophysiologic mechanisms involved are largely unknown. We
have previously reported that simultaneous delivery of allergen
to the lung and gastrointestinal tract induces experimental eosinophilic
esophagitis (EE). We aimed to determine whether delivery of a
Th2 cytokine (interleukin [IL]-13) to the lung was sufficient
for induction of EE. Methods: IL-13 was delivered intratracheally
to wild-type, signal transducer and activator of transcription
(STAT) 6, eotaxin-1, or IL-5-deficient mice. Eosinophil levels
and 5´-bromodeoxyuridine (BrdU) incorporation were examined
by immunohistochemical staining. Results: Intratracheal
delivery of IL-13 induced dose-dependent eosinophil accumulation
in the esophagus (but not the stomach). In addition, intratracheal
IL-13 induced esophageal epithelial hyperplasia. The ability
of IL-13 to induce EE was abolished in STAT6-deficient mice.
IL-13-induced EE was nearly completely ablated in IL-5-deficient
mice (37.3 ± 11.6 vs. 3.3 ± 3.2 eosinophils/mm2
in wild-type and IL-5-deficient mice, respectively). Additionally,
IL-13-induced EE was significantly diminished in eotaxin-1-deficient
mice (48.7 ± 10.3 vs. 14.1 ± 12.5 eosinophils/mm2
in wild-type and eotaxin-1-deficient mice, respectively). Conclusions:
IL-13 delivery to the lung induces EE by an IL-5, eotaxin-1,
and STAT6-dependent mechanism. These results further establish
an intimate connection between respiratory and esophageal inflammation.
Genetic background modifies intestinal pseudo-obstruction
and the expression of a reporter gene in Hox11L1/
mice
Melissa A. Parisi, Audrey E. Baldessari, Malissa H.K. Iida, Christine
M. Clarke, Barbara Doggett, Senji Shirasawa, Raj P. Kapur
Background & Aims: The transcription factor Hox11L1
is expressed by enteric neurons. Two groups mutated murine Hox11L1,
and reported lethal intestinal pseudo-obstruction and colonic
hyperganglionosis in many, but not all, homozygous null mutants.
We investigated the regulation of Hox11L1 and factors
that influence the penetrance of pseudo-obstruction in Hox11L1-null
mice. Methods: Expression of -galactosidase (lacZ),
under control of putative Hox11L1 regulatory sequences,
was assessed in transgenic mice wild-type, heterozygous, and
null for native Hox11L1. Transgene expression and signs
of pseudo-obstruction were compared in null mice with different
genetic backgrounds. Results: In enteric neurons and other
parts of the nervous system, the transgene was expressed in a
pattern consistent with native Hox11L1. Enteric -galactosidase
activity initiated in the proximal small intestine and spread
cranially and caudally in a subset of postmitotic enteric neurons.
Hox11L1-lacZ transgene expression persisted in Hox11L1-null
animals, suggesting that Hox11L1 is not required cell autonomously
for neuronal survival. Genetic background dramatically affected
the phenotypes of Hox11L1-null animals, with complete
penetrance of severe proximal colonic distention on a predominantly
C57BL/6J (B6) background and very low penetrance of dysmotility
on a 129SvJ (129) background. Coincidently, Hox11L1-lacZ
expression by most enteric neurons, but not CNS neurons, was
lost on a 129 background. Conclusions: Cis-acting, 5´
regulatory elements are sufficient to regulate site-specific
expression of Hox11L1 in vivo. Expression of the transgene
by enteric neurons and penetrance of pseudo-obstruction in Hox11L1-null
animals are influenced by one or more modifier genes, counterparts
of which may play a similar role in human disease.
Basic-liver, Pancreas, and Biliary Tract
Analysis of pulmonary heme oxygenase-1 and nitric oxide
synthase alterations in experimental hepatopulmonary syndrome
Junlan Zhang, Yiqun Ling, Bao Luo, Liping Tang, Stefan W. Ryter,
Cecil R. StockarD, Willam E. Grizzle, Michael B. Fallon
Background & Aims: Cirrhosis and portal hypertension
due to chronic common bile duct ligation reproduce the features
of human hepatopulmonary syndrome, whereas portal hypertension
alone due to partial portal vein ligation does not. Nitric oxide
contributes to experimental hepatopulmonary syndrome, but the
nitric oxide synthase forms involved remain controversial. Recently,
increased pulmonary heme oxygenase-1 expression and carbon monoxide
production have also been found after common bile duct ligation.
Our aim was to explore the role of the heme oxygenase-1/carbon
monoxide pathway in the pathogenesis of experimental hepatopulmonary
syndrome. Methods: Pulmonary heme oxygenase-1 expression
and distribution were assessed in sham; 3-week partial portal
vein ligation; and 1-, 2-, 3-, 4-, and 5-week common bile duct
ligation animals by Northern, Western and immunohistochemical
analysis relative to endothelial and inducible nitric oxide synthase
levels and to hepatopulmonary syndrome development. In vivo heme
oxygenase enzyme inhibition with tin protoporphyrin IX in common
bile duct ligation animals was used to define effects on intrapulmonary
vasodilatation and arterial blood gases. Results: Heme
oxygenase-1 expression in pulmonary intravascular monocytes/macrophages
and arterial carboxyhemoglobin levels increased progressively
from 3 to 5 weeks after common bile duct ligation relative to
controls (5-week protein levels were 15.94 ± 1.75-fold
those of sham animals; P < 0.001). Inducible nitric
oxide synthase increased transiently in pulmonary intravascular
monocytes/macrophages in 3-week common bile duct ligation animals,
whereas pulmonary microvascular endothelial nitric oxide synthase
increases began at 2 weeks and correlated with the onset of hepatopulmonary
syndrome. Tin protoporphyrin treatment normalized carboxyhemoglobin
and improved arterial blood gases and intrapulmonary vasodilatation,
reflecting partial reversal of hepatopulmonary syndrome. Conclusions:
The heme oxygenase-1/carbon monoxide system is an important contributor
to the progression of experimental hepatopulmonary syndrome in
addition to alterations in the endothelial nitric oxide synthase/nitric
oxide pathway.
Mesenteric vasoconstriction triggers nitric oxide overproduction
in the superior mesenteric artery of portal hypertensive rats
Ming-Hung Tsai, Yasuko Iwakiri, Gregory Cadelina, William C.
Sessa, Roberto J. Groszmann
Background & Aims: Vasoconstriction of the superior
mesenteric artery (SMA) is the earliest hemodynamic event occurring
after partial portal vein ligation (PVL). We tested the hypothesis
that this early vasoconstriction of the SMA may initiate eNOS
up-regulation in PVL. Methods: Portal hypertension with
or without mesenteric vasoconstriction was induced by differentially
calibrated stenosis of the portal vein (PVL-20G and PVL-18G,
respectively). In a separate group of rats, mesenteric vasoconstriction
was achieved by renal artery ligation. Sham-operated rats were
used as controls. Effects of vasoconstriction of the SMA in PVL
and RAL rats were evaluated by measuring perfusion pressure changes
in isolated SMA beds in response to methoxamine, nitric oxide
synthase activity, and eNOS protein expression. Mean arterial
pressure, portal pressure, and SMA blood flow were measured by
catheterization and Doppler flowmetry. SMA vascular resistance
was calculated from arterial pressure, portal pressure, and SMA
flow. Results: There was a significant increase in SMA
vascular resistance in PVL-20G (2.33 ± 0.13 vs. 1.22 ±
0.03 mm Hg/% flow; P < 0.05) and RAL (2.32 ±
0.18 vs. 1.18 ± 0.02 mm Hg/% flow; P < 0.05)
but not in PVL-18G, showing mesenteric vasoconstriction in both
PVL-20G and RAL groups. The mesenteric vasculature of PVL-20G
and RAL animals showed hyporeactivity to methoxamine (P
< 0.01). Whereas both PVL groups were portal hypertensive
(P < 0.01), RAL rats were not. The SMA hyporeactivity
of PVL-20G and RAL rats was corrected by NG-monomethyl-L-arginine,
and nitric oxide synthase enzyme activity was significantly higher
in PVL-20G and RAL rats (P < 0.05). Conclusions:
Mesenteric arterial vasoconstriction plays a triggering role
in up-regulation of eNOS catalytic activity in the SMA of portal
hypertensive rats.
Case Report
Familia liver adenomatosis associated with hepatocyte nuclear
factor 1 inactivation
Yannick Bacq, Emmanuel Jacquemin, Charles Balabaud, Emmanuelle
Jeannot, Beatrice Scotto, Sophie Branchereau, Christophe Laurent,
Pascal Bourlier, Daniele Pariente, Anne De Muret, Monique Fabre,
Paulette Bioulac-Sage, Jessica Zucman-Rossi
Background & Aims: Germline mutations in hepatocyte
nuclear factor 1 (TCF1/HNF-1) are associated with maturity-onset
diabetes of the young type 3 (MODY3), and somatic biallelic inactivations
of the gene are found in hepatocellular adenomas and liver adenomatosis.
This study investigated cosegregation of HNF-1 germline mutations
with diabetes and liver adenomatosis in 2 families. Methods:
Two unrelated patients with liver adenomatosis and harboring
HNF-1 germline and somatic mutations were studied. Subsequently,
we screened 9 relatives in the 2 independent families for diabetes,
hepatocellular adenomas, and HNF-1 germline mutations. Results:
In family A, a father and his son presented with an intraperitoneal
hemorrhagic rupture of a liver adenomatosis without diabetes.
A heterozygous R229X germline mutation was identified in HNF-1
in the father and his son and also in his second 27-year-old
son without hepatocellular adenomas. In family B, a diagnosis
of liver adenomatosis was made fortuitously in a 14-year-old
girl. A heterozygous G55fsX57 germline mutation in HNF-1 was
identified in this patient, her diabetic father, and her 2 sisters.
Systematic exploration showed liver adenomatosis in the 2 sisters.
Somatic inactivation of the second HNF-1 allele was found in
liver tumors in both families. Conclusions: This study
describes familial liver adenomatosis and shows the association
with germline HNF-1 mutations in adults and children. It also
highlights the importance of screening for hepatocellular adenomas,
diabetes, and HNF-1 germline mutations in relatives of patients
with liver adenomatosis. Finally, prevalence of liver adenomatosis
remains to be evaluated in MODY3 subjects.
Pediatric-onset primary biliary cirrhosis
Yaser Dahlan, Leslie Smith, Doug Simmonds, Larry Douglas Jewell,
Ian Wanless, E. Jenny Heathcote, Vincent Gordon Bain
Unlike other autoimmune liver diseases, primary biliary cirrhosis
(PBC) has not been reported in childhood. We report 2 cases of
PBC diagnosed at 16 and 15 years of age, respectively. The first
girl was noted to have increased liver enzyme levels at 16 years
of age. Antimitochondrial antibody (AMA) was strongly positive,
and serum quantitative immunoglobulin M level was 8.26 g/L (normal,
0.63 g/L). A liver biopsy specimen showed stage II PBC.
Despite treatment with ursodeoxycholic acid, she developed progressive
cholestasis, intractable pruritus, and a significant sensory
neuropathy and weight loss eventually requiring liver transplantation.
Her mother had PBC/autoimmune overlap syndrome and underwent
successful liver transplantation at 34 years of age. The second
girl had persistently elevated liver enzyme levels following
cholecystectomy at 15 years of age for symptomatic cholelithiasis.
Endoscopic retrograde cholangiopancreatography showed no abnormalities.
AMA was positive at 1:160, and serum quantitative immunoglobulin
was 6.96 g/L. A liver biopsy specimen showed stage II PBC, and
her liver enzyme levels almost normalized after starting treatment
with ursodeoxycholic acid. In conclusion, we present 2 liver
biopsy-confirmed cases of pediatric-onset AMA-positive PBC. With
increased awareness of early-onset PBC, further pediatric cases
may be discovered.
Special Report and Review
Recent insights on the mechanisms of liver preconditioning
Rita Carini, Emanuele Albano
Ischemia/reperfusion is the main cause of hepatic damage consequent
to temporary clamping of the hepatoduodenal ligament during liver
surgery as well as graft failure after liver transplantation.
In recent years, a number of animal studies have shown that pre-exposure
of the liver to transient ischemia, hyperthermia, or mild oxidative
stress increases the tolerance to reperfusion injury, a phenomenon
known as hepatic preconditioning. The development of hepatic
preconditioning can be differentiated into 2 phases. An immediate
phase (early preconditioning) occurs within minutes and involves
the direct modulation of energy supplies, pH regulation, Na+
and Ca2+ homeostasis, and caspase activation. The subsequent
phase (late preconditioning) begins 1224 hours after the
stimulus and requires the synthesis of multiple stress-response
proteins, including heat shock proteins HSP70, HSP27, and HSP32/heme
oxygenase 1. Hepatic preconditioning is not limited to parenchymal
cells but ameliorates sinusoidal perfusion, prevents postischemic
neutrophil infiltration, and decreases the production of proinflammatory
cytokines by Kupffer cells. This latter effect is important in
improving systemic disorders associated with hepatic ischemia/reperfusion.
The signals triggering hepatic preconditioning have been partially
characterized, showing that adenosine, nitric oxide, and reactive
oxygen species can activate multiple protein kinase cascades
involving, among others, protein kinase C and p38 mitogen-activated
protein kinase. These observations, along with preliminary studies
in humans, give a rationale to perform clinical trials aimed
at verifying the possible application of hepatic preconditioning
in preventing ischemia/reperfusion injury during liver surgery.
Ghrelin for the gastroenterologist: History and potential
Charles D.R. Murray, Michael A. Kamm, Stephen R. Bloom, Anton
V. Emmanuel
Ghrelin, a novel 28-amino acid orexigenic peptide discovered
in 1999, has given us further insights into the control of energy
homeostasis and growth hormone secretion. As a natural endogenous
ligand of the growth hormone secretagogue receptor, it potently
stimulates growth hormone release but is also implicated in many
other homeostatic mechanisms. Released from the stomach, it stimulates
lactotroph and corticotroph secretion, increases appetite and
adiposity, has beneficial hemodynamic effects, has prokinetic
and gastric acid secretory functions in the stomach, and may
even be implicated in sleep. As advances in the understanding
of appetite and obesity are made, it is timely to review the
possibly central role of ghrelin in these physiological and pathophysiological
states. This review will discuss the recent literature concerning
this exciting novel neuropeptide and discuss the possible therapeutic
possibilities it may open up to us.
Copyright © 2001-2003 European Association
for the Study of the Liver. All rights reserved.
Table of Contents for Volume
39, Issue 5, November 2003
Biliary Tract and Cholestasis
Hemopoietic progenitor cells and bone marrow stromal cells
in patients with autoimmune hepatitis type 1 and primary biliary
cirrhosis
Despina S. Kyriakou et al.
Background/Aims: Autologous hematopoietic stem cell transplantation
has been used in severe cases of autoimmunity. We investigated
whether hemopoietic progenitor cells and/or bone marrow (BM)
microenvironment are affected in autoimmune hepatitis type-1
(AIH-1) and primary biliary cirrhosis (PBC).Methods: We
studied 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls
(CC) and ten healthy controls (HC). Flow cytometry, expansion
cultures, long-term BM cultures and clonogenic progenitor cell
assays were used. Stromal cell function was assessed in long-term
BM cultures recharged with normal CD34+ cells.Results:
AIH-1 had increased CD34+, CD34+/CD38+ and CD34+/CD38 cells compared
to all groups (P<0.001). PBC had lower progenitor cells
compared to controls (P<0.005). No differences were
found between CC and HC. Committed progenitor cells in non-adherent
cell fraction were increased in AIH-1 (P<0.05) but
decreased in PBC compared to controls (P<0.05). Granulocyte-macrophage
colony forming units (CFU) and erythroid-burst CFU were increased
in AIH-1 compared to all groups (P<0.001). PBC had
these CFUs decreased compared to controls (P<0.005).
Stromal cells failed to support normal hemopoiesis in PBC.Conclusions:
We demonstrated for the first time that AIH-1 had increased hemopoietic
progenitor cells and normal stromal function. In PBC, progenitor
cells and BM microenvironment were defective. Further studies
will determine the significance of these novel findings.
Keywords: Stem cell transplantation; Autoimmunity; Autoimmune
liver diseases; Autoimmune hepatitis; Primary biliary cirrhosis;
Hemopoietic progenitor cells; Stromal cells
Hepatic artery malformations associated with a primary
defect in intrahepatic bile duct development
Frédéric Clotman et al.
Background/Aims: The portal tracts contain bile
ducts associated with branches of the portal vein and of the
hepatic artery. Hepatic artery malformations are found in diseases
in which fetal biliary structures persist after birth (ductal
plate malformations). Here we investigated how hepatic artery
malformations relate to abnormal bile duct development.Methods:
Hepatic artery and biliary development was analyzed in fetuses
with Jeune syndrome or Meckel syndrome, which show ductal plate
malformations. We also analyzed hepatic artery development in
transgenic mice which exhibit biliary anomalies following inactivation
of the genes for hepatocyte nuclear factor (HNF)-6 or HNF-1,
two transcription factors expressed in biliary cells, but not
in arteries.Results: We show that arterial anomalies
occurred in fetuses with Jeune syndrome or Meckel syndrome. We
provide the first description of hepatic artery branch development
in the mouse and show that inactivation of the Hnf6 or
Hnf1 gene results in anomalies of the hepatic artery branches.
In the transgenic mice and in the human syndromes, the biliary
anomalies preceded the arterial anomalies.Conclusions:
A primary defect in biliary epithelial cells is associated with
hepatic artery malformations in mice. Our data provide a model
to interpret and study hepatic artery anomalies in humans.
Keywords: Biliary epithelial cell; Hepatic artery; Ductal
plate malformation; Jeune syndrome; Meckel syndrome; HNF-6; HNF-1
Changes in the expression and localization of hepatocellular
transporters and radixin in primary biliary cirrhosis
Hideyuki Kojima et al.
Background/Aims: Expression and localization of
human hepatocellular transporters and of radixin, cross-linking
actin with some membrane transporters, may change in cholestatic
liver diseases. Methods: We investigated the uptake
transporters OATP2 (SLC21A6), OATP8 (SLC21A8), and NTCP (SLC10A1),
the export pumps MRP2 (ABCC2), MRP3 (ABCC3), MRP6 (ABCC6), and
P-glycoproteins (ABCB1, ABCB4, ABCB11), and radixin, in non-icteric
primary biliary cirrhosis (PBC stages I-III) and control human
liver needle-biopsies using immunofluorescence microscopy and
semi-quantitative RT-PCR. Results: Expression and
localization of all transporters were unchanged in PBC I-II.
Immunostaining intensities of uptake transporters decreased in
PBC III with a concomitant decrease in mRNA levels. Immunostaining
intensities and mRNA levels of export pumps were similar in controls
and PBC I-III, however, irregular MRP2 immunostaining suggested
redistribution of MRP2 into intracellular structures in PBC III.
Areas of irregular MRP2 immunostaining showed largely reduced
radixin immunostaining, whereas normal hepatocytes had MRP2 and
radixin confined to the canalicular membrane. Disrupted localization
of radixin and MRP2 supports the concept that radixin contributes
to the canalicular localization of MRP2. Conclusions:
Down-regulation of uptake transporters may contribute to the
impaired hepatobiliary elimination in advanced PBC, and partially
altered localization of MRP2 may reflect the onset of changes
leading to icteric PBC.
Keywords: Organic anion transporters; Radixin; Multidrug
resistance protein; Primary biliary cirrhosis; Immunofluorescence;
RT-PCR; Cholestatic liver disease
Cell Biology, Metabolism and Transport
The ubiquitously expressed MURR1 protein is absent in canine
copper toxicosis
Adriana E.M. Klomp, Bart van de Sluis, Leo W.J. Klomp and Cisca
Wijmenga
Background/Aims: Copper toxicosis (CT) in Bedlington terriers
is an autosomal recessive disorder characterized by massive lysosomal
copper accumulation in livers of affected dogs, and a defect
in the biliary excretion of this metal. We propose that MURR1,
the gene defective in canine CT, has a role in the regulation
of copper excretion into bile during copper overload. Methods:
Polyclonal antibodies raised against full-length recombinant
human MURR1 were used for immunoblot analysis and indirect immunofluorescence
studies. Results: Using Western blot analysis, these antibodies
abundantly detected MURR1 as a 23 kDa protein in liver extracts
of mice and dogs, but MURR1 was undetectable in the livers of
affected Bedlington terriers. MURR1 was also detected in different
tissues and cell lines; in cell lines the protein was found both
in cytosol and membrane preparations. Consistent with this observation,
indirect immunofluorescence staining revealed that in some cells
MURR1 was associated with a vesicular compartment diffusely localized
throughout the cell. Conclusions: The genomic deletion
in MURR1 results in complete absence of MURR1 protein.
Based on the unanticipated subcellular localization, our results
suggest a role for MURR1 in the regulation of vesicular copper
sequestration during copper overload.
Keywords: MURR1; Canine copper toxicosis; Bedlington terrier;
Liver; Copper accumulation; Vesicular compartment; Wilson disease
The role of the iron responsive element in the control
of ferroportin1/IREG1/MTP1 gene expression
Athina Lymboussaki et al.
Background/Aims: MTP1/Ferroportin1/IREG1, the product
of the SLC40A1 gene, is a main iron export protein in
mammals. However, the way this gene is regulated by iron is still
unclear. The aim of this study was to investigate the functional
role of genomic SLC40A1 elements in response to iron.
Methods: Vectors containing either 2.6 kb 5´
flanking region or deletion constructs, including one devoid
of an iron responsive element (SLC40A1-IRE-Luc), were
analyzed by luciferase reporter gene in transfected HepG2, CaCO2
and U937 cells. Expression of iron genes and activity of the
iron regulatory protein were also studied. Results:
Iron increased and desferrioxamine decreased luciferase activity
in all the cell types using both the full-length construct and
the promoter deletion constructs, in the absence of changes in
SLC40A1 or luciferase mRNA levels. To test the role of
the SLC40A1 5´ untranslated region, we first demonstrated
that wild type and not SLC40A1-IRE-Luc could bind iron
regulatory protein. Then, in cells transfected with SLC40A1-IRE-Luc,
we found that, in spite of iron regulatory protein activation,
the response to iron manipulation was lost. Conclusions:
We demonstrate that the iron responsive element in the SLC40A1
gene is functional and that it controls gene expression through
the cytoplasmic iron regulatory protein system.
Keywords: Iron; Hemochromatosis; Gene promoter; Metabolism
Rapid formation of hepatic organoid in collagen sponge
by rat small hepatocytes and hepatic nonparenchymal cells
Keisuke Harada et al.
Background/Aims: Hybrid bioartificial liver devices
supporting a large mass of metabolically active hepatocytes are
thought to be necessary for the successful treatment of patients
with severe acute liver failure. However, it is very difficult
to obtain cells with both growth activity and differentiated
functions. Rat small hepatocytes (SHs), which are hepatic progenitor
cells, can differentiate into mature hepatocytes and reconstruct
a hepatic organoid by interacting with hepatic nonparenchymal
cells (NPCs). Methods: Colonies of SHs were collected
and replated on a collagen sponge. Hepatic functions were examined
by ELISA, immunoblotting, and Northern blotting. Cells in the
sponge were characterized by immunocytochemistry and transmission
electron microscopy. Urea synthesis was measured and metabolization
of fluorescein diacetate was examined. Results:
SHs could proliferate and expand to form a hepatic organoid in
the sponge. Albumin secretion and other hepatic protein production
of the cells in the sponge increased with time in culture and
the amounts were much larger than for those obtained from cells
grown on dishes. Morphologically and functionally differentiated
hepatocytes were observed and some CK19-positive cells formed
duct-like structures within the sponge. Excretion of fluorescein
was observed in bile canaliculi. Conclusions: Hepatic
organoids can be rapidly reconstructed in a collagen sponge by
rat SHs and NPCs.
Keywords: Rat small hepatocytes; Maturation; Duct-like structure;
Collagen sponge; Scaffold
Cirrhosis and its Complications
Intrapulmonary vascular dilatation and nitric oxide in
hypoxemic rats with chronic bile duct ligation
Xue-Jun Zhang et al.
Background/Aims: Nitric oxide (NO) has been suggested
as the major cause of pulmonary vascular dilatation and hypoxemia
in hepatopulmonary syndrome (HPS). The aim of this study was
to assess the effect of NO on arterial oxygenation in rats with
common bile duct ligation (CBDL rats), a model of HPS. Methods:
Arterial blood gases were measured in 44 CBDL rats and 44 Sham
rats under unrestrained conditions. Intrapulmonary shunting was
assessed with 141Ce-labeled microspheres (15-µm diameter)
and serum nitrate/nitrite levels were measured by HPLC. The effect
of NOS inhibition on A-aDO2 was studied using L-NAME. Results:
A decrease of PaO2 below 82.7 mmHg (the mean value2 in Sham rats)
was seen in 43% of CBDL rats. Intrapulmonary shunting was greater
in CBDL rats than in Sham rats (P<0.001). A correlation
between the extent of shunting and A-aDO2 was found in all animals
studied (r=0.89, P<0.001, n=16). Serum
levels of nitrate/nitrite increased significantly across the
lungs, and the increase was significantly correlated with A-aDO2
in the total population of animals studied. Administration of
L-NAME to CBDL rats achieved a significant improvement of A-aDO2.
Conclusions: These results suggest that pulmonary
vascular dilatation due to NO leads to hypoxemia in CBDL rats.
Inflammation and Fibrosis
Inhibitory effect of a soluble transforming growth factor
type II receptor on the activation of rat hepatic stellate cells
in primary culture
Xuezhi Cui et al.
Background/Aims: Oxidative stress, including the generation
of reactive oxygen species (ROS) that acts as a signaling mediator
for transforming growth factor (TGF)-, plays a key role in hepatic
fibrosis. Hepatic stellate cells (HSCs) produce and respond to
TGF- in an autocrine manner with increased collagen expression.
It has previously been reported that the adenovirus-mediated
overexpression of a soluble receptor against the extracellular
domain of the TGF- type II receptor prevents hepatofibrogenesis
in vivo, although its inhibitory role and mechanism in HSC activation
remains to be elucidated. Methods: In this study, we report
on an examination of the actual role of TGF- inhibition on oxidative
stress and the activation of cultured rat HSCs, using the adenovirus-mediated
soluble TGF- type II receptor. Results: This soluble receptor
secreted from the adenovirus-infected cells binds to TGF-. Infection
of HSCs with this adenovirus attenuated intracellular levels
of TGF-1 mRNA and protein, NADH oxidative activity, ROS generation
and lipid peroxidation, and prevented HSC activation. Conclusions:
These findings suggest that this adenovirus-mediated soluble
TGF- receptor may lead to an interruption of the TGF- autocrine
loop in activated HSC, in part, by inhibiting oxidative stress.
Keywords: Hepatic stellate cell; Soluble transforming growth
factor- type II receptor; Oxidative stress; Reactive oxygen species;
Hepatic fibrosis
Three-dimensional reconstruction of hepatic bridging fibrosis
in chronic hepatitis C viral infection
Alan Hoofring, John Boitnott and Michael Torbenson
Background/Aims: Fibrosis in chronic hepatitis
C infection begins in portal tracts, and can progress to `bridging
fibrosis' and eventually lead to cirrhosis. All current fibrosis
staging systems are based on these three conceptual fibrosis
stages (portal fibrosis, bridging fibrosis, cirrhosis) and vary
only in how these stages are further subdivided. Despite the
importance of bridging fibrosis, little is known about its three-dimensional
characteristics. Methods: Foci of bridging fibrosis
were digitally reconstructed in three dimensions on two separate
liver specimens with chronic hepatitis C viral infection. The
amount of portal fibrosis was then correlated with bile duct
diameter on trichrome-stained sections. Results:
After three-dimensional reconstruction, the bridges were seen
to represent webs or membranes of fibrosis located at branch
points of the portal tracts that extended between two portal
branches, much like the webbing between the thumb and forefinger
in a baseball-mitt. Direct measurements indicated that early
portal fibrosis was associated with portal tracts containing
bile ducts of approximately 18-19 µm diameter. Conclusions:
Fibrosis bridges are located at branch points of portal tracts
and are composed of three-dimensional webs or membranes that
extend between portal tracts; when viewed as two dimensions on
a glass slide, a `bridge' of fibrosis is seen.
Keywords: Chronic hepatitis C; Liver; Fibrosis; Bridging
A blocking peptide for transforming growth factor-1 activation
prevents hepatic fibrosis in vivo
Hiroki Kondou et al.
Background/Aims: Thrombospondin-1 is a major activator
of transforming growth factor-1 (TGF-1), and a peptide derived
from the latency-associated peptide, Leu-Ser-Lys-Leu (LSKL),
inhibits the activation of TGF-1. In this study, the effects
of LSKL on the hepatocyte damage and fibrogenesis in dimethylnitrosamine
(DMN)-induced rat liver fibrosis were examined. Methods:
Animals were given an intraperitoneal (i.p.) injection of DMN
or saline three times per week for 4 weeks, and treated with
LSKL, a control peptide, or saline i.p. daily. Results:
Liver atrophy caused by DMN-injection was significantly inhibited
in the DMN+LSKL group. The degrees of necrosis/degeneration and
fibrosis scores were significantly lower in the DMN+LSKL group
than in the control groups. The hydroxyproline content was significantly
higher in the control groups than in the DMN+LSKL group. The
amount of active TGF-1 was less in the DMN+LSKL group than in
the control groups, and the active/total TGF-1 ratio in the DMN+LSKL
group was suppressed in the control groups. Phosphorylation of
Smad 2 in the liver was significantly decreased in the DMN+LSKL
group. Conclusions: The LSKL peptide prevented the progression
of hepatic damage and fibrosis through the inhibition of TGF-1
activation and its signal transduction in vivo.
Keywords: Hepatic fibrosis; Transforming growth factor-1;
Thrombospondin-1; Latency-associated peptide; Smad 2
Carbenoxolone inhibits DNA synthesis and collagen gene
expression in rat hepatic stellate cells in culture
Naoki Uyama et al.
Background/Aims: This study using primary-cultured rat
hepatic stellate cells (HSCs) was aimed to reveal the effect
of carbenoxolone and the other gap-junction blockers on the proliferation
and activation of HSCs. Methods: HSC morphology was microscopically
evaluated. DNA synthesis was determined by [3H]thymidine incorporation.
Expression of HSC activation markers and cell cycle-related proteins
was evaluated by Western blot. Collagen 1(I) mRNA expression
was evaluated by quantitative reverse transcription polymerase
chain reaction. Results: Carbenoxolone triggered the morphological
change of activated HSCs without inducing apoptosis. Culture-induced
DNA synthesis was suppressed to 22.6 and 8.51%, respectively,
by 40 and 80 µM carbenoxolone. The other gap-junction blockers
failed to affect the morphology and the DNA synthesis of activated
HSCs. Carbenoxolone decreased the expression of cyclins D1/2
and cyclin-dependent kinases 4/6. Platelet-derived growth factor
(PDGF)-BB-elicited DNA synthesis was reduced to 45.6 and 3.27%,
respectively, by 40 and 80 µM carbenoxolone. Phosphorylation
of c-Raf, MEK and mitogen-activated protein kinase, but not PDGF
receptor , under PDGF-BB stimulation was attenuated by carbenoxolone.
Collagen 1(I) mRNA expression was significantly reduced. In addition,
carbenoxolone suppressed the activation process of quiescent
HSCs. Conclusions: Carbenoxolone reduced the DNA synthesis
and the expression of collagen 1(I) mRNA in activated HSCs independently
of its pharmacological action as gap-junction blocker.
Keywords: Platelet-derived growth factor; Mitogen-activated
protein kinase pathway; Gap junction; DNA synthesis; Collagen
1(I) mRNA
Lipid peroxidation, stellate cell activation and hepatic
fibrogenesis in a rat model of chronic steatohepatitis
Jacob George et al.
Background/Aims: We explored the involvement of
cell types, cytokines and lipid peroxidation in a rat dietary
model of fibrosing steatohepatitis. Methods: Male
rats were fed a high fat diet deficient in methionine and choline
(MCD) for up to 17 weeks. Whole liver, hepatocytes and non-parenchymal
cells were analysed for reduced glutathione (GSH) levels, products
of lipid peroxidation (thiobarbituric acid reactive substances,
TBARS), liver injury, and fibrosis. Results: MCD
diet-fed rats developed hepatic steatosis at week 2 and focal
necroinflammatory change by week 5, while pericellular fibrosis
evolved and progressed between weeks 12 and 17. Collagen 1(1)
gene expression was upregulated by week 5 and increased fivefold
by week 17. Stellate cells were the unique source of collagen
gene expression. TIMP-1 and -2 were increased at week 12. Livers
of MCD diet-fed rats exhibited lowered levels of GSH and elevated
TBARS. Hepatocytes were the source of lipid peroxidation, and
mRNA levels for TGF1 were increased only in this cell type. Conclusions:
The MCD model of `fibrosing steatohepatitis' replicates the histologic
features of human steatohepatitis, and the sequence of steatosis,
inflammatory cell injury and fibrogenesis. The temporal sequence
is consistent with a concept for involvement of oxidative injury
in inflammatory recruitment and pathogenesis of hepatic fibrogenesis.
Keywords: Steatohepatitis; Hepatic fibrosis; Lipid peroxidation;
Collagen; Stellate cells; Hepatocytes; Transforming growth factor-beta
Liver Cell Injury and Liver Failure
Mangafodipir prevents liver injury induced by acetaminophen
in the mouse
Sassia Bedda et al.
Background/Aims: Acute liver failure (ALF), characterized
by massive hepatocyte necrosis, is often caused by drug poisoning,
particularly with acetaminophen (APAP). Hepatocyte necrosis is
consecutive to glutathione depletion by NAPQI, a metabolite of
APAP, and to mitochondrial damages caused by reactive oxygen
species (ROS) overproduction. Considering the structure of Mangafodipir,
a contrast agent currently used in magnetic resonance imaging
of the liver, we hypothesized that this molecule could exert
an antioxidant activity and be possibly used as a treatment of
APAP-induced ALF. Methods/Results: Mangafodipir is endowed
with superoxide dismutase, catalase, and glutathione reductase
activities. It can inhibit ROS production by hepatocytes in culture,
and protect those cells from oxidative stresses induced by exposure
to xanthine oxidase, H2O2, or UV light. Moreover, preventive
or curative administration of Mangafodipir to mice with APAP-induced
ALF significantly increases survival rates, and abrogates aspartate
aminotransferase elevation and histological damage. Conclusions:
Those data point out the potential interest of Mangafodipir in
the treatment of toxic ALF in humans.
Keywords: Mangafodipir; Superoxide dismutase mimic; Acute
liver failure; Mouse Abbreviations: ALF, liver acute failure;
ASAT, aspartate aminotransferase; AU, arbitrary units; APAP,
acetaminophen; CuDIPS, copper[II]diisopropylsalicylate; H2-DCFDA,
2´,7´-dichlorodihydrofluorescein diacetate; MCB,
monochlorobimane; GR, glutathione reductase; GSH, reduced glutathione;
i.p., intraperitoneal(ly); MnDPDP, Mangafodipir trisodium; MnTBAP,
Manganese III tetrakis (5,10,15,20 benzoic acid); NAC, N-acetyl-cysteine;
NS, non-specific; pNA, paranitroaniline; ROS, reactive oxygen
species; SOD, superoxide dismutase
Caspase-dependent apoptosis in fulminant hepatic failure
induced by herpes simplex virus in mice
Koji Hashimoto, Hiroko Minagawa and Yusuke Yanagi
Background/Aims: Herpes simplex virus (HSV) is known to
induce fulminant hepatic failure. The aim of this study is to
clarify the molecular basis of liver injury in HSV-induced hepatitis.
Methods: Immunocompetent mice were inoculated with HSV
or ultraviolet-inactivated HSV (UV-HSV) intravenously. Extent
of liver injury was evaluated by changes of serum enzymes and
histopathology. Apoptosis was assessed by DNA fragmentation and
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL). Immunohistochemistry for caspase-3 and Fas
ligand was performed. Reverse transcriptase-polymerase chain
reaction for Fas ligand was also performed. Results: All
mice died of rapid and massive liver cell death after HSV infection,
whereas no change was observed in mice after UV-HSV inoculation.
The extent of viral replication and DNA fragmentation correlated
well with the severity of liver injury. Caspase-3 was activated
in the liver after HSV infection, but not UV-HSV. Positive reaction
for TUNEL was observed not only in HSV-antigen positive cells
but also in HSV-antigen negative cells. Fas ligand was induced
in the liver infected with HSV, but not with UV-HSV. Conclusions:
Caspase-dependent apoptosis is involved in HSV-associated liver
injury. Not only viral replication but also non-viral factor
such as Fas ligand may facilitate rapid development of this disease.
Keywords: Herpes simplex virus; Fulminant hepatic failure;
Apoptosis; Caspase; Fas ligand
Liver Growth and Cancer
Osteoactivin expressed during cirrhosis development in
rats fed a choline-deficient, L-amino acid-defined diet, accelerates
motility of hepatoma cells
Masaaki Onaga et al.
Background/Aims: Hepatocellular carcinoma (HCC) is closely
associated with chronic liver diseases, particularly cirrhosis.
However, the genes involved in hepatocarcinogenesis in the context
of developing cirrhosis remain unknown. This study aims to identify
genes associated with early cirrhosis-associated hepatocarcinogenesis.
Methods: We examined genes differentially expressed between
the livers of normal rats and rats fed a choline-deficient, L-amino
acid-defined (CDAA) diet using suppression subtractive hybridization.
We examined both the expression in the liver and HCC tissues
of osteoactivin (OA), isolated in this screen, and its effect
on invasiveness and metastasis. Results: OA mRNA was strongly
expressed in the livers of rats fed the CDAA diet for 1-3 months.
Moderate expression was sustained for 18 months. OA overexpression
increased the invasiveness and metastasis of rat hepatoma cells
in vitro and in vivo. In humans, OA expression was not detectable
in normal liver tissues. While OA transcripts were detectable
in cirrhotic nontumorous liver tissues surrounding HCCs, the
majority of HCC tissue samples exhibited higher levels of OA
expression than the surrounding normal tissue. Conclusions:
These results indicate that OA is a novel factor involved in
the progression of HCC via stimulation of tumor invasiveness
and metastatic potential.
Keywords: Hepatocellular carcinoma; Hepatocyte growth factor;
Invasion; Metastasis; Suppression; Subtractive hybridization
Telomere shortening and telomerase reactivation in dysplastic
nodules of human hepatocarcinogenesis
Bong-Kyeong Oh et al.
Backgrounds/Aims: The maintenance of telomere with
telomerase reactivation, vital for carcinogenesis, was studied
in human multistep hepatocarcinogenesis for the characterization
of borderline lesions. Methods: The terminal restriction
fragment length (TRFL) and telomerase activity (TA) were examined
in 3 chronic hepatitis (CH), 10 cirrhosis, 7 large regenerative
nodules (LRNs), 30 low grade dysplastic nodules (LGDNs), 6 high
grade DNs (HGDNs), 3 DNs with hepatocellular carcinoma (HCC)
foci, 11 HCCs, and 4 normal livers by Southern hybridization
and TRAPeze Elisa telomerase detection. Results:The
TRFL and TA showed significant differences between the LGDNs
and HGDNs. Most LGDNs had similar levels of TRFL and TA to those
of the CH, cirrhosis and LRNs, however, 17% of LGDNs revealed
shortening of telomeres up to the levels of HGDNs and 7% of LGDNs
showed high levels of TA. The levels of TRFL and TA in HGDNs
showed no significant differences from those of DNs with HCC
foci and HCCs. Conclusions: The shortening of telomeres
and reactivation of telomerase occur in the DNs during the early
stages of hepatocarcinogenesis, with a significant change in
the transition of LGDNs to HGDNs. The characteristics of HGDNs
are considered to be closer to those of HCCs.
Keywords: Telomere; Telomerase; Dysplastic nodule; Hepatocellular
carcinoma; Large regenerative nodule; Hepatocarcinogenesis; Cirrhosis
Agmatine inhibits the proliferation of rat hepatoma cells
by modulation of polyamine metabolism
Giulia Gardini et al.
Background/Aims: Previous experiments have shown that
agmatine, the product of arginine decarboxylase, is transported
in competition with putrescine into quiescent rat hepatocytes,
where it promotes several effects, including marked decrease
of intracellular polyamines and induction of apoptosis. The primary
aim of the present study was to assess the action of agmatine
on transformed and proliferating hepatic rat cells. Methods:
To assess the effect of agmatine on hepatoma cells, analysis
by flow cytometry, Western blotting, reverse transcription-polymerase
chain reaction, scanning and transmission electron microscopy,
immunofluorescence detection of -actin and -tubulin were performed.
Results: The results showed that agmatine has antiproliferative
effects on the cell lines studied (HTC, JM2, HepG2). Further
experiments were performed on HTC cells. The effect was proportional
to agmatine concentration (in a range between 50 and 500 µM).
It was not correlated with induction of necrosis or apoptosis
and was accompanied by accumulation in G2/M cell cycle phase
and by dramatic modification of cell morphology. Spermidine reversed
these effects, suggesting that the marked decrease of the polyamine
pool is the main target of agmatine. Conclusions: The
results obtained show a relationship between the decrease of
intracellular polyamine content, the rate of cell growth and
the cytoskeleton organization.
Keywords: Agmatine; Polyamine; Hepatoma cell
Viral Hepatitis
Cytochrome P450 2A6: a new hepatic autoantigen in patients
with chronic hepatitis C virus infection
Georgios N. Dalekos et al.
Background/Aims: Cytochromes P4502A6 (CYP2A6) and
P4501A2 (CYP1A2) were described as hepatic autoantigens in the
autoimmune polyglandular syndrome type-1 (APS-1). We evaluated
the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic
diseases in the absence of APS-1. Methods: A radioligand
assay (RLA) based on immunoprecipitation of [35S]-methionine-labeled
CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects
with chronic viral hepatitis (n=185), autoimmune liver
diseases (n=181), autoimmune rheumatic diseases (ARD,
n=31) and healthy (n=33) were tested. Results:
Seven out of 366 patients with liver diseases were anti-CYP2A6
positive. Neither healthy nor ARD patients showed anti-CYP2A6.
One out of 181 patients with autoimmune liver diseases tested
anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6
(P<0.05) was detected with six out of seven patients
positive in the viral hepatitis group. The latter were infected
by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive
sera were positive for anti-LKM-1 by immunofluorescence and for
anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2. Conclusions:
For the first time CYP2A6 is reported as a hepatic autoantigen
in patients with viral hepatitis caused by flaviviruses and in
particular in HCV/anti-LKM-1 positive patients. Multicenter studies
are needed in order to investigate the clinical importance of
this novel finding. This study further supports that anti-CYP2A6
in the absence of flavivirus is rather limited to APS-1.
Keywords: Autoimmune hepatitis; Autoimmunity; Chronic hepatitis;
Cytochrome P450 2A6; Cytochrome P450 1A2; Cytochrome P450 2D6;
Hepatitis C virus; LKM antibody
Lymph node enlargement within the hepatoduodenal ligament
in patients with chronic hepatitis C reflects the immunological
cellular response of the host
Pierre Muller et al.
Background/Aims: Lymph nodes in the hepatoduodenal
ligament seem to be a common ultrasonographic finding in patients
with chronic hepatitis C. Lymphadenopathic enlargement is associated
with the histological hepatic features reflecting the immunological
response of the host, but the correlation between lymphadenopathy,
liver histology and the cellular immunoreactivity of the host
has never been studied. Aim: (1) To specify the
prevalence of lymph nodes within the hepatoduodenal ligament;
and (2) to investigate whether lymphadenopathies might reflect
the immunological response of the host. Methods:
One hundred and eleven patients were enrolled in this study.
Eleven chronic hepatitis B patients and 34 healthy volunteers
served as controls. Results: Lymph nodes were detectable
in 90 out of the 104 chronic hepatitis C patients studied. After
logistic regression, a high CD8 level and the absence of post
hepatitis C cirrhosis were associated with lymph node enlargement.
The total lymph node volume was correlated with transaminase
levels, inflammatory activity, and stage of fibrosis. Conclusions:
(1) The prevalence of lymph nodes within the hepatoduodenal ligament
is high; (2) lymph node enlargement is correlated with the immunological
cellular response of the host; and (3) the total lymph node volume
is correlated with hepatic necroinflammatory markers and the
stage of fibrosis.
Keywords: Chronic hepatitis C; Cirrhosis; Lymph node; Immunological
response; Host
Screening for hepatitis C in genito-urinary medicine clinics:
a cost utility analysis
Ken Stein et al.
Background/Aims: To estimate the cost utility (cost
per QALY) of screening for hepatitis C (HCV) infection in people
attending genito-urinary medicine clinics in England. Methods:
An epidemiological model of screening and diagnosis was combined
with a Markov chain model of treatment with combination therapy
to estimate cost utility. Parameters for the model were informed
by literature review, expert opinion and a survey of current
screening practice. Results: The base case estimate
was about £85,000 per QALY. Selective screening is more
cost effective. If screening is restricted to only 20% or 10%
of attenders, cost utility is estimated as £39,647 and
£34,288 per QALY. If screening is restricted only to those
with a history of injecting drug use, cost utility would be £27,138
per QALY. Estimates are particularly sensitive to acceptance
rates for screening and treatment. Conclusions:
Universal screening for HCV in GUM clinics is unlikely to be
cost effective. There is limited evidence to support screening
of people other than those with a history of injecting drug use
and even this policy should be considered with some care and
in the context of further research.
Keywords: Cost utility; Hepatitis C; Epidemiological model
Antiviral effect of ribavirin in early non-responders to
interferon monotherapy assessed by kinetics of hepatitis C virus
RNA and hepatitis C virus core antigen
Françoise Lunel et al.
Background/Aims: To evaluate the efficacy of ribavirin,
given in second intention in non-responders to interferon alone,
by studying viral kinetics. Methods: We conducted a trial
including 203 patients with chronic hepatitis C, naïve of
treatment. Patients were treated with interferon three times
a week with or without ribavirin and amantadine according to
response. Viral kinetics were assessed by serial measurements
of HCV RNA (bDNA 3.0 and Monitor 2.0) and a new assay, trak-C,
able to quantify total Hepatitis C virus (HCV) core antigen.
Results: A significant initial drop in HCV RNA or HCV
core antigen, under interferon alone, was associated with response
to therapy, 4.85±1.33 log for HCV RNA in sustained responders
versus 1.86±1.53 log for others groups, P<0.001.
In patients receiving ribavirin in second intention, we also
observed a similar drop in HCV RNA and HCV core antigen, predictive
of sustained response, 2.67±1.26 log for HCV RNA in sustained
responders versus 0.44±0.49 log in non-responders, P<0.001.
Conclusions: Ribavirin has probably an additional antiviral
effect in interferon treated patients. Kinetics of HCV RNA and
HCV core antigen under treatment are highly predictive of a sustained
virological response.
Keywords: Chronic hepatitis C virus hepatitis; Interferon;
Ribavirin; Hepatitis C virus RNA; Hepatitis C virus core antigen
The role of inducible nitric oxide synthase in a murine
acute hepatitis B virus (HBV) infection model induced by hydrodynamics-based
in vivo transfection of HBV-DNA
Wen-Wei Chang et al.
Background/Aims: Inducible nitric oxide synthase (iNOS)
is found to have antiviral activity. Its role is evaluated using
a murine acute hepatitis B virus (HBV) infection model. Methods:
pHBV3.6 plasmid containing HBV genome was injected into mice
by hydrodynamics-based in vivo transfection. HBV antigenemia
and serum HBV-DNA were detected by enzyme-linked immunosorbent
assay (ELISA) and polymerase chain reaction, respectively. HBV
replication in liver was analyzed by Northern and Southern blot.
Intrahepatic leukocytes were isolated and analyzed with flow
cytometry. Results: After hydrodynamics injection of pHBV3.6,
HBV genome was synthesized in the liver and HBV-DNA, as well
as hepatitis B surface antigen and hepatitis B e antigen were
secreted into the blood. Anti-HBV antibody responses developed
afterward. A murine acute HBV infection model was established
with hydrodynamics injection of non-transponase based HBV-DNA.
Using this protocol in iNOS deficient or wild type B6 mice, the
level of HBV transcript, replicative intermediate, and antigenemia
were higher in iNOS/ than in B6 mice. The intrahepatic leukocytes
in iNOS/ mice were also affected after transfection. Conclusions:
Our data suggests that the iNOS expression not only affects the
HBV clearance, but also modulates the infiltrating leukocytes
response to HBV antigens.
Keywords: Rodent; Viral; Antibodies; Inducible nitric oxide
synthase; Liver
A randomized trial of consensus interferon in combination
with ribavirin as initial treatment for chronic hepatitis C
Giovanna Fattovich et al.
Background/Aims: The aim of the present, open-labeled,
randomized study was to determine the efficacy and safety of
different doses of consensus interferon plus ribavirin in the
initial treatment of chronic hepatitis C. Methods:
One hundred and one genotype 2/3 patients were randomized to
receive 9 mcg (group A, n=48) or 18 mcg (group
B, n=53) of consensus interferon thrice weekly
plus ribavirin (1000/1200 mg/daily) for 24 weeks and 92 genotype
1 patients to receive 9 mcg (group C, n=47) or
18 mcg (group D, n=45) of consensus interferon
plus ribavirin for 48 weeks. Results: In an intention-to-treat
analysis, the sustained virologic response at 24-week follow-up
was 69% and 66% for group A and B (P=0.77) and
40% and 36% for group C and D (P=0.63). The overall
sustained response was 67% and 38% in patients with genotype
2/3 and 1, respectively. Among genotype 1 patients the sustained
virologic response was 39% and 41% for high or low baseline viremia
levels. Conclusions: Higher consensus interferon
dose does not increase sustained virologic response. Naive genotype
1 patients may achieve significant response rate of approximately
40% if treated with 9 mcg of consensus interferon plus ribavirin
for 48 weeks.
Keywords: Chronic hepatitis C; HCV genotype 1; Consensus
interferon; Ribavirin; Initial therapy
Clinical outcome and virologic profiles of severe hepatitis
B exacerbation due to YMDD mutations
Man-Fung Yuen et al.
Background/Aims: To study the outcome and the virologic
profiles of severe hepatitis exacerbations due to YMDD mutants
in lamivudine-treated patients. Methods: Eighteen lamivudine-treated
patients with severe hepatitis exacerbations due to YMDD mutants
were recruited. Laboratory and clinical parameters were monitored.
Viral genotypes and YMDD mutations were determined. Results:
None of the 18 patients had YMDD wild-type during exacerbations.
Three (17%) and 15 (83%) patients had genotypes B and C, respectively.
Elevated bilirubin levels and prolonged prothrombin time were
found in 11 (61%) and six patients (33%) respectively. Three
patients (17%) had adverse outcome with the development of ascites
and/or encephalopathy. One of these patients required liver transplantation
and one died. Both patients had evidence of cirrhosis before
treatment and hepatitis B e antigen (HBeAg) seroreversion from
anti-HBe positivity. The remaining 16 patients (89%) have no
evidence of pre-existing cirrhosis. Thirty seven percent of patients
had normal alanine aminotransferase levels at the last follow-up.
The median HBV DNA level at the last follow-up was significantly
lower than the pre-treatment level (P=0.009).
Conclusions: Though the majority of patients with severe
hepatitis exacerbations due to YMDD mutants had uneventful course,
early liver transplantation should be considered in patients
with pre-existing cirrhosis and HBeAg seroreversion.
Keywords: Severe hepatitis B exacerbation; YMDD mutation;
Hepatitic decompensation; Mortality
Letter to the Editor
Lamivudine `as needed': a novel therapeutic approach for
Hße-minus chronic hepatitis
Marcello Persico et al.
The management of acute hepatitis C virus (HCV) infection after
renal transplantation (RT) remains controversial, due to the
potential risk of interferon-induced graft dysfunction. There
is little experience with combined interferon and ribavirin therapy
in this group of patients. We treated four consenting RT recipients
who developed acute de novo HCV infection with a combination
of interferon-alpha 2b and ribavirin. After 48 weeks' treatment,
sustained virologic and biochemical remission were achieved in
three patients infected with HCV genotypes 1a, 2, and 6a, respectively.
The median time from treatment onset to ALT normalization was
8 weeks. The fourth patient was a non-responder infected with
genotype 1b. Dose-dependent hemolysis was the most frequent side-effect.
No patient developed allograft dysfunction. Our experience indicates
that the judicious use of combined interferon and ribavirin can
be considered in selected RT recipients with severe acute hepatitis
C infection.
Keywords: Hepatitis C; Kidney transplantation; Interferon;
Ribavirin
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.
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