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 Archives
depuis le 01/09/00 |
Liver Biology and Pathobiology
Obesity-related fatty liver is unchanged in mice deficient
for mitochondrial uncoupling protein 2
György Baffy, Chen-Yu Zhang, Jonathan N. Glickman, Bradford
B. Lowell
Nonalcoholic fatty liver disease (NAFLD), a prevalent condition
associated with obesity, has the potential of evolving into end-stage
liver disease. The biochemical mechanisms that define the progression
of NAFLD are not well known, but reactive oxygen species (ROS)
have been implicated in this process. Uncoupling protein (UCP)
2 is a mitochondrial inner-membrane protein that mediates proton
leak, uncouples adenosine triphosphate (ATP) synthesis, and negatively
regulates ROS production. UCP2 expression is increased in various
animal models of NAFLD. Up-regulation of UCP2 may compromise cellular
ATP levels and worsen liver damage, or it may be protective by
ROS reduction in NAFLD. This study aimed to obtain a definitive
answer as to whether increased UCP2 expression contributes to
NAFLD. UCP2/ mice were exposed to obesity by crossbreeding
with ob/ob mice and by long-term high-fat feeding to study
the effect of UCP2 deficiency on the outcome of NAFLD. Steatohepatitis
score of crossbred mice (ob/ob/ko) was similar to that
of ob/ob mice at 25 weeks. No compensatory increase was
observed in the expression of UCP5 in ob/ob/ko livers.
To unmask the effects of absent leptin and its potential proinflammatory
actions, steatosis was also induced in UCP2/ mice by
a high-fat diet continued for 6 months. Serum alanine aminotransferase
(ALT) levels remained normal, and the steatohepatitis score in
UCP2/ mice was the same as in wild-type controls. We
conclude that increased expression of UCP2 in the livers of mice
with genetically or diet-induced obesity exerts neither protective
nor deleterious effects on the severity of fatty liver disease.
(HEPATOLOGY 2002;35:753-761.)
Leptin in hepatic fibrosis: Evidence for increased collagen
production in stellate cells and lean littermates of ob/ob
mice
Neeraj K. Saxena, Kazuo Ikeda, Don C. Rockey, Scott L. Friedman,
Frank A. Anania
Leptin is a 16-kd hormone that mediates a range of metabolic effects
by using a transduction pathway from the long form of the leptin
receptor, OB-RL, through Janus kinasesignal transducer and
activator of transcription (Jak-Stat) signaling components. Leptin
is produced by hepatic stellate cells (HSCs) but only following
their "activation." Because activation of stellate cells
is a central event in the fibrotic response to liver injury, we
hypothesized that leptin may directly stimulate fibrogenesis in
activated stellate cells via OB-RL. We analyzed leptin receptors
and their signaling partners in a stellate cell line (HSC-T6)
as well as in primary stellate cell isolates. We also examined
the effect of leptin on stellate cell expression of 2(I) collagen
messenger RNA (mRNA) levels by ribonuclease protection analysis
(RPA). Finally, we examined the role of leptin in in vivo
fibrogenesis by inducing a wounding response in ob/ob mice,
which lack functional leptin. HSC-T6 and culture-activated stellate
cells expressed OB-RL. Scatchard analysis verified specific binding
of leptin to HSCs, with an association constant (Kd) equal to
660 ± 5.8 pmol/L. Exposure of HSCs to leptin resulted in
significant increases in 2(I) collagen mRNA expression. Transient
transfection with a promoter reporter construct showed a 3-fold
increase in 2(I) collagen transgene activity. Leptin stimulated
activation of Stat3 in activated HSCs. Finally, lean animals,
but not ob/ob littermates, had significant fibrosis as
assessed by picrosirius red staining and abundant smooth
muscle actin staining. In conclusion, these results indicate that
leptin is profibrogenic in activated HSCs and can signal via the
Jak-Stat pathway. Up-regulation of leptin signaling in liver injury
could contribute to enhanced fibrogenesis, particularly in states
in which leptin levels are high. (HEPATOLOGY 2002;35:762-771.)
NF-B inhibition sensitizes hepatocytes to TNF-induced apoptosis
through a sustained activation of JNK and c-Jun
Hailing Liu, Chau R. Lo, Mark J. Czaja
Hepatocyte resistance to tumor necrosis factor (TNF)-induced apoptosis
is dependent on activation of the transcription factor nuclear
factor B (NF-B). To determine the mechanism by which NF-B protects
against TNF toxicity, the effect of NF-B inactivation on the proapoptotic
c-Jun NH2-terminal kinase (JNK) signaling pathway was examined
in the rat hepatocyte cell line RALA255-10G. Adenovirus-mediated
NF-B inactivation led to a prolonged activation of JNK and increased
activating protein-1 (AP-1) transcriptional activity in response
to TNF treatment. Inhibition of the function of the JNK substrate
and AP-1 subunit c-Jun blocked cell death from NF-B inactivation
and TNF as determined by measures of cell survival, numbers of
apoptotic and necrotic cells, and DNA hypoploidy. Inhibition of
c-Jun function blocked mitochondrial cytochrome c release and
activation of caspase-3 and -7. NF-B therefore blocks the TNF
death pathway through down-regulation of JNK and c-Jun/AP-1. In
conclusion, sustained JNK activation that occurs in the absence
of NF-B initiates apoptosis through a c-Jundependent induction
of the mitochondrial death pathway. (HEPATOLOGY 2002;35:772-778.)
Inhibition of the MAPK and PI3K pathways enhances UDCA-induced
apoptosis in primary rodent hepatocytes
Liang Qiao, Adly Yacoub, Elaine Studer, Seema Gupta, Xin Yan Pei,
Steven Grant, Philip B. Hylemon, Paul Dent
The mechanisms by which bile acids induce apoptosis in hepatocytes
and the signaling pathways involved in the control of cell death
are not understood fully. Here, we examined the impact of mitogen-activated
protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)
signaling on the survival of primary hepatocytes exposed to bile
acids. Treatment of hepatocytes with deoxycholic acid (DCA), chenodeoxycholic
acid (CDCA) or ursodeoxycholic acid (UDCA) caused sustained MAPK
activation that was dependent on activation of the epidermal growth
factor receptor (EGFR). Activation of MAPK was partially blocked
by inhibitors of PI3K. Inhibition of DCA-, CDCA-, and UDCA-stimulated
MAPK activation resulted in ~20%, ~35%, and ~55% apoptosis, respectively.
The potentiation of DCA- and CDCA-induced apoptosis by MEK1/2
inhibitors correlated with cleavage of procaspase 3, which was
blocked by inhibitors of caspase 8 (ile-Glu-Thr-Asp-p-nitroanilide
[IETD]) and caspase 3 (DEVD). In contrast, the potentiation of
UDCA-induced apoptosis weakly correlated with procaspase 3 cleavage,
yet this effect was also blocked by IETD and DEVD. Incubation
of hepatocytes with the serine protease inhibitor AEBSF reduced
the death response of cells treated with UDCA and MEK1/2 inhibitor
to that observed for DCA and MEK1/2 inhibitor. The apoptotic response
was FAS receptor and neutral sphingomyelinasedependent
and independent of FAS ligand expression, and neither chelation
of intracellular and extracellular Ca2+ nor down-regulation of
PKC expression altered the apoptotic effects of bile acids. In
conclusion, bile acid apoptosis is dependent on the production
of ceramide and is counteracted by activation of the MAPK and
PI3K pathways. (HEPATOLOGY 2002;35;779-789.)
Maintaining HNF6 expression prevents AdHNF3-mediated decrease
in hepatic levels of Glut-2 and glycogen
Yongjun Tan, Guy Adami, Robert H. Costa
The hepatocyte nuclear factor 3 (HNF-3) proteins are members of
the Forkhead Box (Fox) family of transcription factors that play
important roles in regulating expression of genes involved in
cellular proliferation, differentiation, and metabolic homeostasis.
In previous studies we increased liver expression of HNF-3 by
using either transgenic mice (transthyretin HNF-3) or recombinant
adenovirus infection (AdHNF3), and observed diminished hepatic
levels of glycogen, and glucose transporter 2 (Glut-2), as well
as the HNF-6, HNF-3, HNF-1, HNF-4, and C/EBP transcription factors.
We conducted the present study to determine whether maintaining
HNF-6 protein expression during AdHNF3 infection prevents reduction
of hepatic levels of glycogen and the earlier-mentioned genes.
Here, we show that AdHNF3- and AdHNF6-infected mouse liver displayed
increased hepatic levels of glycogen, Glut-2, HNF-3, HNF-1, and
HNF-4 at 2 and 3 days postinfection (PI). Furthermore, restoration
of hepatic glycogen levels after AdHNF3 and AdHNF6 coinfection
was associated with increased Glut-2 expression. AdHNF6 infection
alone caused a 2-fold increase in hepatic Glut-2 levels, suggesting
that HNF 6 stimulates in vivo transcription of the Glut-2
gene. DNA binding assays showed that only recombinant HNF-6 protein,
but not the HNF-3 proteins, binds to the mouse 185 to 144
bp Glut-2 promoter sequences. Cotransfection assays in human hepatoma
(HepG2) cells with either HNF-3 or HNF-6 expression vectors show
that only HNF-6 provided significant transcriptional activation
of the Glut-2 promoter. In conclusion, these studies show that
the hepatic Glut-2 promoter is a direct target for HNF-6 transcriptional
activation. (HEPATOLOGY 2002;35:790-798.)
Bone marrow transplantation in mice leads to a minor population
of hepatocytes that can be selectively amplified in vivo
Vincent O. Mallet, Claudia Mitchell, Eva Mezey, Monique Fabre,
Jacques-Emmanuel Guidotti, Laurent Renia, Laure Coulombel, Axel
Kahn, Hélène Gilgenkrantz
Cell-based therapy may some day be a therapeutic alternative to
liver transplantation. Recent observations indicating that hematopoietic
stem cells can differentiate into hepatocytes have opened new
therapeutic prospects. However, the clinical relevance of this
phenomenon is unknown. We have previously developed a strategy
based on the protective effect of Bcl-2 against Fas-mediated apoptosis
to selectively amplify a small number of hepatocytes in vivo.
We now show that this approach can be used to amplify a minor
population of bone marrowderived hepatocytes. Normal mice
were transplanted with unfractionated bone marrow cells from transgenic
animals expressing Bcl-2 under the control of a liver-specific
promoter. Recipients were then submitted to weekly injections
of the anti-Fas antibody, Jo2. Upon sacrifice, the liver of the
recipients showed bone marrowderived clusters of mature hepatocytes
expressing Bcl-2, which showed that the hepatocyte progeny of
a genetically modified bone marrow can be selectively expanded
in vivo. In contrast, no Bcl-2 expression could be detected
without the selective pressure of Jo2, suggesting that differentiation
of bone marrow cells into mature hepatocytes is very inefficient
under physiologic conditions. We conclude that a selection strategy
will be required to achieve a therapeutic level of liver repopulation
with bone marrowderived hepatocytes. (HEPATOLOGY 2002;35:799-804.)
The resistance of P. acnesprimed interferon
deficient mice to low-dose lipopolysaccharide-induced acute
liver injury
Yoshiaki Shimizu, Julie A. Margenthaler, Keith Landeros, Naoki
Otoma, Gerard Doherty, M. Wayne Flye
Endotoxin has been identified as a principal mediator of sepsis,
often with resulting multiple organ failure. Although interferon
(IFN-) has a central role in controlling bacterial infection through
the activation of macrophages and T lymphocytes, it can also enhance
the harmful effects of the inflammatory response. To examine the
role of IFN- in lipopolysaccharide (LPS)-induced injury, we administered
LPS (20 or 800 µg/mouse) alone or as low-dose LPS (20 µg/mouse)
7 days after heat-killed Propionibacterium acnes (P.
acnes) injection into wild-type C57BL/6 (B6) mice or IFN-deficient
(GKO) mice (B6 background). Although low-dose (20 µg) LPS
alone had no effect on survival, the administration of 800 µg
LPS alone resulted in 100% mortality in both B6 and GKO mice without
significant hepatic mononuclear cellular infiltration or differences
in elevated plasma tumor necrosis factor (TNF-), interleukin 6
(IL-6), and IL-12 levels. In contrast, mortality after low-dose
(20 µg) LPS challenge in P. acnes-primed B6 mice
was 100%, but 0% in GKO mice. In vivo plasma cytokine (IFN-,
TNF-, IL-6, and IL-12) levels and in vitro cytokine production
by hepatic mononuclear cells were significantly higher in B6 mice
compared with GKO mice. Associated hepatic mononuclear cellular
infiltration, multifocal liver necrosis, hepatomegaly, and splenomegaly
were found in B6 mice, but not in GKO mice. Finally, the anti-inflammatory
NK1.1+CD4+ cell proportion of hepatic infiltrating mononuclear
cell numbers 7 days after P. acnes administration was significantly
reduced in B6 compared with GKO mice, whereas the proportion of
inflammatory NK1.1+CD4 cells was increased. In conclusion,
these data suggest that IFN- mediates P. acnesprimed
low-dose LPS injury through the hepatic infiltration of mononuclear
cells and the subsequent elevation of inflammatory cytokines after
LPS challenge, whereas the lethal effects of high-dose LPS alone
does not depend on the presence of IFN-. (HEPATOLOGY 2002;35:805-814.)
Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion
injury through p38 MAP kinase pathway
Farin Amersi, Xiu-Da Shen, Dean Anselmo, Judy Melinek, Suhasani
Iyer, Daniel J. Southard, Masamichi Katori, Hans-Dieter Volk,
Ronald W. Busuttil, Roland Buelow, Jerzy W. Kupiec-Weglinski
A direct role of carbon monoxide (CO), an effector-signaling molecule
during heme oxygenase-1 (HO-1) catalysis of heme, in the protection
against hepatic ischemia/reperfusion (I/R) injury needs to be
established. This study was designed to determine the effects
and downstream mechanisms of CO on cold I/R injury in a clinically
relevant isolated perfusion rat liver model. After 24 hours of
cold storage, rat livers perfused ex vivo for 2 hours with
blood supplemented with CO (300 parts per million) showed significantly
decreased portal venous resistance and increased bile production,
as compared with control livers perfused with blood devoid of
CO. These beneficial effects correlated with improved liver function
(serum glutamic oxaloacetic transaminase levels) and diminished
histological features of hepatocyte injury (Banff's scores). The
CO-mediated cytoprotective effects were nitric oxide synthase-
and cyclic guanine monophosphate-independent, but p38 mitogen-activated
protein kinase (MAPK)-dependent. Moreover, adjunctive use of zinc
protoporphyrin, a competitive HO-1 inhibitor, has shown that exogenous
CO could fully substitute for endogenous HO-1 in preventing hepatic
I/R insult. This study performed in a clinically relevant ex
vivo cold ischemia model is the first to provide the evidence
that HO-1-mediated cytoprotection against hepatic I/R injury depends
on the generation of, and can be substituted by, exogenous CO.
The p38 MAPK signaling pathway represents the key downstream mechanism
by which CO prevents the I/R insult. In conclusion, regimens that
employ exogenous CO should be revisited, as they may have potential
applications in preventing/mitigating I/R injury, and thus expanding
the liver donor pool for clinical transplantation. (HEPATOLOGY
2002;35:815-823.)
The in vivo apoptotic effect of interferon alfa-2b
on rat preneoplastic liver involves Bax protein
María de Luján Alvarez, Juan Pablo Cerliani, Juan
Monti, Cristina Carnovale, María Teresa Ronco, Gerardo
Pisani, María Cristina Lugano, María Cristina Carrillo
To determine whether interferon alfa (IFN-) prevents in vivo
oncogenesis in very-early-stage cancer cells, we evaluated the
action of IFN-2b over preneoplastic foci in rats. Animals were
divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine
[DEN] plus 2-acetylaminofluorene [2-AAF]) of preneoplasia development
(group 1), treated with IFN-2b during the 2 phases (group 2),
only during initiation with DEN (group 3), only during administration
of 2-AAF (group 4), subjected only to an initiation stage (group
5), and treated with IFN-2b during this period (group 6). The
numbers of placental form of rat glutathione S-transferase (rGST-P)-positive
foci per liver and the foci as percentage of liver were significantly
reduced in groups 2, 3, and 6 but not in group 4. Rats treated
with IFN-2b showed a higher apoptotic index (AI) in altered hepatic
foci (AHF). Levels of p53 and Bax protein in liver lysates were
significantly increased in those animals. Similarly, levels of
antiapoptotic proteins Bcl-2 and Bcl-xL in mitochondrial fraction
were decreased. Finally, increased levels of Bax protein were
localized in the mitochondria of rats that received IFN-2b, at
least during the DEN phase (groups 2, 3, and 6), whereas mitochondrial
Bax expression was not increased in group 4. In conclusion, the
preneoplastic hepatocytes in rats that received IFN-2b during
the initiation stage undergo programmed cell death as a primary
result of a significant increase in the amount and translocation
to the mitochondria of Bax protein. (HEPATOLOGY 2002;35:824-833.)
Synergistic effect of basic fibroblast growth factor and vascular
endothelial growth factor in murine hepatocellular carcinoma
Hitoshi Yoshiji, Shigeki Kuriyama, Junichi Yoshii, Yasuhide Ikenaka,
Ryuichi Noguchi, Daniel J. Hicklin, James Huber, Toshiya Nakatani,
Hirohisa Tsujinoue, Koji Yanase, Hiroo Imazu, Hiroshi Fukui
The growth of any solid tumor depends on angiogenesis. Among the
known angiogenic factors, basic fibroblast growth factor (bFGF)
and vascular endothelial growth factor (VEGF), are potent and
representative factors involved in tumor development. It has been
reported that bFGF and VEGF showed a synergistic effect in both
in vitro and in vivo angiogenesis. However, the
interaction of these factors on tumor development and angiogenesis,
including hepatocellular carcinoma (HCC), has not yet been elucidated.
In this study, we examined the combined effect of bFGF and VEGF
overexpression by means of a combination of a retroviral tetracycline
(tet)regulated (Retro-Tet) gene expression system, which
can manipulate the gene expression in vivo by providing
tet in the drinking water, and a conventional plasmid gene expression
system. In an allograft study, bFGF and VEGF overexpression synergistically
increased tumor growth and angiogenesis in the murine HCC cells.
This synergistic effect also was found in established tumors.
VEGF messenger RNA (mRNA) expression in the tumor was increased
3.1-fold by bFGF-overexpression, and the bFGF-induced tumor development
was significantly attenuated by treatment with KDR/Flk-1 neutralizing
monoclonal antibody. In conclusion, these results suggest that
bFGF synergistically augments VEGF-mediated HCC development and
angiogenesis at least partly by induction of VEGF through KDR/Flk-1.
(HEPATOLOGY 2002;35:834-842.)
The MDR phenotype is associated with the expression of COX-2
and iNOS in a human hepatocellular carcinoma cell line
Ornella Fantappiè, Emanuela Masini, Iacopo Sardi, Laura
Raimondi, Daniele Bani, Michela Solazzo, Alfredo Vannacci, Roberto
Mazzanti
The presence of multiple drug resistance (MDR1) and angiogenic
phenotypes negatively affect patients' prognosis with cancer even
when treated with drugs that are not transported by the MDR1 gene
product. It is possible to suggest a link between the MDR1 and
angiogenic phenotypes. Because prostaglandins (PGs) and nitric
oxide (NO) have been proposed to be involved in angiogenesis in
vivo, the production of PGs and NO and the behavior of inducible
NO synthase (iNOS), cyclooxygenase 1 (COX-1), and inducible cyclooxygenase
(COX-2) were studied in parental drug-sensitive (P5) liver cancer
cell lines and in P5-derived MDR1 cells P1(0.5). Immunohistochemical
evaluation, Northern and Western blot analysis of COX-2 and iNOS,
and assessment of cell proliferation were performed in basal conditions
and after the exposure to stimulants or to specific inhibitors
of COX-2 and iNOS. The messenger RNA and protein levels of COX-2
and iNOS were in basal conditions higher in P1(0.5) cells than
the parental P5 cells. The exposure to lipopolysaccharide (LPS)
or epidermal growth factor (EGF) determined an increase of PG
and NO production in both cell lines and this increase was strongly
reduced by COX-2 inhibitors such as celecoxib (CLX) and nimesulide
(NIME). The inhibition of NO production by COX-2 inhibitors suggests
cross-talk between COX-2 and iNOS pathways. CLX and NIME also
inhibited cell proliferation, but only in MDR1 cells. A specific
inhibitor of iNOS, N6-(1-iminoethyl)-L-lysine, had only
a mild effect on cell proliferation in both cell lines. In conclusion,
these data support the hypothesis that the MDR1 and angiogenic
phenotypes are linked to each other in human liver cancer cell
lines. (HEPATOLOGY 2002;35:843-852.)
Liver Failure and Liver Disease
A-Fetoprotein mRNA in the circulation as a predictor of
postsurgical recurrence of hepatocellular carcinoma: A prospective
study (*Human Study*)
Masayoshi Ijichi, Tadatoshi Takayama, Masayuki Matsumura, Yasushi
Shiratori, Masao Omata, Masatoshi Makuuchi
fetoprotein (AFP) messenger RNA (mRNA) has been proposed as a
marker of hepatocellular carcinoma (HCC) cells disseminated into
the circulation, but its clinical significance remains controversial.
We prospectively assessed the prognostic value of AFP mRNA in
patients undergoing curative hepatic resection for HCC. Peripheral
blood samples were taken from 87 patients before and after surgery
to determine the presence of AFP mRNA by use of a reverse-transcription
polymerase chain reaction. A primary endpoint was recurrence-free
interval. AFP mRNA was detectable preoperatively in 31 patients
(36%) and postoperatively in 30 patients (34%). With a median
follow-up period of 28 months (range, 3-41 months), HCC recurred
in 46 patients (53%). Among 4 groups separated according to preoperative
and postoperative AFP mRNA status, patients with consistent positivity
of AFP mRNA showed the highest recurrence rate (85%) and trend
to distant or multiple recurrence. The recurrence-free interval
was significantly shorter in patients with postoperative positivity
of AFP mRNA than in those without (53% [95% CI, 36-71] vs. 88%
[95% CI, 79-96] at 1 year, 37% [95% CI, 17-57] vs. 60% [95% CI,
46-75] at 2 years; P = .014), whereas the preoperative
positivity of AFP mRNA provided no significance (P = .100).
Cox's proportional-hazards model identified the postoperative
positivity of AFP mRNA as an independent prognostic factor for
HCC recurrence (relative risk, 2.33; 95% CI, 1.26-4.34; P
= .007). In conclusion, postsurgical recurrence of HCC can be
predicted by detecting AFP mRNAexpressing cells in peripheral
blood. (HEPATOLOGY 2002;35:853-860.)
Tie2 vascular endothelial receptor expression and function
in hepatocellular carcinoma (*Human Study*)
Shinji Tanaka, Keishi Sugimachi, Yo-ichi Yamashita, Takefumi Ohga,
Ken Shirabe, Mitsuo Shimada, Jack R. Wands, Keizo Sugimachi
Hepatocellular carcinoma (HCC) is generally characterized as a
hypervascular tumor of rapid growth. We have previously reported
that angiopoietin (Ang), a ligand for Tie2 vascular endothelial-specific
receptor tyrosine kinase, may play a role in the progression of
human HCC (J Clin Invest 1999;103:341-345) and matrix proteinase
expression (Cancer Res 2001;61:2145-2153). However, the role of
Tie2 receptor in hepatic oncogenesis is unknown. The Tie2 receptor
protein was overexpressed in the neovascular endothelium of 31
of 39 (80%) human HCC tumors by immunohistochemical analysis with
significant correlation to cell dedifferentiation and tumor size
(P < .05). In vitro expression of a dominant-negative
construct, containing a soluble Tie2 ectodomain (sTie2), led to
Ang protein interaction, inhibition of endogenous Tie2 phosphorylation
in vascular endothelial cells and matrix metalloproteinase 9 (MMP-9)
suppression. In conclusion, tumorigenicity with neovascularization
was suppressed by in vivo gene transfer and sTie2 expression
in a murine HCC model, suggesting a possible role for Tie2 expression
in the induction of HCC neovascularization and disease progression.
Inhibition of the Ang/Tie2 signal transduction cascade is a promising
approach for tumor treatment. (HEPATOLOGY 2002;35:861-867.)
Lower risk for alcohol-induced cirrhosis in wine drinkers (*Human
Study*)
Ulrik Becker, Morten Grønbæk, Ditte Johansen, Thorkild
I. A. Sørensen
Although there is a well-known relationship between total alcohol
intake and future risk for cirrhosis, other factors such as the
type of alcohol consumed are sparsely studied. The aim of this
study was to assess the effects of wine compared with other types
of alcoholic beverages on risk for alcohol-induced cirrhosis.
In 3 prospective studies, 30,630 participants from the Copenhagen
area were followed-up for a total observation time of 417,325
person-years. Information on weekly intake of beer, wine, and
spirits, and sex, age, body mass index, smoking habits, and education
was obtained from questionnaires. The primary outcome measures
were first admission or death, with alcohol-induced cirrhosis
obtained from death certificates and from the National Hospital
Discharge Register. Data were analyzed by means of multiplicative
Poisson regression models. We confirmed the increasing risk for
cirrhosis with increasing alcohol intake. Individuals who drank
more than 5 drinks per day had a relative risk of 14 to 20 for
developing cirrhosis compared with non- or light drinkers. However,
compared with individuals who drank no wine (relative risk set
at 1.0), individuals drinking 16% to 30% wine of their total intake
had a relative risk of 0.4 (95% confidence limits, 0.3-0.6) and
those drinking 51% or more of wine had a relative risk of 0.3
(95% confidence limits, 0.2-0.5) for developing cirrhosis. In
conclusion, the results suggest that a high intake of all 3 types
of alcohol conveys an increased risk for cirrhosis, but wine drinkers
are at a lower risk than beer and spirits drinkers. (HEPATOLOGY
2002;35:868-875.)
Acute versus chronic alcohol consumption in acetaminophen-induced
hepatotoxicity (*Human Study*)
Lars E. Schmidt, Kim Dalhoff, Henrik Enghusen Poulsen
The aim of this study was to determine by multivariate analysis
how alcohol and other factors affect the clinical course and outcome
in patients with acetaminophen (paracetamol) poisoning. A total
of 645 consecutive patients admitted from 1994 to 2000 with single-dose
acetaminophen poisoning were studied, giving special attention
to alcohol history, time between overdose and intravenous N-acetylcysteine
(NAC) treatment ("time to NAC"), and other data available
at the time of admittance. Up until 72 hours after ingestion,
time to NAC was the single most important independent risk factor.
With a time to NAC less than 12 hours, the mortality rate was
0.42% (95% CI, 0.05-2.7). When time to NAC exceeded 12, 24, and
48 hours, the mortality rate increased to 6.1%, 13%, and 19%,
respectively. Chronic alcohol abuse was an independent risk factor
of mortality (odds ratio [OR], 3.52; 95% CI, 1.78-6.97). Acute
alcohol ingestion was an independent protective factor regarding
mortality in alcoholic patients (OR, 0.08; 95% CI, 0.01-0.66)
but not in nonalcoholic patients (OR, 0.21; 95% CI, 0.03-1.67).
Patient age and quantity of acetaminophen were independent risk
factors. In conclusion, time to NAC was confirmed as the major
risk factor in acetaminophen-induced hepatotoxicity and mortality.
Chronic alcohol abuse was an independent risk factor that could
be counteracted by concomitant acute alcohol ingestion. We suggest
that patients with chronic alcoholism and suspected acetaminophen
poisoning due to an increased risk of developing hepatotoxicity
should be treated with NAC regardless of risk estimation. (HEPATOLOGY
2002;35:876-882.)
Polymorphism of the N-acetyltransferase 2 gene as
a susceptibility risk factor for antituberculosis druginduced
hepatitis (*Human Study*)
Yi-Shin Huang, Herng-Der Chern, Wei-Juin Su, Jaw-Ching Wu, Shinn-Liang
Lai, Shi-Yi Yang, Full-Young Chang, Shou-Dong Lee
Antituberculosis druginduced hepatitis is one of the most
prevalent drug-induced liver injuries. Isoniazid is the major
drug incriminated in this hepatotoxicity. Isoniazid is mainly
metabolized to hepatotoxic intermediates by N-acetyltransferase
(NAT). However, the association of polymorphic NAT acetylator
status and antituberculosis druginduced hepatitis is debatable.
To determine whether acetylator status is a risk factor for antituberculosis
druginduced hepatitis, we genotyped NAT2 in 224 incident
tuberculosis patients who received antituberculosis treatment.
Antituberculosis druginduced hepatitis was diagnosed based
on a positive isoniazid rechallenge test and exclusion of viral
hepatitis. Acetylator status was determined by genotyping NAT2
in patients using a polymerase chain reaction with restriction
fragment length polymorphism. Univariate analysis and logistic
regression analysis were used to evaluate the risk factors of
isoniazid-induced hepatitis. Thirty-three patients (14.7%) were
diagnosed with antituberculosis druginduced hepatitis. Slow
acetylators had a higher risk of hepatotoxicity than rapid acetylators
(26.4% vs. 11.1%, P = .013). Among patients with hepatotoxicity,
slow acetylators had significantly higher serum aminotransferase
levels than rapid acetylators. Logistic regression showed that
slow-acetylator status (odds ratio [OR], 3.66; 95% CI, 1.58-8.49;
P = .003) and age (OR, 1.09; 95% CI, 1.04-1.14; P
< .001) were the only 2 independent risk factors for antituberculosis
druginduced hepatitis. In conclusion, slow-acetylator status
of NAT2 is a significant susceptibility risk factor for
antituberculosis druginduced hepatitis. Additionally, slow
acetylators are prone to develop more severe hepatotoxicity than
rapid acetylators. Regular monitoring of serum aminotransferase
levels is mandatory in patients receiving antituberculosis treatment,
especially in slow acetylators. (HEPATOLOGY 2002;35:883-889.)
Sustained remission after corticosteroid therapy for type 1
autoimmune hepatitis: A retrospective analysis (*Human Study*)
Albert J. Czaja, K. V. Narayanan Menon, Herschel A. Carpenter
Autoimmune hepatitis commonly relapses after corticosteroid therapy,
and long-term management strategies have been proposed based on
the premise that repeated relapses after drug withdrawal are inevitable.
Our goal was to determine the frequency that remission can be
sustained after its induction and termination of therapy. A total
of 107 patients who had entered remission on conventional regimens
were assessed for sustained remission after initial treatment
and after relapse and re-treatment. Re-treatment strategies included
conventional regimens and long-term maintenance schedules. Twenty-two
patients (21%) achieved a sustained remission after initial treatment,
and 24 of 85 patients who relapsed and were re-treated (28%) had
a similar outcome. The probability of a sustained remission was
47% after 10 years of follow-up. Patients who sustained remission
after initial therapy were distinguished only by a lower serum
-globulin level at entry. Conventional re-treatment schedules
after relapse were able to induce a sustained remission more commonly
then long-term maintenance schedules (59% vs. 12%, P =
.00002). In conclusion, patients who respond to initial corticosteroid
therapy can achieve a sustained remission after treatment withdrawal
or after relapse and re-treatment. All patients are candidates
for this outcome, and withdrawal of medication, even during maintenance
schedules, is necessary to assess its likelihood. (HEPATOLOGY
2002;35:890-897.)
Apolipoprotein synthesis in nonalcoholic steatohepatitis (*Human
Study*)
Michael Charlton, Raghavakaimal Sreekumar, Deborah Rasmussen,
Keith Lindor, K. Sreekumaran Nair
The pathophysiology of hepatic steatosis, a prerequisite of nonalcoholic
fatty liver disease, is poorly understood. Because very-lowdensity
lipoprotein (VLDL) formation is the chief route of hepatic lipid
export, we hypothesized that the synthesis of apoB-100, a rate-determining
step in hepatic VLDL formation, may be altered in patients with
nonalcoholic steatohepatitis (NASH). This study evaluated the
relative synthesis rates of apolipoprotein B-100 (apoB-100) in
patients with NASH and in lean and body mass index (BMI)matched
(obese) controls without NASH. A primed continuous infusion of
L-[1-13C] leucine was used to measure the absolute synthesis rates
(ASR) of apoB-100 and fibrinogen in 7 patients with NASH and compared
them with 7 lean and 7 obese (BMI-matched) controls without NASH.
The ASRs of fibrinogen and albumin also were measured. The mean
ASR of apoB-100 in patients with NASH was lower (31.5 ±
3.4 mg/kg/d) than that of obese (115.2 ± 7.2 mg/kg/d, P
< .001) and lean controls (82.4 ± 4.1 mg/kg/d, P
= .002). In contrast, the mean ASR of fibrinogen was greater in
subjects with NASH than in both control groups. These data indicate
that NASH is associated with markedly altered hepatic synthesis
of apoB-100. The finding that albumin synthesis was not similarly
decreased in patients with NASH shows that the attenuation of
apoB-100 synthesis is not on the basis of globally impaired hepatic
protein synthesis. In conclusion, because apoB-100 synthesis is
a rate-determining step in hepatocyte lipid export, decreased
synthesis of this protein may be an important factor in the development
of hepatic steatosis, a prerequisite for NASH. (HEPATOLOGY 2002;35:898-904.)
Hemolysis and bilirubin conjugation in association with UDP-glucuronosyltransferase
1A1 promoter polymorphism (*Human Study*)
Michael Kaplan, Cathy Hammerman, Firmino F. Rubaltelli, Maria
T. Vilei, Ephrat Levy-Lahad, Paul Renbaum, Hendrik J. Vreman,
David K. Stevenson, Maurizio Muraca
Hemolysis may contribute to hyperbilirubinemia in Gilbert's syndrome.
The authors examined blood carboxyhemoglobin corrected for inspired
CO (COHbc) to index heme catabolism and serum conjugated bilirubin
fractions to reflect bilirubin conjugation. Both parameters were
related to UDP-glucuronosyltransferase 1A1 (UGT) promoter polymorphism,
associated with Gilbert's syndrome, in term male newborns. COHbc
was expressed as percentage of total hemoglobin, and total conjugated
bilirubin (TCB) value as a percentage of serum total bilirubin
(STB), (TCB/STB[%]). A production/conjugation index, COHbc/(TCB/STB[%]),
represented bilirubin production divided by conjugation. UGT promoter
genotype was designated according to the number of promoter TA
insertions in each allele: 6/6, homozygous normal; 6/7, heterozygous;
7/7, homozygous variant. STB and COHbc values were higher in the
7/7 subgroup than the other counterparts (P < .01).
The COHbc/(TCB/STB[%]) was higher in the 7/7 than either the 6/6
or 6/7 subsets (1.93 [1.31-2.88] vs. 0.85 [0.51-1.72] and 0.84
[0.53-1.87], respectively; P < .01). In conclusion,
7/7 UGT promoter polymorphism was associated with increased blood
COHbc values (unexpected finding) as well as diminished serum
total conjugated bilirubin ratios (expected finding). The increased
hemolysis may contribute to the pathogenesis of increased STB
values seen in Gilbert's syndrome, and exacerbate neonatal hyperbilirubinemia
associated with the promoter polymorphism. (HEPATOLOGY 2002;35:905-911.)
Uroporphyria in mice: Thresholds for hepatic CYP1A2 and iron
Nadia Gorman, Kerry L. Ross, Heidi S. Walton, William J. Bement,
Juliana G. Szakacs, Glenn S. Gerhard, Timothy P. Dalton, Daniel
W. Nebert, Richard S. Eisenstein, Jacqueline F. Sinclair, Peter
R. Sinclair
In mice treated with 5-aminolevulinic acid (ALA) and polyhalogenated
aromatic compounds, the levels of both hepatic cytochrome P450
(CYP)1A2 and iron-which can be quite different among inbred strains-are
critical in causing experimental uroporphyria. Here we investigate
the development of uroporphyria as a function of CYP1A2 and iron
levels in the liver of mice having a common C57BL/6 genetic background.
We compared Cyp1a2(/) knockout mice, Cyp1a2(+/)
heterozygotes, Cyp1a2(+/+) wild type, and Cyp1a2(+/+)
mice pretreated with a low dose of 3,3',4,4',5-pentachlorobiphenyl
(PCB126) (4 µg/kg). Cyp1a2(+/) mice contain
about 60% of the hepatic CYP1A2 content of Cyp1a2(+/+)
mice, and the PCB126-pretreated Cyp1a2(+/+) mice have about
twice the wild-type levels of CYP1A2. ALA- and iron-treated Cyp1a2(+/+)
mice are known to accumulate hepatic uroporphyrin; this accumulation
was increased 7-fold by pretreatment with the low dose of PCB126.
ALA- and iron-treated Cyp1a2(+/) heterozygote mice
accumulated no uroporphyrin in 4 weeks, but by 8 weeks accumulated
significant amounts of uroporphyrin. As previously reported, the
ALA- and iron-treated Cyp1a2(/) knockout mouse
has no CYP1A2 and exhibits no detectable uroporphyrin accumulation.
Iron dose-response curves in ALA- and PCB126-treated Cyp1a2(+/+)
mice showed that hepatic iron levels greater than 850 µg/g
liver were required to produce significant uroporphyrin accumulation
in the liver. Other measures of hepatic effects of iron (iron-response
element-binding protein [IRP]-iron response element [IRE] binding
activity and accumulation of protoporphyrin from ALA) decreased
when the level of iron was considerably lower than 850 µg/g
liver. At low iron doses, accumulation of iron was principally
in Kupffer cells, whereas at the higher doses (required to stimulate
uroporphyrin accumulation), more iron was found in parenchymal
cells. We conclude that small changes in hepatic CYP1A2 levels
can dramatically affect uroporphyria in C57BL/6 mice, providing
the animals have been sufficiently loaded with iron; these data
might be clinically relevant to acquired (sporadic) porphyria
cutanea tarda, because humans show greater than 60-fold genetic
differences in hepatic basal CYP1A2. (HEPATOLOGY 2002;35:912-921.)
Viral Hepatitis
Hepatitis B e antigen in sera from individuals infected
with hepatitis B virus of genotype G (*Human Study*)
Hideaki Kato, Etsuro Orito, Robert G. Gish, Natalie Bzowej, Margaret
Newsom, Fuminaka Sugauchi, Seiji Suzuki, Ryuzo Ueda, Yuzo Miyakawa,
Masashi Mizokami
Hepatitis B virus (HBV) genotype G (HBV/G) was detected in sera
from four individuals by polymerase chain reaction with hemi-nested
primers deduced from an insertion of 36 nt in the core gene that
is specific for this genotype. Despite two stop codons in the
precore region characteristic of HBV/G, all patients were positive
for hepatitis B e antigen (HBeAg) in serum. When 10 HBV clones
were propagated from one patient, and sequenced within precore
region and a section of the core gene, 6 clones were HBV/G while
2 were genotype A (HBV/A); a recombination between HBV/G and HBV/A
occurred in the remaining 2 clones. Mixed infection of HBV/G and
HBV/A, as well as the recombination, was demonstrated in the sequence
of preS1 and preS2 regions also. Coinfection with HBV/G and HBV/A
was demonstrated in the other three patients, and their recombination
in two patients. Ten HBV clones were propagated from one patient
at two time points separated by 1 year. Clones of HBV/A, HBV/G
and their recombination were found in 9 : 1 : 0 when the patient
was positive for HBeAg, while the proportion shifted to 0 : 8
: 2 after the patient seroconverted to anti-HBe. In conclusion,
HBV/G is frequently found as a coinfection with HBV/A. This coinfection
would explain the presence of HBeAg in individuals infected with
HBV/G. Along with seroconversion to anti-HBe, HBV/G would be selected
accompanied by the recombination with HBV/A. Further studies should
be performed to confirm these findings. (HEPATOLOGY 2002;35:922-929.)
Viral kinetics in genotype 1 chronic hepatitis C patients during
therapy with 2 different doses of peginterferon alfa-2b plus ribavirin
(*Human Study*)
Maria Buti, Francisco Sanchez-Avila, Yoav Lurie, Carlos Stalgis,
Auristela Valdés, Maria Martell, Rafael Esteban
Pegylated interferon (peginterferon) alfa-2b plus ribavirin achieves
a higher sustained response rate in patients with genotype 1 chronic
hepatitis C virus (HCV) than standard combination therapy. This
study evaluated HCV kinetics throughout therapy with 2 doses of
peginterferon alfa-2b and ribavirin in 55 patients. Twenty-eight
patients were randomized to receive a high once-weekly dose of
peginterferon alfa-2b (3 µg/kg for 1 week, 1.5 µg/kg
for 3 weeks, and 1.0 µg/kg for 44 weeks), and 27 patients
were randomized to receive a low dose (0.5 µg/kg) for 48
weeks. Both groups also received 800 mg ribavirin daily. Mean
baseline HCV RNA load, measured by reverse-transcription polymerase
chain reaction, was similar in both groups (5.32 ± 0.86
log vs. 5.15 ± 1.04 log). The 3-µg/kg dose of peginterferon
alfa-2b inhibited HCV RNA more significantly than the 0.5-µg/kg
dose during the first 48 hours (2.08 ± 0.93 log vs. 1.09
± 0.80 log; P < .001) and both increased at 72
hours (0.54 ± 0.73 log vs. 0.03 ± 0.36 log; P
= not significant [NS]), but the high dose showed a greater reduction
at the end of the week (1.07 ± 0.99 log vs. 0.72 ±
0.73 log). Both doses showed a progressive, slower viral decrease
throughout therapy; however, HCV RNA became undetectable faster
and in more patients with the high dose (22% vs. 7% at week 4,
56% vs. 44% at week 12, 69% vs. 63% at week 24, and 71% vs. 61.5%
at the end of therapy). In conclusion, peginterferon alfa-2b/ribavirin
produces an initial rapid decline in HCV RNA levels, followed
by a slower, progressive decrease, similar to the biphasic kinetic
profile of standard combination therapy. Higher doses of peginterferon
alfa-2b also accelerate viral clearance. (HEPATOLOGY 2002;35:930-936.)
Interaction of hepatitis C virus core protein with retinoid
X receptor modulates its transcriptional activity
Takeya Tsutsumi, Tetsuro Suzuki, Takashi Shimoike, Ryosuke Suzuki,
Kyoji Moriya, Yoshizumi Shintani, Hajime Fujie, Yoshiharu Matsuura,
Kazuhiko Koike, Tatsuo Miyamura
Hepatic steatosis and hepatocellular carcinoma (HCC) are common
and serious features of hepatitis C virus (HCV) infection, and
the core protein has been shown to play distinct roles in the
pathogenesis. Here we report the direct interaction of HCV core
protein with retinoid X receptor (RXR), a transcriptional regulator
that controls many aspects of cell proliferation, differentiation,
and lipid metabolism. The core protein binds to the DNA-binding
domain of RXR, leading to increase the DNA binding of RXR to its
responsive element. In addition, RXR is activated in cells expressing
the core protein as well as in the livers of the core-transgenic
mice that would develop hepatic steatosis and HCC later in their
lives. Using promoter genes of cellular retinol binding protein
II (CRBPII) and acyl-CoA oxidase as reporters, we also show that
the expression of the core protein enhances the transcriptional
activity regulated by the RXR homodimer as well as by the heterodimer
with peroxisome proliferator activated receptor . Furthermore,
expression of the CRBPII gene is also up-regulated in the livers
of HCV core-transgenic mice. In conclusion, these results suggest
that modulation of RXR-controlled gene expression via interaction
with the core protein contributes to the pathogenesis of HCV infection.
(HEPATOLOGY 2002;35:937-946.)
Peripheral blood count abnormalities among patients with hepatitis
C in the United States (*Human Study*)
Michael B. Streiff, Shruti Mehta, David L. Thomas
An estimated 2.7 million people in the United States are infected
with the hepatitis C virus (HCV), yet the influence of HCV infection
on the peripheral blood count remains unknown. To investigate
the prevalence of low peripheral blood counts among HCV-infected
adults in the United States general population, we analyzed data
collected in the third National Health and Nutrition Examination
Survey (NHANES III). The study population consisted of 16,196
individuals age 18 or older who had peripheral blood counts and
data on HCV infection. The lowest fifth percentile of each component
of the peripheral blood was designated a priori as being
low. HCV infection was assessed by antibody reactivity. HCV antibodypositive
individuals were 3-fold more likely to have low neutrophil counts
(HCV positive, 9% vs. HCV negative, 3%, P < .0001) and
2.6-fold more likely to have low platelet counts (HCV positive,
13% vs. HCV negative, 5%, P < .0001) independent of
other evaluated factors. HCV infection was observed in more than
20% of persons with neutrophil counts below 1.0 ¥ 109/L or
platelet counts less than 100 ¥ 109/L. No association was
detected between anti-HCV status and anemia or other peripheral
blood cell components. In conclusion, HCV-infected persons in
the general population of the United States are more likely to
have low neutrophil and platelet counts, and HCV testing should
be considered for persons with unexplained neutrophil counts below
1.0 ¥ 109/L or platelet counts less than 100 ¥ 109/L.
Alternate causes of anemia should be considered for HCV-infected
persons with low red blood cell counts. (HEPATOLOGY 2002;35:947-952.)
SEN virus: Response to interferon alfa and influence on the
severity and treatment response of coexistent hepatitis C (*Human
Study*)
Takeji Umemura, Harvey J. Alter, Eiji Tanaka, Koji Orii, Anthony
E. T. Yeo, J. Wai-Kuo Shih, Akihiro Matsumoto, Kaname Yoshizawa,
Kendo Kiyosawa
The SEN virus (SENV) is a recently identified single-stranded,
circular DNA virus. A strong association between 2 SENV variants
(SENV-D and SENV-H) and transfusion-associated nonA-to-E
hepatitis has been reported. To clarify the effect of SENV infection
on coexisting chronic hepatitis C and the effect of interferon
alfa (IFN-) therapy on SENV replication, SENV DNA was quantitated
by polymerase chain reaction in serum samples from 186 patients
with chronic hepatitis C. Thirty-nine of 186 (21%) patients with
chronic hepatitis C were positive for SENV DNA. There were no
differences in the clinical, virologic and histologic features
between patients with and without SENV infection. Eighteen of
102 patients with chronic hepatitis C who received IFN- were positive
for SENV DNA. The sustained response rate for hepatitis C virus
(HCV) clearance after IFN- treatment did not differ significantly
between patients with SENV (28%) and without SENV infection (39%).
SENV DNA levels decreased during therapy in 15 of 16 patients,
and 11 of the 16 patients (69%) had a sustained loss of SENV DNA
in response to IFN-. In coinfected patients, SENV responses to
IFN- were significantly better in those who failed to clear HCV
RNA than in those who lost HCV RNA (P = .013). In conclusion,
SENV infection was frequently found in patients with chronic hepatitis
C. SENV infection had no apparent influence on the severity of
HCV-related liver disease or the HCV response to IFN-. SENV was
sensitive to IFN- therapy and the majority of patients had a sustained
virologic response. (HEPATOLOGY 2002;35:953-959.)
The molecular classification of the clinical manifestations
of Crohn's disease
T. Ahmad, A. Armuzzi, M. Bunce, K. Mulcahy-Hawes, S. E. Marshall,
T. R. Orchard, J. Crawshaw, O. Large, A. de Silva, J. T. Cook,
M. Barnardo, S. Cullen, K. I. Welsh, D. P. Jewell
Background & Aims: Crohn's disease is a common inflammatory
disorder of the gut characterized by variation in both location
and behavior. Chromosome 16 and the HLA region on chromosome 6
have been implicated in susceptibility to disease. Mutations in
the NOD2/CARD15 gene, recently identified on chromosome 16, have
been associated with disease overall but are found in only 25%
of patients. No data regarding their contribution to specific
disease subtypes exist. Here we report a detailed genotype-phenotype
analysis of 244 accurately characterized patients.
Methods: A total of 244 white patients with Crohn's disease
recruited from a single center in the United Kingdom were studied.
All patients were rigorously phenotyped and followed-up for a
median time of 16 years. By using linkage disequilibrium mapping
we studied 340 polymorphisms in 24 HLA genes and 3 NOD2/CARD15
polymorphisms.
Results: We show that NOD2/CARD15 mutations determine ileal
disease only. We confirm that alleles on specific long-range HLA
haplotypes determine overall susceptibility and describe novel
genetic associations with susceptibility, location, and behavior
of Crohn's disease.
Conclusions: The clinical pattern of Crohn's disease may
be defined by specific genotypes. This study may provide the basis
for a future molecular classification of disease.
The contribution of NOD2 gene mutations to the
risk and site of disease in inflammatory bowel disease
A. P. Cuthbert, S. A. Fisher, M. M. Mirza, K. King, J. Hampe,
P. J. P. Croucher, S. Mascheretti, J. Sanderson, A. Forbes, J.
Mansfield, S. Schreiber, C. M. Lewis, C. G. Mathew
Background & Aims: Mutations in the NOD2 gene
are strongly associated with susceptibility to Crohn's disease
(CD). We analyzed a large cohort of European patients with inflammatory
bowel disease to determine which mutations confer susceptibility,
the degree of risk conferred, their prevalence in familial and
sporadic forms of the disease, and whether they are associated
with site of disease.
Methods: Individuals were genotyped for 4 NOD2 mutations:
P268S, R702W, G908R, and 3020insC. Allelic transmission distortion
to 531 CD and 337 ulcerative colitisaffected offspring
was assessed by the transmission disequilibrium test. Association
was also tested in an independent cohort of 995 patients with
inflammatory bowel disease and 290 controls. Cases were stratified
by disease site and compared across NOD2 genotypes.
Results: R702W, G908R, and 3020insC were strongly associated
with CD but not with ulcerative colitis. Linkage disequilibrium
was observed between P268S and the other mutations, forming 3
independent disease haplotypes. Genotype relative risks were 3.0
for mutation heterozygotes and 23.4 for homozygotes or compound
heterozygotes. The frequency of NOD2 mutations was higher
in cases from families affected only with CD and was significantly
increased in ileal-specific disease cases compared with colon-specific
disease (26.9% vs. 12.7%, P = 0.0004).
Conclusions: The R702W, G908R, and 3020insC mutations are
strong independent risk factors for CD and are associated particularly
with ileal disease.
Clinical Research
The natural history of fistulizing Crohn's disease in Olmsted
County, Minnesota
D. A. Schwartz, E. V. Loftus, Jr., W. J. Tremaine, R. Panaccione,
W. S. Harmsen, A. R. Zinsmeister, W. J. Sandborn
Background & Aims: Little is known about the cumulative
incidence and natural history of fistulas in Crohn's disease in
the community.
Methods: The medical records of all Olmsted County, Minnesota
residents who were diagnosed with Crohn's disease from 1970 to
1993 and who developed a fistula were abstracted for clinical
features and outcomes. Six patients denied research authorization.
The cumulative incidence of fistula from time of diagnosis was
estimated by using the KaplanMeier product-limit method.
Results: At least 1 fistula occurred in 59 patients (35%),
including 33 patients (20%) who developed perianal fistulas. Twenty-six
(46%) developed a fistula before or at the time of formal diagnosis.
Assuming that the 9 patients with fistula before Crohn's disease
diagnosis were instead simultaneous diagnoses, the cumulative
risk of any fistula was 33% after 10 years and was 50% after 20
years (perianal, 21% after 10 years and 26% after 20 years). At
least 1 recurrent fistula occurred in 20 patients (34%). Most
fistulizing episodes (83%) required operations, most of which
were minor. However, 11 perianal fistulizing episodes (23%) resulted
in bowel resection.
Conclusions: Fistulas in Crohn's disease were common in
the community. In contrast to referral-based studies, only 34%
of patients developed recurrent fistulas. Surgical treatment was
frequently required.
Celiac disease in patients with severe liver disease: Gluten-free
diet may reverse hepatic failure
K. Kaukinen, L. Halme, P. Collin, M. Färkkilä, M. Mäki,
P. Vehmanen, J. Partanen, K. Höckerstedt
Background & Aims: Mild liver abnormalities are common
in patients with celiac disease and usually resolve with a gluten-free
diet. We investigated the occurrence of celiac disease in patients
with severe liver failure.
Methods: Four patients with untreated celiac disease and
severe liver disease are described. Further, the occurrence of
celiac disease was studied in 185 adults with previous liver transplantation
using serum immunoglobulin A endomysial and tissue transglutaminase
antibodies in screening.
Results: Of the 4 patients with severe liver disease and
celiac disease, 1 had congenital liver fibrosis, 1 had massive
hepatic steatosis, and 2 had progressive hepatitis without apparent
origin. Three were even remitted for consideration of liver transplantation.
Hepatic dysfunction reversed in all cases when a gluten-free diet
was adopted. In the transplantation group, 8 patients (4.3%) had
celiac disease. Six cases were detected before the operation:
3 had primary biliary cirrhosis, 1 had autoimmune hepatitis, 1
had primary sclerosing cholangitis, and 1 had congenital liver
fibrosis. Only 1 patient had maintained a long-term strict gluten-free
diet. Screening found 2 cases of celiac disease, 1 with autoimmune
hepatitis and 1 with secondary sclerosing cholangitis.
Conclusions: The possible presence of celiac disease should
be investigated in patients with severe liver disease. Dietary
treatment may prevent progression to hepatic failure, even in
cases in which liver transplantation is considered.
The association between hepatitis C infection and survival
after orthotopic liver transplantation
L. M. Forman, J. D. Lewis, J. A. Berlin, H. I. Feldman, M. R.
Lucey
Background & Aims: The effect of hepatitis C viral
(HCV) infection on patient and allograft survival after orthotopic
liver transplantation is controversial. Hepatitis C recurrence
after transplant is inevitable, but studies to date have not found
a survival difference between recipients with and without HCV.
Methods: Using data from the United Network for Organ Sharing,
we performed a retrospective cohort study of 11,036 patients who
underwent 11,791 liver transplants between 1992 and 1998. The
hazard rates of patient and allograft survival for patients who
were HCV-positive as compared with patients who were HCV-negative
were assessed by proportional-hazards analysis, with adjustment
for potential confounding variables, including donor, recipient,
and transplant center characteristics.
Results: Liver transplantation in HCV-positive recipients
was associated with an increased rate of death (hazard ratio,
1.23; 95% confidence interval [CI], 1.121.35) and allograft
failure (hazard ratio, 1.30; 95% CI, 1.211.39), as compared
with transplantation in HCV-negative recipients. This reduction
in survival persisted after adjusting for potential confounders.
There was an interaction between HCV and sex (P < 0.001)
with the effect of HCV on survival being most pronounced in female
recipients (patient survival hazard ratio, 1.56; 95% CI, 1.351.81;
allograft survival hazard ratio, 1.51; 95% CI, 1.341.70).
Conclusions: HCV infection significantly impairs patient
and allograft survival after liver transplantation.
Occurrence of hepatopulmonary syndrome in BuddChiari syndrome
and the role of venous decompression
B. K. De, S. Sen, P. K. Biswas, S. K. Mandal, D. Das, U. Das,
S. Guru, K. Bandyopadhyay
Background & Aims: Hepatopulmonary syndrome (HPS) has
been predominantly detected in cirrhotic patients and rarely in
patients with noncirrhotic portal hypertension. The aim of this
study was to determine the occurrence of HPS in patients with
BuddChiari syndrome (only anecdotal reports available) and
evaluate the role of venous decompression in its reversal.
Methods: Twentynine consecutive cases of BuddChiari
syndrome without primary cardiopulmonary disease were investigated
by air contrast echocardiography and arterial blood gas analysis.
Venous decompression (e.g., by balloon cavoplasty) was attempted
when feasible.
Results: Eight cases (27.6%) of HPS and 9 cases (31.0%)
with positive contrast echocardiography but unimpaired oxygenation
were detected. Duration of disease was longer (P = 0.026)
among those with positive contrast echocardiography. Cavoplasty
reversed 4 of 5 cases of HPS and 2 of 2 cases with positive contrast
echocardiography alone. Venous decompression by drainage of amebic
liver abscess (which was compressing hepatic venous outflow) also
reversed 1 case of HPS. HPS was relieved by venous decompression
in 5 of 6 cases.
Conclusions: HPS developed in a substantial fraction of
our patients with BuddChiari syndrome, with positive contrast
echocardiography occurring mainly in the benign, slowly progressing
variety. Venous decompression showed promise in reversing such
cases.
6-MP metabolite profiles provide a biochemical explanation
for 6-MP resistance in patients with inflammatory bowel disease
M. C. Dubinsky, H. Yang, P. V. Hassard, E. G. Seidman, L. Y. Kam,
M. T. Abreu, S. R. Targan, E. A. Vasiliauskas
Background & Aims: Approximately 40% of inflammatory
bowel disease (IBD) patients fail to benefit from 6-mercaptopurine
(6-MP)/azathioprine (AZA). Recent reports suggest 6-thioguanine
nucleotide (6-TGN) levels (>235) independently correlate with
remission. An inverse correlation between 6-TGN and thiopurine
methyltransferase (TPMT) has been described. The objectives of
this study were to determine whether dose escalation optimizes
both 6-TGN levels and efficacy in patients failing therapy because
of subtherapeutic 6-TGN levels and its effect on TPMT.
Methods: Therapeutic efficacy and adverse events were recorded
at baseline and upon reevaluation after dose escalation in 51
IBD patients. 6-MP metabolite levels and TPMT activity were recorded
blinded to clinical information.
Results: Fourteen of 51 failing 6-MP/AZA at baseline achieved
remission upon dose escalation, which coincided with significant
rises in 6-TGN levels. Despite increased 6-MP/AZA doses, 37 continued
to fail therapy at follow-up. Dose escalation resulted in minor
changes in 6-TGN, yet a significant increase in 6-methylmercaptopurine
ribonucleotides (6-MMPR) (P 0.01) and 6-MMPR:6-TGN ratio
(P < 0.001). 6-MMPR rises were associated with dose-dependent
hepatotoxicity in 12 patients (24%). TPMT was not influenced by
dose escalation.
Conclusions: Serial metabolite monitoring identifies a
novel phenotype of IBD patients resistant to 6-MP/AZA therapy
biochemically characterized by suboptimal 6-TGN and preferential
6-MMPR production upon dose escalation.
Effects of long-term propranolol and octreotide on postprandial
hemodynamics in cirrhosis: A randomized, controlled trial
J. D. Vorobioff, M. Gamen, D. Kravetz, E. Picabea, R. Villavicencio,
J. Bordato, A. Ruf, F. Bessone, G. Romero, J. Palazzi, A. Nicora,
M. Passamonti, H. Tanno
Background & Aims: Postprandial increases in portal
pressure may influence esophageal variceal rupture. The effects
of chronic propranolol and octreotide (100 and 200 µg subcutaneously
in a single dose) on postprandial hemodynamics were evaluated.
Methods: First study: 36 cirrhotic patients were studied
at baseline and 30 and 60 minutes after a standard meal and then
treated with propranolol (139 ± 9 mg/d during 39 ±
2 days). Second study: After baseline measurements, patients were
randomized into 3 groups: (1) placebo, (2) octreotide (100 µg),
or (3) octreotide (200 µg) (n = 12 for each group). Thirty
minutes postinjection a new baseline was established and measurements
were repeated 30 and 60 minutes after the meal.
Results: First study: Baseline portal pressure was 18.1
± 1.2 mm Hg, 30 and 60 minutes after the meal it was 21.5
± 0.8 mm Hg and 20.5 ± 0.8 mm Hg, respectively (both
P < 0.01 vs. baseline). Cardiac index (CI) was 4.5 ±
0.2, 4.8 ± 0.2, and 4.9 ± 0.2 L · min1
· m2, respectively (both P < 0.05 vs. baseline).
Peripheral vascular resistance was 1012 ± 56, 902 ±
51 (P = NS), and 884 ± 49 dynes · sec ·
cm5 (P< 0.05 vs. baseline), respectively. Second
study: Propranolol and placebo did not blunt postprandial increase
in portal pressure. Octreotide (100 µg) partially ameliorated
postprandial increase in portal pressure. Octreotide (200 µg)
significantly enhanced the portal hypotensive effect of propranolol
and blunted the postprandial increase in portal pressure.
Conclusions: Octreotide blunts postprandial increase in
portal pressure not prevented by long-term propranolol administration.
Terlipressin in patients with cirrhosis and type 1 hepatorenal
syndrome: A retrospective multicenter study
R. Moreau, F. Durand, T. Poynard, C. Duhamel, J.-P. Cervoni, P.
Ichaï, A. Abergel, C. Halimi, M. Pauwels, J.-P. Bronowicki,
E. Giostra, C. Fleurot, D. Gurnot, O. Nouel, P. Renard, M. Rivoal,
P. Blanc, D. Coumaros, S. Ducloux, S. Levy, A. Pariente, J.-M.
Perarnau, J. Roche, M. Scribe-Outtas, D. Valla, B. Bernard, D.
Samuel, J. Butel, A. Hadengue, A. Platek, D. Lebrec, J.-F. Cadranel
Background & Aims: Type 1 hepatorenal syndrome (HRS)
is a severe complication of cirrhosis associated with a short
median survival time (<2 weeks). Although the administration
of terlipressin improves renal function, its effect on survival
is unknown. This study investigated predictive factors of survival
in patients with type 1 HRS treated with terlipressin.
Methods: Ninety-nine patients with type 1 HRS treated with
terlipressin in 24 centers were retrospectively studied. Terlipressin-induced
improved renal function was defined as a decrease in serum creatinine
value to <130 µmol/L or a decrease of at least 20% at
the end of treatment.
Results: At inclusion, the ChildPugh score was 11.8
± 1.6 (mean ± SD). Terlipressin (3.2 ± 1.3
mg/day) was administered for 11 ± 12 days. Renal function
improved in 58% of patients (serum creatinine decreased by 46%
± 17% from 272 ± 114 µmol/L). Median survival
time was 21 days. Survival rate was 40% at 1 month. Multivariate
analysis showed that improved renal function and ChildPugh
score 11 at inclusion were independent predictive factors of survival
(P < 0.0001 and 0.02, respectively). Thirteen patients
underwent liver transplantation (92 ± 95 days after HRS
onset), 10 of whom had received terlipressin and had had improved
renal function.
Conclusions: This retrospective uncontrolled study shows
that in patients with type 1 HRS, terlipressin-induced improved
renal function is associated with an increase in survival. Thus,
a randomized trial investigating the effect of terlipressin on
survival in patients with type 1 HRS should be performed.
Effect of iron depletion in carbohydrate-intolerant patients
with clinical evidence of nonalcoholic fatty liver disease
F. S. Facchini, N. W. Hua, R. A. Stoohs
Background & Aims: Increased body iron, genetic hemochromatosis
(GH) mutations, and nonalcoholic fatty liver disease (NAFLD) tend
to cluster in carbohydrate-intolerant patients. In an attempt
to further clarify the interrelationships among these conditions,
we studied 42 carbohydrate-intolerant patients who were free of
the common GH mutations C282Y and H63D, and had a serum iron saturation
lower than 50%.
Methods: We measured body iron stores, and induced iron
depletion to a level of near-iron deficiency (NID) by quantitative
phlebotomy.
Results: In the 17 patients with clinical evidence of NAFLD,
we could not demonstrate supranormal levels of body iron (1.6
± 0.2 vs. 1.4 ± 0.2 g; P = 0.06). However,
at NID, there was a 40%55% improvement (P = 0.050.0001)
of both fasting and glucose-stimulated plasma insulin concentrations,
and near-normalization of serum alanine aminotransferase activity
(from 61 ± 5 to 32 ± 2 IU/L; P < 0.001).
Conclusions: These results reflect the insulin-sparing
effect of iron depletion and indicate a key role of iron and hyperinsulinemia
in the pathogenesis of NAFLD.
Hereditary nonpolyposis colorectal cancer in young colorectal
cancer patients: High-risk clinic versus population-based registry
J. P. Terdiman, T. R. Levin, B. A. Allen, J. R. Gum, Jr., A. Fishbach,
P. G. Conrad, G. A. Miller, V. Weinberg, R. Bachman, J. Bergoffen,
A. Stembridge, N. W. Toribara, M. H. Sleisenger, Y. S. Kim
Background & Aims: Early onset colorectal cancer (CRC)
is an important feature of hereditary nonpolyposis colorectal
cancer (HNPCC). We sought to compare rates of genetically defined
HNPCC among individuals with early onset CRC drawn from a high-risk
clinic and a population-based cancer registry.
Methods: Probands with CRC diagnosed before 36 years of
age were enrolled from a high-risk CRC clinic at the University
of California, San Francisco (UCSF), and a population-based Kaiser
Permanente (KP) Health Plan cancer registry. Probands provided
cancer family histories and tumors for microsatellite instability
(MSI) testing and MSH2/MLH1 protein immunostaining. Germline MSH2
and MLH1 mutational analysis was performed.
Results: Forty-three probands were enrolled from UCSF and
23 from KP. The UCSF and KP probands had similar median age of
onset of CRC (30 vs. 31 years) and the percentage with any personal
or family history of another HNPCC-related cancer (70% vs. 74%).
However, 28 of 40 (70%) of the UCSF tumors were MSI-H compared
with 6 of 18 (33%) of KP tumors (P = 0.01), and 13 germline
MSH2 or MLH1 mutations were found in the UCSF group
compared with 0 in the KP group (P = 0.0001). In a multivariate
analysis, institution (P = 0.002) and the total number
of colorectal cancers in the family (P = 0.0001) were independent
predictors of MSH2 or MLH1 mutation.
Conclusions: Family history of cancer is an important feature
of HNPCC, even among individuals with early onset CRC. Caution
must be undertaken when extrapolating data regarding HNPCC from
high-risk clinic populations to the general population.
Basic Research
Dietary sphingomyelin suppresses intestinal cholesterol
absorption by decreasing thermodynamic activity of cholesterol
monomers
E. R. M. Eckhardt, D. Q.-H. Wang, J. M. Donovan, M. C. Carey
Background & Aims: In humans, cholesterol absorbed
from the intestine contributes appreciably to serum cholesterol
levels. We hypothesized that cholesterol thermodynamic activity
(At) would predict bioavailability of cholesterol monomers in
intestinal content, and that natural dietary phospholipids exhibiting
high affinity for cholesterol would reduce its absorption.
Methods: Cholesterol At was determined by measuring partitioning
of monomeric cholesterol from aqueous solutions of taurocholate,
cholesterol, and either milk sphingomyelin (MSM), dipalmitoyl
phosphatidylcholine (DPPC), or egg yolk phosphatidylcholine (EYPC)
into wafers of polymerized silicone. Cholesterol absorption from
the same mixtures was tested with monolayers of Caco-2 cells.
For in vivo absorption studies (employing male C57L/J mice), we
used the fecal dual isotope method during dietary enrichment with
MSM, DPPC, or EYPC at varying dose levels.
Results: Cholesterol At values were reduced significantly
in MSM- and DPPC-containing systems compared with EYPC and correlated
positively with reduced uptake and esterification of cholesterol
by Caco-2 cells. Mice fed chow absorbed 31.4% ± 6.9% (mean
± SEM) cholesterol, whereas enrichment with MSM or DPPC
led to dose-dependent decreases in cholesterol absorption; even
at 0.1% MSM, cholesterol absorption was reduced by 20.4% ±
15.4% (P < 0.05, n = 6).
Conclusions: Different phospholipids have distinct effects
on micellar cholesterol At, which predicts cholesterol uptake
by enterocytes in vitro as well as in vivo. Natural phospholipids
with high affinity for cholesterol, as evidenced particularly
by sphingomyelin, decrease At and curtail intestinal cholesterol
absorption.
Ivermectin used in percutaneous drug injection method for the
treatment of liver hydatid disease in sheep
M. Hokelek, B. A. Deger, E. Deger, E. Tutar, M. Sunbul
Background & Aims: Ivermectin is a macrocyclic lactone
(avermectins) produced by the actinomycete Streptomyces avermitilis.
In this experimental study, the effectiveness of intracystic injection
of ivermectin was studied as a new approach of percutaneous treatment
of cystic echinococcosis.
Methods: Twelve naturally infected sheep were selected
and divided into 2 subgroups: treatment group (n = 9) and control
group (n = 3). In the treatment group, approximate volume of ivermectin
solution needed to achieve an intracystic concentration of 10
µg/mL was injected into cysts, but in the control group,
sterile distillated water was applied. No reaspiration was performed
at all.
Results: In the following period of 6 months, repeated
sonography revealed a significant decrease in cyst sizes and progressive
solidification of the cysts in the treatment group. In the control
group, volumes of the cysts were increased. No major complications
occurred during or after the procedure. After 6 months, all sheep
were killed and examined for macroscopic and microscopic changes.
Pathologic examination in the treatment group showed pericyst
hyalinization, inflammatory cells in the cyst wall, degeneration
of laminated and germinal membrane, and necrotic material in the
cyst cavity. No viable protoscolices or daughter cysts were observed.
Conclusions: Percutaneous treatment of cystic echinococcosis
with ivermectin as a scolicidal agent seems to be effective in
this animal model.
Heat stress prevents impairment of bile acid transport in endotoxemic
rats by a posttranscriptional mechanism
U. Bolder, A. Schmidt, L. Landmann, V. Kidder, S. Tange, K.-W.
Jauch
Background & Aims: Endotoxemia leads to reduction of
bile acid transporters in the hepatocyte membrane and impaired
bile acid transport. Because heat stress ameliorates other sequelae
of endotoxemia, studies were performed to determine whether heat
stress would correct deficient bile acid transport caused by endotoxin.
Methods: Body temperature of rats was elevated to 42°C
for 10 minutes. Lipopolysaccharide was injected after different
time intervals, and maximal transport for cholyltaurine was measured
in perfused rat livers. Sodium-dependent and -independent uptake
was studied in isolated hepatocytes. Protein expression, messenger
RNA levels, and tissue distribution of the bile acid transporters
sodium taurocholate cotransporting protein (ntcp) and bile
salt export pump (bsep) were also analyzed.
Results: In the perfused liver, cholyltaurine transport
was reduced by 59% by endotoxin, but transport was not reduced
when heat stress was applied 2 hours before injection of lipopolysaccharide.
The protective effect coincided with maximal expression of heat
shock proteins 70 and 25. Sodium-dependent and -independent transport
was preserved by heat stress. Expression of bile acid transporters
in plasma membrane fractions was reduced after injection of lipopolysaccharide
but not if lipopolysaccharide was preceded by heat stress. In
contrast, messenger RNA levels of bile acid transporters were
not preserved by heat stress.
Conclusions: Heat stress preserves bile acid transporters
during endotoxemia by a posttranscriptional mechanism.
Neutrophils and NADPH oxidase mediate intrapancreatic trypsin
activation in murine experimental acute pancreatitis
A. S. Gukovskaya, E. Vaquero, V. Zaninovic, F. S. Gorelick, A.
J. Lusis, M.-L. Brennan, S. Holland, S. J. Pandol
Background & Aims: Intrapancreatic activation of digestive
enzymes is a key event in the parenchymal cell injury of pancreatitis.
We hypothesized that neutrophils recruited to the pancreas during
pancreatitis may contribute to such activation.
Methods: To cause experimental pancreatitis, rats and mice
were treated with high doses of cerulein. Activation of the digestive
enzyme, trypsin, was measured in pancreatic homogenates using
a fluorogenic assay and localized immunocytochemically with antibody
to trypsin-activation peptide (TAP).
Results: Compared with controls, rats depleted of neutrophils
with antineutrophil serum exhibited a marked attenuation in intrapancreatic
trypsin activation and acinar cell TAP labeling induced by high-dose
cerulein. To examine the mechanism, mice deficient in either nicontinamide
adenine dinucleotide phosphate (NADPH) oxidase, or myeloperoxidase
(MPO) were studied for trypsin activation. Mice deficient in NADPH
oxidase exhibited attenuation of the cerulein-induced trypsin
activation, but those deficient in MPO did not. Using measurements
of Western blot analysis, generation of reactive oxygen species,
and immunocytochemistry, we demonstrated the NADPH oxidase activity
is in neutrophils and not pancreatic acinar tissue.
Conclusions: The results demonstrate a novel role for neutrophils
infiltrating the pancreas in pathologic activation of digestive
enzymes in acute pancreatitis and indicate that this effect is
mediated by products of NADPH oxidase.
Bile acids induce cyclooxygenase-2 expression via the epidermal
growth factor receptor in a human cholangiocarcinoma cell line
J.-H. Yoon, H. Higuchi, N. W. Werneburg, S. H. Kaufmann, G. J.
Gores
Background & Aims: Although bile acids have been implicated
in colon cancer development, their role in biliary tract carcinogenesis
remains unexplored. Because receptor tyrosine kinases and cyclooxygenase
(COX)-2 have been implicated in carcinogenesis, we examined the
hypothesis that bile acids modulate these enzymes in KMBC cells,
a human cholangiocarcinoma cell line.
Methods: The effect of bile acids on epidermal growth factor
receptor (EGFR) stimulation, mitogen-activated protein kinase
(MAPK) activation, and COX-2 expression was evaluated.
Results: Bile acids both induced EGFR phosphorylation and
enhanced COX-2 protein expression. Bile acidinduced EGFR
phosphorylation was associated with subsequent activation of MAPK
p42/44, p38, and c-Jun-N-terminal kinase (JNK). The MAPK inhibitors,
PD098059 for MAP or extracellular signal-regulated kinase 1, SB203580
for p38, and BAY 37-9751 for Raf-1, blocked COX-2 induction by
bile acids. However, inhibition of JNK activity did not block
bile acidmediated COX-2 induction.
Conclusions: The results show that EGFR is activated by
bile acids and functions to induce COX-2 expression by an MAPK
cascade. This induction of COX-2 may participate in the genesis
and progression of cholangiocarcinomas.
SNAP-25, a SNARE protein, inhibits two types of K+ channels
in esophageal smooth muscle
J. Ji, A. M. F. Salapatek, H. Lau, G. Wang, H. Y. Gaisano, N.
E. Diamant
Background & Aims: The plasma membraneassociated
soluble N-ethylmaleimidesensitive factors attachment protein
receptors (SNAREs), synaptosome-associated protein of 25 kilodaltons
(SNAP-25), and syntaxin 1A, have been found to physically interact
with and functionally modify membrane-spanning ion channels. Studies
were performed in cat esophageal body and lower esophageal sphincter
(LES) smooth muscle to (1) show the presence of SNAP-25, and (2)
determine whether SNAP-25 affects K+ channel activity.
Methods: Single circular muscle cells from the esophageal
body and sphincter were studied. Cellular localization of SNAP-25
and K+ channel activity were assessed.
Results: SNAP-25 was found in the plasma membrane of all
regions examined. Outward K+ currents in body circular muscle
were mainly composed of large conductance Ca2+-activated channel
currents (KCa, 40.1%) and delayed rectifier K+ channel currents
(KV, 54.2%). Microinjection of SNAP-25 into muscle cells caused
a dose-dependent inhibition of both outward K+ currents, maximal
44% at 108 mol/L. Cleavage of endogenous SNAP-25 by dialyzing
botulinum neurotoxin A into the cell interior resulted in a 35%
increase in outward currents.
Conclusions: SNAP-25 protein is present in esophageal smooth
muscle cells, and inhibits both KV and KCa currents in circular
muscle cells. The findings suggest a role for SNAP-25 in regulation
of esophageal muscle cell excitability and contractility, and
point to potential new targets for treatment of esophageal motor
disorders.
Facilitation and attenuation of a visceral nociceptive reflex
from the rostroventral medulla in the rat
M. Zhuo, G. F. Gebhart
Background & Aims: Noxious inputs from somatic tissue
are subject to biphasic descending modulation from the rostroventral
medulla (RVM). In the present study, we investigated descending
facilitatory and inhibitory influences from the RVM on a visceral
nociceptive reflex.
Methods: The visceromotor response (VMR), a contraction
of peritoneal musculature during noxious colorectal distention
(80 mm Hg, 20 seconds), was quantified as the integrated electromyogram.
Results: At 22 sites in the RVM, electrical stimulation
produced biphasic effects, facilitating the VMR at low (5, 10,
and 25 µA) and inhibiting it at greater (>50 µA)
intensities of stimulation. Electrical stimulation at all intensities
tested (5200 µA) in other sites in the RVM only inhibited
(30 sites) or only facilitated (12 sites) the VMR to colorectal
distention. Activation of glutamatergic receptors in the RVM replicated
the effects of electrical stimulation. Reversible blockage (intraspinal
lidocaine injection) or irreversible transection of spinal funiculi
revealed that descending facilitatory influences from the RVM
were conveyed in the ventrolateral/ventral funiculus, whereas
descending inhibitory influences were contained in the dorsolateral
funiculi.
Conclusions: Spinal visceral nociceptive reflexes are subject
to facilitatory modulation from the RVM, providing the basis for
a mechanism by which visceral sensations can be enhanced from
supraspinal sites.
Interferon- activates multiple STAT signals and down-regulates
c-Met in primary human hepatocytes
S. Radaeva, B. Jaruga, F. Hong, W.-H. Kim, S. Fan, H. Cai, S.
Strom, Y. Liu, O. El-Assal, B. Gao
Background & Aims: Interferon (IFN)- therapy is currently
the primary choice for viral hepatitis and a promising treatment
for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes
respond poorly to IFN- stimulation. Thus, it is very important
to examine the IFN- signal pathway in primary human hepatocytes.
Methods: The IFN-activated signals and genes in primary
human hepatocytes and hepatoma cells were examined by Western
blotting and microarray analyses.
Results: Primary human hepatocytes respond very well to
IFN- stimulation as shown by activation of multiple signal transducer
and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple
genes. The differential response to IFN- stimulation in primary
human and mouse hepatocytes may be caused by expression of predominant
functional IFN- receptor 2c (IFNAR2c) in primary human hepatocytes
vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes.
Microarray analyses of primary human hepatocytes show that IFN-
up-regulates about 44 genes by over 2-fold and down-regulates
about 9 genes by 50%. The up-regulated genes include a variety
of antiviral and tumor suppressors/proapoptotic genes. The down-regulated
genes include c-myc and c-Met, the hepatocyte growth factor (HGF)
receptor. Down-regulation of c-Met is caused by IFN- suppression
of the c-Met promoter through down-regulation of Sp1 binding and
results in attenuation of HGF-induced signals and cell proliferation.
Conclusions: IFN- directly targets human hepatocytes, followed
by activation of multiple STATs and regulation of a wide variety
of genes, which may contribute to the antiviral and antitumor
activities of IFN- in human liver.
Proteinases and proteinase-activated receptor 2: A possible
role to promote visceral hyperalgesia in rats
A.-M. Coelho, N. Vergnolle, B. Guiard, J. Fioramonti, L. Bueno
Background & Aims: PAR-2s are highly expressed throughout
the gastrointestinal tract. These receptors are cleaved by trypsin
and mast cell tryptase and can be activated by peptides corresponding
to the tethered ligand of the receptor (SLIGRL-NH2 for rat). The
aim of this study was to determine whether colonic administration
of PAR-2 agonists affects visceral sensitivity to rectal distention
in conscious rats.
Methods: Abdominal contractions (a criteria of visceral
pain) were recorded in rats equipped with intramuscular electrodes.
Rectal distention was performed at various times after intracolonic
infusion of SLIGRL-NH2 and trypsin. Inflammation parameters and
permeability were followed in the colon after the intracolonic
injections. Fos expression at a spinal level (L4-L6) was also
studied 2 hours after intracolonic injection of SLIGRL-NH2.
Results: Rectal distention significantly increased abdominal
contractions starting at the RD volume of 0.8 mL. Intracolonic
injection of SLIGRL-NH2 (200 µg/rat) and trypsin (200 U/rat),
but not vehicle, LRGILS-NH2 (control peptide), boiled trypsin,
or SLIGRL-NH2 injected IP, significantly increased (P <
0.05) abdominal contractions for high volumes of distention, 10-
and 24-hour postinfusion. SLIGRL-NH2induced hyperalgesia
was inhibited by a NK1 receptor antagonist (SR 140333) but not
by indomethacin. Intracolonic injection of SLIGRL-NH2 elevated
spinal Fos expression and caused increased intestinal permeability
but did not cause detectable inflammation.
Conclusions: Intracolonic infusion of subinflammatory doses
of PAR-2 agonists activated spinal afferent neurons and produced
a delayed rectal hyperalgesia that involves changes in intestinal
permeability and the activation of NK1 receptors. These results
identify a possible role for proteinases and PAR-2 in the genesis
of visceral hyperalgesia.
Clostridium difficile toxin A triggers human colonocyte IL-8
release via mitochondrial oxygen radical generation
D. He, S. Sougioultzis, S. Hagen, J. Liu, S. Keates, A. C. Keates,
C. Pothoulakis, J. T. LaMont
Background & Aims: Clostridium difficile toxin
A causes mitochondrial dysfunction resulting in generation of
oxygen radicals and adenosine triphosphate (ATP) depletion. We
investigated whether mitochondrial dysfunction is involved in
nuclear factor B (NF-B) activation and interleukin (IL)-8 release
from toxin Aexposed enterocytes.
Methods: NF-B activation and IL-8 release in response to
toxin A were correlated with reactive oxygen intermediate (ROI)
generation and ATP production in HT-29 monolayers or HT-29 cells
exposed to ethidium bromide (EB) to inhibit mitochondrial function.
Results: HT-29 cells exposed to EB showed damaged mitochondria
and diminished resting levels of ATP. ROI production in EB-treated
cells exposed to toxin A for 30 minutes was significantly reduced.
Exposure of wild-type HT-29 cells to toxin A resulted in increased
oxygen radical generation and IL-8 production (P < 0.01
vs. control) that was inhibited by antioxidant pretreatment. Degradation
of IB was observed within 30 minutes of toxin exposure, before
ras homologue (Rho) glucosylation, and was followed by nuclear
translocation of NF-B. Toxin A did not increase IL-8 levels in
EB-treated cells, whereas IL-8 release in response to IL-1 was
not affected.
Conclusions: Our data support an early role for mitochondria-derived
ROIs in stimulation of IL-8 release from colonocytes by toxin
A. ROI generation is independent of Rho inactivation and involves
nuclear translocation of NF-B before release of IL-8.
Corticosteroids modulate the secretory processes of the rat
intrahepatic biliary epithelium
D. Alvaro, A. Gigliozzi, L. Marucci, G. Alpini, B. Barbaro, R.
Monterubbianesi, L. Minetola, M. Grazia Mancino, J. F. Medina,
A. F. Attili, A. Benedetti
Background & Aims: We investigated the expression of
glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium
and the role of corticosteroids in the regulation of cholangiocyte
secretion.
Methods: GcR was studied by immunohistochemistry, reverse-transcription
polymerase chain reaction, and Western blots. The effects of dexamethasone
and budesonide on biliary bicarbonate excretion and H+/HCO3
transport processes were investigated in bile fistula rats, isolated
intrahepatic bile duct units (IBDUs), and purified cholangiocytes.
Results: GcRs were expressed by rat cholangiocytes. Although
acute administration of corticosteroids showed no effect, treatment
for 2 days with dexamethasone or budesonide increased (P
< 0.05) biliary bicarbonate concentration and secretion, which
were blocked by the specific GcR antagonist, RU-486. IBDUs isolated
from rats treated with dexamethasone or budesonide showed an increased
(P < 0.05) activity of the Na+/H+ exchanger (NHE1 isoform)
and Cl/HCO3 exchanger (AE2 member), which was blocked
by RU-486. Protein expression of NHE1 and AE2 and messenger RNA
for NH1 but not AE2 were increased (P < 0.05) in isolated
cholangiocytes by dexamethasone treatment.
Conclusions: The intrahepatic biliary epithelium expresses
GcR and responds to corticosteroids by increasing bicarbonate
excretion in bile. This is caused by corticosteroid-induced enhanced
activities and protein expression of transport processes driving
bicarbonate excretion in the biliary epithelium.
Enteroinvasive bacteria alter barrier and transport properties
of human intestinal epithelium: Role of iNOS and COX-2
S. Resta-Lenert, K. E. Barrett
Background & Aims: Various invasive pathogens cause
diarrhea, but the mechanism(s) are poorly understood. We hypothesized
that nitric oxide and prostaglandins might modulate chloride secretory
and barrier properties of the infected intestinal epithelium and
that diarrhea is caused, in part, by altered expression of inducible
NO synthase (iNOS) and cyclooxygenase 2 (COX-2).
Methods: Studies were conducted in human intestinal epithelial
cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected
with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella
dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus
thermophilus [ST] and Lactobacillus acidophilus [LA]).
Infected cells and controls were tested for transepithelial resistance,
chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate
and adenosine 3',5'-cyclic monophosphate, and protein expression.
Results: Cells infected with EIEC or SD, but not uninfected
controls or ST/LA-exposed monolayers, showed a progressive reduction
in transepithelial resistance starting at 612 hours. Infected
HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an
increase in total nitrite. Expression of iNOS, and consequently
COX-2, was also increased, followed by increased production of
prostaglandins and cyclic nucleotides. Furthermore, basal and
stimulated chloride secretory responses to various agonists were
enhanced in HT29/cl.19A and Caco-2 cells after infection with
enteroinvasive bacteria, and this effect was reversed for some
agonists by iNOS or COX-2 inhibitors. Increased expression of
cystic fibrosis transmembrane conductance regulator and NKCC1
was also observed in EIEC or SD-infected cells vs. controls, secondary
to NO synthase activity.
Conclusions: Up-regulation of iNOS and COX-2 by enteroinvasive
bacteria can modulate chloride secretion and barrier function
in intestinal epithelial cells. Thus, these enzymes represent
possible therapeutic targets in infectious diarrhea.
The type II inositol 1,4,5-trisphosphate receptor can trigger
Ca2+ waves in rat hepatocytes
K. Hirata, T. Pusl, A. F. O'Neill, J. A. Dranoff, M. H. Nathanson
Background & Aims: Ca2+ regulates cell functions through
signaling patterns such as Ca2+ oscillations and Ca2+ waves. The
type I inositol 1,4,5-trisphosphate receptor is thought to support
Ca2+ oscillations, whereas the type III inositol 1,4,5-trisphosphate
receptor is thought to initiate Ca2+ waves. The role of the type
II inositol 1,4,5-trisphosphate receptor is less clear, because
it behaves like the type III inositol 1,4,5-trisphosphate receptor
at the single-channel level but can support Ca2+ oscillations
in intact cells. Because the type II inositol 1,4,5-trisphosphate
receptor is the predominant isoform in liver, we examined whether
this isoform can trigger Ca2+ waves in hepatocytes.
Methods: The expression and distribution of inositol 1,4,5-trisphosphate
receptor isoforms was examined in rat liver by immunoblot and
confocal immunofluorescence. The effects of inositol 1,4,5-trisphosphate
on Ca2+ signaling were examined in isolated rat hepatocyte couplets
by using flash photolysis and time-lapse confocal microscopy.
Results: The type II inositol 1,4,5-trisphosphate receptor
was concentrated near the canalicular pole in hepatocytes, whereas
the type I inositol 1,4,5-trisphosphate receptor was found elsewhere.
Stimulation of hepatocytes with vasopressin or directly with inositol
1,4,5-trisphosphate induced Ca2+ waves that began in the canalicular
region and then spread to the rest of the cell. Inositol 1,4,5-Trisphosphateinduced
Ca2+ signals also increased more rapidly in the canalicular region.
Hepatocytes did not express the ryanodine receptor, and cyclic
adenosine diphosphateribose had no effect on Ca2+ signaling
in these cells.
Conclusions: The type II inositol 1,4,5-trisphosphate receptor
establishes a pericanalicular trigger zone from which Ca2+ waves
originate in hepatocytes.
Chemoprevention of esophageal adenocarcinoma by COX-2 inhibitors
in an animal model of Barrett's esophagus
N. S. Buttar, K. K. Wang, O. Leontovich, J. Y. Westcott, R. J.
Pacifico, M. A. Anderson, K. K. Krishnadath, L. S. Lutzke, L.
J. Burgart
Background & Aims: Carcinogenesis in Barrett's esophagus
(BE) is associated with an increased expression of cyclooxygenase
(COX) 2. However, there has been no direct evidence that inhibition
of COX-2 prevents cancer in BE. We studied the effect of MF-Tricyclic,
a selective COX-2 inhibitor, on the development of BE and adenocarcinoma
in a rat model.
Methods: Four weeks after esophagojejunostomy, 105 Sprague-Dawley
rats were randomized to a chow containing MF-Tricyclic or Sulindac,
or a placebo. Ninety-six (92%) rats completed the study and were
sacrificed at 28 ± 2 weeks. The animals were assessed for
the presence of cancer, tumor volume, BE, degree of inflammation,
and COX-2 expression and activity.
Results: MF-Tricyclic and Sulindac reduced the relative
risk of development of esophageal cancer by 55% (95% confidence
interval [CI] = 43%66%, P < 0.008) and by 79% (95%
CI = 68%87%, P < 0.001), respectively, compared
with controls. No significant differences were noted in the risk
of esophageal cancer between the MF-Tricyclic and the Sulindac
group (P = 0.34). The median tumor volume was not significantly
different among the 3 groups (P = 0.081). Moderate to severe
degree of inflammation was significantly more common (P
= 0.005) in the control compared with the MF-Tricyclic and the
Sulindac group; however, the prevalence of BE was not significantly
different between groups (P = 0.98). Rats in the control
group had higher tissue PGE2 level compared with the MF-Tricyclic
and Sulindac groups (P = 0.038).
Conclusions: Selective and nonselective COX-2 inhibitors
can inhibit inflammation, COX-2 activity, and development of adenocarcinoma
induced by reflux. This provides direct evidence that COX-2 inhibitors
may have chemopreventive potential in BE.
p16 Inactivation by methylation of the CDKN2A promoter occurs
early during neoplastic progression in Barrett's esophagus
Y.-S. Bian, M.-C. Osterheld, C. Fontolliet, F. T. Bosman, J. Benhattar
Background & Aims: The potential role of p16 inactivation
by CDKN2A/p16 promoter hypermethylation and/or loss of heterozygosity
(LOH) of the CDKN2A gene was investigated in neoplastic progression
of Barrett's esophagus.
Methods: CDKN2A promoter hypermethylation was studied by
methylation sensitive single-strand conformation analysis and
sequencing using bisulfite modified DNA in Barrett's esophageal
adenocarcinomas, premalignant lesions, and normal squamous esophageal
epithelium. All of the lesions of interest were sampled by microdissection
from paraffin-embedded fixed tissue sections.
Results: No methylation of the CDKN2A promoter was found
in normal esophageal squamous cell epithelia, whereas methylation
was detected in 18 of 22 (82%) adenocarcinomas and 10 of 33 (30%)
premalignant lesions, including 4 of 12 (33%) samples with intestinal
metaplasia only. LOH at the CDKN2A gene locus was found in 68%
of adenocarcinomas and in 55% of premalignant lesions. Of 28 samples
without p16 immunoreactivity, 25 (89%) showed CDKN2A promoter
hypermethylation with or without LOH of CDKN2A. Only 2 (8%) samples
expressing p16 protein were found to be methylated; these showed
a mixture of completely methylated and unmethylated CDKN2A promoters.
In 7 of 19 (37%) informative samples without LOH of CDKN2A, the
CDKN2A promoter was found to be methylated at both alleles. Loss
of p16 protein expression was strongly associated with CDKN2A
promoter hypermethylation (P < 0.00001), but not with
LOH (P = 0.33).
Conclusions: Our results indicate that methylation of the
CDKN2A promoter is the predominant mechanism for p16 inactivation.
This hypermethylation is a very common event in esophageal adenocarcinoma
and occurs as early as metaplasia.
Leukocyte recruitment in colon cancer: Role of cell adhesion
molecules, nitric oxide, and transforming growth factor 1
X. Bessa, J. I. Elizalde, F. Mitjans, V. Piñol, R. Miquel,
J. Panés, J. Piulats, J. M. Piqué, A. Castells
Background & Aims: A deficient leukocyte recruitment
has been suggested in tumor vasculature, but little is known about
the underlying molecular mechanism. To characterize leukocyte-endothelium
interaction in experimental colon cancer, quantify the main endothelial
cell adhesion molecules (CAMs), and evaluate the effect of tumor-derived
products.
Methods: Leukocyte recruitment was assessed by intravital
videomicroscopy in mice bearing HT29derived tumors. Endothelial
CAMs were measured using the dual-radiolabeled antibody technique.
The role of molecules mediating leukocyte rolling (P-, E-, and
L-selectin) or adhesion (intercellular adhesion molecule 1 [ICAM-1]
and vascular cell adhesion molecule 1 [VCAM-1]) carcinoembryonic
antigen (CEA), and transforming growth factor (TGF) 1 was assessed
through immunoblockade, whereas participation of nitric oxide
(NO) and cyclooxygenase (COX) metabolites were evaluated by means
of nonselective and selective inhibition.
Results: Basal and lipopolysaccharide-stimulated leukocyte
rolling and adhesion were markedly reduced in tumor vasculature.
ICAM-1 immunoblockade prevented leukocyte adhesion in both tumor
and nontumor microvessels. Neither baseline nor LPS-induced endothelial
ICAM-1, P-, and E-selectin expression in tumors were reduced with
respect to nontumor vasculature. Although VCAM-1 expression was
reduced in tumor endothelium, immunoneutralization of VCAM-1 failed
to reverse LPS-induced leukocyte recruitment in this setting.
CEA immunoblockade and COX inhibition did not modify the deficient
leukocyte rolling. Nonselective NO inhibition partially reversed
the defective adhesion response in tumor microvessels. Finally,
TGF-1 immunoblockade partially and selectively restored impaired
leukocyte rolling and adhesion in tumor microvessels.
Conclusions: Impaired leukocyte recruitment in tumor vasculature
cannot be attributed to a depressed expression of the main CAMs.
Selective restoration after NO inhibition and TGF-1 immunoblockade
suggests involvement of both molecules in this phenomenon.
Case Report TOP
Acquired myopathic intestinal pseudo-obstruction may be due
to autoimmune enteric leiomyositis
T. H. Ruuska, R. Karikoski, V. V. Smith, P. J. Milla
We describe a previously healthy boy who developed intestinal
pseudo-obstruction following an episode of gastroenteritis at
age 2 years. At presentation, the patient had mildly raised erythrocyte
sedimentation rate and C-reactive protein level, and elevated
antineutrophil cytoplasmic antibodies, antinuclear anti-DNA, and
anti-smooth muscle antibodies. His electrogastrography was myopathic
with no dominant frequency. First full-thickness intestinal biopsies
showed a T lymphocytic myositis, particularly in the circular
muscle. Steroid therapy resulted in clinical remission; cessation
of steroids, in relapse. Further full-thickness biopsies showed
an initial reduction in -smooth muscle actin immunostaining in
circular muscle myocytes and later atrophy and disappearance of
many myocytes. Vascular and the remaining enteric smooth muscle
cells showed HLA-DR and intercellular adhesion molecule 1 expression.
These observations demonstrate the ability of enteric myocytes
to take part in an inflammatory response and to change their phenotype,
allowing them to act as antigen-presenting cells and to activate
T cells. This and possible cytokine production by the myocytes
play a role in their own destruction. This process responded to
immunosuppressive therapy.
Special Reports and Reviews TOP
Systematic review of the comorbidity of irritable bowel syndrome
with other disorders: What are the causes and implications?
W. E. Whitehead, O. Palsson, K. R. Jones
Background & Aims: Comorbid or extraintestinal symptoms
occur frequently with irritable bowel syndrome and account for
up to three fourths of excess health care visits. This challenges
the assumption that irritable bowel is a distinct disorder. The
aims of this study were to (1) assess comorbidity in 3 areas:
gastrointestinal disorders, psychiatric disorders, and nongastrointestinal
somatic disorders; and (2) evaluate explanatory hypotheses.
Methods: The scientific literature since 1966 in all languages
cited in Medline was systematically reviewed.
Results: Comorbidity with other functional gastrointestinal
disorders is high and may be caused by shared pathophysiological
mechanisms such as visceral hypersensitivity. Psychiatric disorders,
especially major depression, anxiety, and somatoform disorders,
occur in up to 94%. The nongastrointestinal nonpsychiatric disorders
with the best-documented association are fibromyalgia (median
of 49% have IBS), chronic fatigue syndrome (51%), temporomandibular
joint disorder (64%), and chronic pelvic pain (50%).
Conclusions: Multivariate statistical analyses suggest
that these are distinct disorders and not manifestations of a
common somatization disorder, but their strong comorbidity suggests
a common feature important to their expression, which is most
likely psychological. Some models explain the comorbidity of irritable
bowel with other disorders by suggesting that each disorder is
the manifestation of varying combinations of interacting physiological
and psychological factors. An alternative hypothesis is that the
irritable bowel diagnosis is applied to a heterogeneous group
of patients, some of whom have a predominantly psychological etiology,
whereas others have a predominantly biological etiology, and that
the presence of multiple comorbid disorders is a marker for psychological
influences on etiology.
Validity of randomized clinical trials in gastroenterology
from 19642000
L. L. Kjaergard, S. L. Frederiksen, C. Gluud
Background & Aims: The internal validity of clinical
trials depends on the adequacy of the reported methodological
quality. We assessed the methodological quality of all 383 randomized
clinical trials published in GASTROENTEROLOGY as original articles
from 1964 to 2000.
Methods: The methodological quality (randomization and
blinding), sample size, publication year, and disease area were
extracted from each trial. Changes during the study period were
analyzed by analysis of variance with adjustments for potential
confounders.
Results: Forty-two percent of all trials reported adequate
generation of the allocation sequence, 39% reported adequate allocation
concealment, and 62% were double blind. The reported methodological
quality improved significantly in the mid-1990s.
Conclusions: The present study shows a positive development,
but the reported methodological quality of trials can still be
improved.
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