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 Archives
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Liver Biology and Pathobiology
Insulin signaling in the transcriptional and posttranscriptional
regulation of CYP2E1 expression
Kimberley J. Woodcroft, Mikehl S. Hafner, Raymond F. Novak
Diabetes has been reported to increase the expression of cytochrome
P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold,
and enhanced expression has been associated with elevated ketone
bodies. Primary cultured rat hepatocytes were used to explore
ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate
and acetoacetate (AC), alone or in combination, either failed
to affect or decreased CYP2E1 mRNA levels by up to 90% relative
to untreated hepatocytes. Insulin produced a concentration-dependent
decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation
showed a correspondence between receptor phosphorylation and the
decrease in CYP2E1 mRNA levels at physiologic levels of insulin.
Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater
than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors
wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase
phosphorylation, ameliorated the insulin-mediated decrease in
CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also
abrogated the insulin-mediated decrease in CYP2E1 mRNA levels.
In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide,
the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059,
and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190
did not affect the insulin-mediated decrease in CYP2E1. CYP2E1
mRNA half-life decreased from ~48 hours in the absence of insulin
to ~15 hours at 10 nmol/L insulin, and this decrease was prevented
by wortmannin. The half-life of CYP2B mRNA was increased by insulin,
whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene
transcription using heterogeneous nuclear RNA (hnRNA) showed that
insulin suppressed CYP2E1 transcription. In conclusion, these
data show involvement of transcriptional and posttranscriptional
mechanisms in the insulin-mediated regulation of CYP2E1 and implicate
PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects.
(HEPATOLOGY 2002;35:263-273.)
S-adenosylmethionine and methylthioadenosine are antiapoptotic
in cultured rat hepatocytes but proapoptotic in human hepatoma
cells
Eduardo Ansorena, Elena R. García-Trevijano, Maria L. Martínez-Chantar,
Zong-Zhi Huang, Lixin Chen, José M. Mato, Maria Iraburu,
Shelly C. Lu, Matías A. Avila
S-adenosylmethionine (AdoMet) is an essential compound in cellular
transmethylation reactions and a precursor of polyamine and glutathione
synthesis in the liver. In liver injury, the synthesis of AdoMet
is impaired and its availability limited. AdoMet administration
attenuates experimental liver damage, improves survival of alcoholic
patients with cirrhosis, and prevents experimental hepatocarcinogenesis.
Apoptosis contributes to different liver injuries, many of which
are protected by AdoMet. The mechanism of AdoMet's hepatoprotective
and chemopreventive effects are largely unknown. The effect of
AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using
primary cultures of rat hepatocytes and human hepatoma cell lines.
AdoMet protected rat hepatocytes from OA-induced apoptosis dose
dependently. It attenuated mitochondrial cytochrome c release,
caspase 3 activation, and poly(ADP-ribose) polymerase cleavage.
These effects were independent from AdoMet-dependent glutathione
synthesis, and mimicked by 5'-methylthioadenosine (MTA), which
is derived from AdoMet. Interestingly, AdoMet and MTA did not
protect HuH7 cells from OA-induced apoptosis; conversely both
compounds behaved as proapoptotic agents. AdoMet's proapoptotic
effect was dose dependent and observed also in HepG2 cells. In
conclusion, AdoMet exerts opposing effects on apoptosis in normal
versus transformed hepatocytes that could be mediated through
its conversion to MTA. These effects may participate in the hepatoprotective
and chemopreventive properties of this safe and well-tolerated
drug. (HEPATOLOGY 2002;35:274-280.)
In vivo cell lineage analysis in cyproterone acetatetreated
rat liver using genetic labeling of hepatocytes
Isabelle Auvigne, Virginie Pichard, Dominique Aubert, Nelly Robillard,
Nicolas Ferry
Genetic labeling using recombinant retroviruses is a powerful
strategy for the study of cell lineage in the liver. However,
this type of vector is only able to infect dividing cells. The
synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic
in the adult rat liver. In this study, we used retroviral vectors
carrying the nuclear targeted -galactosidase gene to selectively
label and follow the fate of hepatocytes dividing on administration
of CPA. Labeled cells as well as those in mitosis were preferentially
located around the portal tract, whereas apoptotic bodies were
predominant in the pericentral area. Labeled hepatocytes did not
disappear after apoptosis, suggesting a preferential elimination
of nontransduced cells. The presence of labeled binucleated hepatocytes
showed the persistence of a binucleation process. Finally, we
performed long-term analysis of labeled cells in transgenic animals
tolerant for -galactosidase and treated with 2-acetylaminofluorene
(2-AAF) to promote the growth of CPA-initiated hepatocytes. The
presence of -galactosidasepositive hepatocyte clones showed
that hepatocytes divided during treatment with 2-AAF. Only 3%
of -galactosidase clones were positive for the placental form
of glutathione S-transferase (GSTp), indicating the absence of
a preferential appearance of preneoplastic foci in the population
of -galactosidaselabeled hepatocytes. In conclusion, our
results show that the mitogenic and tumor-initiating activities
of CPA are directed toward different hepatocyte populations. (HEPATOLOGY
2002;35:281-288.)
Protection against acetaminophen-induced liver injury and lethality
by interleukin 10: Role of inducible nitric oxide synthase
Mohammed Bourdi, Yasuhiro Masubuchi, Timothy P. Reilly, Hamid
R. Amouzadeh, Jackie L. Martin, John W. George, Anjali G. Shah,
Lance R. Pohl
Mechanistic study of idiosyncratic drug-induced hepatitis (DIH)
continues to be a challenging problem because of the lack of animal
models. The inability to produce this type of hepatotoxicity in
animals, and its relative rarity in humans, may be linked to the
production of anti-inflammatory factors that prevent drug-protein
adducts from causing liver injury by immune and nonimmune mechanisms.
We tested this hypothesis by using a model of acetaminophen (APAP)-induced
liver injury in mice. After APAP treatment, a significant increase
was observed in serum levels of interleukin (IL)-4, IL-10, and
IL-13, cytokines that regulate inflammatory mediator production
and cell-mediated autoimmunity. When IL-10 knockout (KO) mice
were treated with APAP, most of these mice died within 24 to 48
hours from liver injury. This increased susceptibility to APAP-induced
liver injury appeared to correlate with an elevated expression
of liver proinflammatory cytokines, tumor necrosis factor (TNF)-,
and IL-1, as well as inducible nitric oxide synthase (iNOS). In
this regard, mice lacking both IL-10 and iNOS genes were protected
from APAP-induced liver injury and lethality when compared with
IL-10 KO mice. All strains, including wild-type animals, generated
similar amounts of liver APAP-protein adducts, indicating that
the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity
was not caused by an enhanced formation of APAP-protein adducts.
In conclusion, these findings suggest that an important feature
of the normal response to drug-induced liver injury may be the
increased expression of anti-inflammatory factors such as IL-10.
Certain polymorphisms of these factors may have a role in determining
the susceptibility of individuals to idiosyncratic DIH. (HEPATOLOGY
2002;35:289-298.)
Mdr P-glycoproteins are not essential for biliary excretion
of the hydrophobic heme precursor protoporphyrin in a griseofulvin-induced
mouse model of erythropoietic protoporphyria
Torsten Plösch, Vincent W. Bloks, Juul F. W. Baller, Rick
Havinga, Henkjan J. Verkade, Peter L. M. Jansen, Folkert Kuipers
Hepatic complications in erythropoietic protoporphyria (EPP)
have been attributed to toxic actions of accumulated protoporphyrin
(PP). PP can only be removed via the bile but transport systems
involved have not been defined. The aim of this study was to gain
insight in the mode of biliary PP excretion, with emphasis on
the potential contribution of the Mdr1 P-glycoprotein export pump
and biliary lipids as PP carriers. Control mice and mice homozygous
for Mdr1a/b (Abcb1) or Mdr2 (Abcb4)
gene disruption, the latter unable to secrete phospholipids and
cholesterol into bile, were treated with griseofulvin to chemically
induce protoporphyria. All groups showed dramatically increased
PP levels in erythrocytes and liver after griseofulvin treatment.
Histologically, massive PP deposits were found in livers of control
and Mdr1a/b/ mice but not in those of Mdr2/
mice. Serum unesterified cholesterol and phospholipids were increased
by griseofulvin because of formation of lipoprotein-X in control
and Mdr1a/b/ mice only. Yet, bile flow was not
impaired in griseofulvin-treated mice, and biliary bile salt,
phospholipid, and cholesterol secretion rates were significantly
increased. Surprisingly, biliary PP excretion was similar in all
3 groups of griseofulvin-treated mice: the observed linear relationship
between hepatic and biliary PP concentrations and identical liver-to-bile
concentration ratios in treated and untreated mice suggest a passive
mode of excretion. In conclusion, the data show that Mdr
P-glycoproteins are not critically involved in biliary removal
of excess PP and indicate that the presence of biliary lipids
is required for formation of intrahepatic PP deposits. (HEPATOLOGY
2002;35:299-306.)
Activation of the Raf-1/MEK/ERK cascade by bile acids occurs
via the epidermal growth factor receptor in primary rat hepatocytes
Yi-Ping Rao, Elaine J. Studer, R. Todd Stravitz, Seema Gupta,
Liang Qiao, Paul Dent, Phillip B. Hylemon
Bile acids have been reported to activate several different
cell signaling cascades in rat hepatocytes. However, the mechanism(s)
of activation of these pathways have not been determined. This
study aims to determine which bile acids activate the Raf-1/MEK/ERK
cascade and the mechanism of activation of this pathway. Taurodeoxycholic
acid (TDCA) stimulated (+235%) the phosphorylation of p74 Raf-1
in a time (5 to 20 minutes) and concentration-dependent (10 to
100 µmol/L) manner. Raf-1 and ERK activities were both significantly
increased by most bile acids tested. Deoxycholic acid (DCA) was
the best activator of ERK (3.6-fold). A dominant negative Ras
(N17) construct expressed in primary hepatocytes prevented the
activation of ERK by DCA. The epidermal growth factor receptor
(EGFR)-specific inhibitor (AG1478) significantly inhibited (~81%)
the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased
phosphorylation of the EGFR (~2-fold) and Shc (~4-fold). A dominant
negative mutant of the EGFR (CD533) blocked the ability of DCA
to activate ERK. In conclusion, these results show that DCA activates
the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily
via an EGFR/Ras-dependent mechanism. (HEPATOLOGY 2002;35:307-314.)
Derivation, characterization, and phenotypic variation of hepatic
progenitor cell lines isolated from adult rats
Li Yin, Mingzeng Sun, Zoran Ilic, Hyam L. Leffert, Stewart Sell
Liver progenitor cells (LPCs) cloned from adult rat livers
following allyl alcohol injury express hematopoietic stem cell
and early hepatic lineage markers when cultured on feeder layers;
under these conditions, neither mature hepatocyte nor bile duct,
Ito, stellate, Kupffer cell, or macrophage markers are detected.
These phenotypes have remained stable without aneuploidy or morphological
transformation after more than 100 population doublings. When
cultured without feeder layers, the early lineage markers disappear,
and mature hepatocyte markers are expressed; mature hepatocytic
differentiation and cell size are also augmented by polypeptide
and steroidal growth factors. In contrast to hepatocytic potential,
duct-like structures and biliary epithelial markers are expressed
on Matrigel. Because they were derived without carcinogens or
mutagens, these bipotential LPC lines provide novel tools for
models of cellular plasticity and hepatocarcinogenesis, as well
as lines for use in cellular transplantation, gene therapy, and
bioreactor construction. (HEPATOLOGY 2002;35:315-324.)
Inhibition of rat liver fibrogenesis through noradrenergic
antagonism
Liliane Dubuisson, Alexis Desmoulière, Boris Decourt, Laetitia
Evadé, Christiane Bedin, Liliane Boussarie, Laurence Barrier,
Michel Vidaud, Jean Rosenbaum
The effect of adrenergic innervation and/or circulating catecholamines
on the function of liver fibrogenic cells is poorly understood.
Our aim was to investigate the effects of noradrenergic antagonism
on carbon tetrachloride (CCl4)-induced liver fibrosis in rats.
Two weeks of CCl4 induced a ~5-fold increase in the area of fibrosis
as compared with controls. The addition of 6-hydroxydopamine (OHDA),
a toxin that destroys noradrenergic fibers, decreased fibrosis
by 60%. After 6 weeks of CCl4, the area of fibrosis increased
about 30-fold in CCl4-treated animals and was decreased by 36%
with OHDA. At 2 weeks, OHDA abrogated the CCl4-induced increase
in mRNA level of tissue inhibitor of matrix metalloproteinases-1
(TIMP-1), an inhibitor of extracellular matrix degradation, and
it greatly reduced it at 6 weeks. Finally, when rats treated with
CCl4 for 2 weeks also received prazosin, an antagonist of 1-adrenergic
receptors, fibrosis was decreased by 83%. In conclusion, destruction
of noradrenergic fibers or antagonism of noradrenergic signaling
through 1 receptors inhibited the development of liver fibrosis.
Because adrenoreceptor antagonists have a very sound safety profile,
they appear as attractive drugs to reduce liver fibrogenesis.
(HEPATOLOGY 2002;35:325-331.)
Phosphorylation of retinoid X receptor suppresses its ubiquitination
in human hepatocellular carcinoma (*Human Study*)
Seiji Adachi, Masataka Okuno, Rie Matsushima-Nishiwaki, Yukihiko
Takano, Soichi Kojima, Scott L. Friedman, Hisataka Moriwaki, Yukio
Okano
Retinoid X receptor (RXR) has emerged as an important nuclear
receptor involved in hepatocarcinogenesis, because its ligand
suppresses the development of hepatocellular carcinoma (HCC) in
both experimental and clinical studies. We have demonstrated that
phosphorylation of RXR at serine 260 interferes with its function
and delays its degradation in cultured human HCC, leading to enhanced
cellular proliferation. Here, we show that in normal liver and
in nonproliferating hepatocyte cultures, RXR is unphosphorylated
and highly ubiquitinated, rendering it sensitive to proteasome-mediated
degradation. On the other hand, phosphoserine 260 RXR is resistant
to ubiquitination and proteasome-mediated degradation in both
human HCC tissues and a human HCC cell line, HuH7. In these tissues
and cells, serine 260 is phosphorylated by mitogen-activated protein
(MAP) kinase. In proliferating normal hepatocytes, similar to
HCC cells, RXR is also phosphorylated at serine 260 and resistant
to ubiquitin-mediated degradation by proteasome, but this ubiquitination
of RXR is differentially regulated between HCC cells and normal
hepatocytes. In proliferating hepatocytes, 9-cis retinoic
acid (9cRA), a ligand to RXR, suppresses MAP kinasemediated
phosphorylation and thereby enhances ubiquitination of RXR, whereas
it fails to exert these effects in HCC cells. In conclusion, switching
of the ubiquitin/proteasome-dependent degradation of RXR by phosphorylation
at serine 260 may be responsible for the aberrant growth of HCC
and its suppression by retinoids. (HEPATOLOGY 2002;35:332-340.)
Absence of nuclear factor B inhibition by NSAIDs in hepatocytes
Nuria A. Callejas, Marta Casado, Lisardo Boscá, Paloma
Martín-Sanz
Stimulation of fetal hepatocytes with proinflammatory cytokines
and lipopolysaccharide promotes the expression of cyclooxygenase-2
(COX-2) and nitric oxide synthase-2 (NOS-2), whereas the hepatoma
cell line HepG2 exhibits a behavior similar to that described
for adult hepatocytes and only expresses NOS-2. The effect of
nonsteroidal anti-inflammatory drugs (NSAIDs) on the inflammatory
onset was analyzed in these cells since in addition to the inhibition
of cyclooxygenase activity, these drugs interfere with other signaling
pathways related with the inflammatory response. Inhibition of
nuclear factor B (NF-B) activation by aspirin and salicylate has
been described in many cells. However, incubation of hepatic cells
with salicylate, aspirin, indomethacin, ibuprofen, or 5,5-dimethyl-3(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone
(DFU), a fluorinated derivative of rofecoxib, failed to impair
IB kinase activity, the processing of NF-B, and the expression
of NF-Bdependent genes, such as NOS-2. Moreover, selective
COX-2 inhibitors did not promote apoptosis in hepatocytes under
inflammatory conditions, suggesting that prostaglandins are not
required to maintain cell viability. In conclusion, these data
indicate that hepatocytes are not sensitive to NF-B inhibition
by NSAIDs and that these drugs, especially the COX-2 selective
inhibitors, do not alter cell viability. (HEPATOLOGY 2002;35:341-348.)
Liver Failure and Liver Disease
Response to steroids in de novo autoimmune hepatitis after
liver transplantation (*Human Study*)
Magdalena Salcedo, Javier Vaquero, Rafael Bañares, Margarita
Rodríguez-Mahou, Emilio Alvarez, Jose Luis Vicario, Alicia
Hernández-Albújar, José Luis R. Tíscar,
Diego Rincón, Sonia Alonso, Alejandro De Diego, Gerardo
Clemente
Graft dysfunction associated with autoimmune phenomena has
been recently described in liver transplant recipients without
previous autoimmune disease. However, the natural history, diagnostic
criteria, and definitive therapeutic approach of de novo
autoimmune hepatitis (de novo AIH) are poorly understood.
We report 12 cases of de novo AIH 27.9 ± 24.5 months
after liver transplantation: the outcome of 7 patients treated
with steroids is compared with a group of 5 nontreated patients.
Nontreated patients lost the graft after 5.8 ± 2.6 months
from de novo AIH onset. All treated patients were alive
after 48.4 ± 14 (29-65) months from de novo AIH
onset, and none of them lost the graft. However, 5 patients relapsed
in relation to steroid tapering. All patients presented an atypical
antiliver/kidney cytosolic autoantibody, associated to classical
autoantibodies in 10 cases. Histological study showed several
degrees of lobular necrosis and inflammatory infiltrate. HLA antigen
frequencies and matching were compared with 2 control groups (16
orthotopic liver transplantation [LTX] patients without de
novo AIH and 929 healthy blood donors); de novo AIH
patients showed a higher prevalence of HLA-DR3 (54.5% vs. 25.9%,
P = .04) than healthy controls, which was not observed
in LTX patients without de novo AIH. In conclusion, this
new disease should be included in the differential diagnosis of
unexplained graft dysfunction. In addition, treatment with steroids
results in a dramatically improved outcome. However, maintenance
therapy is usually required. (HEPATOLOGY 2002;35:349-356.)
Critical flicker frequency for quantification of low-grade
hepatic encephalopathy (*Human Study*)
Gerald Kircheis, Matthias Wettstein, Lars Timmermann, Alfons Schnitzler,
Dieter Häussinger
Subclinical hepatic encephalopathy (SHE) is currently diagnosed
by psychometric tests or neurophysiologic techniques. In view
of its sociomedical relevance, simple and reproducible tests for
routine diagnosis are required. This study evaluates critical
flicker-frequency thresholds for quantification of low-grade hepatic
encephalopathy. A total of 115 patients (92 with cirrhosis, 23
controls) were analyzed for HE severity (mental state, computerized
psychometric tests), and the threshold frequencies at which light
pulses are perceived as fused (fusion frequency) or flickering
light (critical flicker frequency [CFF]). CFF was a highly reproducible
parameter with little age, day-time, and training dependency.
CFFs in cirrhotic patients without HE (HE 0) were not different
from those found in noncirrhotic controls. Significantly lower
CFFs were found in cirrhotic patients with subclinical or manifest
HE, and the various HE groups separated from each other at a high
level of significance (P < .01). By using a CFF cut-off
value of 39 Hz, a 100% separation of patients with manifest HE
from noncirrhotic controls and HE 0 cirrhotic patients was obtained.
SHE patients separated from HE 0 cirrhotic patients with high
sensitivity (55%) and specificity (100%). The HE severitydependent
differences were found in both, alcoholic and posthepatitic cirrhosis.
Statistically significant correlations (P < .01) were
found between CFFs and individual psychometric tests. Aggravation
of preexisting HE after transjugular intrahepatic portosystemic
stent shunt (TIPS) implantation was accompanied by a corresponding
decrease of CFF, whereas improvement of HE increased CFF. In conclusion,
CFF is a sensitive, simple, and reliable parameter for quantification
of low-grade HE severity in cirrhotic patients and may be useful
for the detection and monitoring of SHE. (HEPATOLOGY 2002;35:357-366.)
Nonalcoholic steatohepatitis, insulin resistance, and metabolic
syndrome: Further evidence for an etiologic association (*Human
Study*)
Gianfranco Pagano, Giovanni Pacini, Giovanni Musso, Roberto Gambino,
Fabio Mecca, Nadia Depetris, Maurizio Cassader, Ezio David, Paolo
Cavallo-Perin, Mario Rizzetto
This study aims to determine the presence of the components
of the metabolic syndrome in primary nonalcoholic steatohepatitis
(NASH) and to assess the role of liver disease in the genesis
of peripheral hyperinsulinemia. Nineteen patients (18 men and
1 woman; mean age, ± SD, 38 ± 10 years; body mass
index [BMI], 26 ± 2 kg/m2) with histologic evidence of
NASH were enrolled; 19 age- and sex-matched normal subjects were
investigated as controls. Plasma glucose, insulin, and C-peptide
levels were measured during an oral glucose tolerance test, and
a frequently sampled intravenous glucose tolerance test (FSIGT),
analyzed by minimal modeling technique, was performed. Compared
with controls, the NASH group had lower insulin sensitivity (3.84
± 2.44 vs. 7.48 ± 3.01 104 ¥ min1/µU/mL;
P = .0003) and higher total insulin secretion (21 ±
13 vs. 10 ± 3 nmol/L in 240 minutes; P = .001).
Hepatic insulin extraction was similar in both groups (69.8% ±
16.1% vs. 70.2% ± 18.3%; P = .854). According to
the results of the oral glucose tolerance test, no patient was
classified as diabetic, 5 were classified as glucose intolerant,
and 1 was classified as having impaired fasting glycemia. Nine
patients (47%) had at least the 2 minimum criteria required to
define the metabolic syndrome according to the European Group
for the Study of Insulin Resistance (EGIR). In conclusion, hyperinsulinemia
and insulin resistance occur frequently in patients with NASH;
these conditions do not stem from a reduced hepatic insulin extraction
but from an enhanced pancreatic insulin secretion compensatory
to reduced insulin sensitivity. The derangement of insulin regulation,
often associated with the metabolic syndrome, may play a causal
role in the pathogenesis of NASH. (HEPATOLOGY 2002;35:367-372.)
NASH and insulin resistance: Insulin hypersecretion and specific
association with the insulin resistance syndrome (*Human Study*)
Shivakumar Chitturi, Shehan Abeygunasekera, Geoffrey C. Farrell,
Jane Holmes-Walker, Jason M. Hui, Caroline Fung, Rooshdiya Karim,
Rita Lin, Dev Samarasinghe, Christopher Liddle, Martin Weltman,
Jacob George
Nonalcoholic steatohepatitis (NASH) is often linked with disorders
that are clearly associated with insulin resistance (IR): obesity,
type 2 diabetes mellitus, and hypertriglyceridemia. We tested
the hypotheses that (1) IR is an essential requirement for the
development of NASH and (2) a high association between IR and
liver disease is relatively specific for NASH. We measured body
mass index (BMI), waist/hip ratio, and fasting serum lipid, insulin,
C-peptide, and glucose levels in 66 patients with NASH (21 with
advanced fibrosis and 45 with mild fibrosis). IR was determined
by the homeostasis model assessment (HOMA). We also determined
the strength of the association of NASH with insulin resistance
syndrome (IRS) as defined by World Health Organization criteria.
To assess whether the finding of IR was relatively specific to
NASH rather than simply to obesity or liver disease, we compared
the results of a subset of 36 patients with less-severe NASH with
36 age- and sex-matched patients with chronic hepatitis C virus
(HCV) of comparable fibrotic severity. IR was confirmed in 65
patients (98%) with NASH, and 55 (87%) fulfilled minimum criteria
for IRS. IR was found in lean as well as in overweight and obese
patients. The IR values and the prevalence of IRS (75% vs. 8.3%)
were significantly higher in those with NASH than in comparable
cases of HCV. Hyperinsulinemia was attributable to increased insulin
secretion rather than decreased hepatic extraction. In conclusion,
most patients with NASH have IRS, and there is a near-universal
association between NASH and IR irrespective of obesity. IR is
present in mild as well as advanced cases of NASH but is unusual
in chronic HCV of similar fibrotic severity. (HEPATOLOGY 2002;35:373-379.)
Effect of liver transplantation on inflammatory bowel disease
in patients with primary sclerosing cholangitis (*Human Study*)
Igor Dvorchik, Michael Subotin, A. Jake Demetris, John J. Fung,
Thomas E. Starzl, Samuel Wieand, Kareem M. Abu-Elmagd
This report investigates the influence of liver transplantation
and concomitant immunosuppression on the course of progression
of inflammatory bowel disease (IBD) and discusses statistical
methodology appropriate for such settings. The data on 303 patients
who underwent liver transplantation for primary sclerosing cholangitis
(PSC) were analyzed using person-time analysis and Cox regression,
with the duration of IBD as the time variable and transplantation
as a segmented time-dependent covariate, to take into account
both posttransplant and pretransplant history of IBD. The need
for colectomy and appearance of colorectal cancer were taken as
outcome measures. The only significant risk factor in the multivariate
model for colectomy was transplantation itself, which increased
the risk of colectomy due to intractable disease (Wald statistic;
P = .001). None of the variables available for analysis
were found to influence the risk of colon cancer significantly.
Graphs showing the dependence of the instantaneous risk of cancer
on the time from onset of IBD and its independence from the latter
in the case of colectomy are presented. The use of a unique statistical
methodology described for the first time in this setting led us
to the somewhat surprising conclusion that transplantation and
concomitant use of immunosuppression accelerate the progression
of IBD. At the same time, transplantation does not affect the
incidence of colorectal cancer. These results confirm the findings
of some recent studies and can potentially shed new light on the
disease pathogenesis. (HEPATOLOGY 2002;35:380-384.)
TIPS versus drug therapy in preventing variceal rebleeding
in advanced cirrhosis: A randomized controlled trial (*Human
Study*)
Àngels Escorsell, Rafael Bañares, Juan Carlos García-Pagán,
Rosa Gilabert, Eduardo Moitinho, Belén Piqueras, Concepció
Bru, Antonio Echenagusia, Alicia Granados, Jaume Bosch
Prevention of variceal rebleeding is mandatory in cirrhotic
patients. We compared the efficacy, safety, and cost of transjugular
intrahepatic portosystemic shunt (TIPS) versus pharmacologic therapy
in preventing variceal rebleeding in patients with advanced cirrhosis.
A total of 91 Child-Pugh class B/C cirrhotic patients surviving
their first episode of variceal bleeding were randomized to receive
TIPS (n = 47) or drug therapy (propranolol + isosorbide-5-mononitrate)
(n = 44) to prevent variceal rebleeding. Mean follow-up was 15
months. Rebleeding occurred in 6 (13%) TIPS-treated patients versus
17 (39%) drug-treated patients (P = .007). The 2-year rebleeding
probability was 13% versus 49% (P = .01). A similar number
of reinterventions were required in the 2 groups; these were mainly
angioplasty ± restenting in the TIPS group (90 of 98) and
endoscopic therapy for rebleeding in the medical group (45 of
62) (not significant). Encephalopathy was more frequent in TIPS
than in drug-treated patients (38% vs. 14%, P = .007).
Child-Pugh class improved more frequently in drug-treated than
in TIPS-treated patients (72% vs. 45%; P = .04). The 2-year
survival probability was identical (72%). The identified cost
of therapy was double for TIPS-treated patients. In summary, medical
therapy was less effective than TIPS in preventing rebleeding.
However, it caused less encephalopathy, identical survival, and
more frequent improvement in Child-Pugh class with lower costs
than TIPS in high-risk cirrhotic patients. This suggests that
TIPS should not be used as a first-line treatment, but as a rescue
for failures of medical/endoscopic treatments (first-option therapies).
(HEPATOLOGY 2002;35:385-392.)
Activation of eNOS in rat portal hypertensive gastric mucosa
is mediated by TNF- via the PI 3-kinaseAkt signaling pathway
Hirofumi Kawanaka, Michael K. Jones, Imre L. Szabo, Dolgor Baatar,
Rama Pai, Kouji Tsugawa, Keizo Sugimachi, I. James Sarfeh, Andrzej
S. Tarnawski
Activation of endothelial nitric oxide synthase (eNOS) in portal
hypertensive (PHT) gastric mucosa leads to hyperdynamic circulation
and increased susceptibility to injury. However, the signaling
mechanisms for eNOS activation in PHT gastric mucosa and the role
of TNF- in this signaling remain unknown. In PHT gastric mucosa
we studied (1) eNOS phosphorylation (at serine 1177) required
for its activation; (2) association of the phosphatidylinositol
3-kinase (PI 3-kinase), and its downstream effector Akt, with
eNOS; and, (3) whether TNF- neutralization affects eNOS phosphorylation
and PI 3-kinaseAkt activation. To determine human relevance,
we used human microvascular endothelial cells to examine directly
whether TNF- stimulates eNOS phosphorylation via PI 3-kinase.
PHT gastric mucosa has significantly increased (1) eNOS phosphorylation
at serine 1177 by 90% (P < .01); (2) membrane translocation
(P < .05) and phosphorylation (P < .05) of
p85 (regulatory subunit of PI 3-kinase) by 61% and 85%, respectively;
(3) phosphorylation (P < .01) and activity (P
< .01) of Akt by 40% and 52%, respectively; and (4) binding
of Akt to eNOS by as much as 410% (P < .001). Neutralizing
antiTNF- antibody significantly reduced p85 phosphorylation,
phosphorylation and activity of Akt, and eNOS phosphorylation
in PHT gastric mucosa to normal levels. Furthermore, TNF- stimulated
eNOS phosphorylation in human microvascular endothelial cells.
In conclusion, these findings show that in PHT gastric mucosa,
TNF- stimulates eNOS phosphorylation at serine 1177 (required
for its activation) via the PI 3-kinaseAkt signal transduction
pathway. (HEPATOLOGY 2002;35:393-402.)
Fine specificity of autoantibodies to soluble liver antigen
and liver/pancreas (*Human Study*)
Johannes Herkel, Birgit Heidrich, Nicole Nieraad, Ingrid Wies,
Michael Rother, Ansgar W. Lohse
Autoantibodies to soluble liver antigen and liver pancreas
(SLA/LP) have been described as specific markers for Autoimmune
Hepatitis (AIH), occurring in about 20% of patients with AIH.
The high degree of specificity for SLA/LP in autoimmune liver
disease suggests a possible role in its pathogenesis. This study
aims to map the exact epitope(s) recognized by SLA/LP autoantibodies
and to assess the role of molecular mimicry between microbial
antigens and self-epitopes. Using SLA/LP-reactive sera of 18 individual
AIH patients and a pool of 15 patient sera, we found the dominant
immune reactivity directed to peptide p395-414 and a less prominent
immune response to 2 other epitopes adjacent to the dominant epitope.
Immunodominance of peptide p395-414 was confirmed by absorption
experiments. The SLA/LP autoantibodies of all tested AIH patients
were mainly of the IgG1 type, suggesting that SLA/LP autoantibodies
may arise by a common and specific underlying immune stimulus.
Based on sequence homologies of the SLA/LP antigenic region with
viral proteins, it was hypothesized that molecular mimicry may
drive autoimmunity to SLA/LP. However, the homologous virus-derived
peptides were not recognized by SLA/LP autoantibodies. Similarly,
the only known procaryotic homologue, MJ0610 of Methanococcus
jannaschii, was only weakly recognized by SLA/LP-positive
sera. Thus, no evidence could be found for molecular mimicry being
the causative mechanism for the development of SLA/LP autoantibodies.
In conclusion, the exquisite epitope specificity and IgG subtype
are evidence for the maturity of the SLA/LP autoantibody response;
a specific autoantigen-driven process underlying the immunopathogenesis
is likely. (HEPATOLOGY 2002;35:403-408.)
Primary biliary cirrhosis with additional features of autoimmune
hepatitis: Response to therapy with ursodeoxycholic acid (*Human
Study*)
Supriya Joshi, Karen Cauch-Dudek, Ian R. Wanless, Keith D. Lindor,
Roberta Jorgensen, Kenneth Batts, E. Jenny Heathcote
Patients with primary biliary cirrhosis (PBC) may have additional
features of autoimmune hepatitis (AIH). Corticosteroids usually
contraindicated in PBC have been advocated for these patients.
Patients with antimitochondrial antibody (AMA)-positive PBC from
two previous randomized, controlled trials were assessed for features
of AIH. Their biochemical, immunologic, and histologic responses
to ursodeoxycholic acid (UDCA) versus placebo were compared with
those without AIH features. The survival of patients testing positive
or negative for antinuclear antibodies (ANA) was also examined.
Features of AIH were defined by the presence of 2 or more of the
following: 1) alanine transaminase (ALT) > 5¥ the upper
limit of normal (ULN); 2) immunoglobulin G (IgG) > 2¥ ULN
or positive anti-smooth muscle antibody (ASMA); and 3) moderate
to severe lobular inflammation on pretreatment liver biopsy. Testing
for AMA, ASMA, and ANA was done by immunofluorescence. The change
in serum bilirubin, alkaline phosphatase (ALP), transaminases,
IgM, and IgG from baseline to 2 years was compared. Of the 331
patients randomized, 16 (4.8%) had features of AIH (12 UDCA, 4
placebo). The median percent change in serum biochemistry and
immunoglobulin values were similar in patients with PBC ±
features of AIH after 2 years of therapy with UDCA. Over 2 years,
little change in histologic features of AIH was observed. Survival
was similar for patients with PBC with and without ANA. In conclusion,
features of AIH in PBC may be transient and response to UDCA therapy
similar to patients with PBC without features of AIH. (HEPATOLOGY
2002;35:409-413.)
Histologic scoring of liver biopsy in focal nodular hyperplasia
with atypical presentation (*Human Study*)
Aurélie Fabre, Pascale Audet, Valérie Vilgrain,
Bich N. Nguyen, Dominique Valla, Jacques Belghiti, Claude Degott
The contribution of radio-guided transcutaneous biopsy in the
diagnosis of focal nodular hyperplasia (FNH) of the liver was
compared with the findings on surgical specimens to assess its
contribution in clinical and radiologic atypical cases. This retrospective
study involved 30 patients with atypical tumors on imaging who
underwent liver biopsy and then surgery. All surgical specimens
were diagnosed as FNH, either classical (n = 18) or nonclassical
(n = 12). Imaging data were reviewed according to 4 radiologic
criteria on magnetic resonance imaging (MRI) and/or computed tomography
(CT) scan (hypervascularity, homogeneity, nonencapsulation, and
presence of a central scar), and classified depending on the number
of criteria found (group I, 4 of 4; group II, 3 of 4; group III,
2 or fewer). Histologic assessment of ultrasound (US)-guided liver
biopsy recorded major diagnostic features (fibrous bands, thick-walled
vessels, reactive ductules, and nodularity) and minor features
(sinusoidal dilatation and perisinusoidal fibrosis). "Definite
FNH" (3 or 4 major features) was diagnosed in 14 biopsies,
"possible FNH" (2 major and 1 or 2 minor features) in
7 cases, and "negative for FNH" (2 or fewer major features
without minor features) in 9 cases. The diagnosis of FNH on biopsy
was reached in 14 cases (58.3%) in patients with 2 or fewer imaging
criteria (group III; n = 24). Biopsies with a diagnosis of "possible
FNH" corresponded to a large proportion of telangiectatic-type
FNH on the specimen. In conclusion, liver biopsy does not appear
to be necessary in cases in which imaging is typical. However,
the absence of radiologic diagnostic criteria in FNH does not
preclude a positive diagnosis on liver needle biopsy. Using the
proposed histologic scoring system, surgical management may be
avoided in these cases. (HEPATOLOGY 2002;35:414-420.)
Adenosine triphosphate infusion increases liver energy status
in advanced lung cancer patients: An in vivo 31P
magnetic resonance spectroscopy study
Susanne Leij-Halfwerk, Hendrik J. Agteresch, Paul E. Sijens, Pieter
C. Dagnelie
We recently observed inhibition of weight loss in patients
with advanced nonsmall-cell lung cancer after intravenous infusion
of ATP. Because liver ATP levels were found to be decreased in
lung cancer patients with weight loss, the present 31P magnetic
resonance spectroscopy (MRS) study was aimed at investigating
whether ATP infusion restores liver energy status in these patients.
Nine patients with advanced nonsmall-cell lung cancer (stage IIIB/IV)
were studied 1 week before (baseline) and at 22 to 24 hours of
continuous ATP infusion (37-75 µg/kg/min). Localized hepatic
31P MR spectra (repetition time 15 seconds), obtained in the overnight-fasted
state, were analyzed for ATP and Pi content. Ten healthy subjects
(without ATP infusion) served as control. Liver ATP levels in
lung cancer patients increased from 8.8 ± 0.7% (relative
to total MR-detectable phosphate; mean ± SE) at baseline
to 12.2 ± 0.9% during ATP infusion (P < .05),
i.e., a level similar to that in healthy subjects (11.9
± .9%). The increase in ATP level during ATP infusion was
most prominent in patients with 5% weight loss (baseline: 7.9
± 0.7%, during ATP infusion: 12.8 ± 1.0%, P
< .01). In conclusion, ATP infusion restores hepatic energy
levels in patients with advanced lung cancer, especially in weight-losing
patients. These changes may contribute to the previously reported
beneficial effects of ATP infusion on the nutritional status of
lung cancer patients. (HEPATOLOGY 2002;35:421-424.)
Viral Hepatitis
Expression of interferon alfa signaling components in human
alcoholic liver disease (*Human Study*)
Van-Anh Nguyen, Bin Gao
Interferon alfa (IFN-) is currently the only well-established
therapy for viral hepatitis. However, its effectiveness is much
reduced (<10%) in alcoholic patients. The mechanism underlying
this resistance is not fully understood. In this study, we examined
the expression of IFN- signaling components and its inhibitory
factors in 9 alcoholic liver disease (ALD) and 8 healthy control
liver tissues. In comparison with normal control livers, expression
of IFN-, IFN- receptor 12, Jak1, and Tyk2 remained unchanged in
ALD livers, whereas expression of IFN-, signal transducer and
activator of transcription factor 1 (STAT1), and p48 were up-regulated
and expression of STAT2 was down-regulated. Expression of antiviral
MxA a karyophilic 75 kd protein induced by IFN in mouse cells
carrying the influenza virus resistance allele Mx+ and 2'-5' oligoadenylate
synthetase (OAS) proteins was not regulated, whereas expression
of double-stranded RNA-activated protein kinase (PKR) was decreased
by 55% in ALD livers. Three families of inhibitory factors for
the JAK-STAT signaling pathway were examined in ALD livers. Members
of the suppressor of cytokine signaling (SOCS) family, including
SOCS 1, 2, 3, and CIS, and the protein tyrosine phosphatases,
including Shp-1, Shp-2, and CD45, were not up-regulated in ALD
livers, whereas the phosphorylation of and protein levels of p42/44
mitogen-activated protein kinase (p42/44MAP kinase) were increased
about 3.9- and 3.2-fold in ALD livers in comparison with normal
control livers, respectively. In conclusion, these findings suggest
that chronic alcohol consumption down-regulates STAT2 and PKR,
but up-regulates p42/44 mitogen-activated protein kinase (p42/44MAP
kinase), which may cause down-regulation of IFN- signaling in
the liver of ALD patients. (HEPATOLOGY 2002;35:425-432.)
Hepatitis C and cognitive impairment in a cohort of patients
with mild liver disease (*Human Study*)
Daniel M. Forton, Howard C. Thomas, Christine A. Murphy, Joanna
M. Allsop, Graham R. Foster, Janice Main, Keith A. Wesnes, Simon
D. Taylor-Robinson
Patients with chronic hepatitis C virus (HCV) infection frequently
report fatigue, lassitude, depression, and a perceived inability
to function effectively. Several studies have shown that patients
exhibit low quality-of-life scores that are independent of disease
severity. We therefore considered whether HCV infection has a
direct effect on the central nervous system, resulting in cognitive
and cerebral metabolite abnormalities. Twenty-seven viremic patients
with biopsy-proven mild hepatitis due to HCV and 16 patients with
cleared HCV were tested with a computer-based cognitive assessment
battery and also completed depression, fatigue, and quality-of-life
questionnaires. The HCV-infected patients were impaired on more
cognitive tasks than the HCV-cleared group (mean [SD]: HCV-infected,
2.15 [1.56]; HCV-cleared, 1.06 [1.24]; P = .02). A factor
analysis showed impairments in power of concentration and speed
of working memory, independent of a history of intravenous drug
usage (IVDU), depression, fatigue, or symptom severity. A subgroup
of 17 HCV-infected patients also underwent cerebral proton magnetic
resonance spectroscopy (1H MRS). The choline/creatine ratio was
elevated in the basal ganglia and white matter in this group.
Patients who were impaired on 2 or more tasks in the battery had
a higher mean choline/creatine ratio compared with the unimpaired
patients. In conclusion, these preliminary results demonstrate
cognitive impairment that is unaccounted for by depression, fatigue,
or a history of IVDU in patients with histologically mild HCV
infection. The findings on MRS suggest that a biological cause
underlies this abnormality. (HEPATOLOGY 2002;35:433-439.)
Neuropsychological impairment in patients with chronic hepatitis
C (*Human Study*)
Robin C. Hilsabeck, William Perry, Tarek I. Hassanein
Hepatitis C is the most common cause of chronic liver disease
in the United States and it significantly reduces quality of life.
The role of cognitive deficits contributing to the morbidity of
this disease has not been well characterized. The purpose of this
study was to examine cognitive functioning in patients with chronic
hepatitis C and to investigate relationships among parameters
of disease severity and performance on neuropsychological tests.
Sixty-six patients with chronic hepatitis C and 14 patients with
other chronic liver diseases were administered a brief battery
of neuropsychological tests assessing attention, visuoconstructional
ability, learning, memory, and psychomotor speed. Cognitive impairment
in patients with chronic hepatitis C ranged from 0% on a visuoconstructional
task to 82% on a measure of sustained attention and concentration.
Test scores of patients with chronic hepatitis C did not differ
from those of patients with other chronic liver diseases. Hence,
patients with and without chronic hepatitis C experience cognitive
deficits, especially in tasks requiring attention and psychomotor
speed. In addition, there was a significant relationship between
fibrosis stage and test performance, with greater fibrosis associated
with poorer performance. However, both patients with and without
cirrhosis exhibited cognitive dysfunction. In conclusion, these
findings suggest that progressive hepatic injury may result in
cognitive problems even before the development of cirrhosis. Future
studies need to determine the effect of this decrease in cognitive
function on quality of life. (HEPATOLOGY 2002;35:440-446.)
Interferon and amantadine in naive chronic hepatitis C: A double-blind,
randomized, placebo-controlled trial (*Human Study*)
Beat Helbling, Ivan Stamenic, Francesco Viani, Jean-Jacques Gonvers,
Jean-Francois Dufour, Jurg Reichen, Gieri Cathomas, Michael Steuerwald,
Jan Borovicka, Markus Sagmeister, Eberhard L. Renner, for the
Investigators of the Swiss Association for the Study of the Liver
(SASL)
Recent controlled trials on the efficacy of an amantadine/interferon
combination in treatment-naive patients with chronic hepatitis
C yielded contradictory results. We therefore conducted a large,
double-blind, placebo-controlled, multicenter trial in naive patients
with chronic hepatitis C: 246 patients were randomized to receive
interferon alfa-2a (6 MIU sc thrice weekly for 20 weeks, then
3 MIU sc thrice weekly) and either amantadine sulphate (2 ¥
100 mg po QD) or placebo. Treatment continued for a total of 52
weeks, if HCV-RNA in serum polymerase chain reaction (PCR) had
fallen below detection limit (1,000 copies/mL) at treatment week
10, and stopped otherwise. All patients were followed for 24 weeks
off therapy. After 10 weeks of treatment, 66/121 patients treated
with amantadine (55%) and 78/125 treated with placebo (62%) had
lost HCV-RNA (n.s.). After 24 weeks of follow-up, 25 patients
in the amantadine (21%) and 17 (14%) in the placebo group remained
HCV-RNA negative (n.s.). During therapy, virologic breakthroughs
occurred less often in the amantadine than in the placebo group
[14 (12%) vs. 27 (22%) patients; P = .04]. Multivariate
logistic regression analysis revealed genotype, viremia level,
age, and amantadine therapy [risk ratio 0.4 (95%CI 0.2-1.0), P
= .05] as predictors of sustained virologic response. Adverse
events and impact of therapy on quality of life were similar in
amantadine and placebo treated patients. Compared with current
standard treatment (interferon/ribavirin), the interferon/amantadine
combination was not cost-effective. In conclusion, amantadine
does not add to a clinically relevant extent to the treatment
of naive patients with chronic hepatitis C. (HEPATOLOGY 2002;35:447-454.)
Variants of two major T cell epitopes within the hepatitis
B surface antigen are not recognized by specific T helper cells
of vaccinated individuals (*Human Study*)
Tanja Bauer, Klaus Weinberger, Wolfgang Jilg
Several naturally occurring variants of immunogenic T cell
epitopes were identified within the hepatitis B surface antigen
(HBsAg). The effect of these variants on the cellular immune response
was studied in individuals vaccinated against HBV. Class-II restricted
T-cell responses of 30 vaccinees were analyzed after stimulation
of peripheral blood mononuclear cells (PBMCs) with 4 synthetic
peptides representing the 4 T-cell epitopes of HBsAg known as
of yet. The 2 epitopes P1 (aa 16-33) and P4 (aa 213-226) could
be identified as the dominant ones in our vaccinees by proliferation
assays and enzyme-linked immunospot assays. Responses to these
epitopes were compared with responses to their naturally occurring
variants found in HBV isolates of chronic virus carriers. Three
of 11 variants of epitope P4 led to a complete loss of T-cell
reactivity in 4 of 10 donors, all of whom reacted well to the
corresponding wild-type sequence. The remaining 6 donors recognized
these variants as well as the vaccine epitope. Similarly, 3 P1-variants
of the 12 found induced only a significantly reduced reactivity
in 4 of 10 donors, whereas they led to a normal response in the
other 6 individuals. Stimulation of T cells also induced the secretion
of antibody to HBsAg (anti-HBs) by specific B cells; however,
those peptides that failed to activate T cells were also unable
to cause any significant anti-HBs production. In conclusion, our
results suggest an immune escape of certain mutant strains of
HBV in vaccinated individuals could exist at the T-cell level.
(HEPATOLOGY 2002;35:455-465.)
Complex HBV populations with mutations in core promoter, C
gene, and pre-S region are associated with development of cirrhosis
in long-term renal transplant recipients (*Human Study*)
Petra Preikschat, Stephan Günther, Simone Reinhold, Hans
Will, Klemenz Budde, Hans H. Neumayer, Detlev H. Krüger,
Helga Meisel
Long-term immunosuppressed renal transplant recipients with
chronic hepatitis B virus (HBV) infection often develop liver
cirrhosis (LC) and end-stage liver disease (ESLD). This study
investigated accumulation and persistence of specific HBV mutants
in relation to the clinical course in these patients (n = 38;
mean follow-up, 3.5 years). HBV was analyzed longitudinally via
length polymorphism of polymerase chain reaction (PCR) fragments
(median, 6.5 serum samples per patient) as well as by cloning
and partial sequencing of 346 full-length HBV genomes. Fourteen
patients (group 1) developed LC or died from ESLD, whereas 24
patients (group 2) showed no evidence of LC during follow-up.
Development of LC and ESLD was associated with persistence of
HBV mutant populations characterized by deletions/insertions in
core promoter plus deletions in the C gene and/or deletions in
the pre-S region (86% of group 1 vs. 17% of group 2; P
< .0001). HBV without these mutations or with core promoter
mutations alone were predominantly found in group 2 (14% of group
1 vs. 75% of group 2). In patients infected with core promoter
mutants, the additional appearance and persistence of deletions
in the C gene and/or the pre-S region were accompanied or followed
by development of LC and ESLD. The mutations were distributed
on individual genomes in various combinations, leading to a high
complexity of the virus population. In conclusion, these data
suggest that accumulation and persistence of specific HBV populations
characterized by mutations in 3 subgenomic regions play a role
in pathogenesis of LC and ESLD in long-term renal transplant recipients.
(HEPATOLOGY 2002;35:466-477.)
Vagal Stimulation Rapidly Increases Leptin Secretion in
Human Stomach
IRADJ SOBHANI, MARION BUYSE, HÉLÈNE GOÏOT,
NINA WEBER, JEAN-PIERRE LAIGNEAU, DOMINIQUE HENIN, JEAN-CLAUDE
SOULÉ, and ANDRÉ BADO
Gastroenterology 2002 122: 259-263.
Background & Aims: Leptin production has
been reported in the rat and in human stomach. It initiates intestinal
nutrient absorption. In this study, we analyzed the effect of
vagal stimulation on leptin release in the human stomach. Methods:
We studied the secretion of gastric acid and leptin on stimulation
with insulin (a stimulant of vagal pathways via hypoglycemia)
and pentagastrin in 11 healthy men (normal endoscopy and
normal histological gastric mucosa), 5 with previous highly
selective vagotomy (HSV), and 6 without HSV. Fundic biopsies
were performed for immunostaining of leptin. Results: There
was no difference between the 2 groups with respect to age,
body mass index, basal leptin (4.8 ± 1.2 ng/15
minutes) and gastric acid (0.7 ± 0.2 mmol/15
minutes) outputs. Leptin-immunoreactivity was found in the fundic
glands, and its distribution and density were similar in 2 groups.
Insulin caused a rapid (15-minute) increase in leptin output in
men without HSV (31 ± 9 ng/15 minutes),
but not in those with HSV (7.7 ± 3.2 ng/15
minutes). Insulin-stimulated gastric leptin was biphasic, with
a rapid increase (15 minutes after injection) followed by
a second steady and sustained increase (39.9 ± 7.6 ng/15
minutes at 120 minutes after injection). Pentagastrin increased
gastric leptin output in individuals with (30 ± 4.9 ng/15
minutes) and without (26 ± 3.2 ng/15 minutes)
HSV. Insulin and pentagastrin did not modify plasma leptin, whatever
HSV status. Conclusions: Vagal stimulation of leptin release
in the human stomach suggests that leptin is released during the
cephalic phase of gastric secretion. Luminal leptin may be involved
in vagus-mediated intestinal functions.
CLINICAL RESEARCH:
Selection of Hepatitis B Virus Polymerase Mutants With Enhanced
Replication by Lamivudine Treatment After Liver Transplantation
C.-THOMAS BOCK, HANS L. TILLMANN, JOSEPH TORRESI, JÜRGEN
KLEMPNAUER, STEPHEN LOCARNINI, MICHAEL P. MANNS, and CHRISTIAN
TRAUTWEIN
Gastroenterology 2002 122: 264-273. Published online Feb 6 2002.
Background & Aims: Lamivudine has become
a main therapeutic option for treating hepatitis B virus (HBV)
infection. Although drug resistance develops, the clinical course
after selection of antiviral-resistant HBV mutants seems to be
benign. However, we observed a severe clinical course of hepatitis
B infection in several liver transplant recipients after the emergence
of lamivudine resistance. This was associated with high viral
load in the blood. Methods: In this report, we characterize
the molecular mechanisms underlying drug-dependent enhanced replication
of particular lamivudine-resistant HBV mutants selected in these
patients, which were associated with sudden onset of liver failure.
Results: The clinical course was characterized by a sudden
rise in serum bilirubin, prothrombin time, and transaminase. HBV
sequence analysis of these patients revealed both mutations in
the "a-determinant" of the envelope and the YMDD (tyrosine,
methionine, aspartate, aspartate) motif (domain C) of the polymerase
protein. Transfection experiments with replication competent vectors
indicated that the "a-determinant" changes were not
associated with resistance, whereas mutations in the YMDD motif
conferred resistance to lamivudine. More importantly, combinations
of mutations in the "a-determinant" and the YMDD motif
in patients with a severe hepatitis were not only resistant to
lamivudine treatment, but showed enhanced replication in vitro
in the presence of lamivudine. This observation was confirmed
in separate laboratories. Conclusions: Severe and fatal
hepatitis B infection can occur during lamivudine therapy and
may be associated with certain HBV mutants selected during sequential
nucleoside and HBIg treatment. The lamivudine-enhanced replication
shown by these mutants suggests that continuation of therapy with
lamivudine could be deleterious in some patients.
Tumor Necrosis Factor Promoter Polymorphisms and Insulin
Resistance in Nonalcoholic Fatty Liver Disease
LUCA VALENTI, ANNA LUDOVICA FRACANZANI, PAOLA DONGIOVANNI, GENNARO
SANTORELLI, ADRIANA BRANCHI, EMANUELA TAIOLI, GEMINO FIORELLI,
and SILVIA FARGION
Gastroenterology 2002 122: 274-280. Published online Feb 6 2002.
Background & Aims: Nonalcoholic fatty
liver disease, which can range from fatty liver alone to nonalcoholic
steatohepatitis and cirrhosis, is related to insulin resistance.
Tumor necrosis factor (TNF-) may induce insulin resistance,
and polymorphisms of its promoter have been associated with an
increased release of this cytokine. We analyzed (1) the prevalence
of insulin resistance, (2) the prevalence of the 238 and
308 TNF- polymorphisms, and (3) the relationship among TNF-
polymorphisms, insulin resistance, and the occurrence of steatohepatitis
in 99 patients with nonalcoholic fatty liver diagnosed by
ultrasonography and confirmed by histologic analysis in the 53 who
underwent biopsy. Methods: Insulin resistance was evaluated
by the homeostatic metabolic assessment insulin resistance indices
and TNF- polymorphisms by polymerase chain reaction and restriction
fragment length polymorphism analysis. Results: Insulin
resistance was detected in almost all of the patients and was
more severe in those with steatohepatitis. The prevalence of the
238, but not of the 308, TNF- polymorphism was higher
in subjects with nonalcoholic fatty liver than in controls (31%
vs. 15%; P < 0.0001), and patients positive
for TNF- polymorphisms had higher insulin resistance indices,
a higher prevalence of impaired glucose tolerance, and a lower
number of associated risk factors for steatosis. Conclusions:
TNF- polymorphisms could represent a susceptibility genotype for
insulin resistance, nonalcoholic fatty liver, and steatohepatitis.
Excess Alcohol Greatly Increases the Prevalence of Cirrhosis
in Hereditary Hemochromatosis
LINDA M. FLETCHER, JEANNETTE L. DIXON, DAVID M. PURDIE, LAWRIE
W. POWELL, and DARRELL H. G. CRAWFORD
Gastroenterology 2002 122: 281-289. Published online Feb 6 2002.
Background & Aims: The progression of
fibrosis to cirrhosis is the most significant prognostic factor
in hereditary hemochromatosis. We aimed to determine the range
of hepatic iron concentration associated with cirrhosis in the
absence of alcohol and other pro-fibrogenic cofactors and to quantify
the contribution of excess alcohol consumption to the development
of cirrhosis. Methods: Liver biopsy data were evaluated
on 224 C282Y homozygous hemochromatosis subjects. To determine
the effect of alcohol alone on the development of fibrosis, subjects
with viral hepatitis or nonalcoholic steatohepatitis were excluded.
Subjects were divided into those who consumed less than 60 g
alcohol per day and those who consumed 60 g per day or more.
Results: Seven percent of subjects who consumed less than
60 g per day had severe fibrosis/cirrhosis compared with
61% of excess alcohol consumers. Conclusions: Hemochromatosis
subjects who drink more than 60 g alcohol per day are approximately
9 times more likely to develop cirrhosis than those who drink
less than this amount, and the range of hepatic iron concentration
associated with cirrhosis in the absence of cofactors was 233-675
µmol/g dry weight.
Cerebral Cortical Registration of Subliminal Visceral Stimulation
MARK K. KERN and REZA SHAKER
Gastroenterology 2002 122: 290-298. Published online Feb 6 2002.
Background & Aims: Although brain registration
of subliminal somatic stimulations such as masked visual stimuli
and their influence on electrical and hemodynamic measures of
cerebral activity have been reported previously, there have been
no reports on cerebral cortical registration of subliminal visceral
stimulation. Because studies evaluating the consequences of subliminal
somatic stimulation have shown that subliminal stimulation can
effect behavior, it is conceivable that such subliminal messages
from the intestine could potentially influence intestinal sensory/motor
function or effect the perception/interpretation of sensory signals
originating from the gut. Methods: We studied the cerebral
cortical functional magnetic resonance imaging (fMRI) response
to subliminal, liminal, and supraliminal rectal distention in
healthy volunteers. Results: Study findings indicate that
subliminal afferent signals originating from the gut are registered
in the cerebral cortex without reaching the level of awareness.
Locations of cortical activity caused by intestinal subliminal
stimulation are similar to those of liminal and supraliminal stimulation
but their intensity and volume are significantly lower (P < 0.05).
Conclusions: Subliminal afferent signals originating from
the gut are registered in the cerebral cortex and induce changes
in measures of brain activity, such as hemodynamic changes detectable
by fMRI.
Acid Exposure Activates the Mitogen-Activated Protein Kinase
Pathways in Barrett's Esophagus
RHONDA F. SOUZA, KENNETH SHEWMAKE, LANCE S. TERADA, and STUART
JON SPECHLER
Gastroenterology 2002 122: 299-307. Published online Feb 6 2002.
Background & Aims: To explore mechanisms whereby
acid reflux might contribute to carcinogenesis in Barrett's esophagus
(BE) we studied: (1) the effects of acid on the mitogen-activated
protein kinase (MAPK) pathways, cell proliferation, and apoptosis
in a Barrett's adenocarcinoma cell line (SEG-1); and (2) the ability
of acid to activate the MAPK pathways in vivo in patients with
BE. Methods: SEG-1 cells were exposed to acidic media for
3 minutes, and the activities of 3 MAPKs (ERK, p38,
and JNK) were determined. Proliferation was assessed using flow
cytometry; cell growth and apoptosis were assessed using cell
counts and an apoptosis ELISA assay. MAPK activation was studied
in biopsy specimens taken from patients with BE before and after
esophageal perfusion for 3 minutes with 0.1N HCl. Results:
Acid-exposed SEG-1 cells exhibited a significant increase in proliferation
and total cell numbers, and a significant decrease in apoptosis.
These effects were preceded by a rapid increase in the activities
of ERK and p38, and a delayed increase in JNK activity. PD 98059 abolished
the acid-induced increase in G0/G1 and decrease in subG0 phases
of the cell cycle. Both SB 203580 and DN-JNK 1/2 inhibited
the acid-induced progression from G0/G1 to G2/M. The acid-induced
decrease in apoptosis was abolished by inhibition of either ERK
or p38. In the patients, acid exposure significantly increased
the activity of p38 in the metaplastic epithelium. Conclusions:
Acid increases proliferation and survival, and decreases apoptosis
in SEG-1 cells by activating the MAPK pathways. Acid also activates
the MAPK pathways in BE in vivo. These findings suggest that acid
might contribute to carcinogenesis in BE through activation of
MAPK pathways.
BASIC RESEARCH:
3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitors
Reduce Human Pancreatic Cancer Cell Invasion and Metastasis
TOSHIYUKI KUSAMA, MUTSUKO MUKAI, TERUO IWASAKI, MASAHARU TATSUTA,
YOSHIROU MATSUMOTO, HITOSHI AKEDO, MASAHIRO INOUE, and HIROYUKI
NAKAMURA
Gastroenterology 2002 122: 308-317. Published online Feb 6 2002.
Background & Aims: Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme
A (HMG-CoA) reductase blocks the mevalonate metabolic pathway,
which is necessary for the isoprenylation of a number of small
guanosine triphosphatases. We examined the effects of HMG-CoA
reductase inhibitors, fluvastatin and lovastatin, on human pancreatic
cancer cell invasion in vitro and experimental liver metastasis
in vivo. Methods: Cell invasion was studied in a modified
Boyden chamber assay. The translocation of RhoA was assessed by
immunoblotting. Experimental liver metastases were induced in
nude mice by intrasplenic inoculation of ASPC-1 human pancreatic
cancer cells. Results: Fluvastatin and lovastatin inhibited
the in vitro cancer cell invasion induced by epidermal growth
factor (EGF) in a manner sensitive to C3 transferase, a specific
inhibitor of Rho. Treatment of ASPC-1 cells with fluvastatin markedly
attenuated the EGF-induced translocation of RhoA from the cytosol
to the membrane fraction and caused cell rounding. The effects
of fluvastatin could be reversed by the addition of all-trans-geranylgeraniol.
Administration of fluvastatin to nude mice reduced both metastatic
tumor formation in the liver and the growth of established liver
metastases at doses recommended for the treatment of hypercholesterolemia
in humans. Conclusions: HMG-CoA reductase inhibitors can
be antimetastatic agents with the potential for useful clinical
applications.
Carbon Dioxide Affects Rat Colonic Na+ Absorption
by Modulating Vesicular Traffic
ALAN N. CHARNEY, RICHARD W. EGNOR, NICHOLAS CASSAI, and GURDIP
S. SIDHU
Gastroenterology 2002 122: 318-330. Published online Feb 6 2002.
Background & Aims: We examined whether CO2
affects colonic Na+ absorption by endosome recycling of the Na+/H+
exchanger NHE3. Methods: Rat distal colon segments exposed
to various acid-base conditions were examined by transmission
electron microscopy at 27,500¥ magnification and subapical
vesicles quantified. Immunocytochemistry was used to identify
vesicular NHE3. Endocytosis was tested for by observing internalization
of apical membrane labeled with fluorescein isothiocyanate-phytohemagglutinin
and Cy-3-NHE3 antibody using confocal microscopy. The effects
of mucosal 5-(N,N-dimethyl)-amiloride (DMA), which inhibits NHE2
and/or NHE3, and wortmannin, which inhibits phosphatidylinositol
3-kinase, on CO2-stimulated Na+ absorption were measured in the
Ussing chamber. Results: The number of (coated and uncoated)
subapical vesicles in epithelial cells was specifically and inversely
related to net colonic Na+ absorption and Pco2. Immunoperoxidase
labeling localized NHE3 on microvilli and vesicle membranes. Under
the confocal microscope, a fluorescent band along apical membranes
at Pco2 70 mm Hg became a subapical haze at Pco2 21 mm
Hg. This pattern was not affected by carbonic anhydrase inhibition
or when pH or [HCO3] was changed, but Pco2 was held constant.
DMA inhibition indicated that NHE3 mediates CO2-stimulated Na+
absorption. Wortmannin inhibited CO2-stimulated vesicle movement
(exocytosis) and Na+ absorption. Conclusions: CO2 affects
Na+ absorption in rat distal colon epithelium in part by modulating
the movement of NHE3-containing vesicles to and from the apical
membrane.
Tumor Necrosis Factor Stimulates Invasion of Src-Activated
Intestinal Cells
NAOKI KAWAI, SHINGO TSUJI, MASAHIKO TSUJII, TOSHIFUMI ITO, MASAKAZU
YASUMARU, YOSHIMI KAKIUCHI, ARATA KIMURA, MASATO KOMORI, YUTAKA
SASAKI, NORIO HAYASHI, SUNAO KAWANO, RAYMOND DUBOIS, and MASATSUGU
HORI
Gastroenterology 2002 122: 331-339. Published online Feb 6 2002.
Background & Aims: Src activation is correlated
with progression of colorectal cancer (CRC). CRCs accompanied
by ulcerative colitis, chronic inflammation in the colon, often
have elevated Src activity, and ulcerative colitis-related CRCs
are more likely to become invasive, whereas Ras activation is
rarely associated with this disease. The aim of this study was
to investigate the effects of a proinflammatory cytokine, tumor
necrosis factor (TNF-), on the invasive properties of epithelial
cells constitutively expressing activated Ras or Src. Methods:
A cell line derived from intestinal epithelia was transfected
with a v-src- or v-H-ras-expressing vector. The
effect of TNF- on morphologic changes in colonies cultured in
soft agar was determined. Src protein kinase activity, peroxide
production, E-cadherin expression levels, and the phosphorylation
status of -catenin and E-cadherin were determined. The invasive
potential of these cells was determined by measuring cell motility
and using an in vitro invasion assay. Results: TNF- altered
the colony morphology of src-, but not ras-expressing cells. TNF-
increased peroxide production, leading to Src protein expression
as well as Src activity in src transfectants. Activation of Src
by TNF- led to reduced E-cadherin levels and enhanced invasion
of src transfectants. Pyrrolidine dithiocarbamate and herbimycin
A inhibited these effects. Conclusion: These results indicate
that Src kinase activation enhances the response of epithelial
cells to TNF- leading to increased invasion through mechanisms
that involve production of reactive oxygen intermediates.
Opioid Agonists Inhibit Excitatory Neurotransmission in
Ganglia and at the Neuromuscular Junction in Guinea Pig Gallbladder
FAY A. GUARRACI, MARIA J. POZO, SARA M. PALOMARES, TRACY A. FIRTH,
and GARY M. MAWE
Gastroenterology 2002 122: 340-351. Published online Feb 6 2002.
Background & Aims: Opiates administered
therapeutically could have an inhibitory effect on the neuromuscular
axis of the gallbladder, and thus contribute to biliary stasis
and acalculous cholecystitis. Methods: Intracellular recordings
were made from gallbladder neurons and smooth muscle, and tension
measurements were made from muscle strips. Opioid receptor-specific
agonists tested: delta, DPDPE; kappa, U-50488H; and mu, DAMGO.
Results: Opioid agonists had no effect on gallbladder neurons
or smooth muscle. Each of the opioid agonists potently suppressed
the fast excitatory synaptic input to gallbladder neurons, in
a concentration-dependent manner with half-maximal effective concentration
values of about 1 pmol/L. Also, each agonist caused a concentration-dependent
reduction in the amplitude of the neurogenic contractile response
(half-maximal effective concentration values: DPDPE, 189 pmol/L;
U-50488H, 472 pmol/L; and DAMGO, 112 pmol/L). These
ganglionic and neuromuscular effects were attenuated by the highly
selective opioid-receptor antagonist, naloxone. Opioid-receptor
activation also inhibited the presynaptic facilitory effect of
cholecystokinin in gallbladder ganglia. Immunohistochemistry with
opioid receptor-specific antisera revealed immunostaining for
all 3 receptor subtypes in nerve bundles and neuronal cell
bodies within the gallbladder, whereas opiate-immunoreactive nerve
fibers are sparse in the gallbladder. Conclusions: These
results show that opiates can cause presynaptic inhibition of
excitatory neurotransmission at 2 sites within the wall of
the gallbladder: vagal preganglionic terminals in ganglia and
neuromuscular nerve terminals. These findings support the concept
that opiates can contribute to gallbladder stasis by inhibiting
ganglionic activity and neurogenic contractions.
Steatosis and Liver Cancer in Transgenic Mice Expressing
the Structural and Nonstructural Proteins of Hepatitis C Virus
HERVÉ LERAT, MASAO HONDA, MICHAEL R. BEARD, KIM LOESCH,
JIAREN SUN, YAN YANG, MICHIARI OKUDA, RAINER GOSERT, SHU-YUAN
XIAO, STEVEN A. WEINMAN, and STANLEY M. LEMON
Gastroenterology 2002 122: 352-365. Published online Feb 6 2002.
Background and Aims: The aim of this study
was to determine whether expression of hepatitis C virus proteins
alters hepatic morphology or function in the absence of inflammation.
Methods: Transgenic C57BL/6 mice with liver-specific expression
of RNA encoding the complete viral polyprotein (FL-N transgene)
or viral structural proteins (S-N transgene) were compared with
nontransgenic littermates for altered liver morphology and function.
Results: FL-N transcripts were detectable only by reverse-transcription
polymerase chain reaction, and S-N transcripts were identified
in Northern blots. The abundance of viral proteins was sufficient
for detection only in S-N transgenic animals. There was no inflammation
in transgenic livers, but mice expressing either transgene developed
age-related hepatic steatosis that was more severe in males. Apoptotic
or proliferating hepatocytes were not significantly increased.
Hepatocellular adenoma or carcinoma developed in older male animals
expressing either transgene, but their incidence reached statistical
significance only in FL-N animals. Neither was ever observed in
age-matched nontransgenic mice. Conclusions: Constitutive
expression of viral proteins leads to common pathologic features
of hepatitis C in the absence of specific anti-viral immune responses.
Expression of the structural proteins enhances a low background
of steatosis in C57BL/6 mice, while additional low level expression
of nonstructural proteins increases the risk of cancer.
Mitochondrial Injury, Oxidative Stress, and Antioxidant
Gene Expression Are Induced by Hepatitis C Virus Core Protein
MICHIARI OKUDA, KUI LI, MICHAEL R. BEARD, LORI A. SHOWALTER, FRANK
SCHOLLE, STANLEY M. LEMON, and STEVEN A. WEINMAN
Gastroenterology 2002 122: 366-375. Published online Feb 6 2002.
Background & Aims: The mechanisms of liver
injury in chronic hepatitis C virus (HCV) infection are poorly
understood. Indirect evidence suggests that oxidative stress and
mitochondrial injury play a role. The aim of this study was to
determine if the HCV core protein itself alters mitochondrial
function and contributes to oxidative stress. Methods:
HCV core protein was expressed in 3 different cell lines,
and reactive oxygen species (ROS) and lipid peroxidation products
were measured. Results: Core expression uniformly increased
ROS. In 2 inducible expression systems, core protein also
increased lipid peroxidation products and induced antioxidant
gene expression as well. A mitochondrial electron transport inhibitor
prevented the core-induced increase in ROS. A fraction of the
expressed core protein localized to the mitochondria and was associated
with redistribution of cytochrome c from mitochondrial to cytosolic
fractions. Sensitivity to oxidative stress was also seen in HCV
transgenic mice in which increased intrahepatic lipid peroxidation
products occurred in response to carbon tetrachloride. Conclusions:
Oxidative injury occurs as a direct result of HCV core protein
expression both in vitro and in vivo and may involve a direct
effect of core protein on mitochondria. These results provide
new insight into the pathogenesis of hepatitis C and provide an
experimental rationale for investigation of antioxidant therapy.
Aminopeptidase N Is Involved in Cell Motility and Angiogenesis:
Its Clinical Significance in Human Colon Cancer
HIROKI HASHIDA, ARIMICHI TAKABAYASHI, MICHIYUKI KANAI, MASASHI
ADACHI, KEIICHI KONDO, NOBUOKI KOHNO, YOSHIO YAMAOKA, and MASAYUKI
MIYAKE
Gastroenterology 2002 122: 376-386. Published online Feb 6 2002.
Background & Aims: The molecular basis of
cell motility is highly complex and is controlled by a number
of molecular systems, whereas angiogenesis is an important biological
component of tumor progression. The aims of this study were to
investigate the possible involvement of proteins at the cell surface
in controlling cell motility and angiogenesis, and to identify
the cell surface molecules involved in gastrointestinal tumors.
Methods: We addressed these issues using functional monoclonal
antibodies, which inhibit cell motility, endothelial cell migration,
and tube formation. Furthermore, we investigated the relationship
between this antigen and colon cancer, and showed the prognostic
significance in human colon cancer. Results: We established
a murine monoclonal antibody MH8-11, which inhibits cell motility
and in vitro angiogenesis. This epitope was a 165-kilodalton protein,
and the sequencing analysis revealed that it was almost identical
to aminopeptidase N (APN)/cluster of differentiation (CD) 13. APN/CD13
expression was associated with tumor status (P = 0.025).
The disease-free and overall survival rate for patients with positive
APN/CD13 expression tumors was significantly lower than that for
patients with negative APN/CD13 expression tumors(P = 0.014, 0.033, respectively).
Among 47 node-positive patients, the survival rate of patients
with negative APN/CD13 expression was better than that of those
with positive APN/CD13 expression. Conclusions: Our data
suggest that APN/CD13 is involved in cell motility and angiogenesis,
and APN/CD13 expression may be a useful indicator of a poor prognosis
for node-positive patients with colon cancer.
5-Lipoxygenase Inhibition Reduces Intrahepatic Vascular
Resistance of Cirrhotic Rat Livers: A Possible Role of Cysteinyl-Leukotrienes
MARIONA GRAUPERA, JUAN-CARLOS GARCÍA-PAGÁN, ESTHER
TITOS, JOAN CLARIA, ANNA MASSAGUER, JAIME BOSCH, and JUAN RODÉS
Gastroenterology 2002 122: 387-393. Published online Feb 6 2002.
Background & Aims: Cysteinyl-leukotrienes
(Cys-LTs) increase intrahepatic vascular resistance in normal
rat livers. CCl4 cirrhotic rat livers have increased Cys-LT production
and 5-lipoxygenase messenger RNA (mRNA) expression. The aim of
this study was to investigate the role of 5-lipoxygenase-derived
eicosanoids regulating intrahepatic vascular tone in control and
CCl4-induced cirrhotic rat livers. Methods: In different
groups of portally perfused control and cirrhotic rat livers,
the following were analyzed: a portal perfusion pressure (PP)
dose-response curve to LTD4; the effects on PP caused by either
vehicle, the selective 5-lipoxygenase inhibitor AA-861, the selective
Cys-LT1 receptor antagonist MK-571, or the dual Cys-LT1 and Cys-LT2
receptor antagonist BAY u9773; and immunohistochemistry for 5-lipoxygenase
in liver sections of cirrhotic and control livers. Results:
Cirrhotic livers have a hyperesponse to LTD4. In control livers,
AA-861 and MK-571 produced a moderate and similar reduction in
PP. In cirrhotic livers, 5-lipoxygenase inhibition produced a
marked and significantly greater reduction in PP than in controls.
However, no effect on PP was observed after MK-571 or BAY u9773.
5-Lipoxygenase-positive cells were markedly increased in cirrhotic
livers. Conclusions: Our resultssuggest that 5-lipoxygenase-derived
eicosanoids may contribute to the increased intrahepatic vascular
resistance of cirrhotic rat livers and therefore the pathogenesis
of portal hypertension.
Experimental Ulcers Alter Voltage-Sensitive Sodium Currents
in Rat Gastric Sensory Neurons
K. BIELEFELDT, N. OZAKI, and G. F. GEBHART
Gastroenterology 2002 122: 394-405. Published online Feb 6 2002.
Background & Aims: Voltage-dependent Na+
currents are important determinants of excitability. We hypothesized
that gastric inflammation alters Na+ current properties in primary
sensory neurons. Methods: The stomach was surgically exposed
in rats to inject the retrograde tracer 1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine
methanesulfonate and saline (control) or 20% acetic acid (ulcer
group) into the gastric wall. Nodose or thoracic dorsal root ganglia
(DRG) were harvested after 7 days to culture neurons and
record Na+ currents using patch clamp techniques. Results:
There were no lesions in the control and 3 ± 1 ulcers
in the ulcer group. Na+ currents recovered significantly more
rapidly from inactivation in nodose and DRG neurons obtained from
animals in the ulcer group compared with controls. This was partially
a result of an increase in the relative contribution of the tetrodotoxin-resistant
to the peak sodium current. In addition, the recovery kinetics
of the tetrodotoxin-sensitive current were faster. In DRG neurons,
gastric inflammation shifted the voltage-dependence of activation
of the tetrodotoxin-resistant current to more hyperpolarized potentials.
Conclusions: Gastric injury alters the properties of Na+
currents in gastric sensory neurons. This may enhance excitability,
thereby contributing to the development of dyspeptic symptoms.
Detection of Dysplastic Intestinal Adenomas Using Enzyme-Sensing
Molecular Beacons in Mice
KATHARINA MARTEN, CHRISTOPH BREMER, KHASHAYARSHA KHAZAIE, MANSOUREH
SAMENI, BONNIE SLOANE, CHING-HSUAN TUNG, and RALPH WEISSLEDER
Gastroenterology 2002 122: 406-414. Published online Feb 6 2002.
Background & Aims: Proteases play key
roles in the pathogenesis of tumor growth and invasion. This study
assesses the expression of cathepsin B in dysplastic adenomatous
polyps. Methods: Aged ApcMin/+ mice served as an experimental
model for familial adenomatous polyposis. The 4 experimental
groups consisted of (a) animals injected with a novel activatable,
cathepsin B sensing near infrared fluorescence (NIRF) imaging
probe; (b) animals injected with a nonspecific NIRF; (c) uninjected
control animals; and (d) non-APCMin/+ mice injected with the cathepsin
B probe. Lesions were analyzed by immunohistochemistry, Western
blotting, reverse transcription-polymerase chain reaction, and
optical imaging. Results: Cathepsin B was consistently
overexpressed in adenomatous polyps. When mice were injected intravenously
with the cathepsin reporter probe, intestinal adenomas became
highly fluorescent indicative of high cathepsin B enzyme activity.
Even microscopic adenomas were readily detectable by fluorescence,
but not light, imaging. The smallest lesion detectable measured
50 µm in diameter. Adenomas in the indocyanine green
and/or noninjected group were only barely detectable above the
background. Conclusions: The current experimental study
shows that cathepsin B is up-regulated in a mouse model of adenomatous
polyposis. Cathepsin B activity can be used as a biomarker to
readily identify such lesions, particularly when contrasted against
normal adjacent mucosa. This detection technology can be adapted
to endoscopy or tomographic optical imaging methods for screening
of suspicious lesions and potentially for molecular profiling
in vivo.
Ca2+ Waves Require Sequential Activation of Inositol
Trisphosphate Receptors and Ryanodine Receptors in Pancreatic
Acini
M. FATIMA LEITE, ANGELA D. BURGSTAHLER, and MICHAEL H. NATHANSON
Gastroenterology 2002 122: 415-427. Published online Feb 6 2002.
Background & Aims: The inositol 1,4,5-trisphosphate
(InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are
the principal Ca2+-release channels in cells and are believed
to serve distinct roles in cytosolic Ca2+ (Cai2+) signaling. This
study investigated whether these receptors instead can release
Ca2+ in a coordinated fashion. Methods: Apical and basolateral
Cai2+ signals were monitored in rat pancreatic acinar cells by
time-lapse confocal microscopy. Caged forms of second messengers
were microinjected into individual cells and then photoreleased
in a controlled fashion by either UV or 2-photon flash photolysis.
Results: InsP3 increased Cai2+ primarily in the apical
region of pancreatic acinar cells, whereas the RyR agonist cyclic
adenosine diphosphate ribose (cADPR) increased Cai2+ primarily
in the basolateral region. Apical-to-basal Cai2+ waves were induced
by acetylcholine and initiation of these waves was blocked by
the InsP3R inhibitor heparin, whereas propagation into the basolateral
region was inhibited by the cADPR inhibitor 8-amino-cADPR. To
examine integration of apical and basolateral Cai2+ signals, Ca2+
was selectively released either apically or basolaterally using
2-photon flash photolysis. Cai2+ increases were transient and
localized in unstimulated cells. More complex Cai2+ signaling
patterns, including polarized Cai2+ waves, were observed when
Ca2+ was photoreleased in cells stimulated with subthreshold concentrations
of acetylcholine. Conclusions: Polarized Cai2+ waves are
induced in acinar cells by serial activation of apical InsP3Rs
and then basolateral RyRs, and subcellular release of Ca2+ coordinates
the actions of these 2 types of Ca2+ channels. This subcellular
integration of Ca2+-release channels shows a new level of complexity
in the formation of Cai2+ waves.
Expression of CCK2 Receptors in the Murine Pancreas: Proliferation,
Transdifferentiation of Acinar Cells, and Neoplasia
PASCAL CLERC, STÉPHANE LEUNG-THEUNG-LONG, TIMOTHY C. WANG,
GRAHAM J. DOCKRAY, MICHÈLE BOUISSON, MARIE-BERNADETTE DELISLE,
NICOLE VAYSSE, LUCIEN PRADAYROL, DANIEL FOURMY, and MARLÈNE
DUFRESNE
Gastroenterology 2002 122: 428-437. Published online Feb 6 2002.
Background & Aims: To explore the pancreatic
function of CCK2/gastrin receptor, we created ElasCCK2 transgenic
mice expressing the human receptor in pancreatic exocrine cells.
In previous studies, the transgenic CCK2/gastrin receptor was
demonstrated to mediate enzyme release and protein synthesis.
We now report results of phenotypic and long-term studies. Methods:
Pancreas was characterized using morphometry and immunohistochemistry.
ElasCCK2 mice were crossed with INS-GAS mice expressing gastrin
in pancreatic cells to achieve continuous stimulation of
the CCK2/gastrin receptor. Results: The pancreatic weight
of ElasCCK2 mice was increased by 40% and correlated with an increase
in the area of exocrine tissue. Alterations in pancreatic histology
were apparent from postnatal day 50. Crossing the ElasCCK2
mice with INS-GAS mice resulted in development of morphologic
changes in younger animals. Malignant transformation occurred
in 3 of 20 homozygous ElasCCK2 mice. Although tumors
had different phenotypes, they all developed through an acinar-ductal
carcinoma sequence. Conclusions: Our data show that transgenic
expression of a G protein-coupled receptor can lead to cancer.
This study also supports a key role of the CCK2/gastrin receptor
in the development of pre- and neoplastic lesions of the pancreas.
ElasCCK2 mice provide a model for carcinogenesis by transformation
and dedifferentiation of acinar cells.
Early Cell Transplantation in LEC Rats Modeling Wilson's
Disease Eliminates Hepatic Copper With Reversal of Liver Disease
HARMEET MALHI, ADIL N. IRANI, IRENE VOLENBERG, MICHAEL L. SCHILSKY,
and SANJEEV GUPTA
Gastroenterology 2002 122: 438-447. Published online Feb 6 2002.
Background & Aims: The Long-Evans Cinnamon
(LEC) rat is an excellent model of Wilson's disease with impaired
copper excretion, hypoceruloplasminemia, and copper toxicosis.
We hypothesized that early hepatocyte transplantation would improve
copper excretion and liver disease in Wilson's disease. Methods:
Normal syngeneic Long-Evans Agouti rat hepatocytes were transplanted
intrasplenically into 2-week-old LEC rats. Untreated LEC pups
were controls. Liver repopulation was shown by changes in serum
ceruloplasmin, hepatic atp7b messenger RNA, and bile and liver
copper levels. Histologic analysis of the liver was performed.
Results: Significant copper accumulation and liver disease
were observed in 5-month-old LEC rats, with occasional treated
rats showing increased bile copper excretion. The liver was repopulated
extensively in 10 of 14 treated LEC rats (71%) 20 months
after cell transplantation. In these 10 rats, hepatic copper
content was virtually normal in 6 rats (53 ± 12 µg/g
liver) and substantially less in 4 others (270 ± 35 µg/g)
compared with elevated liver copper levels in untreated LEC rats
(1090 ± 253 µg/g) (P < 0.001).
Changes in serum ceruloplasmin levels, bile copper excretion capacity,
and liver histology were in concordance with decreases in liver
copper levels. Conclusions: Transplanted cells proliferated
subsequent to the onset of liver injury, and the liver was repopulated
over an extended period. Cell transplantation eventually restored
copper homeostasis and reversed liver disease without hepatic
preconditioning in LEC rats.
NF-B Activation in Pancreas Induces Pancreatic and Systemic
Inflammatory Response
XUEQING CHEN, BAOAN JI, BING HAN, STEPHEN A. ERNST, DIANE SIMEONE,
and CRAIG D. LOGSDON
Gastroenterology 2002 122: 448-457. Published online Feb 6 2002.
Background & Aims: The role of nuclear
factor B (NF-B) activation in acute pancreatitis is uncertain.
The transcription factor NF-B is activated early in acute pancreatitis,
and NF-B is widely considered a key element in inflammatory responses
based on its ability to regulate the expression of inflammatory
mediators in vitro. However, its role in vivo in specific diseases
remains unclear, and the current data on the role of NF-B in acute
pancreatitis is primarily correlative. Methods: In this
study, NF-B was directly activated within the pancreas using adenoviral-mediated
transfer of an active subunit, RelA/p65 (Adp65), delivered by
intraductal injection. Results: Administration of Adp65
led to the infection of a population of acinar cells within the
pancreas, the activation of NF-B, the expression of NF-B target
genes, and an inflammatory response. Administration of Adp65 increased
the infiltration of neutrophils to the pancreas and lung and caused
widespread damage to pancreatic acinar cells. In contrast, at
the same titer, control adenovirus (AdGFP) had no effect on these
parameters. The level of NF-B activation and the severity of inflammation
were reduced when an adenovirus bearing the inhibitory subunit
IB- was coadministered with Adp65. Conclusions: Thus, activation
of NF-B within the pancreas was sufficient for the initiation
of an inflammatory response in this model. These results help
define the specific role of NF-B activation in acute pancreatitis.
Esophageal Ulceration Activates Keratinocyte Growth Factor
and Its Receptor in Rats: Implications for Ulcer Healing
DOLGOR BAATAR, HIROFUMI KAWANAKA, IMRE L. SZABO, RAMA PAI, MICHAEL
K. JONES, SEIGO KITANO, and ANDRZEJ S. TARNAWSKI
Gastroenterology 2002 122: 458-468. Published online Feb 6 2002.
Background & Aims: Cellular and molecular
mechanisms of esophageal ulcer healing remain unexplored. We studied
the sequential cellular events and the expression of keratinocyte
growth factor (KGF) and its receptor (KGF-R) during the healing
of experimental esophageal ulcers. Methods: Esophageal
ulcers were produced in rats by local application of acetic acid.
Studies included (1) ulcer size, (2) quantitative histology, and
(3) KGF and KGF-R messenger RNA and protein expression by reverse-transcription
polymerase chain reaction, Western blotting, and immunostaining.
In separate groups, ulcer size and esophageal epithelial proliferation
were evaluated after a single injection of recombinant human KGF
(1 mg/kg) around the ulcer. Results: Ulcers were fully
developed 3 days after induction, and 58% of ulcers were
re-epithelialized by 9 days. At 3 days, in esophageal
tissue bordering the ulcers, KGF messenger RNA and protein were
increased by 191% and 151%, respectively, and KGF-R messenger
RNA and protein were increased by 357% and 237%, respectively.
KGF was expressed in stromal cells, whereas KGF-R was expressed
in epithelial cells. At 6 days, epithelial proliferation
at the ulcer margin was increased by 216%, and treatment with
KGF further enhanced cell proliferation and accelerated ulcer
healing. Conclusions: KGF is a likely mediator of esophageal
epithelial proliferation and ulcer healing.
Disruption of Hedgehog Signaling Reveals a Novel Role in
Intestinal Morphogenesis and Intestinal-Specific Lipid Metabolism
in Mice
LI CHUN WANG, FATIHA NASSIR, ZHONG-YING LIU, LEONA LING, FRANK
KUO, THOMAS CROWELL, DIAN OLSON, NICHOLAS O. DAVIDSON, and LINDA
C. BURKLY
Gastroenterology 2002 122: 469-482. Published online Feb 6 2002.
Background & Aims: The hedgehog (hh) signaling
pathway has been shown to play crucial roles in the development
of embryonic gut. However, its role in intestinal development
and function beyond the embryonic stage is still undefined. Methods:
Expression of hh and its receptor, Patched, were examined by Western
blot and X-gal staining. An anti-hh monoclonal antibody was administered
into developing embryos or postnatal mice and histologic analyses
were performed. Effects on lipid metabolism were examined by Oil
Red O and Sudan III stainings, messenger RNA (mRNA) analysis,
and electron microscopy. Serum apolipoprotein IV level, a marker
for lipid absorption, was quantified by Western blot. Results:
Mice receiving anti-hh monoclonal antibody in utero or after birth
exhibited progressive runting and died before weaning. Histology
revealed hyperproliferation of intestinal crypt epithelial cells
and disorganization of the villi with prominent vacuolation and
accumulation of neutral lipid. Fecal fat microscopy revealed numerous
large fat droplets. Intestinal mRNA abundance of 2 candidate
genes involved in lipid transport, mtp and apob,
was unchanged, although serum levels of apolipoprotein A-IV were
reduced. Conclusions: Abnormal villus structure, lipid-filled
enterocytes, and fatty stools in anti-hh monoclonal antibody-treated
mice indicate a novel role for hh signaling in intestinal morphogenesis
and lipid transport in postnatal mice.
Selective Modulation of PKC Isozymes by Inflammation in
Canine Colonic Circular Muscle Cells
IRSHAD ALI and SUSHIL K. SARNA
Gastroenterology 2002 122: 483-494. Published online Feb 6 2002.
Background & Aims: Protein kinase C (PKC)
is a key signaling molecule in excitation-contraction coupling
in several types of smooth muscle cells. We investigated whether
the attenuated contraction in inflamed colon cells is caused by
alterations in the expression, distribution, and activation of
specific PKC isozymes. Methods: Kinase assays, immunofluorescence
imaging, and Western immunoblotting were performed on single circular
smooth muscle cells obtained from the normal dog colon as well
as from colon with experimental colitis induced by mucosal exposure
to ethanol and acetic acid, to determine the distribution, expression,
and activation of PKC isozymes. Results: Classical (, ,
and ), novel ( and ), and the atypical PKC (, , and ) isozymes
were detected in colonic circular muscle cells. The expression
of PKC , , and isozymes was down-regulated, whereas that
of PKC and isozymes was up-regulated; other isozymes
were not affected by inflammation. Acetylcholine (ACh) treatment
translocated only the PKC , , and isozymes from the cytosol
to the membrane in normal cells; this translocation was absent
in inflamed colon cells. Immunofluorescence imaging confirmed
the translocation of PKC from the cytosol to the membrane
in response to ACh in normal cells. PKC inhibitors, chelerythrine,
and myristoylated peptides to , , and isozymes inhibited
the contractile response to ACh in normal, but not in inflamed,
cells. PKC and did not participate in the contractile
response to ACh. Conclusions: ACh-induced contraction is
mediated by PKC , , and isozymes in normal colonic circular
muscle cells. Contractile dysfunction in inflamed colon cells
is, in part, caused by decreased expression and impaired activation
of specific PKC isozymes.
CASE REPORTS:
Intestinal Ischemia and Peripheral Gangrene in a Patient
With Chronic Renal Failure
JESÚS RIVERA-NIEVES, GIORGOS BAMIAS, JONATHAN ALFERT, STEPHEN
J. BICKSTON, CHRISTOPHER A. MOSKALUK, and FABIO COMINELLI
Gastroenterology 2002 122: 495-499. Published online Feb 6 2002.
Gastrointestinal complications are common in patients with
renal failure and result in significant morbidity and mortality.
Systemic calciphylaxis is an uncommon complication of renal failure,
characterized by disseminated intravascular calcification and
associated with progressive vascular compromise. We describe the
case of a 63-year-old woman who presented with abdominal pain,
elevated transaminases, and skin manifestations consistent with
a vasculitic process. Hand films and skin biopsies showed extensive
vascular calcification, and a computerized tomography scan confirmed
colonic perforation and disseminated visceral vascular calcification.
Histologic analysis of the resected skin and colonic tissues revealed
extensive ischemic damage and mural calcification of medium to
large vessels. Gastrointestinal involvement has been reported
in only 3 prior cases of calciphylaxis; consequently, gastroenterologists
are often unaware of this disease entity and may fail to recognize
it, even in patients with the classical presentation. Prompt diagnosis
is crucial, as parathyroidectomy may result in clinical improvement
in up to two thirds of patients who present with elevated parathyroid
hormone levels.
Dominant Negative Action of an Abnormal Secretin Receptor
Arising From mRNA Missplicing in a Gastrinoma
WEI-QUN DING, SUSAN KUNTZ, MICHAEL BÖHMIG, BERTRAM WIEDENMANN,
and LAURENCE J. MILLER
Gastroenterology 2002 122: 500-511. Published online Feb 6 2002.
Background & Aims: The provocative secretin-stimulation
test has an important role in the diagnosis and management of
gastrin-secreting neuroendocrine tumors. The aim of the present
study was to explore the molecular basis for positive and false-negative
secretin-stimulation test results in patients with these tumors.
Methods: One of the rare patients with this histologically
proven tumor who had a normal serum gastrin level and a negative
secretin-stimulation test result, and 2 more typical patients
with this syndrome were investigated using immunohistochemistry,
reverse-transcription polymerase chain reaction, receptor binding,
and signaling assays. Results: We confirmed the molecular
nature of the secretin receptor in the gastrinomas with a positive
provocative test result and identified a novel mechanism for a
false-negative result. Tumor expression of the class B G protein-coupled
secretin receptor mediates a positive result. The false-negative
result was explained by messenger RNA missplicing, resulting in
a receptor variant missing exon 3 that encodes residues 44-79
in the amino-terminal tail of the mature receptor. This variant
with an in-frame deletion was shown to be synthesized and to traffic
to the cell surface normally, where it could neither bind secretin
nor mediate a secretin-stimulated adenosine 3',5'-cyclic monophosphate
response. It was able to act as a dominant negative inhibitor
of wild-type secretin receptor function. Conclusions: These
data may explain some of the atypical presentations of this syndrome
and provide important insights into basic mechanisms of disease.
Biological Actions and Therapeutic Potential of the Glucagon-like
Peptides
DANIEL J. DRUCKER
Gastroenterology 2002 122: 531-544. Published online Feb 6 2002.
The Banting and Best Diabetes Centre, Department of Medicine, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada
The glucagon-like peptides (GLP-1 and GLP-2) are proglucagon-derived
peptides cosecreted from gut endocrine cells in response to nutrient
ingestion. GLP-1 acts as an incretin to lower blood glucose via
stimulation of insulin secretion from islet cells. GLP-1
also exerts actions independent of insulin secretion, including
inhibition of gastric emptying and acid secretion, reduction in
food ingestion and glucagon secretion, and stimulation of -cell
proliferation. Administration of GLP-1 lowers blood glucose and
reduces food intake in human subjects with type 2 diabetes.
GLP-2 promotes nutrient absorption via expansion of the mucosal
epithelium by stimulation of crypt cell proliferation and inhibition
of apoptosis in the small intestine. GLP-2 also reduces epithelial
permeability, and decreases meal-stimulated gastric acid secretion
and gastrointestinal motility. Administration of GLP-2 in the
setting of experimental intestinal injury is associated with reduced
epithelial damage, decreased bacterial infection, and decreased
mortality or gut injury in rodents with chemically induced enteritis,
vascular-ischemia reperfusion injury, and dextran sulfate-induced
colitis. GLP-2 also attenuates chemotherapy-induced mucositis
via inhibition of drug-induced apoptosis in the small and large
bowel. GLP-2 improves intestinal adaptation and nutrient absorption
in rats after major small bowel resection, and in humans with
short bowel syndrome. The actions of GLP-2 are mediated by a distinct
GLP-2 receptor expressed on subsets of enteric nerves and enteroendocrine
cells in the stomach and small and large intestine. The beneficial
actions of GLP-1 and GLP-2 in preclinical and clinical studies
of diabetes and intestinal disease, respectively, has fostered
interest in the potential therapeutic use of these gut peptides.
Nevertheless, the actions of the glucagon-like peptides are limited
in duration by enzymatic inactivation via cleavage at the N-terminal
penultimate alanine by dipeptidyl peptidase IV (DP IV). Hence,
inhibitors of DP IV activity, or DP IV-resistant glucagon-like
peptide analogues, may be alternative therapeutic approaches for
treatment of human diseases.
Clinical course of hepatitis C virus during the first decade
of infection: cohort study
Helen E Harris, Mary E Ramsay, Nick Andrews, and Keith P Eldridge
[Full Text]
Objective: To determine the clinical course of hepatitis
C virus in the first decade of infection in a group of patients
who acquired their infections on a known date.
Design: Cohort study.
Setting: Clinical centres throughout the United Kingdom.
Participants: 924 transfusion recipients infected with
the hepatitis C virus (HCV) traced during the HCV lookback programme
and 475 transfusion recipients who tested negative for antibodies
to HCV (controls).
Main outcome measures: Clinical evidence of liver disease
and survival after 10 years of infection.
Results: All cause mortality was not significantly different
between patients and controls (Cox's hazards ratio 1.41, 95%
confidence interval 0.95 to 2.08). Patients were more likely
to be certified with a death related to liver disease than were
controls (12.84, 1.73 to 95.44), but although the risk
of death directly from liver disease was higher in patients than
controls this difference was not significant (5.78, 0.72 to
46.70). Forty per cent of the patients who died directly from
liver disease were known to have consumed excess alcohol. Clinical
follow up of 826 patients showed that liver function was
abnormal in 307 (37.2%), and 115 (13.9%) reported physical
signs or symptoms of liver disease. Factors associated with developing
liver disease were testing positive for HCV ribonucleic acid (odds
ratio 6.44, 2.67 to 15.48), having acquired infection
when older (at age 40 years; 1.80, 1.14 to
2.85), and years since transfusion (odds ratio 1.096 per
year, 1.00 to 1.20). For patients with severe disease, sex
was also significant (odds ratio for women 0.38, 0.17 to
0.88). Of the 362 patients who had undergone liver biopsy,
328 (91%) had abnormal histological results and 35 (10%)
of these were cirrhotic.
Conclusions: Hepatitis C virus infection did not have a
great impact on all cause mortality in the first decade of infection.
Infected patients were at increased risk of dying directly from
liver disease, particularly if they consumed excess alcohol, but
this difference was not statistically significant.
Better surveillance needed for second colorectal cancers
Marilynn Larkin
Despite intensive surveillance after treatment of an initial
cancer, the incidence of second primary colorectal cancers remains
high in patients with a history of the disease, US researchers
report this week. "The risk is higher than we might have
expected from previous information", asserts lead researcher
Robert Green (Helen and Harry Gray Cancer Institute, West Palm
Beach, FL, USA). "These patients need close follow-up, and
at the very least, they need to adhere to the guidelines that
are out there", he emphasises. "They should have a complete
visualisation of the colon shortly after an initial diagnosis
to ensure that synchronous cancers aren't missed, and have regular
colonoscopies thereafter." Green and co-workers analysed
data from 3278 patients with resected stage II or stage III colorectal
cancer who participated in a multicentre adjuvant chemotherapy
trial that included surveillance based on current, but unproven
guidelines: colonoscopy 1 year after treatment and every 3-5 years
if results are normal. During 15 245 patient-years of follow-up,
42 cases of second primary colorectal cancers developed. This
number translates to a rate of 274 cases per 100 000 patient-years,
which is significantly higher than expected from previous studies.
The standardised incidence ratio was 1·6 compared with
the Surveillance, Epidemiology, and End Results (SEER) programme,
which monitors the US general population, and 6·8 compared
with the National Polyp Study (Ann Intern Med 2002; 136:
261-69). Although Green concedes that the reason for the higher
rate isn't clear, he adds that "our findings raise the very
important issue of whether surveillance in the real world is as
accurate as it was in the very controlled environment of the National
Polyp Study. The rate of second colorectal cancers in our study--even
though it was part of a clinical trial--is probably a very good
reflection of the 'best-case' scenario of what is really going
on." Paul Limburg (Mayo Clinic, Rochester, MN, USA) warns
that the high rate of second cancers reflects "a failure
in patient management", and stresses that both physicians
and patients "need to do a better job of adhering to existing
guidelines" for the detection of synchronous and second cancers.
Colonoscopy is the preferred method, he notes, but computed tomography
colonography [virtual colonoscopy] is an option if colonoscopy
can't be completed because of an obstructing tumour. The researchers
note that their study does not prove that more frequent colonoscopy
would have improved patients' outcomes, and that any recommendations
for changes in surveillance strategies must take into account
the complications and cost of the technique.
Blood lactate as an early predictor of outcome in paracetamol-induced
acute liver failure: a cohort study
William Bernal, Nora Donaldson, Duncan Wyncoll, Julia Wendon [Full
Text]
Background Although the King's College Hospital (KCH)
selection criteria for emergency liver transplantation in paracetamol-induced
acute liver failure are widely used, strategies to improve sensitivity
and facilitate earlier transplantation are required. We investigated
the use of arterial blood lactate measurement for the identification
of transplantation candidates. Methods In a single-centre
study, we measured arterial blood lactate early (median 4 h) and
after fluid resuscitation (median 12 h) in patients admitted to
a tertiary-referral intensive-care unit. Threshold values that
best identified individuals likely to die without transplantation
were derived in a retrospective initial sample of 103 patients
with paracetamol-induced acute liver failure and applied to a
prospective validation sample of 107 patients. Predictive value
and speed of identification were compared with those of KCH criteria.
Findings In the initial sample, median lactate was significantly
higher in non-surviving patients than in survivors both in the
early samples (8·5 [range 1·7-21·0] vs
1·4 [0·53-7·9] mmol/L, p<0·0001)
and after fluid resuscitation (5·5 [1·3-18·6]
vs 1·3 [0·26-3·2], p<0·0001).
Applied to the validation sample, a threshold value of 3·5
mmol/L early after admission had sensitivity 67%, specificity
95%, positive likelihood ratio 13, and negative likelihood ratio
0·35; the corresponding values for a threshold of 3·0
mmol/L after fluid resuscitation were 76%, 97%, 30, and 0·24.
Combined early and postresuscitation lactate concentrations had
similar predictive ability to KCH criteria but identified non-surviving
patients earlier (4 [3-13] vs 10 [3·5-19·5]
h, p=0·01). Addition of postresuscitation lactate concentration
to KCH criteria increased sensitivity from 76% to 91% and lowered
negative likelihood ratio from 0·25 to 0·10. Interpretation
Arterial blood lactate measurement rapidly and accurately
identifies patients who will die from paracetamol-induced acute
liver failure. Its use could improve the speed and accuracy of
selection of appropriate candidates for transplantation. Lancet
2002; 359: 558-63
Reflux of gastric juice and glue ear in children
Andrea Tasker, Peter W Dettmar, Marguerite Panetti, James
A Koufman, John P Birchall, Jeffery P Pearson
Otitis media with effusion (glue ear) is the most frequent
cause of deafness in children. We investigated the role of gastric
juice reflux in this disease. We measured pepsin concentrations
in middle ear effusions from children using ELISA and enzyme activity
assays. 45 (83%) of 54 effusions contained pepsin/pepsinogen at
concentrations of up to 1000-fold greater than those in serum.
Our data suggest that reflux of gastric juice could be a major
cause of glue ear in children.
Laparoscopic living donor hepatectomy for liver transplantation
in children [Full
Text]
Daniel Cherqui, Olivier Soubrane, Emmanuel Husson, Eric Barshasz,
Olivier Vignaux, Mourad Ghimouz, Sophie Branchereau, Christophe
Chardot, Frédéric Gauthier, Pierre-Louis Fagniez,
Didier Houssin.
Summary Background Because cadaveric organ donors are in short supply, living donors are increasingly being used in transplantations. We have developed a safe and reproducible method for laparoscopic liver resection. Methods Left hepatic lobectomy (resection of segments 2 and 3) was done by laparoscopy in one woman aged 27 years and one man aged 31 years. The grafts were prepared under laparoscopy, without any vascular clamping, and were externalised through a suprapubic Pfannenstiel incision. Both grafts were transplanted conventionally to the patients' respective sons, who were both aged 1 year and had biliary atresia. Findings Donor operations lasted 7 h for the woman and 6 h for the man, and warm ischaemia times were 4 and 10 min, respectively. Blood loss was 150 and 450 mL, respectively, and no transfusions were required. Neither patient had complications during or after sugery; and hospital stay was 7 and 5 days, respectively. Both recipients are alive and have excellent graft function. Interpretation We have shown the feasibility of laparoscopic living donor hepatectomy from parent to child. If the safety and feasbility of this procedure can be shown in larger series, laparoscopic donor left lobectomy could become a new option for paediatric living donor liver transplantation. Lancet 2002; 359: 392-96
Detection of proximal colorectal cancers through analysis
of faecal DNA
Giovanni Traverso, Anthony Shuber, Louise Olsson, Bernard
Levin, Constance Johnson, Stanley R Hamilton, Kevin Boynton, Kenneth
W Kinzler, Bert Vogelstein
Detection of mutations in faecal DNA represents a promising,
non-invasive approach for detecting colorectal cancers in average-risk
populations. One of the first practical applications of this technology
involves the examination of microsatellite markers in sporadic
cancers with mismatch-repair deficiencies. Since such cancers
nearly always occur in the proximal colon, this test might be
useful as an adjunct to sigmoidoscopy, which detects only distal
colorectal lesions. We report here the first in-depth analysis
of faecal DNA from patients with proximal cancers to determine
the feasibility, sensitivity, and specificity of this approach.
Using a sensitive method for microsatellite mutation detection,
we found that 18 of 46 cancers had microsatellite alterations
and that identical mutations could be identified in the faecal
DNA of 17 of these 18 cases.
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