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 Archives depuis le 01/09/00 |
Concise Review in Mechanisms of Disease
Molecular aspects of iron absorption: Insights into the
role of HFE in hemochromatosis
Caroline C. Philpott
Hereditary hemochromatosis is the most common genetic disorder
occurring in persons of northern European descent, and the clinical
hallmark of the disease is the gradual accumulation of iron in
internal organs, especially the liver, heart, and pancreas, which
ultimately leads to organ failure. HFE, the gene that is defective
in the majority of cases, was identified in 1996 and, although
the exact role that HFE plays in the uptake and utilization of
iron is not yet clear, important aspects of HFE function are emerging.
Identification and studies of new proteins involved in the absorption
of iron in the gut and in somatic cells has led to a clearer picture
of how humans absorb iron from the diet and regulate this absorption
to meet metabolic needs and to balance body iron stores. This
review focuses on the molecular aspects of iron absorption and
the role that HFE may play in these processes. (HEPATOLOGY 2002;35:993-1001.)
Liver Biology and Pathobiology
Hypoxia-induced VEGF and collagen I expressions are associated
with angiogenesis and fibrogenesis in experimental cirrhosis
Christophe Corpechot, Veronique Barbu, Dominique Wendum, Nils
Kinnman, Colette Rey, Raoul Poupon, Chantal Housset, Olivier Rosmorduc
Cirrhosis consists of hepatocyte nodules surrounded by a highly
vascularized fibrous tissue. We previously showed that the development
of biliary cirrhosis in the rat is associated with the occurrence
of hepatocellular hypoxia and the induction of hepatic angiogenesis.
We herein examined the occurrence of hypoxia in an experimental
model of diethylnitrosamine (DEN)-induced cirrhosis. We also determined
whether hypoxia directly affects the expression of vascular endothelial
growth factor (VEGF), of VEGF receptors (Flt-1, Flk-1), and of
type I and type IV collagens in activated hepatic stellate cells
(HSCs) and the expression of VEGF in hepatocytes. Our results
show that in DEN-treated rats, although the progression of liver
fibrosis is associated with hepatocellular hypoxia and angiogenesis,
VEGF and Flt-1 expressions in the liver are increased and correlated
with the density of microvessels. In vitro, hypoxia induces
the expression of VEGF, Flt-1, and type I collagen in activated
HSCs and that of VEGF in hepatocytes. In addition, we show that
hypoxia-induced type I collagen expression in HSCs may occur independently
of transforming growth factor 1 (TGF-1) overexpression. In conclusion,
the present study provides further evidence that hepatocellular
hypoxia and angiogenesis progress together with fibrogenesis after
liver injury and that hypoxia directly contributes to the progression
of liver fibrosis. (HEPATOLOGY 2002;35:1010-1021.)
Dose-dependent inhibition of hepatic fibrosis in mice by
a TGF- soluble receptor: Implications for antifibrotic therapy
Yutaka Yata, Philip Gotwals, Victor Koteliansky, Don C. Rockey
Transforming growth factor (TGF) isoforms (in particular, TGF-1)
play a central role in the fibrogenic response to injury in many
organs, including the liver. Although TGF- is clearly important
in fibrogenesis, a number of issues related to therapeutic antagonism
have emerged. For example, the long-term effect of TGF- antagonism
is unknown; furthermore, controversy exists as to appropriate
levels of TGF- inhibition. Therefore, we aimed to examine TGF-
in models of chronic liver injury and to determine whether an
in vivo dose-response relationship exists for inhibition
of TGF-. Liver injury was induced in BALB/c mice by administering
carbon tetrachloride for 4 or 8 weeks. TGF- binding was inhibited
with a soluble TGF- type II receptor (STR) construct, administered
intraperitoneally over a dose range of 4.0, 1.0, 0.4, or 0.1 mg/kg
twice weekly during fibrogenesis. Fibrogenesis was assessed by
measurement of type I collagen messenger RNA (mRNA) expression
and by quantitative morphometric analysis. In the 4-week study,
STR at concentrations of 4.0, 1.0, and 0.1 mg/kg reduced type
I collagen mRNA expression by 31%, 49%, and 60% compared with
immunoglobulin (Ig) G controls, respectively. In the 8-week study,
lower concentrations of STR (0.1 mg/kg) also had the greatest
effect on type I collagen mRNA expression. Quantitative morphometrics
similarly showed that lower concentrations of STR were the most
antifibrogenic. In conclusion, the results confirm the antifibrotic
effect of inhibiting TGF- in chronic hepatic wounding and, moreover,
show that its in vivo effect in the mouse is dose dependent.
Such findings have major translational implications for therapeutic
strategies aimed at TGF-. (HEPATOLOGY 2002;35:1022-1030.)
Transport of the sulfated, amidated bile acid, sulfolithocholyltaurine,
into rat hepatocytes is mediated by Oatp1 and Oatp2
Ling-Jie Meng, Pijun Wang, Allan W. Wolkoff, Richard B. Kim, Rommel
G. Tirona, Alan F. Hofmann, K. Sandy Pang
The uptake of the sulfated bile acid sulfolithocholyltaurine
(SLCT) was investigated in isolated rat hepatocytes and in HeLa
cells transfected with complementary DNAs (cDNAs) of organic anion
transporting polypeptides (Oatps) 1 and 2 cloned from rat liver.
In hepatocytes, transport of SLCT was greatly reduced by bromosulfophthalein
(BSP), estrone sulfate, the precursor bile acids cholyltaurine
and lithocholyltaurine, and 4,4'-diisothiocyanostilbene-2-2'-disulfonic
acid (DIDS). However, SLCT transport was insensitive to 4-methylumbelliferyl
sulfate, harmol sulfate, digoxin, fexofenadine, and lack of sodium
ion. Because the estimation of kinetic constants was enhanced
with use of inhibitors, BSP (1-50 µmol/L) was added to isolated
rat hepatocytes to assess the various transport components for
SLCT uptake. The resulting data showed a nonsaturable pathway
and at least 2 pathways of different Michaelis-Menten constants
(Km) (70 and 6 µmol/L) and similar maximum velocities
(Vmax) (1.73 and 1.2 nmol/min/mg protein) and inhibition
constants of 0.63 and 10.3 µmol/L for BSP. In expression
systems, SLCT was taken up by Oatp1 and Oatp2 expressed in HeLa
cells with similar Km values (12.6 ± 6.2 and 14.6
± 1.9 µmol/L). These Km values were comparable
to that observed for the high-affinity pathway in rat hepatocytes.
In conclusion, the results suggest that transport of SLCT into
rat liver is mediated in part by Oatp1 and Oatp2, high-affinity
pathways, a lower-affinity pathway of unknown origin, and a nonsaturable
pathway that is compatible with a transport system of high Km
and/or passive diffusion. (HEPATOLOGY 2002;35:1031-1040.)
Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte
growth and secretion of BDL rats through activation of PKC alpha
Gianfranco Alpini, Leonardo Baiocchi, Shannon Glaser, Yoshiyuki
Ueno, Marco Marzioni, Heather Francis, Jo Lynne Phinizy, Mario
Angelico, Gene LeSage
Accumulating bile acids (BA) trigger cholangiocyte proliferation
in chronic cholestasis. The aim of this study was to determine
if ursodeoxycholate (UDCA) or tauroursodeoxycholate (TUDCA) chronic
feeding prevents the increased cholangiocyte growth and secretion
in bile ductligated (BDL) rats, if UDCA and TUDCA effects
are associated with increased cholangiocyte apoptosis, and to
determine if this inhibition is dependent on increased intracellular
Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) alpha.
Immediately after BDL, rats were fed UDCA or TUDCA (both 275 µmol/d)
for 1 week. We determined the number of bile ducts in liver sections,
cholangiocyte proliferation (by measurement of H3 histone and
proliferating cellular nuclear antigen in isolated cholangiocytes),
and ductal secretion. In purified cholangiocytes from 1-week BDL
rats, we evaluated if UDCA and TUDCA directly inhibit cholangiocyte
proliferation and secretin-stimulated adenosine 3', 5'-monophosphate
levels. We determined if UDCA and TUDCA activate PKC, increase
[Ca2+]i, and alter the apical BA transporter (ABAT) expression
in cholangiocytes. UDCA and TUDCA inhibited in vivo the
cholangiocyte proliferation, secretion, and ABAT expression. In
vitro UDCA and TUDCA inhibition of cholangiocyte growth and
secretion required increased [Ca2+]i and PKC alpha. In conclusion,
activation of Ca2+-dependent PKC alpha is required for UDCA and
TUDCA inhibition of cholangiocyte growth and secretion. Reduced
cholangiocyte ABAT may decrease endogenous BA stimulation of cholangiocyte
growth and secretion. (HEPATOLOGY 2002;35:1041-1052.)
Mallory body-A disease-associated type of sequestosome (*Human
Study*)
Cornelia Stumptner, Andrea Fuchsbichler, Hans Heid, Kurt Zatloukal,
Helmut Denk
Mallory bodies (MBs) consist of abnormal keratins, ubiquitin,
heat shock proteins, and the protein p62. p62 is encoded by an
immediate-early response gene that rapidly responds to a variety
of extracellular signals involved in cell proliferation, differentiation,
and particularly oxidative stress. It acts as an adapter in signal
transduction and binds noncovalently to ubiquitin, possibly being
involved in the regulation of the fate of ubiquitinated proteins
by segregation (i.e., sequestosome or aggresome formation).
The presence of p62 together with ubiquitinated abnormal keratins
in the MB characterizes MBs as a disease-associated type of sequestosome.
A detailed study on the expression of p62 and its relationship
to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC)-treated mouse liver is reported based on immunohistochemical,
immunoblot, and Northern blot analyses. The results indicate that
p62 is rapidly induced in hepatocytes of intoxicated animals preceding
MB formation. As suggested by experiments with short-term DDC-treated
naive mice and mice refed DDC after recovery from long-term DDC
treatment (primed mice), p62 does not exert an initiating effect
on MB formation but the appearance of MBs requires the presence
of abnormal keratins, which associate with p62 after ubiquitination.
The rapid induction of p62 and its association with MBs further
support the role of oxidative stress in MB formation. In conclusion,
the constant presence of p62 in MBs suggests that binding of p62
to abnormal keratins may allow hepatocytes to dispose potentially
harmful proteins in a biologically inert manner. (HEPATOLOGY 2002;35:1053-1062.)
The p21Cip1 protein, a cyclin inhibitor, regulates the levels
and the intracellular localization of CDC25A in mice regenerating
livers
Maribel Jaime, Maria Jesús Pujol, Joan Serratosa, Cristina
Pantoja, Núria Canela, Oriol Casanovas, Manuel Serrano,
Neus Agell, Oriol Bachs
Liver cells from p21Cip1/ mice subjected to partial
hepatectomy (PH) progress into DNA synthesis faster than those
from wild-type mice. These cells also show a premature induction
of cyclin E/cyclin-dependent kinase (CDK) 2 activity. We studied
the mechanisms whereby cells lacking p21Cip1 showed a premature
induction of this activity. Whereas the levels of CDK2, cyclin
E, and p27Kip1 were similar in both wild-type and p21Cip1/
mice, those of the activator CDC25A were much higher in p21Cip1/
quiescent and regenerating livers than in wild-type animals. Moreover,
p21Cip1/ cells also showed a premature translocation
of CDC25A from cytoplasm into the nucleus. The ectopic expression
of p21Cip1 into mice embryo fibroblasts from p21Cip1/
mice decreased the levels of CDC25A and delayed its nuclear translocation.
The levels of CDC25A messenger RNA in p21Cip1/ cells
were higher than in wild-type cells, suggesting that this increase
might be responsible, at least in part, for the high levels of
CDC25A protein in these cells. Thus, the results reported here
indicate that p21Cip1 regulates the levels and the intracellular
localization of CDC25A. We also found a good correlation between
CDC25A nuclear translocation and cyclin E/CDK2 activation. In
conclusion, premature translocation of CDC25A to the nucleus might
be involved in the advanced induction of cyclin E/CDK2 activity
and DNA replication in cells from animals lacking p21Cip1. (HEPATOLOGY
2002;35:1063-1071.)
A tamoxifen-inducible chimeric Cre recombinase specifically
effective in the fetal and adult mouse liver
Mounia Tannour-Louet, Arlette Porteu, Sophie Vaulont, Axel Kahn,
Mireille Vasseur-Cognet
The spatiotemporal control of somatic mutagenesis in mice is
considered a promising step to determine the function of a given
gene product in a defined population of cells at any given time
during animal life and also to generate better mouse models of
human diseases. To introduce defined mutations in a temporally
controlled manner in the liver, we established transgenic mice
expressing a tamoxifen-inducible Cre recombinase under the control
of the transthyretin promoter (TTR-Cre ind). The recombinase
activity was examined on 2 different floxed alleles by crossing
TTR-Cre ind mice with either the reporter strain ROSA
26 or with homozygous mice carrying floxed catalytic 2 subunit
of the adenosine monophosphate (AMP)-activated protein kinase
gene. By placing 2 mutated hormone-binding domains of murine estrogen
receptor (Mer) at both termini of the Cre, we show that the fusion
protein is active only on administration of the synthetic estrogen
antagonist 4-hydroxytamoxifen (4-OHT) without any background in
the absence of the inducing agent. The recombination is specific
of the fetal and adult liver, and we show that the efficiency
of recombination reached 80% to 100% after treatment with 4-OHT.
In conclusion, TTR-Cre ind transgenic mice represent a
valuable tool for temporally controlling the desired gene modifications
in vivo in the fetal and adult liver. This would certainly
help to understand the physiologic functions of genes in the liver,
to create various mouse models mimicking human diseases, and to
contribute to liver cancerspecific suicide gene therapy studies.
(HEPATOLOGY 2002;35:1072-1081.)
Extended preservation of rat liver graft by induction of
heme oxygenase-1
Claudio A. Redaelli, Ying-Hua Tian, Thomas Schaffner, Monika Ledermann,
Hans U. Baer, Jean-François Dufour
Livers can be preserved only for a short period without jeopardizing
the transplantation outcome. Heat shock proteins (HSPs) protect
against ischemia and reperfusion injury. We studied whether their
induction and, in particular, the induction of heme oxygenase
1 (HO-1), improves transplantation survival after an extended
time of cold storage. Rats were subjected to heat preconditioning
(42°C for 20 minutes). Livers were harvested 24 hours later,
preserved in cold University of Wisconsin solution for 44 hours,
and transplanted in isogeneic rats (arterialized transplantation).
HO-1 was specifically induced and inhibited by cobalt protoporphyrin
and tin protoporphyrin, respectively. All animals receiving a
graft without preconditioning and subjected to 44 hours of cold
preservation died within 3 days, whereas 89% of rats who received
a graft exposed to heat survived for 3 weeks (P = .0004).
Preconditioning reduced serum aspartate transaminase (AST) and
lactate dehydrogenase activities after reperfusion, improved bile
flow, and decreased the histologic lesions of reperfusion injury.
These significant effects of heat preconditioning were prevented
by administration of tin protoporphyrin and could be reproduced
by administration of cobalt protoporphyrin. In grafts without
preconditioning, only a small fraction (<5%) of hepatocytes
were positive with the terminal deoxynucleotide transferase-mediated
dUTP nick-end labeling (TUNEL) assay, and even less expressed
activated caspase 3. Preconditioning tended to reduce the number
of positive cells and to stimulate the expression of antiapoptotic
Bcl-XL. In conclusion, heat preconditioning and, specifically,
overexpression of HO-1 improve posttransplantation survival and
graft function after prolonged cold ischemia preservation. The
mechanism underlying these beneficial effects does not appear
to be prevention of apoptosis. (HEPATOLOGY 2002;35:1082-1092.)
Role of CCR2 in macrophage migration into the liver during
acetaminophen-induced hepatotoxicity in the mouse
Donna M. Dambach, Linda M. Watson, Kevin R. Gray, Stephen K. Durham,
Debra L. Laskin
The biological effects of monocyte chemoattractant protein
(MCP) 1 are mediated by binding to C-C chemokine receptor (CCR)
2. In the present studies, we used CCR2 knockout (CCR2/)
mice to examine the role of MCP-1 in acetaminophen-induced macrophage
accumulation in the liver, expression of inflammatory cytokines,
and hepatotoxicity. We found that hepatic expression of CCR2 and
MCP-1 was increased 10-fold and 20-fold, respectively, 12 to 72
hours after administration of acetaminophen to wild-type mice.
Expression of these proteins was localized in centrilobular regions
of the liver. Whereas MCP-1 was expressed by both hepatocytes
and macrophages, CCR2 was identified in inflammatory macrophages.
F4/80 is a marker of mature macrophages expressed in large quantities
by Kupffer cells. In wild-type mice, a 75% decrease in F4/80-positive
macrophages was observed 24 to 48 hours after administration of
acetaminophen. In contrast, expression of macrosialin (CD68),
a marker of activated macrophages, increased 2-fold 24 to 72 hours
after administration of acetaminophen and was associated with
inflammatory cells. Although there was a decrease in the overall
severity of inflammation and in the number of macrosialin-positive
macrophages 72 hours after administration of acetaminophen in
CCR2/ mice, the number of F4/80-positive cells did not
change. Loss of CCR2 was also found to alter acetaminophen-induced
expression of tumor necrosis factor , monocyte chemoattractant
protein 3, and KC/gro. However, the overall outcome of acetaminophen-induced
hepatic injury was not affected. In conclusion, these data indicate
that MCP-1 and CCR2 contribute to the recruitment of a subset
of activated macrophages into the liver during acetaminophen-induced
hepatotoxicity that may be important in resolution of tissue injury.
(HEPATOLOGY 2002;35:1093-1103.)
Experimental Liver Cancer
Endostatin inhibits murine colon carcinoma sinusoidal-type
metastases by preferential targeting of hepatic sinusoidal endothelium
Miren S. Solaun, Lorea Mendoza, Marco De Luca, Virginia Gutierrez,
Mari-Paz López, Elvira Olaso, B. Kim Lee Sim, Fernando
Vidal-Vanaclocha
An angiogenic response originating from peritumoral sinusoids
and portal tracts that leads to the formation of metastases with
sinusoidal- and portal-type angiogenic patterns, respectively,
occurs during the course of liver colonization by murine 51b colon
carcinoma (51b-CC) cells. We found a 5-fold increase in endogenous
endostatin levels from hepatic blood over baseline (25 ±
6 ng/mL) when micrometastatic foci had a detectable size and a
14-fold increase when macrometastases were developed. Despite
this endogenous endostatin production, subcutaneous administration
of recombinant human endostatin (rh-E; 50 mg/kg) decreased metastasis
number by 60% when dosed from days 1 to 20 after 51b-CC cell injection,
by 40% when given from days 10 to 20, and by 30% when administered
as a single dose 30 minutes before 51b-CC cell injection compared
with controls. In addition, administration of rh-E from days 10
to 20 decreased overall metastasis volume by 90% compared with
controls. rh-E increased the number of necrotic sinusoidal-type
metastases by 7-fold and decreased their intrametastatic CD31+-microvessel
density by 80% without affecting portal-type metastases. Flow
cytometry showed rh-E binding to mouse liver sinusoidal cells
but not to CD45+ cells (leukocytes and Kupffer cells) or 51b-CC
cells. Furthermore, rh-E induced sinusoidal endothelium cell apoptosis.
In conclusion, despite the direct correlation between metastasis
development and endogenous endostatin generation in the liver,
administration of rh-E inhibited micrometastasis generation and
macrometastasis growth very efficiently. The antiangiogenic mechanism
was selective for sinusoidal-type metastases, in which the neovasculature
originating from sinusoidal endothelium cells was targeted by
rh-E. (HEPATOLOGY 2002;35:1104-1116.)
Aspirin and NS-398 inhibit hepatocyte growth factorinduced
invasiveness of human hepatoma cells (*Human Study*)
Seigou Abiru, Kazuhiko Nakao, Tatsuki Ichikawa, Kiyoshi Migita,
Masaya Shigeno, Miyuki Sakamoto, Hiroki Ishikawa, Keisuke Hamasaki,
Keisuke Nakata, Katsumi Eguchi
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase
(COX) activity and are considered to exert antitumor actions in
a variety of cancer cells, although the effects are unlikely entirely
due to COX inhibition. Because clinical observations suggest that
hepatocyte growth factor (HGF) can promote metastasis of hepatoma
cells while stimulating tumor invasiveness, we investigated the
effect of aspirin and NS-398, a selective COX-2 inhibitor, on
HGF-mediated invasiveness of HepG2 human hepatoma cells. HGF stimulated
the invasiveness of HepG2 cells in Matrigel cell invasion assay,
together with increased expression of matrix metalloproteinase
(MMP) 9. Addition of aspirin or NS-398, similar to PD98059, which
acts as a specific inhibitor of mitogen-activated protein kinase/extracellular
signalregulated kinase (MEK), an upstream kinase regulating
extracellular signalregulated kinase (ERK)1/2, abrogated
such actions of HGF without affecting cell viability. Aspirin
and NS-398, in contrast to PD98059, did not suppress ERK1/2 phosphorylation
induced by HGF. However, both agents inhibited the kinase activity
of ERK1/2 induced by HGF and repressed HGF-induced phosphorylation
of 90-kd ribosomal S6 kinase (RSK) and Elk-1, key downstream substrates
of ERK1/2, resulting in the suppression of transcriptional activity
of Elk-1 as well as nuclear factor B (NF-B) and AP-1, which are
involved in MMP-9 gene regulation. In conclusion, our results
suggest that aspirin and NS-398 inhibit HGF-induced invasiveness
of HepG2 human hepatoma cells through ERK1/2. (HEPATOLOGY 2002;35:1117-1124.)
Effects of Sho-saiko-to on hepatocarcinogenesis and 8-hydroxy-2'-deoxyguanosine
formation
Goshi Shiota, Yoshiko Maeta, Tomoyuki Mukoyama, Atsushi Yanagidani,
Akihide Udagawa, Kenji Oyama, Kazuo Yashima, Yosuke Kishimoto,
Yoichiro Nakai, Tetsuo Miura, Hisao Ito, Yoshikazu Murawaki, Hironaka
Kawasaki
Oxidative stress plays an important role in hepatocarcinogenesis.
Although Sho-saiko-to (TJ-9), a Japanese herbal medicine which
has been recently administered to patients with chronic liver
disease in Japan, prevents hepatocarcinogenesis, the mechanism
by which TJ-9 protects against cancer development is not fully
understood. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a DNA adduct
by reactive oxygen species, is known as a parameter of genetic
risk for hepatocarcinogenesis. To clarify whether the preventive
effect on hepatocarcinogenesis by TJ-9 is dependent on 8-OHdG,
the effect on 8-OHdG levels by TJ-9 was examined by using high-performance
liquid chromatographymass spectrometry (LC-MS) in a diethylnitrosamine
(DEN)-induced hepatocarcinogenesis model of male Fisher rats.
TJ-9 reduced the number of preneoplastic cells, detected as the
glutathione S transferase P (GST-P)positive hepatocytes,
and inhibited the development of liver tumors. TJ-9 also significantly
decreased the formation of 8-OHdG, as indicated by LC-MS and immunohistochemical
analysis. In addition, ornithine decarboxylase (ODC) activity
and the number of proliferating cell nuclear antigen (PCNA)-positive
cells were not altered. An electron paramagnetic resonance spin-trapping
technique showed that TJ-9 scavenges hydroxyl radicals in a dose-dependent
manner. In conclusion, the results of the present study suggest
that TJ-9 prevents hepatocarcinogenesis in association with inhibition
of 8-OHdG formation. (HEPATOLOGY 2002;35:1125-1133.)
Functional and genomic implications of global gene expression
profiles in cell lines from human hepatocellular cancer
Ju-Seog Lee, Snorri S. Thorgeirsson
Global gene expression profiles in cancer have impacted both
classification of tumors and definition of molecular pathways
in neoplasia. To explore the possibility of employing human tumor
cell lines to obtain information on the functional genomics of
the early stages of tumorigenesis, we have characterized variation
in gene-expression patterns in a cytogenetically well-defined
series of cell lines derived from human hepatocellular carcinoma
(HCC). Microarrays containing 6,720 sequence-verified human cDNAs
were used in this study. Nineteen well-characterized HCC cell
lines were analyzed, and a nontumorigenic liver-derived epithelial
cell line (Chang) was used as a reference. Each sample was examined
at least twice by switching fluorescent dyes, Cy-5 and Cy-3, and
average values of 2 experiments on each sample were used for further
analysis. Analysis of the clustered data revealed 2 distinctive
subtypes of gene-expression patterns among the 19 cell lines,
suggesting a degree of heterogeneity among the gene-expression
profiles of cell lines. Remarkably, expression of -fetoprotein
(AFP) was highly correlated with the molecular subtypes of HCC.
Although the 3 most distinctive gene-expression modules represented
the signatures of 2 different subgroups of HCC, most of the cell
lines shared many coexpressed genes. However, sets of coexpressed
genes that are specific for the subtypes of HCC were identified.
Furthermore, our results indicate that the comparison between
gene-expression patterns and structural alterations in chromosomes
is potentially useful in identifying genes critical in early stages
of tumorigenesis. In conclusion, these results not only identified
unrecognized subtypes of HCC, but also provided potential molecular
markers for each subtype that can be useful for diagnostic and/or
therapeutic purposes. (HEPATOLOGY 2002;35:1134-1143.)
Naked DNA injection for liver metastases treatment in rats
(*Human Study*)
Patrick Baqué, Valérie Pierrefite-Carle, Adolfo
Gavelli, Nicole Brossette, Daniel Benchimol, André Bourgeon,
Pascal Staccini, Marie-Christine Saint-Paul, Bernard Rossi
The cytosine deaminase (CD) gene converts the nontoxic prodrug,
5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU). We previously
showed that injection of CD-bearing cancer cells followed by 5-FC
treatment can act as an autologous tumor vaccine in a syngenic
liver metastasis model in rats. In the present work, we analyzed
the antitumor efficiency of a direct intratumoral injection of
a CD-expressing plasmid. In rats bearing microscopic or macroscopic
metastases in right and left liver lobes, an injection of a CD-expressing
plasmid was performed in the left lobe tumor, followed by 5-FC
treatment of the animals. A significant regression of the DNA-injected
tumor was observed in 5-FCtreated rats, both in microscopic
(P = .007) or advanced (P < .0001) tumor models.
Moreover, this treatment also induced a potent distant bystander
effect on untreated controlateral liver tumors and extrahepatic
metastases, resulting in an increased survival compared with control
animals in both tumor models (P < .05). In conclusion,
these data suggest that direct intratumoral injection of a CD-expressing
plasmid, associated to 5-FC administration, can constitute a powerful
and innocuous alternative treatment for unresectable liver metastases
from colon carcinoma. (HEPATOLOGY 2002;35:1144-1152.)
Liver Failure and Liver Disease
M6P/IGF2R tumor suppressor gene mutated in hepatocellular
carcinomas in Japan (*Human Study*)
Yoshihiko Oka, Robert A. Waterland, J. Keith Killian, Catherine
M. Nolan, Hong-Seok Jang, Keiji Tohara, Seigo Sakaguchi, Tsuneyoshi
Yao, Akinori Iwashita, Yutaka Yata, Terumi Takahara, Shin-ichiro
Sato, Kazuyuki Suzuki, Tomoyuki Masuda, Randy L. Jirtle
Mannose 6-phosphate/insulin-like growth factor II receptor
(M6P/IGF2R) tumor suppressor gene mutation is an early
event in human hepatocellular carcinoma (HCC) formation in the
United States, but its role in hepatocarcinogenesis in Japan is
unclear. We therefore determined M6P/IGF2R mutation frequency
in HCCs from patients who resided in the southern, central, and
northern regions of Japan. Ten single nucleotide polymorphisms
were used to identify HCCs and dysplastic liver nodules with M6P/IGF2R
loss of heterozygosity. The retained allele in these tumors was
also assessed for point mutations and deletions in the M6P/IGF2R
ligand binding domains by direct sequencing of polymerase chain
reaction (PCR) amplified DNA products. Fifty-eight percent (54
of 93) of the patients were heterozygous at the M6P/IGF2R
locus, and 67% (43 of 64) of the HCCs and 75% (3 of 4) of the
dysplastic nodules had loss of heterozygosity. The remaining allele
in 21% of the HCCs contained either M6P/IGF2R missense
mutations or deletions, whereas such mutations were not found
in the dysplastic lesions. In conclusion, M6P/IGF2R is
mutated in HCCs from throughout Japan with a frequency similar
to that in the United States. Loss of heterozygosity in dysplastic
liver nodules provides additional evidence that M6P/IGF2R
haploid insufficiency is an early event in human hepatocarcinogenesis.
(HEPATOLOGY 2002;35:1153-1163.)
Randomized controlled trial of transarterial lipiodol chemoembolization
for unresectable hepatocellular carcinoma (*Human Study*)
Chung-Mau Lo, Henry Ngan, Wai-Kuen Tso, Chi-Leung Liu, Chi-Ming
Lam, Ronnie Tung-Ping Poon, Sheung-Tat Fan, John Wong
This randomized, controlled trial assessed the efficacy of
transarterial Lipiodol (Lipiodol Ultrafluide, Laboratoire Guerbet,
Aulnay-Sous-Bois, France) chemoembolization in patients with unresectable
hepatocellular carcinoma. From March 1996 to October 1997, 80
out of 279 Asian patients with newly diagnosed unresectable hepatocellular
carcinoma fulfilled the entry criteria and randomly were assigned
to treatment with chemoembolization using a variable dose of an
emulsion of cisplatin in Lipiodol and gelatin-sponge particles
injected through the hepatic artery (chemoembolization group,
40 patients) or symptomatic treatment (control group, 40 patients).
One patient assigned to the control group secondarily was excluded
because of unrecognized systemic metastasis. Chemoembolization
was repeated every 2 to 3 months unless there was evidence of
contraindications or progressive disease. Survival was the main
end point. The chemoembolization group received a total of 192
courses of chemoembolization with a median of 4.5 (range, 1-15)
courses per patient. Chemoembolization resulted in a marked tumor
response, and the actuarial survival was significantly better
in the chemoembolization group (1 year, 57%; 2 years, 31%; 3 years,
26%) than in the control group (1 year, 32%; 2 years, 11%; 3 years,
3%; P = .002). When adjustments for baseline variables
that were prognostic on univariate analysis were made with a multivariate
Cox model, the survival benefit of chemoembolization remained
significant (relative risk of death, 0.49; 95% CI, 0.29-0.81;
P = .006). Although death from liver failure was more frequent
in patients who received chemoembolization, the liver functions
of the survivors were not significantly different. In conclusion,
in Asian patients with unresectable hepatocellular carcinoma,
transarterial Lipiodol chemoembolization significantly improves
survival and is an effective form of treatment. (HEPATOLOGY 2002;35:1164-1171.)
Hepatocellular carcinoma and survival in patients with primary
biliary cirrhosis (*Human Study*)
Akitaka Shibuya, Katsuaki Tanaka, Hiroshi Miyakawa, Minoru Shibata,
Masao Takatori, Kazuhiko Sekiyama, Naoaki Hashimoto, Shuuichi
Amaki, Tatsuji Komatsu, Toshio Morizane, for the PBC Forum 21
The incidence of hepatocellular carcinoma (HCC) in patients
with primary biliary cirrhosis (PBC) is not well known. The aims
of this study are to determine HCC incidence and survival, and
to identify risk factors associated with these outcomes in patients
with PBC. We collected information on 396 patients with PBC at
enrollment and followed-up from 6 to 271 months. They were all
negative for hepatitis B and C virus markers. HCC was detected
by scanning with ultrasonography, computed tomography, or both
every 4 to 6 months. Life expectancy (LE) was approximated with
the declining exponential approximation of LE. A total of 14 patients
developed HCC. The cumulative appearance rate of HCC in patients
with advanced-stage PBC (Scheuer's stage III or IV) was significantly
higher than that for patients with early-stage (stage I or II)
(12.3% and 7.7% by the tenth year, respectively. P = .021).
Proportional hazards analysis showed 3 factors are independently
associated with the development of HCC: age at the time of diagnosis,
male gender, and history of blood transfusion. Age, male gender,
and advanced-stage PBC were associated with survival, but HCC
development was not. The disease-specific annual mortality rate
was estimated to be 0.008 for women and 0.028 for men with advanced-stage
PBC. In conclusion, HCC develops in old patients with advanced-stage
PBC, but HCC does not affect the patients' survival. (HEPATOLOGY
2002;35:1172-1178.)
Pretransplant renal function predicts survival in patients
undergoing orthotopic liver transplantation (*Human Study*)
Satheesh Nair, Sumita Verma, Paul J. Thuluvath
The objective of this study was to determine the impact of
pretransplant renal function on graft and patient survival rates
after orthotopic liver transplantation (OLT) using the United
Network for Organ Sharing (UNOS) database for adults who underwent
OLT between 1988 and 1996. Based on calculated creatinine clearance
(CCr) at the time of OLT, patients were classified arbitrarily
into those with normal renal function (>70 mL/min) and mild
(40-69.9 mL/min), moderate (20-39.9 mL/min), and severe (<20
mL/min) renal insufficiency. Of the 20,281 patients who underwent
transplantation, complete data were available for 19,261 patients.
Of these, 12,778 (67%) had normal CCr (mean, 118 ± 50 mL/min)
and 4,419 (22%) had mild (56 ± 8.5 mL/min), 1,560 (8%)
had moderate (30 ± 5.7 mL/min), and 504 (3%) had severe
(14 ± 3.6 mL/min) renal failure. UNOS status 1 was more
common in patients with moderate and severe renal failure. Primary
graft nonfunction and 30-day mortality rates were higher and 1-,
2-, and 5-year graft and patient survival rates were lower in
patients with moderate or severe renal failure. Multiple regression
analysis showed that renal failure was an independent predictor
of 30-day and 2-year mortality after adjusting for the recipient's
age, sex, etiology of liver disease, diabetes status, body mass
index, cold ischemic time, and UNOS status. CCr less than 40 mL/min
was associated with significantly lower short-term and long-term
graft and patient survival rates. In conclusion, our findings
suggest that when Mayo End-Stage Liver Disease (MELD) score is
used to prioritize organ allocation, lower-than-expected graft
and patient survival rates may be seen. (HEPATOLOGY 2002;35:1179-1185.)
Bilirubin induces apoptosis via the mitochondrial pathway
in developing rat brain neurons
Cecília M. P. Rodrigues, Susana Solá, Dora Brites
Increased levels of unconjugated bilirubin, the end-product
of heme catabolism, are detrimental to the central nervous system.
To examine the role of apoptosis in bilirubin-induced toxicity
and to characterize the biochemical pathway of cell death, we
exposed developing rat brain neurons to purified unconjugated
bilirubin at concentrations below and above saturation of human
serum albumin. Isolated neurons treated with bilirubin showed
increased levels of apoptosis. Mitochondrial cytochrome c
was extensively released and accumulated in cytosol. Consistent
with this observation, caspase-3 was activated and the full-length
substrate poly(ADP)ribose polymerase (PARP) degraded, even in
the presence of very modestly elevated concentrations of bilirubin.
In parallel, all events were prevented in cells preincubated with
ursodeoxycholate. Further experiments showed that bilirubin diminished
mitochondrial transmembrane potential (m) and increased mitochondrial-associated
Bax protein levels, while directly disrupting membrane lipid and
protein structure. In conclusion, bilirubin induces mitochondrial
depolarization and Bax translocation via physical interaction
with membranes, mediating the mitochondrial pathway of apoptosis
in neurons exposed to bilirubin. These results provide a novel
insight into the mechanism of bilirubin-induced toxicity. (HEPATOLOGY
2002;35:1186-1195.)
Relationship between acetaldehyde levels and cell survival
in ethanol-metabolizing hepatoma cells
Dahn L. Clemens, Andrew Forman, Thomas R. Jerrells, Michael F.
Sorrell, Dean J. Tuma
We have created a number of recombinant Hep G2 cell lines,
designated VA cells, that constitutively express alcohol dehydrogenase.
Oxidation of ethanol by the VA cells results in the production
and accumulation of acetaldehyde, and a dramatic increase in the
nicotinamide adenine dinucleotide, reduced (NADH)/nicotinamide
adenine dinucleotide (NAD+) ratio (redox-state). It is believed
that production of acetaldehyde, and the increase in the redox-state
of hepatocytes, are responsible for many of the dysfunctions associated
with alcoholic liver disease. When the VA cells were cultured
in the presence of ethanol, we observed a dramatic reduction in
cell accumulation. This reduction was more pronounced in cells
that metabolized ethanol more efficiently. Inhibition of alcohol
dehydrogenase activity abolished this reduction, demonstrating
that ethanol oxidation was required for this dysfunction. Subsequent
investigations indicated that this ethanol oxidationmediated
reduction in cell accumulation was the result of both cytotoxicity
and impaired DNA synthesis. To dissociate the increase in the
cellular redox-state from acetaldehyde production, VA cells were
cultured in the presence of isopropanol. The oxidation of isopropanol
results in similar redox changes, but the metabolic by-product
of isopropanol oxidation is acetone. The metabolism of isopropanol
by VA cells resulted in very little reduction in cell number.
Furthermore, treatment of ethanol-metabolizing VA cells with the
aldehyde dehydrogenase inhibitor, cyanamide, increased the levels
of acetaldehyde and resulted in an additional reduction in cell
number. In conclusion, these studies indicated that exposure to
acetaldehyde caused cytotoxicity, as well as the ethanol oxidationmediated
reduction in cell number. (HEPATOLOGY 2002;35:1196-1204.)
Mitochondrial oxidative stress and CD95 ligand: A dual mechanism
for hepatocyte apoptosis in chronic alcoholism
Juan B. Miñana, Luis Gómez-Cambronero, Ana Lloret,
Federico V. Pallardó, Juan Del Olmo, Amparo Escudero, José
M. Rodrigo, Antonio Pellín, Juan R. Viña, José
Viña, Juan Sastre
Apoptosis plays an important role in the progression of alcohol-induced
liver disease to cirrhosis. Oxidative stress is an early event
in the development of apoptosis. The major aim of this study was
to study the conditions in which oxidative stress occurs in chronic
alcoholism and its relationship with apoptosis of hepatocytes.
We have found that oxidative stress is associated with chronic
ethanol consumption in humans and in rats, in the former independently
of the existence of alcohol-induced liver disease. Ethanol or
acetaldehyde induces apoptosis in hepatocytes isolated from alcoholic
rats, but not in those from control rats. Inhibition of aldehyde
dehydrogenase, but not of cytochrome P450 2E1, prevents ethanol-induced
cell death. Ethanol-induced apoptosis is caused by increased reactive
oxygen species (ROS) driven by increased availability of the reduced
form of nicotinamide-adenine dinucleotide (NADH) owing to mitochondrial
acetaldehyde metabolism and it is prevented by blocking the opening
of mitochondrial permeability transition (MPT) pores with cyclosporine
A. Inhibition of nitric oxide (NO) synthase or addition of antioxidant
vitamins C and E completely prevented ethanol-induced apoptosis.
Mitochondrial oxidative stress, which occurs during chronic alcoholism,
renders hepatocytes susceptible to apoptosis. On the other hand,
the CD95 ligand expression was up-regulated by acetaldehyde. In
conclusion, ethanol induces apoptosis via 2 different pathways:
MPT and up-regulation of the expression of CD95-Fas ligand. The
overproduction of ROS by mitochondria, driven by acetaldehyde
metabolism, is a common trigger of both mechanisms. (HEPATOLOGY
2002;35:1205-1214.)
Viral Hepatitis
Characterization of two hepatitis B virus populations isolated
from a hepatitis B surface antigennegative patient (*Human
Study*)
Damien Jeantet, Isabelle Chemin, Bernard Mandrand, Fabien Zoulim,
Christian Trepo, Alan Kay
In a study of surface antigen-negative, but weakly hepatitis
B virus (HBV) DNA-positive, patients, we were able to amplify
and clone whole HBV genomes from the serum of a cirrhotic patient.
Sequencing showed that the patient harbored two different HBV
populations, one of genotype A and the other of genotype D, with
the genotype D genome apparently predominating. The surface antigen
of the genotype A virus is heavily mutated, especially in the
extracellular determinant a region, with several mutations that
have not been previously described. The genotype D virus is a
precore mutant. Both genomes possess the common A1762T-G1764A
double mutation of the basal core promoter (BCP), and the genotype
D virus is also mutated in the TATA box of the large surface antigen
promoter. Biological characterization showed that the genotype
A genome was fully replication-competent, whereas the genotype
D genome replicated poorly. The small surface antigen of the genotype
A virus was only very weakly recognized by commercial tests. The
small surface antigen of the genotype D virus could be recognized
by the tests, but it was mainly retained within transfected cells,
probably because of an excess of large surface antigen. In conclusion,
the cryptic nature of this double HBV infection is characterized
by the predominance of the replication-deficient genotype D virus
over the replication-competent genotype A virus. (HEPATOLOGY 2002;35:1215-1224.)
Intrahepatic and circulating HLA class IIrestricted, hepatitis
C virusspecific T cells: Functional characterization in patients
with chronic hepatitis C (*Human Study*)
Amalia Penna, Gabriele Missale, Vincenzo Lamonaca, Massimo Pilli,
Cristina Mori, Paola Zanelli, Albertina Cavalli, Gianfranco Elia,
Carlo Ferrari
To compare the functional features of circulating and intrahepatic
hepatitis C virus (HCV)-specific CD4+ T cells in chronic HCV infection,
peripheral blood and liver-infiltrating lymphocytes from 29 patients
with chronic hepatitis C were stimulated with structural and nonstructural
HCV proteins to produce antigen-specific T-cell lines and clones.
Antigen specificity, fine specificity, phenotype, cytokine production,
and T-cell receptor (TCR)-v chain expression were analyzed. The
results indicate a hierarchy of stimulatory capacity by the different
HCV proteins, core being the antigen most frequently recognized
by CD4+ intrahepatic lymphocytes, followed by NS4 and NS5. The
CD4 response was directed simultaneously against different HCV
proteins in individual patients, but fine-specificity analysis
indicated that the response was generally focused on a limited
number of immunodominant epitopes. Although the narrowly focused
nature of this response may favor the emergence of escape mutations,
this event was not observed by following-up over time the sequence
of 2 epitopes strongly immunodominant for intrahepatic CD4 cells
of a patient with chronic HCV infection. In conclusion, simultaneous
analysis of peripheral blood and intrahepatic CD4 cells in the
same patients indicated a predominant Th1 profile of HCV-specific
CD4 cells and suggests a specific compartmentalization of virus-specific
T cells into the liver. (HEPATOLOGY 2002;35:1225-1236.)
Cellular response to conditional expression of hepatitis C
virus core protein in Huh7 cultured human hepatoma cells
Kui Li, Tarl Prow, Stanley M. Lemon, Michael R. Beard
Data suggesting that the hepatitis C virus (HCV) core protein
influences normal cellular processes remain controversial. To
determine the effects of core on cellular gene expression in hepatocytes,
we developed a human hepatoma (Huh7)-derived cell line with tightly
regulated core expression under the control of a tetracycline-regulated
promoter. Cells expressing core did not have impaired proliferative
abilities. Changes in gene expression profiles in response to
core expression were determined using commercial oligonucleotide
microarrays (Affymetrix GeneChip). Significant increases were
observed in the abundance of mRNA-encoding members of the metallothionein
(MT) family, as well as nicotinamide N-methyltransferase
(NNMT) and glutathione peroxidaselike protein (GPLP). These
changes did not result from removal of tetracycline from growth
media, and were confirmed in reverse-transcription polymerase
chain reaction (RT-PCR) assays. They suggest that core protein
expression leads to intracellular oxidative stress, and that vital
cellular functions are, in turn, protected by up-regulation of
cellular antioxidant defense mechanisms. In conclusion, these
findings can explain many potentially conflicting prior observations
concerning the effects of core on cellular physiology, and are
of relevance to the role of core protein in the pathogenesis of
HCV-related fibrosis and hepatocellular carcinoma. (HEPATOLOGY
2002;35:1237-1246.)
Severity and correlates of liver disease in hepatitis C virusinfected
injection drug users (*Human Study*)
Rudra Rai, Lucy E. Wilson, Jacquie Astemborski, Frank Anania,
Michael Torbenson, Charles Spoler, David Vlahov, Steffanie A.
Strathdee, John Boitnott, Kenrad E. Nelson, David L. Thomas
Between May 1996 and June 1998, 210 members of a cohort of
1,667 hepatitis C virus (HCV)-infected injection drug users (IDUs)
were selected for liver biopsy procedure after stratification
based on 2 consecutive serum alanine transaminase (ALT) levels.
Liver histology, which could be fully evaluated for 207 subjects,
was classified by using the modified Ishak scores. At the time
of biopsy, the median age of subjects was 41.3 years and the median
estimated duration of HCV infection was 20.7 years; 94% were African
American; 78% men; 31% were human immunodeficiency virus (HIV)
seropositive; and 76% had HCV genotype 1a or 1b. Total modified
histologic activity index (MHAI) scores ranged from 0 to 9, and
26.6% had a total MHAI score of 5 or greater. Persons with a total
MHAI score of 5 or greater were more likely to be HIV infected
(P = .04). Higher fibrosis, indicated by Ishak modified
fibrosis scores of 3 to 6, was present in 10.1% of subjects and
was found more often in those older than 46 years of age (the
highest quartile) (P < .01). Both fibrosis scores of
3 or greater and total scores of 5 or greater were associated
with elevated ALT, aspartate transaminase (AST), and -glutamyl
transpeptidase (GGT) levels (P < .01). When serial values
were considered, the results of liver enzyme testing could reduce
the probability of an IDU having a fibrosis score of 3 or greater
from 10% to 3%. In conclusion, these data indicate that severe
liver disease is uncommon in this urban, HCV-infected IDU cohort,
especially in younger persons and those with repeatedly normal
liver enzymes. (HEPATOLOGY 2002;35:1247-1255.)
The pathobilogy of biliary epithelia
Gianfranco Alpini, James M. McGill, Nicholas F. LaRusso
The morbidity and mortality from chronic biliary diseases (i.e.,
the cholangiopathies) remains substantial. End-stage liver disease
from biliary causes of cirrhosis (e.g., primary biliary
cirrhosis [PBC], and primary sclerosing cholangitis) account for
approximately one third of patients referred for liver transplantation.
A single-topic conference sponsored by the American Association
for the Studies of Liver Diseases entitled "The Pathobiology
of Biliary Epithelia" brought together investigators to review
the status of the field of cholangiocyte pathobiology, identify
new areas of interest, and propose future directions. This information
was presented in 6 sessions: "Structural and Functional Characteristics
of the Bile Duct System," "Biological Topics from Nonbiliary
Epithelia," "Malignant Transformation of Cholangiocytes,"
"Cholangiocyte Proliferation and Death," "Transport
Mechanisms in Bile Duct Epithelia," and "Pathobiology
of Biliary Epithelia." In the 7 years since the first symposium
on this topic, major advances have been made in our understanding
of ductal bile formation, including, greater insight into the
hormones, intracellular signaling mechanisms, and effector proteins
responsible for bile secretion and absorption. More sophisticated
imaging technologies have increased our understanding of the polarity
of cholangiocytes, their embryology and ultrastructural anatomy,
and in vivo human secretory responses to current medical
therapy. Information on mediators of inflammation permeated many
sessions, having potentially important roles in malignant transformation
of cholangiocytes, cholangiocyte apoptosis, fluid and electrolyte
transport, and have begun to be specifically characterized for
certain biliary diseases, e.g., acquired immunodeficiency
syndrome (AIDS) cholangiopathy and graft-versus-host disease (GVHD).
(HEPATOLOGY 2002;35:1256-1268.)
Clinical Research
Activated virus-specific T cells are early indicators of
anti-CMV immune reactions in liver transplant patients
C. Benz, O. Utermöhlen, A. Wulf, B. Villmow, V. Dries, T.
Goeser, U. Koszinowski, D. H. Busch
Background & Aims: Cytomegalovirus (CMV) infection
represents the most common infectious complication after liver
transplantation. Because CMV-associated complications in liver
transplantation patients are often liver-restricted and clinically
unrecognized, diagnosis of early infection or reactivation is
still very difficult. Because cytotoxic T cells (CTLs) are crucial
for the immune control of CMV, analysis of virus-specific CTLs
could contribute to diagnosis and management of CMV infection.
Methods: Major histocompatibility complex class I tetramers
and intracellular cytokine staining were used to determine frequencies
and phenotypes of peripheral blood CMV/pp65-specific CD8+ T cells
in HLA-A2, -B7, and -B35 positive liver transplantation patients
and in healthy individuals.
Results: After liver transplantation (633 months after
liver transplantation), frequencies of CMV-specific T cells were
significantly elevated compared with healthy individuals. In contrast
to immunoglobulin (Ig) Mnegative patients and healthy blood
donors, patients with increasing CMV IgM titers or IgG seroconversion
had high percentages of activated (CD38high) CMV-specific T cells.
In recently transplanted patients, activation of CMV-specific
T cells was associated with increased transaminases and histopathological
abnormalities in the absence of positive CMVpolymerase chain
reaction results from peripheral blood.
Conclusions: These data indicate that T-cell analysis based
on MHC tetramer staining may be a valuable parameter in the early
diagnosis of CMV-induced, liver-restricted complications after
liver transplantation.
Eosinophilic esophagitis in children: Immunopathological
analysis and response to fluticasone propionate
J. E. Teitelbaum, V. L. Fox, F. J. Twarog, S. Nurko, D. Antonioli,
G. Gleich, K. Badizadegan, G. T. Furuta
Background & Aims: Eosinophilic esophagitis (EE)
shares symptoms with gastroesophageal reflux disease but has distinctive
pathologic features and unknown immunopathology. Treatments with
antigen restriction or systemic immunosuppression pose problems
with compliance and side effects. Topically applied steroids offer
an attractive alternative treatment. The aims of this study were
to determine the immunopathologic features of EE and the effectiveness
of antigen-specific diet restriction (DR) and topical immunosuppression.
Methods: A prospective trial was conducted examining the
impact of DR and swallowed fluticasone propionate (FP) on pediatric
patients with EE. Clinicopathologic features, including immunohistochemical
analysis of the esophageal mucosa, were measured before and after
treatment.
Results: Immunohistochemical analysis of 11 prospectively
identified children showed a significantly greater number of mucosal
CD3 and CD8 lymphocytes, as well as CD1a antigen-presenting cells
compared with normal controls. DR did not induce clinical improvement
in any patients, whereas all children who completed treatment
with FP had resolution of symptoms. Posttreatment analysis of
proximal and distal esophageal mucosa showed a significant reduction
in the number of eosinophils, as well as CD3+ and CD8+ lymphocytes
compared with pretreatment sections.
Conclusions: EE is characterized by immunologically active
esophageal mucosa. FP, not DR, effectively relieves symptoms.
FP significantly reduces mucosal inflammation associated with
EE.
On-site screening sigmoidoscopy promotes long-term utilization
but fails as a venue for training primary care endoscopists
P. C. Schroy, III, T. Heeren, C. M. Bliss, C. M. Bliss, Jr., J.
Pincus, S. Wilson, M. Prout
Background & Aims: "Academic detailing"
is an effective strategy for promoting the use of screening sigmoidoscopy
by primary care physicians. The primary objectives of this study
were to determine whether the sustained presence of an "outside"
university-based gastroenterologist performing on-site screening
sigmoidoscopy promoted long-term utilization and whether the provision
for on-site sigmoidoscopy was an effective venue for training
primary care endoscopists.
Methods: Nine urban community health centers, including
4 intervention and 5 control sites, participated in a nonrandomized
controlled trial conducted over 3 years.
Results: By the end of year 3, overall self-reported use
of screening sigmoidoscopy increased by 61% for the intervention
group vs. only 25% for the comparison group (P = 0.001).
Ninety-seven percent of those reporting compliance referred 1
or more asymptomatic average-risk patients for screening examinations.
Only 2 of 83 (2.4%) eligible providers completed on-site training
and continued performing screening examinations independently.
The major barriers to participation included lack of interest,
lack of time to learn or perform sigmoidoscopy, concerns about
technical competence, and lack of need because of on-site availability.
Conclusions: Maintenance of on-site screening sigmoidoscopy
services performed by an outside gastroenterologist promotes long-term
utilization but fails as venue for training primary care endoscopists.
Alternative strategies for expanding capacity are needed.
The true impact of fatigue in primary biliary cirrhosis: A
population study
J. Goldblatt, P. J. S. Taylor, T. Lipman, M. I. Prince, A. Baragiotta,
M. F. Bassendine, O. F. W. James, D. E. J. Jones
Background & Aims: Patient surveys suggest that
fatigue is a common problem in primary biliary cirrhosis (PBC).
The actual extent of the problems caused by fatigue in PBC has
yet to be determined as previous studies addressing this question
have tended to use selected patient subgroups and subjective or
non-quantitative fatigue assessment tools. Here, we have attempted
to more accurately assess the extent of fatigue in PBC, and the
specificity of the symptom for this disease, by the application
of an objective measure of fatigue impact (the fatigue impact
score [FIS]) to a geographically based patient cohort, age- and
sex-matched normal controls, and chronic liver disease controls.
Methods: Postal completion of the FIS and linked symptom
assessment tools.
Results: Median FIS was significantly higher in patients
(n = 136) than community controls (40 [0138] vs. 28 [0156];
P < 0.0001) and chronic liver disease controls (n =
38) (20.5 [0145]; P < 0.05). Fatigue scores in
the 11 patients who had undergone liver transplantation (median
3.5 years previously) were the same as those in non-transplanted
patients with advanced disease.
Conclusions: Fatigue is a significant and specific problem
in PBC. It is not, however, universal and affects fewer patients
than has previously been thought to be the case based on data
from selected patient cohorts. This definition of the "normal
range" for fatigue in PBC will assist in future studies of
etiology and therapy.
The value of serologic markers in indeterminate colitis: A
prospective follow-up study
S. Joossens, W. Reinisch, S. Vermeire, B. Sendid, D. Poulain,
M. Peeters, K. Geboes, X. Bossuyt, P. Vandewalle, G. Oberhuber,
H. Vogelsang, P. Rutgeerts, J.-F. Colombel
Background & Aims: In the absence of pathognomonic
markers for Crohn's disease (CD) and ulcerative colitis (UC),
the diagnosis of inflammatory bowel disease depends on a compendium
of clinical, radiographic, endoscopic, and histologic criteria
that bears imperfect specificity to the individual disorders.
In 10% of cases of colitis, no differentiation can be made between
CD and UC; these patients are diagnosed with indeterminate colitis
(IC). We evaluated the value of antiSaccharomyces cerevisiae
antibodies (ASCA) and perinuclear antineutrophil cytoplasmic antibodies
(pANCA) to increase diagnostic accuracy in categorizing IC.
Methods: Since 1996, 97 patients with IC from 3 centers
(Leuven, Lille, and Vienna) were enrolled, analyzed for pANCA
and ASCA, and followed up prospectively.
Results: A definitive diagnosis has been reached for 31
of 97 patients (32%). In these patients, ASCA+/pANCA correlated
with CD in 8 of 10 patients, whereas ASCA/pANCA+ correlated
with UC in 7 of 11 patients. The remaining 4 cases became CD,
clinically behaving as UC-like CD. Almost half of the patients
(47 of 97 [48.5%]) were negative for ASCA and pANCA, and 40 remain
diagnosed with IC to date. Only 7 seronegative cases (14.9%) became
CD or UC compared with 48% (24 of 50) of seropositive patients
(P < 0.001).
Conclusions: Results so far show that ASCA+/pANCA
predicts CD in 80% of patients with IC and ASCA/pANCA+ predicts
UC in 63.6%. Interestingly, 48.5% of patients do not show antibodies
against ASCA or pANCA. Most of these patients remain diagnosed
with IC during their further clinical course, perhaps reflecting
a distinct clinicoserological entity.
Dietary nitrate generates potentially mutagenic concentrations
of nitric oxide at the gastroesophageal junction
K. Iijima, E. Henry, A. Moriya, A. Wirz, A. W. Kelman, K. E. L.
McColl
Background & Aims: Twenty-five percent of absorbed
dietary nitrate is re-secreted in saliva, and 30% of this is reduced
to nitrite by buccal bacteria. When saliva is swallowed, the acidic
gastric juice reduces the nitrite to nitric oxide. The aim of
this study was to examine the anatomic distribution of nitric
oxide generation within the lumen of the upper gastrointestinal
tract under basal conditions and after ingesting nitrate equivalent
to that in salad portion.
Methods: Using custom-made sensors, the dissolved luminal
nitric oxide concentration and pH were measured at 1-cm increments
for 2 minutes throughout the length of the stomach and distal
esophagus in 15 Helicobacter pylorinegative healthy
volunteers with and without ingestion of 2 mmol potassium nitrate.
Serum nitrate and saliva nitrite concentrations were also monitored.
Results: The nitrate ingestion increased mean (range) serum
nitrate from 30 µmol/L (1849) to 95 µmol/L (32152),
mean salivary nitrite from 36 µmol/L (19153) to 252
µmol/L (32600), and mean peak luminal nitric oxide
concentration from 4.7 µmol/L (1.47.8) to 23.2 µmol/L
(2.150) (P < 0.05 for each). After nitrate, the
peak nitric oxide concentration occurred in 11 of the 15 (73%)
subjects within 1 cm distal to the gastroesophageal pH step-up
point. The mean nitric oxide concentration over the 1-cm segment
immediately distal to the gastroesophageal pH step-up after nitrate
was 7.5 µmol/L (range, 0.530.7) and was significantly
higher than at all other sites. Nitric oxide concentrations greater
than 50 µmol/L were observed at the precise location where
neutral esophageal pH fell to acidic gastric pH.
Conclusions: Luminal generation of nitric oxide from dietary
nitrate via salivary nitrite is maximal at the gastroesophageal
junction and cardia. The high concentrations of nitric oxide generated
may contribute to the high incidence of mutagenesis and neoplasia
at this site.
Abnormal esophageal motility in children with congenital central
hypoventilation syndrome
C. Faure, F. Viarme, G. Cargill, J. Navarro, C. Gaultier, H. Trang
Background & Aims: Congenital central hypoventilation
syndrome, an unexplained disorder of the central control of breathing
that may reflect widespread dysfunction of brainstem structures,
is regarded as a form of neuro cristopathy. Because swallowing-induced
peristalsis is centrally controlled and depends on neural crest-derived
esophageal innervation, we looked for esophageal dysmotility in
patients with congenital central hypoventilation syndrome.
Methods: Seven patients without dysphagia or any other
upper gastrointestinal tract symptoms were studied prospectively
(5 girls and 2 boys; median age, 14 years; range, 1118 years).
They were compared with 7 age- and sex-matched controls. Esophageal
manometry was performed using a low-compliance infusion system
and the station pull-through technique. At least 10 wet swallows
were analyzed in each subject.
Results: Pressure wave propagation was abnormal in all
7 patients (median percentage of swallows propagated, 18%, and
range, 066; controls, 90% and 80100; P < 0.001).
Lower esophageal sphincter relaxation was abnormal in 5 patients
(patients, 73% and 53100; controls, 95% and 90100; P
= 0.01). In 2 patients, lower esophageal sphincter pressure was
above the 95th percentile of control values.
Conclusions: These abnormalities are strong evidence of
lower esophageal dysfunction in congenital central hypoventilation
syndrome. We speculate that the underlying mechanism may be dysfunction
of the central structures that control swallowing.
HLA DRB1*0405-DQB1*0401 haplotype is associated with autoimmune
pancreatitis in the Japanese population
S. Kawa, M. Ota, K. Yoshizawa, A. Horiuchi, H. Hamano, Y. Ochi,
K. Nakayama, Y. Tokutake, Y. Katsuyama, S. Saito, O. Hasebe, K.
Kiyosawa
Background & Aims: Autoimmune pancreatitis is a
distinctive disease entity characterized by high serum immunoglobulin
G4 concentrations. Because of the close association between some
autoimmune diseases and particular alleles of major histocompatibility
complex genes, we investigated the association between HLA alleles
and autoimmune pancreatitis.
Methods: HLA-A, -B, -C, -DR, and -DQ gene typing and HLA-DRB1,
-DQB1, and -DPB1 allele typing were performed by the polymerase
chain reaction sequence-specific primers method and the restriction
fragment length polymorphism method, respectively, in 40 patients
with autoimmune pancreatitis, 43 patients with chronic calcifying
pancreatitis, and 201 healthy subjects.
Results: In patients with autoimmune pancreatitis compared
with healthy subjects, we found a significant increase in DR4
(73% vs. 44%, corrected P = 0.01) and DRB1*0405 (58% vs.
21%, corrected P = 0.000026) and DQ4 (58% vs. 26%, corrected
P = 0.001) and DQB1*0401 (58% vs. 21%, corrected P
= 0.000017). The DRB1*0405-DQB1*0401 haplotype in autoimmune pancreatitis
showed no significant association with any HLA class I antigens,
in contrast to the B54-DRB1*0405-DQB1*0401 haplotype reported
in autoimmune hepatitis. The frequencies of DRB1*0405 and DQB1*0401
were significantly high in patients with autoimmune pancreatitis
compared with chronic calcifying pancreatitis.
Conclusions: It is probable that DRB1*0405-DQB1*0401 haplotype
is associated with autoimmune pancreatitis in the Japanese population.
T(11;18) is a marker for all stage gastric MALT lymphomas that
will not respond to H. pylori eradication
H. Liu, H. Ye, A. Ruskone-Fourmestraux, D. de Jong, S. Pileri,
C. Thiede, A. Lavergne, H. Boot, G. Caletti, T. Wündisch,
T. Molina, B. G. Taal, S. Elena, T. Thomas, P. L. Zinzani, A.
Neubauer, M. Stolte, R. A. Hamoudi, A. Dogan, P. G. Isaacson,
M.-Q. Du
Background & Aims: Eradication of Helicobacter
pylori leads to cure of gastric mucosa-associated lymphoid
tissue (MALT) lymphoma in 75% of localized cases. However, prolonged
follow-up is necessary to determine whether a lymphoma responds
to therapy. In a small series of cases, we showed that t(11;18)(q21;q21)-positive
MALT lymphomas failed to respond to H. pylori eradication.
The present study aimed to verify this finding in a large cohort
and confirm whether the translocation predicts the response of
stage IE tumors, for which clinical staging has little prognostic
value.
Methods: A total of 111 patients with H. pyloripositive
gastric MALT lymphoma treated with antibiotics were studied. Clinical
staging was undertaken before therapy. The response of lymphoma
to H. pylori eradication was determined by histologic examination
of gastric biopsy specimens. Diagnostic biopsy specimens were
analyzed for t(11;18)(q21;q21) by reverse-transcription polymerase
chain reaction of the API2-MALT1 transcript.
Results: Forty-seven of the 48 patients who showed complete
regression had lymphoma at stage IE, whereas 43 of the 63 nonresponsive
cases were at stage IE and the remaining cases at stage IIE or
above. t(11;18)(q21;q21) was detected in 2 of 48 complete-regression
cases, and these positive cases showed relapse of lymphoma in
the absence of H. pylori reinfection. In contrast, the
translocation was present in 42 of the 63 nonresponsive cases,
including 26 of 43 (60%) at stage IE.
Conclusions: t(11;18)(q21;q21)-positive gastric MALT lymphomas,
including those at stage IE, do not respond to H. pylori
eradication. Detection of the translocation should help the clinical
management of patients with gastric MALT lymphoma.
Clinical and pathologic findings in hemochromatosis type 3
due to a novel mutation in transferrin receptor 2 gene
D. Girelli, C. Bozzini, A. Roetto, F. Alberti, F. Daraio, R. Colombari,
O. Olivieri, R. Corrocher, C. Camaschella
Background & Aims: Although most patients with hereditary
hemochromatosis are homozygous for a single mutation of the HFE
gene on chromosome 6p, accumulating evidence indicates that the
disease is genetically heterogeneous. Type 3 hemochromatosis,
recently described in 4 families, is linked to mutations of the
gene encoding transferrin receptor 2 on chromosome 7q22. Here
we report data from a family carrying a new mutation of the transferrin
receptor 2 gene.
Methods: Detailed clinical and histopathologic documentation
was available for most family members. The entire coding sequence
and exon/intron boundaries of the transferrin receptor 2 gene
were analyzed by direct sequencing.
Results: A 12-nucleotide deletion in exon 16, causing the
loss of 4 amino acids (AVAQ 594597 del), was detected at
the homozygous state in the 3 patients with histologically proven
iron overload. The deletion segregated with the disease within
the family and was not found in 100 healthy controls. Some clinical
and pathologic characteristics, such as low penetrance in the
premenopausal woman, and early iron deposition in periportal hepatocytes
resembled those of classic, HFE-related hemochromatosis.
Conclusions: Our data support the role of the transferrin
receptor 2 gene in hemochromatosis type 3 as well as its critical
involvement in the maintenance of iron homeostasis in humans.
Impact of pegylated interferon alfa-2b and ribavirin on liver
fibrosis in patients with chronic hepatitis C
T. Poynard, J. McHutchison, M. Manns, C. Trepo, K. Lindsay, Z.
Goodman, M.-H. Ling, J. Albrecht, for the PEG-FIBROSIS Project
Group
Background & Aims: Liver fibrosis is an important
prognostic factor in patients with hepatitis C. The effect of
pegylated (PEG) interferon alone or its combination with ribavirin
on fibrosis has not been established.
Methods: We pooled individual data from 3010 naive patients
with pretreatment and posttreatment biopsies from 4 randomized
trials. Ten different regimens combining standard interferon,
PEG interferon, and ribavirin were compared. The impact of each
regimen was estimated by the percentage of patients with at least
1 grade improvement in the necrosis and inflammation (METAVIR
score), the percentage of patients with at least 1 stage worsening
in fibrosis METAVIR score, and by the fibrosis progression rate
per year.
Results: Necrosis and inflammation improvement ranged from
39% (interferon 24 weeks) to 73% (optimized PEG 1.5 and ribavirin;
P < 0.001). Fibrosis worsening ranges from 23% (interferon
24 weeks) to 8% (optimized PEG 1.5 and ribavirin; P <
0.001). All regimens significantly reduced the fibrosis progression
rates in comparison to rates before treatment. The reversal of
cirrhosis was observed in 75 patients (49%) of 153 patients with
baseline cirrhosis. Six factors were independently associated
with the absence of significant fibrosis after treatment: baseline
fibrosis stage (odds ratio [OR] = 0.12; P < 0.0001),
sustained viral response (OR = 0.36; P < 0.0001), age
< 40 years (OR = 0.51; P < 0.001), body mass index
< 27 kg/m2 (OR = 0.65; P < 0.001), no or minimal
baseline activity (OR = 0.70; P = 0.02), and viral load
< 3.5 millions copies per milliliter (OR = 0.79; P =
0.03).
Conclusions: PEG-interferon and ribavirin combination significantly
reduces the rate of fibrosis progression in patients with hepatitis
C.
Rehabilitation of swallowing by exercise in tube-fed patients
with pharyngeal dysphagia secondary to abnormal UES opening
R. Shaker, C. Easterling, M. Kern, T. Nitschke, B. Massey, S.
Daniels, B. Grande, M. Kazandjian, K. Dikeman
Background & Aims: We evaluated the effect of a
novel rehabilitative exercise on restoration of deglutition in
a group of patients with deglutitive failure caused by abnormal
upper esophageal sphincter (UES) opening manifested by postswallow
residue and aspiration necessitating percutaneous tube feeding.
Methods: We studied a total of 27 patients by videofluoroscopy
and functional assessment of swallowing scores before and after
6 weeks of a head-raising exercise program. Seven of 27 patients,
assigned randomly, participated in a sham exercise before entering
the tested exercise program. Eleven of 27 were randomized to the
real exercise program.
Results: Although there was no change in swallow function
and biomechanics after the sham exercise, following 6 weeks of
real exercise, all 11 patients exhibited a significant improvement
in their UES opening, anterior laryngeal excursion (P <
0.01), as well as resolution of postdeglutitive aspiration and
were able to resume oral feeding. Similar results were found when
the 7 patients in the sham group were crossed over to the real
exercise group. Comparison of before and after exercise values
for anteroposterior UES opening (P < 0.01) and laryngeal
anterior excursion (P < 0.05), as well as functional
outcome assessment of swallowing (P < 0.05) in the entire
group of 27 patients also showed significant improvement. Etiology
and duration of dysphagia did not affect the outcome.
Conclusions: The proposed suprahyoid muscle strengthening
exercise program is effective in restoring oral feeding in some
patients with deglutitive failure because of abnormal UES opening.
Basic Research
H+/dipeptide absorption across the human intestinal epithelium
is controlled indirectly via a functional Na+/H+ exchanger
D. T. Thwaites, D. J. Kennedy, D. Raldua, C. M. H. Anderson, M.
E. Mendoza, C. L. Bladen, N. L. Simmons
Background & Aims: For optimal nutrient absorption
to occur, the enterocyte must express a range of specialist ion-driven
carrier proteins that function cooperatively in a linked and mutually
dependent fashion. Thus, absorption via the human intestinal H+-coupled
di/tripeptide transporter (hPepT1) is dependent on maintenance
of the trans-apical driving force (the H+-electrochemical gradient)
established, in part, by brush-border Na+/H+ exchanger (NHE3)
activity. This study aimed to examine whether physiologic regulation
of NHE3 activity can limit hPepT1 capacity and, therefore, protein
absorption after a meal.
Methods: hPepT1 and NHE3 activities were determined in
intact human intestinal epithelial Caco-2 cell monolayers by measurements
of [14C]glycylsarcosine transport and uptake, 22Na+-influx, H+-influx,
and H+-efflux. Expression of NHE regulatory factors was determined
by reverse-transcriptase polymerase chain reaction.
Results: Optimal dipeptide transport was observed in the
presence of a transapical pH gradient and extracellular Na+. At
apical pH 6.5, and only in Na+-containing media, protein kinase
A activation (by forskolin or vasoactive intestinal peptide) or
selective NHE3 inhibition (by S1611) reduced transepithelial dipeptide
transport and cellular accumulation by a reduction in the capacity
(without effect on affinity) of dipeptide uptake.
Conclusions: Protein kinase Amediated modulation of
intestinal dipeptide absorption is indirect via effects on the
apical Na+/H+ exchanger.
Critical role of caspases in the regulation of apoptosis and
proliferation of mucosal T cells
A. Sturm, S. Mohr, C. Fiocchi
Background & Aims: Caspases are critical mediators
of apoptosis and proliferation of peripheral blood T cells (PBT),
but their role in lamina propria T cells (LPT), a cell population
highly susceptible to apoptosis, has not been explored.
Methods: RA+, RO+ PBT, and LPT were activated with CD3,
CD2, and CD28 antibodies, and caspase activity, apoptosis, and
proliferation were measured by a fluorometric assay, DNA content,
and thymidine incorporation, respectively. Levels of FLIP, an
endogenous inhibitor of caspase 8, were measured by immunoblotting.
Results: In RA+ and RO+ PBT, activation leads to significant
increase of caspase activity but not cell death, whereas in LPT
a lower elevation of caspase activity was followed by a marked
degree of apoptosis. Based on the results of its inhibition, caspase
8 seemed to be essential for LPT apoptosis but, in contrast to
RA+ PBT, had no effect on proliferation. In addition, compatible
with their differential susceptibility to apoptosis, levels of
FLIP were lower in LPT than PBT.
Conclusions: The high susceptibility of LPT to apoptosis
is associated with a distinct regulation of caspase 8 activity,
which seems to reflect their mucosal origin rather than simply
their memory status. This unique behavior may allow proper control
of mucosal T-cell proliferation while still permitting elimination
by apoptosis in the face of excessive antigenic pressure.
Monoassociation of SCID mice with Helicobacter muridarum, but
not four other enterics, provokes IBD upon receipt of T cells
H.-Q. Jiang, N. Kushnir, M. C. Thurnheer, N. A. Bos, J. J. Cebra
Background & Aims: Recently, a number of animal
models for different aspects of inflammatory bowel disease (IBD)
have been developed. The aim of this study was to use one of these
to determine whether particular, ostensibly innocuous, intestinal
bacteria could provoke or exacerbate IBD.
Methods: Conventionally reared C.B17 SCID mice were compared
with germ-free and gnotobiotic mice, monoassociated with 1 of
5 intestinal bacteria, after transfer of CD45RBhigh CD4+ T cells
from conventionally reared congenic BALB/c mice. Recipient mice
were monitored over 712 weeks for clinical signs of IBD,
and tissues were analyzed by histology/flow cytometry for abnormal
inflammation and CD4+ T cell outgrowth.
Results: Neither germ-free mice nor mice monoassociated
with segmented filamentous bacteria, Ochrobactrum anthropi,
a nonpathogenic mutant of Listeria monocytogenes, or Morganella
morganii developed any signs of IBD. In contrast, mice monoassociated
with Helicobacter muridarum displayed an accelerated development
of IBD in 56 weeks compared with 812 weeks observed
in conventionally reared mice. The outgrowth of CD4+ T cells in
spleen and large intestine of H. muridarum monoassociated
mice, as well as in conventionally reared mice was significantly
higher than that in the other monoassociated mice.
Conclusions: Among the intestinal bacteria tested, H.
muridarum can serve as a provocateur of IBD in this model.
NO sensitizes rat hepatocytes to proliferation by modifying
S-adenosylmethionine levels
E. R. García-Trevijano, M. L. Martínez-Chantar,
M. U. Latasa, J. M. Mato, M. A. Avila
Background & Aims: Liver regeneration is a fundamental
response of this organ to injury. Hepatocyte proliferation is
triggered by growth factors, such as hepatocyte growth factor.
However, hepatocytes need to be primed to react to mitogenic signals.
It is known that nitrous oxide (NO), generated after partial hepatectomy,
plays an important role in hepatocyte growth. Nevertheless, the
molecular mechanisms behind this priming event are not completely
known. S-adenosylmethionine (AdoMet) synthesis by methionine adenosyltransferase
is the first step in methionine metabolism, and NO regulates hepatocyte
S-adenosylmethionine levels through specific inhibition of this
enzyme. We have studied the modulation of hepatocyte growth factorinduced
proliferation by NO through the regulation of S-adenosylmethionine
levels.
Methods: Studies were conducted in cultured rat hepatocytes
isolated by collagenase perfusion, which triggers NO synthesis.
Results: The mitogenic response to hepatocyte growth factor
was blunted when inducible NO synthase was inhibited; this process
was overcome by the addition of an NO donor. This effect was dependent
on methionine concentration in culture medium and intracellular
S-adenosylmethionine levels. Accordingly, we found that S-adenosylmethionine
inhibits hepatocyte growth factorinduced cyclin D1 and D2
expression, activator protein 1 induction, and hepatocyte proliferation.
Conclusions: Together our findings indicate that NO may
switch hepatocytes into a hepatocyte growth factorresponsive
state through the down-regulation of S-adenosylmethionine levels.
The role of transforming growth factor beta-2, beta-3 in mediating
apoptosis in the murine intestinal mucosa
N. Dünker, K. Schmitt, N. Schuster, K. Krieglstein
Background & Aims: Apoptosis is especially relevant
in the gastrointestinal tract because the mammalian intestinal
mucosa undergoes continual epithelial regeneration. Most recently,
we confirmed the proapoptotic role of endogenous transforming
growth factor (TGF)- in the developing chick retina as well as
in chick ciliary, dorsal root, and spinal motor neurons. In the
present study, we determined to establish the role of TGF-2 and
TGF-3 in mediating apoptosis in non-neuronal tissue by analyzing
the intestinal mucosa of Tgf2+/ and Tgf3+/
heterozygous mice.
Methods: Intestinal localization of TGF-2 and TGF-3 isoforms
and antiapoptotic molecules Bcl-xL and Bcl-2 was examined immunocytochemically
and by Western blot analysis. Apoptosis was detected by enzyme-linked
immunosorbent assay and terminal deoxynucleotidyl transferasemediated
deoxyuridine triphosphate nick-end labeling, and proliferation
was detected by proliferating cell nuclear antigen stains.
Results: TGF-2 was detected in endocrine cells, whereas
TGF-3 was predominantly found in goblet cells. Programmed cell
death was significantly reduced in the intestinal mucosa of Tgf2+/
and Tgf3+/ heterozygous mice. This decrease in apoptosis
was accompanied by an increase in villus length; proliferation,
however, seemed to remain unchanged. The level of Bcl-xL and Bcl-2
was significantly up-regulated in Tgf2+/ and Tgf3+/
mice.
Conclusions: Our data show that TGF-2 and TGF-3 play an
important role in mediating apoptosis in the intestinal mucosa
and regulating apoptosis-associated proteins Bcl-xL and Bcl-2
in vivo.
CpG island methylation sporadic colorectal cancers and its
relationship to microsatellite instability
N. Hawkins, M. Norrie, K. Cheong, E. Mokany, S.-L. Ku, A. Meagher,
T. O'Connor, R. Ward
Background & Aims: Methylation of CpG islands is
increasingly recognized as an important event in colorectal carcinogenesis.
We evaluated the extent of CpG island methylation in 426 sporadic
colorectal cancers to define its relationship to microsatellite
instability and to describe its clinicopathologic and genetic
features.
Methods: Fresh cancer tissue was obtained from 417 consecutive
individuals undergoing curative surgery for sporadic colorectal
cancer. Methylation of p16 and hMLH1 promoters was determined
by methylation-specific polymerase chain reaction (PCR), whereas
methylation at MINT 1, 2, 12, and 31 loci was assessed by bisulfite
PCR. Microsatellite instability and K-ras and p53 status
were determined using microsatellite PCR, restriction enzymemediated
PCR, and immunohistochemistry, respectively.
Results: Individual loci were commonly methylated, but
locus-specific phenotypic changes were not seen. CpG island methylation
was associated with right-sided location, female sex, and older
age, as well as high tumor grade, mucinous type, wild-type P53,
microsatellite instability, and K-ras mutations. More than
half of tumors showing CpG island methylation were microsatellite
stable. Compared with microsatellite unstable cancers, they were
more commonly left-sided, had fewer intraepithelial lymphocytes,
presented later, and had a worse outcome.
Conclusions: Colorectal cancers with CpG island methylation
have distinct clinicopathologic features and in some cases lead
to sporadic microsatellite unstable cancers.
Expression and regulation of nonsteroidal anti-inflammatory
drugactivated gene (NAG-1) in human and mouse tissue
K.-S. Kim, S. J. Baek, G. P. Flake, C. D. Loftin, B. F. Calvo,
T. E. Eling
Background & Aims: Nonsteroidal anti-inflammatory
drugs (NSAIDs) induce NSAID-activated gene 1 (NAG-1), which has
proapoptotic and antitumorigenic activities. However, NAG-1 expression
and its relationship with apoptosis in human and mouse intestinal
tract have not been determined.
Methods: NAG-1 expression in human and mouse tissue was
determined by immunohistochemistry, and apoptosis was estimated
by in situ apoptosis detection. Apoptosis in NAG-1 overexpressing
HCT-116 cells was examined with flow cytometry after cell sorting
by green fluorescence protein. NAG-1 regulation in mouse cells
was examined by Northern blot analysis, comparing sulindac-treated
and nontreated mice.
Results: Apoptosis was higher in NAG-1 overexpressing cells
compared with controls. Human NAG-1 protein was localized to the
colonic surface epithelium where cells undergo apoptosis, and
higher expression was observed in the normal surface epithelium
than in most of the tumors. This localization and lower expression
in tumors was similar to that in the Min mouse, in which
NSAIDs were also shown to regulate the expression of NAG-1 in
mouse cells. Sulindac treatment of mice increased the NAG-1 expression
in the colon and liver.
Conclusions: Based on these results, we propose that NAG-1
acts as a mediator of apoptosis in intestinal cells and may contribute
to cancer chemoprevention by NSAIDs.
Leptin receptormediated signaling regulates hepatic fibrogenesis
and remodeling of extracellular matrix in the rat
K. Ikejima, Y. Takei, H. Honda, M. Hirose, M. Yoshikawa, Y.-J.
Zhang, T. Lang, T. Fukuda, S. Yamashina, T. Kitamura, N. Sato
Background & Aims: In this study, we investigated
the role of leptin and its receptors (Ob-R) in profibrogenic responses
in the liver using Zucker (fa/fa) rats, a natural occurring Ob-Rdeficient
animal.
Methods: Male Zucker (fa/fa) rats and their lean (+/?)
littermates were given intraperitoneal injections of thioacetamide
(TAA) (200 mg/kg body wt, 3 times/wk) for 48 weeks, and progression
of hepatic fibrosis was evaluated. In vitro transactivation of
hepatic stellate cells (HSCs) isolated from Zucker rats was evaluated
by Western blotting and immunocytochemistry for -smooth muscle
actin and type I collagen. Further, a long-form Ob-R (Ob-Rb) in
sinusoidal endothelial cells (SECs) and Kupffer cells was identified
by reverse-transcription polymerase chain reaction. Moreover,
transforming growth factor (TGF)-1 messenger RNA in LSE cells,
a human SEC-derived cell line, was measured by Northern blotting.
Results: Although the normal liver does not produce leptin,
activated HSCs produced leptin in vivo during fibrogenesis caused
by TAA. In Zucker rats, TAA-induced hepatic fibrosis was prevented
almost completely, whereas induction of TGF-1 and activation of
HSCs were abolished. It is less likely, however, that leptin plays
an essential role in the activation of HSCs as a strong autocrine
regulator, because HSCs isolated from Zucker rats undergo normal
transactivation process in vitro. In contrast, SECs and Kupffer
cells contain Ob-Rb, through which leptin up-regulates the expression
of matrix remodeling genes including TGF-1.
Conclusions: Collectively, these findings indicated that
leptin and its functional receptors (Ob-Rb) play a pivotal role
in profibrogenic responses in the liver.
Taurolithocholic acid-3 sulfate induces CD95 trafficking and
apoptosis in a c-Jun N-terminal kinasedependent manner
D. Graf, A. K. Kurz, R. Fischer, R. Reinehr, D. Häussinger
Background & Aims: Prevention of bile acidinduced
apoptosis is of therapeutic interest and requires the understanding
of underlying mechanisms.
Methods: The effect of tauroursodeoxycholate (TUDC) on
taurolithocholic acid-3 sulfate (TLCS)-induced apoptosis was studied
in cultured rat hepatocytes.
Results: TLCS induced activation of caspases 8, 9, and
3 and hepatocyte apoptosis. These effects were abolished by TUDC
in a PI 3-kinase/protein kinase B (PKB)-, p38MAPK-, and extracellular
signalregulated kinase-2 (Erk-2)-independent manner. These
protein kinases were activated by both TLCS and TUDC, however,
with different kinetics. TLCS, but not TUDC, led to a sustained
activation of c-Jun N-terminal kinase (JNK) and CD95 trafficking
to the plasma membrane; both TLCS effects were prevented by TUDC.
Inhibition of JNK1 or protein kinase C prevented TLCS-induced
CD95 membrane trafficking and blunted the apoptotic response.
The apoptotic potency of other bile acids paralleled their ability
to induce sustained JNK activation.
Conclusions: Protection by TUDC against TLCS-induced apoptosis
starts upstream of caspase 8 activation and is independent of
a PI 3-kinasedependent survival pathway. JNK activation may
be important for bile acidinduced apoptosis by triggering
ligand-independent CD95 surface trafficking and activation of
apoptosis.
Immunostimulatory DNA ameliorates experimental and spontaneous
murine colitis
D. Rachmilewitz, F. Karmeli, K. Takabayashi, T. Hayashi, L. Leider-Trejo,
J. Lee, L. M. Leoni, E. Raz
Background & Aims: Impaired mucosal barrier, cytokine
imbalance, and dysregulated CD4+ T cells play important roles
in the pathogenesis of experimental colitis and human inflammatory
bowel disease. Immunostimulatory DNA sequences (ISS-DNA) and their
synthetic oligonucleotide analogs (ISS-ODNs) are derived from
bacterial DNA, are potent activators of innate immunity at systemic
and mucosal sites, and can rescue cells from death inflicted by
different agents. We hypothesized that these combined effects
of ISS-DNA could inhibit the damage to the colonic mucosa in chemically
induced colitis and thereby limit subsequent intestinal inflammation.
Methods: The protective and the anti-inflammatory effect
of ISS-ODN administration were assessed in dextran sodium sulfateinduced
colitis and in 2 models of hapten-induced colitis in Balb/c mice.
Similarly, these effects of ISS-ODN were assessed in spontaneous
colitis occurring in IL-10 knockout mice.
Results: In all models of experimental and spontaneous
colitis examined, ISS-ODN administration ameliorated clinical,
biochemical, and histologic scores of colonic inflammation. ISS-ODN
administration inhibited the induction of colonic proinflammatory
cytokines and chemokines and suppressed the induction of colonic
matrix metalloproteinases in both dextran sodium sulfate
and hapteninduced colitis.
Conclusions: As the colon is continuously exposed to bacterial
DNA, these findings suggest a physiologic, anti-inflammatory role
for immunostimulatory DNA in the GI tract. Immunostimulatory DNA
deserves further evaluation for the treatment of human inflammatory
bowel disease.
Constitutive expression and function of cyclooxygenase-2 in
murine gastric muscles
C. Porcher, B. Horowitz, O. Bayguinov, S. M. Ward, K. M. Sanders
Background & Aims: Cyclooxygenase enzymes (COX)
generate intermediates in the prostaglandin (PG) cascade. COX-1
is constitutively expressed in many cells, and COX-2 is typically
thought to be an inducible isoform.
Methods: We evaluated constitutive expression and function
of COX-2 in murine gastric muscles.
Results: Immunohistochemistry showed COX-2like immunoreactivity
(COX-2LI) in myenteric neurons. Half the neurons with COX-2LI
expressed nitric oxide synthase (NOS). COX-2LI was not observed
in smooth muscle cells. Interstitial cells of Cajal within muscle
layers (IC-IM) expressed COX-2LI, suggesting a novel role
for IC-IM. Molecular studies verified expression of COX-2 in gastric
muscles. Quantitative polymerase chain reaction (PCR) showed equal
expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant
in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased
antral phasic contractions and potentiated responses to ACh. Indomethacin,
but not GR253035X, increased contractions and potentiated responses
in tissues of COX-2 knockout mice. Indomethacin and GR253035X
reduced tone in the fundus.
Conclusions: COX-2 is constitutively expressed by IC-IM
and neurons in the stomach and at levels similar to COX-1. Prostanoids
produced by COX-2 regulate mechanical activities of fundus and
antral muscles.
Inactivation of the transcription factor Elf3 in mice results
in dysmorphogenesis and altered differentiation of intestinal
epithelium
A. Y.-N. Ng, P. Waring, S. Ristevski, C. Wang, T. Wilson, M. Pritchard,
P. Hertzog, I. Kola
Background & Aims: The mammalian small intestine
is lined by a highly specialized epithelium that functions in
the digestion and absorption of nutrients. The molecular mechanisms
that direct intestinal epithelial cell morphogenesis and terminal
differentiation are poorly understood. We have previously identified
Elf3 (E74-like factor-3) as a member of the ETS
transcription factor family strongly expressed in small intestinal
epithelium. The aim of this study is to investigate the biological
roles of Elf3 in vivo.
Methods: Mice with a null mutation of Elf3 were
generated through targeted gene disruption. Characterization of
intestinal development was performed by histologic and immunohistochemical
techniques.
Results: Targeted disruption of Elf3 resulted in
fetal lethality of about 30% at around embryonic day 11.5. Seventy
percent of the Elf3-deficent progeny were born and displayed
severe alterations of tissue architecture in the small intestine,
manifested by poor villus formation and abnormal morphogenesis
and terminal differentiation of absorptive enterocytes and mucus-secreting
goblet cells. Crypt cell proliferation, however, appeared intact
in Elf3-deficient mice.Elf3-deficient enterocytes
express markedly reduced levels of the transforming growth factor
type II receptor (TGF- RII), an inducer of intestinal epithelial
differentiation.
Conclusions: Elf3 is an important regulator of morphogenesis
and terminal differentiation of epithelial cell lineages in the
small intestine.
Novel genes and functional relationships in the adult mouse
gastrointestinal tract identified by microarray analysis
M. D. Bates, C. R. Erwin, L. P. Sanford, D. Wiginton, J. A. Bezerra,
L. C. Schatzman, A. G. Jegga, C. Ley-Ebert, S. S. Williams, K.
A. Steinbrecher, B. W. Warner, M. B. Cohen, B. J. Aronow
Background & Aims: A genome-level understanding
of the molecular basis of segmental gene expression along the
anterior-posterior (A-P) axis of the mammalian gastrointestinal
(GI) tract is lacking. We hypothesized that functional patterning
along the A-P axis of the GI tract could be defined at the molecular
level by analyzing expression profiles of large numbers of genes.
Methods: Incyte GEM1 microarrays containing 8638 complementary
DNAs (cDNAs) were used to define expression profiles in adult
mouse stomach, duodenum, jejunum, ileum, cecum, proximal colon,
and distal colon. Highly expressed cDNAs were classified based
on segmental expression patterns and protein function.
Results: 571 cDNAs were expressed 2-fold higher than reference
in at least 1 GI tissue. Most of these genes displayed sharp segmental
expression boundaries, the majority of which were at anatomically
defined locations. Boundaries were particularly striking for genes
encoding proteins that function in intermediary metabolism, transport,
and cell-cell communication. Genes with distinctive expression
profiles were compared with mouse and human genomic sequence for
promoter analysis and gene discovery.
Conclusions: The anatomically defined organs of the GI
tract (stomach, small intestine, colon) can be distinguished based
on a genome-level analysis of gene expression profiles. However,
distinctions between various regions of the small intestine and
colon are much less striking. We have identified novel genes not
previously known to be expressed in the adult GI tract. Identification
of genes coordinately regulated along the A-P axis provides a
basis for new insights and gene discovery relevant to GI development,
differentiation, function, and disease.
Bile acids regulate the ontogenic expression of ileal bile
acid binding protein in the rat via the farnesoid X receptor
S. T. Hwang, N. L. Urizar, D. D. Moore, S. J. Henning
Background & Aims: In the rat, an increase in ileal
bile acid binding protein (IBABP) expression occurs during the
third postnatal week. In vitro studies suggest that bile acids
(BAs) increase IBABP transcription by activating the BA receptor,
farnesoid X receptor (FXR). Thus, we investigated the role of
BAs on the ontogenic expression of IBABP and whether FXR may mediate
these effects.
Methods: Suckling rats were gavage-fed taurocholate for
3 days or were allowed to develop normally. Ileums were collected
for Northern and Western blot analyses. Electrophoretic mobility
shift assays for functional FXR were performed using nuclear extracts
from ileums of both adult and developing rats.
Results: Taurocholate gavage significantly elevated IBABP
messenger RNA and protein levels in suckling animals. Gelshift
assays using adult ileal nuclear extracts incubated with a radiolabeled
consensus inverted repeat-1 oligonucleotide (response element
for FXR) revealed a high-molecular weight DNA/protein complex.
Cold competition and supershift assays showed that this complex
is sequence specific and confirmed that FXR is a component of
the complex. Gelshift assays with nuclear extracts from rat ileum
at different ages revealed absence of the DNA/protein complex
in the second postnatal week when there is lack of IBABP expression
and presence of these complexes at later ages when there is normally
high expression. Western blot analyses showed FXR and its heterodimer
partner, retinoid X receptor , protein levels are low in the ileum
during the suckling period and increase during the third postnatal
week.
Conclusions: BAs play a role in the normal developmental
expression of IBABP through FXR activation, and decreased functional
FXR in ileal nuclei during the suckling period may account, in
part, for the lack of IBABP expression at this time.
Case Report
Familial gastrointestinal stromal tumors associated with
dysphagia and novel type germline mutation of KIT gene
S. Hirota, T. Nishida, K. Isozaki, M. Taniguchi, K. Nishikawa,
A. Ohashi, A. Takabayashi, T. Obayashi, T. Okuno, K. Kinoshita,
H. Chen, Y. Shinomura, Y. Kitamura
A family with multiple gastrointestinal stromal tumors (GISTs),
a new type of germline mutation of KIT gene, and dysphagia is
reported. The mutation was observed at Asp-820 in tyrosine kinase
(TK) II domain. Mutations in TK II domain have been found in mast
cell and germ cell tumors but not in GISTs, and the present family
members are the first reported cases of GISTs with TK II domain
mutations, including sporadic GISTs. Because interleukin 3dependent
Ba/F3 murine lymphoid cells transfected with the mutant KIT complementary
DNA grew autonomously without any growth factors and formed tumors
in nude mice, the mutation was considered to be gain-of-function
type. Family members with the germline KIT mutation reported dysphagia,
but those without the mutation did not. The mechanism of dysphagia
was examined with gastrointestinal fiberscopy, endoscopic ultrasonography,
and esophageal manometry. No mechanical obstruction was found,
and the esophagus was not remarkably dilated. In the family members
with dysphagia, endoscopic ultrasonography at the esophagocardiac
junction showed a thickened hyperechoic layer between the circular
and longitudinal muscle layers, suggesting hyperplasia of interstitial
cells of Cajal at the myenteric plexus layer. Manometry showed
low resting lower esophageal sphincter pressure and abnormal simultaneous
contractions of the esophagus without normal peristalsis. These
findings indicate that the dysphagia of the present family is
different from typical achalasia. This is the first report of
familial dysphagia caused by germline gain-of-function mutation
of the KIT gene at the TK II domain.
Special Reports and Reviews
The burden of selected digestive diseases in the United
States
R. S. Sandler, J. E. Everhart, M. Donowitz, E. Adams, K. Cronin,
C. Goodman, E. Gemmen, S. Shah, A. Avdic, R. Rubin
Background & Aims: Gastrointestinal (GI) and liver
diseases inflict a heavy economic burden. Although the burden
is considerable, current and accessible information on the prevalence,
morbidity, and cost is sparse. This study was undertaken to estimate
the economic burden of GI and liver disease in the United States
for use by policy makers, health care providers, and the public.
Methods: Data were extracted from a number of publicly
available and proprietary national databases to determine the
prevalence, direct costs, and indirect costs for 17 selected GI
and liver diseases. Indirect cost calculations were purposefully
very conservative. These costs were compared with National Institutes
of Health (NIH) research expenditures for selected GI and liver
diseases.
Results: The most prevalent diseases were nonfood-borne
gastroenteritis (135 million cases/year), food-borne illness (76
million), gastroesophageal reflux disease (GERD; 19 million),
and irritable bowel syndrome (IBS; 15 million). The disease with
the highest annual direct costs in the United States was GERD
($9.3 billion), followed by gallbladder disease ($5.8 billion),
colorectal cancer ($4.8 billion), and peptic ulcer disease ($3.1
billion). The estimated direct costs for these 17 diseases in
1998 dollars were $36.0 billion, with estimated indirect costs
of $22.8 billion. The estimated direct costs for all digestive
diseases were $85.5 billion. Total NIH research expenditures were
$676 million in 2000.
Conclusions: GI and liver diseases exact heavy economic
and social costs in the United States. Understanding the prevalence
and costs of these diseases is important to help set priorities
to reduce the burden of illness.
Biliary Tract and Cholestasis
Natural history and outcome in 32 Swedish patients with
small duct primary sclerosing cholangitis (PSC)
Ulrika
Broomé et al.
Background/Aims: This study aims at describing
the natural history and outcome of small duct primary sclerosing
cholangitis (PSC). Methods: Thirty-two patients
with small duct PSC were studied. The average time taken for diagnosis
was 69 (1-168) months. The median follow-up time was 63 (1-194)
months. Results: All patients including one who
underwent liver transplantation because of end-stage liver disease
and hepatocellular carcinoma were alive at follow-up. None developed
cholangiocarcinoma. In 27 patients repeated cholangiographic examinations
were done after a median time of 72 (12-192) months from first
ERCP. Four developed features of large duct PSC.Conclusions:
Small duct PSC rarely progresses to large bile duct PSC and it
seems to have a benign course in most patients and no development
of cholangiocarcinoma was found.
Cell Biology, Metabolism and Transport
Regulation of cultured rat hepatocyte proliferation by stellate
cells
Naoki Uyama et al.
Background/Aims: This study using primary culture
models was aimed to reveal the stellate cell-derived factors that
regulate hepatocyte proliferation. Methods: Rat
hepatocytes and stellate cells were cultured in serum-free Williams-E
medium. We prepared hepatocyte mono-culture and two different
co-cultures of hepatocytes and stellate cells; (1) co-culture
on the same surface (Co-mix.) and (2) co-culture without contact
between hepatocytes and stellate cells using a culture insert
(Co-sep.). The change in the number and the DNA synthesis of hepatocytes
was evaluated. Results: The number of hepatocytes
decreased to 76% of the original number after 48h of starting
mono-culture, while it remained at 106% in mixed co-culture (Co-mix.)
and increased to 135% in separated co-culture (Co-sep.). The hepatocyte
DNA synthesis was enhanced by carbenoxolone in Co-mix. and reduced
by NK1 in each co-culture. PD153035 had no effect. Heparitinase-I
(20mU/ml) and sodium chrolate (25mM) reduced the hepatocyte DNA
synthesis in Co-sep. to 71.8 and 61.6%, respectively. Activation
of mitogen-activated protein kinase was induced in hepatocytes
stimulated by conditioned mediums. Conclusions:
Hepatocyte proliferation was stimulated in the presence of stellate
cells through hepatocyte growth factor, extracellular heparan
sulfate (HS), and HS proteoglycan, and might be negatively regulated
by gap junction-dependent mechanism.
Cirrhosis and its Complications
Effect of 1-week losartan administration on bile duct-ligated
cirrhotic rats with portal hypertension
Ying-Ying Yang et al.
Background/Aims: Nitric oxide and angiotensin play
important roles in the pathogenesis of the hemodynamic derangement
in cirrhosis and portal hypertension. The hemodynamic effects
of losartan, an angiotensin II type 1 receptor antagonist, in
cirrhotic patients with portal hypertension are conflicting. This
study was undertaken to explore the possible mechanism of action
of losartan on portal hypertension in cirrhotic rats produced
by bile duct ligation (CBL). Methods: Three weeks
after surgery, CBL and sham-operated rats randomly received vehicle
or losartan (3 mg/kg per 12 h by gavage) for 1 week. Hemodynamic
values, hormone levels, and aortic eNOS protein expression were
measured after drug administration. Results: In
CBL rats, 1-week losartan treatment decreased portal pressure
and ameliorated hyperdynamic circulation associated with a blunted
vascular response to N-nitro-L-arginine methyl ester infusion.
The hematocrit increased and the plasma volume, aldosterone, plasma
renin activity, norepinephrine, and nitrate and nitrite levels
decreased. The eNOS protein expression was reduced in CBL rats
receiving losartan compared with those receiving vehicle. Conclusions:
One-week losartan treatment in CBL rats decreased portal pressure
and ameliorated hyperdynamic circulation. In addition to the suppression
of renin-angiotensin axis, the reduced aortic eNOS protein expression
may play a partial role for the mechanism of action of losartan
in CBL rats.
Inflammation and Fibrosis
Expression of reelin in hepatic stellate cells and during
hepatic tissue repair: a novel marker for the differentiation
of HSC from other liver myofibroblasts
Dominik Kobold et al.
Background/Aims: Hepatic stellate cells (HSC) and
rat liver myofibroblasts (rMF), two similar but not identical
cell populations, play a major role during hepatic tissue repair.
Methods: To identify marker proteins for the different
fibroblastic cell populations, m-RNA-profiling technology was
employed using c-DNAs prepared from HSC and rMF. Results/Conclusions:
The extracellular matrix protein reelin was identified through
its presence in HSC and absence in rMF derived samples. As confirmed
by Northern blot analysis and by immunoprecipitation, reelin expression
was present in similar amounts in resting and activated HSC and
was not detectable in rMF. Therefore reelin is the only marker
presently available to distinguish HSC at any stage of differentiation
from rMF. Following a single CCl4 mediated liver injury,
reelin specific mRNAs were induced early, were elevated up to
24h following CCl4 dosage and were diminished afterwards.
Hepatocytes and non-parenchymal liver cells located in the damaged
areas were identified as the main cellular source of enhanced
reelin expression. Although reelin expression was upregulated
during liver injury, reelin deficient mice recovered completely
suggesting either a more distinct role in tissue repair reactions
or a case of redundancy through the action of related proteins.
Liver Cell Injury and Liver Failure
Liver injury due to sequential activation of natural killer
cells and natural killer T cells by carrageenan
Tetsuya Abe et al.
Background/Aims: Carrageenan is a high molecular
weight polysaccharide and is widely used as a food additive for
the solidification of plant oils and the thickening of many beverages.
It is known that acute toxicity of carrageenan is possibly induced
by the activation of phagocytic cells. We investigated other effects
of carrageenan on lymphocytes in this study. Methods:
Carrageenan was intraperitoneally injected once into mice and
phenotypic and functional characterizations were conducted in
various immune organs. Results: Natural killer (NK)
cells were prominently activated in the liver, lungs, and spleen.
A time-kinetic study showed sequential activation of NK and natural
killer T (NKT) cells in the liver on days 3-10 after the injection.
In parallel with the activation of NK and NKT cells in number,
NK and NKT cytotoxicities were augmented. At this time, liver
injury was induced, accompanied by massive hepatic necrosis and
the elevation of transaminases. The in vivo elimination of NK
cells reduced the liver injury induced by carrageenan. Direct
binding of carrageenan onto NK cells was also demonstrated. Such
a binding then induced a subsequent production of IFN. Perforin
molecules of NK cells were responsible for this liver injury.
Conclusions: These results suggest that not only
phagocytic cells but also primitive lymphocyte (mainly NK cells)
subsets might be important targets for the acute toxicity of carrageenan.
The role of endogenous heme oxygenase in the initiation of
liver injury following limb ischemia/reperfusion
Robert G. Nie et al.
Background/Aims: Heme oxygenase (HO) derived liver
protection was tested in mice following 1h bilateral hindlimb
ischemia and either 1.5 or 3h reperfusion. Methods:
Groups consisted of limb ischemia/reperfusion (I/R), sham (no
I/R), I/R+chromium mesoporphyrin (I/R+CrMP;40µmol/kg, i.p.),
or I/R+hemin (10mg/kg, i.p.). The vital dye propidium iodide (PI),
was used to measure hepatocellular death (#/0.1mm3), while the
number of sinusoids perfused by red blood cells (SPRBC) were measured
from the periportal (Pp) and pericentral (Pc) zones of liver acini
using intravital microscopy. Whole organ injury was estimated
from serum alanine aminotransferase (ALT). Results:
SPRBC reduced within 1.5h with no further decline following 3h.
CrMP resulted in a dramatic loss of SPRBC following 3h only. Hemin
restored perfusion in both zones. Hepatocellular death and organ
injury increased at 1.5 and 3h. At 1.5h, CrMP further increased
cell death in the Pc zone, as well as whole organ injury, while
hemin restored cell viability. Increased HO mRNA, protein and
activity suggested induction within 3h. Conclusions:
HO does not protect perfusion during the early stage (1.5h), but
becomes increasingly important in preserving liver perfusion and
cell viability during the later stage (3h) of liver injury.
4-Nitrobenzylidene malononitrile reduces apoptosis-mediated
liver injury in mice
Alexey Vanichkin, Miriam Patya, Irina Lagovsky, Asher Meshorer
and Abraham Novogrodsky
Background: Apoptosis plays a role in experimental
and clinically related liver damage. Inhibitors of tyrosine kinases
were shown to modulate apoptosis induced by different agents in
various cell types. Aims: Investigation of the effect
of 4-nitrobenzylidene malononitrile (belonging to the tyrphostins
family which are selective inhibitors of protein tyrosine kinases)
on apoptosis-mediated acute liver injury. Methods:
Two murine experimental models exhibiting apoptosis-mediated liver
injury were used: (1) mice treated with tumor necrosis factor-
and d-galactosamine; and (2) mice treated with anti-Fas
antibody. Liver injury was assessed by serum levels of transaminases
and by microscopic analysis. Apoptosis was assessed by labeling
of apoptotic cells in the liver by the TUNEL assay and by determination
of caspase-3 activity. Results: Pretreatment of
mice with 4-nitrobenzylidene malononitrile reduced tumor necrosis
factor-/d-galactosamine-induced hepatotoxicity. TUNEL positive
cells in sections from livers treated with vehicle (control),
4-nitrobenzylidene malononitrile, tumor necrosis factor-/d-galactosamine
and tumor necrosis factor-/d-galactosamine and 4-nitrobenzylidene
malononitrile, were >0.2, >0.2, 49±2.3 and 4±0.2
per mm2, respectively. 4-Nitrobenzylidene malononitrile also reduced
hepatotoxicity induced by anti-Fas antibody. Caspase-3 activation
induced by either tumor necrosis factor-/d-glactosamine
or by anti-Fas treatment, was reduced by pretreatment with N-nitrobenzylidene
malononitrile Conclusions: The findings may provide a base for
development of a new therapeutic modality to reduce apoptosis-mediated
liver damage.
Liver Growth and Cancer
Inhibition of urokinase-type plasminogen activator delays
expression of c-jun, activated transforming growth factor 1, and
matrix metalloproteinase 2 during post-hepatectomy liver regeneration
in mice
Kazuhiko Nomura et al.
Background/Aims: Although urokinase-type
plasminogen activator (u-PA) is suggested to initiate various
factors in liver regeneration after hepatectomy, no corroborative
evidence has been reported. In the present study, we investigated
the effect of u-PA on liver regeneration after hepatectomy. Methods:
Mice were placed into either a control group or a u-PA-inhibited
group that received an in vivo u-PA inhibitor, p-aminobenzamidine.
After we had removed two-thirds of the liver, we examined the
expressions of c-jun mRNA and activated transforming growth factor
1 (TGF-1), matrix metalloproteinase-2 (MMP-2) activity, and the
level of hepatocyte and non-parenchymal cell proliferation in
the two groups. Results: In the u-PA-inhibited group,
the delays in c-jun mRNA expression, hepatocyte proliferation,
activated TGF-1 expression, and expression of MMP-2 activity,
were 2h, 1, 2, and 1 day, respectively, and the sinusoid architecture
was not restored by 10 days after hepatectomy. Conclusions:
u-PA inhibition delays the expression of c-jun mRNA, hepatocyte
proliferation, and restoration of the sinusoid architecture, suggesting
that u-PA plays important roles in liver regeneration after hepatectomy
through control of a transcription factor, c-jun expression.
Liver Growth and Cancer
Steatosis is not sufficient to cause an impaired regenerative
response after partial hepatectomy in rats
Christian Picard et al.
Background/Aims: Fatty liver is known to be associated
with increased mortality and morbidity after liver resection.
The ability of fatty liver to regenerate after two-thirds partial
hepatectomy was studied in three different models of steatosis
in rats: obese Zucker rats, orotic acid-fed Wistar rats and Wistar
rats fed a methionine-low, choline-deficient diet. Methods:
Liver regeneration was assessed 24 h after partial hepatectomy
by bromodeoxyuridine incorporation (immunohistochemistry), proliferating
cell nuclear antigen, cyclin E and cyclin-dependent kinase 2 protein
expression (Western blot analysis) and cyclin-dependent kinase
2 activity (kinase assays using histone H1 as a substrate). Results:
No significant difference of proliferative response was found
between orotic acid or methionine-low, choline-deficient diet-fed
and control Wistar rats 24 h after partial hepatectomy. In contrast,
hepatocyte proliferation in obese Zucker rats after partial hepatectomy
was significantly reduced when compared with their lean controls.
Conclusions: Steatosis per se does not impair liver
regeneration. The reduced liver regeneration observed in obese
Zucker rats may not be due to fatty infiltration itself but to
other factors such as leptin receptor dysfunction.
Contribution of bone marrow cells to liver regeneration after
partial hepatectomy in mice
Hideaki Fujii et al.
Background/Aims: We examined whether bone marrow
(BM) cells can commit to liver-consisting cells during liver regeneration
after partial hepatectomy, using mice transplanted with green
fluorescent protein (GFP) positive BM from GFP transgenic mice.
Methods: Partial hepatectomy or sham operation was
performed. Lineage marker analysis of GFP positive liver cells
was by immunostaining and flow cytometry. DiI-labeled acetylated
low-density lipoprotein uptake or microsphere phagocytosis was
examined in vitro. Lineage marker expression in BM and peripheral
blood (PB) cells, and the vascular endothelial growth factor (VEGF)
concentration in the liver were also examined. Results:
In hepatectomized mice, significantly more GFP positive cells
participated in liver sinusoid than in sham-operated mice, expressing
CD31 but not albumin. The percentage of cells that incorporated
acetylated low-density lipoprotein but not microspheres was 69.5±3.4%,
while 28.3±2.6% incorporated both, revealing sinusoidal
endothelial and Kupffer cells, respectively. Increased expression
of the CD31 and CD16/CD32 on GFP positive liver cells was also
detected. The elevation of the VEGF concentration during liver
regeneration and the increase in the CD34 and Flk-1 expression
in the liver, BM, and PB cells suggested endothelial progenitor
cell mobilization. Conclusions: GFP cell-marking
provided direct evidence of the BM cells participation in liver
regeneration after hepatectomy, where the majority was committed
to sinusoidal endothelial cells probably through endothelial progenitor
cell mobilization.
Acyclic retinoid induces partial differentiation, down-regulates
telomerase reverse transcriptase mRNA expression and telomerase
activity, and induces apoptosis in human hepatoma-derived cell
lines
Ichiro Yasuda et al.
Background/Aims: Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic
acid) prevented hepatocarcinogenesis in animal models and in a
randomized clinical trial by eradicating premalignant and latent
malignant clones of transformed cells from the liver. We investigated
the possible mechanism of this clonal deletion at the cellular
level. Methods: Human hepatoma-derived cell lines,
PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion
of albumin and that of lectin-reactive isoform of -fetoprotein
(AFP-L3) were measured as markers of differentiation and dedifferentiation
of the cells, respectively. Telomerase reverse transcriptase (TERT)
mRNA expression and telomerase activity were measured by reverse
transcriptase polymerase chain reaction (RT-PCR) and stretch PCR
assay, respectively. Caspase activities were measured by colorimetric
protease assay. Mitochondrial membrane permeability transition
was examined by Rhodamine staining. Results: Production
of albumin was recovered while that of AFP-L3 was reduced after
exposure of the cells to 10µM AR for 2 days. This differentiation
was maintained for another 2 days without retinoid. In parallel,
both TERT mRNA expression and telomerase activity were down-regulated.
The cells subsequently died due to apoptosis after 4-6 experimental
days. Serial increases in mitochondrial membrane permeability
and caspase-9 and -3 activities induced apoptosis. Conclusions:
AR first induces differentiation and reduces telomerase activity.
Subsequent apoptosis may contribute to the eradication of the
clone.
Viral Hepatitis
Daily or three times a week interferon alfa-2b in combination
with ribavirin or interferon alone for the treatment of patients
with chronic hepatitis C
Victor de Lédinghen et al.
Background/Aims: Data on hepatitis C virus (HCV)
viral dynamics and on the effect of interferon in blocking virion
production have suggested a rationale for daily administration
of interferon in patients with chronic hepatitis C infection.
We compared the efficacy and safety of daily interferon alfa-2b
in combination with ribavirin with those of interferon alfa-2b
three times a week alone or in combination with ribavirin. Methods:
We randomly assigned 321 patients with chronic hepatitis C to
receive standard-dose interferon alfa-2b alone or in combination
with ribavirin for 48 weeks or daily interferon alfa-2b (3 million
units per day for 12 weeks then 3 million units three times per
week for 24 weeks) and ribavirin (36 week treatment). Results:
The rate of sustained virologic response (defined as an undetectable
serum HCV-RNA level 72 weeks after initiation of treatment) was
higher in patients who received combination therapy with three
times weekly interferon (51.7%) or daily interferon (46.1%) than
in patients who received interferon alone (25%) (P=0.0001
and P=0.002, respectively). Independent predictive
factors for sustained virologic response were combination therapy,
weight, genotype and viral load. In conclusion, in patients with
chronic hepatitis C, combination therapy with induction treatment
(daily interferon for 12 weeks) and shorter duration of treatment
was not different from combination therapy for 48 weeks without
induction treatment. Conclusions: Induction treatment
with interferon for 12 weeks and combination therapy for a total
duration of 36 weeks could therefore be cost effective.
The evolution of hepatitis B virus serological patterns and
the clinical relevance of isolated antibodies to hepatitis B core
antigen in HIV infected patients
Lionel Piroth et al.
Background/Aims: The evolution of hepatitis B virus
(HBV) serological patterns and the clinical relevance of isolated
anti-HBc pattern are not well established in HIV infected patients.
Methods: A cohort of 240 patients was followed for
6.9±3.4 years, with iterative HBV serologic assays performed
(mean interval of 2.2 years). Results: Five patients
without HBV markers at baseline subsequently developed positive
anti-HBs (incidence 0.66/100 patient-year), as did two patients
with chronic HBs antigenemia (incidence 1.66/100 patient-year).
Only one patient with isolated anti-HBc pattern developed HBs
chronic antigenemia. Persistent isolated anti-HBc pattern was
observed in 37 patients (13 with detectable blood HBV DNA) and
was strongly associated with positive hepatitis C virus (HCV)
viremia (hazard ratio=9.5, confidence interval 95%: 4.5-20.0,
P<0.0001). Hepatic lesions were more severe in
HCV infected patients with persistent isolated anti-HBc pattern
than in those without (Knodell score 9.2±4.6 versus 6.7±5.0,
P=0.04). In time updated analysis, this pattern
was not associated with an increased risk of hepatotoxicity, by
contrast with HCV infection or positive HBs antigenemia. Conclusions:
In HIV infected patients, HBV serological status must be systematically
and regularly assessed, and systematic HBV vaccination must be
proposed in those without HBV marker. Isolated anti-HBc pattern
must be considered in the management of hepatitis C, but not for
antiretroviral therapy.
Accelerated hepatic fibrosis in patients with combined hereditary
hemochromatosis and chronic hepatitis C infection
Hari H. Diwakaran, Alex S. Befeler, Robert S. Britton, Elizabeth
M. Brunt and Bruce R. Bacon
Background/Aims: Hereditary hemochromatosis (HH)
and chronic hepatitis C virus (HCV) infection can both result
in hepatic fibrosis and cirrhosis. It has been proposed that iron
overload and HCV may have potentiating effects on hepatic fibrogenesis.
This study determined if HH patients with HCV would present with
hepatic fibrosis/cirrhosis at a younger age and at a lower hepatic
iron concentration compared to patients with HH or HCV alone.
Methods: Ten patients with combined HCV and HH were
compared to 13 patients who had HH alone and 24 patients who had
HCV alone. All patients had advanced fibrosis/cirrhosis on liver
biopsy. All HH patients were homozygous for the C282Y mutation.
Results: At presentation with advanced fibrosis/cirrhosis,
the mean age of the HH/HCV group was significantly lower than
that of the HH group and the HCV group. The mean hepatic iron
concentration was lower in the combined HH/HCV group compared
to that of the HH group. Conclusions: HH patients
with HCV present with advanced fibrosis/cirrhosis at a younger
age and at a lower hepatic iron concentration compared to HH patients
without HCV. These findings support the concept that the combination
of HH-induced iron overload and HCV has a potentiating effect
on hepatic fibrogenesis.
Case Report
Progressive reversion of clinical and molecular phenotype
in a child with liver mitochondrial DNA depletion
Pierre-Henri Ducluzeau et al.
Mitochondrial DNA depletion is a well established cause of severe
liver failure in infancy. The autosomal inheritance of this quantitative
mitochondrial DNA defect supports the involvement of a nuclear
gene in the control of mitochondrial DNA level. We previously
described a case of a 28-month-old child presenting with a progressive
liver fibrosis due to a mitochondrial DNA depletion (85% at 12
months of age). As this syndrome was clinically liver-restricted,
a liver transplant was initially discussed. We report the clinical,
biochemical and molecular follow-up of this child, now 6 years
old. The patient displayed a spontaneous gradual improvement of
his liver function with continuous increment of clotting factor
values since 32 months of age. A marked reduction of the previous
extensive fibrosis was evidenced on a liver biopsy performed at
46 months of age associated with a dramatic decrease of the mitochondrial
DNA depletion (35%). Consequently, an almost complete restoration
of respiratory chain activities containing mitochondrial DNA-encoded
subunits was observed. This is the first report of a revertant
phenotype in liver mitochondrial DNA depletion syndrome.
RAS
Volume 346:1706-1713 May 30, 2002 Number 22
Clinical Trial of Lamivudine in Children with Chronic Hepatitis
B
Maureen M. Jonas, M.D., Deirdre A. Kelley, M.D., Jacek
Mizerski, M.D., Isabel B. Badia, M.D., Jorge A. Areias, M.D.,
Kathleen B. Schwarz, M.D., Nancy R. Little, B.S., Martin J. Greensmith,
Ph.D., Stephen D. Gardner, M.S.P.H., M. Steve Bell, B.Sc., Etienne
M. Sokal, M.D., for the International Pediatric Lamivudine Investigator
Group
Background : Lamivudine therapy is effective for chronic hepatitis B infection in adults. We evaluated the efficacy and tolerability of lamivudine as a treatment for chronic infection with hepatitis B virus (HBV) in children. Methods Children with chronic hepatitis B were randomly assigned in a 2:1 ratio to receive either oral lamivudine (3 mg per kilogram of body weight; maximum, 100 mg) or placebo once daily for 52 weeks. The primary end point was virologic response (defined by the absence of serum hepatitis B e antigen and serum HBV DNA) at week 52 of treatment. Results : Of the 403 children screened, 191 were randomly assigned to receive lamivudine and 97 to receive placebo. The rate of virologic response at week 52 was higher among children who received lamivudine than among those who received placebo (23 percent vs. 13 percent, P=0.04). Lamivudine therapy was well tolerated and was also associated with higher rates of seroconversion from hepatitis B e antigen to hepatitis B e antibody, normalization of alanine aminotransferase levels, and suppression of HBV DNA. Conclusions : In children with chronic hepatitis B, 52 weeks of treatment with lamivudine was associated with a significantly higher rate of virologic response than was placebo.
Maintenance infliximab for Crohn's disease: the ACCENT I
randomised trial
Stephen B Hanauer, Brian G Feagan, Gary R Lichtenstein, Lloyd
F Mayer, S Schreiber, Jean Frederic Colombel, Daniel Rachmilewitz,
Douglas C Wolf, Allan Olson, Weihang Bao, Paul Rutgeerts, and
the ACCENT I Study Group*
Summary :
Background We did a randomised controlled trial to assess the benefit of maintenance infliximab therapy in patients with active Crohn's disease who respond to a single infusion of infliximab. Methods 573 patients with a score of at least 220 on the Crohn's disease activity index (CDAI) received a 5 mg/kg intravenous infusion of infliximab at week 0. After assessment of response at week 2, patients were randomly assigned repeat infusions of placebo at weeks 2 and 6 and then every 8 weeks thereafter until week 46 (group I), repeat infusions of 5 mg/kg infliximab at the same timepoints (group II), or 5 mg/kg infliximab at weeks 2 and 6 followed by 10 mg/kg (group III). The prespecified co-primary endpoints were the proportion of patients who responded at week 2 and were in remission (CDAI <150) at week 30 and the time to loss of response up to week 54 in patients who responded. Analyses of the co-primary endpoints were by intention to treat. Findings 335 (58%) patients responded to a single infusion of infliximab within 2 weeks. At week 30, 23 of 110 (21%) group I patients were in remission, compared with 44 of 113 (39%) group II (p=0·003) and 50 of 112 (45%) group III (p=0·0002) patients. Thus, patients in groups II and III combined were more likely to sustain clinical remission than patients in group I (odds ratio 2·7, 95% CI 1·6-4·6). Throughout the 54-week trial, the median time to loss of response was 38 weeks (IQR 15 to >54) and more than 54 weeks (21 to >54) for groups II and III, respectively, compared with 19 weeks (10-45) for group I (p=0·002 and p=0·0002, respectively). Infliximab safety was consistent with that seen in other trials of infliximab in Crohn's disease and rheumatoid arthritis. In particular, the incidence of serious infections was similar across treatment groups. Interpretation Patients with Crohn's disease who respond to an initial dose of infliximab are more likely to be in remission at weeks 30 and 54, to discontinue corticosteroids, and to maintain their response for a longer period of time, if infliximab treatment is maintained every 8 weeks. Lancet 2002; 359: 1541-49
Comparison of survival, palliation, and quality of life
with three chemotherapy regimens in metastatic colorectal cancer:
a multicentre randomised trial
T S Maughan, R D James, D J Kerr, J A Ledermann, C McArdle,
M T Seymour, D Cohen, P Hopwood, C Johnston, R J Stephens, for
the British MRC Colorectal Cancer Working Party*
Summary
Background This randomised trial compared three chemotherapy regimens in the first-line treatment of advanced colorectal cancer, in terms of their effect on overall and progression-free survival; other endpoints included toxicity, symptom palliation, and quality of life. Methods 905 patients were randomly assigned the de Gramont regimen (n=303; folinic acid 200 mg/m2, fluorouracil bolus 400 mg/m2, and infusion 600 mg/m2 on days 1 and 2, repeated every 14 days), the Lokich regimen (n=301; protracted venous infusion of fluorouracil 300 mg/m2 daily), or raltitrexed (n=301; 3 mg/m2 intravenously every 21 days). Analyses were by intention to treat. Findings Median follow-up of survivors was 67 weeks. For the de Gramont, Lokich, and raltitrexed groups, respectively, median survival was 294, 302, and 266 days. The hazard ratios for overall survival were 0·88 (95% CI 0·70-1·12, p=0·17) for de Gramont versus Lokich, and 0·99 (0·79-1·25, p=0·94) for de Gramont versus raltitrexed. An increase in treatment-related deaths was seen on raltitrexed (de Gramont one, Lokich two, raltitrexed 18) due to combined gastrointestinal and haematological toxicity. Patients' assessment of quality of life showed that raltitrexed was inferior to the fluorouracil-based regimens, especially in terms of palliation and functioning. Interpretation The deGramont and Lokich regimens were similar in terms of survival, quality of life, and response rates. The Lokich regimen was associated with more central line complications and hand-foot syndrome. Raltitrexed showed similar response rates and overall survival to the de Gramont regimen and was easier to administer, but resulted in greater toxicity and inferior quality of life. Lancet 2002: 359: 1555-63
New treatment for inflammatory bowel disease could soon
enter clinical trials
Jane Bradbury
Immunostimulatory bacterial DNA sequences could provide a new treatment for inflammatory bowel disease (IBD), an international team of researchers suggests this week. Daniel Rachmilewitz (Shaare Zedek Medical Center, Jerusalem, Israel) and colleagues report that in three experimental and one spontaneous mouse model of colitis, administration of immunostimulatory bacterial DNA reduced colonic inflammation. In addition, the same DNAs inhibited in-vitro release of proinflammatory mediators from biopsy material from patients with either Crohn's disease or ulcerative colitis (Gastroenterology 2002; 122: 1428-41). "I think these data justify a speedy move into clinical studies", says senior researcher Eyal Raz (University of California, San Diego, CA, USA), adding that his Israeli colleagues are actively planning such trials. IBD comprises two related inflammatory disorders of the intestinal tract--Crohn's disease and ulcerative colitis. The exact cause of IBD remains unclear, but susceptibility genes and environmental factors have been implicated in its aetiology. Progress in the overall management of IBD is being made, but to date few innovative treatments have been described. In their search for such a new treatment, the researchers investigated the effects of small synthetic DNAs containing CpG motifs from bacterial genomes previously shown to have an immunostimulatory effect. "It was somewhat counterintuitive to try these molecules --ISS-ODNs [immunostimulatory oligonucleotides]--but we discovered that they had a strong anti-inflammatory effect in animal models for IBD", explains Raz. ISS-ODN was effective when injected or given orally, but a single dose did not provide long-term protection against colitis. "In clinical terms, this might mean that patients would need to take the drug orally every week or two", says Raz. "This is interesting work", comments Sander van Deventer (Academic Medical Center, Amsterdam, Netherlands), "and DNAs of this type have been used as immunostimulators in several diseases. But", he cautions, "there remain many unknowns in relation to the use of ISS-ODNs in IBD. For example, it is unclear which cells in the intestinal mucosa are stimulated by administration of ISS-ODNs." Raz agrees that it is unclear how immunostimulatory DNAs work in IBD but suggests that they could be both stimulating innate immunity and helping to protect the mucosal barrier. "ISS-ODNs could limit the invasion of commensal bacteria and/or their inflammatory products into the colonic mucosa and thus reduce mucosal inflammation. ISS-ODNs", he adds, "might actually work in an equivalent way to probiotics, which have been shown to be effective against colitis in clinical trials and could have the potential for providing a more controlled therapy for IBD".
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