ISSN 1007-9327

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World Journal of Gastroenterology

World Journal of
Gastroenterology

Volume 12 Number 22
June 14, 2006

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2005-2006

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Number 22 Gastroenterology
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Indexed and Abstracted in: Volume 12 Number 22
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2006 June 14; 12(22): 3461-3624
2006

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ISSN 1007-9327 CN 14-1219/R Local Post Offices Code No. 82-261 A Weekly Journal of Gastroenterology and Hepatology
 World Journal of
Gastroenterology

National Journal Award Volume 12 Number 22 Supported by NSFC

2005 2005-2006
June 14, 2006
Contents

EDITORIAL 3461 Role of microbubble ultrasound contrast agents in the non-invasive

assessment of chronic hepatitis C-related liver disease
Grier S, Lim AKP, Patel N, Cobbold JFL, Thomas HC, Cox IJ, Taylor-Robinson SD

TOPIC HIGHLIGHT 3466 Calcium signaling in cholangiocytes

Minagawa N, Ehrlich BE, Nathanson MH

3471 Nervous and Neuroendocrine regulation of the pathophysiology of cholestasis

and of biliary carcinogenesis
Marzioni M, Fava G, Benedetti A

3481 Bioinformatic approach for understanding the heterogeneity of cholangiocytes

Fukushima K, Ueno Y

3487 Ursodeoxycholic acid treatment of vanishing bile duct syndromes

Pusl T, Beuers U

3496 Cholangiocyte anion exchange and biliary bicarbonate excretion

Banales JM, Prieto J, Medina JF

3512 Biliary wound healing, ductular reactions, and IL-6/gp130 signaling in the
development of liver disease
Demetris AJ, Lunz JG , Specht S, Nozaki I

3523 Heterogeneity of the intrahepatic biliary epithelium

Glaser S, Francis H, DeMorrow S, LeSage G, Fava G, Marzioni M, Venter J, Alpini G

3537 Estrogens and the pathophysiology of the biliary tree

Alvaro D, Mancino MG, Onori P, Franchitto A, Alpini G, Francis H, Glaser S, Gaudio E

3546 Cholangiocytes and blood supply

Gaudio E, Franchitto A, Pannarale L, Carpino G, Alpini G, Francis H, Glaser S, Alvaro D,
Onori P

3553 Bile acid interactions with cholangiocytes

Xia X, Francis H, Glaser S, Alpini G, LeSage G

REVIEW 3564 Synchronous and metachronous occurrence of gastric adenocarcinoma and

gastric lymphoma: A review of the literature
Hamaloglu E, Topaloglu S, Ozdemir A, Ozenc A

LIVER CANCER 3575 Hepatocellular carcinoma in Lebanon: Etiology and prognostic factors
associated with short-term surviva
Yaghi C, Sharara AI, Rassam P, Moucari R, Honein K, BouJaoude J, Slim R, Noun R,
Abdul-Baki H, Khalifeh M, Ramia S, Sayegh R

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 World Journal of Gastroenterology
Contents Volume 12 Number 22 June 14, 2006

COLORECTAL CANCER 3581 Apoptosis of human colon carcinoma HT-29 cells induced by ceramide
Zhang XF, Li BX, Dong CY, Ren R

BASIC RESEARCH 3585 Gene expression profiling defined pathways correlated with fibroblast cell
proliferation induced by Opisthorchis viverrini excretory/secretory product
Thuwajit C, Thuwajit P, Uchida K, Daorueang D, Kaewkes S, Wongkham S, Miwa M

CLINICAL RESEARCH 3593 Effect of abdominal trauma on hemorrhagic shock-induced acute lung injury
in rats
Kilicoglu B, Eroglu E, Kilicoglu SS, Kismet K, Eroglu F

RAPID COMMUNICATION 3597 The vital threat of an upper gastrointestinal bleeding: Risk factor analysis of
121 consecutive patients
Schemmer P, Decker F, Dei-Anane G, Henschel V, Buhl K, Herfarth C, Riedl S

3602 Roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of

immunoproliferative small intestinal disease
Vaiphei K, Kumari N, Sinha SK, Dutta U, Negi B, Joshi K, Singh K

3609 Expression of E-selectin, integrin 1 and immunoglobulin superfamily member

in human gastric carcinoma cells and its clinicopathologic significance
Ke JJ, Shao QS, Ling ZQ

CASE REPORTS 3612 Peliosis hepatis as an early histological finding in idiopathic portal
hypertension: A case report
Berzigotti A, Magalotti D, Zappoli P, Rossi C, Callea F, Zoli M

LETTERS TO THE EDITOR 3616 Noni juice is not hepatotoxic

West BJ, Jensen CJ, Westendorf J

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 World Journal of Gastroenterology
Contents Volume 12 Number 22 June 14, 2006

ACKNOWLEDGMENTS 3620 Acknowledgments to Reviewers of World Journal of Gastroenterology

APPENDIX 3621 Meetings

3622 Instructions to authors

FLYLEAF I-V Editorial Board

INSIDE FRONT COVER Online Submissions

INSIDE BACK COVER International Subscription

COPY EDITOR FOR THIS ISSUE: Yogesh K Chawla and Atif Iqbal, MD

World Journal of Gastroenterology ( World J Gastroenterol , WJG ), a leading international journal in gastroenterology and hepatology, has an established reputation
for publishing first class research on esophageal cancer, gastric cancer, liver cancer, viral hepatitis, colorectal cancer, and Helicobacter pylori infection, providing
a forum for both clinicians and scientists, and has been indexed and abstracted in Index Medicus, MEDLINE, PubMed, Chemical Abstracts, EMBASE,
Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. WJG is a weekly journal published by The
WJG Press. The publication date is on 7th, 14th, 21st, and 28th every month. The WJG is supported by The National Natural Science Foundation of China, No.
30224801 and No.30424812, which was founded with a name of China National Journal of New Gastroenterology on October 1,1995, and renamed as WJG on
January 25, 1998.

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EDITORIAL
Role of microbubble ultrasound contrast agents in the
non-invasive assessment of chronic hepatitis C-related
liver disease
Scott Grier, Adrian KP Lim, Nayna Patel, Jeremy FL Cobbold, Howard C Thomas, Isobel J Cox, Simon D Taylor-Robinson
Scott Grier, Nayna Patel, Jeremy FL Cobbold, Howard C
Thomas, Simon D Taylor-Robinson, Liver Unit, St Marys
Hospital Campus, Faculty of Medicine, Imperial College London,
London, United Kingdom
Adrian KP Lim, Isobel J Cox, Department of Imaging Sciences,
Hammersmith Hospital Campus, Faculty of Medicine, Imperial
College London, London, United Kingdom
Supported by the United Kingdom Department of Health, British
Medical Research Council, Grant No. G99000178 and the United
Kingdom National Health Service Research and Development
Initiative
Correspondence to: Dr. Simon Taylor-Robinson, Liver Unit,
Department of Medicine A, Imperial College London, 10th Floor,
QEQM Building, St Marys Hospital Campus, South Wharf Road,
London W2 1NY, United Kingdom. s.taylor-robinson@imperial.ac.uk
Telephone: +44-207-8866454 Fax: +44-207-7249369
Received: 2006-01-27 Accepted: 2006-02-18
Abstract
Patients who are chronically infected with the hepatitis
C virus often develop chronic liver disease and assess-
ment of the severity of liver injury is required prior to
considering viral eradication therapy. This article exam-
ines the various assessment methods currently available
from gold standard liver biopsy to serological markers
and imaging. Ultrasound is one of the most widely used
imaging modalities in clinical practice and is already a
first-line diagnostic tool for liver disease. Microbubble ul-
trasound contrast agents allow higher resolution images
to be obtained and functional assessments of microvas-
cular change to be carried out. The role of these agents
in quantifying the state of hepatic injury is discussed as
a viable method of determining the stage and grade of
liver disease in patients with hepatitis C. Although cur-
rently confined to specialist centres, the availability of
microbubble contrast-enhanced ultrasound will inevitably
increase in the clinical setting.
2006 The WJG Press. All rights reserved.
Key words: Hepatitis C; Microbubble contrast agents;
Ultrasound
Grier S, Lim AKP, Patel N, Cobbold JFL, Thomas HC, Cox IJ,
Taylor-Robinson SD. Role of microbubble ultrasound contrast
agents in the non-invasive assessment of chronic hepatitis
C-related liver disease. World J Gastroenterol 2006; 12(22):
3461-3465
World J Gastroenterol 2006 June 14; 12(22): 3461-3465
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
http://www.wjgnet.com/1007-9327/12/3461.asp
INTRODUCTION
It is estimated that 170 to 200 million people (around 3%
of the global population) are chronically infected with the
hepatitis C virus (HCV), while around 4 million people are
newly infected each year[1]. There is a great degree of vari-
ation in prevalence, ranging from less than 1% in the Unit-
ed Kingdom to more than 20% in Egypt. In the developed
world, the relatively low prevalence is mostly the result of
intravenous drug abuse, while a World Health Organisa-
tion (WHO) anti-schistosomal vaccination programme
in Egypt in the 1970s and other vaccination programmes
in Mongolia and Bolivia have led to much higher rates in
those countries[2]. Between 55% and 85% of those infect-
ed go on to develop chronic liver disease and even then,
many remain asymptomatic, while the rest suffer primarily
from chronic fatigue[3]. Around 20% of patients with fi-
brosis develop cirrhosis and 5% of these are susceptible to
developing hepatocellular cancer each year (Figure 1)[4]. A
number of factors have been linked to the progression of
chronic hepatitis, with some being implicated not only in
advancing the progression, but also worsening the overall
prognosis[5]. These factors include age at infection greater
than 55 years, male sex, excessive alcohol consumption, a
long history of cigarette smoking and the presence of he-
patic steatosis, obesity and diabetes mellitus (the metabolic
syndrome)[5].
ASSESSING CHRONIC HEPATITIS C-RE-
LATED LIVER DISEASE
An assessment of the severity of hepatic involvement is
obviously required prior to considering antiviral treatment
regimens with interferon and ribavirin in patients with
hepatitis C. In addition, owing to the variable disease pro-
gression, the potentially life-threatening complications of
end-stage liver disease and the development of hepatocel-
lular carcinoma, it is also imperative to monitor hepatitis
C-infected patients with established cirrhosis.
LIVER BIOPSY
For many years, the gold standard for assessing chronic
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 3462 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Table 1 Commonly available serological markers for assessing
[8]
liver injury
Group Marker/panel Comments
1 Prothrombin index Highest diagnostic accuracy (cirrhosis)
86%
1 PGAA Diagnostic accuracy 80%
1 AST/ALT ratio Increase strongly correlated with decrease
in hepatic function
AST/ALT > 1 can diagnose cirrhosis with
sensitivity of 77.8% and specificity 96.9%,
but misclassified 7% as having cirrhosis
and failed to diagnose 11%
1 FibroTest Sensitivity 75%, specificity 85%
1 APRI Predicts fibrosis in 51% patients
Predicts cirrhosis in 81% patients
2 Hyaluronic acid (HA) Only group 2 marker to show encouraging
results, with a positive predictive value
similar to Child-Pugh
hepatitis has been histological examination of liver biopsy
material. However, this invasive procedure is not without
risks with a small, but defined mortality rate and it has a
significant error rate, sampling less than 1/50000th of the
liver volume even in cases of diffuse disease[6-8]. As a result
of the problems associated with biopsy, there has been
a steady drive in recent times to find an effective non-
invasive method of evaluating liver injury, which has led to
major developments in both serological markers of disease
and imaging.
SEROLOGICAL MARKERS
An ideal serological marker for assessing liver injury
should be liver-specific, easy to use and allow measure-
ment of the stage of disease using the degree of fibrosis,
the activity of matrix deposition and the activity of matrix
removal[8]. These can be broadly classified into two groups.
The first (Group 1 in Table 1) are those which are indirect
markers of fibrosis, reflecting alterations in hepatic func-
tion, and include PGAA {prothrombin index, gamma
glutamyl transpeptidase ( GT), apolipoprotein A 1 and
2-macroglobulin}[8], AST/ALT (aspartate aminotrans-
ferase and alanine aminotransferase) [9], FibroTest ( 2-
macroglobulin, 2-globulin, -globulin, apolipoprotein A1,
[10]
GT and total bilirubin) and APRI (AST to platelet ratio
[11]
index) . The second (Group 2 in Table 1) are those which
are direct markers of fibrosis, reflecting extracellular matrix
metabolism (Group 2 in Table 1)[8]. The current issue is
that many of these proposed panels have not been widely
validated around the world and often remain championed
largely by the research groups that initially proposed them.
Currently, none fulfils all the criteria for an ideal serological
marker and none has yet achieved the levels of sensitivity
and specificity seen with liver biopsy.
IMAGING
Of all the proposed non-invasive alternatives to liver biop-
sy, imaging has shown some promise. Using routine grey-
scale ultrasound, computed tomography (CT) or magnetic
resonance imaging (MRI), it has been shown that a small
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June 14, 2006 Volume 12 Number 22
Minimal Moderate/severe Cirrhosis Hepatocellular
hepatitis hepatitis (5-25 yr) (15-35 yr) cancer
(1-15 yr)
Asymptomatic/ Asymptomatic until
Asymptomatic symptomatic decompensation:
Ascites
Bleeding varices
Hepatic encephalopathy
Poor synthetic function
Poor metabolic function
Figure 1 Time scale for progression of hepatitis C-related liver disease[3,4].
right hepatic lobe, with relatively enlarged left and caudate
lobes, are reliable markers of cirrhosis, having a sensitivity
of 84%, specificity of 100% and diagnostic accuracy of
94%. These figures are well beyond any achieved by the
many serological markers[8].
ULTRASOUND IMAGING OF THE LIVER
Ultrasound is one of the most widely used imaging mo-
dalities in clinical practice. The major attraction is that it
is already the first line of imaging in the diagnosis of liver
disease and is of particular interest, as it has been shown
to be complementary in patients with equivocal biopsy
results or where biopsy is contraindicated, but cirrhosis is
suspected[8]. It is also used to screen patients with chronic
hepatitis for hepatocellular cancer, enabling detection and
characterisation of tumors[12]. In patients with cirrhosis,
it is possible to demonstrate nodularity of the liver sur-
face and parenchyma and sometimes atrophy of the right
lobe[13], but for the most part, standard grey-scale imaging
does not render much information on the extent of fibro-
sis in pre-cirrhotic disease. The most accurate diagnoses
which can be made on grey-scale imaging are those of
hepatic steatosis, demonstrated by parenchymal hyper-
echogenicity (Figure 2), and the presence of a nodular
surface in cirrhosis (Figure 3). The need to discriminate
pre-cirrhotic liver disease non-invasively has driven the de-
velopment of additional ultrasound techniques, including
elastography and a group of microbubble ultrasound con-
trast agents, in order to achieve better resolution images
and allow a degree of hepatic functional assessment.
Transient elastography
Transient elastography has recently been used to charac-
terize the stiffness of liver tissue in vivo, which has been
correlated with the histological grade of fibrosis in hepa-
titis C[14]. A probe vibrating at a predetermined amplitude
and frequency is placed against skin overlying the liver.
The resultant shear wave is propagated through tissue at
a velocity dependent on its stiffness. The velocity can be
measured by one-dimensional ultrasound along the axis of
the transmitted wave. Although correlation is good over-
all, there is significant overlap of stiffness as measured by
elastography between the different stages of fibrosis. Elas-
tography has been used in conjunction with the serological
markers, FibroTest (Table 1 for details) and the aspartate
aminotransferase (AST) to platelet ratio (APRI) to evaluate
 Grier S et al. Microbubble ultrasound in hepatitis C 3463
RLL
Figure 2 Ultrasound image of hepatic steatosis, showing increased echotexture
of the liver parenchyma in comparison to the right kidney.
liver fibrosis. There was correlation with histology, but sig-
nificant overlap of elastography values between stages of
fibrosis[15,16]. Further larger scale studies are required using
this methodology.
Ultrasound contrast imaging using microbubbles
In the late 1960s, a group of echocardiographers discov-
ered, quite by accident, that the microscopic bubbles of air
which enter the circulation during intravenous injections
produce strong echoes, where normally none is seen[17,18].
This discovery led to the development of the first type of
microbubble contrast agents for ultrasound, which became
available in the mid-1990s. Microbubbles are tiny, gas-
filled bubbles with a shell (made from denatured albumin,
phospholipids, surfactant or cyanoacrylate) which measure
less than 10 m in diameter and are small enough to pass
through the cardiopulmonary circulation and also cross
capillary beds[19].
In simple terms, microbubbles are echo-enhancers,
which enhance both grey-scale and Doppler ultrasound
signals by up to 25 dB, giving an increase of more than 300
folds[20]. The first microbubble agent to be licensed for use
in the United Kingdom was Levovist in 1997. Although
it exhibited a degree of liver specificity, it had a short half-
life and the bubbles were mostly destroyed by liver, mean-
ing that only one pass through the liver was possible. The
introduction of SonoVue, a third-generation microbubble
agent, in 2001 allowed real-time contrast-enhanced imag-
ing and multiple passes through the liver.
Safety of microbubbles
As with all pharmaceutical agents, microbubble agents
have to pass rigorous safety tests in order for them to be
licensed. Extensive trials have established an excellent
overall safety record with few significant adverse effects[20].
However, there are some concerns that disruption of their
outer shells produces local bioeffects such as sonoporation
(subcellular membrane damage) and cell lysis at diagnostic
frequencies and that these effects are enhanced by per-
fluorocarbon gases. Despite this, no effects of these proc-
esses have been observed in humans[20]. Concern about ad-
ministering volumes of gas into the blood stream has been
shown not to be an issue as the amount given is under 200
[21]
L, too small to exhibit an effect . Overall, it has been
Figure 3 Ultrasound image of a typical cirrhotic liver with a shrunken right lobe,
a nodular surface (white arrow), surrounding ascites and a heterogeneous
echotexture.
shown that the safety of microbubbles compares favour-
ably to that of conventional radiographic contrast agents
and those used in contrast-enhanced MRI[21].
Applications of microbubbles to the assessment of liver
disease
Microbubbles have a multitude of clinical uses, but are
best known for imaging the vasculature, contrast-enhanced
echocardiography, imaging the liver and functional studies.
In these functional studies, following an intravenous bolus
injection, their passage can be tracked through a tissue or
organ and used to generate transit time curves in much
the same way as those in nuclear medicine, CT and MRI
are produced[20].
One of the major applications of transit times is in the
assessment of liver disease and the non-invasive diagnosis
of cirrhosis[21]. Severe liver disease leads to characteristic
haemodynamic changes affecting hepatic blood flow. The
development of a hyperdynamic circulation (increased car-
diac output, reduced systemic vascular resistance, altered
circulating vasoactive factors-glucagons and prostagland-
ins), pulmonary arteriovenous shunts, venous collaterals
(due to increased portal pressure), intrahepatic shunts
(between hepatic artery, portal vein and hepatic veins), and
arterialisation of the liver capillary beds, results in the early
arrival of a bolus of microbubbles injected peripherally
(Figure 4). The hepatic vein transit time (HVTT) is the
difference between the hepatic artery and the hepatic vein
arrival times.
Many of the published studies[22-24] assessing HVTT in
patients with chronic liver disease have employed measure-
ments using the spectral Doppler technique, where the
arrival time is calculated as a rise in Doppler intensity 10%
above baseline. This requires specialized software to evalu-
ate the HVTT, but is relatively straight-forward, as shown
in Figure 5. The figure demonstrates a normal hepatic
vein transit time (43 s) in a patient without significant liver
disease (upper graph, Figure 5) and a comparative early
HVTT in a patient with cirrhosis (14 s) (lower graph, Fig-
ure 5). There is a much shorter HVTT in the patient with
cirrhosis. It is important to note that 20 s of baseline is
collected before the injection of the microbubble contrast
agent. The continuous horizontal line denotes a Doppler
intensity 10% above baseline and hence the HVTT is taken
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 3464 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

b 0.5

Doppler signal intensity

Figure 2
0.4 Normal (late HVTT)

0.3 Injection

Injection of
0.2
microbubble 1 0.1
a
0
2 2 -20 -10 0 10 20 30 40 50 60 70 80
d
t /s

0.5

Doppler signal intensity

Cirrhosis (early HVTT)
0.4
c
e 0.3 Injection

0.2
Figure 4 Characteristic haemodynamic changes seen in severe chronic liver 0.1
disease which lead to the early arrival of microbubbles in the liver. Time is
measured from injection (1) to arrival in the liver (2). 0
-20 -10 0 10 20 30 40 50 60 70 80
t /s

Figure 5 Hepatic vascular transit times (HVTT) following a bolus injection of

as the intersection of this line with that of the Doppler
intensity trace (Figure 5).
The development of newer more stable microbubbles,
such as SonoVue (Bracco, Milan, Italy) and improvement
in ultrasound capabilities have allowed real-time evalu-
ation of the transit times which can be readily visualized
arriving in the hepatic arteries and veins (Figures 5 and 6)
without the need for off-line processing.
A typical, simplified transit time assessment would now
a microbubble contrast agent, measured in a volunteer without significant liver
disease (upper graph) and a patient with cirrhosis (lower graph). There is a much
shorter HVTT in the patient with cirrhosis. Adapted from Lim et al[23].
50
40
Mean [HVTT] s +/- 2 s.e.

involve the injection of a bolus of the microbubble agent

into an antecubital fossa vein and measurement of the
time taken for the contrast agent to be seen in the liver. 30

The first flash of microbubbles seen in the hepatic arteries

is recorded as the hepatic artery arrival time. A continuous
stream of microbubbles in the hepatic vein is recorded as
the hepatic vein arrival time. The hepatic vascular transit
time (HVTT) is thus the difference between the hepatic
artery and the hepatic vein arrival times.
It has been shown that these transit times can be linked
to progression in the Child-Pugh score, becoming shorter
as the liver disease worsens[22]. Pre-cirrhotic disease due
to hepatitis C can now be classified into mild ( 28 s) or
moderate to severe ( 22 s, < 28 s) according to transit
times[20]. A recent study has shown that a transit time of
21 s is 100% sensitive and 96% specific for cirrhosis,
making it a viable alternative when liver biopsy is contrain-
dicated. It is also perhaps a method of monitoring disease
progression in response to treatment[23]. Figure 6 is adapted
from a recent study by Lim et al[23] which shows a progres-
sive shortening of the HVTT with increasing severity of
liver disease in a cohort of patients with HCV-related liver
disease. There is clear separation between the groups (cat-
egorisation of patients was carried out according to the
Ishak histological scoring system[25]). Importantly, there is a
clear separation, not only between the patients with mod-
erate hepatitis from those with cirrhosis, but also in the
pre-cirrhotic groups, where there is good separation of pa-
tients with mild hepatitis from those with moderate hepa-
titis[23]. This may be clinically relevant and demonstrates for
the first time the possibility that ultrasound techniques can
actually monitor the severity of pre-cirrhotic disease non-
invasively.
20
10
n = 20 20 30 28
Control Mild Moderate/severe Cirrhosis
Severity of hepatitis
Figure 6 Graph showing hepatic vascular transit times (HVTT) in patients with
mild hepatitis, moderate to severe hepatitis and established cirrhosis. There is
progressive shortening of the HVTT with increasing severity of liver disease.
Adapted from Lim et al[23].
Newer microbubble contrast agents
Levovist is at present the most commonly used micro-
bubble agent, but there are several newer agents, such as
SonoVue (Bracco, Milan, Italy) or Definity (Bristol-My-
ers-Squibb, New York, USA), which have different chemi-
cal properties to Levovist, but also provide enhanced
Doppler intensity when they arrive in the hepatic veins. It
remains to be shown whether the HVTT for these agents
are similar and whether they can be used interchangeably.
A benefit of using these newer agents is that their arrival
within a hepatic vein can be observed in real-time, using
low acoustic power two-dimensional harmonic scanning
modes. The main benefit of using these modes is that it
would not require computer-aided post-processing of the
data to calculate the HVTT, thus making it accessible to
any department with a modern ultrasound machine.

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 Grier S et al. Microbubble ultrasound in hepatitis C 3465
CONCLUSION
There are clearly a number of very different methods cur-
rently available for the monitoring of hepatitis C-related
liver disease. Although liver biopsy remains the gold stand-
ard, it is a highly invasive procedure that is not without risk
and is open to sampling error, particularly in disease where
there is patchy involvement. There is a continual drive
towards minimally or non-invasive testing, and therefore,
alternatives to liver biopsy are being actively sought.
The use of serological markers remains somewhat con-
fused, with prothrombin index having the highest diagnos-
tic accuracy, despite many other panels being suggested.
Further research by independent teams is required to un-
derstand exactly what role these markers can perform.
Developments in imaging technology in the past decade
have yielded sensitivities and specificities similar to that of
liver biopsy for the first time. Ultrasound continues to be
the primary imaging modality of choice when investigating
liver disease. Although currently limited to a few special-
ist centers, microbubble contrast agents will inevitably
become much more widely used. Hepatic vein transit time
measurement using an ultrasound microbubble agent is
a simple, non-invasive test which can be used to stage
and grade pre-cirrhotic liver disease with clear separation
between mild hepatitis and cirrhosis, and statistically sig-
nificant differences between these two groups and moder-
ate to severe hepatitis. The response to treatment using
hepatic vein transit times needs to be assessed in future
longitudinal studies, but the methodology is potentially
translatable as an efficient, quick and discriminative test
for the assessment of disease severity in hepatitis C to any
ultrasound department anywhere in the world.
REFERENCES
1 World Health Organisation. Hepatitis C. Fact Sheet No. 164,
revised October 2000. Available from: URL: http//www.who.
int/mediacentre/factsheets/fs164/en/
2 Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur
RR, Magder LS, El Khoby T, Abdel-Wahab Y, Aly Ohn ES,
Anwar W, Sallam I. The role of parenteral antischistosomal
therapy in the spread of hepatitis C virus in Egypt. Lancet
2000; 355: 887-891
3 Hoofnagle JH. Course and outcome of hepatitis C. Hepatology
2002; 36: S21-S29
4 National Institute of Clinical Excellence (N.I.C.E.). Guidance
on the use of ribavirin and interferon alpha for hepatitis C.
October 2000. Available from: URL: http//www.nice.org.uk/
pdf/RIBAVIRIN-guidance14.pdf
5 Marcellin P, Asselah T, Boyer N. Fibrosis and disease
progression in hepatitis C. Hepatology 2002; 36: S47-S56
6 Poynard T, Imbert-Bismut F, Munteanu M, Messous D,
Thabut D, Ratziu V, Benhamou Y. Biomarkers as non-
invasive assessment of hepatic fibrosis in chronic hepatitis C. J
Gastroenterol Hepatol 2004; 19: S236-S245
7 Fontana RJ, Lok AS. Noninvasive monitoring of patients with
chronic hepatitis C. Hepatology 2002; 36: S57-S64
8 Afdhal NH, Nunes D. Evaluation of liver fibrosis: a concise
review. Am J Gastroenterol 2004; 99: 1160-1174
9 Giannini E, Risso D, Botta F, Chiarbonello B, Fasoli A,
Malfatti F, Romagnoli P, Testa E, Ceppa P, Testa R. Validity
and clinical utility of the aspartate aminotransferase-alanine
aminotransferase ratio in assessing disease severity and
prognosis in patients with hepatitis C virus-related chronic
liver disease. Arch Intern Med 2003; 163: 218-224
10 Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou
Y, Poynard T. Biochemical markers of liver fibrosis in patients
with hepatitis C virus infection: a prospective study. Lancet
2001; 357: 1069-1075
11 Wai CT, Greenson JK, Fontana RJ, Kalbfleisch JD, Marrero JA,
Conjeevaram HS, Lok AS. A simple noninvasive index can
predict both significant fibrosis and cirrhosis in patients with
chronic hepatitis C. Hepatology 2003; 38: 518-526
12 Kono Y, Mattrey RF. Ultrasound of the liver. Radiol Clin North
Am 2005; 43: 815-826, vii
13 Tchelepi H, Ralls PW, Radin R, Grant E. Sonography of
diffuse liver disease. J Ultrasound Med 2002; 21: 1023-1032; quiz
1033-1034
14 Ziol M, Handra-Luca A, Kettaneh A, Christidis C, Mal
F, Kazemi F, de Ldinghen V, Marcellin P, Dhumeaux D,
Trinchet JC, Beaugrand M. Noninvasive assessment of liver
fibrosis by measurement of stiffness in patients with chronic
hepatitis C. Hepatology 2005; 41: 48-54
15 Castra L, Vergniol J, Foucher J, Le Bail B, Chanteloup
E, Haaser M, Darriet M, Couzigou P, De Ldinghen V.
Prospective comparison of transient elastography, Fibrotest,
APRI, and liver biopsy for the assessment of fibrosis in chronic
hepatitis C. Gastroenterology 2005; 128: 343-350
16 Colletta C, Smirne C, Fabris C, Toniutto P, Rapetti R, Minisini
R, Pirisi M. Value of two noninvasive methods to detect
progression of fibrosis among HCV carriers with normal
aminotransferases. Hepatology 2005; 42: 838-845
17 Blomley M, Cosgrove D. Microbubble echo-enhancers: a new
direction for ultrasound? Lancet 1997; 349: 1855-1856
18 Becher H, Burns PN. Handbook of contrast echocardiography.
LV function and myocardial perfusion (Electronic Version).
Springer, 2000: 2-42. Available from: URL: http//www.
sunnybrook.utoronto.ca/EchoHandbook/
19 Harvey CJ, Pilcher JM, Eckersley RJ, Blomley MJ, Cosgrove
DO. Advances in ultrasound. Clin Radiol 2002; 57: 157-177
20 Harvey CJ, Blomley MJ, Eckersley RJ, Cosgrove DO.
Developments in ultrasound contrast media. Eur Radiol 2001;
11: 675-689
21 Blomley MJ, Cooke JC, Unger EC, Monaghan MJ, Cosgrove
DO. Microbubble contrast agents: a new era in ultrasound.
BMJ 2001; 322: 1222-1225
22 Blomley MJ, Lim AK, Harvey CJ, Patel N, Eckersley RJ,
Basilico R, Heckemann R, Urbank A, Cosgrove DO, Taylor-
Robinson SD. Liver microbubble transit time compared with
histology and Child-Pugh score in diffuse liver disease: a cross
sectional study. Gut 2003; 52: 1188-1193
23 Lim AK, Taylor-Robinson SD, Patel N, Eckersley RJ, Goldin
RD, Hamilton G, Foster GR, Thomas HC, Cosgrove DO,
Blomley MJ. Hepatic vein transit times using a microbubble
agent can predict disease severity non-invasively in patients
with hepatitis C. Gut 2005; 54: 128-133
24 Albrecht T, Blomley MJ, Cosgrove DO, Taylor-Robinson SD,
Jayaram V, Eckersley R, Urbank A, Butler-Barnes J, Patel N.
Non-invasive diagnosis of hepatic cirrhosis by transit-time
analysis of an ultrasound contrast agent. Lancet 1999; 353:
1579-1583
25 Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat
F, Denk H, Desmet V, Korb G, MacSween RN. Histological
grading and staging of chronic hepatitis. J Hepatol 1995; 22:
696-699
S- Editor Wang J L- Editor Kumar M E- Editor Bi L
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 PO Box 2345, Beijing 100023, China
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TOPIC HIGHLIGHT
Gianfranco D Alpini, PhD, Series Editor
Calcium signaling in cholangiocytes
Noritaka Minagawa, Barbara E Ehrlich, Michael H Nathanson
Noritaka Minagawa, Barbara E Ehrlich, Departments of Medi-
cine and Pharmacology Yale University School of Medicine, New
Haven, CT, United States
Michael H Nathanson, Section of Digestive Diseases, Yale
University School of Medicine, 1 Gilbert Street, Room TAC
S241D, New Haven, CT 06519, United States
Supported by NIH grants DK61747, DK45710, DK57751,
and DK34989, and by a grant-in-aid from the American Heart
Association
Correspondence to: Michael H Nathanson, Section of Digestive
Diseases, Yale University School of Medicine, 1 Gilbert Street,
Room TAC S241D, New Haven, CT 06519,
United States. michael.nathanson@yale.edu
Telephone: +1-203-7857312 Fax: +1-203-7854306
Received: 2006-01-01 Accepted: 2006-01-24
Abstract
2+
Cytosolic Ca is an important second messenger in virtu-
2+
ally every type of cell. Moreover, Ca generally regulates
multiple activities within individual cells. This article re-
views the cellular machinery that is responsible for Ca
2+
2+
signaling in cholangiocytes. In addition, two Ca -medi-
ated events in cholangiocytes are discussed: bicarbonate
secretion and apoptosis. Finally, emerging evidence is
2+
reviewed that Ca signaling is involved in the pathogen-
2+
esis of diseases affecting the biliary tree and that Ca
signaling pathways can be manipulated to therapeutic
advantage in the treatment of cholestatic disorders.
2006 The WJG Press. All rights reserved.
Key words: Ca2+; Cholangiocyte; Inositol trisphosphate;
Bile secretion; Cholestasis; Apoptosis
Minagawa N, Ehrlich BE, Nathanson MH. Calcium signaling
in cholangiocytes. World J Gastroenterol 2006; 12(22):
3466-3470
http://www.wjgnet.com/1007-9327/12/3466.asp
Introduction
Cytosolic Ca2+ is an important second messenger in virtu-
ally every type of cell. Ca2+ regulates a wide range of cell
functions, including contraction, gene transcription, cell
growth, differentiation, apoptosis, membrane fusion, ion
channel activation, and secretion[1]. Moreover, Ca2+ gen-
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World J Gastroenterol 2006 June 14; 12(22): 3466-3470
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
erally regulates multiple activities within individual cells.
Temporal Ca2+ signaling patterns, such as Ca2+ oscillations,
and spatial signaling patterns, such as Ca2+ gradients and
waves, permit Ca2+ to exhibit this differential regulation
of cell function. Therefore, the manner in which cells or-
ganize their Ca2+ signals is critical for the types of signals
they can produce, and thus is critical for the ways in which
Ca2+ can regulate cell function. There are certain com-
mon mechanisms for generating Ca2+ signals among cell
types, but each cell type has distinct features of signaling
machinery as well. Here we will review the machinery that
is responsible for Ca2+ signaling in cholangiocytes. We also
will discuss in detail two Ca2+-mediated events in cholan-
giocytes, bicarbonate secretion and apoptosis. Finally, we
will review emerging evidence that Ca2+ signaling plays an
important role in the pathogenesis of diseases affecting the
biliary tree and in the treatment of cholestatic disorders.
Ca 2+ SIGNALING MACHINERY IN CHO-
LANGIOCYTES
Cytosolic Ca2+ signals originate from two general sources,
release from intracellular Ca2+ stores and influx across the
plasma membrane. The initial component of Ca2+ signals
in cholangiocytes and other epithelia is due entirely to Ca2+
release from intracellular stores, since this component
is unchanged in Ca 2+-free medium [2,3]. In contrast, the
role of extracellular Ca2+ is to maintain cytosolic signals
over longer time intervals and to replenish intracellular
stores. This review will therefore focus on intracellular
Ca2+ release mechanisms. There are two intracellular Ca2+
release channels that are principally responsible for Ca2+
release in nearly every cell type, the ryanodine receptor
(RyR) and the inositol 1,4,5-trisphosphate (InsP3) receptor
(InsP3R)[1]. The RyR is most heavily expressed in excitable
cells such as myocytes and to a lesser extent in neurons.
The RyR also is expressed in some nonexcitable cells,
including certain polarized epithelia, such as pancreatic
acinar cells [4]. The RyR is localized to sarcoplasmic or
endoplasmic reticulum (ER), where most intracellular
Ca2+ is stored. Activation of the receptor allows Ca2+ to
be released from the ER into the cytosol. The RyR can be
activated in several different ways, including through direct
coupling with certain plasma membrane Ca2+ channels, via
stimulation with the second messenger cyclic ADP-ribose,
or by stimulation with Ca 2+ itself (Ca 2+-induced Ca 2+
release). Many epithelia, including cholangiocytes[5] as well
as hepatocytes[6], do not express the RyR.
 Minagawa N et al. Ca2+ signaling in cholangiocytes 3467
McL-1 InsP3R
The InsP3R is expressed in virtually every cell type, is
the predominant Ca2+ release channel in epithelia, and is
the only intracellular Ca2+ release channel in cholangio-
cytes[5]. Like the RyR, the InsP3R is expressed in the ER.
The endogenous ligand for the InsP3R is InsP3, which is
formed from phospholipase C (PLC)-induced hydrolysis
of phosphatidyl inositol bisphosphate (PIP2)[7]. This bio-
chemical pathway can be activated by stimulation of either
G protein-coupled receptors or receptor tyrosine kinases[1].
Therefore, stimulation of cholangiocytes with certain
hormones or growth factors results in InsP3-mediated
increases in cytosolic Ca2+. Hormone-induced Ca2+ signal-
ing is mediated entirely by InsP3 in cholangiocytes, since
Ca2+ signaling is blocked by microinjection of the InsP3R
inhibitor heparin[2]. There are three InsP3R isoforms, each
of which has distinct biophysical properties [8,9]. These
properties result in different effects on Ca2+ signaling. For
example, in DT40 cells engineered to express only a single
InsP3R isoform, activation of the type InsP3R results
in transient Ca2+ oscillations, while the type InsP3R
induces sustained oscillations, and the type isoform
results in a single, transient increase in Ca2+[10]. Similarly, in
CHO cells in which expression of each one of the three
isoforms has been silenced, ATP-induced Ca 2+ signals
are preferentially converted from oscillations to either
sustained or transient increases in Ca2+[11]. Many cell types
express multiple InsP3R isoforms, and some cells[12,13], in-
cluding cholangiocytes[5], express all three isoforms. Even
among cell types that express all three isoforms, there is
considerable variability in terms of how much of each iso-
form is expressed, and in terms of the subcellular region
in which each isoform is expressed. In cholangiocytes, the
type InsP3R accounts for about 80% of InsP3Rs, while
the types and isoforms each account for about 10%.
Moreover, the type InsP3R is most concentrated in the
apical region (Figure 1), while the other two isoforms are
not distributed in a polarized fashion[5]. Hormone-induced
Ca2+ signals in cholangiocytes begin as apical-to-basal Ca2+
waves[5], similar to what is observed in other polarized epi-
thelia[6,14]. In cholangiocytes, this polarized Ca2+ signaling
pattern is likely due to the apical concentration of the type
InsP3R, although it is not clear if this is because the re-
ceptor has properties that permit it to trigger Ca2+ waves[8],
or simply because of the increased InsP3R concentration
in the apical region[15]. Ca2+ signals also can spread among
neighboring cholangiocytes. This effect is mediated by gap
junctions in most epithelia[16], including cholangiocytes[17].
The predominant gap junction protein in cholangiocytes
Figure 1 Distribution of the InsP3
receptor and Mcl-1 in human bile ducts.
This confocal immunofluorescence
image was obtained from a paraffin-
embedded biopsy specimen from
a normal liver. The specimen was
double-labeled with antibodies against
Mcl-1 (green) and the type III InsP3R
(red). Note that the Mcl-1 is distributed
diffusely throughout the cytosol, while
the InsP3R is most concentrated in the
apical region.
Merge
is connexin 43, although connexin 32 is expressed in these
cells to a lesser extent[17]. Permeability of cholangiocyte
gap junctions is decreased by protein kinases A and C[17],
which provides additional ways in which Ca2+ signals can
be modulated in intact bile ducts.
Intercellular messengers that act
through Ca2+
Several intercellular messengers have been identified that
increase cytosolic Ca2+ in cholangiocytes. Each messenger
molecule interacts with specific G protein-coupled recep-
tors that increase Ca2+ via activation of PLC and forma-
tion of InsP3. Acetylcholine (ACh) increases Ca 2+ by
stimulation of M3 muscarinic receptors, which have been
identified on cholangiocytes by RT-PCR, immunoblot,
and immunochemistry[18]. These receptors are expressed
on the basolateral membrane[2]. ACh-induced increases in
Ca2+ can have both indirect and direct effects on down-
stream signaling pathways. ACh-induced increases in Ca2+
activate calcineurin, which potentiates increases in cAMP
that are induced by secretin[18]. Stimulation of cholangio-
cytes with ACh can result in a sustained, transient, or os-
cillatory increase in cytosolic Ca2+[2]. The likelihood that a
particular pattern of Ca2+ signal will be elicited is not dose-
dependent, though. ATP also increases Ca2+ in cholangio-
cytes[2]. Like ACh, ATP can induce a sustained, transient,
or oscillatory increase in cytosolic Ca2+. Unlike ACh, lower
concentrations of ATP predominantly induce Ca2+ oscilla-
tions, while higher concentrations induce single (either sus-
tained or transient) increases in Ca2+[2]. RT-PCR evidence
suggests that cholangiocytes express P2Y1, P2Y2, P2Y4,
P2Y6, and P2X4 nucleotide receptors[19]. Pharmacologic
evidence using microperfused bile duct segments isolated
form rat liver suggests that each of the four P2Y subtypes
are expressed on the apical membrane of cholangiocytes.
P2Y receptors also are expressed on the basolateral mem-
brane, but the actions of ATP in that region are attenuated
by ecto-nucleotidases expressed at that membrane[19] or by
nearby portal fibroblasts[20]. Pharmacological studies sug-
gest that there is little or no functional expression of P2X
receptors in cholangiocytes[19]. These findings suggest that
the principal route for signaling to cholangocytes through
ATP is via bile. ATP may be released from cholangiocytes
to signal in an autocrine or paracrine fashion[21], and this
pathway is important for cell volume regulation[22]. ATP
also can be secreted into bile from hepatocytes[21,23], which
provides a pathway through which hepatocytes can signal
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 3468 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
to cholangiocytes[24]. The manner in which this signaling
pathway is regulated has not been established, but certain
bile acids appear to activate this pathway in order to stimu-
late ductular bicarbonate secretion[23].
Cholangiocytes also express receptors for a number
of neuropeptides that act through Ca2+ signaling. Cholan-
giocytes express both the serotonin 1a and 1b receptor on
their basolateral membrane[25]. Stimulation of this receptor
inhibits the bile duct proliferation that occurs following
bile duct ligation. This is likely mediated through Ca 2+
signaling, since receptor stimulation is associated with an
increase in InsP3 and the effects on cholangiocyte prolif-
eration are blocked by chelation of cytosolic Ca2+[25]. Work
in cholangiocyte cell lines has shown that these cells also
express CCK-B gastrin receptors. As with serotonin recep-
tors, stimulation of gastrin receptors inhibits cholangiocyte
growth. The effect of gastrin also is blocked by chelation
of cytosolic Ca2+[26]. Cholangiocytes express alpha-1 adren-
ergic receptors, primarily on their basolateral membrane.
The alpha-1 adrenergic agonist phenylephrine increases
secretin-stimulated ductal secretion, as measured in both
intact rat liver and isolated rat bile duct units. Phenyleph-
rine also potentiates the effects of secretin on cAMP pro-
duction, and directly increases cytosolic Ca2+ and InsP3 in
cholangiocytes[27]. Pharmacologic evidence suggests that
cholangiocytes express D2 but not D1 or D3 dopamine re-
ceptors. Stimulation of these receptors inhibits rather than
stimulates secretin-induced ductular secretion and cAMP
production. This is thought to be because D2 receptor
stimulation co-activates PKC-gamma, whereas gastrin
receptor stimulation co-activates PKC-alpha[28]. Thus, al-
though several types of receptors link to Ca2+ signaling in
cholangiocytes, they can have distinct downstream actions
because of differential effects on Ca2+ signaling patterns as
well as because of differential activation of related signal-
ing pathways.
Regulation of secretion via Ca2+
Cholangiocytes comprise less than 5% of normal liver, but
play an important role in the process of bile secretion[29,30].
Cholangiocytes can be stimulated to increase bile flow by
up to 50%[31], although cholangiocytes typically affect the
pH rather than the volume of bile[32]. Two parallel signaling
pathways exist to regulate ductular bicarbonate secretion,
which are mediated by either cAMP or Ca2+. Evidence in
isolated cholangiocytes and bile duct units suggests that
cAMP stimulates chloride secretion via the cystic fibro-
sis transmembrane conductance regulator (CFTR), and
that bicarbonate secretion then occurs via an associated
chloride-bicarbonate exchanger. Some evidence in isolated
cell systems suggests that Ca2+ analogously stimulates chlo-
ride secretion via a Ca2+-activated chloride channel, and
that bicarbonate secretion then occurs via an associated
chloride-bicarbonate exchanger[33]. Alternative evidence
instead suggests that Ca2+ stimulates ductular secretion
by calcineurin-mediated potentiation of the cAMP path-
way[18]. The mechanism of ductular secretion has also been
investigated in the intact liver, by bivascular perfusion of
the isolated liver via both the hepatic artery and the portal
vein[32]. This bivascular perfusion model is necessary to
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June 14, 2006 Volume 12 Number 22
use because the blood supply of cholangiocytes is derived
via the hepatic artery in normal liver. Thus, for example,
the effects of secretin on ductular secretion can be read-
ily detected when this hormone is infused via the hepatic
artery, but are difficult to detect when infused via the por-
tal vein[32]. These studies show that ductular bicarbonate
secretion induced by the Ca2+ agonist ACh depends upon
both chloride channels and chloride-bicarbonate exchange.
This observation supports the idea that Ca2+-mediated
bicarbonate secretion results from serial activation of
chloride channels, and then chloride-bicarbonate exchange.
However, bivascular perfusion studies show that cAMP-
mediated bicarbonate secretion depends upon chloride
channels but not chloride-bicarbonate exchange[32]. This
observation supports the idea that ductular bicarbonate
secretion induced by cAMP may occur directly via CFTR,
similar to what has been described in intestine[34,35].
Regulation of apoptosis via Ca2+
Apoptosis in all cell types is regulated in part through Ca2+
signaling pathways, and there are special considerations in
cholangiocytes. Apoptotic pathways are modulated through
mitochondrial permeabilization, which in turn is regulated
through Ca2+ signaling. The interrelationship between mi-
tochondria and Ca2+ signaling results from the close prox-
imity between mitochondria and InsP3Rs[36]. As a result
of this physical association, mitochondria are subjected to
more intense increases in Ca2+ than typically occur in cyto-
sol[36]. Excessive increases in mitochondrial Ca2+ result in
increases in mitochondrial permeability that are associated
with leakage of cytochrome c into the cytosol, which leads
to apoptosis[37]. Cytochrome c released from mitochondria
binds directly to nearby InsP3Rs, which potentiates release
of Ca2+ from the receptor[38]. This leads to a positive feed-
back loop that further increases mitochondrial permeabil-
ity and promotes apoptosis[38]. On the other hand, the anti-
apoptotic Bcl-2 family members Bcl-xL and Bcl-2 inhibit
apoptosis by inhibiting InsP3-mediated Ca2+ release. Bcl-xL
decreases expression of InsP3R, which reduces InsP3-me-
diated Ca2+ release in order to protect against apoptosis[39].
Bcl-2 itself increases leakage of Ca2+ from the ER, which
also has the net effect of decreasing Ca2+ signals in both
the cytosol and mitochondria[40]. Mcl-1 is the principal
Bcl-2 family member in cholangiocytes[41] (Figure 1). Like
other Bcl-2 family members, it appears to inhibit apoptosis
through effects on Ca2+ signaling. Unlike Bcl-xL or Bcl-2,
however, Mcl-1 does not affect either InsP3R expression
or ER Ca2+ stores[42]. Instead, Mcl-1 inhibits Ca2+ signaling
directly within mitochondria[42].
All three isoforms of the InsP3R can induce apopto-
sis[11]. However, each isoform has a different propensity
to induce apoptosis[11]. Recent evidence suggests that this
may be due in part to their differential distribution relative
to mitochondria. Specifically, in cells that co-express each
InsP3R isoform, the type InsP3R is most effective in
transmitting Ca2+ signals to mitochondira as well as in in-
ducing apoptosis[11]. Furthermore, the type isoform co-
localizes most strongly with mitochondria at the light lev-
el[11]. Thus, the type InsP3R has a particular propensity
to form signaling microdomains with mitochondria. The
 Minagawa N et al. Ca2+ signaling in cholangiocytes 3469
type InsP3R is most concentrated in the apical region
of cholangiocytes[5], as described above, but little is known
about the factors that regulate this subcellular targeting.
It also remains unknown whether apical, type InsP3Rs
associate with mitochondria in cholangiocytes, or whether
such an association is needed to mediate apoptosis in bile
ducts.
Ca2+ signaling in health and disease
Ca2+ signaling is universal among cells, so one might expect
altered Ca2+ signaling to play an important role in patho-
genesis of disease. In fact, the spectrum of human dis-
eases that had been found to result from alterations in Ca2+
signaling has been remarkably limited. These have included
a mutation in the RyR, which results in malignant hyper-
thermia[43]; mutations of the Ca2+-ATPase pump, which are
associated with deafness[44]; and an animal model of loss
of the types and InsP3Rs, which together results in
pancreatic insufficiency and failure to thrive[45]. However,
it now appears that loss of InsP3Rs is a common event
in human cholestatic disorders. Specifically, expression
of the type InsP3R is decreased in the cholangiocytes
of patients with primary biliary cirrhosis, sclerosing chol-
angitis, biliary atresia, and ductular obstruction[46]. This
loss of InsP3R expression also occurs in animal models
of cholestasis, where it is associated with impaired Ca2+
signaling, as well as impaired Ca2+-induced secretion in
cholangiocytes[46]. However, cAMP-mediated secretion is
maintained in cholangiocytes despite the loss of InsP3Rs.
Interestingly, the loss of InsP3Rs that is seen in patients
with cholangiopathies is not seen in patients with hepatitis
C viral infections, even though such infections are associ-
ated with inflammation in the region of the portal triad[46].
This suggests that loss of InsP3Rs from cholangiocytes is
not merely a nonspecific effect of local inflammation. To-
gether, these findings suggest that Ca2+ signaling pathways
are necessary for ductular secretion as it occurs in vivo, and
that loss of InsP3Rs results in cholestasis, although direct
support for this hypothesis is lacking.
Just as impaired Ca2+ signaling in cholangiocytes is as-
sociated with the development of cholestasis, stimulation
of Ca2+ signals may be beneficial for the treatment of cho-
lestasis. Ursodeoxycholic acid (UDCA) and its taurine con-
jugate (TUDCA) are of therapeutic benefit in a range of
cholestatic disorders[47]. Recent evidence suggests that these
but not other bile acids selectively induce hepatocytes to
secrete ATP into bile[23]. In addition, luminal perfusion of
isolated intrahepatic bile duct segments with ATP increases
Ca2+ in cholangiocytes and stimulates ductular bicarbonate
secretion, while perfusion with TUDCA does not[23]. These
findings suggest that UDCA and TUDCA may promote
ductular secretion through a paracrine signaling pathway
whereby hepatocytes release ATP to activate apical pu-
rinergic receptors on cholangiocytes, which link to Ca2+-
mediated secretion of chloride and bicarbonate into bile.
Further work is needed to understand how these bile acids
induce hepatocytes to secrete ATP, and to demonstrate
that this action is in part responsible for their therapeutic
effects.
Conclusion
Ca2+ signaling is a ubiquitous mechanism for regulation of
cell function. A range of neuroendocrine and paracrine
messenger molecules exert their effects on cholangiocytes
through Ca2+ signaling. It is already established that these
effects include regulation of bicarbonate secretion and
regulation of apoptosis. By extension from what has been
shown in other cell types, it is likely that Ca2+ signaling also
regulates other aspects of ductular secretion, including
fluid secretion and vesicular targeting and exocytosis, as
well as other aspects of cell proliferation, including gene
transcription and progression through the cell cycle.
Future areas of investigation are likely to more fully
identify the range of actions of Ca2+ in cholangiocytes.
Such information may in turn reveal specific ways in
which impaired Ca2+ signaling leads to cholangiopathies
and related disease states, which then may lead to targeted
therapies that are based on correcting such impairments.
References
1 Berridge MJ, Lipp P, Bootman MD. The versatility and
universality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1:
11-21
2 Nathanson MH, Burgstahler AD, Mennone A, Boyer JL.
Characterization of cytosolic Ca2+ signaling in rat bile duct
epithelia. Am J Physiol 1996; 271: G86-G96
3 Nathanson MH, Burgstahler AD, Fallon MB. Multistep
mechanism of polarized Ca2+ wave patterns in hepatocytes.
Am J Physiol 1994; 267: G338-G349
4 Leite MF, Dranoff JA, Gao L, Nathanson MH. Expression
and subcellular localization of the ryanodine receptor in rat
pancreatic acinar cells. Biochem J 1999; 337(Pt 2): 305-309
5 Hirata K, Dufour JF, Shibao K, Knickelbein R, O'Neill AF,
Bode HP, Cassio D, St-Pierre MV, Larusso NF, Leite MF,
Nathanson MH. Regulation of Ca(2+) signaling in rat bile duct
epithelia by inositol 1,4,5-trisphosphate receptor isoforms.
Hepatology 2002; 36: 284-296
6 Hirata K, Pusl T, O'Neill AF, Dranoff JA, Nathanson MH. The
type II inositol 1,4,5-trisphosphate receptor can trigger Ca2+
waves in rat hepatocytes. Gastroenterology 2002; 122: 1088-1100
7 Leite MF, Nathanson MH. Calcium signaling in the
hepatocyte. In: Arias IM. The Liver: Biology and Pathobilogy.
4th ed. Philadelphia: Raven, 2001
8 Hagar RE, Burgstahler AD, Nathanson MH, Ehrlich BE.
Type III InsP3 receptor channel stays open in the presence of
increased calcium. Nature 1998; 396: 81-84
9 Ramos-Franco J, Fill M, Mignery GA. Isoform-specific function
of single inositol 1,4,5-trisphosphate receptor channels. Biophys
J 1998; 75: 834-839
10 Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kurosaki T,
Iino M. Encoding of Ca2+ signals by differential expression of
IP3 receptor subtypes. EMBO J 1999; 18: 1303-1308
11 Mendes CC, Gomes DA, Thompson M, Souto NC, Goes
TS, Goes AM, Rodrigues MA, Gomez MV, Nathanson
MH, Leite MF. The type III inositol 1,4,5-trisphosphate
receptor preferentially transmits apoptotic Ca2+ signals into
mitochondria. J Biol Chem 2005; 280: 40892-40900
12 Wojcikiewicz RJ. Type I, II, and III inositol 1,4,5-trisphosphate
receptors are unequally susceptible to down-regulation and
are expressed in markedly different proportions in different
cell types. J Biol Chem 1995; 270: 11678-11683
13 Sugiyama T, Yamamoto-Hino M, Wasano K, Mikoshiba K,
Hasegawa M. Subtype-specific expression patterns of inositol
1,4,5-trisphosphate receptors in rat airway epithelial cells. J
Histochem Cytochem 1996; 44: 1237-1242
14 Leite MF, Burgstahler AD, Nathanson MH. Ca2+ waves
www.wjgnet.com
 3470 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
require sequential activation of inositol trisphosphate receptors
and ryanodine receptors in pancreatic acini. Gastroenterology
2002; 122: 415-427
15 Marchant JS, Parker I. Role of elementary Ca(2+) puffs in
generating repetitive Ca(2+) oscillations. EMBO J 2001; 20:
65-76
16 Leite MF, Hirata K, Pusl T, Burgstahler AD, Okazaki K,
Ortega JM, Goes AM, Prado MA, Spray DC, Nathanson MH.
Molecular basis for pacemaker cells in epithelia. J Biol Chem
2002; 277: 16313-16323
17 Bode HP, Wang L, Cassio D, Leite MF, St-Pierre MV,
Hirata K, Okazaki K, Sears ML, Meda P, Nathanson MH,
Dufour JF. Expression and regulation of gap junctions in rat
cholangiocytes. Hepatology 2002; 36: 631-640
18 Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C, Fraioli
F, Romeo R, Marucci L, Le Sage G, Glaser SS, Benedetti A. Role
and mechanisms of action of acetylcholine in the regulation of
rat cholangiocyte secretory functions. J Clin Invest 1997; 100:
1349-1362
19 Dranoff JA, Masyuk AI, Kruglov EA, LaRusso NF, Nathanson
MH. Polarized expression and function of P2Y ATP receptors
in rat bile duct epithelia. Am J Physiol Gastrointest Liver Physiol
2001; 281: G1059-G1067
20 Dranoff JA, Kruglov EA, Robson SC, Braun N, Zimmer-
mann H, Svigny J. The ecto-nucleoside triphosphate
diphosphohydrolase NTPDase2/CD39L1 is expressed in a
novel functional compartment within the liver. Hepatology
2002; 36: 1135-1144
21 Chari RS, Schutz SM, Haebig JE, Shimokura GH, Cotton
PB, Fitz JG, Meyers WC. Adenosine nucleotides in bile. Am J
Physiol 1996; 270: G246-G252
22 Wang Y, Roman R, Lidofsky SD, Fitz JG. Autocrine signaling
through ATP release represents a novel mechanism for
cell volume regulation. Proc Natl Acad Sci U S A 1996; 93:
12020-12025
23 Nathanson MH, Burgstahler AD, Masyuk A, Larusso NF.
Stimulation of ATP secretion in the liver by therapeutic bile
acids. Biochem J 2001; 358: 1-5
24 Schlosser SF, Burgstahler AD, Nathanson MH. Isolated rat
hepatocytes can signal to other hepatocytes and bile duct cells
by release of nucleotides. Proc Natl Acad Sci U S A 1996; 93:
9948-9953
25 Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A,
Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J,
Fava G, Lesage GD, Alpini G. Autocrine/paracrine regulation
of the growth of the biliary tree by the neuroendocrine
hormone serotonin. Gastroenterology 2005; 128: 121-137
26 Kanno N, Glaser S, Chowdhury U, Phinizy JL, Baiocchi
L, Francis H, LeSage G, Alpini G. Gastrin inhibits cholan-
giocarcinoma growth through increased apoptosis by
activation of Ca2+-dependent protein kinase C-alpha. J Hepatol
2001; 34: 284-291
27 LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L,
Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani
S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic
receptor agonists modulate ductal secretion of BDL rats via
Ca(2+)- and PKC-dependent stimulation of cAMP. Hepatology
2004; 40: 1116-1127
28 Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H,
Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino
MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-
stimulated choleresis by increased PKC-gamma expression
and decrease of PKA activity. Am J Physiol Gastrointest Liver
Physiol 2003; 284: G683-G694
29 Boyer JL. Bile duct epithelium: frontiers in transport physi-
ology. Am J Physiol 1996; 270: G1-G5
30 Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
cholestasis. N Engl J Med 1998; 339: 1217-1227
31 Nathanson MH, Burgstahler AD, Mennone A, Dranoff JA,
Rios-Velez L. Stimulation of bile duct epithelial secretion by
glybenclamide in normal and cholestatic rat liver. J Clin Invest
1998; 101: 2665-2676
32 Hirata K, Nathanson MH. Bile duct epithelia regulate biliary
bicarbonate excretion in normal rat liver. Gastroenterology 2001;
121: 396-406
33 Boyer JL, Nathanson MH. Bile formation. In: Schiff ER,
Scrrell MF, Maddrey WC. Diseases of the liver. Philadelphia:
Lippincott-Raven, 1999: 146-199
34 Poulsen JH, Fischer H, Illek B, Machen TE. Bicarbonate
conductance and pH regulatory capability of cystic fibrosis
transmembrane conductance regulator. Proc Natl Acad Sci U S
A 1994; 91: 5340-5344
35 Seidler U, Blumenstein I, Kretz A, Viellard-Baron D,
Rossmann H, Colledge WH, Evans M, Ratcliff R, Gregor M.
A functional CFTR protein is required for mouse intestinal
cAMP-, cGMP- and Ca(2+)-dependent HCO3- secretion. J
Physiol 1997; 505(Pt 2): 411-423
36 Rizzuto R, Brini M, Murgia M, Pozzan T. Microdomains with
high Ca2+ close to IP3-sensitive channels that are sensed by
neighboring mitochondria. Science 1993; 262: 744-747
37 Ichas F, Jouaville LS, Mazat JP. Mitochondria are excitable
organelles capable of generating and conveying electrical and
calcium signals. Cell 1997; 89: 1145-1153
38 Boehning D, Patterson RL, Sedaghat L, Glebova NO,
Kurosaki T, Snyder SH. Cytochrome c binds to inositol (1,4,5)
trisphosphate receptors, amplifying calcium-dependent
apoptosis. Nat Cell Biol 2003; 5: 1051-1061
39 Li C, Fox CJ, Master SR, Bindokas VP, Chodosh LA, Thomp-
son CB. Bcl-X(L) affects Ca(2+) homeostasis by altering
expression of inositol 1,4,5-trisphosphate receptors. Proc Natl
Acad Sci U S A 2002; 99: 9830-9835
40 Bassik MC, Scorrano L, Oakes SA, Pozzan T, Korsmeyer SJ.
Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and
apoptosis. EMBO J 2004; 23: 1207-1216
41 Taniai M, Grambihler A, Higuchi H, Werneburg N, Bronk SF,
Farrugia DJ, Kaufmann SH, Gores GJ. Mcl-1 mediates tumor
necrosis factor-related apoptosis-inducing ligand resistance
in human cholangiocarcinoma cells. Cancer Res 2004; 64:
3517-3524
42 Minagawa N, Kruglov EA, Dranoff JA, Robert ME, Gores
GJ, Nathanson MH. The anti-apoptotic protein Mcl-1 inhibits
mitochondrial Ca2+ signals. J Biol Chem 2005; 280: 33637-33644
43 Lynch PJ, Tong J, Lehane M, Mallet A, Giblin L, Heffron
JJ, Vaughan P, Zafra G, MacLennan DH, McCarthy TV. A
mutation in the transmembrane/luminal domain of the
ryanodine receptor is associated with abnormal Ca2+ release
channel function and severe central core disease. Proc Natl
Acad Sci U S A 1999; 96: 4164-4169
44 Street VA, McKee-Johnson JW, Fonseca RC, Tempel BL,
Noben-Trauth K. Mutations in a plasma membrane Ca2+-
ATPase gene cause deafness in deafwaddler mice. Nat Genet
1998; 19: 390-394
45 Futatsugi A, Nakamura T, Yamada MK, Ebisui E, Nakamura K,
Uchida K, Kitaguchi T, Takahashi-Iwanaga H, Noda T, Aruga
J, Mikoshiba K. IP3 receptor types 2 and 3 mediate exocrine
secretion underlying energy metabolism. Science 2005; 309:
2232-2234
46 Shibao K, Hirata K, Robert ME, Nathanson MH. Loss of
inositol 1,4,5-trisphosphate receptors from bile duct epithelia
is a common event in cholestasis. Gastroenterology 2003; 125:
1175-1187
47 Beuers U, Boyer JL, Paumgartner G. Ursodeoxycholic acid in
cholestasis: potential mechanisms of action and therapeutic
applications. Hepatology 1998; 28: 1449-1453
S- Editor Wang J E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
www.wjgnet.com
wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Nervous and Neuroendocrine regulation of the
pathophysiology of cholestasis and of biliary carcinogenesis
Marco Marzioni, Giammarco Fava, Antonio Benedetti
Marco Marzioni, Giammarco Fava, Antonio Benedetti,
Department of Gastroenterology, Universit Politecnica delle
Marche, via Tronto 10, 60020 Ancona, Italy
Supported by the MIUR grant, No. 2003060137_004 and by
the Fondazione Cariverona 2002 grant "Ambiente e sviluppo
sostenibile" to Prof. Benedetti; by the "Premio S.I.G.E. 2004"
to Dr. Marzioni; by the Universit Politecnica delle Marche
intramural grants ATBEN00205 to Professor. Benedetti and
ATMAR01105 to Dr. Marzioni, by the Premio S.I.G.E. 2006 to
Dr Fava
Co-first-authors: Dr. Giammarco Fava
Co-correspondents: Dr. Giammarco Fava
Correspondence to: Dr. Marco Marzioni, Assistant Professor,
Department of Gastroenterology, Universit Politecnica delle
Marche, Nuovo Polo Didattico, III piano, Via Tronto 10, 60020
Ancona, Italy. marco.marzioni@tele2.it
Telephone: +39-071-883885 Fax: +39-071-883886
Received: 2006-01-13 Accepted: 2006-02-18
Abstract
Cholangiocytes, the epithelial cells lining the biliary
ducts, are the target cells in several liver diseases.
Cholangiopathies and cholangiocarcinoma generate
interest in many scientists since the genesis. The
developing mechanisms, and the therapeutic tools
of these diseases are still undefined. Several studies
demonstrate that many hormones, neuropeptides and
neurotransmitters regulate malignant and non-malignant
cholangiocyte pathophysiology in the course of chronic
biliary diseases. The aim of this review is to present
the findings of several studies published in the recent
years that contributed to clarifying the role of nervous
and neuroendocrine regulation of the pathophysiologic
events associated with cholestasis and cholangiocarcino-
ma development. This manuscript is organized into two
parts. The first part offers an overview of the innervation
of the liver and the origin of neuroendocrine hormones,
neurotransmitters and neuropeptides affecting cholan-
giocyte function and metabolism. The first section also
reviews the effects played by several neuroendocrine
hormones and nervous system on cholangiocyte growth,
survival and functional activity in the course of cholesta-
sis. In the second section, we summarize the results of
some studies describing the role of nervous system and
neuroendocrine hormones in the regulation of malignant
cholangiocyte growth.
World J Gastroenterol 2006 June 14; 12(22): 3471-3480
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
2006 The WJG Press. All rights reserved.
Key words: Cholangiocyte; Neuroendocrine hormones;
Neurotransmitters; Neuropeptides; Cholestasis; Nervous
System; Biliary carcinogenesis; Pathophysiology; Cholan-
giocarcinoma; Proliferation
Marzioni M, Fava G, Benedetti A. Nervous and Neuro-
endocrine regulation of the pathophysiology of cholestasis
and of biliary carcinogenesis. World J Gastroenterol 2006;
12(22): 3471-3480
http://www.wjgnet.com/1007-9327/12/3471.asp
INTRODUCTION
Cholangiocytes are the epithelial cells that line the intra-
hepatic biliary tree. Their physiologic role is to modify
the bile of canalicular origin, through a wide array of
absorbtive and secretive processes[1,2]. Indeed, although
in normal conditions they represent approximately the
4%-5% of the liver mass, they contribute for at least the
10%-30% of the bile flow[1].
Cholangiocytes are the target of chronic diseases,
termed cholangiopathies[3,4], that are commonly character-
ized by dysregulation of the balance between cell growth
and survival. In the course of such diseases there is im-
paired proliferative response to duct injury and increased
cell death by apoptosis, that leads to vanishing of bile
ducts[3] and functional liver failure at the end stage. Chol-
angiopathies thus represent a daily challenge for the clini-
cians, since definitive medical treatments are not available
yet. As a consequence, 20% of liver transplants among
adults and 50% of those among pediatric patients are due
to these disorders[5].
The malignant transformation of cholangiocytes gives
origin to cholangiocarcinoma[6]. Cholangiocarcinoma is of-
ten diagnosed at late stages; its surgical resection has given
very limited success, as response of this neoplasm to con-
ventional chemotherapy[6] is minimal. These features make
cholangiocarcinoma a malignancy characterized by a very
poor prognosis. In addition, the incidence and prevalence
of cholangiocarcinoma are increasing worldwide[7].
Many of the problems that arise in the management
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 3472 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol July 7, 2006 Volume 12 Number 25

of cholangiopathies and of cholangiocarcinoma can at Table 1 Neuroendocrine hormones affecting cholangiocyte

least in part be ascribed to the poor knowledge about biology
their pathophysiology. Very little is in fact known on
which factors are able to affect cholangiocyte biology, Hormone Receptor Effect on cholangiocyte biology Reference
with particular regard to those endogenous molecules
or systems that affect either cholangiocyte proliferation, Secretin SR Stimulates HCO3- secretion through 1, 34-43
survival or functional activity[4]. the increase of cAMP/PKA and
opening of CFTR
Bile acids are endogenous factors that have been
historically investigated more in this regard, and their Somatostatin SSTR2 Counteracts the effect of secretin; 44, 45
stimulates bile absorption
properties in modulating cholangiocyte biology and
cholangiocarcinoma development are now unanimously Insulin IR Counteracts the effect of secretin 46

accepted[8-13]. More recently, several studies demonstrated
that many hormones, neuropeptides and neurotransmitters
are able to affect malignant and non malignant cho-
langiocyte biology as well. This data showed that the
biliary epithelium is similar to the gastric or intestinal
epithelia, for which the key role played by nerves and
neuroendocrine hormones has been known for years[14].
Moreover, it has been shown that in the course of chronic
cholestasis, cholangiocytes acquire a neuroendocrine
phenotype, that is not proper for these cells in normal
conditions [15]. Immunohistochemical studies have also
reported that many biliary tract carcinomas (up to the 70%
of the cases) show a neuroendocrine differentiation[16,17].
The purpose of this review is therefore to summarize
the evidences from the several studies published in the
recent years that contributed to clarify the role of nervous
and neuroendocrine regulation of the pathophysiologic
events associated with cholestasis and cholangiocarcinoma
development. In particular, we will focus on how these
factors affect cholangiocyte cell biology. We suggest those
who might be interested to learn the effects of nerves and
neuroendocrine hormones on other liver cell type biology
to other reviews recently published on this subject[18-20].
ORIGIN OF NEUROENDOCRINE
HORMONES, NEUROTRANSMITTERS
AND NEUROPEPTIDES AFFECTING
CHOLANGIOCYTES
Neuroendocrine hormones are secreted by neuroendocrine
cells, that are diffusely present in the whole gastrointestinal
tract, in particular the stomach, small bowel[21], as well
as pancreas [22]. If these cells are considered the main
source of the neuroendocrine hormones, in this review
some studies suggest that cholangiocytes them-selves
can synthesize some of these peptides in the course of
cholestasis.
The liver is innervated by both sympathetic and para-
sympathetic nerves, whose fibers are located around the
hepatic artery, portal vein, intrahepatic and extrahepatic
bile ducts [23,24]. Sympathetic nerves originate from the
celiac ganglion, whereas the parasympathetic from the
vagus nerve[23,24]. Besides catecholamines and acetylcholine,
autonomic fibers that innervate the liver can also release
other neurotransmitters, like neuropeptide Y (NPY)[25,26],
calcitonin gene related peptide (CGRP), somatostatin,
vasoactive intestinal polypeptide (VIP), enkephalin and
bombesin [27-30]. Nerve terminations mostly follow the
vascular structures of the portal tract, and have been
ET-1 ETA-ETB Counteracts the effect of secretin; 47
reduces the SR expression
VIP unspecified Increases HCO3- and water secretion 48, 49
Bombesin unspecified Increases HCO3- and water secretion 50
Gastrin CCK-B/ Counteracts the effect of secretin; 40, 51
gastrin reduces cell proliferation
Estrogens ER-ER Stimulate cell proliferation 52-55
Serotonin 5HT1A- Synthesized and secreted by 58
5HT1B cholangiocytes in the course of
cholestasis. Counteracts the effect of
secretin and reduces cell growth
GH/IGF-1 GH-R-IGF- Stimulate cell proliferation 59
1R
identified around bile ducts and vessels. Many of the
above mentioned neurotransmitters have been shown to
modulate intrahepatic hemodynamics[31-33].
NERVOUS AND NEUROENDOCRINE
REGULATION OF THE PATHOPHYSIOLOGY
OF CHOLESTASIS
Regulation of cholangiocyte growth, survival and func-
tional activity by neuroendocrine hormones (Table 1)
The close link between cholangiocytes and neuroendocrine
hormones has its basis on the early studies that showed the
hormone secretin as the most potent regulator of cholan-
giocyte functional activity[34,35]. In 1988, Alpini et al demon-
strated that in a rat model of cholestasis (induced by bile
duct ligation, BDL) the infusion of secretin was associated
with a marked increase of the bile flow and bicarbonate
biliary excretion[35]. After that "landmark" manuscript, a
large series of investigations later defined the intracellular
mechanisms by which secretin induces such a potent func-
tional stimulus. It is now known that secretin stimulates
ductal secretion[1,34-36] by selective interaction with secretin
receptors, expressed only by cholangiocytes in rat liver[37].
The interaction of secretin with its own receptors [37]
leads to an increase in intracellular cAMP levels[1,36,38-40],
activation of PKA[41], opening of CFTR Cl- channels[42]
with activation of the Cl-/HCO3- exchanger[1,36,41,43], which
leads to secretion of bicarbonate into bile[35].
Other neuroendocrine hormones were then discovered
to affect cholangiocyte choleretic activity. One of the first
molecules studied was somatostatin. It was demonstrated
that cholangiocytes express the SSTR 2 receptor; the
interaction of somatostatin with this receptor markedly
diminished the effect of secretin on the biliary excretion

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 Marzioni M et al. Nervous and Neuroendocrine regulation of cholangiopathies
of water and bicarbonate by cholangiocytes in cholestatic
conditions [44]. Such an effect of somatostatin was due
to the fact that the activation of the SSTR 2 receptor
prevented the increase of the adenylyl-cyclase elicited by
secretin[44]. In later studies conduced in IBDUs isolated
from wild type and SSTR2-knock out mice, it was also
found that somatostatin not only reduces cholangiocyte
choleresis, but it also stimulates ductal bile absorption[45].
Cholangiocytes also express at the apical pole the
receptor for insulin [46]. Upon its activation, a marked
reduction of the secretin-induced choleresis was observed,
both if the hormone was administered in vivo to BDL rats
and if microinjected into the lumen of IBDUs isolated
from animals with cholestasis. It was observed that the
activation of the insulin receptor determined a cascade
of intracellular events that resulted in the inhibition
of secretin-stimulated cAMP and PKA activity. Such
a chain of events seemed to have its core event in the
enhancement of the intracellular Ca 2+ levels and the
consequent activation of the Ca2+-dependent PKC[46].
An effect similar to the one of insulin has been described
for endothelin-1 (ET-1), which interacts with the specific
receptors expressed in the biliary epithelium (ETA and
ETB) and blunts the secretin-induced choleresis of the
BDL rat and reduces the expression of the secretin
receptor on cholangiocytes[47].
In contrast to somatostatin and insulin, vasoactive
intestinal polypeptide (VIP) and bombesin have been
found to be able to enhance cholangiocyte choleresis. Both
the hormones induced a potent fluid and bicarbonate
excretion in IBDUs, but not in hepatocytes, isolated from
normal and cholestatic rats. Interestingly, neither VIP
nor bombesin had any significant effect on modulating
intracellular cAMP levels [48-50] . Detailed pH studies
indicated that the underlying intracellular mechanism, at
least for bombesin, is its ability to stimulate the activity of
Cl-/HCO3- exchange in association with a counterbalancing
secondar y activation of electrog enic Na + /HCO 3 -
symport[50].
In more recent studies, increasing evidence regarding
the ability of neuroendocrine hormones to affect cholan-
giocyte growth, and functional activity, hvee been reported.
If the infusion of gastrin to BDL rats is associated
with the reduction of the choleretic response to secretin
by cholangiocytes[40], its chronic administration through
an intraperitoneal minipump resulted not only in reduced
functional activity, but also in a marked decrease of the
bile duct mass [51]. Cholangiocytes indeed express the
CCK-B/gastrin receptors [51], which, upon activation,
elicit the intracellular Ca 2+ release, the increase of IP 3
levels, and the membrane translocation (e.g. activation)
of the Ca 2+-dependent PKC [51]. In turn, the gastrin-
activated PKC, as above mentioned, is able to interfere
with the secretin signaling and modulate the adenyl-
cyclase activity, thus reducing the intracellular cAMP levels
and PKA activity. These observations, demonstrated
that bile acids also activate this intracellular pathway to
modulate cholangiocyte growth[8], contribute to elucidate
which intracellular signalings sustain the proliferative
response of cholangiocytes to cholestasis and by which
mechanisms it is possible to modulate them. Moreover,
3473
since cAMP/PKA resulted the key molecules implicated
in cholangiocyte proliferation, this helped to explain the
association between increase of duct mass and enhanced
biliary choleresis[8,35].
A major contribution to this field of research has
been given by the studies that clarified the effects of
estrogens on the biliary epithelium. Both the estrogen
receptor (ER) and were observed in cholangiocytes[52],
their expression being up-regulated after BDL[53]. When
cholangiocytes were stimulated in vitro with 17--estradiol,
their proliferation was markedly increased, as a con-
sequence of the ER-dependent activation of Src/
Shc/ERK1/2 intracellular pathway [54]. To demonstrate
the physiological and pathophysiological relevance of
estrogens on cholangiocyte proliferative response to
cholestasis, when BDL male rats were treated in vivo with
antiestrogens like tamoxifen or Ici 182 780[52] or when BDL
female rats were subjected to ovariectomy[53], the growth
of the biliary tree was blunted and the biliary epithelium
underwent programmed cell death by apoptosis [52,53] .
This evidence suggested that estrogens are required for
a separative response of the biliary tree to injury. These
studies produced in the last few years by Alvaro represent
a significant change in the knowledge of the role played
by the neuroendocrine system on the biliary cell biology.
Indeed, primary biliary cirrhosis (PBC), the most common
from of cholangiopathies[4], is much more frequent in
women than in men, and has its clinical outcome typically
after menopause, when the endogenous estrogen levels
suddenly drop[55]. Therefore, these observations by Alvaro
seem to provide the biological confirmation of the role
of estrogens in the progression of PBC, a role that was,
earlier, only hypothesized on the basis of epidemiological
data. To further support this concept, Alvaro also
demonstrated that the ER expression in cholangiocytes
is markedly reduced in late stages of PBC[55]. Altogether,
Alvaros studies made clear that gaining knowledge on
the role of neuroendocrine hormones on cholangiocyte
biology might also be an effective strategy to further
understand the pathophysiology of the cholangiopathies
and thus eventually to design novel therapeutic strategies.
The neuroendocrine hor mone serotonin has
been hypothesized to be involved in the g enesis
of certain clinical features of PBC, like fatigue and
pruritus [56,57]. Interestingly, it has been recently shown
that cholangiocytes express the serotonin 1A and 1B
receptors[58]. When they are activated by selective agonists,
the proliferation of BDL cholangiocytes are dramatically
reduced, both in vivo and in vitro[58], in association with
the loss of the response to secretin of the markers of
cholangiocyte functional activity[58], like the bile flow, the
bicarbonate excretion and the intracellular cAMP levels[34].
Such an effect seems more to be mediated by the cross-
talk between the Ca2+ and the cAMP/PKA signalings. If
the serotonin 1A and 1B receptors are activated, there
is an increase of Ca 2+, IP 3 and PKC levels, with the
consequent reduction of the intracellular cAMP levels
and PKA activity. Most interestingly, it was observed that
hyperplastic cholangiocytes isolated from BDL rats are
able to synthesize and secrete serotonin.[58] If, in vitro or
in vivo, cholangiocyte-secreted serotonin is neutralized,
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 3474 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Cholestasis
Cholangiocytes
Neuroendocrine hormone
Neuropeptide
Figure 1 Proposed working model for the existence of an autocrine/paracrine
loop of neuroendocrine hormones and neuropeptides that regulate the proliferative
response of cholangiocytes to cholestasis. A chronic cholestatic condition induces
cholangiocyte proliferation and their transdifferentiation in neuroendocrine-like cell.
This allows the biliary epithelium to start to synthesize and secrete a number of
peptides that aim to counterbalance the effects of cholestasis itself on cell growth,
as a sort of negative feed-back.
cholangiocyte proliferation in response to cholestasis
further increases[58]. This study, therefore, proposes an
additional novel working model (as shown in Figure 1):
in the course of cholestasis, cholangiocytes acquire a
neuroendocrine phenotype that allow them to synthesize
some neuroendocrine hormones, like serotonin. Once
secreted, these hormones aim to counterbalance the
excessive proliferative response of the biliary epithelium. It
is thus possible to postulate the existence of an autocrine/
paracrine loop of peptides and neuroendocrine hormones
that modulate with a negative (or an eventually positive)
feed-back the response of cholangiocytes to liver injury[58].
The investigation of the presence of other peptides and
hormones that are likely to participate with serotonin
in this autocrine/paracrine loop might thus be needed
to understand certain still obscure clinical aspects of
cholangiopathies.
Recent evidence demonstrated that a stimulatory
autocrine/paracrine loop should also exist. It has been
found that cholangiocytes are the target of the growth
hormone (GH)-insulin like growth factor (IGF)-1 axis[59]:
GH induces IGF-1 expression and release in isolated
cholangiocytes, with the consequent stimulation of cell
growth by IGF-1[59].
Regulation of cholangiocyte growth, survival and
functional activity by neurotransmitters (Table 2)
A number of studies showed that cholinergic nerves
regulate bile secretion [60,61] . In bile-fistula dogs with
interrupted enterohepatic circulation, distal stimulation
of the vagus nerve increases bile bicarbonate secretion,
whereas vag otomy decreases basal bile f low and
bicarbonate output[60,61].
In early studies [62] it seemed that cholangiocytes
might express both the M1 and the M3 acetylcholine
(ACh) receptor subtypes and that the cholinergic agonist,
carbachol, stimulates bile flow of the isolated perfused rat
liver. These observations were not confirmed by successive
investigations. In 1996, Nathanson et al demonstrated
that, in accordance with the concept that cholangiocytes,
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July 7, 2006 Volume 12 Number 25
Table 2 Neurotransmitters/neurotrophins affecting cholangi-
ocyte biology
Peptide Receptor Effect on cholangiocyte biology Reference
Acetylcholine M3 Potentiates the effect of secretin. 43, 60,
Required for cholangiocyte response 61, 64,
to BDL: sustains cholangiocyte 65
proliferation and prevents apoptosis
Epinephrine/ 1 Potentiates the effect of secretin 67
Norephinephrine
Epinephrine/ 1-2 Required for cholangiocyte response 70
Norephinephrine to BDL: sustains cholangiocyte
proliferation and prevents apoptosis
Dopamine D2 Inhibits secretin-induced ductal 73
secretion
NGF TrkA Secreted by hyperplastic, cholestatic 74
cholangiocytes; sustains the
rpoliferative response to BDL
but not hepatocytes, express ACh receptors, ACh did
not affect the functions of hepatocytes, but elicited Ca2+
increase and oscillation in IBDUs, due to both an influx
of extracellular Ca2+ and the mobilization of thapsigargin-
sensitive Ca2+ stores[63]. A year later, Alvaro et al showed
that cholangiocytes express, at the basolateral domain, M3
(but not M1 and M2) Ach receptors.[43] In that study it was
shown that ACh has no effect on the basal activity of the
Cl-/HCO3- exchanger, but it significantly potentiates the
stimulatory effect of secretin on this anion exchanger[43].
Presumably, the significance of ACh-regulation of
bicarbonate secretion by bile ducts is to be researched in
the need of bicarbonate-enriched secretion in the small
intestine during the digestive phase, when, indeed, the
parasympathetic system activity is high. By selectively
interacting with M3 receptor subtypes, ACh induces a Ca2+-
calcineurin mediated potentiation of the secretin-induced
adenylyl cyclase activity, which leads to activation of the
Cl -/HCO 3- exchanger with bicarbonate secretion into
bile[43]. Furthermore, recent studies have shown that ACh
sustains cholangiocyte proliferation, since interruption of
the cholinergic innervation by vagotomy induces a marked
decrease in total bile duct mass caused by both impaired
cholangiocyte proliferative capacity and intracellular cAMP
levels, and enhanced cell death by apoptosis[64,65]. These
studies also show that maintenance of intracellular cAMP
levels (by chronic administration of forskolin) prevents
the effects of vagotomy on cholangiocyte apoptosis,
proliferation and secretion[65].
There is growing information regarding the role of
adrenergic and dopaminergic innervation in the regulation
of cholangiocyte proliferation and secretion. An intact
sympathetic inner vation is required for hepatocyte
and cholangiocyte proliferation following partial hepa-
tectomy [66] . Recent studies demonstrated that: (1)
cholangiocytes from BDL rats express alpha-1, alpha-2,
beta-1 and beta-2 adrenergic receptors; and (2) alpha-1
(but not beta-1) adrenergic receptor agonists increase
secretin-stimulated ductal secretion[67]. Similar to what is
shown in the gut, adrenergic innervation may play a role
in counterbalancing the stimulatory effects of cholinergic
 Marzioni M et al. Nervous and Neuroendocrine regulation of cholangiopathies
nerves[65] on ductal bile secretion in chronic cholestatic
liver diseases.
In support of the concept that adrenergic innervation
plays an important role in the regulation of cholangiocyte
functions, administration of a single intraportal injection
of 6-hydroxidopamine, which induces degeneration of
dopaminergic terminal fibers[68,69], blunts the cholangiocyte
functional and proliferative response to cholestasis and
induces cell death by apoptosis[70]. Chronic administration
of clenbuterol (a beta-2 adrenergic agonist) [71] and
dobutamine (a beta-1 adrenergic agonist) [72] prevents
the decrease in cAMP levels and secretion induced by
6-OHDA, maintains cholangiocyte proliferation and
decreases cholangiocyte apoptosis due to 6-OHDA [70].
Furthermore, it has been shown that cholangiocytes
express the D2 (but not the D1 and D3) dopaminergic
receptors and that the D2 dopaminergic ag onist,
quinelorane inhibits secretin-induced ductal secretion in
BDL rats through activation of the Ca2+-dependent PKC
gamma (but not PKC alpha, beta I and II) and inhibition
of secretin-stimulated cAMP levels and PKA activity[73].
The relationship between the biliary epithelium
and nerves appears to be more than what was thought
at the beginning. Cholangiocytes express the receptor
for neurotrophin Ner ve Growth Factor (NGF), the
activation of which strongly promotes biliar y cell
growth[74]. Moreover, it has been found that cholangiocytes
themselves can produce and secrete NGF and that when
this neurotrophin is immunoneutralized the growth of the
biliary tree in the BDL rat is strongly diminished[74].
Altogether, these data suggest that the autonomic
innervation plays a substantial role in the regulation of
cholangiocyte biology, thus enlightening the need of
studying whether the liver denervation after transplantation
might affect the functions of the grafted biliary tree.
Of major interest would also be to investigate whether
other endogenous factors, instead of nerves, can support
cholangiocyte functions in the denervated organ. In
this view, some evidence suggest that bile acids could
be important. It has been found that administration of
taurocholic[64] or ursodeoxycholic acid[75] counteracts the
loss of bile ducts induced by cholinergic denervation in
the BDL rat. Similarly, taurocholic acid administration
also prevents the loss of bile ducts induced by adrenergic
denervation[76].
NERVOUS AND NEUROENDOCRINE REGU-
LATION OF BILIARY CARCINOGENESIS
Cholangiocarcinoma, the primary cancer that originates
from the epithelial cells lining the bile ducts, is a
devastating malignancy characterized by poor prognosis
and high mortality[6,77]. The only curative treatment, even
if only possible at an early stage of this neoplasm, is
surgical. However, patients often present this tumor at
an advanced stage, when a curative surgery is unlikely.
In fact, at the time of diagnosis, more than two-thirds
of patients affected by biliary tract cancer present as an
unresectable disease [78] and patients with an operable
tumor have a high rate of recurrence. Overall survival rate,
3475
Table 3 Neuroendocrine hormones affecting cholangio-
carcinoma cell biology
Hormone Receptor Effect on cholangiocarcinoma cell Reference
biology
Estrogens ER Stimulated SK-ChA-1 human 81
cholangiocarcinoma cell growth
Tamoxifen induced dose-dependent 81
growth inhibition of OZ and SK-ChA-1
human cells in vitro; reduced growth
of a SK-ChA-1 tumor cell xenografts
implanted in athymic nude mice
Tamoxifen stimulated SK-ChA-1 82
human cholangiocarcinoma apoptotic
cell death
Tamoxifen induced human cholangi- 83, 84
ocarcinoma cell apoptosis in vitro and
inhibited tumor xenograft growth after
pretreatment with IFN-gamma
Gastrin CCK-B Inhibited Mz-ChA-1, HuH-28, and 90
/gastrin TFK-1 human cell lines proliferation
and induced Mz-ChA-1 cell apoptosis
CCK CCK Reduced the growth of SLU-132 93
human tumor xenografts implanted in
nude mice; stimulated the release of
carcinoembryonic antigen (CEA) by
SLU-132 cells
including resected patients is quite poor, with less than 5%
of patients surviving 5 years, a rate which has remained
unchanged over the past 30 years[77]. Chemotherapy and
radiation therapy have been used in an attempt to control
this disease and improve survival and quality of life
of patients with unresectable, recurrent and metastatic
cholangiocarcinoma, but these therapies have not shown
to be effective in prolonging long-term survival. For these
reasons there is a compelling need to discover novel mol-
ecules and agents to target the cholangiocarcinoma cells
and to regulate their destructive growth[79]. There is grow-
ing information regarding the role of nerves and neuro-
peptides as modulators of cholangiocyte function and me-
tabolism[14]. Moreover, several reports suggest that nervous
stimuli are involved in the regulation of growth of biliary
malignancies[16,17,80].
Regulation of malignant cholangiocyte growth by
neuroendocrine hormones (Table 3,Table 4)
The involvement of neuroendocrine system in regulating
cholangiocarcinoma cell proliferation has been suggested
by several studies. Tamoxifen, an estrogen antagonist,
cause a dose-dependent decrease of proliferation of two
human cholangiocarcinoma cell lines in vitro[81]. On the
other hand, 17- estradiol stimulate human cholangiocarci-
noma cell growth in vitro[81]. In addition, tamoxifen induced
a growth reduction of a cholangiocarcinoma tumor
implanted in athymic nude mice[81]. Tamoxifen exerts its
inhibitory effect in human cholangiocarcinoma cells by
stimulating apoptotic cell death and this is likely mediated
through the Fas/APO-1 (CD95) signaling pathway via a
calmodulin-dependent mechanism[82].
A further study showed that tamoxifen exposure to
human cholangiocarcinoma after pre-treatment with
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 3476 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
IFN-gamma allows for induction of apoptosis in vitro
and significant inhibition of tumor growth[83]. Thus, the
combination of IFN-gamma and estrogen antagonists,
including tamoxifen, could be useful to have a sustained
and valid inhibitory effect on cholangiocarcinoma cell
growth[83,84].
Gastrointestinal polypeptide hormones regulate the
growth of various normal gastrointestinal tissues as well
as certain visceral cancers. Gastrin, which belongs to
the superfamily of cholecystokinin receptors (CCK-A,
CCK-B/gastrin receptors), is a trophic factor within the
normal gastrointestinal tract and is also a mitogen for a
number of gastrointestinal and non-gastrointestinal tumors
such as gastric, colonic and pancreatic[85-88]. In contrast,
CCK-B/gastrin receptor signaling in the human pancreatic
cell lines MiaPaca-2 and Panc-1 leads to inhibition of cell
growth[89]. It has been shown that malignant cholangiocytes
express gastrin receptors[90] and that gastrin, interacting
with CCK-B/gastrin receptors, inhibited the proliferation
of Mz-ChA-1, HuH-28 and TFK-1 cholangiocarcinoma
cell lines, also inducing apoptosis in Mz-ChA-1 cells by
activation of the Ca2+-dependent PKC- signaling[90]. The
blockage of CCK-B receptors did not totally reverse the
inhibitory effect of gastrin on Mz-ChA-1 cell growth.
This finding could be explained by the fact the gastrin may
exert its effect not only through CCK-B/gastrin receptors,
but also through other receptor types, as suggested by
studies in other carcinomas cell lines (e.g. colonic cancer
cells)[91]. This partial inhibitory effect by CCK-B/gastrin
receptor inhibitors might also be due to altered processing
of the CCK/B receptor by malignant transfor med
cells, as previously described [92]. A previous study by
Hudd et al showed that human cholangiocarcinoma
cells expressed CCK receptors and chronic treatment
with CCK octapeptide reduced the growth of human
cholangiocarcinoma tumors implanted in nude mice[93].
Somatostatin receptors (SS), most commonly the SS
receptor type 2 (SSTR2), have been described in several
neuroendocrine and epithelial malignancies[94], such as
in malignant cholangiocytes[95,96]. Studies demonstrated
that somatostatin and its analogues inhibit in vitr o
cholangiocarcinoma cell proliferation. Indeed, chronic
administration of lanreotide, a long-acting SS analogue,
reduces the growth of human cholangiocarcinoma cells
when implanted in athymic mice[95,96] and the inhibitory
effect of somatostatin in cholangiocarcinoma cell growth
was accompanied by no changes in cellular cyclic adenosine
monophosphate (cAMP) or calcium intracellular levels.
Furthermore, Zhao et al showed that octreotide inhibits
cholangiocarcinoma cells growth through G0/G1 cell
cycle arrest rather than through the process of apoptosis.
These effects were partially mediated by enhancing the
expression of p27kip1, and by decreasing the amounts of
cyclin E-CDK2 complex[95]. Taken together, these findings
suggested possible use of SS analogues in the diagnosis or
therapy of cholangiocarcinoma. However, a subsequent
phase II study showed absence of therapeutic efficacy of
the somatostatin analogue lanreotide in the treatment of
advanced primary hepatic cholangiocellular cancer and
gallbladder adenocarcinoma, despite in vivo somatostatin-
receptor expression[97].
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July 7, 2006 Volume 12 Number 25
Table 4 Neuroendocrine hormones affecting cholangio-
carcinoma cell biology
Hormone Receptor Effect on cholangiocarcinoma cell biology Reference
Somatostatin, SSTR2 Inhibited human cholangiocarcinoma 96
analogues cell proliferation in vitro and reduced
(octreotide, human cholangiocarcinoma cell
lanreotide) growth implanted in athymic mice
Inhibited RBE, NEC, QBC939, and 95
SSP-25 cell proliferation in vitro
through cell cycle arrest and QBC939
xenografts growth
GABA GABAA,-B,-C Inhibited human Mz-ChA-1, HuH-28 98
and TFK-1 cell proliferation in vitro;
reduced malignant cholangiocyte
migration. Reduced Mz-ChA-1
xenograft tumor growth implanted in
athymic mice
NPY NPY-Y5 Inhibited Mz-ChA-1 cell proliferation 99
in vitro
Liver represents the most important site of GABA
synthesis and metabolism outside the central nervous
system. Recently, our group demonstrated that cholangio-
carcinoma cells express GABAA,-B,-C recep-tors and respond
to GABA stimulation with growth inhibition. GABA
inhibitory effect on malignant cholangiocyte proliferation
was evident in vitro and also in vivo, by reducing the growth
of cholangiocarcinoma tumors injected subcutaneously in
nude mice. Moreover, GABA has been shown to be able
to inhibit malignant cholangiocyte migration, a peculiar
characteristic of the cholangiocarcinoma cells[98].
Another neurotransmitter available in the liver
parenchyma and biliary tract is the neuropeptide Y
(NPY) [99] . Recent preliminar y data from our group
showed that NPY inhibits cholangiocarcinoma growth
by interaction with a G-protein coupled receptor
by C a 2 + d e p e n d e n t m o d u l a t i o n o f S r c / E R K 1 / 2
phosphorylation[99].
Regulation of malignant cholangiocyte growth by
neurotransmitters (Table 5)
Autonomic nervous system regulates the growth of several
tumors[100-102]. For example, alpha-blockers, terazosin and
doxazosin, suppress prostate growth by inducing apopto-
sis among the epithelial cells in the benign and malignant
prostate [103]. Also, phenylephrine reduced HepG 2 cell
growth through alpha1B-adrenergic receptors activation[104].
Moreover, activation of a -2 adrenergic receptor/Gs
fusion protein leads to the inhibition of cAMP-sensitive
S49 lymphoma and carcinoma carB cells proliferation
in vitro[105].
A specific role of sympathetic nervous system in the
regulation of cholangiocarcinoma growth has been de-
scribed by Kanno et al, that showed that cholangiocarci-
noma cell lines Mz-ChA-1 and TFK-1 express the 2A-,
[80]
2B-, 2C- adrenergic receptor subtypes . Furthermore,
stimulation of malignant cholangiocytes by UK14, 304, an
2-adrenoreceptor agonist, causes up-regulation of cAMP,
which inhibits EGF-induced MAPK activity through
 Marzioni M et al. Nervous and Neuroendocrine regulation of cholangiopathies
Table 5 Neurotransmitters/neurotrophins affecting
cholangiocarcinoma cell biology
Peptide Receptor Effect on cholangiocarcinoma
cell biology
Epinephrine/ 2 UK 14,304 inhibited human Mz-
Norephinephrine ChA-1 and TFK-1 cell proliferation
in vitro
Acetylcholine M1 Carbachol produced an increase of
IP3 and Ca2+ intracellular levels in
Mz-ChA-1 cells
an acute increase of Raf-1 and sustained activation of
B-Raf [80], thus reducing tumor cell proliferation. Since
inhibition of cholangiocarcinoma cell growth through ac-
tivation of 2-adrenergic receptor occurred downstream
to Ras, this study suggested that adrenergic stimulation or
other stimulants of cAMP may overcome the Ras muta-
tions present in malignant cholangiocytes and offer a new
therapeutic approach in patients with cholangiocarcino-
ma[80].
Other studies showed that Mz-ChA-1 cells express
muscarinic acetylcholine (ACh) receptors[62]. In support
of the hypothesis that such receptors modulate cho-
langiocarcinoma cell growth, stimulation of malignant
biliary cells with carbachol, a muscarinic acetylcholine
receptor agonist, triggers IP3 formation and a subsequent
increase of intracellular Ca 2+ levels [62]. Even if several
studies show IP3 and Ca2+ levels play an important role
in the inhibition of cholangiocarcinoma cell growth after
stimulation with several molecules [13,90,98]. the specific
action of parasympathetic nervous system in modulating
cholangiocarcinoma cell growth is still unknown.
However, recent data showed that muscarinic receptors
are directly activated by other molecules. In fact, recent
studies showed that bile acids, interacting with M3 Ach
receptors, transactivate EGFR and stimulate colon cancer
cell proliferation by inducing p90RSK phosphorylation via
a calcium-, MEK- and MAPK-dependent pathways[106].
Recent findings suggest that cholangiocarcinoma
and other liver tumors could arise from activated hepatic
progenitor cells [107-109]. Several evidences indicate that
hepatic progenitor cell activation in diseased liver is
regulated by neural and neuroendocrine factors such
as vagal inner vation [110,111] , sug gesting an important
involvement of autonomic nervous system in developing
biliary carcinogenesis.
SUMMARY AND FUTURE PERSPECTIVES
The amount of evidences indicating the substantial
role of neuroendeocrine hormones, neuropeptides and
neurotransmitters in modulating malignant and non
malignant cholangiocyte biology is rapidly increasing.
There is still much to understand on how actually these
peptides create a network that affects the response of
biliary cells to liver injury. On the other hand the other
frontier is no doubt represented by the identification of
the most proper way of modulating such a network of
peptides. Will we be able to interfere with it in order to
3477
divert or relent the progression of cholangiopathies and
cholangiocarcinoma?
Reference
REFERENCES
[80] 1 Alpini G, McGill JM, Larusso NF. The pathobiology of biliary
epithelia. Hepatology 2002; 35: 1256-1268
2 Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G, Al-
[62] pini G. Functional heterogeneity of cholangiocytes. Semin Liver
Dis 2002; 22: 227-240
3 Desmet VJ. Vanishing bile duct disorders. Prog Liver Dis 1992;
10: 89-121
4 Lazaridis KN, Strazzabosco M, Larusso NF. The cholangiopa-
thies: disorders of biliary epithelia. Gastroenterology 2004; 127:
1565-1577
5 2001 Annual Report of the U.S. Organ Procurement and Trans-
plantation Network and the Scientific Registry for Transplant
Recipients: Transplant Data 1991-2000. Rockville, MD: US
Department of Health and Human Services, Health Resources
and Services Administration, Office of Special Programs, Divi-
sion of Transplantation; Richmond, VA: United Network for
Organ Sharing; Ann Arbor, MI: University Renal Research and
Education Association
6 Lazaridis KN, Gores GJ. Cholangiocarcinoma. Gastroenterology
2005; 128: 1655-1667
7 Patel T. Worldwide trends in mortality from biliary tract ma-
lignancies. BMC Cancer 2002; 2: 10
8 Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Francis H,
Phinizy JL, Angelico M, Lesage G. Ursodeoxycholate and tau-
roursodeoxycholate inhibit cholangiocyte growth and secre-
tion of BDL rats through activation of PKC alpha. Hepatology
2002; 35: 1041-1052
9 Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H,
Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, Lesage
G. Bile acid depletion and repletion regulate cholangiocyte
growth and secretion by a phosphatidylinositol 3-kinase-de-
pendent pathway in rats. Gastroenterology 2002; 123: 1226-1237
10 Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis
H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD. Bile acid
feeding induces cholangiocyte proliferation and secretion: evi-
dence for bile acid-regulated ductal secretion. Gastroenterology
1999; 116: 179-186
11 Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL, Francis H,
Lesage G. Bile acid feeding increased proliferative activity and
apical bile acid transporter expression in both small and large
rat cholangiocytes. Hepatology 2001; 34: 868-876
12 Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE,
Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional ex-
pression of the apical Na+-dependent bile acid transporter in
large but not small rat cholangiocytes. Gastroenterology 1997;
113: 1734-1740
13 Alpini G, Kanno N, Phinizy JL, Glaser S, Francis H, Taffetani
S, LeSage G. Tauroursodeoxycholate inhibits human cholan-
giocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent
pathways. Am J Physiol Gastrointest Liver Physiol 2004; 286:
G973-G982
14 Shetzline MA, Liddle RA. Gastrointestinal hormones and
neurotransmitters. In: Feldman M, Friedman LS, Sleisenger
MH, Sleisenger & Fordtrans Gastrointestinal and Liver Dis-
ease. Philadelphia, U.S.: Saunders, 2002: 3-20
15 Roskams T, van den Oord JJ, De Vos R, Desmet VJ. Neuroen-
docrine features of reactive bile ductules in cholestatic liver
disease. Am J Pathol 1990; 137: 1019-1025
16 Chan C, Medina-Franco H, Bell W, Lazenby A, Vickers S.
Carcinoid tumor of the hepatic duct presenting as a Klatskin
tumor in an adolescent and review of world literature. Hepato-
gastroenterology 2000; 47: 519-521
17 Hsu W, Deziel DJ, Gould VE, Warren WH, Gooch GT, Staren
ED. Neuroendocrine differentiation and prognosis of extra-
hepatic biliary tract carcinomas. Surgery 1991; 110: 604-610;
discussion 610-611
18 Bioulac-Sage P, Lafon ME, Saric J, Balabaud C. Nerves and
www.wjgnet.com
 3478 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
perisinusoidal cells in human liver. J Hepatol 1990; 10: 105-112
19 Oda M, Han JY, Nakamura M. Endothelial cell dysfunction
in microvasculature: relevance to disease processes. Clin
Hemorheol Microcirc 2000; 23: 199-211
20 Pschel GP. Control of hepatocyte metabolism by sympathet-
ic and parasympathetic hepatic nerves. Anat Rec A Discov Mol
Cell Evol Biol 2004; 280: 854-867
21 Schneider DA, Sayegh AI. Gastrointestinal neuroendocrinol-
ogy. Vet Clin North Am Equine Pract 2002; 18: 205-217
22 Le Douarin NM. On the origin of pancreatic endocrine cells.
Cell 1988; 53: 169-171
23 Reilly FD, McCuskey PA, McCuskey RS. Intrahepatic distri-
bution of nerves in the rat. Anat Rec 1978; 191: 55-67
24 Tsuneki K, Ichihara K. Electron microscope study of verte-
brate liver innervation. Arch Histol Jpn 1981; 44: 1-13
25 Gulbenkian S, Wharton J, Hacker GW, Varndell IM, Bloom
SR, Polak JM. Co-localization of neuropeptide tyrosine (NPY)
and its C-terminal flanking peptide (C-PON). Peptides 1985; 6:
1237-1243
26 Lundberg JM, Terenius L, Hkfelt T, Martling CR, Tatemoto
K, Mutt V, Polak J, Bloom S, Goldstein M. Neuropeptide Y
(NPY)-like immunoreactivity in peripheral noradrenergic neu-
rons and effects of NPY on sympathetic function. Acta Physiol
Scand 1982; 116: 477-480
27 Costa M, Furness JB. Somatostatin is present in a subpopula-
tion of noradrenergic nerve fibres supplying the intestine.
Neuroscience 1984; 13: 911-919
28 Gibbins IL, Furness JB, Costa M, MacIntyre I, Hillyard CJ,
Girgis S. Co-localization of calcitonin gene-related peptide-like
immunoreactivity with substance P in cutaneous, vascular and
visceral sensory neurons of guinea pigs. Neurosci Lett 1985; 57:
125-130
29 Jul Y, Clerc N, Niel JP, Condamin M. [Met]- and
[Leu]enkephalin-like immunoreactive cell bodies and nerve
fibres in the coeliac ganglion of the cat. Neuroscience 1986; 18:
487-498
30 Schultzberg M, Dalsgaard CJ. Enteric origin of bombesin im-
munoreactive fibres in the rat coeliac-superior mesenteric gan-
glion. Brain Res 1983; 269: 190-195
31 Burt AD, Tiniakos D, MacSween RN, Griffiths MR, Wisse
E, Polak JM. Localization of adrenergic and neuropeptide
tyrosine-containing nerves in the mammalian liver. Hepatology
1989; 9: 839-845
32 Inoue N, Sakai H, Magari S, Sakanaka M. Distribution and
possible origins of substance P-containing nerve fibers in the
rat liver. Ann Anat 1992; 174: 557-560
33 Goehler LE, Sternini C, Brecha NC. Calcitonin gene-related
peptide immunoreactivity in the biliary pathway and liver of
the guinea-pig: distribution and colocalization with substance
P. Cell Tissue Res 1988; 253: 145-150
34 Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholan-
giocyte bicarbonate secretion. Am J Physiol Gastrointest Liver
Physiol 2001; 281: G612-G625
35 Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology
in rats with bile ductular cell hyperplasia. Evidence for a se-
cretory function of proliferated bile ductules. J Clin Invest 1988;
81: 569-578
36 Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV,
LeSage G, LaRusso NF. Morphological, molecular, and func-
tional heterogeneity of cholangiocytes from normal rat liver.
Gastroenterology 1996; 110: 1636-1643
37 Alpini G, Ulrich CD 2nd, Phillips JO, Pham LD, Miller LJ,
LaRusso NF. Upregulation of secretin receptor gene expres-
sion in rat cholangiocytes after bile duct ligation. Am J Physiol
1994; 266: G922-G928
38 Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV,
Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and
functional heterogeneity of cholangiocytes from rat liver after
bile duct ligation. Am J Physiol 1997; 272: G289-G297
39 Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri
A, LeSage G, LaRusso NF. Heterogeneity of the proliferative
capacity of rat cholangiocytes after bile duct ligation. Am J
Physiol 1998; 274: G767-G775
www.wjgnet.com
July 7, 2006 Volume 12 Number 25
40 Glaser SS, Rodgers RE, Phinizy JL, Robertson WE, Lasater J,
Caligiuri A, Tretjak Z, LeSage GD, Alpini G. Gastrin inhibits
secretin-induced ductal secretion by interaction with spe-
cific receptors on rat cholangiocytes. Am J Physiol 1997; 273:
G1061-G1070
41 Alvaro D, Mennone A, Boyer JL. Role of kinases and phos-
phatases in the regulation of fluid secretion and Cl-/HCO3-
exchange in cholangiocytes. Am J Physiol 1997; 273: G303-G313
42 Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regula-
tion of membrane chloride currents in rat bile duct epithelial
cells. J Clin Invest 1993; 91: 319-328
43 Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C, Fraioli
F, Romeo R, Marucci L, Le Sage G, Glaser SS, Benedetti A.
Role and mechanisms of action of acetylcholine in the regula-
tion of rat cholangiocyte secretory functions. J Clin Invest 1997;
100: 1349-1362
44 Tietz PS, Alpini G, Pham LD, Larusso NF. Somatostatin inhib-
its secretin-induced ductal hypercholeresis and exocytosis by
cholangiocytes. Am J Physiol 1995; 269: G110-G118
45 Gong AY, Tietz PS, Muff MA, Splinter PL, Huebert RC,
Strowski MZ, Chen XM, LaRusso NF. Somatostatin stimulates
ductal bile absorption and inhibits ductal bile secretion in mice
via SSTR2 on cholangiocytes. Am J Physiol Cell Physiol 2003;
284: C1205-C1214
46 Lesage GD, Marucci L, Alvaro D, Glaser SS, Benedetti A,
Marzioni M, Patel T, Francis H, Phinizy JL, Alpini G. Insulin
inhibits secretin-induced ductal secretion by activation of
PKC alpha and inhibition of PKA activity. Hepatology 2002; 36:
641-651
47 Caligiuri A, Glaser S, Rodgers RE, Phinizy JL, Robertson W,
Papa E, Pinzani M, Alpini G. Endothelin-1 inhibits secretin-
stimulated ductal secretion by interacting with ETA receptors
on large cholangiocytes. Am J Physiol 1998; 275: G835-G846
48 Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a po-
tent regulator of bile secretion from rat cholangiocytes. Gastro-
enterology 1999; 117: 420-428
49 Cho WK, Mennone A, Rydberg SA, Boyer JL. VIP is a potent
stimulus of bicarbonate and fluid secretion in bile ducts. Hepa-
tology 1995; 22: A752
50 Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin stim-
ulates bicarbonate secretion from rat cholangiocytes: implica-
tions for neural regulation of bile secretion. Gastroenterology
1997; 113: 311-321
51 Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno
N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G,
Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-
ligated rats by interaction with cholecystokinin-B/Gastrin
receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and
protein kinase C alpha-dependent mechanisms. Hepatology
2000; 32: 17-25
52 Alvaro D, Alpini G, Onori P, Perego L, Svegliata Baroni G,
Franchitto A, Baiocchi L, Glaser SS, Le Sage G, Folli F, Gaudio E.
Estrogens stimulate proliferation of intrahepatic biliary epithe-
lium in rats. Gastroenterology 2000; 119: 1681-1691
53 Alvaro D, Alpini G, Onori P, Franchitto A, Glaser S, Le Sage G,
Gigliozzi A, Vetuschi A, Morini S, Attili AF, Gaudio E. Effect
of ovariectomy on the proliferative capacity of intrahepatic rat
cholangiocytes. Gastroenterology 2002; 123: 336-344
54 Alvaro D, Onori P, Metalli VD, Svegliati-Baroni G, Folli F,
Franchitto A, Alpini G, Mancino MG, Attili AF, Gaudio E.
Intracellular pathways mediating estrogen-induced cholangio-
cyte proliferation in the rat. Hepatology 2002; 36: 297-304
55 Alvaro D, Invernizzi P, Onori P, Franchitto A, De Santis A,
Crosignani A, Sferra R, Ginanni-Corradini S, Mancino MG,
Maggioni M, Attili AF, Podda M, Gaudio E. Estrogen recep-
tors in cholangiocytes and the progression of primary biliary
cirrhosis. J Hepatol 2004; 41: 905-912
56 Jones EA, Bergasa NV. The pathogenesis and treatment of
pruritus and fatigue in patients with PBC. Eur J Gastroenterol
Hepatol 1999; 11: 623-631
57 Swain MG, Maric M. Improvement in cholestasis-associated
fatigue with a serotonin receptor agonist using a novel rat
model of fatigue assessment. Hepatology 1997; 25: 291-294
 Marzioni M et al. Nervous and Neuroendocrine regulation of cholangiopathies
58 Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Al-
varo D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J,
Fava G, Lesage GD, Alpini G. Autocrine/paracrine regulation
of the growth of the biliary tree by the neuroendocrine hor-
mone serotonin. Gastroenterology 2005; 128: 121-137
59 Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A, Bar-
baro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili AF,
Gaudio E. The intrahepatic biliary epithelium is a target of the
growth hormone/insulin-like growth factor 1 axis. J Hepatol
2005; 43: 875-883
60 Kaminski DL, Dorighi J, Jellinek M. Effect of electrical vagal
stimulation on canine hepatic bile flow. Am J Physiol 1974; 227:
487-493
61 Tanturi C, Ivy A. On the existence of secretory nerves in the
vagi for and the reflex excitation and inhibition of bile secre-
tion. Am J Physiol 1938; 121: 270-283
62 Elsing C, Hbner C, Fitscher BA, Kassner A, Stremmel W.
Muscarinic acetylcholine receptor stimulation of biliary epithe-
lial cells and its effect on bile secretion in the isolated perfused
liver [corrected]. Hepatology 1997; 25: 804-813
63 Nathanson MH, Burgstahler AD, Mennone A, Boyer JL. Char-
acterization of cytosolic Ca2+ signaling in rat bile duct epithe-
lia. Am J Physiol 1996; 271: G86-G96
64 Marzioni M, LeSage GD, Glaser S, Patel T, Marienfeld C,
Ueno Y, Francis H, Alvaro D, Tadlock L, Benedetti A, Marucci
L, Baiocchi L, Phinizy JL, Alpini G. Taurocholate prevents the
loss of intrahepatic bile ducts due to vagotomy in bile duct
ligated rats. Am J Physiol Gastrointest Liver Physiol 2003; 284:
G837-852
65 LeSagE G, Alvaro D, Benedetti A, Glaser S, Marucci L,
Baiocchi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis
H, Alpini G. Cholinergic system modulates growth, apoptosis,
and secretion of cholangiocytes from bile duct-ligated rats.
Gastroenterology 1999; 117: 191-199
66 Iwai M, Shimazu T. Alteration in sympathetic nerve activity
during liver regeneration in rats after partial hepatectomy. J
Auton Nerv Syst 1992; 41: 209-214
67 LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L, Ros-
kams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani S, Bar-
baro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic receptor
agonists modulate ductal secretion of BDL rats via Ca(2+)-
and PKC-dependent stimulation of cAMP. Hepatology 2004; 40:
1116-1127
68 Elliott KJ, Jones JM, Sacaan AI, Lloyd GK, Corey-Naeve J.
6-hydroxydopamine lesion of rat nigrostriatal dopaminergic
neurons differentially affects nicotinic acetylcholine receptor
subunit mRNA expression. J Mol Neurosci 1998; 10: 251-260
69 Lindfeldt J, Zoucas E, Ekelund M, Kullendorff CM, Holmin
T. The effect of intraportal six-hydroxy-dopamine on hemor-
rhage after standardized liver trauma in rats. J Trauma 1987;
27: 312-314
70 Glaser S, Alvaro D, Francis H, Ueno Y, Marucci L, Benedetti A,
De Morrow S, Marzioni M, Mancino MG, Phinizy JL, Reichen-
bach R, Fava G, Summers R, Venter J, Alpini G. Adrenergic re-
ceptor agonists prevent bile duct injury induced by adrenergic
denervation by increased cAMP levels and activation of Akt.
Am J Physiol Gastrointest Liver Physiol 2006; 290: G813-G826
71 Hinkle RT, Hodge KM, Cody DB, Sheldon RJ, Kobilka BK,
Isfort RJ. Skeletal muscle hypertrophy and anti-atrophy effects
of clenbuterol are mediated by the beta2-adrenergic receptor.
Muscle Nerve 2002; 25: 729-734
72 Barnes E, Dutka DP, Khan M, Camici PG, Hall RJ. Effect of
repeated episodes of reversible myocardial ischemia on myo-
cardial blood flow and function in humans. Am J Physiol Heart
Circ Physiol 2002; 282: H1603-H1608
73 Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H,
Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino
MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-
stimulated choleresis by increased PKC-gamma expression
and decrease of PKA activity. Am J Physiol Gastrointest Liver
Physiol 2003; 284: G683-G694
74 Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD, Gla-
ser SS, Francis H, Mancino MG, Ueno Y, Barbaro B, Benedetti
3479
A, Attili AF, Alvaro D. Nerve growth factor modulates the
proliferative capacity of the intrahepatic biliary epithelium in
experimental cholestasis. Gastroenterology 2004; 127: 1198-1209
75 Marzioni M, Francis H, Benedetti A, Ueno Y, Fava G, Venter J,
Reichenbach R, Mancino MG, Summers R, Alpini G, Glaser S.
Ca2+-dependent cytoprotective effects of ursodeoxycholic and
tauroursodeoxycholic acid on the biliary epithelium in a rat
model of cholestasis and loss of bile ducts. Am J Pathol 2006;
168: 398-409
76 Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Taf-
fetani S, Alvaro D, Phinizy JL, Baumann B, Venter J, Ueno Y,
Alpini G. Taurocholate feeding prevents the functional dam-
age of intrahepatic bile ducts induced by adrenergic denerva-
tion in a PI3K dependent manner. Hepatology 2003; 38: A28
77 Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD.
Cholangiocarcinoma. Lancet 2005; 366: 1303-1314
78 Thongprasert S. The role of chemotherapy in cholangiocarci-
noma. Ann Oncol 2005; 16 Suppl 2: ii93-ii96
79 Sirica AE. Cholangiocarcinoma: molecular targeting strategies
for chemoprevention and therapy. Hepatology 2005; 41: 5-15
80 Kanno N, Lesage G, Phinizy JL, Glaser S, Francis H, Alpini
G. Stimulation of alpha2-adrenergic receptor inhibits cholan-
giocarcinoma growth through modulation of Raf-1 and B-Raf
activities. Hepatology 2002; 35: 1329-1340
81 Sampson LK, Vickers SM, Ying W, Phillips JO. Tamoxifen-
mediated growth inhibition of human cholangiocarcinoma.
Cancer Res 1997; 57: 1743-1749
82 Pan G, Vickers SM, Pickens A, Phillips JO, Ying W, Thompson
JA, Siegal GP, McDonald JM. Apoptosis and tumorigenesis in
human cholangiocarcinoma cells. Involvement of Fas/APO-1
(CD95) and calmodulin. Am J Pathol 1999; 155: 193-203
83 Vickers SM, Jhala NC, Ahn EY, McDonald JM, Pan G, Bland
KI. Tamoxifen (TMX)/Fas induced growth inhibition of hu-
man cholangiocarcinoma (HCC) by gamma interferon (IFN-
gamma). Ann Surg 2002; 235: 872-878
84 Ahn EY, Pan G, Oh JH, Tytler EM, McDonald JM. The com-
bination of calmodulin antagonists and interferon-gamma in-
duces apoptosis through caspase-dependent and -independent
pathways in cholangiocarcinoma cells. Am J Pathol 2003; 163:
2053-2063
85 Ishizuka J, Martinez J, Townsend CM Jr, Thompson JC. The
effect of gastrin on growth of human stomach cancer cells.
Ann Surg 1992; 215: 528-534
86 Yassin RR, Clearfield HR, Little KM. Gastrins trophic effect
in the colon: identification of a signaling pathway mediated by
protein kinase C. Peptides 1993; 14: 1119-1124
87 Seva C, Dickinson CJ, Yamada T. Growth-promoting effects of
glycine-extended progastrin. Science 1994; 265: 410-412
88 Watson SA, Steele RJ. Gastrin antagonists in the treatment of
gastric cancer. Anticancer Drugs 1993; 4: 599-604
89 Detjen K, Fenrich MC, Logsdon CD. Transfected cholecysto-
kinin receptors mediate growth inhibitory effects on human
pancreatic cancer cell lines. Gastroenterology 1997; 112: 952-959
90 Kanno N, Glaser S, Chowdhury U, Phinizy JL, Baiocchi L,
Francis H, LeSage G, Alpini G. Gastrin inhibits cholangio-
carcinoma growth through increased apoptosis by activation
of Ca2+-dependent protein kinase C-alpha. J Hepatol 2001; 34:
284-291
91 Bold RJ, Alpard S, Ishizuka J, Townsend CM Jr, Thompson
JC. Growth-regulatory effect of gastrin on human colon cancer
cell lines is determined by protein kinase a isoform content.
Regul Pept 1994; 53: 61-70
92 Cookson MS, Reuter VE, Linkov I, Fair WR. Glutathione
S-transferase PI (GST-pi) class expression by immunohisto-
chemistry in benign and malignant prostate tissue. J Urol 1997;
157: 673-676
93 Hudd C, Euhus DM, LaRegina MC, Herbold DR, Palmer DC,
Johnson FE. Effect of cholecystokinin on human cholangio-
carcinoma xenografted into nude mice. Cancer Res 1985; 45:
1372-1377
94 Eden PA, Taylor JE. Somatostatin receptor subtype gene ex-
pression in human and rodent tumors. Life Sci 1993; 53: 85-90
95 Zhao B, Zhao H, Zhao N, Zhu XG. Cholangiocarcinoma cells
www.wjgnet.com
 3480 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
express somatostatin receptor subtype 2 and respond to oc-
treotide treatment. J Hepatobiliary Pancreat Surg 2002; 9: 497-502
96 Tan CK, Podila PV, Taylor JE, Nagorney DM, Wiseman GA,
Gores GJ, LaRusso NF. Human cholangiocarcinomas express
somatostatin receptors and respond to somatostatin with
growth inhibition. Gastroenterology 1995; 108: 1908-1916
97 Fiebiger WC, Scheithauer W, Traub T, Kurtaran A, Gedlicka
C, Kornek GV, Virgolini I, Raderer M. Absence of therapeutic
efficacy of the somatostatin analogue lanreotide in advanced
primary hepatic cholangiocellular cancer and adenocarcinoma
of the gallbladder despite in vivo somatostatin-receptor ex-
pression. Scand J Gastroenterol 2002; 37: 222-225
98 Fava G, Marucci L, Glaser S, Francis H, De Morrow S, Bene-
detti A, Alvaro D, Venter J, Meininger C, Patel T, Taffetani S,
Marzioni M, Summers R, Reichenbach R, Alpini G. gamma-
Aminobutyric acid inhibits cholangiocarcinoma growth by
cyclic AMP-dependent regulation of the protein kinase A/ex-
tracellular signal-regulated kinase 1/2 pathway. Cancer Res
2005; 65: 11437-11446
99 Fava G, Glaser S, Francis H, Phinizy J, Venter J, Reichenbach
R, Taffetani S, Marzioni M, Marucci L, Benedetti A, Alpini G.
Neuropeptide Y (NPY) inhibits cholangiocarcinoma growth
by interaction with a G-protein coupled receptor by a Ca2+
dependent modulation of Src/ERK1/2 phosphorylation. Gas-
troenterology 2004; 126 Suppl 2
100 Cardani R, Zavanella T. Decreased density of beta1-adrenergic
receptors in preneoplastic and neoplastic liver lesions of F344
rats. Histol Histopathol 2005; 20: 843-850
101 Fernando MA, Heaney AP. Alpha1-adrenergic receptor antag-
onists: novel therapy for pituitary adenomas. Mol Endocrinol
2005; 19: 3085-3096
102 Roumegure T. [Novel therapeutics strategies in benign hy-
pertrophy of prostate management]. Rev Med Brux 2005; 26:
S407-S411
103 Kyprianou N, Chon J, Benning CM. Effects of alpha(1)-adre-
www.wjgnet.com
July 7, 2006 Volume 12 Number 25
noceptor (alpha(1)-AR) antagonists on cell proliferation and
apoptosis in the prostate: therapeutic implications in prostatic
disease. Prostate Suppl 2000; 9: 42-46
104 Auer KL, Spector MS, Tombes RM, Seth P, Fisher PB, Gao B,
Dent P, Kunos G. Alpha-adrenergic inhibition of proliferation
in HepG2 cells stably transfected with the alpha1B-adrenergic
receptor through a p42MAPkinase/p21Cip1/WAF1-depen-
dent pathway. FEBS Lett 1998; 436: 131-138
105 Bertin B, Jockers R, Strosberg AD, Marullo S. Activation of a
beta 2-adrenergic receptor/Gs alpha fusion protein elicits a
desensitization-resistant cAMP signal capable of inhibiting
proliferation of two cancer cell lines. Receptors Channels 1997; 5:
41-51
106 Cheng K, Raufman JP. Bile acid-induced proliferation of a
human colon cancer cell line is mediated by transactivation of
epidermal growth factor receptors. Biochem Pharmacol 2005; 70:
1035-1047
107 Lee JH, Rim HJ, Sell S. Heterogeneity of the oval-cell re-
sponse in the hamster liver during cholangiocarcinogenesis
following Clonorchis sinensis infection and dimethylnitrosa-
mine treatment. J Hepatol 1997; 26: 1313-1323
108 Robrechts C, De Vos R, Van den Heuvel M, Van Cutsem E,
Van Damme B, Desmet V, Roskams T. Primary liver tumour of
intermediate (hepatocyte-bile duct cell) phenotype: a progeni-
tor cell tumour? Liver 1998; 18: 288-293
109 Steinberg P, Steinbrecher R, Radaeva S, Schirmacher P, Dienes
HP, Oesch F, Bannasch P. Oval cell lines OC/CDE 6 and OC/
CDE 22 give rise to cholangio-cellular and undifferentiated
carcinomas after transformation. Lab Invest 1994; 71: 700-709
110 Cassiman D, Libbrecht L, Sinelli N, Desmet V, Denef C, Ros-
kams T. The vagal nerve stimulates activation of the hepatic
progenitor cell compartment via muscarinic acetylcholine re-
ceptor type 3. Am J Pathol 2002; 161: 521-530
111 Libbrecht L, Roskams T. Hepatic progenitor cells in human
liver diseases. Semin Cell Dev Biol 2002; 13: 389-396
S- Editor Wang J L- Editor Kumar M E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Bioinformatic approach for understanding the heterogeneity
of cholangiocytes
Koji Fukushima, Yoshiyuki Ueno
Koji Fukushima, Yoshiyuki Ueno, Division of Gastroenterology,
Tohoku University Graduate School of Medicine, Seiryo, Aobaku,
Sendai, 980-8574, Japan
Supported by Health and Labour Sciences Research Grants
from the Ministry of Health, Labour and Welfare of Japan for the
Research on Measures for Intractable Diseases and GRANT-IN-
AID FOR SCIENTIFIC RESEARCH C (16590573) from JSPS
Correspondence to: Yoshiyuki Ueno, MD, PhD, Division
of Gastroenterology, Tohoku University Graduate School of
Medicine, 1-1Seiryo, Aobaku, Sendai, 980-8574,
Japan. yueno@mail.tains.tohoku.ac.jp
Telephone: +81-22-7177171 Fax: +81-22-7177177
Received: 2006-01-18 Accepted: 2006-02-18
Abstract
It is remarkable that microarray technologies have
nearly reached a pinnacle. Establishment of further
analysis and management of enormous data derived
from microarray technology is currently the highest
priority. The heterogeneous functions of cholangiocytes
regulate the pathophysiology of the biliary epithelium
in relation to secretory, proliferative and apoptotic
activities. Distinct expression profiles of two murine
cholangiocyte lines, termed small and large have been
revealed by microarray analysis. The features of the two
cholangiocyte cell lines, categorized partly according to
gene ontology, indicate the specific physiological role of
each cell line. The large cholangiocytes are characterized
as transport and immune/ inflammatory responses.
In contrast, small cholangiocytes are associated with
properties of limited physiological functional ability and
proliferating/migrating potential with specific molecules
like Eph receptors, comparable to mesenchymal cells.
'Omic study will be of great help in understanding the
heterogeneity of cholangiocytes.
2006 The WJG Press. All rights reserved.
Key words: Cholangiocytes; Heterogeneity
Fukushima K, Ueno Y. Bioinformatic approach for
understanding the heterogeneity of cholangiocytes. World J
Gastroenterol 2006; 12(22): 3481-3486
http://www.wjgnet.com/1007-9327/12/3481.asp
World J Gastroenterol 2006 June 14; 12(22): 3481-3486
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
Introduction
Several research avenues have established novel scientific
theories. We initially examined phenotypic manifestations
of various forms of induction, analogy and abduction.
Subsequent preliminary studies resulted in a firm-working
hypothesis. We then tested this hypothesis in order to
establish our theoretical groundwork. However, recent
advances in the field of bioinformatics have become
routine, especially in regards to the initial speculative step
in designing a rational approach. Since the initial DNA
microarray analysis experiment[1], microarray has developed
rapidly and is now well established, with manufacturers
meeting market demand [2] . The number of scientific
papers with "microarray" as a key word steadily increased
on PubMed from 2001 to 2004, reaching a total of 8603
papers. Moreover, an enormous quantity of raw data
potentially contained in "microarray" papers is anticipated
in the near future, and the estimate is based on the number
of the genes analyzed in each paper (e.g. 30 000-40 000
genes expressed in mammalian cells). Therefore, it is
essential for future advances to reliably preserve a database
containing all the raw data. The establishment of data
analysis system as well as data mining and data sharing,
is anticipated to be the highest priority in this field. We
will focus on such issues pertaining to general microarray
experiments as well as the methods and the results of
microarray analyses designed to characterize cholangiocytes
in this chapter.
Microarray analysis
We will simply give an overview of the current status,
analytic method and data-sharing aspects of microarray
analysis, partly in accordance with the description of
Knudsen [3]. One of the currently popular microarray
systems is the Affymetrix DNA Chip, which employs
in situ oligonucleotide synthetic technology as well as
mass-production compatible to that of silicon chips.
Several microarrays and expression analysis services are
commercially available from several companies including
Amersham, Clontech (TAKARA), Invitrogen and so on.
As in other experiments, the quantity of RNA samples
should be standardized, otherwise correction steps
will be necessary for the evaluation of the results by
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 3482 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
"scaling" generally employing internal controls. Moreover,
verification of the linearity of spot intensity-sample
quantity curves for all applied spots is also necessary
in microarray experiments. The companies supplying
ready-made products have overcome these problems and
established quality control, making their products popular
with researchers applying commercial products to limited
numbers of samples. Although array technologies at the
bench have nearly reached a zenith during the past several
years, there are some in silico systems under development.
The parametric t-test and non-parametric Wilcoxon's rank-
sum/ Mann-Whitney tests are the most popular methods
applicable to test the significance of differences between
pairs of samples. The normality of each spot intensity for
the samples becomes an issue if the t-test or ANOVA is
used with a routine microarray having thousands of spots.
The most conservative statistical method, Bonferroni's
correction, which defines the level of significance more
strictly according to the number of tested items, might
be applicable even to microarray experiments[4]. Even if
we employ other less conservative approaches[5], we may
need to verify the differential expressions of the specific
molecules by other methods in some cases. Several
software programs are available, including some that are
free like Cluster (http://rana.lbl.gov/EisenSoftware.htm)[6],
for analysis of microarray data. Cluster analysis categorizes
each gene into distinct clusters according to Euclidian
distance based on the variation among the samples at
multiple time-points or under the different conditions.
Each gene in a cluster shows a similar expression pattern
under different conditions, which may indicate a common
transcription pathway, RNA degradation process, and/ or
similarities of gene function in a cluster. In other words,
microarray technology holds promise for elucidating the
complex regulatory mechanisms of RNA transcription as
well as unknown gene functions. Every gene is currently
being systematically defined, allowing organization
according to gene ontology (http://www.geneontology.
org/). The gene ontology project was originally designed
to create a thesaurus-like hierarchy of gene properties,
based on molecular function, biological processes and
cellular components, in order to integrate databases in a
unified way. The combination of raw microarray data and
this unified classification will greatly facilitate developing
the speculative groundwork for our experiments. This
approach differs from the usual methodology based on
molecular properties including domain structure, 3D
structure, evolution or expression patterns. Therefore,
raw microarray data can be a source of new hypotheses,
regardless of the original aim of the experiment. This
may make it difficult to share raw microarray data. The
unified approach, termed MIAME (http://www.mged.
org/Workgroups/MIAME/miame.html), for submission
of microarray data is described in the guidelines of each
paper contributing data to be shared and for peer review.
The statement that "MIAME is neither a dogma, nor a
legal document-it assumes a cooperative data provider and
a fair reviewer" suggests potential difficulty in data sharing,
probably stemming from the complexities of ownership
and intellectual property rights to data. Microarray data is
clearly precious, even in the current era of non-sharing,
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June 14, 2006 Volume 12 Number 22
and should be carefully stored by each lab, organization
or government. Such data should be viewed as common
property, with potential future benefits to mankind.
Features of cholangiocytes
Bile f lows fr om canalicular spaces, encircled by
hepatocytes, through the canals of Hering, interlobular
bile ducts (branches of which are 20-100 m in diameter
and lined by cuboidal epithelium), and septal bile ducts
(which are more than 100 m in diameter and lined by a
simple tall columnar epithelium) to hilar intrahepatic bile
ducts[7]. The cholangiocytes that line the interlobular and
bolder bile ducts [7] also contribute to bile secretion [8,9].
The mechanism underlying this phenomenon, described
elsewhere in detail, is explained briefly by out-intraluminal
vectorial passive transport of water caused by the
osmotic gradient which is formed by active transport
of several substrates driven by cholangiocytes [10,11] .
This physiological function, conserved even in cultured
cell lines, characterizes the specialized cholangiocytes.
Isolated rat cholangiocytes are classified as large or small,
depending on their size. Large cholangiocytes, regarded
as representative functional biliary epithelial cells in
vivo, respond to secretin with increased choleresis[12]. In
contrast, little is known about the properties of small
cholangiocytes. Cholangiocytes are sometimes altered in
a disease-specific manner. For example, primary biliary
cirrhosis (PBC), a potentially fatal cholestatic disorder due
to ductopenia, is a representative disease characterized
by specific destruction of interlobular bile ducts. CD8+
cytotoxic T cells are suspected to play a major role in
this type of destruction. Tetramer technology reveals the
existence of an E2 subunit of the pyruvate dehydrogenase
complex (PDC-E2)159-167-specific to autoreactive cytotoxic
T cells in PBC[13]. Hypothetically, there are several specific
properties (e.g. adhesion molecules, MHC antigens [14],
specific autoantigens, variations in apoptosis and so
on) of cholangiocytes lining interlobular bile ducts,
which make them vulnerable to attack by cytotoxic
lymphocytes. A novel disease etiology was proposed by
Gershwin et al[15], who demonstrated that IgA class anti-
mitochondrial antibodies, transported via transcytosis
from the basolateral to the apical surface co-localize
with PDC-E2 in cholangiocytes. Moreover, molecules
that bind PDC-E2 monoclonal antibody are expressed
on the apical membranes of PBC cholangiocytes [15] .
These observations indicate the cytotoxicity associated
with functional impairment of cholangiocytes to be
caused by interaction with IgA-PDC-E2. In this regard,
the mechanism of transcytosis may specifically dictate
the underlying disease process. Such a hypothesis could
be explained by cholangiocyte heterogeneity based on
the recognition that large and small cholangiocytes are
derived from bile ducts of corresponding diameters[16].
The susceptibility of bile ducts to pathological conditions
(e.g. chronic ductopenic rejection[17], GVHD[18], ischemic
cholangitis[19], PBC or ductopenia resulting from other
pathologies) is due not only to the specific destruction of
cholangiocytes, but also to the difficulty in regeneration of
the bile ducts i.e. with normal structures, in contrast to the
 Fukushima K et al. The heterogeneity of cholangiocytes 3483

Table 1 Differences in cDNA expression between small and large cholangiocyte lines

1 2 3
GO annotation/ Gene name Gene accession GO accession Small Large Ratio (L/S)

Immune response GO:0006955

CD1d1 antigen NM_007639 107 156 1.46

CD1d2 antigen NM_007640 80 131 1.64
CD86 antigen; B7-2; CD28 antigen ligand 2 (CD28L2) L25606 52 17 0.33
Histocompatibility 2, class II antigen E alpha NM_010381 132 407 3.08
Histocompatibility 2, class II antigen A, beta 1 NM_010379 586 832 1.42
Histocompatibility 2, class II antigen E beta NM_010382 3579 4481 1.25
Interleukin 2 receptor, beta chain NM_008368 540 2604 4.83
Cell adhesion GO:0007155

Intercellular adhesion molecule 1 (ICAM1) X52264 39 20 0.51

Vascular cell adhesion molecule 1 (VCAM1) M84487 17 11 0.65
Cadherin 1 (CDH1); epithelial cadherin (E-cadherin; E-CAD); uvomorulin (UM) X06115 32 42 1.29
Cytoskeleton GO:0005856

Cytoplasmic beta-actin (ACTB) M12481 455 275 0.60

Vimentin (VIM) X51438 260 73 0.28
Proteolysis GO:0006508
Matrix metalloproteinase 14 (MMP14); membrane-type matrix matalloproteinase 1 X83536 2585 1339 0.52
(MTMMP1)
Cell death GO:0008219

Fas antigen ligand (FASL); apoptosis antigen ligand (APTL; APT1LG1); tumor U06948 37 21 0.57
necrosis factor superfamily member 6 (TNFSF6); generalized lymphoproliferative
disease protein (GLD)
Fas antigen; fasL receptor; apoptosis antigen 1 (APO1; APT1); CD95 antigen M83649 18 20 1.07
Fas death domain-associated protein NM_007829 392 103 0.26
Fas-associating protein with death domain NM_010175 484 188 0.39
B-cell leukemia/lymphoma protein 2 (BCL2) M16506 31 18 0.56
Caspase 9 NM_015733 318 996 3.14
Anti-apoptosis
Insulin-like growth factor 1 receptor alpha subunit (IGFIR-alpha) U00182 GO:0006916 95 176 1.86
Inflammatory response GO:0006954
Tumor necrosis factor NM_013693 130 275 2.11
Tumor necrosis factor (ligand) superfamily, member 4 NM_009452 98 227 2.32
Tumor necrosis factor (ligand) superfamily, member 7 NM_011617 133 239 1.80
Tumor necrosis factor (ligand) superfamily, member 8 NM_009403 189 514 2.73
Tumor necrosis factor (ligand) superfamily, member 9 NM_009404 278 305 1.10
Tumor necrosis factor (ligand) superfamily, member 19 NM_011615 164 88 0.53
Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A); tumor X57796 58 41 0.72
necrosis factor receptor 1 (TNFR1)
Tumor necrosis factor receptor superfamily member 1B2 (TNFRSF1B2); tumor M59378 18 17 0.92
necrosis factor receptor 2 (TNFR2)
Interleukin 6 receptor alpha subunit (IL6R-alpha; IL6RA) X51975 57 64 1.13
Cytokine activity GO:0005125

Interleukin 6 (IL6) X06203 36 25 0.70

Oncostatin M (OSM) D31942 79 509 6.42
Cellular metabolism GO:0044237

PDC-E2 NT NT
PDC-E3BP NT NT
Transport GO:0006810

Solute carrier family 4 (anion exchanger), member 2 NM_009207 80 145 1.82

Solute carrier family 4 (anion exchanger), member 3 NM_009208 52 120 2.31
Solute carrier family 4 (anion exchanger), member 1 NM_011403 995 2146 2.16
Voltage-dependent anion channel 1 NM_011694 111 146 1.32
Voltage-dependent anion channel 2 NM_011695 87 62 0.71
Solute carrier family (organic anion transporter) member 1 NM_013797 76 114 1.50
Aquaporin 1 NM_007472 257 245 0.95
Aquaporin 2 NM_009699 198 276 1.39
Aquaporin 3 NM_016689 124 204 1.64
Aquaporin 5 NM_009701 396 222 0.56
Aquaporin 8 NM_007474 2160 8728 4.04

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 3484 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

Solute carrier family 10 (sodium/bile acid cotransporter family), member 1 NM_011387 208 402 1.93
G-protein coupled purinergic receptor P2Y1 (P2RY1) U22829 346 652 1.89
Purinergic receptor P2X, ligand-gated ion channel 4 NM_011026 711 2229 3.13
Detection of Stimulus GO0051606
Toll-like receptor 2 NM_011905 95 68 0.72
Toll-like receptor 5 NM_016928 168 114 0.68
Toll-like receptor 6 NM_011604 255 569 2.23
Cell proliferation GO:0008283
FMS-like tyrosine kinase 1 (FLT1); vascular endothelial growth factor receptor 1 L07297 36 23 0.65
(VEGFR1)
Epidermal growth factor (EGF) J00380 88 116 1.32
Kinase insert domain receptor (KDR); vascular endothelial growth factor receptor X70842 93 58 0.62
2 (VEGFR2); FLK1
Vascular endothelial growth factor (VEGF); vascular permeability factor (VPF) M95200 165 152 0.92
Heparin-binding growth factor 5 (HBGF5); fibroblast growth factor 5 (FGF5) M30643 355 152 0.43
Fibroblast growth factor receptor 3 (FGFR3); heparin-binding growth factor M81342 192 485 2.53
receptor (HBGFR)
Transforming growth factor beta 1 (TGF-beta 1; TGFB1) M13177 69 105 1.51
Transforming growth factor beta receptor 1 (TGF-beta receptor 1; TGFR1); ESK2 D25540 44 29 0.65
Acetylcholine receptor M3 NM_033269 NT NT
Cell cycle GO:0007049
Proliferating cell nuclear antigen (PCNA); cyclin X53068 211 124 0.59
Cell growth
Estrogen receptor 1 (ESTR1); estrogen receptor alpha (ER-alpha; ESTRA) M38651 GO:0016049 22 33 1.48
Others
Nerve growth factor receptor AF105292 GO:0007411 N.T. N.T.
Neurotrophic tyrosine kinase receptor type1 XM283871 GO:0042490 N.T. N.T.
Neurotrophic tyrosine kinase receptor type 2 M33385 GO:0042490 93 18 0.20
Neurotrophic tyrosine kinase, receptor, type 3 NM_008746 GO:0042490 2923 3827 1.31
Interferon gamma receptor (IFN-gamma receptor; IFNGR) M28233 GO:0005615 51 50 0.98

The entire dataset obtained from the Atlas Glass Array mouse (Clontech, Takara Bio Inc., Shiga, Japan) was analyzed by ArrayGauge software (Fuji Photo Film
Co., Ltd., Tokyo). 1gene ontology, 2,3spot intensities of all samples from small or large cholangiocytes.

rapid regeneration of hepatocytes in cases with acute liver
injury. The proliferation of cholangiocytes that potentiate
the regeneration of bile ducts occurs in a characteristic
manner, i.e. secretin, somatostatin or bile duct ligation
results in proliferation of large cholangiocytes[20], whereas
chemical injury of bile ducts with CCl 4 increases the
numbers of small cholangiocytes [21,22] . Therefore, to
clarify the mechanisms sustaining cholangiocyte growth
it is essential to understand the regulation of bile duct
regeneration. We identified an Eph receptor, a membrane
bound-type tyrosine kinase, as one of the key molecules
for reorganization/proliferation of cholangiocytes, and a
subtype of this family, which is expressed mainly in small
cholangiocytes [23]. Thus, the heterogeneous expression
profile of cholangiocytes is expected to facilitate further
study of these cells.
Analysis of heterogeneous cholan-
giocytes
For the evaluation of genes that are expressed by
small and large cholangiocytes see Table1. Several liver
diseases, including non-alcoholic fatty liver disease [24],
liver cirrhosis [25] , hepatocellular carcinoma [26,27] and
cholangiocellular carcinoma [28] have been studied
extensively using microarray techniques. Some studies have
examined whole liver samples, consisting of various cell
types, probably for the purpose of disease classification.
Sample size is critical for the detection of subtle changes
in the expression of meaningful genes [29] , a possible
problem in assessing rare diseases. Another problem in
the study of biliary diseases like PBC by microarray is that
cholangiocytes account for only 3% of the cell population
even in the nor mal liver [30] . Over 75% of the cell
population is regarded as necessary to test the significance
of differences in expression levels [2] . Therefore, for
the purpose of analyzing chlangiocytes, isolation [31] or
microdissection [32] is necessary prior to analysis. Our
study goals were to characterize the physiological role of
biliary epithelia in the mouse and to analyze cholangiocyte
heterogeneity. Due to the complexities of the isolation
steps, maintaining quality control or reproducibility was
possible when obtaining a large sample of RNA from
freshly isolated cholangiocytes. Moreover, given the
necessity of conducting further functional assays, we
immortalized and subcloned the isolated Balb-c mouse
cholangiocytes by introducing the SV40 large T antigen[33].
The established large and small cholangiocyte cell lines
were evaluated by their morphologies and responsiveness
to secretin. We revealed 230 genes (4.74%) showing
different expression patterns in the two cells lines, among
4800 genes tested by combining two types of ready-
made microarrays [34]. Our large cholangiocyte line was
characterized by gene ontology, transport and immune/
inflammatory responses, which were apparent, even
without the statistical tests presented in the table. The term

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 Fukushima K et al. The heterogeneity of cholangiocytes
"transport" includes movement of anions, water and bile
acids, and thus represents the physiological functions of
bile ducts. These observations indicate a special feature of
large cholangiocytes which presumably play a role in local
immune reactions. In contrast, our small cholangiocytes
are categorized into a subgroup characterized by rapid
cell cycle turnover as well as poor physiological functional
ability. In addition to these fundamental properties,
our small cholangiocytes are characterized by abundant
expressions of actin and vimentin and poor expression of
E-cadherin. Together with rich spindle-type cell processes,
small cholangiocytes have a feature in common with
mesenchymal cells probably originating from epithelial-
mesenchymal transition[35]. This remains a controversial
issue. Another important feature of cholangiocyte is
its capability of the responsiveness to hormones and
neuropeptides. Specifically, estrogen receptor [36] and
receptor for IGF-1(insulin like growth factor 1) [37] ,
NGF(nerve growth factor)[38] and acetylcholine (M3)[39]
have been shown to play a major role in modulating
cholangiocyte proliferation. Estrogen receptor expresses at
a minimal level in both types of cell lines. In contrast, IGF
receptor was preferentially expressed in large cholangiocyte
line in our microarray study. The expression of estrogen
receptor inducible under pathological conditions like bile
duct injury[40] may explain the discrepancy between the
experiments results. Predominant expression of IGF
receptor in large cholangiocytes may be a marker of
differentiated biliary epithelial cells as well as a proliferating
effector of maturated cholangiocytes.
Concluding remarks
Microarray is a powerful tool for elucidating functional
cholangiocyte heterogeneity. Although the evaluation
of some crucial biological regulatory processes like
protein modification requires methodologies other than
microarray, the potential of microarray technology is
anticipated to grow with the development of data-analysis
theory for the comprehension of complex networks.
ACKNOWLEDGMENTS
We thank Hiroyuki Izu (Center for Functional Analysis of
DNA, TAKARA BIO Inc.) for his technical advice on the
microarray.
REFERENCES
1 Schena M, Shalon D, Davis RW, Brown PO. Quantitative
monitoring of gene expression patterns with a complementary
DNA microarray. Science 1995; 270: 467-470
2 Imbeaud S, Auffray C. 'The 39 steps' in gene expression profil-
ing: critical issues and proposed best practices for microarray
experiments. Drug Discov Today 2005; 10: 1175-1182
3 Knudsen S. ANALYSIS of DNA MICROARRAY DATA. New
York: John Wiley & Sons Inc, 2002
4 Bender R, Lange S. Adjusting for multiple testing--when and
how? J Clin Epidemiol 2001; 54: 343-349
5 Storey JD, Tibshirani R. Statistical significance for genome-
wide studies. Proc Natl Acad Sci U S A 2003; 100: 9440-9445
6 Li X, Rao S, Wang Y, Gong B. Gene mining: a novel and pow-
erful ensemble decision approach to hunting for disease genes
3485
using microarray expression profiling. Nucleic Acids Res 2004;
32: 2685-2694
7 Sherlock S. Diseases of the Liver and Biliary System. 11th ed.
Malden: Blackwell Science Inc, 2002
8 Nathanson MH, Boyer JL. Mechanisms and regulation of bile
secretion. Hepatology 1991; 14: 551-566
9 Alpini G, Lenzi R, Zhai WR, Slott PA, Liu MH, Sarkozi L,
Tavoloni N. Bile secretory function of intrahepatic biliary epi-
thelium in the rat. Am J Physiol 1989; 257(1 Pt 1): G124-G133
10 Gong AY, Masyuk AI, Splinter PL, Huebert RC, Tietz PS,
LaRusso NF. Channel-mediated water movement across en-
closed or perfused mouse intrahepatic bile duct units. Am J
Physiol Cell Physiol 2002; 283: C338-C346
11 Splinter PL, Masyuk AI, LaRusso NF. Specific inhibition
of AQP1 water channels in isolated rat intrahepatic bile
duct units by small interfering RNAs. J Biol Chem 2003; 278:
6268-6274
12 Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV,
Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and
functional heterogeneity of cholangiocytes from rat liver after
bile duct ligation. Am J Physiol 1997; 272: G289-G297
13 Kita H, Matsumura S, He XS, Ansari AA, Lian ZX, Van de
Water J, Coppel RL, Kaplan MM, Gershwin ME. Quantitative
and functional analysis of PDC-E2-specific autoreactive cyto-
toxic T lymphocytes in primary biliary cirrhosis. J Clin Invest
2002; 109: 1231-1240
14 Hreha G, Jefferson DM, Yu CH, Grubman SA, Alsabeh R, Gel-
ler SA, Vierling JM. Immortalized intrahepatic mouse biliary
epithelial cells: immunologic characterization and immunoge-
nicity. Hepatology 1999; 30: 358-371
15 Gershwin ME, Ansari AA, Mackay IR, Nakanuma Y, Nishio A,
Rowley MJ, Coppel RL. Primary biliary cirrhosis: an orches-
trated immune response against epithelial cells. Immunol Rev
2000; 174: 210-225
16 Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV,
LeSage G, LaRusso NF. Morphological, molecular, and func-
tional heterogeneity of cholangiocytes from normal rat liver.
Gastroenterology 1996; 110: 1636-1643
17 Wiesner RH, Ludwig J, van Hoek B, Krom RA. Current
concepts in cell-mediated hepatic allograft rejection leading
to ductopenia and liver failure. Hepatology 1991; 14(4 Pt 1):
721-729
18 Ueno Y, Ishii M, Yahagi K, Mano Y, Kisara N, Nakamura N,
Shimosegawa T, Toyota T, Nagata S. Fas-mediated cholangi-
opathy in the murine model of graft versus host disease. Hepa-
tology 2000; 31: 966-974
19 Ludwig J, Batts KP, MacCarty RL. Ischemic cholangitis in he-
patic allografts. Mayo Clin Proc 1992; 67: 519-526
20 Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE,
Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional ex-
pression of the apical Na+-dependent bile acid transporter in
large but not small rat cholangiocytes. Gastroenterology 1997;
113: 1734-1740
21 LeSage GD, Glaser SS, Marucci L, Benedetti A, Phinizy JL,
Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Hol-
comb LA, Alpini G. Acute carbon tetrachloride feeding in-
duces damage of large but not small cholangiocytes from BDL
rat liver. Am J Physiol 1999; 276: G1289-G1301
22 LeSage GD, Benedetti A, Glaser S, Marucci L, Tretjak Z, Ca-
ligiuri A, Rodgers R, Phinizy JL, Baiocchi L, Francis H, Lasater J,
Ugili L, Alpini G. Acute carbon tetrachloride feeding selective-
ly damages large, but not small, cholangiocytes from normal
rat liver. Hepatology 1999; 29: 307-319
23 Fukushima K UY, Kanno N, Moritoki Y, Inoue J, Shimosega-
wa T. Susceptible role and distinct distribution of Eph mem-
bers in cholangiocyte. Gastroenterology 2004; 126: A750-750
24 Younossi ZM, Baranova A, Ziegler K, Del Giacco L, Schlauch
K, Born TL, Elariny H, Gorreta F, VanMeter A, Younoszai A,
Ong JP, Goodman Z, Chandhoke V. A genomic and proteomic
study of the spectrum of nonalcoholic fatty liver disease. Hepa-
tology 2005; 42: 665-674
25 Kim S, Park YM. Specific gene expression patterns in liver cir-
rhosis. Biochem Biophys Res Commun 2005; 334: 681-688
www.wjgnet.com
 3486 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
26 Budhu AS, Zipser B, Forgues M, Ye QH, Sun Z, Wang XW.
The molecular signature of metastases of human hepatocellu-
lar carcinoma. Oncology 2005; 69 Suppl 1: 23-27
27 Mao HJ, Li HN, Zhou XM, Zhao JL, Wan DF. Monitoring
microarray-based gene expression profile changes in hepato-
cellular carcinoma. World J Gastroenterol 2005; 11: 2811-2816
28 Obama K, Ura K, Li M, Katagiri T, Tsunoda T, Nomura A,
Satoh S, Nakamura Y, Furukawa Y. Genome-wide analysis of
gene expression in human intrahepatic cholangiocarcinoma.
Hepatology 2005; 41: 1339-1348
29 Wei C, Li J, Bumgarner RE. Sample size for detecting dif-
ferentially expressed genes in microarray experiments. BMC
Genomics 2004; 5: 87
30 Alpini G, McGill JM, Larusso NF. The pathobiology of biliary
epithelia. Hepatology 2002; 35: 1256-1268
31 Ishii M, Vroman B, LaRusso NF. Isolation and morphologic
characterization of bile duct epithelial cells from normal rat
liver. Gastroenterology 1989; 97: 1236-1247
32 Taniai M, Higuchi H, Burgart LJ, Gores GJ. p16INK4a pro-
moter mutations are frequent in primary sclerosing cholangitis
(PSC) and PSC-associated cholangiocarcinoma. Gastroenterol-
ogy 2002; 123: 1090-1098
33 Yahagi K, Ishii M, Kobayashi K, Ueno Y, Mano Y, Niitsuma
H, Igarashi T, Toyota T. Primary culture of cholangiocytes
from normal mouse liver. In Vitro Cell Dev Biol Anim 1998; 34:
512-514
34 Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima
K, Glaser S, LeSage G, Shimosegawa T. Evaluation of differen-
tial gene expression by microarray analysis in small and large
cholangiocytes isolated from normal mice. Liver Int 2003; 23:
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
449-459
35 Boyer B, Valls AM, Edme N. Induction and regulation of
epithelial-mesenchymal transitions. Biochem Pharmacol 2000;
60: 1091-1099
36 Alvaro D, Onori P, Metalli VD, Svegliati-Baroni G, Folli
F, Franchitto A, Alpini G, Mancino MG, Attili AF, Gaudio
E. Intracellular pathways mediating estrogen-induced
cholangiocyte proliferation in the rat. Hepatology 2002; 36:
297-304
37 Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A,
Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili
AF, Gaudio E. The intrahepatic biliary epithelium is a target
of the growth hormone/insulin-like growth factor 1 axis. J
Hepatol 2005; 43: 875-883
38 Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD,
Glaser SS, Francis H, Mancino MG, Ueno Y, Barbaro B,
Benedetti A, Attili AF, Alvaro D. Nerve growth factor
modulates the proliferative capacity of the intrahepatic biliary
epithelium in experimental cholestasis. Gastroenterology 2004;
127: 1198-1209
39 LeSagE G EG, Alvaro D, Benedetti A, Glaser S, Marucci L,
Baiocchi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis
H, Alpini G. Cholinergic system modulates growth, apoptosis,
and secretion of cholangiocytes from bile duct-ligated rats.
Gastroenterology 1999; 117: 191-199
40 Alvaro D, Invernizzi P, Onori P, Franchitto A, De Santis
A, Crosignani A, Sferra R, Ginanni-Corradini S, Mancino
MG, Maggioni M, Attili AF, Podda M, Gaudio E. Estrogen
receptors in cholangiocytes and the progression of primary
biliary cirrhosis. J Hepatol 2004; 41: 905-912
S- Editor Wang J E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Ursodeoxycholic acid treatment of vanishing bile duct
syndromes
Thomas Pusl, Ulrich Beuers
Thomas Pusl, Ulrich Beuers, Department of Medicine ,
Klinikum Grosshadern, University of Munich, Germany
Correspondence to: Ulrich Beuers, MD, Department of
Medicine II, Klinikum Grosshadern, Marchioninistrasse 15, 81377
Munich, Germany. beuers@med.uni-muenchen.de
Telephone: +49-89-70955272 Fax: +49-89-70955271
Received: 2006-01-31 Accepted: 2006-03-14
Abstract
Vanishing bile duct syndromes (VBDS) are characterized
by progressive loss of small intrahepatic ducts caused
by a variety of different diseases leading to chronic
cholestasis, cirrhosis, and premature death from liver
failure. The majority of adult patients with VBDS
suffer from primary biliary cirrhosis (PBC) and primary
sclerosing cholangitis (PSC). Ursodeoxycholic acid
(UDCA), a hydrophilic dihydroxy bile acid, is the only
drug currently approved for the treatment of patients
with PBC, and anticholestatic effects have been reported
for several other cholestatic syndromes. Several potential
mechanisms of action of UDCA have been proposed
including stimulation of hepatobiliary secretion, inhibition
of apoptosis and protection of cholangiocytes against
toxic effects of hydrophobic bile acids.
2006 The WJG Press. All rights reserved.
Key words: Cholestasis; Primary biliary cirrhosis; Primary
sclerosing cholangitis; Secretion; Signaling; Transport;
Ursodeoxycholic acid; Vanishing bile duct syndrome
Pusl T, Beuers U. Ursodeoxycholic acid treatment of va-
nishing bile duct syndromes. World J Gastroenterol 2006;
12(22): 3487-3495
http://www.wjgnet.com/1007-9327/12/3487.asp
INTRODUCTION
Cholestasis is defined as an impairment of bile flow and
failure to adequately secrete cholephilic compounds into
bile often associated with clinical manifestations such as
jaundice and pruritus and biochemical alterations such as
an elevation in serum alkaline phosphatase and g-glutamyl
World J Gastroenterol 2006 June 14; 12(22): 3487-3495
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
transpeptidase. The liver damage observed in chronic
cholestasis has long been attributed to accumulation
and retention of potentially toxic bile acids within
hepatocytes. Chronic cholestasis is the main feature of
vanishing bile duct syndromes (VBDS) characterized by
progressive loss of small intrahepatic ducts caused by a
variety of different diseases. In children, biliary atresia and
Alagille syndrome are rare ductopenic diseases classified
as VBDS. The majority of adult patients with VBDS
suffer from primary biliary cirrhosis (PBC) and primary
sclerosing cholangitis (PSC). Other diseases associated
with ductopenia include autoimmune cholangitis, chronic
hepatic allograft rejection, graft-versus-host disease
(GVHD), chronic cholestatic sarcoidosis and ischemic
(vascular) cholangiopathies. Among the various causes
of VBDS, drugs have an increasing importance. Drug
reactions are related especially to antibiotics, phenothiazine
derivates and carbamazepine. It is increasingly clear that
immunopathogenetic mechanisms involving innate and
adaptive immune responses contribute to ductopenia in
most of these diseases. Prognosis varies from hepatic
failure and death or liver transplantation to resolution of
cholestasis and normal liver function.
Ursodeoxycholic acid (UDCA) is a hydrophilic
dihydroxy bile acid (chemical structure: 3a, 7b-dihydroxy-
5b-cholanoic acid) which was first identified in the bile
of the Chinese black bear[1]. UDCA is a physiologic bile
acid also present in man albeit in a low concentration
of about 3% of the bile acid pool, where it is
formed by 7 b -epimerization of the primary bile acid
chenodeoxycholic acid in the gut by intestinal bacteria[2,3]. It
has been used for centuries in traditional Chinese medicine
for the treatment of liver diseases. Reports from Japan
and Europe first revealed that UDCA was able to dissolve
gallstones[4-6]. In 1985, Leuschner and coworkers observed
improved serum liver tests in patients with chronic active
hepatitis treated with UDCA for gallstone dissolution[7],
and similar observations were made previously in Japan.
Since then, various trials have shown the beneficial effect
of UDCA for different cholestatic syndromes.
Pharmacokinetics and metabolism
of UDCA
After oral administration, UDCA is absorbed by passive
nonionic diffusion mainly in the small intestine (-80%)
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 3488 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
and less in the colon (-20%) following solubilization in
mixed micelles of endogenous bile acids in the proximal
jejunum [2,8]. Its absorption rate is enhanced when it is
given with a meal and may be decreased in patients with
cholestasis and decreased biliary secretion of endogenous
bile acids[9]. After intestinal absorption, UDCA is taken
up from the portal blood by the hepatocytes at their
sinusoidal domain via specific bile acid transporters
namely, NTCP and OATP, conjugated mainly with glycine
and to a lesser extent with taurine and is subsequently
transported across the canalicular domain into the bile
ducts via another bile acid carrier molecule, designated
BSEP [2,10]. UDCA conjugates reach the small intestine
and are reabsorbed mainly from the distal ileum via an
active Na+-dependent transport mechanism undergoing
an effective enterohepatic circulation. Non-absorbed
UDCA and UDCA conjugates pass into the colon and,
after bacterial deamidation of conjugates, are mostly
converted to lithocholic acid by intestinal bacteria and
eliminated via the feces[2]. Only minute amounts of the
insoluble lithocholic acid are reabsorbed via the colonic
mucosa, sulphated in the liver, secreted into bile and
excreted in the feces. Under continuous oral treatment
at pharmacological doses (13-15 mg/kg per day) UDCA
becomes the predominant bile acid in the liver and the
systemic circulation, comprising 40% to 60% of total bile
acid[2,8].
Mechanisms of action of urso-
deoxycholic acid
The mechanisms underlying the beneficial effects of
UDCA in cholestasis are being increasingly unraveled[11-14].
Initial research interest was focused on changes in bile
acid pool composition, hepatocyte membrane protective
effects, immunomodulatory effects, and bicarbonate-rich
hypercholeresis induced by UDCA. Over the past decade,
it has, however, become evident that UDCA is a potent
intracellular signaling agent that induces stimulation of
impaired hepatocellular secretion, anti-apoptotic effects
and may mediate cholangiocyte protection. Depending on
the pathophysiology and the stage of the underlying liver
disease, the predominant mechanisms of action of UDCA
may vary.
Stimulation of hepatobiliary secretion
Cholestatic liver diseases are characterized by an
impairment of hepatobiliary secretion. As a consequence,
bile acids and other potentially toxic cholephiles
accumulate in the hepatocyte and may lead to liver cell
injury, apoptosis and necrosis. UDCA stimulates biliary
secretion of bile acids and other organic compounds
(e.g. bilirubin glucuronides, glutathione conjugates,
bromosulfophthalein) in various experimental models
such as the isolated hepatocytes, isolated perfused rat
liver and bile fistula rat model and counteracts cholestasis
induced by hydrophobic bile acids in rat liver[15-22]. In line
with these observations, biliary secretion of bile acids and
phospholipids is stimulated and elevated serum levels of
the hydrophobic bile acid, chenodeoxycholic acid, and
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June 14, 2006 Volume 12 Number 22
of bilirubin are decreased in patients with PBC and PSC
during UDCA treatment[23-28]. Thus, the beneficial effects
of UDCA in cholestatic liver disease may be partly due to
the enhanced elimination of toxic compounds from the
hepatocytes. The secretory capacity of the hepatocytes
is determined by the number and activity of transporter
proteins in the canalicular membrane. UDCA stimulates
the expression of transpor ter proteins for biliar y
secretion in the liver and the targeting and insertion of
transporter molecules into the canalicular membrane at
a transcriptional and posttranscriptional level[15,29-31]. As
such, UDCA stimulates the overall gene expression of
both canalicular (Mrp2, Bsep) and alternative basolateral
carriers (Mrp3, Mrp4) in mouse liver, which facilitates
alternative efflux of bile salts and other organic anions
into the systemic circulation[32,33]. UDCA also stimulates
murine renal (Mrp2, Mrp4) and intestinal (Mrp2, Mrp3)
efflux transport proteins, resulting in an increased
overall elimination capacity for potentially toxic biliary
compounds[32]. In addition to these transcriptional effects,
UDCA also stimulates vesicular exocytosis and insertion
of transporter proteins into the canalicular membrane
by modulating complex intracellular signalling cascades
including calcium, protein kinase C and different mitogen-
activated protein kinases[15,31,34-36]. Moreover, UDCA may
also directly activate canalicular transporters through
modification of their phosphorylation status [37]. While
effects of UDCA on mRNA and protein levels of
transporters may be important for long-term regulation,
the effects on the insertion into the canalicular membrane
and the activity of transporters may determine short-
term regulation of secretion. In conclusion, UDCA
modulates hepatobiliary secretion by transcriptional and
posttranscriptional mechanisms in experimental models
in vivo and in vitro. Thus, upregulation of synthesis, apical
targeting and insertion, and activation of key canalicular
transporters may represent key mechanisms to explain the
anticholestatic action of UDCA in patients with cholestatic
liver disease.
Inhibition of apoptosis
Apoptosis, or programmed cell death, is an important
mechanism of cell death in cholestatic liver diseases[14,38,39].
For example, apoptotic features causing bile duct loss have
been observed more frequently in liver tissue from patients
with PBC than in normal controls [40]. Toxic bile acids
can induce apoptosis in hepatocytes at concentrations
comparable to those found in chronic cholestasis and
the mechanisms of bile acid-induced apoptosis have
increasingly been understood (reviewed by Higuchi
et al [41] ). Endogenous hydrophobic bile acids such as
glycochenodeoxycholic acid (GCDCA) or glycodeoxycholic
acid (GDCA) induce apoptosis by ligand-independent
activation of the Fas death-receptor, followed by activation
of caspase 8 and Bid, a pro-apoptotic member of the Bcl-2
protein family, which chaperones another pro-apototic
Bcl-2 molecule, Bax, to the mitochondrial membrane
inducing mitochondrial membrane permeability transition
(MMPT). MMPT causes a sudden increase in permeability
of the inner mitochondrial membrane to ions followed by
 Pusl T et al . Ursodeoxycholic acid treatment of VBDS 3489
mitochondrial swelling and release of cytochrome c from
the intermembrane space to the cytosol. In the cytosol,
cytochrome c interacts with the apoptotic protease-
activating factor 1 (APAF-1), which leads to activation of
caspase 9 and subsequently causes apoptotic cell death[40,42].
A number of studies showed that UDCA can
block apoptosis in vitro as well as in vivo in the rat and
in human hepatocytes by interrupting classic pathways
of apoptosis [43-45] . Antiapoptotic effects of UDCA
were associated with a reduction of the MMPT and
mitochondrial cytochrome c release[14,45]. UDCA protects
rat hepatocytes against bile acid-induced apoptosis by
preventing bile acid-induced, c-Jun N-terminal kinase-
dependent Fas trafficking to the plasma membrane [46].
Apoptosis can not only be inhibited by blocking pro-
apoptotic pathways, but also by activation of intracellular
survival signals. Indeed, UDCA was shown to induce
a survival signal in hepatocytes via activation of the
epidermal growth factor receptor (EGFR) and mitogen-
activated protein kinase (MAPK) that may contribute to
the antiapoptotic effect[47]. Thus, UDCA appears to be a
bile acid that partially activates pro-apoptotic signals, but
also simultaneously a strong MAPK-dependent survival
signal, which makes UDCA an overall non-toxic bile
acid. Although intriguing, the impact of antiapoptotic
mechanisms for the beneficial effects of UDCA in
cholestatic liver disease remains unclear at present.
Protection of cholangiocytes against toxic effects of
hydrophobic bile acids
Hydrophobic bile acids damage cell membranes of
hepatocytes and cholangiocytes and exert extracellular
cytotoxicity at millimolar concentrations present in
bile [48-50]. Indeed, levels of hydrophobic bile acids are
about 1000-fold higher in bile than in serum even under
physiological conditions. The proposed mechanisms of
bile acid-induced cell damage extend from simply binding
to plasma membranes to the induction of apoptosis or
even necrosis. UDCA has been shown to counteract
hydrophobic bile acid-induced membrane disruption
in vitro presumably by alteration of the structure and
composition of simple and mixed micelles rather than
by direct membrane interactions[50,51]. UDCA treatment
at therapeutic doses of 13-15 mg/kg per day renders the
bile acid composition of bile less hydrophobic leading to
a relative enrichment of UDCA to about 40%-50% of
total biliary bile acids[8]. To achieve this effect in isolated
cells or whole organ a high (millimolar) concentration
of UDCA is required. Thus, the membrane protective
effects of UDCA play a role mainly at the bile duct level,
since high concentrations of bile acids are only present
within the biliary tree[52]. UDCA decreases the degree of
cholangiocellular injury, portal inflammation and ductular
proliferation in the Mdr2-knockout mouse, an animal
model of cholestasis which shares morphological features
with PSC resulting from the toxic effect of bile acids
on the biliary epithelium in the absence of protective
mixed micelle formation with phospholipids[53,54]. In line
with these observations, the (peri-) portal inflammatory
reaction is less severe in patients with PBC and PSC
under UDCA treatment as compared to those treated
with placebo[27,28,55,56]. The effects of UDCA conjugates
on cholangiocytes are apparently mediated by Ca2+- and
protein kinase Ca-dependent mechanisms which have been
implicated in stimulation of biliary secretion in cholestatic
hepatocytes as outlined above. Although the protection
against the consequences of bile duct destruction is likely
to be one mechanism of action of UDCA, the underlying
pathophysiologic events leading to the ongoing bile duct
destruction are probably not influenced by UDCA.
UDCA in vanishing bile duct syn-
dromes
Primary biliary cirrhosis
Primary biliary cirrhosis (PBC) is a chronic cholestatic
liver disease characterized by chronic inflammation and
destruction of intrahepatic bile ductules, which leads to
progressive ductopenia. ultimately resulting in fibrosis
and biliary cirrhosis. The prevalence differs considerably
in different geographic areas, ranging from 40 to 400
per million. It primarily affects women, with a female
to male ratio as high as 10 to 1[57,58]. Current theories on
the pathogenesis of PBC favor the hypothesis that the
disease develops as a result of an inappropriate immune
response following stimulation by an environmental or
infectious agent. The pathogenetic mechanism is believed
to be caused by a defect in immunologic tolerance,
resulting in the activation and expansion of self-antigen
specific T and B lymphocyte clones and the production
of circulating autoantibodies in addition to a myriad of
cytokines and other inflammatory mediators. The serologic
hallmark of PBC is the presence of autoantibodies to
mitochondria, especially to the E2 component of the
pyruvate dehydrogenase complex (PDC) [59] . 50% to
60% of patients are asymptomatic at diagnosis, and the
disease is initially detected on the basis of screening liver-
biochemistry profiles. Fatigue and pruritus are the most
common presenting symptoms [60], occurring in up to
78% and 70% of patients, respectively. Jaundice develops
usually later, often associated with progression of the
disease[61]. Eventually, complications of cirrhosis and portal
hypertension, such as ascites, variceal bleeding, and hepatic
encephalopathy, develop.
UDCA is now the mainstay of therapy for PBC. In
1987 Poupon and co-workers reported benefit from
UDCA treatment [62] . Since then, several randomized
double-blind placebo-controlled studies have shown
that UDCA improves biochemical parameters includ-
ing serum bilirubin levels which are used as a prognostic
marker for PBC[25,27,55,63-66]. Indeed, it has been shown that
the mean survival in patients with serum bilirubin levels
above 34 mmol/L is 4 years; in those with values of more
than 103 mmol/L only 2.1 years[67]. This prognostic value
of serum bilirubin is similar in UDCA-treated patients as
in non-treated patients. The benefit from UDCA therapy
on liver fibrosis progression in PBC has been shown by
Corpechot et al[68] using Markov modeling: A fivefold de-
crease in progression rate from early-stage disease toward
extensive fibrosis or cirrhosis was found in UDCA-treated
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 3490 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
patients compared with placebo-treated patients. At 4
years, the probability of UDCA-treated patients to remain
in early stage disease was 76% (95% confidence interval:
58%-88%), as compared with 29% (15%-52%) in placebo-
treated patients. These findings are consistent with recent
data showing that a 2-year treatment with UDCA reduces
periportal necroinflammatory lesions, improves ductular
proliferation, and delays the progression of histologic
stage when given at the earlier stages (stages I-II) of the
disease[69]. When started later in the disease course, UDCA
can still ameliorate inflammation and ductular proliferation
but is not capable of reversing fibrosis.
What is still more debatable is the effect of UDCA
treatment on long-term survival. A combined analysis
of three larger randomized trials, in which patients with
primary biliary cirrhosis were randomly assigned to re-
ceive UDCA (n = 273) or placebo (n = 275) suggested
that survival free of liver transplantation was significantly
improved in patients who received UDCA treatment for
4 years[70]. This benefit was seen in patients with moderate
to severe disease but not in those with mild disease (serum
bilirubin concentration < 23.9 mmol/L or histologic stage
I or II) probably because progression to end-stage disease
did not occur in the short time interval of the study in pa-
tients with mild disease[70]. Lindor et al showed that survival
of patients treated up to 6 years with UDCA was increased
as compared with a placebo group or the predicted sur-
vival from the Mayo model[71]. In a large cohort of UDCA-
treated PBC patients (225 patients) followed for up to 10
years survival free of liver transplantation was significantly
higher than survival predicted by the Mayo model and
10-year survival among UDCA-treated patients is slightly
lower than that of an age- and sex-matched population,
the difference being mainly explained by mortality among
cirrhotic patients[72]. However, the efficacy of UDCA in
PBC has also been questioned. The extended follow-up of
another clinical trial[73], as well as a randomized controlled
trial with the longest follow-up[55], did not show a favor-
able effect of UDCA on the incidence of death or liver
transplantation. In addition, a systematic review and meta-
analysis of 11 randomized controlled trials, including 1272
patients, and six reports of the switch-over phases did not
find a beneficial effect of UDCA on the incidence of liver-
related death, liver transplantation, or the development of
complications of liver disease[74]. Similarly, another meta-
analysis of 16 randomized clinical trials evaluating UDCA
against placebo (n = 15) or no intervention (n = 1) in 1422
patients did not detect a significant effect of UDCA on
mortality[75]. However, these meta-analyses included trials
of different length of follow-up, mostly only up to 24 mo,
which were performed with various doses of UDCA, in
part less than 13-15 mg/kg per day, a dose currently re-
garded as optimal for PBC. Thus, these meta-analyses have
to be interpreted with caution, because survival analyses
in a disease with a very long natural history over decades
are ideally based on longer follow-up periods. In a further
analysis including five studies with a follow-up of at least
4 years, a 32% reduction in the risk of death or need of
liver transplantation was reported in patients treated with
UDCA [76]. Nonetheless, to clarify the true efficacy of
UDCA in the long-term treatment of PBC, additional data
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June 14, 2006 Volume 12 Number 22
are needed. At present, the general recommendation is to
treat PBC patients with UDCA at a dose of 13-15 mg/kg
per day. Treatment should be started as early as possible,
since patients with mild or moderate disease are likely to
have a greater benefit from therapy than those with ad-
vanced disease.
Primary sclerosing cholangitis (PSC)
Primary sclerosing cholangitis (PSC) is a rare chronic cho-
lestatic disease of the intra- and extrahepatic bile ducts
that is generally progressive and leads to end-stage liver
disease with a median survival from diagnosis to death or
liver transplantation, currently estimated at 12 years[77-80].
PSC is characterized by diffuse inflammation, concentric
obliterative fibrosis and progressive stricturing and dilata-
tion of the biliary tree. The etiology and pathogenesis
is unknown, but as in PBC, immune dysregulation plays
an important role in the development of the disease. In
Western populations, the estimated prevalence of PSC is
8.5 cases per 100000 persons[81] and 70%-80% of PSC pa-
tients have or will develop inflammatory bowel disease. Of
importance, patients with PSC have an increased risk of
developing cholangiocarcinoma with an estimated yearly
incidence of 1.5% after diagnosis of PSC[82-85] and patients
with both PSC and ulcerative colitis may be at higher risk
for developing colorectal aneuploidy, dysplasia, or cancer
than UC patients without PSC[86,87]. Based on the beneficial
effect of UDCA in patients with PBC, UDCA was evaluat-
ed in a number of small trials for patients with PSC in the
early 1990s[28,88,89]. Although small, these studies observed
significant biochemical and histologic improvements for
patients receiving UDCA. Similar to the treatment of
PBC, no major side effects were reported. In contrast to
these promising results, the first large randomized placebo-
controlled study including 105 patients with PSC could not
find a clinical benefit of UDCA treatment with respect to
time to treatment failure or histologic findings in a dose of
13-15 mg/kg per day during a mean follow-up period of 2.2
years[26]. A meta-analysis of six randomized clinical trials
showed no difference between UDCA and placebo in the
effects of incidence on death, treatment failure (including
liver transplantation, varices, ascites, and encephalopathy),
liver histological deterioration or liver cholangiographic de-
terioration[90]. However, most trials were small with an av-
erage sample size of 37 patients and the follow-up period
might have been too short to show a clinical benefit, since
PSC has usually a long natural history of over a decade. A
large percentage of the patients had advanced disease and
as in PBC, these patients might respond less to medical
treatment than patients in earlier stages of disease. Finally,
UDCA therapy alone will not suffice to treat the bile duct
strictures typical for PSC, but may need additional me-
chanical intervention. Indeed, major bile duct strictures
develop during UDCA treatment[91]. Based on these obser-
vation, UDCA treatment was combined with endoscopic
dilatation of bile duct strictures in an 8-year prospective
study. The survival of patients receiving this combination
therapy was significantly better than the calculated survival
without treatment[92]. As biliary enrichment of UDCA is
expected to be lower in cholestasis and because of dis-
couraging results with standard doses of UDCA (13 -15
 Pusl T et al . Ursodeoxycholic acid treatment of VBDS 3491
mg/kg per day), use of high doses of UDCA in PSC has
a rationale and has been evaluated by three groups[93-95].
The first study compared high-dose UDCA (20 mg/kg per
day) (n = 13) to placebo (n = 13) for 2 years in 26 patients
with PSC and demonstrated a significant improvement in
serum levels of AP and g-GT (no effect on bilirubin and
albumin levels), and a significant reduction in progression
of cholangiographic features and liver fibrosis as assessed
by disease staging without significant side effects. UDCA
did not improve symptoms[94]. Harnois et al[93] reported
similar results in a 1-year open-label study of 30 PSC pa-
tients treated with UDCA at a dose of 25-30 mg/kg per
day. Changes in the Mayo risk score at 1 year of treatment
and projected survival at 4 year were compared with that
observed in patients randomized to placebo or UDCA at
a dose of 13-15 mg/kg per day in a previous study. A sig-
nificant improvement in serum alkaline phosphatase activ-
ity, AST, albumin and total bilirubin occurred at 1 year of
therapy with high-dose UDCA. The expected survival at
4 years was significantly improved for patients in the high-
UDCA group when compared with a historical placebo
control, but not between the dose of 13-15 mg/kg per day
and placebo. As in the first study, high-dose UDCA was
well tolerated. Finally, in the biggest placebo-controlled
prospective study in PSC ever performed, a total of 219
patients were randomized to 17 to 23 mg/kg per day of
UDCA (n = 110) or placebo (n = 109) for 5 years[95]. No
statistically significant benefit from 20 mg/kg UDCA on
survival without liver transplantation or prevention in chol-
angiocarcinoma in PSC was shown in this study. However,
there was a tendency to improved survival in the UDCA-
treated patients and the study was underpowered to show
a possible positive effect.
UDCA has also been shown to have colon cancer
chemopreventive effects in preclinical studies [96,97]. In
addition, two clinical studies suggested that UDCA also
decreases the risk for developing colorectal neoplasia
in UC patients with PSC[98,99]. In a cross-sectional study
by Tung et al involving 59 patients with ulcerative colitis
and primary sclerosing cholangitis who were undergoing
colonoscopic surveillance for colonic dysplasia, UDCA
use was strongly associated with decreased prevalence
of colonic dysplasia (odds ratio, 0.18, 95% confidence
inter val, 0.05 to 0.61; P = 0.005) [98] . In a blinded,
prospective study by Pardi et al [99] , 52 patients with
ulcerative colitis and PSC were randomized to receive
UDCA or placebo and were followed-up for a total of 355
person-years. Those originally assigned to receive UDCA
had a relative risk of 0.26 for the development of dysplasia
or cancer (95% confidence interval, 0.06-0.92; P = 0.034)
suggesting a significant chemoprotective effect for UDCA
in these patients.
In conclusion, UDCA therapy (13-20 mg/kg per day)
of PSC is warranted and should be initiated as soon as
possible and be continued life-long. In addition, major
strictures should be dilated regularly in centers with
experienced endoscopists. In patients with PSC and
ulcerative colitis, who carry a considerable lifetime risk for
colorectal neoplasia, UDCA may act as a chemopreventive
drug. Due to the unresolved issues of optimal dosing and
the true long-term benefit of UDCA in PSC, patients
should be included in trials if possible.
Intrahepatic cholestasis of pregnancy
Intrahepatic cholestasis of pregnancy (ICP) is the most
common pregnancy-related liver disorder characterized
by pruritus associated with a mild or moderate increase
in ser um aminotransferases and ser um bile acids
starting in the second or third trimester of pregnancy
and disappearing after delivery. ICP is associated with
increased risk of intrauterine fetal death and premature
delivery [100,101]. There is increasing evidence that gen-
etically determined dysfunction in the canalicular ABC
transporters bile salt export pump and multidrug resistance
protein 3 might be risk factors for ICP development[102].
In small controlled trials and several observational trials,
UDCA improved maternal pruritus and serum liver
chemistries such as serum bilirubin and transaminases,
and diminished the number of premature deliveries. The
medication seems to be well tolerated by pregnant women
and no adverse effects in mothers or newborns have been
detected [103-105]. A recent prospective, randomized trial
(n = 84) comparing cholestyramine (8 g daily) and UDCA
(8-10 mg/kg daily) for the treatment of ICP further
supported these findings. Pruritus was more effectively
reduced by UDCA than cholestyramine. Newborns were
delivered significantly closer to term by patients treated
with UDCA, than those treated with cholestyramine.
Serum alanine and aspartate aminotransferase activities
were markedly reduced by 78.5% and 73.8%, respectively,
after ursodeoxycholic acid, but by only 21.4%, each, after
cholestyramine therapy. Ursodeoxycholic acid, but not
cholestyramine was free of adverse effects [106]. In ICP,
the relief of cholestasis by UDCA has been suggested to
be due to stimulation of vesicular exocytosis resulting in
mobilization of an increased number of transport proteins
to the canalicular membrane and, thereby, stimulation
of transport systems involved in the biliary secretion of
steroid mono- and disulfates. In conclusion, data that are
available support the use of UDCA as first-line therapy for
ICP in the third trimester.
Hepatic complications of allogeneic bone marrow
transplantation
Hepatic graft-versus-host disease (GVHD) is a frequent
complication after bone-marrow transplantation usually
associated with a cholestatic picture characterized by
elevated serum alkaline phosphatase and hyperbili-
rubinemia attributed to damage of the small bile ducts.
UDCA has been reported to be effective in the treatment
of manifest GVHD [107,108] . In an open-label study 12
patients with refractory chronic GVHD of the liver
after allogeneic bone marrow transplantation were given
UDCA (10 to 15 mg/kg per day) for 6 wk. Serum tests
of cholestatic liver injury showed improvement compared
with baseline. After discontinuation of UDCA therapy,
11 patients were followed for 6 additional weeks. All
showed significant worsening in liver function test results.
Symptom scores were unchanged throughout the study.
No adverse effects were observed [107]. In addition, the
efficacy of UDCA has also been reported as a prophylaxis
for veno-occlusive disease (VOD) of the liver after
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 3492 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
allogeneic bone marrow transplantation. In a randomized,
double-blind, placebo-controlled study including 67
consecutive patients undergoing transplantation with
allogeneic bone marrow with a preoperative regimen of
busulfan plus cyclophosphamide were randomly assigned
to receive UDCA 300 mg twice daily or placebo. The
incidence of VOD was 40% in placebo recipients and
15% in the UDCA-treated recipients (P = 0.03)[109]. Thus,
further studies are certainly needed, but UDCA may be
considered for prophylaxis of VOD and treatment of
GVHD of the liver.
Cystic fibrosis
Cystic fibrosis (CF) is an autosomal recessive disorder
caused by mutations in the CFTR (cystic fibrosis trans-
membrane conductance regulator) gene resulting in the
secretion of viscous bile. This may lead to the formation
of bile duct plugs within small bile ducts, biliary obstruc-
tion and ultimately result in (focal to multilobular) biliary
cirrhosis. Hepatobiliary complications increase with pa-
tient age and up to 7% of children and young adults with
CF may present with liver cirrhosis[110]. Early uncontrolled
studies showed that UDCA significantly improved labora-
tory tests and nutritional status of patients with CF[111-113].
A small randomized, placebo-controlled study showed
clinical, biochemical and nutritional improvement in CF
patients treated with UDCA (15 mg/kg per day) for one
year[114]. Improvement of liver histology of CF patients
treated with UDCA (10-15 mg/kg per day) for 2 years has
been reported as well[115]. A higher dose of 20 mg/kg per
day may be more efficious than lower doses (5-15 mg/kg
per day)[111,116]. Thus, UDCA may be considered as poten-
tially effective in CF patients. However, whether UDCA af-
fects the natural history of liver disease in CF-particularly
amelioration of the complications of portal hypertension,
need for liver transplantation, or a measurable survival is
unknown and further well-designed, adequately powered,
randomized controlled trials should address these issues.
Stimulation of biliary secretion may be the major mecha-
nism for the therapeutic effect. Interestingly, it has been
hypothesized that the therapeutic effect of UDCA is me-
diated in part by stimulation of ATP release into bile and
subsequent ATP-induced bicarbonate secretion by bile-
duct epithelia via calcium-dependent chloride channels dis-
tinct from the CFTR[117].
References
1 Hagey LR, Crombie DL, Espinosa E, Carey MC, Igimi H, Hof-
mann AF. Ursodeoxycholic acid in the Ursidae: biliary bile
acids of bears, pandas, and related carnivores. J Lipid Res 1993;
34: 1911-1917
2 Hofmann AF. Pharmacology of ursodeoxycholic acid, an en-
terohepatic drug. Scand J Gastroenterol Suppl 1994; 204: 1-15
3 Poupon R, Poupon RE. Ursodeoxycholic acid therapy of
chronic cholestatic conditions in adults and children. Pharma-
col Ther 1995; 66: 1-15
4 Sugata F, Shimizu M. [Retrospective studies on gallstone dis-
appearance (author's transl)]. Nihon Shokakibyo Gakkai Zasshi
1974; 71: 75-80
5 Bachrach WH, Hofmann AF. Ursodeoxycholic acid in the
treatment of cholesterol cholelithiasis. part I. Dig Dis Sci 1982;
27: 737-761
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
6 Makino I, Shinozaki K, Yoshino K, Nakagawa S. [Dissolu-
tion of cholesterol gallstones by long-term administration of
ursodeoxycholic acid]. Nihon Shokakibyo Gakkai Zasshi 1975; 72:
690-702
7 Leuschner U, Leuschner M, Sieratzki J, Kurtz W, Hbner K.
Gallstone dissolution with ursodeoxycholic acid in patients
with chronic active hepatitis and two years follow-up. A pilot
study. Dig Dis Sci 1985; 30: 642-649
8 Rubin RA, Kowalski TE, Khandelwal M, Malet PF. Ursodiol
for hepatobiliary disorders. Ann Intern Med 1994; 121: 207-218
9 Sauer P, Benz C, Rudolph G, Klters-Plachky P, Stremmel W,
Stiehl A. Influence of cholestasis on absorption of ursodeoxy-
cholic acid. Dig Dis Sci 1999; 44: 817-822
10 Kullak-Ublick GA, Stieger B, Hagenbuch B, Meier PJ. Hepatic
transport of bile salts. Semin Liver Dis 2000; 20: 273-292
11 Beuers U, Boyer JL, Paumgartner G. Ursodeoxycholic acid in
cholestasis: potential mechanisms of action and therapeutic
applications. Hepatology 1998; 28: 1449-1453
12 Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic
liver disease: mechanisms of action and therapeutic use revis-
ited. Hepatology 2002; 36: 525-531
13 Trauner M, Graziadei IW. Review article: mechanisms of ac-
tion and therapeutic applications of ursodeoxycholic acid in
chronic liver diseases. Aliment Pharmacol Ther 1999; 13: 979-996
14 Lazaridis KN, Gores GJ, Lindor KD. Ursodeoxycholic acid
'mechanisms of action and clinical use in hepatobiliary disor-
ders'. J Hepatol 2001; 35: 134-146
15 Beuers U, Bilzer M, Chittattu A, Kullak-Ublick GA, Keppler D,
Paumgartner G, Dombrowski F. Tauroursodeoxycholic acid
inserts the apical conjugate export pump, Mrp2, into canalicu-
lar membranes and stimulates organic anion secretion by pro-
tein kinase C-dependent mechanisms in cholestatic rat liver.
Hepatology 2001; 33: 1206-1216
16 Kitani K, Ohta M, Kanai S. Tauroursodeoxycholate prevents
biliary protein excretion induced by other bile salts in the rat.
Am J Physiol 1985; 248: G407-G417
17 Schlmerich J, Baumgartner U, Miyai K, Gerok W. Taurourso-
deoxycholate prevents taurolithocholate-induced cholestasis
and toxicity in rat liver. J Hepatol 1990; 10: 280-283
18 Heuman DM, Mills AS, McCall J, Hylemon PB, Pandak
WM, Vlahcevic ZR. Conjugates of ursodeoxycholate protect
against cholestasis and hepatocellular necrosis caused by
more hydrophobic bile salts. In vivo studies in the rat.
Gastroenterology 1991; 100: 203-211
19 Heuman DM, Pandak WM, Hylemon PB, Vlahcevic ZR.
Conjugates of ursodeoxycholate protect against cytotoxicity of
more hydrophobic bile salts: in vitro studies in rat hepatocytes
and human erythrocytes. Hepatology 1991; 14: 920-926
20 Ohiwa T, Katagiri K, Hoshino M, Hayakawa T, Nakai T.
Tauroursodeoxycholate and tauro-beta-muricholate exert
cytoprotection by reducing intrahepatocyte taurocheno-
deoxycholate content. Hepatology 1993; 17: 470-476
21 Deroubaix X, Coche T, Depiereux E, Feytmans E. Saturation of
hepatic transport of taurocholate in rats in vivo. Am J Physiol
1991; 260: G189-G196
22 Galan AI, Jimenez R, Muoz ME, Gonzalez J. Effects of
ursodeoxycholate on maximal biliary secretion of bilirubin in
the rat. Biochem Pharmacol 1990; 39: 1175-1180
23 Jazrawi RP, de Caestecker JS, Goggin PM, Britten AJ, Joseph
AE, Maxwell JD, Northfield TC. Kinetics of hepatic bile acid
handling in cholestatic liver disease: effect of ursodeoxycholic
acid. Gastroenterology 1994; 106: 134-142
24 Poupon RE, Chrtien Y, Poupon R, Paumgartner G. Serum
bile acids in primary biliary cirrhosis: effect of ursodeoxycholic
acid therapy. Hepatology 1993; 17: 599-604
25 Heathcote EJ, Cauch-Dudek K, Walker V, Bailey RJ, Blendis
LM, Ghent CN, Michieletti P, Minuk GY, Pappas SC, Scully
LJ. The Canadian Multicenter Double-blind Randomized
Controlled Trial of ursodeoxycholic acid in primary biliary
cirrhosis. Hepatology 1994; 19: 1149-1156
26 Lindor KD. Ursodiol for primary sclerosing cholangitis. Mayo
Primary Sclerosing Cholangitis-Ursodeoxycholic Acid Study
Group. N Engl J Med 1997; 336: 691-695
 Pusl T et al . Ursodeoxycholic acid treatment of VBDS 3493
27 Poupon RE, Balkau B, Eschwge E, Poupon R. A multicenter,
controlled trial of ursodiol for the treatment of primary biliary
cirrhosis. UDCA-PBC Study Group. N Engl J Med 1991; 324:
1548-1554
28 Beuers U, Spengler U, Kruis W, Aydemir U, Wiebecke B,
Heldwein W, Weinzierl M, Pape GR, Sauerbruch T, Paum-
gartner G. Ursodeoxycholic acid for treatment of primary
sclerosing cholangitis: a placebo-controlled trial. Hepatology
1992; 16: 707-714
29 Medina JF, Martnez-Ans JJ, Prieto J. Decreased anion
exchanger 2 immunoreactivity in the liver of patients with
primary biliary cirrhosis. Hepatology 1997; 25: 12-17
30 Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C,
Zenz R, Lammert F, Stieger B, Meier PJ, Zatloukal K, Denk H,
Trauner M. Effects of ursodeoxycholic and cholic acid feeding
on hepatocellular transporter expression in mouse liver.
Gastroenterology 2001; 121: 170-183
31 Kurz AK, Graf D, Schmitt M, Vom Dahl S, Hussinger
D. Tauroursodesoxycholate-induced choleresis involves
p38(MAPK) activation and translocation of the bile salt export
pump in rats. Gastroenterology 2001; 121: 407-419
32 Zollner G, Fickert P, Fuchsbichler A, Silbert D, Wagner M,
Arbeiter S, Gonzalez FJ, Marschall HU, Zatloukal K, Denk H,
Trauner M. Role of nuclear bile acid receptor, FXR, in adaptive
ABC transporter regulation by cholic and ursodeoxycholic
acid in mouse liver, kidney and intestine. J Hepatol 2003; 39:
480-488
33 Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of
cholestasis. N Engl J Med 1998; 339: 1217-1227
34 Beuers U, Nathanson MH, Isales CM, Boyer JL. Taurourso-
deoxycholic acid stimulates hepatocellular exocytosis and
mobilizes extracellular Ca++ mechanisms defective in
cholestasis. J Clin Invest 1993; 92: 2984-2993
35 Beuers U, Throckmorton DC, Anderson MS, Isales CM,
Thasler W, Kullak-Ublick GA, Sauter G, Koebe HG,
Paumgartner G, Boyer JL. Tauroursodeoxycholic acid activates
protein kinase C in isolated rat hepatocytes. Gastroenterology
1996; 110: 1553-1563
36 Schliess F, Kurz AK, vom Dahl S, Hussinger D. Mitogen-
activated protein kinases mediate the stimulation of bile
acid secretion by tauroursodeoxycholate in rat liver. Gastro-
enterology 1997; 113: 1306-1314
37 Noe J, Hagenbuch B, Meier PJ, St-Pierre MV. Characterization
of the mouse bile salt export pump overexpressed in the
baculovirus system. Hepatology 2001; 33: 1223-1231
38 Yoon JH, Gores GJ. Death receptor-mediated apoptosis and
the liver. J Hepatol 2002; 37: 400-410
39 Guicciardi ME, Gores GJ. Ursodeoxycholic acid cytoprotection:
dancing with death receptors and survival pathways.
Hepatology 2002; 35: 971-973
40 Harada K, Ozaki S, Gershwin ME, Nakanuma Y. Enhanced
apoptosis relates to bile duct loss in primary biliary cirrhosis.
Hepatology 1997; 26: 1399-1405
41 Higuchi H, Gores GJ. Mechanisms of liver injury: an overview.
Curr Mol Med 2003; 3: 483-490
42 Faubion WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ,
Svingen PA, Kaufmann SH, Gores GJ. Toxic bile salts induce
rodent hepatocyte apoptosis via direct activation of Fas. J Clin
Invest 1999; 103: 137-145
43 Benz C, Angermller S, Tx U, Klters-Plachky P, Riedel HD,
Sauer P, Stremmel W, Stiehl A. Effect of tauroursodeoxycholic
acid on bile-acid-induced apoptosis and cytolysis in rat
hepatocytes. J Hepatol 1998; 28: 99-106
44 Benz C, Angermller S, Otto G, Sauer P, Stremmel W, Stiehl
A. Effect of tauroursodeoxycholic acid on bile acid-induced
apoptosis in primary human hepatocytes. Eur J Clin Invest
2000; 30: 203-209
45 R o d r i g u e s C M, F a n G , W o n g P Y , K r e n B T , S t e e r C J .
Ursodeoxycholic acid may inhibit deoxycholic acid-induced
apoptosis by modulating mitochondrial transmembrane
potential and reactive oxygen species production. Mol Med
1998; 4: 165-178
46 Graf D, Kurz AK, Fischer R, Reinehr R, Hussinger D.
Taurolithocholic acid-3 sulfate induces CD95 trafficking and
apoptosis in a c-Jun N-terminal kinase-dependent manner.
Gastroenterology 2002; 122: 1411-1427
47 Qiao L, Yacoub A, Studer E, Gupta S, Pei XY, Grant S,
Hylemon PB, Dent P. Inhibition of the MAPK and PI3K
pathways enhances UDCA-induced apoptosis in primary
rodent hepatocytes. Hepatology 2002; 35: 779-789
48 Hofmann AF. Bile Acids: The Good, the Bad, and the Ugly.
News Physiol Sci 1999; 14: 24-29
49 Gldtuna S, Zimmer G, Imhof M, Bhatti S, You T,
Leuschner U. Molecular aspects of membrane stabilization by
ursodeoxycholate [see comment]. Gastroenterology 1993; 104:
1736-1744
50 Heuman DM, Bajaj RS, Lin Q. Adsorption of mixtures of bile
salt taurine conjugates to lecithin-cholesterol membranes:
implications for bile salt toxicity and cytoprotection. J Lipid Res
1996; 37: 562-573
51 Heuman DM, Bajaj R. Ursodeoxycholate conjugates protect
against disruption of cholesterol-rich membranes by bile salts.
Gastroenterology 1994; 106: 1333-1341
52 Heuman DM. Hepatoprotective properties of ursodeoxycholic
acid. Gastroenterology 1993; 104: 1865-1870
53 Van Nieuwkerk CM, Elferink RP, Groen AK, Ottenhoff
R, Tytgat GN, Dingemans KP, Van Den Bergh Weerman
MA, Offerhaus GJ. Effects of Ursodeoxycholate and cholate
feeding on liver disease in FVB mice with a disrupted mdr2
P-glycoprotein gene. Gastroenterology 1996; 111: 165-171
54 Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Weiglein
AH, Lammert F, Marschall HU, Tsybrovskyy O, Zatloukal
K, Denk H, Trauner M. Ursodeoxycholic acid aggravates
bile infarcts in bile duct-ligated and Mdr2 knockout mice
via disruption of cholangioles. Gastroenterology 2002; 123:
1238-1251
55 Pars A, Caballera L, Rods J, Bruguera M, Rodrigo L, Garca-
Plaza A, Berenguer J, Rodrguez-Martnez D, Mercader
J, Velicia R. Long-term effects of ursodeoxycholic acid in
primary biliary cirrhosis: results of a double-blind controlled
multicentric trial. UDCA-Cooperative Group from the Spanish
Association for the Study of the Liver. J Hepatol 2000; 32:
561-566
56 Stiehl A, Walker S, Stiehl L, Rudolph G, Hofmann WJ,
Theilmann L. Effect of ursodeoxycholic acid on liver and bile
duct disease in primary sclerosing cholangitis. A 3-year pilot
study with a placebo-controlled study period. J Hepatol 1994;
20: 57-64
57 Metcalf J, James O. The geoepidemiology of primary biliary
cirrhosis. Semin Liver Dis 1997; 17: 13-22
58 Kim WR, Lindor KD, Locke GR 3rd, Therneau TM,
Homburger HA, Batts KP, Yawn BP, Petz JL, Melton LJ 3rd,
Dickson ER. Epidemiology and natural history of primary
biliary cirrhosis in a US community. Gastroenterology 2000; 119:
1631-1636
59 Nishio A, Keeffe EB, Gershwin ME. Immunopathogenesis of
primary biliary cirrhosis. Semin Liver Dis 2002; 22: 291-302
60 Bergasa NV. Pruritus and fatigue in primary biliary cirrhosis.
Clin Liver Dis 2003; 7: 879-900
61 Sherlock S, Scheuer PJ. The presentation and diagnosis of 100
patients with primary biliary cirrhosis. N Engl J Med 1973; 289:
674-678
62 Poupon R, Chrtien Y, Poupon RE, Ballet F, Calmus Y, Darnis
F. Is ursodeoxycholic acid an effective treatment for primary
biliary cirrhosis? Lancet 1987; 1: 834-836
63 Leuschner U, Fischer H, Kurtz W, Gldtuna S, Hbner K,
Hellstern A, Gatzen M, Leuschner M. Ursodeoxycholic acid in
primary biliary cirrhosis: results of a controlled double-blind
trial. Gastroenterology 1989; 97: 1268-1274
64 Combes B, Carithers RL Jr, Maddrey WC, Lin D, McDonald
MF, Wheeler DE, Eigenbrodt EH, Muoz SJ, Rubin R, Garcia-
Tsao G. A randomized, double-blind, placebo-controlled trial
of ursodeoxycholic acid in primary biliary cirrhosis. Hepatology
1995; 22: 759-766
65 Battezzati PM, Podda M, Bianchi FB, Naccarato R, Orlandi F,
Surrenti C, Pagliaro L, Manenti F. Ursodeoxycholic acid for
www.wjgnet.com
 3494 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
symptomatic primary biliary cirrhosis. Preliminary analysis of
a double-blind multicenter trial. Italian Multicenter Group for
the Study of UDCA in PBC. J Hepatol 1993; 17: 332-338
66 Lindor KD, Dickson ER, Baldus WP, Jorgensen RA, Ludwig
J, Murtaugh PA, Harrison JM, Wiesner RH, Anderson ML,
Lange SM. Ursodeoxycholic acid in the treatment of primary
biliary cirrhosis. Gastroenterology 1994; 106: 1284-1290
67 Shapiro JM, Smith H, Schaffner F. Serum bilirubin: a
prognostic factor in primary biliary cirrhosis. Gut 1979; 20:
137-140
68 Corpechot C, Carrat F, Bonnand AM, Poupon RE, Poupon R.
The effect of ursodeoxycholic acid therapy on liver fibrosis
progression in primary biliary cirrhosis. Hepatology 2000; 32:
1196-1199
69 Poupon R, Chazouillres O, Balkau B, Poupon RE. Clinical
and biochemical expression of the histopathological lesions of
primary biliary cirrhosis. UDCA-PBC Group. J Hepatol 1999;
30: 408-412
70 Poupon RE, Lindor KD, Cauch-Dudek K, Dickson ER, Poupon
R, Heathcote EJ. Combined analysis of randomized controlled
trials of ursodeoxycholic acid in primary biliary cirrhosis.
Gastroenterology 1997; 113: 884-890
71 Lindor KD, Therneau TM, Jorgensen RA, Malinchoc M,
Dickson ER. Effects of ursodeoxycholic acid on survival in
patients with primary biliary cirrhosis. Gastroenterology 1996;
110: 1515-1518
72 Poupon RE, Bonnand AM, Chrtien Y, Poupon R. Ten-year
survival in ursodeoxycholic acid-treated patients with primary
biliary cirrhosis. The UDCA-PBC Study Group. Hepatology
1999; 29: 1668-1671
73 Kilmurry MR, Heathcote EJ, Cauch-Dudek K, O'Rourke K,
Bailey RJ, Blendis LM, Ghent CN, Minuk GY, Pappas SC,
Scully LJ, Steinbrecher UP, Sutherland LR, Williams CN,
Worobetz LJ. Is the Mayo model for predicting survival useful
after the introduction of ursodeoxycholic acid treatment for
primary biliary cirrhosis? Hepatology 1996; 23: 1148-1153
74 Goulis J, Leandro G, Burroughs AK. Randomised controlled
trials of ursodeoxycholic-acid therapy for primary biliary
cirrhosis: a meta-analysis. Lancet 1999; 354: 1053-1060
75 Gluud C, Christensen E. Ursodeoxycholic acid for primary
biliary cirrhosis. Cochrane Database Syst Rev 2002; (1): CD000551
76 Lindor KD, Poupon R, Poupon R, Heathcote EJ, Therneau T.
Ursodeoxycholic acid for primary biliary cirrhosis. Lancet 2000;
355: 657-658
77 Wiesner RH, Grambsch PM, Dickson ER, Ludwig J, MacCarty
RL, Hunter EB, Fleming TR, Fisher LD, Beaver SJ, LaRusso
NF. Primary sclerosing cholangitis: natural history, prognostic
factors and survival analysis. Hepatology 1989; 10: 430-436
78 Farrant JM, Hayllar KM, Wilkinson ML, Karani J, Portmann
BC, Westaby D, Williams R. Natural history and prognostic
variables in primary sclerosing cholangitis. Gastroenterology
1991; 100: 1710-1717
79 Broom U, Olsson R, Lf L, Bodemar G, Hultcrantz R, Dan-
ielsson A, Prytz H, Sandberg-Gertzn H, Wallerstedt S, Lind-
berg G. Natural history and prognostic factors in 305 Swedish
patients with primary sclerosing cholangitis. Gut 1996; 38:
610-615
80 Boberg KM, Rocca G, Egeland T, Bergquist A, Broom U,
Caballeria L, Chapman R, Hultcrantz R, Mitchell S, Pares A,
Rosina F, Schrumpf E. Time-dependent Cox regression model
is superior in prediction of prognosis in primary sclerosing
cholangitis. Hepatology 2002; 35: 652-657
81 Boberg KM, Aadland E, Jahnsen J, Raknerud N, Stiris M, Bell
H. Incidence and prevalence of primary biliary cirrhosis, pri-
mary sclerosing cholangitis, and autoimmune hepatitis in a
Norwegian population. Scand J Gastroenterol 1998; 33: 99-103
82 Brandsaeter B, Isoniemi H, Broom U, Olausson M, Bckman L,
Hansen B, Schrumpf E, Oksanen A, Ericzon BG, Hckerstedt
K, Mkisalo H, Kirkegaard P, Friman S, Bjro K. Liver trans-
plantation for primary sclerosing cholangitis; predictors and
consequences of hepatobiliary malignancy. J Hepatol 2004; 40:
815-822
83 Lee YM, Kaplan MM. Primary sclerosing cholangitis. N Engl J
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
Med 1995; 332: 924-933
84 Bergquist A, Ekbom A, Olsson R, Kornfeldt D, Lf L, Da-
nielsson A, Hultcrantz R, Lindgren S, Prytz H, Sandberg-
Gertzn H, Almer S, Granath F, Broom U. Hepatic and
extrahepatic malignancies in primary sclerosing cholangitis. J
Hepatol 2002; 36: 321-327
85 Portincasa P, Vacca M, Moschetta A, Petruzzelli M, Palasciano
G, van Erpecum KJ, van Berge-Henegouwen GP. Primary scle-
rosing cholangitis: updates in diagnosis and therapy. World J
Gastroenterol 2005; 11: 7-16
86 Broom U, Lfberg R, Veress B, Eriksson LS. Primary scleros-
ing cholangitis and ulcerative colitis: evidence for increased
neoplastic potential. Hepatology 1995; 22: 1404-1408
87 Marchesa P, Lashner BA, Lavery IC, Milsom J, Hull TL, Strong
SA, Church JM, Navarro G, Fazio VW. The risk of cancer and
dysplasia among ulcerative colitis patients with primary scle-
rosing cholangitis. Am J Gastroenterol 1997; 92: 1285-1288
88 Stiehl A. Ursodeoxycholic acid therapy in treatment of pri-
mary sclerosing cholangitis. Scand J Gastroenterol Suppl 1994;
204: 59-61
89 O'Brien CB, Senior JR, Arora-Mirchandani R, Batta AK, Salen
G. Ursodeoxycholic acid for the treatment of primary scleros-
ing cholangitis: a 30-month pilot study. Hepatology 1991; 14:
838-847
90 Chen W, Gluud C. Bile acids for primary sclerosing cholangi-
tis. Cochrane Database Syst Rev 2003; (2): CD003626
91 Stiehl A, Rudolph G, Klters-Plachky P, Sauer P, Walker S.
Development of dominant bile duct stenoses in patients with
primary sclerosing cholangitis treated with ursodeoxycholic
acid: outcome after endoscopic treatment. J Hepatol 2002; 36:
151-156
92 Stiehl A, Rudolph G, Sauer P, Benz C, Stremmel W, Walker S,
Theilmann L. Efficacy of ursodeoxycholic acid treatment and
endoscopic dilation of major duct stenoses in primary scleros-
ing cholangitis. An 8-year prospective study. J Hepatol 1997;
26: 560-566
93 Harnois DM, Angulo P, Jorgensen RA, Larusso NF, Lindor
KD. High-dose ursodeoxycholic acid as a therapy for patients
with primary sclerosing cholangitis. Am J Gastroenterol 2001;
96: 1558-1562
94 Mitchell SA, Bansi DS, Hunt N, Von Bergmann K, Fleming
KA, Chapman RW. A preliminary trial of high-dose ursode-
oxycholic acid in primary sclerosing cholangitis. Gastroenterol-
ogy 2001; 121: 900-907
95 Olsson R, Boberg KM, de Muckadell OS, Lindgren S, Hult-
crantz R, Folvik G, Bell H, Gangsy-Kristiansen M, Matre J,
Rydning A, Wikman O, Danielsson A, Sandberg-Gertzn H,
Ung KA, Eriksson A, Lf L, Prytz H, Marschall HU, Broom
U. High-dose ursodeoxycholic acid in primary sclerosing chol-
angitis: a 5-year multicenter, randomized, controlled study.
Gastroenterology 2005; 129: 1464-1472
96 Earnest DL, Holubec H, Wali RK, Jolley CS, Bissonette M,
Bhattacharyya AK, Roy H, Khare S, Brasitus TA. Chemopre-
vention of azoxymethane-induced colonic carcinogenesis by
supplemental dietary ursodeoxycholic acid. Cancer Res 1994;
54: 5071-5074
97 Ikegami T, Matsuzaki Y, Shoda J, Kano M, Hirabayashi N,
Tanaka N. The chemopreventive role of ursodeoxycholic acid
in azoxymethane-treated rats: suppressive effects on enhanced
group II phospholipase A2 expression in colonic tissue. Cancer
Lett 1998; 134: 129-139
98 Tung BY, Emond MJ, Haggitt RC, Bronner MP, Kimmey MB,
Kowdley KV, Brentnall TA. Ursodiol use is associated with
lower prevalence of colonic neoplasia in patients with ulcer-
ative colitis and primary sclerosing cholangitis. Ann Intern
Med 2001; 134: 89-95
99 Pardi DS, Loftus EV Jr, Kremers WK, Keach J, Lindor KD.
Ursodeoxycholic acid as a chemopreventive agent in patients
with ulcerative colitis and primary sclerosing cholangitis. Gas-
troenterology 2003; 124: 889-893
100 Bacq Y, Sapey T, Brchot MC, Pierre F, Fignon A, Dubois F.
Intrahepatic cholestasis of pregnancy: a French prospective
study. Hepatology 1997; 26: 358-364
 Pusl T et al . Ursodeoxycholic acid treatment of VBDS 3495
101 Alsulyman OM, Ouzounian JG, Ames-Castro M, Goodwin
TM. Intrahepatic cholestasis of pregnancy: perinatal outcome
associated with expectant management. Am J Obstet Gynecol
1996; 175: 957-960
102 Jacquemin E. Role of multidrug resistance 3 deficiency in
pediatric and adult liver disease: one gene for three diseases.
Semin Liver Dis 2001; 21: 551-562
103 Palma J, Reyes H, Ribalta J, Hernndez I, Sandoval L, Almuna
R, Liepins J, Lira F, Sedano M, Silva O, Toh D, Silva JJ. Urso-
deoxycholic acid in the treatment of cholestasis of pregnancy:
a randomized, double-blind study controlled with placebo. J
Hepatol 1997; 27: 1022-1028
104 Jenkins JK, Boothby LA. Treatment of itching associated with
intrahepatic cholestasis of pregnancy. Ann Pharmacother 2002;
36: 1462-1465
105 Burrows RF, Clavisi O, Burrows E. Interventions for treat-
ing cholestasis in pregnancy. Cochrane Database Syst Rev 2001;
CD000493
106 Kondrackiene J, Beuers U, Kupcinskas L. Efficacy and safety
of ursodeoxycholic acid versus cholestyramine in intrahepatic
cholestasis of pregnancy. Gastroenterology 2005; 129: 894-901
107 Fried RH, Murakami CS, Fisher LD, Willson RA, Sullivan KM,
McDonald GB. Ursodeoxycholic acid treatment of refractory
chronic graft-versus-host disease of the liver. Ann Intern Med
1992; 116: 624-629
108 Wulffraat NM, Haddad E, Benkerrou M, Spliet WG, Patey N,
Fischer A, de Graeff-Meeder BR. Hepatic GVHD after HLA-
haploidentical bone marrow transplantation in children with
severe combined immunodeficiency: the effect of ursodeoxy-
cholic acid. Br J Haematol 1997; 96: 776-780
109 Essell JH, Schroeder MT, Harman GS, Halvorson R, Lew V,
Callander N, Snyder M, Lewis SK, Allerton JP, Thompson JM.
Ursodiol prophylaxis against hepatic complications of allo-
geneic bone marrow transplantation. A randomized, double-
blind, placebo-controlled trial. Ann Intern Med 1998; 128:
975-981
110 Feigelson J, Anagnostopoulos C, Poquet M, Pecau Y, Munck
A, Navarro J. Liver cirrhosis in cystic fibrosis--therapeutic
implications and long term follow up. Arch Dis Child 1993; 68:
653-657
111 Colombo C, Castellani MR, Balistreri WF, Seregni E, Assaisso
ML, Giunta A. Scintigraphic documentation of an improve-
ment in hepatobiliary excretory function after treatment with
ursodeoxycholic acid in patients with cystic fibrosis and asso-
ciated liver disease. Hepatology 1992; 15: 677-684
112 Colombo C, Crosignani A, Assaisso M, Battezzati PM, Podda
M, Giunta A, Zimmer-Nechemias L, Setchell KD. Ursodeo-
xycholic acid therapy in cystic fibrosis-associated liver disease:
a dose-response study. Hepatology 1992; 16: 924-930
113 Cotting J, Lentze MJ, Reichen J. Effects of ursodeoxycholic
acid treatment on nutrition and liver function in patients with
cystic fibrosis and longstanding cholestasis. Gut 1990; 31:
918-921
114 Colombo C, Battezzati PM, Podda M, Bettinardi N, Giunta A.
Ursodeoxycholic acid for liver disease associated with cystic
fibrosis: a double-blind multicenter trial. The Italian Group for
the Study of Ursodeoxycholic Acid in Cystic Fibrosis. Hepatol-
ogy 1996; 23: 1484-1490
115 Lindblad A, Glaumann H, Strandvik B. A two-year prospec-
tive study of the effect of ursodeoxycholic acid on urinary bile
acid excretion and liver morphology in cystic fibrosis-associat-
ed liver disease. Hepatology 1998; 27: 166-174
116 van de Meeberg PC, Houwen RH, Sinaasappel M, Heijerman
HG, Bijleveld CM, Vanberge-Henegouwen GP. Low-dose ver-
sus high-dose ursodeoxycholic acid in cystic fibrosis-related
cholestatic liver disease. Results of a randomized study with
1-year follow-up. Scand J Gastroenterol 1997; 32: 369-373
117 Nathanson MH, Burgstahler AD, Masyuk A, Larusso NF.
Stimulation of ATP secretion in the liver by therapeutic bile
acids. Biochem J 2001; 358: 1-5
S- Editor Pan BR E- Editor Bai SH
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TOPIC HIGHLIGHT
Gianfranco D Alpini, PhD, Series Editor
Cholangiocyte anion exchange and biliary bicarbonate
excretion
Jess M Banales, Jess Prieto, Juan F Medina
Jess M Banales, Jess Prieto, Juan F Medina, Laboratory of
Molecular Genetics, Division of Gene Therapy and Hepatology,
University of Navarra School of Medicine, Clnica Universitaria
and CIMA, Pamplona, Spain
Supported by the UTE for CIMA project as well as by a grant
from the Instituto de Salud Carlos III (PI051098). J. M. B. has
a grant from the Spanish Ministry of Science and Technology
Correspondence to: Juan F Medina, Division of Gene Therapy
and Hepatology, University of Navarra School of Medicine,
Clnica Universitaria and CIMA, Avda. Po XII 55, E-31008
Pamplona, Spain. jfmedina@unav.es
Telephone: +34-948-194700 Fax: +34-948-194717
Received: 2006-01-20 Accepted: 2006-02-18
Abstract
Primary canalicular bile undergoes a process of fluidi-
zation and alkalinization along the biliary tract that is
influenced by several factors including hormones, in-
nervation/neuropeptides, and biliary constituents. The
excretion of bicarbonate at both the canaliculi and the
bile ducts is an important contributor to the generation
of the so-called bile-salt independent flow. Bicarbon-
ate is secreted from hepatocytes and cholangiocytes
through parallel mechanisms which involve chloride ef-
-
flux through activation of Cl channels, and further bi-
- -
carbonate secretion via AE2/SLC4A2-mediated Cl /HCO3
exchange. Glucagon and secretin are two relevant hor-
mones which seem to act very similarly in their target
cells (hepatocytes for the former and cholangiocytes for
the latter). These hormones interact with their specific G
protein-coupled receptors, causing increases in intracel-
lular levels of cAMP and activation of cAMP-dependent
- -
Cl and HCO3 secretory mechanisms. Both hepatocytes
and cholangiocytes appear to have cAMP-responsive in-
tracellular vesicles in which AE2/SLC4A2 colocalizes with
-
cell specific Cl channels (CFTR in cholangiocytes and not
yet determined in hepatocytes) and aquaporins (AQP8 in
hepatocytes and AQP1 in cholangiocytes). cAMP-induced
coordinated trafficking of these vesicles to either cana-
licular or cholangiocyte lumenal membranes and further
exocytosis results in increased osmotic forces and pas-
sive movement of water with net bicarbonate-rich hydro-
choleresis.
2006 The WJG Press. All rights reserved.
Key words: AE2 anion exchanger; Bile salt-independent
flow; Biliary bicarbonate excretion; Regulation of
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World J Gastroenterol 2006 June 14; 12(22): 3496-3511
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
intracellular pH; Hydroionic fluxes in cholangiocytes
Banales JM, Prieto J, Medina JF. Cholangiocyte anion exchange
and biliary bicarbonate excretion. World J Gastroenterol 2006;
12(22): 3496-3511
http://www.wjgnet.com/1007-9327/12/3496.asp
Introduction
Hepatocytes and cholangiocytes are the epithelial cells
in the liver, and they both participate in the production
of bile. The hepatocytes, the major liver cell population
(65%), generate the primary bile at their canaliculi [1].
As an important complement, the epithelial cells lining
intrahepatic bile ducts or cholangiocytes (5% of the
liver cell population) exert a series of reabsorptive and
secretory processes which dilute and alkalinize the
bile flow during its passage along the biliary tract [2-6].
Modifications of ductal bile appear to be tightly regulated
by the action of nerves, biliary constituents, and some
peptide hormones like secretin. Accordingly, it is possible
to distinguish between three different bile flow fractions: (1)
the canalicular bile salt-dependent flow (30% to 60% of
spontaneous basal bile flow) that is driven by concentrative
secretion of bile acids by the hepatocytes followed by a
facilitated efflux of water[7,8]; (2) the canalicular bile salt-
independent flow (another 30% to 60% of spontaneous
basal bile flow)[1,8-10], which is also created by hepatocytes
but through active secretion of both inorganic and organic
compounds (mainly bicarbonate[11,12] and glutathione[13],
respectively); and (3) the ductal bile flow, that is the bile
salt-independent flow fraction modified and contrib-
uted by cholangiocytes, mainly through production of a
bicarbonate-rich fluid in response to secretin[2,14-17] and
other regulatory factors[2,5,6,17,18].
In the last decade, the use of molecular- and cell-biology
tools and the availability of suitable experimental models
have greatly facilitated our knowledge on the processes
involved in bile generation and modification. This review
summarizes some of the experimental models employed
in biliary studies, and focuses on the biliary excretion of
bicarbonate, the chief factor responsible for ductal bile al-
kalinization and fluidization, and the role and interactions
of regulatory factors.
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion 3497

Study Models was developed by using positron emission tomography

(PET) [38]. This imaging technique allows evaluation of
Biliary studies started over one hundred years ago. In 1902
baseline and stimulated organ functions after intravenous
Bayliss and Starling discovered the hormone secretin as
injection of short half-live positron emitting isotopes[39,40].
an agent with stimulatory effects on pancreatic secretion
Thus, 2-3 min after bicarbonate labeled with carbon-11
and bile flow[19]. About 25 years later secretin was reported
(half live of 20.4 min) was given to healthy volunteers,
as a cholagogue, i.e. an agent capable of stimulating the
label uptake was obser ved in the abdominal region
flow of bile into the duodenum[20], as well as a choleretic
corresponding to the liver parenchyma and hepatic
agent that could stimulate the production of bile in the
hilum. Interestingly, administration of secretin increased
liver[21]. In spite of this, although the role of hepatocytes
bicarbonate uptake in the parenchymal region, this being
for bile generation was widely recognized quite early,
followed by accumulation of the label in the perihilar
the contribution of cholangiocytes to the production of
area[38]. Currently, the availability of micro-PET systems
bile with an adequate composition was accepted more
may also facilitate non-invasive in vivo studies of biliary
recently. Knowledge of the cholangiocyte contribution was
bicarbonate secretion in small laboratory animals.
facilitated by the availability of experimental models and
Animal models of bile ductal cell hyperplasia have
the development of both in vivo and in vitro sophisticated
also been developed, mainly in rodents, to study the
procedures.
pathophysiology of bile ducts [41-43]. These models are
closely associated with increased secretin-stimulated
In vivo models ductal secretion. The increased responsiveness seems to
The initial experimental studies on the bile flow and bile occur regardless of the procedure employed to obtain
composition were carried out by using in vivo models of the hyperplasia of cholangiocytes: partial hepatectomy[44],
conscious or anesthetized mammals with biliary fistulas. chronic feeding of bile acids[45] or -naphthylisothiocyanate
In the case of total biliary fistulas, there was a major
(ANIT) [46] , chronic administration of phenobarbital
concern because of the interruption of the enterohepatic
plus CCl4 to rats[47] or just CCl4 in mice[48], or bile-duct
circulation and the subsequent depletion of the bile acid
ligation (BDL) [41,49]. In contrast, a decreased secretin-
pool. However, this can be overcome by continuous
stimulated ductal secretion can be observed in BDL rats
intravenous or intraduodenal administration of exogenous
with ductopenia following interruption of the cholinergic
bile acids at controlled rates. On the other hand, partial
innervation by total vagotomy[50], or selective damage of
biliary fistula allows controlled interruption of the
large (but not small) cholangiocytes by acute feeding with
enterohepatic circulation, and a portion of bile collected
CCl4[51,52].
can be returned to the stomach or duodenum, thereby
The development of genetically modified murine mod-
replenishing the pool of natural bile acids [10]. Fistula
els has contributed to ascertain the role of selective genes,
animals have also been useful to estimate canalicular bile
like those for the cystic fibrosis transmembrane conduct-
flow by measuring the biliary clearance of selected inert
ance regulator Cftr[53,54] and the P-glycoprotein PGY3/
solutes (mainly erythritol and mannitol). This procedure
MDR2-3/ABCB4 Mdr2/Abcb4[55], among others. In Cftr-/-
assumes that these solutes are sufficiently permeable to
mice, for instance, induction of colitis has been shown
enter the canalicular bile by passive processes while being
to result in increased bile duct injury[53]. In Mdr2/Abcb4-/-
unable to cross the ductal epithelium[22,23]. It appears that
mice, a multistep process of bile-duct damage leading to
this might be the case in some animal species but not
sclerosing cholangitis has been described[55]. Spontaneous
in all [24]. Moreover, bile duct cannulation allows direct
mutant animals can also serve as useful in vivo models. The
interventions in the biliary tract through retrograde
PCK rat, a model of the autosomal recessive polycystic
intrabiliar y injection [25-28] ; this procedure has been
kidney and hepatic disease (Pkhd), has been used for
employed to obtain animal models of hepatitis [29] and
studies on possible trigger factors of biliary cystogenesis.
toxic-induced biliary disease[30], as well as for experimental
This mutant was spontaneously developed in the rat strain
gene therapy[31,32]. Retrograde intrabiliary injection has also
Crj:CD/Sprague-Dawley because of a germ line mutation
been employed to assess the effects of toxic substances
of the Pkhd1 gene[56,57]. The TR- rat model is widely used
or inhibitors on the bicarbonate-rich choleresis upon
for studying canalicular secretion of organic anions. This
stimulation. For instance, ursodeoxycholate-induced
mutant lacks the functional canalicular isoform of the
bicarbonate-rich choleresis has been shown to be sensitive
conjugate export pump MRP2/ABCC2 because of one-
to intrabiliary phenol [33] . More recently, secretion of
nucleotide deletion in the Mrp2/Abcc2 gene[58].
bicarbonate in secretin-stimulated rats has been found to
be sensitive to intrabiliary administration of particular ion-
transport blockers[17]. In vitro models
In humans, in vivo assessment of biliary bicarbonate Isolated perfused liver preparations are useful experimen-
secretion has employed cumbersome maneuvers like tal models to evaluate the liver effects of single factors in
nasobiliary drains in hepatic bile ducts[34]. In a context of a manner independent of systemic or humoral effects[59].
surgical interventions, invasive procedures similar to those There are two modalities of these preparations, as the liver
employed in animals (for instance T-tube insertion into can be isolated and perfused in situ or ex situ, i.e. attached
the common bile duct[35,36] or percutaneous transhepatic to or removed from the animal body. Livers can be iso-
cholangio-drainage [37]) were also used. Recently, non- lated from many animal species (rat[1,59], hamster[60], guinea
invasive assessment of biliary bicarbonate secretion pig[61], cat[62], rabbit[63], dog[64], sheep[65], calf[66], pig[67], and

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 3498 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
monkey[68]), the isolated perfused rat liver (IPRL) being the
model most widely used[69]. All these models allow repeated
collection of both the perfusate and the bile, and permit
easy exposure of the liver to different concentrations of
test substances. Test substances may be given intravascu-
larly through just the portal vein[1,59-61,70], which provides
flow to essentially all hepatocytes[71], or through both the
portal vein and the hepatic artery (i.e. isolated bivascularly
perfused liver)[72,73]. The latter bivascularly perfused model
is particularly adequate to investigate ductal physiology be-
cause the predominant blood supply to the bile ducts is via
the hepatic artery[74,75]. For studies on ductal secretion, test
substances and drugs may also be administered intrabiliary
(as retrograde intrabiliary injection[76]). Altogether, these in
vitro preparations of isolated perfused liver had provided
very valuable data on liver physiology and pathophysiol-
ogy, the regulation of bile secretion included. However,
extrapolation of that data to the in vivo situation should be
cautious, as in vivo effects of local factors may be tightly
influenced by systemic players such as humoral factors and
innervation.
Membrane vesicle preparations derived from rat liver
have been useful to distinguish transport across the cell
membrane from intracellular events. They may be selectively
enriched in basolateral or apical plasma membrane, or
in intracellular membranes[77]. The first identification of
liver anion exchange activity was carried out in canalicular
plasma membrane vesicles[78].
While isolated couplets of hepatocytes was particularly
useful as primary secretory units to study canalicular
secretion[79], the development in the last decade of the
model of isolated bile duct units (IBDU), mainly from
rat [16,80,81] , but also from mouse [82] is making a great
contribution to better study the regulation of ductal
bile secretion[83]. Both models permit micropuncture for
electrophysiological studies as well as video microscopic
optical planimetry to determine bile secretion [16,81,84].
Isolated cholangiocytes [85-89] are also widely employed
for transport studies. The preparation of different size
subpopulations of cholangiocytes and IBDU isolated
from specific portions of the rat intrahepatic biliary tree
has made it possible to define the functional heterogeneity
of cells lining specific sized intrahepatic ducts [2,90] .
Furthermore, knockout and mutant animal models are
being used to isolate cholangiocytes and bile duct units to
carry out different in vitro studies[91-93]. Moreover, studies
have been performed in isolated cholangiocytes from
patients with cytic fibrosis (i.e. patients with mutations in
the CFTR gene)[94,95]. Finally, a major advance is coming
from the development of polarized primary cultures
of intrahepatic cholangiocytes from both rat [96,97] and
humans[98-101].
As the excretion of bicarbonate to bile involves a
change in intracellular pH (pH i) and a counterbalance
through ion transporters in the responsible epithelial cells,
several procedures and maneuvers have been developed
to study pHi regulation. The main strategy is based on the
pHi recovery towards its initial values after loading cells
with acid or alkali. This type of experiment requires tech-
niques for rapid and efficient monitoring of the pHi. Al-
though the most direct and accurate method seems to be
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June 14, 2006 Volume 12 Number 22
the insertion of double-barreled microelectrodes sensitive
to [H+]i into the cell, this has important limitations; tested
cells should have large dimensions and a special ability to
manipulate the electrodes. Thus a valuable alternative is
microfluorimetry, a procedure based on the sensitivity of
intracellular fluorescent dyes to pHi. The indicator most
widely employed is the membrane-permeable compound
2, 7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-
methylester (BCECF-AM)[102-104]. Uncharged BCECF-AM
rapidly enters the cell, where it is enzymatically cleaved
into its charged, membrane impermeant, fluorescent form
BCECF. When bound to H+ ions, BCECF exhibits a shift
in its emission fluorescence spectrum, and the ratio be-
tween the pH-sensitive emission at 495 nm wavelength,
and the pH-insensitive (background) emission at the
isosbestic point (440 nm) can be estimated. Once proper
adjustments are set up (correcting for cellular autofluores-
cence, minimizing dye leakage, bleaching, or compartmen-
tation, and preventing photodynamic damage to the cells),
this methodology allows for continuous measurements of
pHi, with a very rapid response time. The technique can be
applied to studies with a fluorimeter, flow cytometer, in-
verted epifluorescent microscope connected to a photon-
counting photometer and a TV camera attachment plus
a digital image-processing software[105]. An example of
successful use of this methodology is the measurement
of the Na+-independent Cl-/HCO3- anion exchange (AE)
activity in both cell clusters and single cells. After inducing
intracellular alkalinization by administration and further
withdrawal of the cell-permeant weak acid propionate in
Krebs-Ringer bicarbonate buffer (KRB), it is possible to
estimate the anion exchange activity as the rate of spon-
taneous recovery of pHi[106]. Rates of pHi recovery can be
measured as pHi/t from the tangent to the experimental
plot; transmembrane acid fluxes (or equivalent transmem-
brane base fluxes, i.e. JOH-) are usually calculated as tot
pHi/t, tot being the total intracellular buffering power in
the presence of CO2/HCO3-, estimated as described[107,108].
The collection of additional important methodologies
which enabe a continuous progress in bile secretion
pathophysiology is still large. Thus, microcomputed
tomography [109] , scanning and transmission electron
microscopy [89,110], immunoelectron microscopy [111], and
dual labeled immunogold [112,113] are only a few among
those techniques deserving a brief mention. Moreover,
concerning molecular biological tools, the techniques
of gene silencing through RNA-interference show
great potential for clarifying the function of selective
genes in bile duct cells. Currently, RNA-interference
may be achieved with small interfering RNAs (siRNA)
and through microRNAs (miRNA) and short/small
hairpin RNA (shRNA) [114] . siRNAs had been used in
normal rat IBDU to examine the role of fibrocystin in
ciliary morphology and biliary cystogenesis [57] and that
of aquaporin-1 (AQP1) in the transport of water by
biliary epithelia [115]. Also siRNA experiments had been
carried out in cholangiocarcinoma cells to identify factors
involved, for example, in the growth of these cells[116] or
their resistance to tumor necrosis factor-related apoptosis-
inducing ligand (TRAIL)[117]. Usual transfection protocols
are not very efficient to internalize the siRNAs in cultured
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
cholangiocytes. Viral vectors that incorporate shRNA
expression cassettes have thus been developed. Recently,
a recombinant adenovirus with this design has been
constructed and employed to efficiently infect normal rat
cholangiocytes in culture and test the function of AE2/
SLC4A2 anion exchanger in these cells[17].
Mechanisms involved
Bile formation is regarded as an osmotic water flow in
response to active solute transport. Bile salts are secreted
in the canaliculi through a specific export pump referred
to as BSEP/SPGP/ABCB11 (from bile salt export pump,
sister of P-glycoprotein, and ATP-binding cassette,
subfamily B, member 11, respectively)[118-120], allowing for
the generation of the bile salt-dependent flow fraction.
Remaining bile-salt independent flow fractions can be
driven by supplementary solutes secreted at both the
canaliculi (the so-called canalicular bile salt-independent
flow), and the bile ducts (named as ductal bile salt-
independent flow)[1,2,10,14,121]. Estimates for the magnitude
of each bile flow fraction in humans are shown in Table 1.
Canalicular bile-salt independent flow
In addition to bile acid secretion, the canalicular
membranes of hepatocytes show active secretion of other
organic and inorganic compounds, mainly glutathione[13]
and bicarbonate [11,12,122], respectively. Glutathione can
be secreted via the organic anion transporter MRP2/
ABCC2[123], while the efflux of bicarbonate occurs through
a DIDS-sensitive Na+-independent Cl-/HCO3- exchange
in association with other ion transport systems (Figure
1) [78,124-127]. Both glutathione and bicarbonate seem to
have an equivalent major input in canalicular bile flow
generation, each driving up to 50% of the bile salt-
independent fraction [11]. For this to be accomplished,
resultant osmotic forces need to be associated with
aquaporin-mediated transcellular movement of water
molecules from plasma to the bile canaliculi[128]. In any
event, the relative contribution of the canalicular bile salt-
independent fraction to the whole canalicular bile flow
may vary substantially between animal species (Table 2).
Canalicular bicarbonate excretion has been reported to
be regulated by glucagon[134,135], a hormonal oligopeptide
secreted by the pancreatic -cells. This hor mone is
encoded by the gene GCG, which belongs to the same
multigene family as secretin (SCT), vasoactive intestinal
peptide (VIP), and gastric inhibitory peptide (GIP) genes,
among others. Following its synthesis in the pancreas,
glucagon may reach the liver and stimulate the hepatocytes
via its interaction with the glucagon receptor (Figure
2). Interestingly, this receptor and the receptors for
secretin, VIP, GIP and other small peptidic hormones, are
included in a superfamily of receptors characterized by a
7-transmembrane domain structure and by their coupling
to adenylate cyclase (ADCY) via GTP-binding proteins
(G proteins). Glucagon-glucagon receptor interaction in
hepatocytes leads to increased intracellular levels of cAMP,
PKA activation, stimulation of canalicular Cl -/HCO 3-
exchange activity[126,134,135], and enhanced AQP8-mediated
water permeability at the canalicular membrane[128,136-138].
3499
Table 1 Estimated bile flow rates in humans
[10]
Bile flow fraction Flow rate
Canalicular bile acid-dependent flow 0.15-0.16 mL/min
Canalicular bile acid-independent flow 0.16-0.17 mL/min
Ductal secretion of bile flow 0.11 mL/min
Daily total bile flow 620 mL/d
Table 2 Estimations of canalicular bile-salt independent flow in
different species
Species Flow rate
[122]
Percentage of the
(L/min/kg) canalicular bile flow
Humans 2 30%[128,129]
Monkey 7
Dog 5 20%[9]
Rat 70 > 60%[1,131,132]
Rabbit 60 > 50%[133]
Current data strongly suggests that these choleretic effects
of glucagon are microtubular-dependent and involve
mobilization of intracellular vesicles[126,128,136,139,140]. These
effects of glucagon in hepatocytes resemble those of
secretin in cholangiocytes (Figure 2). After their interaction
with their specific G-protein-coupled receptors, both
hormones appear to use a similar cAMP-dependent PKA
pathway to co-redistribute cell-type specific intracellular
vesicles with flux proteins towards the apical plasma
membrane of the target cell. But some of the flux
proteins involved in bile formation may differ between
hepatocytes and cholangiocytes. While AQP8 is the
involved water chanel in hepatocytes[128,136,137,140], it changes
to AQP1 in cholangiocytes[112,141,142]. Moreover, there are
data suggesting that in hepatic cells in baseline situation,
the water channel AQP8, the glutathione carrier MRP2/
ABCC2 and the chloride bicarbonate exchanger AE2/
SLC4A2[138,140] are present in pericanalicular vesicles that
might migrate to the canalicular membrane upon glucagon
stimulation[128]. These data, together with previous findings
on AE2/SLC4A2 expression at the canaliculi and the
pericanalicular area[143,144], point towards the canalicular
bicarbonate excretion occurring via an AE2/SLC4A2-
mediated Cl-/HCO3- exchange[127]. Although a number of
observations suggest that this excretion of bicarbonate
in exchange for chloride functions in connection with
an apical chloride channel that maintains favorable Cl -
gradients[78,124,127], the specific chloride channel(s) involved
need yet to be identified (see Figures 1 and 2). While in
cholangiocytes AE2/SLC4A2 colocalizes with CFTR[112],
this particular cAMP-responsive Cl - channel is not
expressed in hepatocytes. Therefore, it might be expected
that a Cl- channel other than CFTR[145-148] be physically
and/or functionally associated with AE2/SLC4A2 in
hepatocytes. Moreover, previous findings have established
an important link between bicarbonate excretion to bile
and changes in pHi. Canalicular Cl-/HCO3- exchange is a
major hepatic acid loading mechanism; at resting pHi and
in the absence of any stimulation it shows low activity,
but it is rapidly activated by intracellular alkalosis, whereas
cAMP stimulation leads to intracellular acidification
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 3500 ISSN 1007-9327 CN 14-1219/ R
A B
Figure 1 The major ion carriers implicated in pHi regulation and secretion of
bicarbonate to bile from liver cells. A: Hepatocytes; B: Cholangiocytes.
which requires appropriate counterbalance[124,127]. Figure
1 summarizes several carriers putatively involved in these
events in hepatocytes (as well as in cholangiocytes),
including acid/base transporters and related proteins.
A brief recall on bicarbonate loaders -and therefore
acid extruders- highlights the role of electrogenic Na+-
HCO 3- cotransporters (NBCe) [149-151] and the carbonic
anhydrase/CA-CO2 pathway[152,153]. Intracellular load of
bicarbonate may increase upon hydration of intracellular
CO 2 by carbonic anhydrase(s) [154] and subsequent H +
efflux via Na + /H + exchange (NHE) (Figure 1). The
involved exchanger is mainly the basolateral NHE1/
SLC9A1[155-157], but NHE4/SLC9A4[158] and the canalicular
NHE3/SLC9A3 [159] may also participate. The other
relevant way of loading bicarbonate into hepatocytes, i.e.
through electrogenic Na+-HCO3- cotransport, functions
upon membrane depolarization following intracellular
acidification [160]. The specific proteins mediating this
cotransport in hepatocytes remain to be defined. The only
two members of the SLC4 family of bicarbonate trans-
porters[161] known to be electrogenic are NBCe1/SLC4A4
and NBCe2/NBC4/SLC4A5 [162] . NBCe1/SLC4A4 is
widely expressed in most tissues, but its expression is
negligible in the liver[163,164], while mRNA expression for
NBCe2/NCB4/SLC4A5 is high in this tissue[165]. Of the
known human NBCe2/NCB4/SLC4A5 variants, only
the rat NBC4c ortholog can be detected in the rat liver
by RT-PCR[164]. Moreover, NBC4c immunoreactivity had
been observed at the basolateral membrane of rat hepato-
cytes[164]. Altogether these findings suggest that the basola-
teral NBC4c variant might be a relevant bicarbonate loader
that enables hepatocytes for canalicular secretion of bicar-
bonate through the apical AE2/SLC4A2 anion exchanger.
Ductal bile
As previously mentioned, primar y canalicular bile
undergoes a process of fluidization and alkalinization
along the biliary tract that is influenced by several factors
including hormones (mainly secretin in most species[2,14-17]),
innervation/neuropeptides[2,50,166,167], and biliary constitu-
ents[2,5,6,17,18]. This process results in net ductal secretion
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
A B
Figure 2 Hormonal stimulation of bicarbonate excretion from hepatocytes and
cholangiocytes. A: In hepatocytes, glucagon may induce an exocytic trafficking
of vesicles with the anion exchanger AE2/SLC4A2 and the water channel AQP8,
which can results in canalicular bicarbonate-rich hydrocholeresis. MRP2/ABCC2,
while being a carrier for organic anions like glutathione rather than an inorganic ion
transporter, is hereby depicted because of its possible colocalization with the other
two flux proteins; B: In cholangiocytes, secretin induces trafficking of vesicles with
the chloride channel CFTR, the anion exchanger AE2/SLC4A2, and the water
channel AQP1, and further exocytosis to the lumenal membrane of these cells,
which results in ductal bicarbonate-rich hydrocholeresis.
of a bicarbonate-rich watery fluid. The magnitude of the
ductal contribution varies between species, representing
30% of basal bile flow in humans and 10% in rats [6].
Cholangiocytes are provided with specific transport
systems that participate in bile modifications[2,168]. They
are able to take up bile salts via an apical Na+-dependent
transpor ter (SLC10A2, for merly ASBT/ISBT and
occasionaly ABAT) [45,169-172] and release them through
a basolateral truncated isoform of the same carrier[173].
Indeed, transcellular transport through these carriers is
specially important under cholestatic situations. In any case
under normal conditions, biliary transport of bicarbonate
appears as a relevant function of the bile duct epithelium.
It is accomplished by specific acid/base carriers and
related transporters that enable cholangiocytes to regulate
their pHi[105,174] (Figure 1).
Cholangiocyte acid extruders
The vectorial transport of HCO3- from cholangiocytes to
duct lumen starts with the accumulation of HCO3- in the
cells via mechanisms which vary between animal species. In
rat cholangiocytes, for instance, HCO3- loading is mediated
by transport systems similar to those in hepatocytes, i.e. by
electrogenic Na+-HCO3- cotransport activity[168,174-176] and
the carbonic anhydrase/CA-CO2 pathway coupled to sub-
sequent carrier-mediated H+ extrusion[153,154]. Carbonic an-
hydrases (CAs) catalyze the hydratation of carbon dioxide,
CO2 + H2O HCO3- + H+ (reviewed in ref. 153). Thus
far, several CA isoenzymes have been identified which dif-
fer in organ distribution, subcellular location, and function
(cf. Table I in ref. 153). Compared with other secretory
organs, the mammalian liver contains relatively low levels
of total CA activity. Thus, the cytoplasmic CA-II (CA2)
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
is mainly expressed in bile duct cells (but it may also be
found in hepatocytes), and appears to be involved in the
production of HCO3- for its further secretion to bile[153,177].
Also, the biliary epithelial cells express the membrane as-
sociated CA-IV and CA-IX, which on the basis of their
location might be involved in acidification and concentra-
tion of the bile, even though the exact mechanisms have
not been described[153].
Among electrogenic Na+-HCO3- cotransporters[161,162]
only the NBC4c variant of NBCe2/NBC4/SLC4A5 was
found to be expressed in the rat liver, being immunolocal-
ized to both hepatocytes and cholangiocytes[164]. Interest-
ingly, NBC4c immunoreactivity was basolateral in hepato-
cytes but apical in cholangiocytes, suggesting a potential
role for this cotransporter in the luminal fluid secretion
and/or absorption[164]. In humans cholangiocytes, however,
Na+-HCO3- cotransport is not active in the physiological
range of pH i , being active only at very low pH i , and
bicarbonate influx occurs mainly through electroneutral
Na+-dependent Cl-/HCO3- anion exchange[176,178]. Thus far,
NDCBE/SLC4A8 is the only Na+-dependent Cl-/HCO3-
exchanger cloned in humans[179]. Although Northern blot
analysis could not detect its messenger RNA in the whole
liver[179], this cannot rule out that NDCBE/SLC4A8 be
expressed in cholangiocytes (which account for just 5% of
the liver cell population).
In both rat and human cholangiocytes, H+ extrusion
takes place essentially through NHE activity (Figure 1)
-while in pig cholangiocytes H+ extrusion is mediated by a
cAMP-activated H+-ATPase[180]. Thus far, several members
of the NHE/SLC9 family have been described in rat
cholangiocytes. They include NHE1/SLC9A1, restricted to
the basolateral membrane and highly sensitive to amiloride,
and the amiloride-insensitive isoform NHE2/SLC9A2,
which is likely to be active on the side facing the lumen[106].
Moreover, NHE3/SLC9A3 has been immunolocalized
to the apical membrane of rat cholangiocytes, where it
may play an important role in fluid absorption from the
bile duct lumen [159]. Also NHE4/SLC9A4 -an isoform
seemingly activated by hypertonicity and with K +/H +
exchange activity[181], was identified in whole liver extracts
by Western blot [158]. Some findings in the stomach of
Nhe4/Slc9a4-/- mice had led to speculations on a possible
functional coupling of NHE4/SLC9A4 with the AE2/
SLC4A2 anion exchanger in parietal cells[182]. In any case,
the specific cellular expression and function of this NHE
isoform in the liver remains to be determined.
Cholangiocytes acid loaders
As previously reported in hepatocytes, the main acid loader
mechanism in bile duct cells is the apical Na+-independent
Cl-/HCO3- exchange[17,106,174,176]. Such an AE activity might
secrete HCO3- into the lumen which is exchanged for Cl-
influx. This exchange is electroneutral, being facilitated
by the outside to inside transmembrane gradient of Cl- at
relatively high intracellular concentration of HCO3-, specially
upon secretin stimulation[16,81,106,175,183] (Figure 2). Actually,
several bicarbonate transporters have been described as
exerting Na+-independent Cl-/HCO3- exchange activity.
This is the case for the SLC4 anion exchangers (AE1/
SLC4A1, AE2/SLC4A2 and AE3/SLC4A3) [161,184] as
3501
well as several members of the SLC26 gene family of
multifunctional anion exchangers (DRA/CLD/SLC26A3,
PDS/DFNB4/SLC26A4, and SLC26A6 [185] and more
controversially SL26A7[186,187]). But none of these carriers
except AE2/SLC4A2 had been described to occur in the
liver. Moreover, AE2/SLC4A2 was localized not only to
the canaliculi but also to the lumenal membrane of bile
duct cells[143]. Recent experiments of RNA intereference
with recombinant adenovirus expressing shRNA have
shown that AE2/SL4A2 is indeed the main effector of
both basal and stimulated Na+-independent Cl-/HCO3-
exchange in rat cholangiocytes[17].
Other ion-loaders/extruders
Besides acid/base transporters cholangiocytes possess
other ion carriers like those for Cl-, Na+, and K+, which
greatly contribute to pH i regulation and bicarbonate
secretion (Figure 1). Thus, the cAMP-responsive Cl -
channel CFTR had been localized at the apical side, where
it plays a role in biliary excretion of HCO3-[188,189]. Although
HCO3- permeability through activated CFTR has been
shown in several cell systems[190-194], its main contribution
to biliary bicarbonate secretion appears to occur through
a coordinated action with AE2/SL4A2[17,175,195]. In addition
to CFTR, cholangiocytes possess a dense population of
Ca2+-activated Cl- channels. These channels are responsive
to interaction of the purinergic-2 (P2) receptors with
nucleotides (mainly ATP or UTP)[196-199], and they appear
to be related to the Ca2+-calmodulin-dependent protein
kinase II[188,200,201]. Resultant Ca2+-stimulated Cl- efflux might
be up to 2-fold greater than that mediated by cAMP[202].
Additional high conductance anion channels which are
insensitive to both Ca2+ and cAMP were identified in rat
bile duct cells [203], but their specific role remains to be
defined. The efflux of Cl - from cholangiocytes to the
ductular lumen is counterbalanced not only via apical
Cl-/HCO3- exchange but also through other Cl- uptake
systems, mainly the basolateral Na+-K+-2Cl- cotransporter
NKCC1/SLC12A2 [196,204]. In addition to maintain high
intracellular Cl - concentration facilitating apical Cl -
excretion, this forskolin-stimulable cotransporter has been
reported to participate in cell volume homeostasis[205] and
cell proliferation[204,206,207]. Because the NKCC1/SLC12A2-
mediated influx of Cl- occurs together with the entry of
Na+ and K+, cholangiocytes possess other systems that
sustain the gradients of these cations and the membrane
potential difference. Thus a basolateral sodium pump
(the Na+/K+-ATPase)[208,209], extrudes Na+ and uptakes K+
with a 3:2 stoichiometry[210]. Accumulation of K+ within
cholangiocytes is prevented by its exit through K+ channels.
Intracellular cAMP and/or Ca 2+ concentration may
activate basolateral K+ conductance that hyperpolarizes the
cell[211]. The small conductance K+ channel SK2/KCNN2
has been identified in rat and human hepatocytes[212] and in
rat and human biliary epithelia[202,212], the activity of which
is stimulated by small increases in intracellular Ca2+ in an
apamin-sensitive manner [213]. Although SK2/KCNN2
immunoreactivity has been localized in cholangiocytes at
both apical and basolateral membranes, functional studies
in polarized preparations have demonstrated a significantly
greater basolateral Ca2+-stimulated K+ conductance[202].
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 3502 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Because the nonselective K+ channel blocker Ba2+ is able
to cause a greater degree of inhibition than apamin[202],
apamin-insensitive channels not yet identified may also be
involved in the conductance of K+.
In a context of cAMP- and/or Ca 2+-stimulated Cl -
channels, cell hyperpolarization due to K+ conductance
facilitates the transcellular Cl- movement into the lumen.
Recycling of K+ can be held up through the basolateral
Na +-K +-2Cl - cotransport and the Na +/K +-ATPase. All
these ionic fluxes across cholangiocyte membranes may
ultimately lead to biliary excretion of bicarbonate via its
exchange with lumenal chloride, which is facilitated by the
outside to inside transmembrane gradient of chloride at
relatively high intracellular concentration of bicarbonate[16,
81,106,175,183]
. The apical fluxes of anions result in increased
osmotic forces in the bile duct lumen which in the pres-
ence of aquaporins contribute to the generation of ductal
bile flow. This view has been strongly supported by the
finding that AE2/SLC4A2 and CFTR both colocalize with
AQP1 in cholangiocyte intracellular vesicles which co-
redistribute to the apical cholangiocyte membrane upon
both cAMP and secretin stimulations[112].
Water transporters or aquaporins
Aquaporins are water channels that mediate a bidirectional
passive movement of water molecules across epithelial
cells in response to osmotic gradients established by ions
and solutes. Thus far, rodent cholangiocytes have been
described to possess two types of aquaporins. Thus, in
addition to the AQP1 locating in the intracellular vesicles
which may traffic to the apical membrane upon agonist
stimulation [112], there occurs another water channel at
the basolateral membrane of cholangiocytes named
AQP4, which is not sensitive to secretin [214,215] (Figures
1 and 2). However, there can be differences between
species. For instance, immunohistochemical analysis in
liver pig has shown the presence of AQP1 and AQP9 in
cholangiocytes, while AQP3, AQP4, AQP7, and AQP8
were absent in these cells[216].
Regulatory factors
The bile duct epithelium is constantly regulated by the
action of multiples factors that contribute to the formation
of bile flow with an adequate final composition. Among
these factors secretin is a relevant hormone peptide
which may induce bicarbonate-rich hydrocholeresis in
many animal species. Thus, secretin regulation of ductal
bile flow and the concurrence of other factors are briefly
summarized as follows:
Relevance of secretin stimulation
In 1902, Bayliss and Starling[19] described an active agent
originating from the intestinal mucosa, which they referred
to as secretin because of its capacity to stimulate pancreatic
secretion in the dog. They also used the word hormone
to designate this sort of compound that can be produced
in an organ and carried through the circulation to exert
its effect on another organ. Some years passed before
the choleretic effect of secretin in the liver was reported
in several animal species and humans[217-219]. Secretin was
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June 14, 2006 Volume 12 Number 22
found to stimulate bile flow together with a decrease in
the concentration of bile salts[217-219]. In the late 1960s the
role of bile ducts in secretin-induced hydrocholeresis was
postulated because of the observation that this secretin
effect was associated with reciprocal changes in the biliary
concentration of bicarbonate and chloride anions[35].
Meanwhile secretin was purified from most species,
first in 1962 from pig[220,221] and then from humans, dog,
goat, guinea pig, rabbit, rat, mouse and chicken. Secretin
has only 27 amino acids, which allowed for its chemical
synthesis as early as 1968[222]. There is close homology
between mammalian secretins, but also between the
regulatory peptides (more than 10) currently grouped in
the secretin/glucagon/ VIP superfamily.
Secretin is produced in many organs but mainly in
the mucosa of upper small intestine (duodenum and
jejunum)[223,224]. Its release to blood occurs mainly in the
postprandial period, being stimulated by gastric acid
delivered into duodenal lumen[225], as well as by pancreatic
and intestinal secretions [226,227]. Secretin release appears
to be mediated by a luminal secretin-releasing peptide
contained in these gastrointestinal juices[228-230]. In addition
to its mentioned bilary and pancreatic effects, secretin
may function as feedback inhibitor of gastrin (GAST)
release and gastric acid secretion [231] , and may also
regulate gastric motility[232]. The physiological activities of
secretin are subjected to hormone-hormone and neuro-
hormonal interactions. Inhibition of gastric acid secretion
by secretin is thus mediated by somatostatin (SST) and
prostagladins [233,234], and secretin inhibition of gastric
motility involves a vagal afferent pathway[235,236].
Secretin exerts its physiological actions via interaction
with the N-terminal extracellular tail of its specific
glycoprotein receptor SCTR [237-239]. Like the glucagon
receptor and other members of the same receptor super-
family, SCTR is coupled to adenylate cyclase/ADCY
through an oligomeric GTP-binding protein. In the liver
SCTR is exclusively expressed at the basolateral membrane
of cholangiocytes[240,241]. As previously noted, the action
of secretin in cholangiocytes runs parallel with the
choleretic effect of glucagon in hepatocytes (Figure 2).
Thus, both glucagon and secretin may stimulate the bile
salt-independent bile flow, but each at a different level, i.e.
canalicular for the former and ductular for the latter(cf. ref.
242).
Secretin-SCTR interaction in cholangiocytes results
in increased intracellular levels of cAMP[15,16,243]. Further
PKA activation[175,183] can induce microtubule-dependent
co-redistribution of the intracellular vesicles with AE2-
CFTR-AQP1 flux carriers to the apical membrane [112].
Additionally, the CFTR is phosphorylated and activated[244],
thus resulting in Cl - efflux to the ductular lumen. In
consequence, Cl - secreted by CFTR can be exchanged
with HCO 3- through AE2/SLC4A2. This mechanistic
model has been consistently confirmed in vitro using rat
IBDU [16,175] and cholangiocytes [174]. For in vivo studies,
most experiments with rats have used some of the
aforementioned models with bile duct proliferation[41,43,46,
112,245,246]
, since normal rats appear to respond very poorly
to secretin (likewise rabbit, but in contrast with guinea pig
among rodent species)[15,41,46,47,247]. Indeed the expression of
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
SCTR is increased in BDL rat cholangiocytes[245,246] but the
receptor is also expressed in normal rat cholangiocytes[240].
In the model of IPRL[248], intra-arterial secretin increased
biliary concentration of bicarbonate, but had no effect
on the net bile flow. Because the effect of secretin was
blocked only by the CFTR inhibitor NPPB and not by the
anion exchanger inhibitor DIDS (both administered intra-
arterially), it was proposed that Cl-/HCO3- exchange would
have no role in the ductal secretion of bicarbonate in the
normal rat[248]. However, recent experiments indicate that
secretin does increase bile flow and biliary Cl- and HCO3-
excretions in the normal rat, but when they maintain the
bile acid pool via continuous infusion with taurocholate[17].
Moreover, these effects of secretin were distinctively
blocked by the inhibitors given by intrabiliary retrograde
injection. While secretin effects were all blocked by
intrabiliary NPPB, DIDS only inhibited secretin-induced
increases in bile flow and bicarbonate excretion but not
the increased chloride excretion[17]. These findings provide
evidence for the role of biliary Cl-/HCO3- exchange in
secretin-induced bicarbonate-rich choleresis in the normal
rat model.
The role of the bile acid pool
In line with former findings in earlier experiments with
dogs[14,249], the aforementioned observation that secretin
also has effects on the normal rat when infused continu-
ously with taurocholate[17] confirms the notion that bile
acids are relevant for secretin actions. In a previous study
in normal rats secretin and taurocholate were tested and
no secretin effects were observed [41]. But in that study,
taurocholate infusion was interrupted before secretin ad-
ministration[41]. It is already known that bile acids can enter
into cholangiocytes through the carrier ASBT[45,169-172] and
exert their effects on these cells (reviewed in refs. 2 and
250). For instance, it has been recently reported, activation
of CFTR by ASBT-mediated bile salt absorption, which is
seemingly independent from cAMP or cGMP signaling[251].
The bile salt-dependent canalicular flow is related to the
osmotic activity of bile acids, but some bile acids such as
ursodeoxycholic acid (UDCA), 23-nor-UDCA, and 23-nor-
chenodeoxycholate[252-254], have a higher choleretic effect
than can be accounted for by their secretion into bile (the
so-called hypercholeretic effect). This hypercholeretic
effect is associated with a marked stimulation of
bicarbonate secretion into bile. A classic hypothesis
referred to as cholehepatic shunt pathway[253] claimed
that the hypercholeretic effect may involve intraductal
protonation of unconjugated bile salts which results in the
formation of bicarbonate anions derived from hydrated
CO2 (i.e. H2CO3 or H+ plus HCO3-). Passive diffussion
of uncharged bile acids through cholangiocytes to the
periductular vessels and further uptake in hepatocytes
could be followed by their canalicular resecretion as
unconjugated bile salts (reviewed in ref. 250). After the
identification of apical and basolateral bile acid carriers in
cholangiocytes (ASBT and tASBT, respectively[45,169-173]), the
cholehepatic shunt hypothesis has received a boost, being
updated for the conjugated bile salts[250]. ASBT activity
acutely increases upon secretin stimulation [172], which
may accentuate the cholehepatic bile acid shunting in the
3503
postprandial period.
Neurovegetative liver innervation
The liver is directly regulated by the neurovegetative
innervation. Indeed, the release of the neurotransmitter
acetylcholine from the intrahepatic parasympatic terminals
induces, via selectively interaction with M3 Ach receptors
on cholangiocytes, an increase in both secretin-stimulated
cholangiocyte cAMP synthesis and Cl-/HCO3- exchanger
activity by Ca2+-calcineurin-mediated PKC-independent
modulation of adenylate cyclase/ADCY [255,256]. In fact,
infusion of acetylcholine in the IPRL model was found
to potentiate the effect of secretin on biliary HCO 3-
excretion[248]. Furthermore, interruption of parasympatic
innervation in BDL rats by vagotomy has been reported
to inhibit secretin-stimulated ductal secretion, as well as to
decrease cholangiocyte intracellular cAMP levels[50].
The intrahepatic biliary epithelium also receives
dopaminergic innervation [257,258], but in contrast to the
cholinergic system, the dopaminergic system inhibits
secretin-induced choleresis. Although both systems exert
their functions through increased intracellular ions (1, 4, 5)
P3 and Ca2+, the cholinergic system acts via calmodulin and
calcineurin but without recruitment of PKC[256], whereas
the D2 dopaminergic system inhibits secretin-stimulated
ductal secretion through increased expression and activa-
tion of PKC-[259].
Moreover, cholangiocyte secretion can also be
regulated by the action of the adrenergic system[260,261].
The 2-adrenergic receptor agonist UK-14304 has been
reported to inhibit cholangiocarcinoma growth through
time course-dependent modulation of Raf-1 and B-Raf
activities[260], and the 1-adrenergic agonist phenylephrine
can potentiate secretin-stimulated ductal secretion through
the amplification of the ADCY system via a Ca2+- and
PKC-dependent mechanism[261].
Hormones
In addition to secretin, there are gastrointestinal hormones
and neuropeptides such as bombesin/gastrin releasing
peptide (BN/GRP), VIP, endothelin-1 (ET1/EDN1),
somatostatin/SST, and gastrin/GAST, which may also
modulate the ductular bile salt-independent flow (reviewed
in ref. 2). Some of these factors operate through a
secretin-independent mechanism, while others influence
the release of secretin or interact with the secretin
signaling in cholangiocytes, depending very much on the
animal species. For instance, the neuropeptide bombesin/
BN/GRP can act either by increasing the secretin release
in dogs[262,263], or inducing ductal secretion with activated
Cl-/HCO3- exchange via secretin-independent mechanisms
in isolated rat cholangiocytes[2,264-266]. The effect of VIP on
cholangiocytes depends also on the animal species[2,267-271].
While VIP appears to increase secretin-stimulated bile
flow and bicarbonate excretion in humans[267,268], studies
in rat IBDU show that VIP can stimulate basal fluid and
bicarbonate secretion through a cAMP-independent
pathway[2,269].
The cyclic tetradecapeptide somatostatin/SST is able
to inhibit basal and secretin-stimulated bicarbonate-rich
choleresis via its interaction with the SSTR2 receptor
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 3504 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
subtype[2,272]. In rat, inhibition of secretin-stimulated ductal
secretion by SST is associated with decreased expression of
the secretin receptor SCTR in cholangiocytes and reduced
secretin-stimulated cAMP levels[49,272]. In mice SST has also
been shown to stimulate ductal fluid absorption, a process
involving intracellular cGMP synthesis and inhibition of
secretin-stimulated cAMP synthesis[273]. Moreover, in dogs,
SST has been shown to diminish the acid-induced release
of secretin from the duodenal mucosa[262]. Similarly SST,
the gastrointestinal hormone gastrin/GAST may also
modulate cholangiocyte secretion[2]. In BDL rats, GAST
has been reported to inhibit secretin-stimulated ductal
secretion by reducing both SCTR expression and secretin-
induced cAMP levels[274].
Other peptide hormones like insulin and insulin-like
growth factor 1 (IGF1) can also modulate the biliary epi-
thelium. Insulin was reported to inhibit secretin-induced
secretion in BDL rats through activation of PKC and in-
hibition of secretin-stimulated cAMP and PKA activity[275].
On the other hand, studies using a liver cell line (though
from hepatoma rather than cholangiocytic type), showed
that insulin may stimulate membrane turnover with in-
creased exocytosis/endocytosis of vesicles containing ion
channels[276]. In rats IGF1 was found to stimulate cholere-
sis[277] as well as cholangiocyte proliferation[278]. IGF1 can
be synthesized and released from cholangiocytes under
the control of the growth hormone (GH)[278]. Biliary IGF1
may in turn interact with its receptor (IGF-R) located at
the apical pole of cholangiocytes[278]. Expression of both
IGF1-R and IGF1 is markedly enhanced in cholangiocytes
following bile duct ligation[278].
Also steroidal hormones like corticosteroids and estro-
gens have effects on the biliary epithelium. Corticosteroids
are choleretic and increase biliary bicarbonate excre-
tion[277,279]. However, estrogen-induced cholestasis results
in diminished biliary bicarbonate excretion[280]. Reduced bi-
carbonate excretion might be caused by a reflux of biliary
bicarbonate via leaky tight junctions as it is not associated
with impaired activity of the Cl-/HCO3- exchanger[280].
Purinergic stimulation
Both he patocytes and cholangiocytes are able to
release ATP and UTP, which leads to increased biliary
concentration of nucleotides and nucleosides (the latter
being a result of nucleotide dephosphor ylation by
membrane-associated nucleotidases) [281-283]. Stimulation
of the different subtypes of purinergic receptors (PR)
at the lumenal membrane of cholangiocytes by either
extracellular nucleosides (P1 family receptors) or
extracellular nucleotides (P2 family receptors) may control
cholangiocyte secretion and ion channel activities[281,283-286].
Most subtypes of purinergic receptors are G protein-
coupled receptors except the P2X subtypes which are
ligand-gated channels [281,283] . P2 activation stimulates
cholangiocyte biliary efflux of K+, HCO3-, and Cl-, and
reabsorption of Na+[281,283,286]. Whereas Cl- efflux seems to
be mediated by a calcium-stimulated Cl- channel[284], HCO3-
is secreted from cholangiocytes via an AE2/SLC4A2
mediated Cl -/HCO 3- exchange [17,105,174,284,287]. Of notice,
the P2Y11 subtype receptor has been reported to mediate
secretion via cAMP in pancreatic duct epithelial cells[288],
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June 14, 2006 Volume 12 Number 22
which suggests that a similar mechanism may also occur
in cholangiocytes (as well as in hepatocytes). Finally, P2
receptors may stimulate the basolateral NHE1/SLC9A1
activity in cholangiocytes[287].
Cytokines and related factors
Some cytokines such as IL5 and the combination of the
proinflammatory cytokines IL6, IFN-, IL1, and TNF-
can inhibit secretin-induced choleresis[289,290]. Moreover,
proinflammatory cytokines can impair the barrier function
of biliary epithelia[290], and stimulate the biliary epithelium
to generate NO, via induction of inducible nitric oxide
synthase 2A (NOS2A/INOS). Resultant reactive nitro-
gen oxide species (RNOS) may cause ductular cholestasis
through inhibition of both the soluble adenylate
cyclase (SAC) and the cAMP-dependent HCO3- and Cl-
secretory mechanisms[195]. Such a pathogenetic sequence
may contribute to ductal cholestasis in inflammatory
cholangiopathies[195].
In conclussion, biliary secretion of bicarbonate is an
important contributor to the generation of the bile-salt
independent flow. Bicarbonate is secreted from both he-
patocytes and cholangiocytes through parallel mechanisms
which involve chloride efflux through activation of Cl-
channels, and further bicarbonate excretion via AE2/
SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and
secretin are two relevant hormones which act very simi-
larly in hepatocytes and cholangiocytes, respectively. These
hormones interact with their specific receptor, resulting
in increased intracellular cAMP levels and activation of
cAMP-dependent Cl- and HCO3- secretory mechanisms.
Both hepatocytes and cholangiocytes seem to have cAMP-
responsive intracellular vesicles in which AE2/SLC4A2
may colocalize with cell specific Cl- channels (CFTR in
cholangiocytes and thus far undetermined in hepatocytes)
and aquaporins (AQP1 in cholangiocytes and AQP8 in
hepatocytes). cAMP-induced coordinated trafficking of
these vecicles to either canalicular or cholangiocyte lume-
nal membranes and further exocytosis results in increased
osmotic forces and passive movement of water with net
bicarbonate-rich hydrocholeresis.
REFERENCES
1 Boyer JL. Canalicular bile formation in the isolated perfused
rat liver. Am J Physiol 1971; 221: 1156-1163
2 Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholan-
giocyte bicarbonate secretion. Am J Physiol Gastrointest Liver
Physiol 2001; 281: G612-G625
3 Boyer JL. Bile duct epithelium: frontiers in transport physiol-
ogy. Am J Physiol 1996; 270: G1-G5
4 Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, La-
sater J, LeSage GD. Large but not small intrahepatic bile ducts
are involved in secretin-regulated ductal bile secretion. Am J
Physiol 1997; 272: G1064-G1074
5 Baiocchi L, LeSage G, Glaser S, Alpini G. Regulation of chol-
angiocyte bile secretion. J Hepatol 1999; 31: 179-191
6 Tavoloni N. The intrahepatic biliary epithelium: an area
of growing interest in hepatology. Semin Liver Dis 1987; 7:
280-292
7 Sperber I. Biliary secretion of organics anions and its influence
on bile flow. In: Taylor W, eds. The Biliary System, Oxford:
Blackwell, 1965: 457-467
8 Erlinger S, Dhumeaux D. Mechanisms and control of secre-
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
tion of bile water and electrolytes. Gastroenterology 1974; 66:
281-304
9 Wheeler HO, Ross ED, Bradley SE. Canalicular bile produc-
tion in dogs. Am J Physiol 1968; 214: 866-874
10 Erlinger S. Bile flow. In: Arias IM, Boyer JL, Fausto N, Jakoby
WB, Schachter D, Shafritz DA, eds. The liver: biology and
pathobiology, New York: Raven Press, 1994: 769-786
11 Hardison WG, Wood CA. Importance of bicarbonate in bile
salt independent fraction of bile flow. Am J Physiol 1978; 235:
E158-E164
12 Van Dyke RW, Stephens JE, Scharschmidt BF. Effects of ion
substitution on bile acid-dependent and -independent bile for-
mation by rat liver. J Clin Invest 1982; 70: 505-517
13 Ballatori N, Truong AT. Relation between biliary glutathione
excretion and bile acid-independent bile flow. Am J Physiol
1989; 256: G22-G30
14 Wheeler HO, Mancusi-Ungaro PL. Role of bile ducts during
secretin choleresis in dogs. Am J Physiol 1966; 210: 1153-1159
15 Lenzen R, Alpini G, Tavoloni N. Secretin stimulates bile
ductular secretory activity through the cAMP system. Am J
Physiol 1992; 263: G527-G532
16 Roberts SK, Kuntz SM, Gores GJ, LaRusso NF. Regulation
of bicarbonate-dependent ductular bile secretion assessed by
lumenal micropuncture of isolated rodent intrahepatic bile
ducts. Proc Natl Acad Sci U S A 1993; 90: 9080-9084
17 Banales JM, Arenas F, Rodrguez-Ortigosa CM, Sez E,
Uriarte I, Doctor RB, Prieto J, Medina JF. Bicarbonate-rich
choleresis induced by secretin in normal rat is taurocholate-
dependent and involves AE2 anion exchanger. Hepatology
2006; 43: 266-275
18 Strazzabosco M, Zsembery A, Fabris L. Electrolyte transport
in bile ductular epithelial cells. J Hepatol 1996; 24 Suppl 1: 78-87
19 Bayliss WM, Starling EH. The mechanism of pancreatic
secretion. J Physiol 1902; 28: 325-353
20 Mellanby J. Bile salts and secretin as cholagogues. J Physiol
1928; 64: 331-340
21 Lueth HC, Kloster G. The effect of purified secretin on bile
flow from the liver. Ame J Physiol 1928; 85: 389
22 Forker EL. Two sites of bile formation as determined by
mannitol and erythritol clearance in the guinea pig. J Clin
Invest 1967; 46: 1189-1195
23 Forker EL. Bile formation in guinea pigs: analysis with inert
solutes of graded molecular radius. Am J Physiol 1968; 215:
56-62
24 Tavoloni N. Biliary clearance of inert carbohydrates.
Expectations and reality. Gastroenterology 1988; 94: 217-228
25 Peterson RE, Fujimoto JM. Retrograde intrabiliary injection:
absorption of water and other compounds from the rat biliary
tree. J Pharmacol Exp Ther 1973; 185: 150-162
26 Bockman DE. Route of flow and micropathology resulting
from retrograde intrabiliary injection of India ink and ferritin
in experimental animals. A combined light- and electron-
microscopic study. Gastroenterology 1974; 67: 324-332
27 Olson JR, Fujimoto JM. Evaluation of hepatobiliary function
in the rat by the segmented retrograde intrabiliary injection
technique. Biochem Pharmacol 1980; 29: 205-211
28 Ballatori N, Jacob R, Boyer JL. Intrabiliary glutathione
hydrolysis. A source of glutamate in bile. J Biol Chem 1986; 261:
7860-7865
29 Takehara T, Hayashi N, Miyamoto Y, Yamamoto M, Mita E,
Fusamoto H, Kamada T. Expression of the hepatitis C virus
genome in rat liver after cationic liposome-mediated in vivo
gene transfer. Hepatology 1995; 21: 746-751
30 Goetz M, Lehr HA, Neurath MF, Galle PR, Orth T. Long-term
evaluation of a rat model of chronic cholangitis resembling
human primary sclerosing cholangitis. Scand J Immunol 2003;
58: 533-540
31 Tominaga K, Kuriyama S, Yoshiji H, Deguchi A, Kita Y,
Funakoshi F, Masaki T, Kurokohchi K, Uchida N, Tsujimoto T,
Fukui H. Repeated adenoviral administration into the biliary
tract can induce repeated expression of the original gene
construct in rat livers without immunosuppressive strategies.
Gut 2004; 53: 1167-1173
3505
32 Kuriyama S, Yoshiji H, Deguchi A, Nakai S, Ogawa M,
Nonomura T, Kimura Y, Inoue H, Kinekawa F, Tsujimoto
T, Masaki T, Kurokohchi K, Uchida N. Safe and efficient
transgene expression in rat hepatocytes induced by adenoviral
administration into the biliary tract. Oncol Rep 2005; 13:
825-830
33 Dumont M, DHont C, Moreau A, Mbape H, Feldmann G,
Erlinger S. Retrograde injections of formaldehyde into the
biliary tree induce alterations of biliary epithelial function in
rats. Hepatology 1996; 24: 1217-1223
34 Knyrim K, Vakil N. The effects of synthetic human secretin on
calcium carbonate solubility in human bile. Gastroenterology
1990; 99: 1445-1451
35 Waitman AM, Dyck WP, Janowitz HD. Effect of secretin and
acetazolamide on the volume and electrolyte composition of
hepatic bile in man. Gastroenterology 1969; 56: 286-294
36 Lenzen R, Bhr A, Eichstdt H, Marschall U, Bechstein WO,
Neuhaus P. In liver transplantation, T tube bile represents to-
tal bile flow: physiological and scintigraphic studies on biliary
secretion of organic anions. Liver Transpl Surg. 1999; 5: 8-15
37 Fukumoto Y, Okita K, Yasunaga M, Konishi T, Yamasaki T,
Ando M, Shirasawa H, Fuji T, Takemoto T. A new therapeutic
trial of secretin in the treatment of intrahepatic cholestasis.
Gastroenterol Jpn 1989; 24: 298-307
38 Prieto J, Garca N, Mart-Climent JM, Peuelas I, Richter JA,
Medina JF. Assessment of biliary bicarbonate secretion in hu-
mans by positron emission tomography. Gastroenterology 1999;
117: 167-172
39 Clark JC, Buckingham PD. The preparation and storage of
carbon-11 labelled gases for clinical use. Int J Appl Radiat Isot
1971; 22: 639-646
40 Shields AF, Graham MM, Kozawa SM, Kozell LB, Link JM,
Swenson ER, Spence AM, Bassingthwaighte JB, Krohn KA.
Contribution of labeled carbon dioxide to PET imaging of
carbon-11-labeled compounds. J Nucl Med 1992; 33: 581-584
41 Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology
in rats with bile ductular cell hyperplasia. Evidence for a se-
cretory function of proliferated bile ductules. J Clin Invest 1988;
81: 569-578
42 GOLDFARB S, SINGER EJ, POPPER H. BILIARY DUCTULES
AND BILE SECRETION. J Lab Clin Med 1963; 62: 608-615
43 Kountouras J, McKavanagh S, Burmicky M, Billing BH. The
effect of secretin on bile flow and bile acid and bilirubin excre-
tion following relief of prolonged bile duct obstruction in the
rat. J Hepatol 1987; 4: 198-205
44 Lesage G, Glaser SS, Gubba S, Robertson WE, Phinizy JL, La-
sater J, Rodgers RE, Alpini G. Regrowth of the rat biliary tree
after 70% partial hepatectomy is coupled to increased secretin-
induced ductal secretion. Gastroenterology 1996; 111: 1633-1644
45 Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis
H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD. Bile acid
feeding induces cholangiocyte proliferation and secretion: evi-
dence for bile acid-regulated ductal secretion. Gastroenterology
1999; 116: 179-186
46 Alpini G, Lenzi R, Zhai WR, Slott PA, Liu MH, Sarkozi L,
Tavoloni N. Bile secretory function of intrahepatic biliary epi-
thelium in the rat. Am J Physiol 1989; 257: G124-G133
47 Knuchel J, Krhenbhl S, Zimmermann A, Reichen J. Effect
of secretin on bile formation in rats with cirrhosis of the liver:
structure-function relationship. Gastroenterology 1989; 97:
950-957
48 Alpini G, Elias I, Glaser SS, Rodgers RE, Phinizy JL, Robertson
WE, Francis H, Lasater J, Richards M, LeSage GD. gamma-In-
terferon inhibits secretin-induced choleresis and cholangiocyte
proliferation in a murine model of cirrhosis. J Hepatol 1997; 27:
371-380
49 Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri
A, LeSage G, LaRusso NF. Heterogeneity of the proliferative
capacity of rat cholangiocytes after bile duct ligation. Am J
Physiol 1998; 274: G767-G775
50 LeSagE G, Alvaro D, Benedetti A, Glaser S, Marucci L, Baioc-
chi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis H,
Alpini G. Cholinergic system modulates growth, apoptosis,
www.wjgnet.com
 3506 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
and secretion of cholangiocytes from bile duct-ligated rats.
Gastroenterology 1999; 117: 191-199
51 LeSage GD, Glaser SS, Marucci L, Benedetti A, Phinizy JL,
Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Hol-
comb LA, Alpini G. Acute carbon tetrachloride feeding in-
duces damage of large but not small cholangiocytes from BDL
rat liver. Am J Physiol 1999; 276: G1289-G1301
52 LeSage GD, Benedetti A, Glaser S, Marucci L, Tretjak Z, Ca-
ligiuri A, Rodgers R, Phinizy JL, Baiocchi L, Francis H, Lasater J,
Ugili L, Alpini G. Acute carbon tetrachloride feeding selective-
ly damages large, but not small, cholangiocytes from normal
rat liver. Hepatology 1999; 29: 307-319
53 Blanco PG, Zaman MM, Junaidi O, Sheth S, Yantiss RK, Nass-
er IA, Freedman SD. Induction of colitis in cftr-/- mice results
in bile duct injury. Am J Physiol Gastrointest Liver Physiol 2004;
287: G491-G496
54 Durie PR, Kent G, Phillips MJ, Ackerley CA. Characteristic
multiorgan pathology of cystic fibrosis in a long-living cystic
fibrosis transmembrane regulator knockout murine model. Am
J Pathol 2004; 164: 1481-1493
55 Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg
H, Krause R, Lammert F, Langner C, Zatloukal K, Marschall
HU, Denk H, Trauner M. Regurgitation of bile acids from
leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4)
knockout mice. Gastroenterology 2004; 127: 261-274
56 Lager DJ, Qian Q, Bengal RJ, Ishibashi M, Torres VE. The
pck rat: a new model that resembles human autosomal domi-
nant polycystic kidney and liver disease. Kidney Int 2001; 59:
126-136
57 Masyuk TV, Huang BQ, Ward CJ, Masyuk AI, Yuan D, Splin-
ter PL, Punyashthiti R, Ritman EL, Torres VE, Harris PC,
LaRusso NF. Defects in cholangiocyte fibrocystin expression
and ciliary structure in the PCK rat. Gastroenterology 2003; 125:
1303-1310
58 Mller M, Roelofsen H, Jansen PL. Secretion of organic anions
by hepatocytes: involvement of homologues of the multidrug
resistance protein. Semin Liver Dis 1996; 16: 211-220
59 MILLER LL, BLY CG, WATSON ML, BALE WF. The domi-
nant role of the liver in plasma protein synthesis; a direct
study of the isolated perfused rat liver with the aid of lysine-
epsilon-C14. J Exp Med 1951; 94: 431-453
60 Beije B, Jenssen D, Arrhenius E, Zetterqvist MA. Isolated liver
perfusion--a tool in mutagenicity testing for the evaluation of
carcinogens. Chem Biol Interact 1979; 27: 41-57
61 Demisch K, Ammedick U, Staib W. Testosterone metabolism
in the isolated perfused human foetal liver. Horm Metab Res
1969; 1: 43
62 KRUHFFER P, MUNTZ JA. Carbohydrate metabolism of
the isolated, perfused cat liver as studied by labelled glucose
and fructose. Acta Physiol Scand 1954; 30: 258-274
63 BENHAMOU JP, AFIFI FH, LOVERDO A, FAUVERT R.
[Fixation of colloidal radioactive gold by isolated perfused
rabbit liver. I. Measurement of the efficacy of purification]. C R
Seances Soc Biol Fil 1957; 151: 442-444
64 AXELROD LR, MILLER LL. The metabolism of hydrocorti-
sone in the isolated perfused dog liver. Arch Biochem Biophys
1956; 60: 373-378
65 ANDREWS WH, BRITTON HG, HUGGETT AS. Fructose me-
tabolism in the isolated perfused liver of the foetal and new-
born sheep. J Physiol 1960; 153: 199-208
66 MARTINIS AJ, GOLDSWORTHY PD, JONES TW, NYHUS
LM, DEVITO RV, VOLWILER W, HARKINS HN. Studies of
hepatic physiology in the isolated, perfused calf liver. Surg Fo-
rum 1958; 9: 489-493
67 EISEMAN B, MOORE TC, NORMELL L. HISTAMINE
METABOLISM IN THE ISOLATED PERFUSED PIG LIVER.
Surg Gynecol Obstet 1964; 118: 69-74
68 Sparks JW, Lynch A, Chez RA, Glinsmann WH. Glycogen
regulation in isolated perfused near term monkey liver. Pediatr
Res 1976; 10: 51-56
69 Gores GJ, Kost LJ, LaRusso NF. The isolated perfused rat
liver: conceptual and practical considerations. Hepatology 1986;
6: 511-517
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
70 Reichen J, Paumgartner G. Kinetics of taurocholate uptake by
the perfused rat liver. Gastroenterology 1975; 68: 132-136
71 Wanless IR. Physioanatomic considerations. In: Schiff ER, Sor-
rell MF, Maddrey WC, eds. Shiffs diseases of the liver, New
York: Lippincott-Raven, 1999: 3-37
72 Ahmad AB, Bennett PN, Rowland M. Influence of route of he-
patic administration on drug availability. J Pharmacol Exp Ther
1984; 230: 718-725
73 Zimmermann T, Gardemann A, Machnik G, Dargel R, Junger-
mann K. Metabolic and hemodynamic responses of bivascu-
larly perfused rat liver to nerve stimulation, noradrenaline,
acetylcholine and glucagon in thioacetamide-induced mi-
cronodular cirrhosis. Hepatology 1992; 15: 464-470
74 Gaudio E, Onori P, Pannarale L, Alvaro D. Hepatic micro-
circulation and peribiliary plexus in experimental biliary
cirrhosis: a morphological study. Gastroenterology 1996; 111:
1118-1124
75 Nakanuma Y, Hoso M, Sanzen T, Sasaki M. Microstructure
and development of the normal and pathologic biliary tract
in humans, including blood supply. Microsc Res Tech 1997; 38:
552-570
76 Imamura T, Fujimoto JM. Transit patterns of marker com-
pounds given by segmented retrograde intrabiliary injection
(SRII) in the isolated in situ perfused rat liver. J Pharmacol Exp
Ther 1980; 215: 110-115
77 Meier PJ, Sztul ES, Reuben A, Boyer JL. Structural and func-
tional polarity of canalicular and basolateral plasma mem-
brane vesicles isolated in high yield from rat liver. J Cell Biol
1984; 98: 991-1000
78 Meier PJ, Knickelbein R, Moseley RH, Dobbins JW, Boyer JL.
Evidence for carrier-mediated chloride/bicarbonate exchange
in canalicular rat liver plasma membrane vesicles. J Clin Invest
1985; 75: 1256-1263
79 Graf J, Gautam A, Boyer JL. Isolated rat hepatocyte couplets:
a primary secretory unit for electrophysiologic studies of bile
secretory function. Proc Natl Acad Sci U S A 1984; 81: 6516-6520
80 Benedetti A, Marucci L, Bassotti C, Mancini R, Contucci S,
Jezequel AM, Orlandi F. Tubulovesicular transcytotic pathway
in rat biliary epithelium: a study in perfused liver and in iso-
lated intrahepatic bile duct. Hepatology 1993; 18: 422-432
81 Mennone A, Alvaro D, Cho W, Boyer JL. Isolation of small
polarized bile duct units. Proc Natl Acad Sci U S A 1995; 92:
6527-6531
82 Cho WK, Mennone A, Boyer JL. Isolation of functional polar-
ized bile duct units from mouse liver. Am J Physiol Gastrointest
Liver Physiol 2001; 280: G241-G246
83 Boyer JL. Isolated hepatocyte couplets and bile duct units--
novel preparations for the in vitro study of bile secretory func-
tion. Cell Biol Toxicol 1997; 13: 289-300
84 Gautam A, Ng OC, Strazzabosco M, Boyer JL. Quantitative
assessment of canalicular bile formation in isolated hepatocyte
couplets using microscopic optical planimetry. J Clin Invest
1989; 83: 565-573
85 Gall JA, Bhathal PS. The isolation of intrahepatic biliary epi-
thelial cells from normal rat livers. Cell Biol Int Rep 1985; 9:
315-322
86 Parola M, Cheeseman KH, Biocca ME, Dianzani MU, Slater
TF. Isolation and characterization of biliary epithelial cells
from normal rat liver. J Hepatol 1988; 6: 175-186
87 Kumar U, Jordan TW. Isolation and culture of biliary epitheli-
al cells from the biliary tract fraction of normal rats. Liver 1986;
6: 369-378
88 Grisham JW. Cell types in rat liver cultures: their identifica-
tion and isolation. Mol Cell Biochem 1983; 53-54: 23-33
89 Ishii M, Vroman B, LaRusso NF. Isolation and morphologic
characterization of bile duct epithelial cells from normal rat
liver. Gastroenterology 1989; 97: 1236-1247
90 Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G, Al-
pini G. Functional heterogeneity of cholangiocytes. Semin Liver
Dis 2002; 22: 227-240
91 Cho WK, Siegrist VJ, Zinzow W. Impaired regulatory volume
decrease in freshly isolated cholangiocytes from cystic fibrosis
mice: implications for cystic fibrosis transmembrane conduc-
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
tance regulator effect on potassium conductance. J Biol Chem
2004; 279: 14610-14618
92 Spirl C, Fiorotto R, Song L, Santos-Sacchi J, Okolicsanyi L,
Masier S, Rocchi L, Vairetti MP, De Bernard M, Melero S, Poz-
zan T, Strazzabosco M. Glibenclamide stimulates fluid secre-
tion in rodent cholangiocytes through a cystic fibrosis trans-
membrane conductance regulator-independent mechanism.
Gastroenterology 2005; 129: 220-233
93 Nozaki I, Lunz JG 3rd, Specht S, Park JI, Giraud AS, Murase N,
Demetris AJ. Regulation and function of trefoil factor family 3
expression in the biliary tree. Am J Pathol 2004; 165: 1907-1920
94 Grubman SA, Fang SL, Mulberg AE, Perrone RD, Rogers LC,
Lee DW, Armentano D, Murray SL, Dorkin HL, Cheng SH.
Correction of the cystic fibrosis defect by gene complementa-
tion in human intrahepatic biliary epithelial cell lines. Gastro-
enterology 1995; 108: 584-592
95 Zsembery A, Jessner W, Sitter G, Spirl C, Strazzabosco M,
Graf J. Correction of CFTR malfunction and stimulation of Ca-
activated Cl channels restore HCO3- secretion in cystic fibrosis
bile ductular cells. Hepatology 2002; 35: 95-104
96 Vroman B, LaRusso NF. Development and characterization of
polarized primary cultures of rat intrahepatic bile duct epithe-
lial cells. Lab Invest 1996; 74: 303-313
97 Salter KD, Roman RM, LaRusso NR, Fitz JG, Doctor RB. Mod-
ified culture conditions enhance expression of differentiated
phenotypic properties of normal rat cholangiocytes. Lab Invest
2000; 80: 1775-1778
98 Joplin R, Strain AJ, Neuberger JM. Immuno-isolation and
culture of biliary epithelial cells from normal human liver. In
Vitro Cell Dev Biol 1989; 25: 1189-1192
99 Joplin R, Strain AJ, Neuberger JM. Biliary epithelial cells from
the liver of patients with primary biliary cirrhosis: isolation,
characterization, and short-term culture. J Pathol 1990; 162:
255-260
100 Joplin R, Hishida T, Tsubouchi H, Daikuhara Y, Ayres R,
Neuberger JM, Strain AJ. Human intrahepatic biliary epithe-
lial cells proliferate in vitro in response to human hepatocyte
growth factor. J Clin Invest 1992; 90: 1284-1289
101 Ishida Y, Smith S, Wallace L, Sadamoto T, Okamoto M, Auth
M, Strazzabosco M, Fabris L, Medina J, Prieto J, Strain A,
Neuberger J, Joplin R. Ductular morphogenesis and functional
polarization of normal human biliary epithelial cells in three-
dimensional culture. J Hepatol 2001; 35: 2-9
102 Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracel-
lular pH measurements in Ehrlich ascites tumor cells utilizing
spectroscopic probes generated in situ. Biochemistry 1979; 18:
2210-2218
103 Rink TJ, Tsien RY, Pozzan T. Cytoplasmic pH and free Mg2+
in lymphocytes. J Cell Biol 1982; 95: 189-196
104 Graber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E.
Characteristics of fluoroprobes for measuring intracellular pH.
Anal Biochem 1986; 156: 202-212
105 Strazzabosco M, Boyer JL. Ion Transporters that regulate
intracellular pH and secretion in bile duct epithelial cells. In:
Sirica EA, Longnecker DS, eds. Biliary and pancreatic ductal
epithelia: Pathobiology and pathophysiology, New York: Mar-
cel Dekker, Inc, 1997: 85-106
106 Spirl C, Granato A, Zsembery K, Anglani F, Okolicsnyi L,
LaRusso NF, Crepaldi G, Strazzabosco M. Functional polarity
of Na+/H+ and Cl-/HCO3- exchangers in a rat cholangiocyte
cell line. Am J Physiol 1998; 275: G1236-G1245
107 Chaillet JR, Amsler K, Boron WF. Optical measurements of
intracellular pH in single LLC-PK1 cells: demonstration of Cl-
HCO3 exchange. Proc Natl Acad Sci U S A 1986; 83: 522-526
108 Boron WF. Regulation of intracellular pH. Adv Physiol Educ
2004; 28: 160-179
109 Masyuk TV, Ritman EL, LaRusso NF. Quantitative assessment
of the rat intrahepatic biliary system by three-dimensional re-
construction. Am J Pathol 2001; 158: 2079-2088
110 Grisham JW, Nopanitaya W, Compagno J, Ngel AE. Scan-
ning electron microscopy of normal rat liver: the surface struc-
ture of its cells and tissue components. Am J Anat 1975; 144:
295-321
3507
111 Ishii M, Vroman B, LaRusso NF. Morphologic demonstration
of receptor-mediated endocytosis of epidermal growth factor
by isolated bile duct epithelial cells. Gastroenterology 1990; 98:
1284-1291
112 Tietz PS, Marinelli RA, Chen XM, Huang B, Cohn J, Kole J,
McNiven MA, Alper S, LaRusso NF. Agonist-induced coor-
dinated trafficking of functionally-related transport proteins
for water and ions in cholangiocytes. J Biol Chem 2003; 278:
20413-20419
113 McCaffery JM, Farquhar MG. Localization of GTPases by in-
direct immunofluorescence and immunoelectron microscopy.
Methods Enzymol 1995; 257: 259-279
114 Cullen BR. Derivation and function of small interfering RNAs
and microRNAs. Virus Res 2004; 102: 3-9
115 Splinter PL, Masyuk AI, LaRusso NF. Specific inhibition
of AQP1 water channels in isolated rat intrahepatic bile
duct units by small interfering RNAs. J Biol Chem 2003; 278:
6268-6274
116 Obama K, Ura K, Satoh S, Nakamura Y, Furukawa Y. Up-
regulation of PSF2, a member of the GINS multiprotein com-
plex, in intrahepatic cholangiocarcinoma. Oncol Rep 2005; 14:
701-706
117 Taniai M, Grambihler A, Higuchi H, Werneburg N, Bronk SF,
Farrugia DJ, Kaufmann SH, Gores GJ. Mcl-1 mediates tumor
necrosis factor-related apoptosis-inducing ligand resistance
in human cholangiocarcinoma cells. Cancer Res 2004; 64:
3517-3524
118 Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L,
Roth J, Hofmann AF, Meier PJ. The sister of P-glycoprotein
represents the canalicular bile salt export pump of mammalian
liver. J Biol Chem 1998; 273: 10046-10050
119 Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N,
Arnell H, Sokal E, Dahan K, Childs S, Ling V, Tanner MS,
Kagalwalla AF, Nmeth A, Pawlowska J, Baker A, Mieli-
Vergani G, Freimer NB, Gardiner RM, Thompson RJ. A gene
encoding a liver-specific ABC transporter is mutated in pro-
gressive familial intrahepatic cholestasis. Nat Genet 1998; 20:
233-238
120 Wang R, Salem M, Yousef IM, Tuchweber B, Lam P, Childs SJ,
Helgason CD, Ackerley C, Phillips MJ, Ling V. Targeted inac-
tivation of sister of P-glycoprotein gene (spgp) in mice results
in nonprogressive but persistent intrahepatic cholestasis. Proc
Natl Acad Sci U S A 2001; 98: 2011-2016
121 Nathanson MH, Boyer JL. Mechanisms and regulation of bile
secretion. Hepatology 1991; 14: 551-566
122 Erlinger S. Physiology of bile secretion and enterohepatic
circulation. In: Johnson LR, eds. Physiology of the gastrointes-
tinal tract, New York: Raven Press, 1987: 1557-1580
123 Meier PJ, Stieger B. Molecular Mechanisms in Bile Formation.
News Physiol Sci 2000; 15: 89-93
124 Benedetti A, Strazzabosco M, Corasanti JG, Haddad P, Graf J,
Boyer JL. Cl(-)-HCO3- exchanger in isolated rat hepatocytes:
role in regulation of intracellular pH. Am J Physiol 1991; 261:
G512-522
125 Boyer JL, Graf J, Meier PJ. Hepatic transport systems regulat-
ing pHi, cell volume, and bile secretion. Annu Rev Physiol 1992;
54: 415-438
126 Benedetti A, Strazzabosco M, Ng OC, Boyer JL. Regulation of
activity and apical targeting of the Cl-/HCO3- exchanger in
rat hepatocytes. Proc Natl Acad Sci U S A 1994; 91: 792-796
127 Strazzabosco M, Boyer JL. Regulation of intracellular pH in
the hepatocyte. Mechanisms and physiological implications. J
Hepatol 1996; 24: 631-644
128 Gradilone SA, Garca F, Huebert RC, Tietz PS, Larocca MC,
Kierbel A, Carreras FI, Larusso NF, Marinelli RA. Glucagon
induces the plasma membrane insertion of functional aqua-
porin-8 water channels in isolated rat hepatocytes. Hepatology
2003; 37: 1435-1441
129 Boyer JL, Bloomer JR. Canalicular bile secretion in man. Stud-
ies utilizing the biliary clearance of (14C)mannitol. J Clin Invest
1974; 54: 773-781
130 Prandi D. Canalicular bile production in man. Eur J Clin Invest
1975; 5: 1-6
www.wjgnet.com
 3508 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
131 Berthelot P, Erlinger S, Dhumeaux D, Preaux AM. Mechanism
of phenobarbital-induced hypercholeresis in the rat. Am J
Physiol 1970; 219: 809-813
132 Boyer JL, Klatskin G. Canalicular bile flow and bile secretory
pressure. Evidence for a non-bile salt dependent fraction in the
isolated perfused rat liver. Gastroenterology 1970; 59: 853-859
133 Erlinger S, Dhumeaux D, Berthelot P, Dumont M. Effect of
inhibitors of sodium transport on bile formation in the rabbit.
Am J Physiol 1970; 219: 416-422
134 Lenzen R, Hruby VJ, Tavoloni N. mechanism of glucagon cho-
leresis in guinea pigs. Am J Physiol 1990; 259: G736-744
135 Alvaro D, Della Guardia P, Bini A, Gigliozzi A, Furfaro S, La
Rosa T, Piat C, Capocaccia L. Effect of glucagon on intracel-
lular pH regulation in isolated rat hepatocyte couplets. J Clin
Invest 1995; 96: 665-675
136 Garca F, Kierbel A, Larocca MC, Gradilone SA, Splinter P,
LaRusso NF, Marinelli RA. The water channel aquaporin-8 is
mainly intracellular in rat hepatocytes, and its plasma mem-
brane insertion is stimulated by cyclic AMP. J Biol Chem 2001;
276: 12147-12152
137 Huebert RC, Splinter PL, Garcia F, Marinelli RA, LaRusso NF.
Expression and localization of aquaporin water channels in rat
hepatocytes. Evidence for a role in canalicular bile secretion. J
Biol Chem 2002; 277: 22710-22717
138 Gradilone SA, Tietz PS, Splinter PL, Marinelli RA, LaRusso
NF. Expression and subcellular localization of aquaporin wa-
ter channels in the polarized hepatocyte cell line, WIF-B. BMC
Physiol 2005; 5: 13
139 Bruck R, Benedetti A, Strazzabosco M, Boyer JL. Intracellular
alkalinization stimulates bile flow and vesicular-mediated
exocytosis in IPRL. Am J Physiol 1993; 265: G347-G353
140 Tietz P, Jefferson J, Pagano R, Larusso NF. Membrane micro-
domains in hepatocytes: potential target areas for proteins
involved in canalicular bile secretion. J Lipid Res 2005; 46:
1426-1432
141 Marinelli RA, Tietz PS, Pham LD, Rueckert L, Agre P, LaRus-
so NF. Secretin induces the apical insertion of aquaporin-1
water channels in rat cholangiocytes. Am J Physiol 1999; 276:
G280-286
142 Tietz PS, McNiven MA, Splinter PL, Huang BQ, Larusso NF.
Cytoskeletal and motor proteins facilitate trafficking of AQP1-
containing vesicles in cholangiocytes. Biol Cell 2006; 98: 43-52
143 Martnez-Ans E, Castillo JE, Dez J, Medina JF, Prieto J. Im-
munohistochemical detection of chloride/bicarbonate anion
exchangers in human liver. Hepatology 1994; 19: 1400-1406
144 Aranda V, Martnez I, Melero S, Lecanda J, Banales JM, Prieto J,
Medina JF. Shared apical sorting of anion exchanger isoforms
AE2a, AE2b1, and AE2b2 in primary hepatocytes. Biochem Bio-
phys Res Commun 2004; 319: 1040-1046
145 Wang Y, Roman R, Lidofsky SD, Fitz JG. Autocrine signal-
ing through ATP release represents a novel mechanism for
cell volume regulation. Proc Natl Acad Sci U S A 1996; 93:
12020-12025
146 Roman RM, Bodily KO, Wang Y, Raymond JR, Fitz JG.
Activation of protein kinase Calpha couples cell volume to
membrane Cl- permeability in HTC hepatoma and Mz-ChA-1
cholangiocarcinoma cells. Hepatology 1998; 28: 1073-1080
147 Schfl C, Ponczek M, Mader T, Waring M, Benecke H, von zur
Mhlen A, Mix H, Cornberg M, Bker KH, Manns MP, Wag-
ner S. Regulation of cytosolic free calcium concentration by
extracellular nucleotides in human hepatocytes. Am J Physiol
1999; 276: G164-G172
148 Feranchak AP, Fitz JG, Roman RM. Volume-sensitive puriner-
gic signaling in human hepatocytes. J Hepatol 2000; 33: 174-182
149 Renner EL, Lake JR, Scharschmidt BF, Zimmerli B, Meier PJ.
Rat hepatocytes exhibit basolateral Na+/HCO3- cotransport. J
Clin Invest 1989; 83: 1225-1235
150 Gleeson D, Smith ND, Boyer JL. Bicarbonate-dependent and
-independent intracellular pH regulatory mechanisms in rat
hepatocytes. Evidence for Na+-HCO3- cotransport. J Clin In-
vest 1989; 84: 312-321
151 Fitz JG, Persico M, Scharschmidt BF. Electrophysiological
evidence for Na+-coupled bicarbonate transport in cultured rat
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
hepatocytes. Am J Physiol 1989; 256: G491-500
152 Buanes T, Grotmol T, Veel T, Landsverk T, Ridderstrle Y,
Raeder MG. Importance of carbonic anhydrase for canalicular
and ductular choleresis in the pig. Acta Physiol Scand 1988; 133:
535-544
153 Kivel AJ, Kivel J, Saarnio J, Parkkila S. Carbonic anhydrases
in normal gastrointestinal tract and gastrointestinal tumours.
World J Gastroenterol 2005; 11: 155-163
154 Henry RP. Multiple roles of carbonic anhydrase in cellular
transport and metabolism. Annu Rev Physiol 1996; 58: 523-538
155 Orlowski J, Kandasamy RA, Shull GE. Molecular cloning
of putative members of the Na/H exchanger gene family.
cDNA cloning, deduced amino acid sequence, and mRNA
tissue expression of the rat Na/H exchanger NHE-1 and two
structurally related proteins. J Biol Chem 1992; 267: 9331-9339
156 Moseley RH, Meier PJ, Aronson PS, Boyer JL. Na-H exchange
in rat liver basolateral but not canalicular plasma membrane
vesicles. Am J Physiol 1986; 250: G35-G43
157 Grinstein S, Woodside M, Waddell TK, Downey GP, Orlowski
J, Pouyssegur J, Wong DC, Foskett JK. Focal localization of the
NHE-1 isoform of the Na+/H+ antiport: assessment of effects
on intracellular pH. EMBO J 1993; 12: 5209-5218
158 Pizzonia JH, Biemesderfer D, Abu-Alfa AK, Wu MS, Exner
M, Isenring P, Igarashi P, Aronson PS. Immunochemical
characterization of Na+/H+ exchanger isoform NHE4. Am J
Physiol 1998; 275: F510-F517
159 Mennone A, Biemesderfer D, Negoianu D, Yang CL, Abbiati
T, Schultheis PJ, Shull GE, Aronson PS, Boyer JL. Role of
sodium/hydrogen exchanger isoform NHE3 in fluid secretion
and absorption in mouse and rat cholangiocytes. Am J Physiol
Gastrointest Liver Physiol 2001; 280: G247-G254
160 Fitz JG, Lidofsky SD, Xie MH, Scharschmidt BF. Trans-
membrane electrical potential difference regulates Na+/
HCO3- cotransport and intracellular pH in hepatocytes. Proc
Natl Acad Sci U S A. 1992; 89: 4197-4201
161 Romero MF, Fulton CM, Boron WF. The SLC4 family of HCO
3 - transporters. Pflugers Arch 2004; 447: 495-509
162 Kurtz I, Petrasek D, Tatishchev S. Molecular mechanisms of
electrogenic sodium bicarbonate cotransport: structural and
equilibrium thermodynamic considerations. J Membr Biol 2004;
197: 77-90
163 Abuladze N, Lee I, Newman D, Hwang J, Boorer K, Pushkin
A, Kurtz I. Molecular cloning, chromosomal localization,
tissue distribution, and functional expression of the human
pancreatic sodium bicarbonate cotransporter. J Biol Chem 1998;
273: 17689-17695
164 Abuladze N, Pushkin A, Tatishchev S, Newman D, Sassani
P, Kurtz I. Expression and localization of rat NBC4c in liver
and renal uroepithelium. Am J Physiol Cell Physiol 2004; 287:
C781-C789
165 Pushkin A, Abuladze N, Newman D, Lee I, Xu G, Kurtz I.
Cloning, characterization and chromosomal assignment of
NBC4, a new member of the sodium bicarbonate cotransporter
family. Biochim Biophys Acta 2000; 1493: 215-218
166 Kaminski DL, Dorighi J, Jellinek M. Effect of electrical vagal
stimulation on canine hepatic bile flow. Am J Physiol 1974; 227:
487-493
167 Cucchiaro G, Branum GD, Farouk M, Mansour G, Kuhn CM,
Anthony DC, Meyers WC. The effects of liver denervation on
the regulation of hepatic biliary secretion. Transplantation 1992;
54: 129-136
168 Strazzabosco M. New insights into cholangiocyte physiology.
J Hepatol 1997; 27: 945-952
169 Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC,
Levine S, Dawson PA, LaRusso NF. Rat cholangiocytes
absorb bile acids at their apical domain via the ileal sodium-
dependent bile acid transporter. J Clin Invest 1997; 100:
2714-2721
170 Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE,
Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional
expression of the apical Na+-dependent bile acid transporter
in large but not small rat cholangiocytes. Gastroenterology 1997;
113: 1734-1740
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
171 Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H,
Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, Lesage
G. Bile acid depletion and repletion regulate cholangiocyte
growth and secretion by a phosphatidylinositol 3-kinase-
dependent pathway in rats. Gastroenterology 2002; 123:
1226-1237
172 Alpini G, Glaser S, Baiocchi L, Francis H, Xia X, Lesage G.
Secretin activation of the apical Na +-dependent bile acid
transporter is associated with cholehepatic shunting in rats.
Hepatology 2005; 41: 1037-1045
173 Lazaridis KN, Tietz P, Wu T, Kip S, Dawson PA, LaRusso NF.
Alternative splicing of the rat sodium/bile acid transporter
changes its cellular localization and transport properties. Proc
Natl Acad Sci U S A 2000; 97: 11092-11097
174 Strazzabosco M, Mennone A, Boyer JL. Intracellular pH regu-
lation in isolated rat bile duct epithelial cells. J Clin Invest 1991;
87: 1503-1512
175 Alvaro D, Cho WK, Mennone A, Boyer JL. Effect of secretion
on intracellular pH regulation in isolated rat bile duct epithe-
lial cells. J Clin Invest 1993; 92: 1314-1325
176 Strazzabosco M, Joplin R, Zsembery A, Wallace L, Spirl C,
Fabris L, Granato A, Rossanese A, Poci C, Neuberger JM,
Okolicsnyi L, Crepaldi G. Na(+)-dependent and -indepen-
dent Cl-/HCO-3 exchange mediate cellular HCO3- transport
in cultured human intrahepatic bile duct cells. Hepatology 1997;
25: 976-985
177 Ueno Y, Ishii M, Takahashi S, Igarashi T, Toyota T, LaRusso
NF. Different susceptibility of mice to immune-mediated chol-
angitis induced by immunization with carbonic anhydrase II.
Lab Invest 1998; 78: 629-637
178 Grubman SA, Perrone RD, Lee DW, Murray SL, Rogers LC,
Wolkoff LI, Mulberg AE, Cherington V, Jefferson DM. Regula-
tion of intracellular pH by immortalized human intrahepatic
biliary epithelial cell lines. Am J Physiol 1994; 266: G1060-G1070
179 Grichtchenko II, Choi I, Zhong X, Bray-Ward P, Russell JM,
Boron WF. Cloning, characterization, and chromosomal map-
ping of a human electroneutral Na(+)-driven Cl-HCO3 ex-
changer. J Biol Chem 2001; 276: 8358-8363
180 Villanger O, Veel T, Raeder MG. Secretin causes H+ secretion
from intrahepatic bile ductules by vacuolar-type H(+)-ATPase.
Am J Physiol 1993; 265: G719-G724
181 Burckhardt G, Di Sole F, Helmle-Kolb C. The Na +/H + ex-
changer gene family. J Nephrol 2002; 15 Suppl 5: S3-21
182 Gawenis LR, Greeb JM, Prasad V, Grisham C, Sanford LP,
Doetschman T, Andringa A, Miller ML, Shull GE. Impaired
gastric acid secretion in mice with a targeted disruption of the
NHE4 Na+/H+ exchanger. J Biol Chem 2005; 280: 12781-12789
183 Alvaro D, Mennone A, Boyer JL. Role of kinases and pho-
sphatases in the regulation of fluid secretion and Cl-/HCO3-
exchange in cholangiocytes. Am J Physiol 1997; 273: G303-G313
184 Alper SL. Molecular physiology of SLC4 anion exchangers.
Exp Physiol 2006; 91: 153-161
185 Mount DB, Romero MF. The SLC26 gene family of multifunc-
tional anion exchangers. Pflugers Arch 2004; 447: 710-721
186 Petrovic S, Ju X, Barone S, Seidler U, Alper SL, Lohi H, Kere
J, Soleimani M. Identification of a basolateral Cl-/HCO3- ex-
changer specific to gastric parietal cells. Am J Physiol Gastroin-
test Liver Physiol 2003; 284: G1093-1103
187 Kim KH, Shcheynikov N, Wang Y, Muallem S. SLC26A7 is
a Cl- channel regulated by intracellular pH. J Biol Chem 2005;
280: 6463-6470
188 Fitz JG, Basavappa S, McGill J, Melhus O, Cohn JA. Regula-
tion of membrane chloride currents in rat bile duct epithelial
cells. J Clin Invest 1993; 91: 319-328
189 Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz
JG. Localization of the cystic fibrosis transmembrane conduc-
tance regulator in human bile duct epithelial cells. Gastroenter-
ology 1993; 105: 1857-1864
190 Gray MA, Pollard CE, Harris A, Coleman L, Greenwell JR, Ar-
gent BE. Anion selectivity and block of the small-conductance
chloride channel on pancreatic duct cells. Am J Physiol 1990;
259: C752-C761
191 Illek B, Yankaskas JR, Machen TE. cAMP and genistein stimu-
3509
late HCO3- conductance through CFTR in human airway epi-
thelia. Am J Physiol 1997; 272: L752-L761
192 Choi JY, Lee MG, Ko S, Muallem S. Cl(-)-dependent HCO3-
transport by cystic fibrosis transmembrane conductance regu-
lator. JOP 2001; 2: 243-246
193 Poulsen JH, Fischer H, Illek B, Machen TE. Bicarbonate con-
ductance and pH regulatory capability of cystic fibrosis trans-
membrane conductance regulator. Proc Natl Acad Sci U S A
1994; 91: 5340-5344
194 Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA,
Bridges RJ. Bicarbonate and chloride secretion in Calu-3 hu-
man airway epithelial cells. J Gen Physiol 1999; 113: 743-760
195 Spirl C, Fabris L, Duner E, Fiorotto R, Ballardini G, Roskams
T, Larusso NF, Sonzogni A, Okolicsanyi L, Strazzabosco M.
Cytokine-stimulated nitric oxide production inhibits adenylyl
cyclase and cAMP-dependent secretion in cholangiocytes. Gas-
troenterology 2003; 124: 737-753
196 Basavappa S, Middleton J, Mangel AW, McGill JM, Cohn JA,
Fitz JG. Cl- and K+ transport in human biliary cell lines. Gas-
troenterology 1993; 104: 1796-1805
197 McGill JM, Basavappa S, Mangel AW, Shimokura GH, Mid-
dleton JP, Fitz JG. Adenosine triphosphate activates ion per-
meabilities in biliary epithelial cells. Gastroenterology 1994; 107:
236-243
198 Schlenker T, Fitz JG. Ca(2+)-activated C1- channels in a hu-
man biliary cell line: regulation by Ca2+/calmodulin-depen-
dent protein kinase. Am J Physiol 1996; 271: G304-G310
199 Clarke LL, Harline MC, Gawenis LR, Walker NM, Turner JT,
Weisman GA. Extracellular UTP stimulates electrogenic bicar-
bonate secretion across CFTR knockout gallbladder epitheli-
um. Am J Physiol Gastrointest Liver Physiol 2000; 279: G132-G138
200 McGill JM, Yen MS, Basavappa S, Mangel AW, Kwiatkowski
AP. ATP-activated chloride permeability in biliary epithelial
cells is regulated by calmodulin-dependent protein kinase II.
Biochem Biophys Res Commun 1995; 208: 457-462
201 Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG. En-
dogenous ATP release regulates Cl- secretion in cultured
human and rat biliary epithelial cells. Am J Physiol 1999; 276:
G1391-G1400
202 Feranchak AP, Doctor RB, Troetsch M, Brookman K, Johnson
SM, Fitz JG. Calcium-dependent regulation of secretion in bili-
ary epithelial cells: the role of apamin-sensitive SK channels.
Gastroenterology 2004; 127: 903-913
203 McGill JM, Basavappa S, Fitz JG. Characterization of high-
conductance anion channels in rat bile duct epithelial cells. Am
J Physiol 1992; 262: G703-G710
204 Singh SK, Mennone A, Gigliozzi A, Fraioli F, Boyer JL. Cl(-)-
dependent secretory mechanisms in isolated rat bile duct epi-
thelial units. Am J Physiol Gastrointest Liver Physiol 2001; 281:
G438-G446
205 Geck P, Pfeiffer B. Na+ + K+ + 2Cl- cotransport in animal
cells--its role in volume regulation. Ann N Y Acad Sci 1985; 456:
166-182
206 Panet R, Markus M, Atlan H. Bumetanide and furosemide
inhibited vascular endothelial cell proliferation. J Cell Physiol
1994; 158: 121-127
207 Russell JM. Sodium-potassium-chloride cotransport. Physiol
Rev 2000; 80: 211-276
208 Scoazec JY, Bringuier AF, Medina JF, Martnez-Ans E, Veis-
siere D, Feldmann G, Housset C. The plasma membrane
polarity of human biliary epithelial cells: in situ immunohisto-
chemical analysis and functional implications. J Hepatol 1997;
26: 543-553
209 Hammerton RW, Krzeminski KA, Mays RW, Ryan TA, Woll-
ner DA, Nelson WJ. Mechanism for regulating cell surface
distribution of Na+,K(+)-ATPase in polarized epithelial cells.
Science 1991; 254: 847-850
210 Rakowski RF, Gadsby DC, De Weer P. Stoichiometry and
voltage dependence of the sodium pump in voltage-clamped,
internally dialyzed squid giant axon. J Gen Physiol 1989; 93:
903-941
211 McRoberts JA, Beuerlein G, Dharmsathaphorn K. Cyclic AMP
and Ca2+-activated K+ transport in a human colonic epithelial
www.wjgnet.com
 3510 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
cell line. J Biol Chem 1985; 260: 14163-14172
212 Roman R, Feranchak AP, Troetsch M, Dunkelberg JC, Kilic G,
Schlenker T, Schaack J, Fitz JG. Molecular characterization of
volume-sensitive SK(Ca) channels in human liver cell lines.
Am J Physiol Gastrointest Liver Physiol 2002; 282: G116-122
213 Ishii TM, Maylie J, Adelman JP. Determinants of apamin and
d-tubocurarine block in SK potassium channels. J Biol Chem
1997; 272: 23195-23200
214 Marinelli RA, Pham LD, Tietz PS, LaRusso NF. Expression of
aquaporin-4 water channels in rat cholangiocytes. Hepatology
2000; 31: 1313-1317
215 Splinter PL, Masyuk AI, Marinelli RA, LaRusso NF. AQP4
transfected into mouse cholangiocytes promotes water trans-
port in biliary epithelia. Hepatology 2004; 39: 109-116
216 Talbot NC, Garrett WM, Caperna TJ. Analysis of the expres-
sion of aquaporin-1 and aquaporin-9 in pig liver tissue: com-
parison with rat liver tissue. Cells Tissues Organs 2003; 174:
117-128
217 Still EU, McBean JW, Ries FA. Studies on the physiology of
secretin. IV. Effect on the secretion of bile. Ame J Physiol 1931;
99: 94
218 Mellanby JM, Suffolk FS. Quantitative investigation into en-
terohepatic circulation of bile salts in the cat. Proc R Soc 1938;
126: 287
219 GROSSMAN MI, JANOWITZ HD. The effect of secretin on
bile formation in man. Gastroenterology 1949; 12: 133-138
220 JORPES JE, MUTT V, MAGNUSSON S, STEELE BB. Amino
acid composition and N-terminal amino acid sequence of por-
cine secretin. Biochem Biophys Res Commun 1962; 9: 275-279
221 Mutt V, Jorpes JE, Magnusson S. Structure of porcine secretin.
The amino acid sequence. Eur J Biochem 1970; 15: 513-519
222 Ondetti MA, Narayanan VL, von Saltza M, Sheehan JT, Sabo
EF, Bodanszky M. The synthesis of secretin. 3. The fragment-
condensation approach. J Am Chem Soc 1968; 90: 4711-4715
223 Joffe SN, Bloom SR, Polak JM, Welbourn RB. Proceedings: Re-
lease of secretin for S cells of porcine duodenum and jejunum
by acid. Gut 1975; 16: 398
224 Polak JM, Bloom SR, Kuzio M, Brown JC, Pearse AG. Cellular
localization of gastric inhibitory polypeptide in the duodenum
and jejunum. Gut 1973; 14: 284-288
225 Kim MS, Lee KY, Chey WY. Plasma secretin concentrations in
fasting and postprandial states in dog. Am J Physiol 1979; 236:
E539-E544
226 Sun G, Lee KY, Chang TM, Chey WY. Effect of pancreatic
juice diversion on secretin release in rats. Gastroenterology 1989;
96: 1173-1179
227 Shiratori K, Jo YH, Lee KY, Chang TM, Chey WY. Effect of
pancreatic juice and trypsin on oleic acid-stimulated pancreat-
ic secretion and plasma secretin in dogs. Gastroenterology 1989;
96: 1330-1336
228 Li P, Lee KY, Chang TM, Chey WY. Mechanism of acid-
induced release of secretin in rats. Presence of a secretin-
releasing peptide. J Clin Invest 1990; 86: 1474-1479
229 Li P, Song Y, Lee KY, Chang TM, Chey WY. A secretin releas-
ing peptide exists in dog pancreatic juice. Life Sci 2000; 66:
1307-1316
230 Li JP, Lee KY, Chang TM, Chey WY. MEK inhibits secretin re-
lease and pancreatic secretion: roles of secretin-releasing pep-
tide and somatostatin. Am J Physiol Gastrointest Liver Physiol
2001; 280: G890-G896
231 Chey WY, Kim MS, Lee KY, Chang TM. Secretin is an entero-
gastrone in the dog. Am J Physiol 1981; 240: G239-G244
232 Jin HO, Lee KY, Chang TM, Chey WY, Dubois A. Secretin: a
physiological regulator of gastric emptying and acid output in
dogs. Am J Physiol 1994; 267: G702-G708
233 Chung I, Li P, Lee K, Chang T, Chey WY. Dual inhibitory
mechanism of secretin action on acid secretion in totally iso-
lated, vascularly perfused rat stomach. Gastroenterology 1994;
107: 1751-1758
234 Shimizu K, Li P, Lee KY, Chang TM, Chey WY. The mecha-
nism of inhibitory action of secretin on gastric acid secretion in
conscious rats. J Physiol 1995; 488( Pt 2): 501-508
235 Raybould HE, Holzer H. Secretin inhibits gastric emptying
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
in rats via a capsaicin-sensitive vagal afferent pathway. Eur J
Pharmacol 1993; 250: 165-167
236 Lu Y, Owyang C. Secretin at physiological doses inhibits gas-
tric motility via a vagal afferent pathway. Am J Physiol 1995;
268: G1012-G1016
237 Holtmann MH, Ganguli S, Hadac EM, Dolu V, Miller LJ. Mul-
tiple extracellular loop domains contribute critical determi-
nants for agonist binding and activation of the secretin recep-
tor. J Biol Chem 1996; 271: 14944-14949
238 Pang RT, Ng SS, Cheng CH, Holtmann MH, Miller LJ, Chow
BK. Role of N-linked glycosylation on the function and expres-
sion of the human secretin receptor. Endocrinology 1999; 140:
5102-5111
239 Gardner JD, Jensen RT. Regulation of pancreatic enzyme se-
cretion in vitro. In: Schultz SF, editor. Physiology of the gastro-
intestinal tract. Vol. 2. New York: Raven Press, 1981: 831-837
240 Farouk M, Vigna SR, McVey DC, Meyers WC. Localization
and characterization of secretin binding sites expressed by rat
bile duct epithelium. Gastroenterology 1992; 102: 963-968
241 Farouk M, Vigna SR, Haebig JE, Gettys TW, McVey DC, Chari
R, Pruthi RS, Meyers WC. Secretin receptors in a new prepara-
tion of plasma membranes from intrahepatic biliary epithe-
lium. J Surg Res 1993; 54: 1-6
242 Lenzen R, Elster J, Behrend C, Hampel KE, Bechstein WO,
Neuhaus P. Bile acid-independent bile flow is differently
regulated by glucagon and secretin in humans after orthotopic
liver transplantation. Hepatology 1997; 26: 1272-1281
243 Levine RA, Hall RC. Cyclic AMP in secretin choleresis. Evi-
dence for a regulatory role in man and baboons but not in
dogs. Gastroenterology 1976; 70: 537-544
244 McGill JM, Basavappa S, Gettys TW, Fitz JG. Secretin acti-
vates Cl- channels in bile duct epithelial cells through a cAMP-
dependent mechanism. Am J Physiol 1994; 266: G731-G736
245 Alpini G, Ulrich CD 2nd, Phillips JO, Pham LD, Miller LJ,
LaRusso NF. Upregulation of secretin receptor gene expres-
sion in rat cholangiocytes after bile duct ligation. Am J Physiol
1994; 266: G922-G928
246 Tietz PS, Hadac EM, Miller LJ, LaRusso NF. Upregulation of
secretin receptors on cholangiocytes after bile duct ligation.
Regul Pept 2001; 97: 1-6
247 Mutt V. Secretin: isolation, structure and function. In: Glass
GBJ, editor. Gastrointestinal hormones. New York: Raven
Press, 1980: 85-126
248 Hirata K, Nathanson MH. Bile duct epithelia regulate biliary
bicarbonate excretion in normal rat liver. Gastroenterology 2001;
121: 396-406
249 PREISIG R, COOPER HL, WHEELER HO. The relationship
between taurocholate secretion rate and bile production in the
unanesthetized dog during cholinergic blockade and during
secretin administration. J Clin Invest 1962; 41: 1152-1162
250 Alpini G, Glaser S, Francis H, Marzioni M, Venter J, LeSage G.
Bile acid interaction with cholangiocytes. In: Alpini G, Alvaro
D, Marzioni M, LeSage G, LaRusso N, editors. The Patho-
physiology of the Biliary Epithelia. Georgetown, TX: Landes
Bioscience, 2004: 112-126
251 Bijvelds MJ, Jorna H, Verkade HJ, Bot AG, Hofmann F, Agel-
lon LB, Sinaasappel M, de Jonge HR. Activation of CFTR by
ASBT-mediated bile salt absorption. Am J Physiol Gastrointest
Liver Physiol 2005; 289: G870-G879
252 Dumont M, Erlinger S, Uchman S. Hypercholeresis induced
by ursodeoxycholic acid and 7-ketolithocholic acid in the rat:
possible role of bicarbonate transport. Gastroenterology 1980;
79: 82-89
253 Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL,
Steinbach JH. Effect of side-chain shortening on the physi-
ologic properties of bile acids: hepatic transport and effect on
biliary secretion of 23-nor-ursodeoxycholate in rodents. Gas-
troenterology 1986; 90: 837-852
254 Palmer KR, Gurantz D, Hofmann AF, Clayton LM,
Hagey LR, Cecchetti S. Hypercholeresis induced by
norchenodeoxycholate in biliary fistula rodent. Am J Physiol
1987; 252: G219-G228
255 Nathanson MH, Burgstahler AD, Mennone A, Boyer JL.
 Banales JM et al. Involvement of AE2 anion exchanger in biliary bicarbonate excretion
Characterization of cytosolic Ca2+ signaling in rat bile duct
epithelia. Am J Physiol 1996; 271: G86-G96
256 Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C, Fraioli
F, Romeo R, Marucci L, Le Sage G, Glaser SS, Benedetti A. Role
and mechanisms of action of acetylcholine in the regulation of
rat cholangiocyte secretory functions. J Clin Invest 1997; 100:
1349-1362
257 Mann R, Bhathal PS, Bell C. Aminergic innervation of the gall
bladder in man and dog. Clin Auton Res 1991; 1: 205-213
258 Mann R, Bhathal PS, Bell C. Sympathetic innervation of the
liver in man and dog: an immunohistochemical study. Clin
Auton Res 1991; 1: 141-145
259 Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H,
Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino
MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-
stimulated choleresis by increased PKC-gamma expression
and decrease of PKA activity. Am J Physiol Gastrointest Liver
Physiol 2003; 284: G683-G694
260 Kanno N, Lesage G, Phinizy JL, Glaser S, Francis H, Alpini
G. Stimulation of alpha2-adrenergic receptor inhibits
cholangiocarcinoma growth through modulation of Raf-1 and
B-Raf activities. Hepatology 2002; 35: 1329-1340
261 LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L,
Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani
S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic
receptor agonists modulate ductal secretion of BDL rats via
Ca(2+)- and PKC-dependent stimulation of cAMP. Hepatology
2004; 40: 1116-1127
262 Kaminski DL, Deshpande YG. Effect of somatostatin and
bombesin on secretin-stimulated ductular bile flow in dogs.
Gastroenterology 1983; 85: 1239-1247
263 Kortz WJ, Nashold JR, Delong E, Meyers WC. Effects of
bombesin on fasting bile formation. Ann Surg 1986; 203: 1-7
264 Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin
stimulates bicarbonate secretion from rat cholangiocytes:
implications for neural regulation of bile secretion.
Gastroenterology 1997; 113: 311-321
265 Cho WK, Mennone A, Boyer JL. Intracellular pH regulation in
bombesin-stimulated secretion in isolated bile duct units from
rat liver. Am J Physiol 1998; 275: G1028-G1036
266 Cho WK, Boyer JL. Characterization of ion transport
mechanisms involved in bombesin-stimulated biliary secretion
in rat cholangiocytes. J Hepatol 1999; 30: 1045-1051
267 Nyberg B, Einarsson K, Sonnenfeld T. Evidence that
vasoactive intestinal peptide induces ductular secretion of bile
in humans. Gastroenterology 1989; 96: 920-924
268 Nyberg B, Sonnenfeld T, Einarsson K. Vasoactive intestinal
peptide and secretin: effects of combined and separate
intravenous infusions on bile secretion in man. Scand J
Gastroenterol 1991; 26: 109-118
269 Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a
potent regulator of bile secretion from rat cholangiocytes.
Gastroenterology 1999; 117: 420-428
270 Harada E, Niiyama M, Syuto B. Hepatic bile and pancreatic
exocrine secretions evoked by gastrointestinal peptides in
sheep. Comp Biochem Physiol A Comp Physiol 1986; 85: 729-734
271 Makhlouf GM, Yau WM, Zfass AM, Said SI, Bodanszky
M. Comparative effects of synthetic and natural vasoactive
intestinal peptide on pancreatic and biliary secretion and
on glucose and insulin blood levels in the dog. Scand J
Gastroenterol 1978; 13: 759-765
272 Tietz PS, Alpini G, Pham LD, Larusso NF. Somatostatin
inhibits secretin-induced ductal hypercholeresis and exocytosis
by cholangiocytes. Am J Physiol 1995; 269: G110-G118
273 Gong AY, Tietz PS, Muff MA, Splinter PL, Huebert RC,
Strowski MZ, Chen XM, LaRusso NF. Somatostatin stimulates
ductal bile absorption and inhibits ductal bile secretion in mice
3511
via SSTR2 on cholangiocytes. Am J Physiol Cell Physiol 2003;
284: C1205-C1214
274 Glaser SS, Rodgers RE, Phinizy JL, Robertson WE, Lasater J,
Caligiuri A, Tretjak Z, LeSage GD, Alpini G. Gastrin inhibits
secretin-induced ductal secretion by interaction with specific
receptors on rat cholangiocytes. Am J Physiol 1997; 273:
G1061-G1070
275 Lesage GD, Marucci L, Alvaro D, Glaser SS, Benedetti A,
Marzioni M, Patel T, Francis H, Phinizy JL, Alpini G. Insulin
inhibits secretin-induced ductal secretion by activation of
PKC alpha and inhibition of PKA activity. Hepatology 2002; 36:
641-651
276 Kilic G, Doctor RB, Fitz JG. Insulin stimulates membrane
conductance in a liver cell line: evidence for insertion of ion
channels through a phosphoinositide 3-kinase-dependent
mechanism. J Biol Chem 2001; 276: 26762-26768
277 Mabuchi M, Kawamura I, Fushimi M, Takeshita S, Takakura S,
Hirosumi J, Mutoh S. Choleretic actions of insulin-like growth
factor-I, prednisolone, and ursodeoxycholic acid in rats. Dig
Dis Sci 2003; 48: 1398-1405
278 Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A,
Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili
AF, Gaudio E. The intrahepatic biliary epithelium is a target
of the growth hormone/insulin-like growth factor 1 axis. J
Hepatol 2005; 43: 875-883
279 Alvaro D, Gigliozzi A, Marucci L, Alpini G, Barbaro B,
Monterubbianesi R, Minetola L, Mancino MG, Medina
JF, Attili AF, Benedetti A. Corticosteroids modulate the
secretory processes of the rat intrahepatic biliary epithelium.
Gastroenterology 2002; 122: 1058-1069
280 Alvaro D, Gigliozzi A, Piat C, Carli L, Fraioli F, Romeo
R, Francia C, Attili AF, Capocaccia L. Inhibition of biliary
bicarbonate secretion in ethinyl estradiol-induced cholestasis
is not associated with impaired activity of the Cl-/HCO-3
exchanger in the rat. J Hepatol 1997; 26: 146-157
281 Roman RM, Fitz JG. Emerging roles of purinergic signaling in
gastrointestinal epithelial secretion and hepatobiliary function.
Gastroenterology 1999; 116: 964-979
282 Feranchak AP, Fitz JG. Adenosine triphosphate release and
purinergic regulation of cholangiocyte transport. Semin Liver
Dis 2002; 22: 251-262
283 Bucheimer RE, Linden J. Purinergic regulation of epithelial
transport. J Physiol. 2004; 555: 311-321
284 Dranoff JA, Nathanson MH. Its swell to have ATP in the
liver. J Hepatol 2000; 33: 323-325
285 Dranoff JA. Purinergic regulation of bile ductular secretion.
In: Alpini G, Alvaro D, Marzioni M, LeSage G, LaRusso N,
editors. The Pathophysiology of the Biliary Epithelia. George-
town, TX: Landes Bioscience, 2004: 96-104
286 Leipziger J. Control of epithelial transport via luminal P2 re-
ceptors. Am J Physiol Renal Physiol 2003; 284: F419-F432
287 Zsembery A, Spirl C, Granato A, LaRusso NF, Okolicsanyi
L, Crepaldi G, Strazzabosco M. Purinergic regulation of acid/
base transport in human and rat biliary epithelial cell lines.
Hepatology 1998; 28: 914-920
288 Nguyen TD, Meichle S, Kim US, Wong T, Moody MW.
P2Y(11), a purinergic receptor acting via cAMP, mediates se-
cretion by pancreatic duct epithelial cells. Am J Physiol Gastro-
intest Liver Physiol 2001; 280: G795-G804
289 McGill JM, Yen MS, Cummings OW, Alpini G, LeSage G, Pol-
lok KE, Miller B, Engle SK, Stansfield AP. Interleukin-5 inhibi-
tion of biliary cell chloride currents and bile flow. Am J Physiol
Gastrointest Liver Physiol 2001; 280: G738-G745
290 Spirl C, Nathanson MH, Fiorotto R, Duner E, Denson LA,
Sanz JM, Di Virgilio F, Okolicsanyi L, Casagrande F, Strazza-
bosco M. Proinflammatory cytokines inhibit secretion in rat
bile duct epithelium. Gastroenterology 2001; 121: 156-169
S- Editor Pan BR E- Editor Bi L
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TOPIC HIGHLIGHT
Gianfranco D Alpini, PhD, Series Editor
Biliary wound healing, ductular reactions, and IL-6/gp130
signaling in the development of liver disease
A J Demetris, John G Lunz , Susan Specht, Isao Nozaki
A J Demetris, John G Lunz , Susan Specht, The Thomas
E. Starzl Transplantation Institute, Departments of Pathology,
Division of Transplantation, University of Pittsburgh Medical
Center, Pittsburgh, PA 15213, United States
Isao Nozaki, National Shikoku Cancer Center Hospital, Depart-
ment of Surgery, 13 Horinouchi, Matsuyama, 790-0007, Japan
Supported by NIH grant R01 DK49615 to AJD
Correspondence to: A J Demetris, MD, University of Pittsburgh
Medical Center, UPMC-Montefiore E-741, 200 Lothrop Street,
Pittsburgh, PA 15213-2582, United States. demetrisaj@upmc.edu
Telephone: +1-412-6472067 Fax: +1-412-6472084
Received: 2006-01-21 Accepted: 2006-02-18
Abstract
Basic and translational wound healing research in the
biliary tree lag significantly behind similar studies on the
skin and gastrointestinal tract. This is at least partly at-
tributable to lack of easy access to the biliary tract for
study. But clinical relevance, more interest in biliary epi-
thelial cell (BEC) pathophysiology, and widespread avail-
ability of BEC cultures are factors reversing this trend. In
the extra-hepatic biliary tree, ineffectual wound healing,
scarring and stricture development are pressing issues.
In the smallest intra-hepatic bile ducts either impaired
BEC proliferation or an exuberant response can contrib-
ute to liver disease. Chronic inflammation and persistent
wound healing reactions in large and small bile ducts of-
ten lead to liver cancer. General concepts of wound heal-
ing as they apply to the biliary tract, importance of cellu-
lar processes dependent on IL-6/gp130/STAT3 signaling
pathways, unanswered questions, and future directions
are discussed.
2006 The WJG Press. All rights reserved.
Key words: Biliary wound healing; Ductular reactions;
IL-6/gp130 signaling
Demetris AJ, Lunz JG , Specht S, Nozaki I. Biliary wound
healing, ductular reactions, and IL-6/gp130 signaling in the
development of liver disease. World J Gastroenterol 2006;
12(22): 3512-3522
http://www.wjgnet.com/1007-9327/12/3512.asp
www.wjgnet.com
World J Gastroenterol 2006 June 14; 12(22): 3512-3522
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
INTRODUCTION
Wound healing in the biliary tract significantly contributes
to the development of liver disease. For example,
ineffectual wound healing, mural scarring, and stricture
development in the large extra-hepatic bile ducts are
responsible for diseases such as extra-hepatic biliary atresia
and primary sclerosing cholangitis. These are leading
indications for liver transplantation (http://www.optn.
org). A similar problem occurs in 10%-15% of all liver
allografts-the biliary sludge syndrome. Conversely, an
exuberant wound healing response, or ductular reaction,
contributes to the development of cirrhosis from a variety
of causes[1-3].
BEC lining the extra-hepatic and large intra-hepatic
bile ducts have a different embryologic origin and are
distinct phenotypically from BEC lining small intrahepatic
bile ducts. But considerations with respect to wound
healing are similar to each other and to wound healing at
other sites. Unique aspects of the biliary tract that have
the potential to impact significantly on wound healing
include: (1) anatomy and physiology; (2) exposure to high
concentration of bile; and (3) triggering of reactions in
the smallest intra-hepatic bile ducts by insults other than,
or in addition to, direct injury and ulceration. In addition,
chronic inflammation and persistent wound healing
reactions in either the large or small bile ducts often
precede the development of cancers.
In the skin and intestines, mechanisms of wound
repair depend on the depth of injury. Superficial wounds
or simple erosions are healed primarily by a two-
step process: restitution and regeneration. Restitution
begins immediately after creating a superficial wound in
barrier epithelia. Cells near the edge of the defect lose
close contacts with neighboring cells, undergo shape
changes, spread, migrate, and then contract to close the
hole. However, restitution is necessarily limited because
remaining cells can cover only so much of the denuded
surface area. In large wounds, regeneration or proliferation
of the remaining epithelial cells is also needed. Eventually
the epithelium is restored to a nearly original state [4,5],
although surviving cells may carry a legacy of DNA
damage and senescence-related changes[6]. Deeper wounds
involve the epithelia and underlying stroma. Processes such
as angiogenesis; activation, migration, and proliferation
 Demetris AJ et al. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling
of (myo-) fibroblasts and endothelial cells; formation of
granulation tissue; and wound contraction are needed to
close these larger/deeper defects. These more extensive
wounds are also frequently inflamed and, in general,
stromal involvement and inflammation greatly increase the
risk of subsequent scarring[7-10].
Epithelial aspects of wound repair are often studied,
in vitro, by producing linear wound tracks in confluent
epithelial monolayers. The restitution phase is isolated
by treating the cultures with chemical mito-inhibitors to
prevent cell proliferation from contributing to wound
closure. Distances migrated by the epithelial cells from
the edge of the wound at predetermined time points
measure the effectiveness of restitution. We developed a
BEC model using a collagen-matrix substrate to prevent
premature BEC senescence, which occurs routinely when
BEC are plated on plastic or collagen-coated plates, from
interfering with the assay[11].
Front row cells, or those epithelial cells nearest
the defect, experience dissolution of epithelial cell-cell
contacts and changes in cell shape [5,12]. They can also
acquire some mesenchymal characteristics and, under
some circumstances, can undergo complete epithelial-
mesenchymal transition (EMT)[13,14]. Changes in front row
cells and EMT function to disaggregate epithelial units and
reshape the epithelia for movement. Epithelia in transition
lose polarity, adherens junctions, tight and gap junctions,
desmosomes, and down-regulate cytokeratin intermediate
filaments in order to rearrange their F-actin stress fibers
and express filopodia and lamellopodia[5,12-14]. When wound
closure is complete and proliferation has replenished the
lost cells, re-establishment of inter-epithelial junctions
restores the barrier. Coordinating these processes is
critically important for barrier adaptation and wound
healing[13,14].
POTENTIAL INFLUENCES OF BILIARY TREE
PHYSIOLOGY AND ANATOMY ON WOUND
HEALING
The biliary tree can be thought of as a delicate, relatively
complex, self-contained organ that communicates with,
and is enveloped by, the liver (Figure 1). It monitors, alters
the composition of, and triages bile into the intestine.
The tenuous only arterial blood supply can be damaged
easily by diseases and by surgical procedures. A close
relationship and cross-talk between BEC and periductal
(myo-)fibroblasts exists throughout the entire biliary
tree: damage to one population usually results in reactive
changes in the other. For example, periductal (myo-)
fibroblasts often undergo activation and proliferation
in response to significant BEC growth, injury, and bile
leakage from the small[15-21] or large bile ducts [22-24]. In
extra-hepatic and large intra-hepatic bile ducts, this results
in mural stricturing and luminal narrowing. In smaller
intra-hepatic bile ducts, liver fibrosis and/or obliteration
of the bile duct lumen can occur. A rich lymphatic
network envelopes bile ducts that drains into regional hilar
lymph nodes[25,26]. Numerous intramural peribiliary glands
in the extra-hepatic bile ducts can become walled off after
trauma and produce mucoceles. All of these potential
3513
Hepatic artery branch Peri-biliary lymphatics
Peribiliary glands
? Progenitor cells
Peribiliary capillary plexus
Smooth muscle cells
Figure 1 Diagram of the extra-hepatic and large intra-hepatic bile ducts
highlighting some important anatomic and physiologic considerations that can
potentially impact wound healing. Except for the peribiliary glands and some
features of the BEC, small intra-hepatic bile ducts have a similar anatomy. All of
the bile ducts, including the small intra-hepatic bile ducts are supplied only by
the hepatic artery and the peribiliary vascular plexus, shown in red. All bile ducts
also contain either smooth muscle cells and/or facultative myofibroblasts in their
wall. Deep wounds to the biliary tree result in activation and/or transformation of
myofibroblasts that greatly increase the risk of wound contraction, fibrosis, and
stricture formation.
sources of problems contribute to the well-deserved
moniker of the biliary tree as the Achilles heel of liver
transplantation.
Bile contains bile salts that can induce[27-30] or protect
BEC from apoptosis[31], cross-activate EGFR via TGF
ligand binding[32], induce COX-2 expression[33], or trigger
BEC IL-6 and other cytokine production [34]. Bile also
normally contains several growth factors (e.g. HGF),
cytokines (IL-6), and other molecules[35]. Understanding
the effect of various bile constituents on wound healing
(esp. restitution) is critical because bile composition
can be altered therapeutically (e.g. treating patients with
ursodeoxycholic acid) [36,37].
BEC lining the smallest intra-hepatic bile ducts are
derived from hepatoblasts and are thought to contain a
population of liver stem cells that can differentiate into
either hepatocytes or BEC [38-41]. Changes in the intra-
hepatic environment other than, or in addition to, direct
injury and ulceration can trigger wound repair reactions
in these smallest ducts. These ductular reactions are
recognized as BEC and surrounding myofibroblasts
at the interface zone of diseased livers [3,42,43]. Ductular
reactions can be provoked by: (1) local BEC injury and
inflammation[44,45]; (2) increased intra-biliary tract pressure,
and (3) the combination of: (a) a strong liver regenerative
stimulus, such as partial hepatectomy or chronic necro-
inflammatory liver disease and (b) hepatocyte mito-
inhibition because of carcinogen exposure or chronic
oxidative stress [1-3]. Insufficient BEC regeneration in
the smallest ducts leads to liver diseases such as chronic
ductopenic rejection and drug-induced ductopenia[44].
WOUND HEALING IN THE EXTRA-HEPATIC
BILIARY TREE
Importance of arterial blood flow and wound depth
Study of the biliary sludge syndrome in liver allografts
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 3514 ISSN 1007-9327 CN 14-1219/ R
A B
Biliary sludge
Hepatic
artery
Lumen
Inflammed
Biliary epithelium
granulation tissue
Peribiliary
in restitution phase
glands Nearly obliterated lumen
C D
has been particularly illustrative of pathophysiologic
mech a n ism s i nvo l ve d i n b i l i a r y wo u n d h e a l i n g.
This relatively common and particularly frustrating
complication affects about 10% of all liver allografts.
There are many potential causes of ineffectual biliary
wound healing including abnormal anatomy created by
the operation; suboptimal arterial blood flow because of
technical problems, anastomotic narrowing, thrombosis,
or antibody mediated rejection; recurrent ascending
cholangitis; recurrent primary sclerosing cholangitis;
and ischemic-preservation injury [46]. Regardless of the
cause, impediments to bile drainage results in progressive
intrahepatic fibrosis, which in turn, increases morbidity
and decreases organ half-life.
The extra-hepatic biliary tree is sustained only by the
hepatic artery, which drains into three terminal classical
capillary microvascular networks that supply: (1) the bile
ducts (peribiliary plexus), (2) the connective tissue of the
portal tracts, and (3) the hilar and perihilar structures[47].
The allograft biliary tree is especially vulnerable to arterial
ischemia for the first several months after the operation.
Preservation injury damages the microvasculature of the
peribiliary plexus. The transplant operation can injure
the arterial blood supply and it can also disrupt the
normal collateral circulation typical of the arterial cascade
arrangement supplying all gastrointestinal organs, including
the liver[46]. Interference with arterial flow at any level can
result in ischemic cholangitis - a succinct phrase used
to describe the common association between poor arterial
flow and biliary ischemia that manifests as persistent
ulcers, inflammation, sludge, and strictures[48,49].
Cold ischemic-preservation injury depletes energy
stores in microvascular endothelial cells and BEC. This
results in activation of metalloproteinases, detachment
of endothelium and BEC from the underlying matrix,
and in the microvasculature, predisposition to thrombosis
after reperfusion [50,51] . Reperfusion with blood after
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
Figure 2 A: Cross-section of an extra-hepatic bile duct
(outlined by dashed lines) from a liver allograft that failed
because of the biliary sludge syndrome. Note the hepatic
artery branch traveling along the outer wall, the peribiliary
glands, mucosal ulceration, and that the lumen is nearly
obliterated. Ulceration exposes the underlying stroma to
Large intrahepatic
bile ducts
bile, which results in inflammation, granulation tissue, and
myofibroblast activation and proliferation in the underlying
stroma. The inset shows at higher magnification the area
near the arrow; note the bile sludge, BEC in restitution phase,
and stromal inflammation and granulation tissue (arrows); B:
Cross-section of several large intra-hepatic bile ducts from
the same liver. Note that the same processes are occurring
in these ducts. The duct lumen in the lower left is nearly
obliterated by inflamed granulation tissue and myofibroblasts
Ductular (inset); C: The final stages of pathologic wound healing
reaction
in the intra-hepatic ducts can result in complete fibrous
obliteration of the bile duct lumen by concentric rings of
fibrous tissue, as shown here; D: Sections from the periphery
of the same liver show a prominent ductular reaction
consisting of BEC and periductal myofibroblasts (inset). This
occurs because of increased pressure in the biliary tree distal
to the site of luminal obliteration. Notice that the cholangioles
and myofibroblasts form a wedge of tissue that arises from
the portal tract and distorts the liver architecture (outlined on
the right side of image).
transplantation also delivers leukocytes that become
activated by tissue damage. Activated leukocytes release
effector molecules, which in turn, cause more tissue
damage and further promote thrombogenesis [50,51] .
Hydrophobic bile salts remaining in the biliary tree also
further damage marginally viable BECs [50,52] already
weakened by preservation injury. Damaged BEC are
sloughed into the bile[53]. Although patient and allograft
survival during the first several weeks after transplantation
are dependent primarily on parenchymal function, long
term allograft viability is determined primarily by biliary
wound healing and adequate bile drainage[54,55]. Allografts
that eventually fail show biliary sludge, mucosal ulcers, and
inflamed granulation tissue and myofibroblast activation/
proliferation in the wall of extra-hepatic and large intra-
hepatic bile ducts[55] (Figure 2). Exposure of the underlying
stroma to bile appears to serve as a nidus for crystallization
of biliary sludge and a stimulus for inflammation and
activation of myofibroblasts. This leads to wound
contraction and fibrosis, and eventually, to strictures in
large caliber ducts and to luminal obliteration of smaller
caliber ducts (Figure 2C).
Two observations illustrate the critical and primary
importance to wound healing of an adequate arterial blood
supply. First, any insult that directly interferes with the
arterial flow is usually associated with large bile duct ulcers
and strictures. Second, perfusion of the hepatic artery and
peribiliary plexus with low viscosity preservation solutions
before transplantation dramatically decreases the incidence
of biliary complications after transplantation in otherwise
susceptible extended criteria donor livers with long cold
ischemic times [56]. This maneuver is thought to flush
thrombogenic material from the peribiliary plexus and
facilitate reperfusion and oxygenation after transplantation.
As with many clinical observations, confirmation of this
mechanism is lacking. But it is reasonable to conclude that
without sufficient arterial flow biliary wound healing is
 Demetris AJ et al. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling
unlikely to proceed normally. Once adequate arterial flow is
ascertained, other factors that also significantly contribute
to BEC wound repair can then be studied in greater detail.
Deep wounds of extra-hepatic bile ducts precipitate
stromal involvement in wound healing. Granulation tissue
and inflammation, local production of interferon-[57] and
transforming growth factor beta (TGF-)[58] at this site
are of importance in wound contraction and scarring.
Our laboratory has focused primarily on BEC aspects of
wound repair with an emphasis on interleukin-6/gp130
signaling-dependent cellular processes. This signaling
pathway is also critically important for wound healing in
the gastrointestinal tract[59] and skin[60,61].
Cellular and molecular mechanisms of BEC repair
involving IL-6/gp130 signaling
IL-6/gp130 is pleiotropic cytokine signaling system that
has diverse effects in many different organ systems and
cell types(reviewed in [62,63]). gp130 is one of the most
promiscuous cytokine receptors [62], binding to many
different ligands including, interleukin (IL)-6, IL-11,
leukemia inhibitory factor (LIF), oncostatin M (OSM),
ciliary neurotrophic factor (CNTF), cardiotrophin-1
(CT-1), and cardiotrophin-like cytokine (CLC)[62,63]. Ligand
binding to gp130 generates two major signaling pathways:
(1) Jak/STAT3 and (2) SHP2/ERK/MAPK and there is
reciprocal negative feedback regulation between them[63,64].
Non-canonical STAT3 signaling pathways also exist, but
are not well delineated[65].
In normal livers, IL-6 is produced at low levels by the
BEC, perhaps stimulated by the bile salts[34], and secreted
into the bile[66]. Active IL-6/gp130/STAT3 signaling can
be detected in normal IL-6+/+ but not in normal IL-6-/-
mice livers, as evidenced by the detection of phospho-
STAT3 by Western blotting and immunohistochemistry.
Biliary tree pSTAT3 in normal liver localizes to occasional
BEC lining large bile ducts, but more prevalent expression
is seen in BEC lining the peribiliary glands[11].
Virtually any bile duct insult, such as obstruction[67-69],
infection [69,70], or immunologic damage [66,71,72] triggers
sharp increases in IL-6 mRNA and protein production
by BEC and peribiliary hematolymphoid cells[3]. This, in
essence, alerts BEC to environmental stimuli and leads to
subsequent autocrine, paracrine, and juxtacrine gp130/
STAT3 signaling in BEC at the sites of injury[11,67,68,73]. As
in the gastrointestinal tract[59] and skin[60,61], an absence
of IL-6 in IL-6-deficient (IL-6-/-) mice leads to impaired
wound healing[11] and biliary tree integrity[11,73,74]. For the
last several years our laboratory has focused on cellular
and molecular mechanisms that might contribute to
impaired BEC wound healing and biliary barrier defects
in the IL-6-/- mice and how the findings might apply to
humans. Using a combination of knowledge gained from
the gastrointestinal tract and skin, mRNA microarray
expression analyses, and pathophysiologic studies, we
first searched for genes that were: (1) expressed in BECs,
(2) regulated by IL-6/gp130/STAT3 signaling, and (3)
possibly involved in barrier function and/or repair. Two
of the most interesting candidates, studied in greater
detail, included intestinal trefoil family factors (TFF)[59,75-78]
and small protein rich proteins (SPRR)[11,73]. The reader is
3515
referred to recent reviews of IL-6/gp130/ SHP2/ERK/
MAPK signaling and other growth factors and cytokines
involved in the regenerative phase of BEC wound
healing[3,67,68,79-81].
Trefoil family factor (TFF) proteins are comprised of
one or more trefoil motifs, which consist of 6 cysteine
residues. TFF proteins increase mucous viscosity and
thereby contribute to optimal protection of the intestinal
mucosa from injury [77,82-84] . By enhancing intestinal
epithelial spreading and migration, TFF proteins also
stimulate the restitution phase of wound healing [76,85].
Each of the three known TFF proteins is differentially
regulated in the gastrointestinal tract [59,75]: TFF1 and
TFF2 are expressed primarily in the stomach[86,87]. TFF3
predominates in the small and large intestines [78] and
in the biliary tree [11] of mouse and human livers [88-91].
IL-6/gp130/STAT3 signaling is crucial for BEC TFF3
expression. In normal liver, pSTAT3 and TFF3 mRNA
and protein expression are significantly higher in IL-6+/+
than in IL-6-/- mice livers. Constitutive expression of TFF3
and pSTAT3 localizes to mucin secreting BEC lining large
intrahepatic and extrahepatic bile ducts and peribiliary
glands[11]. Medium and small-sized intra-hepatic bile ducts
are generally negative for mucin secreting BEC, pSTAT3,
and TFF3 expression[11,91,92].
IL-6 +/+ BEC consistently show higher levels of
TFF3 mRNA and protein expression and significantly
better migration and wound healing than IL-6-/- BEC[11],
in vitro. Defective migration in the IL-6 -/- BEC can be
partially, but significantly, reversed by treatment with
recombinant TFF peptides [11]. In vivo, biliary TFF3 is
dynamically regulated by various factors after bile duct
ligation. Included are the reciprocal negative regulation
known to exist between the STAT3 and MAPK signaling
pathways[59], and other cytokines and growth factors, such
as HGF and TGF-, which can down-regulated BEC
TFF3 expression [11]. However, a chronic deficiency of
pSTAT3 signaling during bile duct injury, as seen in IL-6-/-
mice after bile duct ligation, leads to a chronic deficiency
of biliary TFF3 expression and impaired biliary barrier
function[11]. In humans, p-STAT3 and TFF3 are newly
co-expressed in BEC involved in florid duct lesions in
primary biliary cirrhosis and at other sites of BEC injury,
but not in similarly-sized normal bile ducts from the same
livers[11,89,92]. This likely constitutes a primitive or innate
mucosal defense system that guards against injury and
stimulates repair.
Our BEC TFF3 studies are consistent with studies
focused on the colon and carried out in mice harboring
mutations that selectively block all gp130-mediated STAT
activity (gp130STAT), but preserve gp130-mediated MAPK
signaling. These mice show decreased colonic TFF3
expression, increased sensitivity to sodium dextran sulfate-
induced colitis, and impaired mucosal wound healing[59,75].
Thus, it is reasonable to conclude that IL-6/gp130/
STAT3 signaling contributes significantly to normal
BEC cytoprotective mechanisms and to migration during
wound healing, at least in part, by stimulating BEC TFF3
expression[11].
Small proline-rich proteins (SPRR) are encoded
by a tandemly arranged four-member gene family
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 3516 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
contained within a 170-kilobase region of the epidermal
differentiation complex (EDC). The EDC is a cluster of
more than 50 genes located on chromosome 1q21[93-95]
whose products are involved in terminal differentiation
of the human epidermis. Included are formation of the
cornified envelope that is an effective barrier against the
external environment [93,96]. The four SPRR gene families,
SPRR1-4, are distinguished on the basis of the number
of amino acids in the repeats of the proteins central
domain and the consensus of that sequence[95]. There are
two Sprr1 genes and one copy each of Sprr3 and Sprr4
genes[95]. SPRR2 genes are the most diversified family:
there are seven in humans and eleven in mice[95]. In the
skin and other squamous epithelia, SPRR genes are usually
regulated coordinately as part of the EDC (i.e. high
expression of most EDC genes, as in papillomas, or very
low expression of most genes, as in newborn skin). SPRR
genes encode for a series of highly homologous proteins
that function primarily as critical cross-linkers. They
form bridges among other EDC proteins, intermediate
filaments, and cornified envelope constituents[94,95,97], such
as desmoplakin, loricrin, and tricohyalin, through the
catalytic action of transglutaminases[98] .
The diverse SPRR2 genes are also non-coordinately
expressed, or expressed preferentially, without similar
upregulation of other EDC family members [73,97]. This
occurs most commonly in non-keratinizing epithelia in
curious situations that cannot be explained by squamous
differentiation or formation of a cornified envelope.
Examples include greater than 100-fold increases of
SPRR2A in the intestine after small bowel resection[99]
or after introduction of commensal bacteria into germ-
free mice [100,101] or after intentional infection with
intestinal parasites [102]. In uterine epithelium SPRR2A
mRNA and protein expression is, at least in part,
regulated by estrogen[103]. Therefore, it is highly and non-
coordinately upregulated during certain stages of the
oestrous cycle[104,105] and is especially high at the blastocyst
implantation site [103]. SPRR2A mRNA and protein are
also expressed in bronchial and intestinal epithelium
during allergic reactions [106]. Barrier remodeling, as a
response to stress [94,105], inflammation, and/or growth,
is a common condition of these diverse circumstances.
Potential molecular and cellular processes affected by
non-coordinate SPRR2A expression are currently under
investigation in our laboratory.
In the liver, we have shown that SPRR2A mRNA and
protein are not expressed in normal mouse liver, but
are non-coordinately upregulated only in BEC after the
stress of bile duct ligation[73]. Expression after bile duct
ligation is not related to squamous metaplasia and shows
strong dependence on IL-6/gp130/STAT3 signaling. In
BEC lining the large bile ducts, SPRR2 protein localizes
subjacent to the apical plasma membrane. SPRR2
expression is more diffusely distributed throughout
the cytoplasm of cholangioles participating in ductular
reactions and in larg e duct BEC eng ag ed in the
restitution phase of mucosal wound healing. Deficient
BEC SPRR2A expression in IL-6-/- mice after bile duct
ligation is associated with impaired barrier function[73].
IL-6 replacement therapy restores SPRR2A expression to
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June 14, 2006 Volume 12 Number 22
levels seen in wild type controls and reverses the barrier
defect in IL-6-/- mice. In a series of ongoing investigations,
preliminary data suggest that BEC SPRR2A expression is
associated with BEC restitution.
WOUND HEALING IN SMALL INTRA-HEPATIC
BILE DUCTULES-DUCTULAR REACTIONS
Wound healing responses can be triggered in BEC lining
the smallest intrahepatic bile ducts by environmental
changes other than, or in addition to, direct injury
and ulceration. This often occurs in chronic necro-
inflammatory liver disease regardless of the underlying
cause. For several years we were puzzled by the observation
that ductular reactions represent a survival advantage for
BEC and myofibroblasts over hepatocytes, yet hepatocytes
and BEC share the same responses to many cytokines and
growth factors that are upregulated in chronic liver disease
(e.g. HGF, EGF, IL-6, TGF, etc.[81,107]). Why then do BEC
and myofibroblasts survive preferentially under these
circumstances? To answer this question it is helpful to view
chronic necro-inflammatory liver disease as a Darwinian
selection pressure applied to the liver. A survival advantage
for BEC can occur because of a relative increase in the
rate of proliferation, a relative decrease in the rate of
death, transformation of hepatocytes into BEC, or various
combinations of the above. Regardless of the mechanism,
the end result is a relative decrease in volume percentage
of hepatocytes and a relative increase in biliary epithelial
cells and myofibroblasts-a pattern typical of evolving
cirrhosis[1,74]. When combined with sufficient time[1,2], even
a small deficit of hepatocyte survival is enough to evoke a
ductular reaction that distorts the hepatic architecture.
Using an established mouse model of decompensated
biliary cirrhosis[74] and p21-deficient mice, we tested the
hypothesis that hepatocyte mito-inhibition combined
with the regenerative stimulus of bile duct ligation
would accentuate the ductular reaction and accelerate
architectural distortion. Results showed that after long-
term (12-wk) ligation mice prone to decompensation show
significantly more oxidative stress and hepatocyte nuclear
p21 expression, a cyclin dependent kinase inhibitor and
important mediator of hepatocyte mito-inhibition[1]. As
expected, mice prone to decompensation also showed less
hepatocyte proliferation, an exaggerated ductular reaction,
and accelerated architectural distortion compared with
compensation-prone controls[1]. We next subjected p21
deficient mice to bile duct ligation for 12 wk with the
expectation that p21 deficient mice would be better able
than wild-type controls to compensate for long-term BDL
because of significantly greater hepatocyte proliferation.
Indeed, results of these experiments showed that p21-
deficient mice showed a larger liver mass because of more
hepatocyte proliferation, a less florid ductular reaction, and
less architectural distortion than wild type controls[1].
We next wanted to determine whether this concept was
applicable to other, non-cholestatic or non-biliary, liver
diseases. To accomplish this task, we first showed that
hepatocyte nuclear p21 expression in humans awaiting liver
replacement directly correlated with pathological disease
stage and model of end-stage liver disease scoring[1]. We
 Demetris AJ et al. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling
also engaged in a collaborative study with Clouston et al
who had previously shown that HCV-related liver disease
progresses more rapidly when there is co-existent hepatic
steatosis[108]. Liver biopsies from 115 patients with HCV
scored for steatosis, inflammation, and fibrosis showed
a strong correlation between (a) a ductular reaction and
portal fibrosis and (b) steatosis and impaired hepatocyte
replication[2]. Steatosis correlated with the ductular reaction
and greater hepatic progenitor cell proliferation, but was
not an obligate feature. The highly significant correlation
between the ductular reaction area and fibrosis stage
remained even after multivariate analysis [2]. Impaired
hepatocyte replication, as determined by p21 expression,
was independently associated with hepatic progenitor
cell expansion, increased body mass index, and lobular
inflammation.
The observation that ductular reactions often appear
when hepatocyte mito-inhibition is combined with
a liver regenerative stimulus is not new. It was made
originally years ago while treating experimental animals
with genotoxic carcinogens and then subjecting them to
partial hepatectomy (reviewed in[3,109,110]). These maneuvers
stimulate oval cell, or liver epithelial progenitor cell[111],
expansion/proliferation at the interface zone-a ductular
reaction. However, in carcinogenesis experiments, the
ductular reaction eventually resolves without fibrosis.
Our studies show that this concept is applicable
to ductular reactions associated with chronic necro-
inflammatory liver diseases and the development of
fibrosis/cirrhosis [1] (Figure 3). Hepatocytes are more
susceptible to injury and mito-inhibition during chronic
necro-inflammatory liver disease because they: (1) produce
and secrete bile and are the major site of bile stasis; (2)
are more complex metabolically and able preferentially
to store lipids and metals, such as iron and copper, which
are generators and/or catalysts for free oxygen radical
formation; and, (3) support HBV and HCV replication,
and (4) maybe most importantly, contain many more
mitochondria than BEC and mitochondria are the major
site of superoxide production[112]. Diverse disorders such
as cholestasis [1], HCV replication [113,114], steatosis [2,115],
copper deposition[116], and alcohol[117] preferentially stress
or injure hepatocytes and this causes nuclear expression
of the cyclin-dependent kinase inhibitor, p21[1,2], which in
turn, inhibits hepatocyte proliferation.
When small intrahepatic bile ductules are destroyed
(ductopenia), due to drugs or chronic allograft rejection,
classical cirrhosis usually does not develop [45,109,118,119].
Despite ongoing immunologic liver injury and fibrosis,
regenerative nodularity and the associated complications
of portal hypertension rarely occur. BEC survival and
proliferation in response to injury in these small ductules
is related to a combination of immunologic injury,
environmental influences, and importantly, arterial blood
flow[120], as in the large bile ducts. Perhaps the arterial disease
also inhibits regenerative nodule formation (Figure 4).
Progenitor Cell Activation, Persis-
tent Wound Repair and Cancer
Persistent ductular reactions in human chronic necro-
3517
= oxidative stress
p21 up-regulation B
Cholangioles
? Progenitor cells
Bile ducts
A
Portal
vein
Figure 3 This diagram illustrates the peripheral aspects of the biliary tree,
including the portal tract and periportal hepatic parenchyma in normal (A: bottom
of figure) and in diseased livers (B: top of figure). A: Rare liver progenitor, or stem
cells, are thought to reside within the Canals of Herring, or the portion of the
bile ductules that connects BECs to hepatocytes. The same close relationship
between BEC, the arterial supply, and periductal myofibroblasts seen in the large
ducts continues to the biliary tree periphery, as shown here. B: During chronic
necro-inflammatory liver diseases a variety of insults, such as cholestasis[1], HCV
replication[114,115], steatosis[2,116] copper deposition[117], and alcohol[118] can cause
oxidative stress, preferentially in hepatocytes. The stress upregulates hepatocyte
nuclear p21 expression (illustrated by solid nuclei at top of diagram), which in
turn, inhibits hepatocytes proliferation and accentuates ductular reactions[1,2]. A
close relationship between BEC and periductal myofibroblasts in the smallest
bile ductules usually results in co-activation of these populations recognized as
ductular reactions, which often precede the development of cirrhosis.
inflammatory diseases can activate progenitor cell popu-
lations, as in the experimental animal carcinogenesis
models, discussed above (Reviewed in refs 38, 39, 41).
Several groups, including ours, have shown that oval cell
expansion in mice is dependent significantly on IL-6/
gp130/STAT3 signaling[121,122]. This raises the possibility
that ductular reactions accelerate the development of
cirrhosis and potentially increase the risk of hepatocellular
carcinoma. Since oval cells eventually differentiate into
hepatocytes [111], exposure to carcinogens or genotoxic
damage from oxidative stress imprint genetic mutations
in putative liver stem cells. These cells then divide,
differentiate, and spread initiated cells more widely
throughout the entire hepatocyte population. Liver cancers
occur when initiated hepatocytes are subjected to tumor
promoters that generally cause hepatocyte proliferation.
Chronic liver disease is an excellent cancer-promoting
environment.
In the skin, STAT3 signaling enables epidermal stem
cells to the escape apoptosis induced by exposure to
cutaneous carcinogens [123]. Initiated stem cells survive,
divide, differentiate, and subsequently give rise to skin
cancers in a promoting environment[123]. Similar processes
might occur in hepatocellular and cholangiocarcinomas.
Interleukin-6/gp130/STAT3 signaling might provide
important survival signals for initiated liver epithelial
progenitor cells that later give rise to liver cancers in
the context of chronic necro-inflammatory disease.
In evolutionary biology [124] this process is referred as
antagonistic pleiotropy - a short term survival benefit at
the expense of long-term increased risk of cancer.
IL-6/gp130/STAT3 signaling is indeed increased in a
very wide variety of neoplasms, including hepatocellular
carcinomas, melanomas, leukemias and myelomas, and
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 3518 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
lung, breast cancer, kidney, prostate, pancreatic, colon,
gastric, cervical, ovarian, and head and neck cancers[125,126].
This signaling pathway participates and/or regulates many
pathways important in oncogenesis including cell-cycle
progression, apoptosis, tumor angiogenesis, tumor-cell
invasion and metastasis, and tumor-cell evasion of the
immune system (reviewed in[125,126]).
Whether this concept is applicable to cancers arising in
extrahepatic and large intrahepatic bile ducts is uncertain
because the mechanisms of BEC renewal at these sites
have not been studied in any great detail. In the larger bile
ducts there are two potential sources of new BEC: (1)
proliferation of mature BEC; and (2) proliferation and/or
maturation of progenitor cell populations. These potential
sources are not mutually exclusive and only one study in
the literature even indirectly addresses this topic. Koike
et al[127] used pulse and continuous DNA labeling studies
to show, in rats, that a proliferative zone, consistent with
a BEC progenitor cell population, localized to peribiliary
glands (called crypts in their study). Long-term follow-
up of the animals showed that labeled BEC migrated
gradually to the surface and were shed into the lumen
with a transit time of about 30-40 d[127]. They concluded
the arrangement and pattern of BEC renewal in the
extra-hepatic bile ducts was similar to the intestine and
colon, but kinetics of BEC turnover was slower[127]. More
definitive work is needed in this area.
UNANSWERED QUESTIONS AND FUTURE
DIRECTIONS
The following are a few examples of the many unanswered
questions in the study of biliary wound healing that have
stimulated research in our laboratory. For example, are
there progenitor cell populations within the peribiliary
glands or crypts of extra-hepatic and large intra-hepatic
bile ducts? If progenitor cells exist in the extra-hepatic
biliary tree, where exactly do they reside, and how are
they recognized? Are they activated during wound repair,
and do they contribute to the development of bile duct
cancers?
EMT contributes significantly to wound healing and
to kidney fibrogenesis. Is EMT an important process in
BEC wound healing and hepatic fibrogenesis? Can, and
do, BEC transform into myo-(fibroblasts)? BEC appear
to migrate and can acquire mesenchymal characteristics
during the ductular reaction and hepatic fibrogenesis.
They transform from polarized cuboidal epithelial cells
into a spindle or dendritic-shaped vimentin-positive cells
after bile duct ligation and in PBC[128,129]. In embryonic
liver, ductal plate BEC invade or migrate into the portal
tract connective tissue to form mature intrahepatic bile
ducts. During migration BEC express vimentin [130] and
down-regulate membranous E-cadherin [131] expression.
Once mature bile ducts are formed BEC revert totally
to an epithelial phenotype. Preliminary studies from our
laboratory suggest that IL-6/gp130/STAT3 signaling
triggers BEC changes under some circumstances that can
induce BEC EMT. The extent to which EMT contributes
to BEC wound healing, however, requires further study.
Several studies show that IL-6 pre-treatment can prevent
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June 14, 2006 Volume 12 Number 22
Ductular reactions
BEC proliferation
Progenitor cell activation/differentiation
Ductular metaplasia of hepatocytes
(Myo-) fibroblasts activation/proliferation
Fibrous oblieration
of ducts
Inadequate BEC restitution or regeneration
Persistent ulcer
Sludge and stone formation
? Activation of progenitor cells
Stromal involvement
Deep wounds with stromal involvement
Inflammation
Myo-fibroblast activation and proliferation
Angiogenesis
Wound contraction and stricture formation
Figure 4 Summary of the most common wound repair reactions that contribute
to the development of chronic liver disease and liver cancers. Although all of the
processes described in the text can occur at any site in the biliary tree, certain types
of responses are more common at certain sites. For example, in the extra-hepatic
and large intra-hepatic bile ducts (lower right), inadequate or ineffectual epithelial
restitution or proliferation results in persistent ulcers and stone and sludge formation.
This, in turn, leads to possible activation of progenitor cells and stromal involvement,
which can also be triggered by deep wounding of the large ducts. Stromal
involvement is usually accompanied by inflammation, angiogenesis, and activation
and proliferation of myofibroblasts. All of these processes contribute to wound
contraction and stricture formation, which is one of the most common and clinically
significant problems in the large ducts.
In the small bile ducts, ductular reactions are the most commonly encountered
wound healing response. Ductular reactions can occur because of BEC
proliferation, progenitor cell activation and differentiation, and ductular metaplasia
of hepatocytes. They also usually trigger myofibroblast activation and proliferation
and together these cell populations form a tissue wedge at the interface zone that
progressively distorts the liver architecture.
In medium-sized bile ducts, fibrous obliteration is probably the most common
serious wound healing response. This occurs because the response to injury of
the periductal myofibroblasts overwhelms the BEC response and the duct lumen is
obliterated.
liver parenchymal ischemic-preservation injury [132-136].
We have shown similar results in intestinal allografts:
IL-6 pretreatment limits epithelial damage and promotes
repair[137]. Would pretreatment of donor livers with IL-6,
particularly in the aortic flush, have beneficial effects on
preservation injury of the peribiliary plexus and promote
wound healing in the biliary tree? We would expect IL-6/
gp130 signaling to help lessen the incidence of biliary
strictures because it upregulates anti-apoptotic molecules
in the microvascular endothelium[133,136], preserves epithelial
integrity [11,137], and stimulates BEC restitution [11,73] and
regeneration[79] after injury.
Several other strategies might be used to prevent biliary
strictures[138,139], such as reducing eosinophil and mast cell
accumulation with Tranilast [140,141] and Captopril [142-144],
reducing activation and transformation of myofibroblasts
with anti-oxidants such alpha tocopherol (vitamin E) and
peroxisome proliferator-activated receptors- (PPAR-)
ligands, such as the thiazolidinedione family of drugs[145-147].
REFERENCES
1 Lunz JG 3rd, Tsuji H, Nozaki I, Murase N, Demetris AJ.
An inhibitor of cyclin-dependent kinase, stress-induced
p21Waf-1/Cip-1, mediates hepatocyte mito-inhibition during
the evolution of cirrhosis. Hepatology 2005; 41: 1262-1271
 Demetris AJ et al. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling
2 Clouston AD, Powell EE, Walsh MJ, Richardson MM,
Demetris AJ, Jonsson JR. Fibrosis correlates with a ductular
reaction in hepatitis C: roles of impaired replication,
progenitor cells and steatosis. Hepatology 2005; 41: 809-818
3 Demetris AJ, Lunz JG, Subbotin V, Wu T, Nozaki I, Contrucci S,
Yin X. Participation of cytokines and growth factors in biliary
epithelial proliferation and mito-inhibition during ductular
reactions. In: Alpini G.,et al, editor. The pathophysiology
of biliary epithelia. Georgetown, TX: Landes Bioscie, 2003:
167-191
4 Taupin D, Podolsky DK. Trefoil factors: initiators of mucosal
healing. Nat Rev Mol Cell Biol 2003; 4: 721-732
5 Jacinto A, Martinez-Arias A, Martin P. Mechanisms of
epithelial fusion and repair. Nat Cell Biol 2001; 3: E117-E123
6 Poli G, Parola M. Oxidative damage and fibrogenesis. Free
Radic Biol Med 1997; 22: 287-305
7 Redd MJ, Cooper L, Wood W, Stramer B, Martin P. Wound
healing and inflammation: embryos reveal the way to perfect
repair. Philos Trans R Soc Lond B Biol Sci 2004; 359: 777-784
8 Mammen JM, Matthews JB. Mucosal repair in the
gastrointestinal tract. Crit Care Med 2003; 31: S532-S537
9 Carlson MA, Longaker MT. The fibroblast-populated collagen
matrix as a model of wound healing: a review of the evidence.
Wound Repair Regen 2004; 12: 134-147
10 Hinz B, Gabbiani G. Cell-matrix and cell-cell contacts of
myofibroblasts: role in connective tissue remodeling. Thromb
Haemost 2003; 90: 993-1002
11 Nozaki I, Lunz JG 3rd, Specht S, Park JI, Giraud AS, Murase N,
Demetris AJ. Regulation and function of trefoil factor family 3
expression in the biliary tree. Am J Pathol 2004; 165: 1907-1920
12 Jacinto A, Martin P. Morphogenesis: unravelling the cell
biology of hole closure. Curr Biol 2001; 11: R705-R707
13 Kalluri R, Neilson EG. Epithelial-mesenchymal transition and
its implications for fibrosis. J Clin Invest 2003; 112: 1776-1784
14 Boyer B, Valls AM, Edme N. Induction and regulation of
epithelial-mesenchymal transitions. Biochem Pharmacol 2000;
60: 1091-1099
15 Miyazaki H, Van Eyken P, Roskams T, De Vos R, Desmet VJ.
Transient expression of tenascin in experimentally induced
cholestatic fibrosis in rat liver: an immunohistochemical study.
J Hepatol 1993; 19: 353-366
16 Tang L, Tanaka Y, Marumo F, Sato C. Phenotypic change in
portal fibroblasts in biliary fibrosis. Liver 1994; 14: 76-82
17 Libbrecht L, Cassiman D, Desmet V, Roskams T. The
correlation between portal myofibroblasts and development
of intrahepatic bile ducts and arterial branches in human liver.
Liver 2002; 22: 252-258
18 Kinnman N, Francoz C, Barbu V, Wendum D, Rey C,
Hultcrantz R, Poupon R, Housset C. The myofibroblastic
conversion of peribiliary fibrogenic cells distinct from hepatic
stellate cells is stimulated by platelet-derived growth factor
during liver fibrogenesis. Lab Invest 2003; 83: 163-173
19 Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg
H, Krause R, Lammert F, Langner C, Zatloukal K, Marschall
HU, Denk H, Trauner M. Regurgitation of bile acids from
leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4)
knockout mice. Gastroenterology 2004; 127: 261-274
20 Ramadori G, Saile B. Portal tract fibrogenesis in the liver. Lab
Invest 2004; 84: 153-159
21 Jhandier MN, Kruglov EA, Lavoie EG, Svigny J, Dranoff
JA. Portal fibroblasts regulate the proliferation of bile duct
epithelia via expression of NTPDase2. J Biol Chem 2005; 280:
22986-22992
22 Sobesky R, Chollet JM, Prat F, Karkouche B, Pelletier G, Frit-
sch J, Choury AD, Allonier C, Bedossa P, Buffet C. Inflamma-
tory pseudotumor of the common bile duct. Endoscopy 2003;
35: 698-700
23 Dehner LP, Coffin CM. Idiopathic fibrosclerotic disorders and
other inflammatory pseudotumors. Semin Diagn Pathol 1998;
15: 161-173
24 Inaba K, Suzuki S, Yokoi Y, Ota S, Nakamura T, Konno H,
Baba S, Takehara Y, Nakamura S. Hepatic inflammatory pseu-
dotumor mimicking intrahepatic cholangiocarcinoma: report
3519
of a case. Surg Today 2003; 33: 714-717
25 Yamamoto K, Phillips MJ. Three-dimensional observation of
the intrahepatic lymphatics by scanning electron microscopy
of corrosion casts. Anat Rec 1986; 214: 67-70
26 Szab G, Magyar Z, Szentirmai A, Jakab F, Mihaly K. Bile
constituents in blood and lymph during biliary obstruction. II.
The absorption and transport of bile acids and bilirubin. Lym-
phology 1975; 8: 36-42
27 Tsuboi K, Tazuma S, Nishioka T, Chayama K. Partial charac-
terization of cytoprotective mechanisms of lecithin against bile
salt-induced bile duct damage. J Gastroenterol 2004; 39: 955-960
28 Barone M, Maiorano E, Ladisa R, Pece A, Berloco P, Strazza-
bosco M, Caruso ML, Valentini AM, Ierardi E, Di Leo A, Fran-
cavilla A. Ursodeoxycholate further increases bile-duct cell
proliferative response induced by partial bile-duct ligation in
rats. Virchows Arch 2004; 444: 554-560
29 Patel T, LaRusso NF, Gores GJ. Interleukin-6 suppresses chol-
angiocyte apoptosis by down-regulation of Bax. Hepatology
1997; 26: 226A
30 Que FG, Gores GJ, LaRusso NF. Development and initial ap-
plication of an in vitro model of apoptosis in rodent cholan-
giocytes. Am J Physiol 1997; 272: G106-G115
31 Komichi D, Tazuma S, Nishioka T, Hyogo H, Une M, Chaya-
ma K. Unique inhibition of bile salt-induced apoptosis by
lecithins and cytoprotective bile salts in immortalized mouse
cholangiocytes. Dig Dis Sci 2003; 48: 2315-2322
32 Werneburg NW, Yoon JH, Higuchi H, Gores GJ. Bile acids ac-
tivate EGF receptor via a TGF-alpha-dependent mechanism in
human cholangiocyte cell lines. Am J Physiol Gastrointest Liver
Physiol 2003; 285: G31-G36
33 Yoon JH, Higuchi H, Werneburg NW, Kaufmann SH, Gores
GJ. Bile acids induce cyclooxygenase-2 expression via the epi-
dermal growth factor receptor in a human cholangiocarcinoma
cell line. Gastroenterology 2002; 122: 985-993
34 Lamireau T, Zoltowska M, Levy E, Yousef I, Rosenbaum J,
Tuchweber B, Desmoulire A. Effects of bile acids on biliary
epithelial cells: proliferation, cytotoxicity, and cytokine secre-
tion. Life Sci 2003; 72: 1401-1411
35 Kristiansen TZ, Bunkenborg J, Gronborg M, Molina H, Thu-
luvath PJ, Argani P, Goggins MG, Maitra A, Pandey A. A
proteomic analysis of human bile. Mol Cell Proteomics 2004; 3:
715-728
36 Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic
liver disease: mechanisms of action and therapeutic use revis-
ited. Hepatology 2002; 36: 525-531
37 Colombo C, Crosignani A, Assaisso M, Battezzati PM, Podda
M, Giunta A, Zimmer-Nechemias L, Setchell KD. Ursodeoxy-
cholic acid therapy in cystic fibrosis-associated liver disease: a
dose-response study. Hepatology 1992; 16: 924-930
38 Strain AJ, Crosby HA, Nijjar S, Kelly DA, Hubscher SG. Hu-
man liver-derived stem cells. Semin Liver Dis 2003; 23: 373-384
39 Roskams TA, Libbrecht L, Desmet VJ. Progenitor cells in dis-
eased human liver. Semin Liver Dis 2003; 23: 385-396
40 Saxena R, Theise N. Canals of Hering: recent insights and cur-
rent knowledge. Semin Liver Dis 2004; 24: 43-48
41 Thorgeirsson SS, Grisham JW. Overview of recent experimental
studies on liver stem cells. Semin Liver Dis 2003; 23: 303-312
42 POPPER H, KENT G, STEIN R. Ductular cell reaction in the
liver in hepatic injury. J Mt Sinai Hosp N Y 1957; 24: 551-556
43 Demetris AJ, Sakamoto T, Liu Z, Yokomuro S, Ezure T,
Murase N, et al. The ductular reaction in liver disease
emphasis on a type I response. In: Fleig WE, editors. Normal
and Malignant Liver Cell Growth. Dordecht: Kluwer
Academic Publishers, 1999: 141155
44 Lunz JG 3rd, Contrucci S, Ruppert K, Murase N, Fung JJ,
Starzl TE, Demetris AJ. Replicative senescence of biliary
epithelial cells precedes bile duct loss in chronic liver allograft
rejection: increased expression of p21(WAF1/Cip1) as a
disease marker and the influence of immunosuppressive
drugs. Am J Pathol 2001; 158: 1379-1390
45 Blakolmer K, Seaberg EC, Batts K, Ferrell L, Markin R,
Wiesner R, Detre K, Demetris A. Analysis of the reversibility
of chronic liver allograft rejection implications for a staging
www.wjgnet.com
 3520 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
schema. Am J Surg Pathol 1999; 23: 1328-1339
46 Demetris A, Crawford J, Nalesnik M, Randhawa P, Wu T,
Minervini M. Transplantation Pathology of the Liver. In: Odze
RD, Goldblum JR, Crawford JM, editors. Surgical Pathology of
the GI Tract, Liver, Biliary Tract, and Pancreas. Philadelphia:
WB Saunders, 2004: 909-966
47 Wanless I. Physioanatomic Considerations. In: Schiff ER,
Sorrell MF, Maddrey WC, editors. Diseases of the Liver.
Philadelphia: Lippincott Williams & Wilkins, 1999: 3-37
48 Demetris AJ. Ischemic cholangitis. Mayo Clin Proc 1992; 67:
601-602
49 Ludwig J, Batts KP, MacCarty RL. Ischemic cholangitis in
hepatic allografts. Mayo Clin Proc 1992; 67: 519-426
50 Kukan M, Haddad PS. Role of hepatocytes and bile duct cells
in preservation-reperfusion injury of liver grafts. Liver Transpl
2001; 7: 381-400
51 Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury:
pathogenic mechanisms and basis for hepatoprotection. J
Gastroenterol Hepatol 2003; 18: 891-902
52 Noack K, Bronk SF, Kato A, Gores GJ. The greater vulnera-
bility of bile duct cells to reoxygenation injury than to anoxia.
Implications for the pathogenesis of biliary strictures after
liver transplantation. Transplantation 1993; 56: 495-500
53 Carrasco L, Sanchez-Bueno F, Sola J, Ruiz JM, Ramirez P,
Robles R, Rodriquez JM, Parrilla P. Effects of cold ischemia
time on the graft after orthotopic liver transplantation. A bile
cytological study. Transplantation 1996; 61: 393-396
54 Busuttil RW, Tanaka K. The utility of marginal donors in liver
transplantation. Liver Transpl 2003; 9: 651-663
55 Demetris AJ, Fontes P, Lunz JG, Specht S, Murase N, Marcos
A. Wound healing in the biliary tree of liver allografts. Cell
Transplant 2006; 15 Suppl 1: S57-S65
56 Pirenne J, Gunson B, Khaleef H, Hubscher S, Afford S,
McMaster P, Adams D. Influence of ischemia-reperfusion
injury on rejection after liver transplantation. Transplant Proc
1997; 29: 366-367
57 Shivakumar P, Campbell KM, Sabla GE, Miethke A, Tiao
G, McNeal MM, Ward RL, Bezerra JA. Obstruction of
extrahepatic bile ducts by lymphocytes is regulated by IFN-
gamma in experimental biliary atresia. J Clin Invest 2004; 114:
322-329
58 Geng ZM, Yao YM, Liu QG, Niu XJ, Liu XG. Mechanism of
benign biliary stricture: a morphological and immunohisto-
chemical study. World J Gastroenterol 2005; 11: 293-295
59 Tebbutt NC, Giraud AS, Inglese M, Jenkins B, Waring P, Clay
FJ, Malki S, Alderman BM, Grail D, Hollande F, Heath JK,
Ernst M. Reciprocal regulation of gastrointestinal homeostasis
by SHP2 and STAT-mediated trefoil gene activation in gp130
mutant mice. Nat Med 2002; 8: 1089-1097
60 Lin ZQ, Kondo T, Ishida Y, Takayasu T, Mukaida N. Essential
involvement of IL-6 in the skin wound-healing process as
evidenced by delayed wound healing in IL-6-deficient mice. J
Leukoc Biol 2003; 73: 713-721
61 Gallucci RM, Simeonova PP, Matheson JM, Kommineni C,
Guriel JL, Sugawara T, Luster MI. Impaired cutaneous wound
healing in interleukin-6-deficient and immunosuppressed
mice. FASEB J 2000; 14: 2525-2531
62 Mller-Newen G. The cytokine receptor gp130: faithfully pro-
miscuous. Sci STKE 2003; 2003: PE40
63 Kamimura D, Ishihara K, Hirano T. IL-6 signal transduction
and its physiological roles: the signal orchestration model. Rev
Physiol Biochem Pharmacol 2003; 149: 1-38
64 Ishihara K, Hirano T. Molecular basis of the cell specificity of
cytokine action. Biochim Biophys Acta 2002; 1592: 281-296
65 Inghirami G, Chiarle R, Simmons WJ, Piva R, Schlessinger
K, Levy DE. New and old functions of STAT3: a pivotal tar-
get for individualized treatment of cancer. Cell Cycle 2005; 4:
1131-1133
66 Yasoshima M, Kono N, Sugawara H, Katayanagi K, Harada K,
Nakanuma Y. Increased expression of interleukin-6 and tumor
necrosis factor-alpha in pathologic biliary epithelial cells: in
situ and culture study. Lab Invest 1998; 78: 89-100
67 Liu Z, Sakamoto T, Ezure T, Yokomuro S, Murase N, Michalo-
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
poulos G, Demetris AJ. Interleukin-6, hepatocyte growth fac-
tor, and their receptors in biliary epithelial cells during a type
I ductular reaction in mice: interactions between the periductal
inflammatory and stromal cells and the biliary epithelium.
Hepatology 1998; 28: 1260-1268
68 Liu Z, Sakamoto T, Yokomuro S, Ezure T, Subbotin V, Murase
N, Contrucci S, Demetris AJ. Acute obstructive cholangiopa-
thy in interleukin-6 deficient mice: compensation by leukemia
inhibitory factor (LIF) suggests importance of gp-130 signaling
in the ductular reaction. Liver 2000; 20: 114-124
69 Rosen HR, Winkle PJ, Kendall BJ, Diehl DL. Biliary interleu-
kin-6 and tumor necrosis factor-alpha in patients undergoing
endoscopic retrograde cholangiopancreatography. Dig Dis Sci
1997; 42: 1290-1294
70 Scotte M, Daveau M, Hiron M, Delers F, Lemeland JF, Teniere
P, Lebreton JP. Interleukin-6 (IL-6) and acute-phase proteins in
rats with biliary sepsis. Eur Cytokine Netw 1991; 2: 177-182
71 Kimmings AN, van Deventer SJ, Obertop H, Rauws EA,
Huibregtse K, Gouma DJ. Endotoxin, cytokines, and endotoxin
binding proteins in obstructive jaundice and after preopera-
tive biliary drainage. Gut 2000; 46: 725-731
72 Akiyama T, Hasegawa T, Sejima T, Sahara H, Seto K, Saito H,
Takashima S. Serum and bile interleukin 6 after percutaneous
transhepatic cholangio-drainage. Hepatogastroenterology 1998;
45: 665-671
73 Nozaki I, Lunz JG 3rd, Specht S, Stolz DB, Taguchi K, Sub-
botin VM, Murase N, Demetris AJ. Small proline-rich proteins
2 are noncoordinately upregulated by IL-6/STAT3 signaling
after bile duct ligation. Lab Invest 2005; 85: 109-123
74 Ezure T, Sakamoto T, Tsuji H, Lunz JG 3rd, Murase N, Fung
JJ, Demetris AJ. The development and compensation of biliary
cirrhosis in interleukin-6- deficient mice. Am J Pathol 2000; 156:
1627-1639
75 Ernst M, Inglese M, Waring P, Campbell IK, Bao S, Clay FJ,
Alexander WS, Wicks IP, Tarlinton DM, Novak U, Heath JK,
Dunn AR. Defective gp130-mediated signal transducer and
activator of transcription (STAT) signaling results in degen-
erative joint disease, gastrointestinal ulceration, and failure of
uterine implantation. J Exp Med 2001; 194: 189-203
76 Kanai M, Mullen C, Podolsky DK. Intestinal trefoil factor in-
duces inactivation of extracellular signal-regulated protein ki-
nase in intestinal epithelial cells. Proc Natl Acad Sci U S A 1998;
95: 178-182
77 Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired
defense of intestinal mucosa in mice lacking intestinal trefoil
factor. Science 1996; 274: 262-265
78 Podolsky DK, Lynch-Devaney K, Stow JL, Oates P, Murgue
B, DeBeaumont M, Sands BE, Mahida YR. Identification of hu-
man intestinal trefoil factor. Goblet cell-specific expression of
a peptide targeted for apical secretion. J Biol Chem 1993; 268:
6694-6702
79 Matsumoto K, Fujii H, Michalopoulos G, Fung JJ, Demetris AJ.
Human biliary epithelial cells secrete and respond to cytokines
and hepatocyte growth factors in vitro: interleukin-6,
hepatocyte growth factor and epidermal growth factor
promote DNA synthesis in vitro. Hepatology 1994; 20: 376-382
80 Yokomuro S, Lunz JG 3rd, Sakamoto T, Ezure T, Murase N,
Demetris AJ. The effect of interleukin-6 (IL-6)/gp130 signal-
ling on biliary epithelial cell growth, in vitro. Cytokine 2000; 12:
727-730
81 Yokomuro S, Tsuji H, Lunz JG 3rd, Sakamoto T, Ezure T,
Murase N, Demetris AJ. Growth control of human biliary epi-
thelial cells by interleukin 6, hepatocyte growth factor, trans-
forming growth factor beta1, and activin A: comparison of a
cholangiocarcinoma cell line with primary cultures of non-
neoplastic biliary epithelial cells. Hepatology 2000; 32: 26-35
82 Tomasetto C, Masson R, Linares JL, Wendling C, Lefebvre O,
Chenard MP, Rio MC. pS2/TFF1 interacts directly with the
VWFC cysteine-rich domains of mucins. Gastroenterology 2000;
118: 70-80
83 Gum JR Jr, Hicks JW, Gillespie AM, Carlson EJ, Kmves L,
Karnik S, Hong JC, Epstein CJ, Kim YS. Goblet cell-specific ex-
pression mediated by the MUC2 mucin gene promoter in the
 Demetris AJ et al. Biliary wound healing, ductular reactions, and IL-6/gp130 signaling
intestine of transgenic mice. Am J Physiol 1999; 276: G666-G676
84 Kindon H, Pothoulakis C, Thim L, Lynch-Devaney K, Podol-
sky DK. Trefoil peptide protection of intestinal epithelial bar-
rier function: cooperative interaction with mucin glycoprotein.
Gastroenterology 1995; 109: 516-523
85 Dignass A, Lynch-Devaney K, Kindon H, Thim L, Podolsky
DK. Trefoil peptides promote epithelial migration through a
transforming growth factor beta-independent pathway. J Clin
Invest 1994; 94: 376-383
86 Tomasetto C, Rio MC, Gautier C, Wolf C, Hareuveni M,
Chambon P, Lathe R. hSP, the domain-duplicated homolog
of pS2 protein, is co-expressed with pS2 in stomach but not in
breast carcinoma. EMBO J 1990; 9: 407-414
87 Rio MC, Bellocq JP, Daniel JY, Tomasetto C, Lathe R, Chenard
MP, Batzenschlager A, Chambon P. Breast cancer-associated
pS2 protein: synthesis and secretion by normal stomach mu-
cosa. Science 1988; 241: 705-708
88 Hertel SC, Chwieralski CE, Hinz M, Rio MC, Tomasetto C,
Hoffmann W. Profiling trefoil factor family (TFF) expression
in the mouse: identification of an antisense TFF1-related tran-
script in the kidney and liver. Peptides 2004; 25: 755-762
89 Sasaki M, Tsuneyama K, Saito T, Kataoka H, Mollenhauer J,
Poustka A, Nakanuma Y. Site-characteristic expression and in-
duction of trefoil factor family 1, 2 and 3 and malignant brain
tumor-1 in normal and diseased intrahepatic bile ducts relates
to biliary pathophysiology. Liver Int 2004; 24: 29-37
90 Sasaki M, Tsuneyama K, Nakanuma Y. Aberrant expression
of trefoil factor family 1 in biliary epithelium in hepatolithiasis
and cholangiocarcinoma. Lab Invest 2003; 83: 1403-1413
91 Srivatsa G, Giraud AS, Ulaganathan M, Yeomans ND, Dow
C, Nicoll AJ. Biliary epithelial trefoil peptide expression is in-
creased in biliary diseases. Histopathology 2002; 40: 261-268
92 Kimura Y, Leung PS, Kenny TP, Van De Water J, Nishioka M,
Giraud AS, Neuberger J, Benson G, Kaul R, Ansari AA, Cop-
pel RL, Gershwin ME. Differential expression of intestinal tre-
foil factor in biliary epithelial cells of primary biliary cirrhosis.
Hepatology 2002; 36: 1227-1235
93 Marenholz I, Zirra M, Fischer DF, Backendorf C, Ziegler A,
Mischke D. Identification of human epidermal differentiation
complex (EDC)-encoded genes by subtractive hybridization of
entire YACs to a gridded keratinocyte cDNA library. Genome
Res 2001; 11: 341-355
94 Cabral A, Voskamp P, Cleton-Jansen AM, South A, Nizetic D,
Backendorf C. Structural organization and regulation of the
small proline-rich family of cornified envelope precursors sug-
gest a role in adaptive barrier function. J Biol Chem 2001; 276:
19231-19237
95 Patel S, Kartasova T, Segre JA. Mouse Sprr locus: a tandem
array of coordinately regulated genes. Mamm Genome 2003; 14:
140-148
96 Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Genes
encoding structural proteins of epidermal cornification and
S100 calcium-binding proteins form a gene complex ("epider-
mal differentiation complex") on human chromosome 1q21. J
Invest Dermatol 1996; 106: 989-992
97 Elder JT, Zhao X. Evidence for local control of gene expression
in the epidermal differentiation complex. Exp Dermatol 2002;
11: 406-412
98 Tarcsa E, Candi E, Kartasova T, Idler WW, Marekov LN, Stein-
ert PM. Structural and transglutaminase substrate properties
of the small proline-rich 2 family of cornified cell envelope
proteins. J Biol Chem 1998; 273: 23297-23303
99 Stern LE, Erwin CR, Falcone RA, Huang FS, Kemp CJ, Wil-
liams JL, Warner BW. cDNA microarray analysis of adapting
bowel after intestinal resection. J Pediatr Surg 2001; 36: 190-195
100 Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG,
Gordon JI. Molecular analysis of commensal host-microbial
relationships in the intestine. Science 2001; 291: 881-884
101 Mueller A, ORourke J, Grimm J, Guillemin K, Dixon MF, Lee
A, Falkow S. Distinct gene expression profiles characterize the
histopathological stages of disease in Helicobacter-induced
mucosa-associated lymphoid tissue lymphoma. Proc Natl Acad
Sci U S A 2003; 100: 1292-1297
3521
102 Knight PA, Pemberton AD, Robertson KA, Roy DJ, Wright
SH, Miller HR. Expression profiling reveals novel innate and
inflammatory responses in the jejunal epithelial compartment
during infection with Trichinella spiralis. Infect Immun 2004;
72: 6076-6086
103 Hong SH, Nah HY, Lee JY, Lee YJ, Lee JW, Gye MC, Kim CH,
Kang BM, Kim MK. Estrogen regulates the expression of the
small proline-rich 2 gene family in the mouse uterus. Mol Cells
2004; 17: 477-484
104 Tan YF, Li FX, Piao YS, Sun XY, Wang YL. Global gene
profiling analysis of mouse uterus during the oestrous cycle.
Reproduction 2003; 126: 171-182
105 Song HJ, Poy G, Darwiche N, Lichti U, Kuroki T, Steinert PM,
Kartasova T. Mouse Sprr2 genes: a clustered family of genes
showing differential expression in epithelial tissues. Genomics
1999; 55: 28-42
106 Zimmermann N, Doepker MP, Witte DP, Stringer KF,
Fulkerson PC, Pope SM, Brandt EB, Mishra A, King NE,
Nikolaidis NM, Wills-Karp M, Finkelman FD, Rothenberg
ME. Expression and regulation of small proline-rich protein
2 in allergic inflammation. Am J Respir Cell Mol Biol 2005; 32:
428-435
107 Michalopoulos GK, DeFrances MC. Liver regeneration.
Science 1997; 276: 60-66
108 Clouston AD, Powell EE. Interaction of non-alcoholic fatty
liver disease with other liver diseases. Best Pract Res Clin
Gastroenterol 2002; 16: 767-781
109 Demetris AJ, Sakamoto T, Liu Z, Yokomuro S, Ezure T,
Murase N, Blakolmer K. The Ductular Reaction in Liver
Disease emphasis on a type I response. In: Fleig WE, Editor.
Normal and Malignant Liver Cell Growth. Dordecht: Kluwer
Academic Publishers, 1999: 141-155
110 Sirica AE, Mathis GA, Sano N, Elmore LW. Isolation, culture,
and transplantation of intrahepatic biliary epithelial cells and
oval cells. Pathobiology 1990; 58: 44-64
111 Zhang M, Thorgeirsson SS. Modulation of connexins during
differentiation of oval cells into hepatocytes. Exp Cell Res 1994;
213: 37-42
112 Martin KR, Barrett JC. Reactive oxygen species as double-
edged swords in cellular processes: low-dose cell signaling
versus high-dose toxicity. Hum Exp Toxicol 2002; 21: 71-75
113 Marshall A, Rushbrook S, Davies SE, Morris LS, Scott IS,
Vowler SL, Coleman N, Alexander G. Relation between
hepatocyte G1 arrest, impaired hepatic regeneration, and
fibrosis in chronic hepatitis C virus infection. Gastroenterology
2005; 128: 33-42
114 Kobayashi S, Matsushita K, Saigo K, Urashima T, Asano
T, Hayashi H, Ochiai T. P21WAF1/CIP1 messenger
RNA expression in hepatitis B, C virus-infected human
hepatocellular carcinoma tissues. Cancer 2001; 91: 2096-2103
115 Torbenson M, Yang SQ, Liu HZ, Huang J, Gage W, Diehl AM.
STAT-3 overexpression and p21 up-regulation accompany
impaired regeneration of fatty livers. Am J Pathol 2002; 161:
155-161
116 Sawada N, Kojima T, Obata H, Isomura H, Atsumi S, Sawaki M,
Tsuzuki N, Tobioka H, Kokai Y, Satoh M, Mori M. Expression
of p21(waf-1/cip-1) is significantly induced in the livers of
LEC rats with chronic liver injury. Jpn J Cancer Res 1996; 87:
1102-1105
117 Koteish A, Yang S, Lin H, Huang J, Diehl AM. Ethanol
induces redox-sensitive cell-cycle inhibitors and inhibits liver
regeneration after partial hepatectomy. Alcohol Clin Exp Res
2002; 26: 1710-1708
118 Blakolmer K, Jain A, Ruppert K, Gray E, Duquesnoy R,
Murase N, Starzl TE, Fung JJ, Demetris AJ. Chronic liver
allograft rejection in a population treated primarily with
tacrolimus as baseline immunosuppression: long-term follow-
up and evaluation of features for histopathological staging.
Transplantation 2000; 69: 2330-2336
119 Demetris A, Adams D, Bellamy C, Blakolmer K, Clouston A,
Dhillon AP, Fung J, Gouw A, Gustafsson B, Haga H, Harrison
D, Hart J, Hubscher S, Jaffe R, Khettry U, Lassman C, Lewin
K, Martinez O, Nakazawa Y, Neil D, Pappo O, Parizhskaya
www.wjgnet.com
 3522 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
M, Randhawa P, Rasoul-Rockenschaub S, Reinholt F,
Reynes M, Robert M, Tsamandas A, Wanless I, Wiesner R,
Wernerson A, Wrba F, Wyatt J, Yamabe H. Update of the
International Banff Schema for Liver Allograft Rejection:
working recommendations for the histopathologic staging
and reporting of chronic rejection. An International Panel.
Hepatology 2000; 31: 792-799
120 Oguma S, Belle S, Starzl TE, Demetris AJ. A histometric
analysis of chronically rejected human liver allografts: insights
into the mechanisms of bile duct loss: direct immunologic and
ischemic factors. Hepatology 1989; 9: 204-209
121 Omori N, Evarts RP, Omori M, Hu Z, Marsden ER,
Thorgeirsson SS. Expression of leukemia inhibitory factor
and its receptor during liver regeneration in the adult rat. Lab
Invest 1996; 75: 15-24
122 Sakamoto T, Ezure T, Lunz J, Murase N, Tsuji H, Fung JJ,
Demetris AJ. Concanavalin A simultaneously primes liver
hematopoietic and epithelial progenitor cells for parallel
expansion during liver regeneration after partial hepatectomy
in mice. Hepatology 2000; 32: 256-267
123 Chan KS, Sano S, Kiguchi K, Anders J, Komazawa N, Takeda
J, DiGiovanni J. Disruption of Stat3 reveals a critical role in
both the initiation and the promotion stages of epithelial
carcinogenesis. J Clin Invest 2004; 114: 720-728
124 Campisi J. Cancer and ageing: rival demons? Nat Rev Cancer
2003; 3: 339-349
125 Haura EB, Turkson J, Jove R. Mechanisms of disease: Insights
into the emerging role of signal transducers and activators of
transcription in cancer. Nat Clin Pract Oncol 2005; 2: 315-324
126 Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3
in inflammation and cancer. Eur J Cancer 2005; 41: 2502-2512
127 Koike N, Saitoh K, Todoroki T, Kawamoto T, Iwasaki Y,
Nakamura K. Cell proliferation kinetics of the bile duct
epithelium of the rat. Cell Prolif 1993; 26: 183-193
128 Nakanuma Y, Kono N. Expression of vimentin in proliferating
and damaged bile ductules and interlobular bile ducts in
nonneoplastic hepatobiliary diseases. Mod Pathol 1992; 5:
550-554
129 Milani S, Herbst H, Schuppan D, Niedobitek G, Kim KY,
Stein H. Vimentin expression of newly formed rat bile duct
epithelial cells in secondary biliary fibrosis. Virchows Arch A
Pathol Anat Histopathol 1989; 415: 237-242
130 Haruna Y, Saito K, Spaulding S, Nalesnik MA, Gerber MA.
Identification of bipotential progenitor cells in human liver
development. Hepatology 1996; 23: 476-481
131 Terada T, Ashida K, Kitamura Y, Matsunaga Y, Takashima
K, Kato M, Ohta T. Expression of epithelial-cadherin, alpha-
catenin and beta-catenin during human intrahepatic bile duct
development: a possible role in bile duct morphogenesis. J
Hepatol 1998; 28: 263-269
132 Camargo CA Jr, Madden JF, Gao W, Selvan RS, Clavien
PA. Interleukin-6 protects liver against warm ischemia/
reperfusion injury and promotes hepatocyte proliferation in
the rodent. Hepatology 1997; 26: 1513-1520
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
133 Gao B. Therapeutic potential of interleukin-6 in preventing
obesity- and alcohol-associated fatty liver transplant failure.
Alcohol 2004; 34: 59-65
134 Hong F, Radaeva S, Pan HN, Tian Z, Veech R, Gao B.
Interleukin 6 alleviates hepatic steatosis and ischemia/
reperfusion injury in mice with fatty liver disease. Hepatology
2004; 40: 933-941
135 Selzner M, Graf R, Clavien PA. IL-6: a magic potion for liver
transplantation? Gastroenterology 2003; 125: 256-259
136 Sun Z, Klein AS, Radaeva S, Hong F, El-Assal O, Pan HN,
Jaruga B, Batkai S, Hoshino S, Tian Z, Kunos G, Diehl AM,
Gao B. In vitro interleukin-6 treatment prevents mortality
associated with fatty liver transplants in rats. Gastroenterology
2003; 125: 202-215
137 Kimizuka K, Nakao A, Nalesnik MA, Demetris AJ, Uchiyama
T, Ruppert K, Fink MP, Stolz DB, Murase N. Exogenous IL-6
inhibits acute inflammatory responses and prevents ischemia/
reperfusion injury after intestinal transplantation. Am J
Transplant 2004; 4: 482-494
138 Safadi R, Friedman SL. Hepatic fibrosis--role of hepatic
stellate cell activation. MedGenMed 2002; 4: 27
139 Tillmann HL, Manns MP, Rudolph KL. Merging models of
hepatitis C virus pathogenesis. Semin Liver Dis 2005; 25: 84-92
140 Watanabe S, Matsuda A, Suzuki Y, Kondo K, Ikeda Y,
Hashimoto H, Umemura K. Inhibitory mechanism of tranilast
in human coronary artery smooth muscle cells proliferation,
due to blockade of PDGF-BB-receptors. Br J Pharmacol 2000;
130: 307-314
141 Ikeda H, Inao M, Fujiwara K. Inhibitory effect of tranilast on
activation and transforming growth factor beta 1 expression
in cultured rat stellate cells. Biochem Biophys Res Commun 1996;
227: 322-327
142 Naftalin R. Alterations in colonic barrier function caused by a
low sodium diet or ionizing radiation. J Environ Pathol Toxicol
Oncol 2004; 23: 79-97
143 Wengrower D, Zanninelli G, Pappo O, Latella G, Sestieri M,
Villanova A, Faitelson Y, Pines M, Goldin E. Prevention of
fibrosis in experimental colitis by captopril: the role of tgf-
beta1. Inflamm Bowel Dis 2004; 10: 536-545
144 Ramos SG, Montenegro AP, Goissis G, Rossi MA. Captopril
reduces collagen and mast cell and eosinophil accumulation in
pig serum-induced rat liver fibrosis. Pathol Int 1994; 44: 655-661
145 Yuan GJ, Zhang ML, Gong ZJ. Effects of PPARg agonist
pioglitazone on rat hepatic fibrosis. World J Gastroenterol 2004;
10: 1047-1051
146 Kawaguchi K, Sakaida I, Tsuchiya M, Omori K, Takami T,
Okita K. Pioglitazone prevents hepatic steatosis, fibrosis, and
enzyme-altered lesions in rat liver cirrhosis induced by a
choline-deficient L-amino acid-defined diet. Biochem Biophys
Res Commun 2004; 315: 187-195
147 Miyahara T, Schrum L, Rippe R, Xiong S, Yee HF, Motomura
K, Anania FA, Willson TM, Tsukamoto H. Peroxisome
proliferator-activated receptors and hepatic stellate cell
activation. J Biol Chem 2000; 275: 35715-35722
S- Editor Pan BR E- Editor Bi L
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Heterogeneity of the intrahepatic biliary epithelium
Shannon Glaser, Heather Francis, Sharon DeMorrow, Gene LeSage, Giammarco Fava, Marco Marzioni,
Julie Venter, Gianfranco Alpini
Shannon Glaser, Department of Medicine, Division of Research
and Education, Scott & White Memorial Hospital and The Texas
A&M University System Health Science Center, College of Medi-
cine, Temple, TX, United States
Heather Francis, Sharon DeMorrow, Division of Research
and Education, Scott & White Memorial Hospital and The Texas
A&M University System Health Science Center, College of Medi-
cine, Temple, TX, United States
Gene LeSage, University of Texas at Houston Medical School,
Houston, TX. United States
Giammarco Fava, Marco Marzioni, Department of Gastroenter-
ology, Universit Politecnica delle Marche, Azienda Ospedaliera
Ospedali Riuniti di Ancona, Ancona, Italy
Julie Venter, Department of Medicine, Scott & White Memorial
Hospital and The Texas A&M University System Health Science
Center, College of Medicine, Temple, TX, United States
Gianfranco Alpini, Central Texas Veterans Health Care System,
Department of Medicine, Systems Biology and Translational
Medicine, Scott & White Memorial Hospital and The Texas A&M
University System Health Science Center, College of Medicine,
Temple, TX, United States
Supported by a grant award from Scott & White Hospital and
The Texas A&M University System Health Science Center, a VA
Merit Award, a VA Research Scholar Award and the NIH grants
DK58411 and DK062975 to Dr. Alpini, by grant awards to Shan-
non Glaser and Heather Francis from Scott & White Hospital.
Correspondence to: Shannon Glaser, MS, Department of
Medicine, Division of R&E, Scott and White Memorial Hospital
and The Texas A&M University System Health Science Center
College of Medicine, MRB, 702 South West H.K. Dodgen Loop,
Temple, Texas 76504, United States. sglaser@neo.tamu.edu
Telephone: +1-254-7427044 Fax: +1-254-7245944
Received: 2006-01-22 Accepted: 2006-05-18
Abstract
The objectives of this review are to outline the recent
findings related to the morphological heterogeneity
of the biliary epithelium and the heterogeneous
pathophysiological responses of different sized bile ducts
to liver gastrointestinal hormones and peptides and liver
injury/toxins with changes in apoptotic, proliferative and
secretory activities. The knowledge of biliary function
is rapidly increasing because of the recognition that
biliary epithelial cells (cholangiocytes) are the targets
of human cholangiopathies, which are characterized by
proliferation/damage of bile ducts within a small range of
sizes. The unique anatomy, morphology, innervation and
vascularization of the biliary epithelium are consistent
with function of cholangiocytes within different regions
World J Gastroenterol 2006 June 14; 12(22): 3523-3536
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
of the biliary tree. The in vivo models [e.g., bile duct
ligation (BDL), partial hepatectomy, feeding of bile acids,
carbon tetrachloride (CCl4) or -naphthylisothiocyanate
(ANIT)] and the in vivo experimental tools [e.g., freshly
isolated small and large cholangiocytes or intrahepatic
bile duct units (IBDU) and primary cultures of small
and large murine cholangiocytes] have allowed us
to demonstrate the morphological and functional
heterogeneity of the intrahepatic biliary epithelium.
These models demonstrated the differential secretory
ac t ivit ie s and t he he t e r oge ne ous apopt o t i c a n d
proliferative responses of different sized ducts. Similar
to animal models of cholangiocyte proliferation/injury
restricted to specific sized ducts, in human liver diseases
bile duct damage predominates specific sized bile ducts.
Future studies related to the functional heterogeneity
of the intrahepatic biliary epithelium may disclose
new pathophysiological treatments for patients with
cholangiopathies.
2006 The WJG Press. All rights reserved.
Key words: cAMP; Gastrointestinal hormones; Growth
factors; Mitosis; Nerves
Glaser S, Francis H, DeMorrow S, LeSage G, Fava G,
Marzioni M, Venter J, Alpini G. Heterogeneity of the
intrahepatic biliary epithelium. World J Gastroenterol 2006;
12(22): 3523-3536
http://www.wjgnet.com/1007-9327/12/3523.asp
Anatomical and Morphological
Characteristics of the Biliary Epi-
thelium
Two kinds of epithelial cells, hepatocytes and cho-
langiocytes, are present in the liver[1-3]. While hepatocytes
i n i t i a l l y s e c r e t e b i l e i n t o t h e b i l e c a n a l i c u l u s [4],
cholangiocytes modify bile of canalicular origin by
a series of coordinated spontaneous and hormone/
peptide regulated secretion/reabsorption of water and
electrolytes before it reaches the small intestine[3,5-7]. For
more information on the mechanisms of bile formation
we refer to recent reviews[4,5]. The human biliary system is
divided into extrahepatic bile ducts and intrahepatic bile
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 3524 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

Top
Table 1 Terminology and relationship between human and rat
A B 14.84 m intrahepatic bile ducts
8.75 m
Terminology for human bile ducts Terminology for rat bile ducts
(diameter in m) (diameter in m)
(Large bile ducts)
Hepatic ducts (> 800)
Segmental ducts (400-800)
Area ducts (300-400)

40 40 (Small bile ducts)

Septal bile ducts (100-300)
Interlobular bile ducts (15-100) Large bile ducts (> 15)
35 35
Bile ductules (cholangioles) (< 15) Small bile ducts (< 15)

30 30 These data have been obtained from studies [8,12,13] aimed to define the
morphological characteristics of the biliary epithelium of rats, and humans.
Reproduced with permission from Ref 2.
25 25
Frequency

Frequency

20 20
(300-400 m), segmental ducts (400-800 m) and hepatic
ducts (> 800 m)[8] (Table 1). Small ductules are lined by
15 15
4-5 cholangiocytes, have a basement membrane, tight junc-
tions between cells and microvilli projecting into the bile
10 10 duct lumen[10,11]. Cholangiocytes are progressively larger
and more columnar in shape in larger bile ducts (lined by
5 5 10-12 cholangiocytes)[10,11].
In rats, morphological studies in liver sections and
0 0
small and large intrahepatic bile duct units (IBDU) have
6 8 10 12 14 16 18 6 8 10 12 14 16 18
Cholangiocyte diameter (m)
shown[2,12-14] that the intrahepatic biliary tree is divided
Cholangiocyte diameter (m)
Small Large into: (1) small ducts (< 15 m in external diameter) lined
by small cholangiocytes (approximately 8 m in diam-
Bottom eter)[12,13]; and (2) and large ducts (> 15 m in diameter)
C
lined by large cholangiocytes (approximately 15 m in
Laser target D
diameter)[12,13] (Figure 1, Table 1). Specifically, we have
shown [12] that the rat intrahepatic biliary epithelium is

Small IBDU formed by ducts of different sizes (5 to 200 m in external
Small IBDU
diameter) and cholangiocytes of different cell areas (3 to
80 m2). Furthermore, a direct relationship exists between
cholangiocyte area and external duct diameter, a finding

Large IBDU Large IBDU
Figure 1 [Top] Isolation of small (A), approximately 8 m diameter] and large
(B), approximately 14 m diameter] cholangiocytes from small and large ducts,
respectively, from normal rats. Small and large cholangiocytes were purified by
counterflow elutriation followed by immunoaffinity purification. Original magn.,
625. Reproduced with permission from Ref[12]. [Bottom] Isolation of small (C) and
large (D) IBDU from normal rat liver. Small (< 15 m in diameter) and large (> 15
m in diameter) IBDU were pruned off from large ducts by a nitrogen pulsed dye
laser and subsequently separated (D) by picking up IBDU with a micromanipulator
micropipet. Original magnification 2000. Reproduced with permission from
Ref 13.
ducts, the latter further sub-divided into large and small
bile ducts[2,3,8]. The intrahepatic bile ducts represent that
part of the biliary tree proximal to the confluence of the
hepatic ducts[9] extending from the canals of Hering to
the large extrahepatic ducts[2,3,8]. In human liver, a study by
Ludwig classified the intrahepatic bile duct system upon
duct diameter[8], small bile ductules (< 15 m), interlobular
ducts (15-100 m), septal ducts (100-300 m), area ducts
that demonstrates that small ducts are lined by small chol-
angiocytes, whereas larger ducts are lined by larger cholan-
giocytes[12-14]. The fact that small and large ducts are lined
by small and large cholangiocytes, respectively, is important
since it allows for the assignment of the secretory, apop-
totic and proliferative functions (achieved in isolated small
and large cholangiocytes) within the different portions
of the intrahepatic biliary epithelium. Recently, Masyuk
et al[15] have reconstructed the intrahepatic biliary epithe-
lium that resembles a tree, with the common and hepatic
ducts corresponding to the trunk, the intrahepatic bile
ducts corresponding to the large branches and the small
ducts corresponding to the smallest tree limbs of a tree.
Studies by Phillips et al[16] have shown that no major
ultrastructural differences exist among cholangiocytes
lining small and large bile ducts. However, in support
of the concept that the intrahepatic biliary epithelium
is morphologically heterogeneous, electron microscopic
studies by Benedetti et al[14] in rat liver sections and IBDU
have demonstrated that large bile ducts are lined by 8-15
cholangiocytes and small ducts by 4-5 cholangiocytes. The
studies also showed that small and large cholangiocytes

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 Glaser S et al. Heterogeneity of bile ducts 3525
have a multilobulated nucleus, numerous vesicles at the
subapical region, tight junctions, high density of microvilli
and lysosomes and a few mitochondria [14]. Other stud-
ies have shown the presence of microvilli and cilia in the
apical plasma membrane of cholangiocytes[17,18], cilia that
play an important role in the regulation of cholangiocyte
functions[19,20]. While large cholangiocytes are columnar
in shape, small cholangiocytes have a cuboidal shape[14].
Abundant Golgi apparatus was observed between the api-
cal pole and the nucleus[14]. Rough endoplasmic reticulum
was inconspicuous in the smallest ducts and increased
only slightly in the largest[14]. While large cholangiocytes
display a small nucleus and conspicuous cytoplasm, small
cholangiocytes possess a high nucleus/cytoplasm ratio[14].
Cholangiocytes have distinct apical and basolateral mem-
branes[14,17,18].Coated pits have also been observed on the
apical and basolateral membranes of cholangiocytes, a
finding suggesting receptor-mediated endocytosis at both
domains of cholangiocytes[21]. Functional tight junctions
are located between adjacent cholangiocytes in proximity
to the apical domain[17].
Innervation
There is growing information regarding the role of the
nervous system in the regulation of the pathophysiology
of the biliary epithelium [3,22-27]. In the liver, adrenergic
and cholinergic nerves are located around the hepatic
artery, portal vein, and the biliary epithelium [28,29]. The
intrahepatic arteries, veins, bile ducts and hepatocytes
are also inner vated [28,29] . In the autonomic ner vous
system, there are a number of regulator y peptides
including neuropeptide tyrosine (NPY) [30,31], calcitonin
gene related peptide (CGRP), somatostatin, vasoactive
intestinal polypeptide (VIP) (mostly associated with
parasympathetic fibers), enkephalin and bombesin[31-35].
NPY-positive nerves are present in extrahepatic bile
ducts[36] and have been suggested to regulate bile flow by
autocrine/paracrine mechanisms[37]. We have shown that
NPY inhibits cholangiocarcinoma growth by interaction
with a G-protein coupled receptor by Ca 2+-dependent
modulation of Src/ERK1/2 phosphorylation[38]. Nerve
fibers containing CGRP and substance P are present
around blood vessels and bile duct radicles within portal
tracts[39,40]. VIP-positive nerve fibers are located in the walls
of hepatic arteries, portal veins and bile ducts[41].
Vascularization
The intrahepatic and extrahepatic bile ducts are nourished
by a complex network of minute vessels [i.e., peribiliary
vascular plexus (PBP)], which originate from branches
of the hepatic artery and flow principally into the hepatic
sinusoids, either directly (lobular branch) or by portal
vein branches (prelobular branches)[42,43]. Since the blood
flows in the opposite direction (from the large towards
the small ducts) to bile flow, the PBP presents a counter-
current stream of biliary reabsorbed substances to hepato-
cytes[44,45]. We have previously shown that the function of
the intrahepatic biliary tree is linked to its vascular supply
sustained by the PBP[44]. Changes in intrahepatic bile duct
Small duct before secretin Large duct before secretin
Small duct after secretin Large duct after secretin
Figure 2 Measurement of H 3 histone gene expression in small and large
cholangiocytes from 1-wk BDL rats and 1-wk BDL rats treated with CCl4 or mineral
oil. H3 histone gene expression in large cholangiocytes decreased on d 2 before
returning to control values on d 7 after CCl4 treatment. H3 histone gene expression
(which was absent in small cholangiocytes from BDL rats) was expressed by small
cholangiocytes on d 1 and 2 before returning to control undetectable values on d
7 after CCl4 treatment. Administration of mineral oil to 1-wk BDL rats did not alter
H3 histone gene expression in large cholangiocytes. The message for H3 histone
gene was absent in small cholangiocytes from oil-treated rats. Comparability
of RNA used was assessed by hybridization for GAPDH (housekeeping gene).
Autoradiograms were quantified by densitometry. Densitometric values are means
of 2 experiments. Reproduced with permission from Ref 50.
mass are associated with changes of the PBP architec-
ture[44]. Following BDL, the PBP undergoes hyperplasia,
thus supporting the increased nutritional and functional
demands from the proliferating bile ducts[44]. In support of
this concept, studies[46] have shown that following chronic
feeding of ANIT (which induces increases in both cholan-
giocyte proliferation/apoptosis)[47], the hepatic artery and
portal vein undergo marked proliferation, presumably to
support the increased nutritional and functional demands
of the proliferated bile ducts[44,46]. However, the prolifera-
tion of the PBP occurs only after the hyperplasia of bile
ducts[44]. Recent studies have shown that small and large
rat bile ducts have a different vascular supply[44]. The PBP
is primarily present around large bile ducts and less vis-
ible around small bile ducts[44], a finding that may partly
explain why large but not small cholangiocytes proliferate
following BDL in rats[48] and why small and large ducts dif-
ferentially proliferate or are damaged in other experimen-
tal models of cholangiocyte proliferation/loss including
chronic feeding of certain bile acids (e.g., taurocholate and
taurolithocholate)[49], [47] or acute gavage administra-
tion of CCl4[50,51] (Figure 2) or partial hepatectomy[52].
General Background on Cholan-
giocyte Functions
The major function of cholangiocytes is to modify
bile of canalicular origin [4] (by basal and hormone/
pe ptide regulated secretion and reabsor ption of
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 3526 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
water and electrolytes) before reaching the small
intestine[3,5,6]. Ductal secretion is coordinately modulated
by gastrointestinal hormones (e.g., secretin, gastrin,
insulin, somatostatin, bombesin and VIP) [3,5-7,12,53-58] ,
gastrointestinal peptides (i.e., endothelin-1, ET-1) [59],
enzymes (e.g., alkaline phosphatase) [60], bile acids (e.g.,
taurocholate, taurolithocholate, taurohyodeoxycholate,
tauroursodeoxycholate, ursodeoxycholate and taurourso
deoxycholate)[49,61-64] and cholinergic[23,26], adrenergic[65,66],
serotoninergic[67] and dopaminergic[25] receptor agonists.
Cholangiocytes, which have a low DNA turnover under
normal physiological conditions[48,52,68], proliferate or are
damaged in response to liver injury/toxins[2,3,6,26,47,48,50,52,68-72].
In rat liver, secretin is of particular importance since
secretin receptors are only expressed by cholangiocytes[53],
and its expression is upregulated under pathological
conditions associated with enhanced cholangiocyte
g rowth (e.g., after BDL) [48,71,72] and downregulated
with cholangiocyte damage/loss (e.g., following acute
CCl 4 administration) [50,51]. Thus, the secretin receptor
is an important pathophysiological tool that allows us
to evaluate the secretory, proliferative and apoptotic
heterogeneity of the intrahepatic biliary epithelium in
response to agonists and liver toxins/injury[2,3,12,13,47-51,54,61,7
1,73]
. Interaction of secretin with its receptor is associated
with increased intracellular cAMP levels [12,13,25,26,48,50-52,54
,59,72]
. Enhanced cAMP levels leads to phosphorylation
of PKA[74], which induces opening of the cystic fibrosis
transmembrane regulator (CFTR) channel[54] leading to the
activation of the Cl-/HCO3- exchanger[12,23,52] resulting in
biliary bicarbonate secretion[6,52].
Experimental Models
A number of in vivo models (e.g., BDL, acute admini-
stration of CCl4, partial hepatectomy, chronic feeding of
ANIT or bile salts)[47-52] demonstrated that the intrahepatic
biliary epithelium is functionally heterogeneous, with
specific sized bile ducts (i.e., small and large) differentially
responding to liver injur y/toxins with changes in
proliferative, apoptotic and secretory activities [2,3,12,47-52,5
4,62,71-73]
. A number of in vitro experimental models (i.e.,
small and large cholangiocytes and IBDU and small
and large immortalized normal murine cholangiocytes)
(Figure 1)[12,13,47,48,50,51,75] have allowed us to suggest that
the intrahepatic biliary epithelium is morphologically
and functionally heterogeneous[2, 3,12,47-52,54,62,71-73]. The very
first approach that was employed and that significantly
contributed to lay down the basis of this field of research
was the purification of small and large cholangiocytes
from rat liver by counterflow elutriation[12,54,76]. Coupling
such a technique to immunoaffinity separation [12,18,54],
it was possible to isolate two distinct subpopulations
of small (approximately 8 m in diameter, obtained
at the centripetal flow rate of 25 ml/min) and large
(approximately 14 m in diameter, collected at the flow
rate of 55 mL/min) cholangiocytes (Figure 1)[12,54]. The
two subpopulations of small and large cholangiocytes are
further purified by immunoaffinity separation [18] using
an antibody against an unidentified antigen (expressed
by all intrahepatic cholangiocytes)[18] and characterized
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June 14, 2006 Volume 12 Number 22
morphologically (by computerized image analysis) (Figure
1)[12,54], phenotypically (expression of -glutamyltransferase
and cytokeratin-19 genes) [12,54] and functionally (by
measurement of gene expression of secretin receptor,
CFTR and Cl-/HCO3- exchanger and basal and secretin-
stimulated cAMP levels, Cl - efflux and Cl - /HCO 3 -
exchanger activity)[12,54].
In addition, we have developed a technique for
isolating small (diameter smaller than 15 m) and large
(diameter greater than 15 m) IBDU from small and large
bile ducts, respectively (Figure 1)[13]. This important tool
allowed us to directly evaluate the differential secretory
responses of different portions of the biliary epithelium
to selected gastrointestinal hormones/peptides[13,25,65,77]. As
shown in Figure 1, the small duct was pruned off from
the large duct by a brief exposure of a laser focused on
the junction between large and small ducts (arrow) leading
to separation of small from large ducts[13]. Small and large
IBDU were characterized by morphometric analysis, gene
expression for secretin receptor, CFTR and Cl-/HCO3-
exchanger, secretin-induced cAMP levels, and secretion by
change in luminal size in response to agonists including
secretin, insulin, the 1-adrenergic receptor agonist, the
2-adrenergic receptor agonist, UK14,304 and the D2
dopaminergic receptor agonist, quinelorane[13,25,65,66,77].
Most recently. we have immortalized, from normal
mice (BALB/c), small and large cholangiocytes by
the introduction of the SV40 large T antigen gene,
that allowed, after cloning, to establish small and large
cholangiocyte cell lines[75]. The characteristics of the two
subpopulations were evaluated by electron microscopy
(EM) and measurement of trans-epithelial electrical
resistance (TER), and secretin-stimulated cAMP levels[75].
EM, TER and differential cAMP response to secretin
are consistent with the concept that small and large
immortalized cholangiocytes originate from small and large
ducts, respectively[75]. Microarray successfully displayed
characteristic differential cDNA expression between small
and large cholangiocytes[75]. Using the above described
methods individually or in tandem, has allowed us to
clearly demonstrate heterogeneity of the intrahepatic
biliar y e pithelium and to dissect the differential
physiological responses of these distinct subpopulations
of cholangiocytes to endogenous stimuli.
Heterogeneous Expression of Pro-
teins
The heterogeneous expression of some enzymes/pro-
teins and membrane transporters/receptors in small and
large ducts from mice, rats and humans is summarized in
Table 2. In human liver, large septal bile ducts mainly
express the sialylated Lewisa blood group antigen[78]. In
normal and diseased human livers, hepatic, segmental,
area, and septal bile ducts, and peribiliary glands express
pancreatic enzymes such as pancreatic lipase, pancreatic
[79,80]
-amylase, and trypsin . By microarray of RNA from
small and large immortalized murine cholangiocytes, we
have demonstrated the heterogeneous expression of ap-
proximately 80 proteins between small and large chol-
angiocytes[75]. The pathophysiological relevance of the
 Glaser S et al. Heterogeneity of bile ducts 3527

Table 2 Expression and function of proteins and surface transporters in small and large ducts from rats and human

Markers Small ducts Large ducts Function References

-glutamyl transpeptidase Not expressed Interlobular large rat bile ducts Glutathione metabolism [81]
Alkaline phosphatase Not expressed Interlobular large rat bile ducts Inhibition of secretin choleresis [81]
Leucine amino peptidase Not expressed Interlobular large rat bile ducts Undefined [81]
Cytochrome P4502E1 Not expressed Expressed by large rat and Dehalogenation of CCl4 [50,81,104,105]
human ducts
Lipase, a-amylase and Human septal ducts Large human ducts, and Biliary tree development [79,80]
trypsin peribiliary glands
Bcl-2 Human small ductules Not expressed Anti-apoptotic protein [109,143]
Secretin receptor Not expressed Expressed by large rat ducts Stimulation of bicarbonate secretion [12,13,48,54]
CFTR Human but not rodent small ducts Expressed by large rat ducts Regulation of Cl--secretion [54]
- -
Cl /HCO exchanger
3 Not expressed Expressed by large rat and Regulation of ductal bicarbonate [12,13,82]
human ducts secretion
Somatostatin receptor Not expressed Expressed by large rat ducts Inhibition of secretin choleresis [48]
D2 dopamine receptors Unknown Expressed by large rat ducts Inhibition of secretin choleresis [25]
a-1 adrenergic receptors Expressed by small rat ducts Expressed by large rat ducts Stimulation of secretin choleresis [65]
Endothelin receptors Expressed by small rat ducts Expressed by large rat ducts Inhibition of secretin choleresis [59]
Na+-dependent ABAT Not expressed Expressed by large rat ducts Regulation of ductal secretion [88]

Heterogeneous expression of proteins and membrane transporters that may play a role in the modulation of the heterogeneous properties of the intrahepatic
biliary tree of rats and human. Modified with permission from Ref 73.

differential expression of these messages remains to be
addressed.
Secretory activity
Recent studies have demonstrated that large bile ducts are
the major anatomical sites of cAMP-dependent ductal
secretion by activation of cAMP/PKA/CFTR/Cl-/
HCO3- exchanger (Figure 3)[3,12,13,48,54]. Specifically, stud-
ies in isolated small and large cholangiocytes and IBDU
from normal and BDL rats have shown that large (but not
small) cholangiocytes express the messages for secretin
receptor, CFTR and Cl-/HCO3- exchanger and respond
to secretin with increases in cAMP levels, Cl- efflux and
Cl-/HCO3- exchanger activity and IBDU lumen expansion
(Figure 3)[12,13,48,54]. In rat liver, large ducts express alkaline
phosphatase and -glutamyltranspeptidase[81]. The expres-
sion of alkaline phosphatase in large ducts is consistent
with our previous studies[81] showing that alkaline phos-
phatase inhibits secretin-stimulated choleresis by blockage
of CFTR activity, which is expressed only in large ducts
(Figure 3)[54]. Furthermore, large cholangiocytes (which
is the only cholangiocyte subpopulation expressing the
somatostatin receptor, SSTR2)[48] are the major anatomi-
cal sites of somatostatin inhibition of secretin-stimulated
ductal secretion (Figure 3)[48,55]. The inhibitory effects of
somatostatin on secretin-stimulated secretion in large chol-
angiocytes are associated with reduced cAMP levels, Cl- ef-
flux and Cl-/HCO3- exchanger activity[48,55,54]. The counter-
regulatory effect of somatostatin on the choleretic effect
of secretin is important in modulating ductal secretion
in pathological conditions associated with cholangiocyte
proliferation/loss[3]. Parallel with the findings observed in
rat bile ducts[3,12,13,48, 4], in human liver secretin-stimulated
duct secretory activity is heterogeneous, since only large
bile interlobular ducts express the Cl-/HCO3- exchanger[82].
We have demonstrated the presence of insulin and
CCK-B/gastrin receptors in large cholangiocytes from
normal and BDL rats and have shown that these two
hormones inhibit secretin-stimulated ductal secretion of
BDL rats by IP 3/Ca 2+/PKC -dependent decrease of
cAMP levels[7,72,77]. Similarly, we found that ETA and ETB
receptors are expressed by large cholangiocytes and that
ET-1 inhibits secretin-stimulated cAMP levels and ductal
bile secretion of BDL rats by interaction with ETA but not
ETB receptors[59]. Furthermore, recent data have shown
that: (1) the D2 dopaminergic receptors are expressed by
large BDL cholangiocytes; and (2) the D2 dopaminergic
receptor agonist, quinelorane, inhibits secretin-stimulated
ductal secretion by activation of the Ca 2+-dependent
PKC [25]. The 2-adrenergic receptor agonist, UK14,
304, inhibits secretin-stimulated cAMP-dependent Cl -
efflux and Cl-/HCO3- in large cholangiocytes and secretin-
stimulated lumen expansion in large IBDU of BDL
rats[66]. The 1-adrenergic receptor agonist, phenylephrine,
stimulates cAMP levels and secretin-stimulated secretion
of large BDL cholangiocytes by IP 3/Ca 2+-dependent
activation of PKC and PKC [65]. We have recently
demonstrated[26] that acetylcholine, by interacting with M3
receptor subtypes, potentiates secretin-stimulated cAMP
levels and Cl -/HCO 3- exchanger activity in IBDU and
purified cholangiocytes by a Ca2+-calcineurin mediated but
PKC independent modulation of adenylyl cyclase.
Following hepatocyte secretion [83] , bile acids are
reabsorbed by the biliary epithelium[84], then they return
via the PBP to the hepatocytes for secretion into bile
(cholehepatic shunting)[85]. As a mechanism for bile acids
entry into cholangiocytes, the apical Na+-dependent bile
transporter, ASBT (structurally identical to the ileal bile
acid transporter) is expressed on the apical membranes of
large cholangiocytes[86]. Consistent with functional activity
for ASBT in cholangiocytes, studies have shown Na +-
dependent and saturable uptake of taurocholate in normal

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 3528 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

Working model

PKA
Small ducts cAMP
Secretin
Express -GT and CK-19 SR
CFTR
Do not express secretin receptor (SR) -
Cl
Do not express STTR2 receptor (+)
Do not participate in secretin or
somatostatin-regulated ductal
bile secretion HCO3
-
(-)
Do not respond to BDL
Proliferrate and secrete in response to CCl4 - SSTR2
Cl
cAMP Somatostatin
PKA
Large cholangiocyte

Large ducts
Express -GT and CK-19
Express secretin receptor (SR)
Physiogical Express STTR2 receptor
function? Participate in secretin and
somatostatin-regulated
ductal bile secretion
Proliferate and secrete in response
to BDL
Small cholangiocyte Damaged in response to CCl4

Figure 3 Working model for the heterogeneity of the intrahepatic biliary epithelium. The model proposes that: (1) bile ducts are morphologically heterogeneous with small
ducts lined by small cholangiocytes and large ducts lined by large cholangiocytes; (2) small and large ducts similarly express both -GT and cytokeratin-19; and (3) large (but
not small) ducts express the secretin and somatostatin receptor, CFTR and Cl-/HCO3- and respond physiologically to these two hormones. The model also shows that following
BDL, only large cholangiocytes proliferate and that a single dose of CCl4 induces damage and loss of large duct function, whereas small cholangiocytes (resistant to CCl4)
de novo proliferate and secrete to compensate for the loss of large duct function. Reproduced with permission from Ref. 73.

cholangiocyte cultures[87] and large cholangiocytes[88]. These
data suggests that after taurocholate and taurolithocholate
enter into large cholangiocytes by ABAT, they stimulate
secretin-stimulated ductal bile flow in these cholangiocyte
subpopulations [88,89] . Other studies have shown that
both taurocholate and taurolithocholate increase
secretin-stimulated cAMP levels in large but not small
cholangiocytes[89]. Chronic feeding of ursodeoxycholate
and tauroursodeoxycholate to BDL rats inhibits secretin-
stimulated ductal secretion in large cholangiocytes[62].
As evidence against the notion that small cholangio-
cytes may be primitive, undifferentiated cells that do
not display secretory activity, recent studies have shown
that in pathological conditions associated with damage
of large cAMP-responsive ducts (e.g., after acute CCl4
administration) (Figure 3) [50,51] , small cholangiocytes
transiently compensate for large cholangiocyte damage
by de novo activation of secretory (including expression
of secretin receptor and secretin-stimulated cAMP
response)[50,51] and proliferative[50,51] (see below) activities.
Following ANIT feeding and partial hepatectomy, small
cholangiocytes proliferate and secrete by the de novo
expression of secretin receptor and activation of cAMP
response [47,52]. Since preliminary data and unpublished
observations (Alpini, 2005) show that small rat and mouse
cholangiocytes express receptors (ETA, CCK-B/gastrin,
1-adrenergic, D2 dopaminergic, insulin, H1 histamine)
signaling by activation of IP3/Ca2+/PKC[59,90], we propose
that there is a secretory gradient in the intrahepatic
biliary tree with small cholangiocytes secreting water and
electrolytes by activation of the IP3/Ca2+/PKC pathway,
whereas large cholangiocytes secrete bile by activation of
the cAMP/PKA/CFTR/Cl-/HCO3- exchanger[2,5,2,13,48,54].
Proliferation and Apoptosis
Cholangiocyte proliferation is coordinately regulated by a
number of factors including gastrointestinal hormones/
peptides, growth factors, cAMP and IP 3 /Ca 2+ /PKC
pathways, nerves and bile acids[2,3,24,49,61,62,68,70-72,91-93]. Recent
studies have shown that different sized cholangiocytes
differentially proliferate or are damaged by apoptosis in
response to injury, toxins, nerve resection and selected
diets [2,26,48-52] . Following BDL, larg e but not small
cholangiocytes proliferate with increases in basal and
secretin-stimulated choleresis (Figure 3)[2,3,48]. We propose
that large cholangiocytes selectively proliferate in response
to BDL due to: (1) the predominant expression of VEGF
in large compared to small cholangiocytes (Alpini et al,
2005, unpublished observation); and (2) the presence
of the PBP mainly around large bile ducts, and less
discernable around small bile ducts[44]. In support of this

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 Glaser S et al. Heterogeneity of bile ducts 3529
concept, in rats with BDL proliferation of the peribiliary
plexus occurs only around large ducts[44]. Furthermore, we
have recently demonstrated[93] that neutralization of VEGF
levels of large cholangiocytes (by administration of a
neutralizing anti-VEGF antibodies) reduces cholangiocyte
growth typical of BDL rats[6]. In support of the concept
that PBP and VEGF play a role in the regulation of large
cholangiocyte function, hepatic artery ligation in BDL rats
is associated with: (1) the disappearance of the PBP; (2)
increased apoptosis and impaired proliferation of large
cholangiocytes; and (3) decreased cholangiocyte VEGF
secretion [92]. The effects of hepatic artery ligation on
PBP and large cholangiocyte function were prevented by
chronic administration of r-VEGF-A that, by maintaining
the integ rity of the PBP and larg e cholangiocyte
proliferation, prevents bile duct damage following ischemic
injury[92].
A number of gastrointestinal hormones/peptides have
been shown to regulate the differential proliferative re-
sponse of small and large cholangiocytes. We have shown
that cholangiocytes express 1, 2, 1 and 2 thyroid of AC activity and lowered intracellular cAMP levels[100].
hormone receptors and that the chronic administration of
the thyroid hormone agonist, 3, 3, 5 L-tri-iodothyronine
to BDL rats reduces in vivo the proliferation of large
cholangiocytes[94], the only cholangiocyte subpopulation
proliferating in this model[48]. In addition, in BDL rats we
have shown that somatostatin inhibits the growth of large
cholangiocytes by a decrease in cAMP levels[48]. Further-
more, gastrin inhibits large cholangiocyte proliferation in
BDL rats by Ca2+/PKC-dependent inhibition of cAMP
levels[72].
We have demonstrated that ovariectomy in BDL female
rats reduces the proliferation of large cholangiocytes and
induces a decrease in the expression of and estrogen
receptors[69]. We propose that estrogens play a role in the
management of chronic cholestatic liver diseases.
Recent studies have shown that nerves regulate the
differential proliferative response of intrahepatic ducts.
We have shown that the activation of serotonin 1 A and
1 B receptors in cholangiocytes leads to the inhibition of
large cholangiocyte proliferation in BDL rats[67]. Serotonin
inhibition of large cholangiocyte proliferation was
associated with activation of the IP3/Ca2+/PKC signaling
pathway and the consequent inhibition of the cAMP/
PKA/Src/ERK 1/2 pathway [67]. Since cholangiocytes
secrete serotonin, we propose that serotonin limits
the growth of intrahepatic bile ducts in the course of
chronic cholestasis by an autocrine mechanism. Similarly,
we have shown that cholangiocytes secrete NGF and
that NGF secretion increases in proliferating BDL
cholangiocytes compared to normal cholangiocytes[24]. In
vivo, immunoneutralization of NGF (with an anti-NGF
antibody) decreased large cholangiocyte proliferation[24].
The data sug gest that NGF regulates cholangiocyte
proliferation by an autocrine mechanism.
We have demonstrated that sensory innervation via
-calcitonin gene related peptide (-CGRP) plays a role
in adaptive proliferative responses of large cholangiocytes
during cholestasis following BDL[95]. Specifically, we have
shown that small and large murine cholangiocytes express
the CGRP receptor components (calcitonin like receptor
or CLR, receptor component protein or RCP and receptor
activity modifying protein or RAMP1)[95]. Large, but not
small, cholangiocytes proliferate in response to -CGRP,
proliferation that was blocked by CGRP [8-37], -CGRP
receptor antagonist [96]. -CGRP stimulation of large
cholangiocyte proliferation was associated with increased
cAMP levels and phosphorylation of PKA and p38[96]. We
observed a decrease in the number of proliferating large
cholangiocytes in BDL knock-out mice (lacking -CGRP)
compared to BDL wild-type mice[95].
T h e r o l e o f t h e s e c o n d m e s s e n g e r, c A M P, i n
the regulation of hepatic cell proliferation has been
demonstrated in a number of animal models that stimulate
hepatocyte and cholangiocyte proliferation via cAMP
dependent mechanisms [26,50-52,54,97,98]. Following partial
hepatectomy, there is an increase in intracellular cAMP
levels in regenerating hepatocytes[99] and cholangiocytes[52].
Activation of Gs coupled receptors leads to activation
of adenylyl cyclase and increased cAMP levels, whereas
activation of Gi coupled receptors results in inhibition
cAMP response elements mediating transcriptional
activation in response to increased intracellular cAMP
levels have been identified[101]. In support of these findings,
we have shown that chronic administration of forskolin to
normal rats increased cAMP levels and the proliferation
of large but not small cholangiocytes compared to
rats receiving saline [70] . In purified cholangiocytes,
forskolin increased large (but not small) cholangiocyte
proliferation [70] , which was blocked by Rp-cAMPs (a
PKA inhibitor)[74], PP2 (a Src inhibitor)[102] and PD98059
(a MEK inhibitor)[103]. The effects of forskolin on large
cholangiocyte proliferation were associated with increased
phosphorylation of PKA, Src Tyr 139 and ERK1/2[70].
Maintenance of cAMP levels by forskolin administration
prevents the effects of vagotomy on large cholangiocyte
apoptosis (activation) and proliferation (inhibition)[26].
The acute administration of CCl4 to normal and BDL
rats induces decreased cAMP levels and loss of function
of large cholangiocytes at d 2 and transient elevation of
cAMP levels in small cholangiocytes[50,51]. In these models,
small cholangiocytes de novo express secretin receptors, a
key component of the biliary proliferative and secretory
mechanisms, suggesting that intracellular cAMP plays a key
role in the: (1) de novo expression of large cholangiocyte
phenotypes by small cholangiocytes (to compensate for
loss of large cholangiocyte function); and (2) perhaps
the differentiation of small cholangiocytes towards a
cholangiocyte subpopulation that has the capacity to
secrete and proliferate by cAMP-dependent pathway[50,51].
Following partial hepatectomy, both small and large
cholangiocytes proliferate and participate in the regenera-
tion of the intrahepatic biliary epithelium[52]. A single ga-
vage dose of CCl4 to normal and BDL rats induces dam-
age of large, cAMP-responsive cholangiocytes, whereas
small cholangiocytes (resistant to CCl4) de novo proliferate
and secrete (by the activation of the secretin receptor and
secretin-stimulated cAMP levels) to compensate for the
damage and loss of functional activity of large cholan-
giocytes[50,51]. The differential resistance of small and large
cholangiocytes to CCl4 is presumably due to the presence
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 3530 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
of cytochrome P4502E1 (the enzyme that converts CCl4
to its radicals)[104] in large but not small cholangiocytes[50,51].
Chronic administration of the toxin, ANIT, induces pro-
liferation of both small and large cholangiocytes, prolifera-
tion that (in contrast to other models including BDL)[26]
was associated with enhanced apoptosis[47]. We propose
that following ANIT or CCl4 feeding, the proliferation
of small cholangiocytes may be due to the presence of
cholangiocyte apoptosis in these models[47,50,51]. We also
propose that the lack of small cholangiocyte proliferation
in BDL rats may be due to the absence of cholangiocyte
apoptosis in this model[26]. Similar to what is observed
following acute CCl 4 administration [50], the differential
responses of small and large cholangiocytes to liver in-
jury/toxins may be due to differential expression of other
enzymes/proteins in small and large cholangiocytes. In
support of this concept, phase I or mixed-function oxy-
genase enzymes (e.g., microsomal cytochrome P-450,
aminopyrine-N-demethylases, G-6-PO4, and NADPH cy-
tochrome C reductase) and phase II or glutathione redox
cycle enzymes (e.g., GSH-peroxidase, UDP-glucuronosyl-
transferase, and glutathione-S-transferase) drug-metaboliz-
ing enzymes are heterogeneously expressed by cholangio-
cytes[50,81,105]. Similarly, since small murine cholangiocytes
express annexin-V[106] (that regulates cell apoptosis)[107], this
finding may explain partly why small ducts are more resis-
tant than large ducts to some hepatic injury/toxins[50, 51]. In
support of this concept, recent studies have shown that
bcl-2 (an anti-apoptotic protein)[108] is expressed by small
bile ducts in normal human liver and human liver with cir-
rhosis and focal nodular hyperplasia[109], a finding that may
also explain partly the greater resistance of small cholan-
giocytes to damage[3,50,51].
In vitro treatment of normal cholangiocytes with tauro-
cholate and taurolithocholate increases the proliferation of
large but not small cholangiocytes[89]. Chronic feeding of
taurocholate and taurolithocholate to normal rats induces
the de novo expression of ASBT and activation of prolifera-
tion of small cholangiocytes, which do not constitutively
express ASBT and are mitotically quiescent, and increases
the proliferation of large cholangiocytes [49]. Prolonged
feeding of ursodeoxycholate and tauroursodeoxycholate
to BDL rats reduces the growth of large cholangiocytes[62]
that selectively proliferate in this hyperplastic model[48].
Furthermore, depletion of endogenous bile acids reduced
large cholangiocyte proliferation compared with BDL
rats[91]. Re-infusion of taurocholate to bile acid-depleted
rats prevented the decrease in cholangiocyte prolifera-
tion that was maintained at levels similar to those of BDL
rats[91].
Histamine, an aminergic neurotransmitter, regulates
many pathophysiological functions. Four G-protein
coupled histamine receptors (H1, H2, H3 and H4) exist[110].
While H 1 histamine receptors act via G q mobilizing
[Ca 2+ ] i [111] , activation of H 2 histamine receptors is
modulated by Gs proteins, coupled to adenylyl cyclase[112].
H3 and H4 histamine receptors couple to Gi/o proteins
that inhibit adenylyl cyclase[113]. Based upon our preliminary
data, we propose a model in which the overall outcome
of histamine on cholangiocyte growth is represented
by a balance between its stimulatory (by activation of
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June 14, 2006 Volume 12 Number 22
H1 and H2 histamine receptors)[90,114] and its inhibitory
(by activation of H3 and H4 histamine receptors)[115, 116]
actions on small and large cholangiocyte proliferation.
Specifically, we have shown that small but not large mouse
cholangiocytes: (1) express the H 1 histamine receptors
and the calcium-dependent CaMK I (but not II or IV)
protein kinase; and (2) proliferate in response to H 1
histamine receptor agonists, proliferation that was blocked
by BAPTA/AM, G6976 and W-7, a CAMK inhibitor[117].
IP 3 (but not cAMP) levels were increased in small
cholangiocytes treated with HTMT dimaleate. Chronic
administration of the specific H3/H4R agonist (RAMH)
to BDL rats decreased large cholangiocyte proliferation
and cAMP levels compared to BDL rats treated with
NaCl[115,116]. This inhibition is mediated through negative
regulation of the cAMP-dependent PKA/ERK1/2
pathway[115,116].
The mechanisms by which different sized ducts
proliferate or are damaged in response to various liver
injury/toxins (e.g., BDL, partial hepatectomy, vagotomy,
feeding of ANIT, bile acids or CCl4)[3,26,47-52] are unclear.
Furthermore, the pathophysiology of small cholangiocytes
is undefined in these models. Based upon preliminary
data and unpublished observations from our laboratory,
we propose that neural/hormonal-dependent (cholinergic
and adrenergic) activation of the Ca2+-dependent NFAT
(Nuclear Factor of Activated T-lymphocytes) stimulates
the proliferative response of small cholangiocytes,
whereas neural/hormonal-dependent activation of the
cAMP-dependent CREB stimulates the proliferation of
large cholangiocytes. NFAT is a ubiquitous transcription
factor that was initially described in T-lymphocytes.
Five isoforms of NFAT have been identified. Four of
these isoforms (NFATc1 to c4) are regulated by Ca 2+
signaling[118]. Preliminary data shows that Ca2+-dependent
activation of NFATc1/c4 stimulates the proliferation
of small cholangiocytes after CCl 4-induced damage of
cAMP-responsive large bile ducts[119]. Specifically, we have
shown that small but not large normal rat cholangiocytes
express the NFAT isoforms, NFAT c1 and c4[119]. CCl4
both in vivo and in vitro increased small cholangiocyte
proliferation that was blocked by BAPTA/AM and
11R-VIVIT (NFAT inhibitor peptide)[120]. Furthermore,
unpublished data from our laboratory show that the de novo
growth of small cholangiocytes is regulated via adrenergic
stimulation of Ca2+-dependent activation of NFATc1/c4
(Ca2+/calcineurin) and Sp1 (Ca2+/PKC). NFAT and Sp1
cooperatively interact to regulate proliferative phenotypes
in other cell types[121].
Recent studies have shown that bile acids have cyto-
protective effects against apoptosis in large cholangiocytes.
Feeding of taurocholate to BDL rats (treated with a single
dose of CCl 4) prevents CCl 4-induced damage of large
cholangiocytes, whereas small cholangiocytes (which are
de novo activated following CCl4-induced damage of large
ducts)[50,51] remained mitotically dormant and unresponsive
to secretin (Figure 3)[122]. In vitro, taurocholate prevented the
inhibitory effects of CCl4 on apoptotic, proliferative and
secretory capacity of large BDL cholangiocytes[122]. The
protective effects of taurocholate against CCl4-induced
damage of large BDL cholangiocytes are due to the
 Glaser S et al. Heterogeneity of bile ducts 3531
activation of PI3-K and AKT expression[122]. Furthermore,
feeding of taurocholate to BDL + vagotomy rats prevented
vagotomy activation of large cholangiocyte apoptosis
and inhibition of large cholangiocyte growth[123], effects
that were abolished by wortmannin, a PI3-K inhibitor[124].
Functional ASBT expression as well as phosphorylation
of Akt were reduced by vagotomy but restored by
taurocholate feeding[123]. Chronic feeding of taurocholate
prevented the increase in cholangiocyte apoptosis and the
damage of large cholangiocyte proliferation induced by
adrenergic denervation by 6-OHDA administration[125].
Taurocholate effects are mediated by the PI3K pathway,
since the simultaneous administration of wortmannin
reverses such effects [125] . In addition, the feeding of
ursodeoxycholate and tauroursodeoxycholate to BDL +
vagotomy rats prevented the activation of apoptosis and
the loss of proliferation of large cholangiocytes observed
in this model[126]. In this study[126], the protective effects of
these two bile acids were neutralized by the simultaneous
administration of BAPTA/AM (an intracellular Ca 2+
chelator) [72] or G6976 (a PKC inhibitor) [65] . Both
ursodeoxycholate and tauroursodeoxycholate increased IP3
and Ca2+ levels, together with enhanced phosphorylation
of PKC- [126] . The data sug gests that bile acids are
important in modulating large cholangiocyte proliferation
in denervated livers.
Heterogeneity in Cholangiopathies
Chronic cholestatic liver diseases (cholangiopathies), which
target intrahepatic and extrahepatic bile ducts, are charac-
terized by the coexistence of cholangiocyte growth/apop-
tosis, inflammation and fibrosis[3,127]. Cholangiopathies dif-
ferentially target the biliary epithelium with heterogeneous
proliferative and apoptotic responses of different sized
ducts[3,47,50,128-130]. Primary biliary cirrhosis is characterized
by the selective proliferation/loss of small interlobular
bile ducts[3,131]. Some studies demonstrated that damage
of interlobular bile ducts is immune mediated[3,132]. The
origin of primary sclerosing cholangitis (PSC), which is
associated with inflammation and fibrosis of bile ducts,
originates from multiple factors including autoimmune,
bacterial, congenital, drug, or viral agents[3,73]. PSC affects
mainly extrahepatic and interlobular or septal bile ducts al-
though smaller bile ducts can be affected[3,73]. Patients with
small duct PSC seem to have a good prognosis in terms
of survival and development of cholangiocarcinoma[133].
Cholangiocarcinoma occurs frequently in patients with
PSC and targets mainly the major bile duct bifurcation[3,134].
Peripheral cholangiocarcinoma occur within the liver rath-
er than within large bile ducts may arise from small bile
ducts[3,134]. Mutations in the CFTR gene are responsible
for causing the human biliary disease, cystic fibrosis, due
to defective transport of water and chloride presumably
by large cholangiocytes expressing CFTR[135]. Our previous
studies in rodent liver has shown that CFTR is expressed
principally in large cholangiocytes and in bile ducts greater
than 15 M diameter[12,13] but in studies of human liver of
cystic fibrosis patients, CFTR was expressed in both large
and small ducts[136].
Defective chloride transport and chloridemediated
bile secretion by large cholangiocytes may be respon-
sible for the reduced fluidity and alkalinity of bile,
leading to bile duct damage. Ca 2+-dependent Cl - cha-
nnels [137,138] (presumably expressed by both small and
large cholangiocytes) may be able to secrete bile, thus
compensating for loss of CFTR functional activity of
CFTR in large cholangiocytes [54]. In polycystic kidney
liver disease (PKLD), the genetic defect results in the
growth of multiple epithelial cysts within the renal, liver
parenchyma and intrahepatic bile ducts[139]. The disease
targets presumably large bile ducts since the cystic ductal
cells also secrete Cl- and HCO3- (as normal large cholangi
ocytes)[2,3,54,71,73] but the secretion is diminished, likely due
to reduced Cl-/HCO3- exchanger activity in cystic ductal
cells as compared with normal cholangiocytes[139]. Biliary
atresia, which is the most common reason of cholestasis in
infants and children, is a destructive, inflammatory process
of the extrahepatic bile ducts but as the disease progresses
smaller intrahepatic bile ducts are also involved[140]. The
pathogenesis of biliary atresia is unknown but infections
or toxic agents combined with genetic/immunologic
susceptibility have been proposed[3, 141, 142].
Summary
In this review, we have summarized the findings demon-
strating that the intrahepatic biliar y epithelium is
heterogeneous regarding: (1) morphological characteristics,
vascularization and innervation; (2) secretory activity
in response to gastrointestinal hor mones/peptides,
nerve receptor agonists and bile salts; and (3) apoptotic
and proliferative responses to liver injury/toxins and
gastrointestinal hormones/peptides. Specifically, the
intrahepatic biliary epithelium is formed by bile ducts
of different sizes with small ducts lined by small
cholangiocytes, whereas larger ducts are lined by larger
cholangiocytes[12-14]. Following a general background on
cholangiocyte functions, we discussed the in vivo and in
vitro experimental models that allowed us to demonstrate
that the biliar y epithelium is morphologically and
functionally heterogeneous. Following a brief review on
the heterogeneous distribution of non-transport related
proteins, we discussed the secretory functions of small and
large cholangiocytes. While large cholangiocytes secrete
water and electrolytes[12,13,48] by changes in cAMP/PKA/
CFTR/Cl-/HCO3-, small cholangiocytes may secrete bile
by a transduction pathway (different from that observed
in large cholangiocytes)[12,13,48] involving activation of IP3/
Ca2+/PKC. We have presented data demonstrating that
small and large cholangiocytes differentially proliferate
or are damaged in response to liver injury/toxins. Small
and large ducts also differ regarding the proliferative and
apoptotic responses to liver injury/toxins[2,71,73]. We propose
that activation of the Ca2+-dependent NFAT stimulates
the proliferation of small cholangiocytes, whereas neural/
hormonal-dependent activation of the cAMP-dependent
CREB stimulates the proliferation of large cholangiocytes.
In the last part of the review, we have briefly outlined the
heterogeneity of the biliary epithelium in relationship to
chronic cholestatic liver diseases targeting different sized
ducts.
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 3532 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Future Perspectives
The concept that the biliary epithelium is functionally het-
erogeneous is clinically relevant since in chronic cholestatic
liver diseases cholangiocyte proliferation/damage is an
event restricted to a specific duct size. Further studies are
needed for understanding the pathophysiology of small
cholangiocytes in the overall contribution of the functions
of the biliary epithelium. However, some preliminary stud-
ies from our laboratory suggest that small cholangiocytes
secrete bile (by a IP3/Ca2+/PKC-dependent mechanism)
and proliferate by activation of the Ca2+-dependent tran-
scription factor, NFAT. Further studies are necessary to
evaluate the role of the nervous system in the regulation
of the heterogeneous secretory, apoptotic and proliferative
responses of different sized bile ducts to gastrointestinal
hormones, injury/toxins and viruses. Since PBP prolifera-
tion is observed only in large proliferating cholangiocytes
from BDL rats, we propose that blood supply and circulat-
ing factors (e.g., vascular endothelial growth factor and pla-
cental growth factor) may be important in the regulation
of the heterogeneous response of cholangiocytes to liver
injury/toxins.
REFERENCES
1 YOKOYAMA HO, WILSON ME, TSUBOI KK, STOWELL RE.
Regeneration of mouse liver after partial hepatectomy. Cancer
Res 1953; 13: 80-85
2 Kanno N, LeSage G, Glaser S, Alvaro D, Alpini G. Functional
heterogeneity of the intrahepatic biliary epithelium. Hepatology
2000; 31: 555-561
3 Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary
epithelia. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Jako-
by W, Schachter D, Shafritz DA, eds. The Liver: Biology &
Pathobiology. 4th ed. Philadelphia, PA: Lippincott Williams &
Wilkins, 2001: 421-435
4 Nathanson MH, Boyer JL. Mechanisms and regulation of bile
secretion. Hepatology 1991; 14: 551-566
5 Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholan-
giocyte bicarbonate secretion. Am J Physiol Gastrointest Liver
Physiol 2001; 281: G612-G625
6 Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology
in rats with bile ductular cell hyperplasia. Evidence for a se-
cretory function of proliferated bile ductules. J Clin Invest 1988;
81: 569-578
7 Glaser SS, Rodgers RE, Phinizy JL, Robertson WE, Lasater J,
Caligiuri A, Tretjak Z, LeSage GD, Alpini G. Gastrin inhibits
secretin-induced ductal secretion by interaction with spe-
cific receptors on rat cholangiocytes. Am J Physiol 1997; 273:
G1061-G1070
8 Ludwig J. New concepts in biliary cirrhosis. Semin Liver Dis
1987; 7: 293-301
9 Ludwig J, Ritman EL, LaRusso NF, Sheedy PF, Zumpe G.
Anatomy of the human biliary system studied by quantitative
computer-aided three-dimensional imaging techniques. Hepa-
tology 1998; 27: 893-899
10 SCHAFFNER F, POPPER H. Electron microscopic studies of
normal and proliferated bile ductules. Am J Pathol 1961; 38:
393-410
11 CARRUTHERS JS, STEINER JW. Studies on the fine structure
of proliferated bile ductules. I. Changes of cytoarchitecture of
biliary epithelial cells. Can Med Assoc J 1961; 85: 1223-1236
12 Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV,
LeSage G, LaRusso NF. Morphological, molecular, and func-
tional heterogeneity of cholangiocytes from normal rat liver.
Gastroenterology 1996; 110: 1636-1643
13 Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, La-
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
sater J, LeSage GD. Large but not small intrahepatic bile ducts
are involved in secretin-regulated ductal bile secretion. Am J
Physiol 1997; 272: G1064-G1074
14 Benedetti A, Bassotti C, Rapino K, Marucci L, Jezequel AM. A
morphometric study of the epithelium lining the rat intrahe-
patic biliary tree. J Hepatol 1996; 24: 335-342
15 Masyuk TV, Ritman EL, LaRusso NF. Quantitative assessment
of the rat intrahepatic biliary system by three-dimensional re-
construction. Am J Pathol 2001; 158: 2079-2088
16 Phillips MJ, Poucell S, Patterson J, Valencia P. The normal
liver. In: Phillips MJ, Powell S, Patterson S, Valencia P, eds.
The liver: an atlas and text of ultrastructural pathology. New
York, NY: Raven Press, 1987: 1-35
17 LaRusso NF, Ishii M, Vroman BT. The ins and outs of mem-
brane movement in biliary epithelia. Trans Am Clin Climatol
Assoc 1991; 102: 245-258; discussion 258-259
18 Ishii M, Vroman B, LaRusso NF. Isolation and morphologic
characterization of bile duct epithelial cells from normal rat
liver. Gastroenterology 1989; 97: 1236-1247
19 Vroman B, LaRusso NF. Development and characterization of
polarized primary cultures of rat intrahepatic bile duct epithe-
lial cells. Lab Invest 1996; 74: 303-313
20 Masyuk TV, Huang BQ, Ward CJ, Masyuk AI, Yuan D, Splin-
ter PL, Punyashthiti R, Ritman EL, Torres VE, Harris PC,
LaRusso NF. Defects in cholangiocyte fibrocystin expression
and ciliary structure in the PCK rat. Gastroenterology 2003; 125:
1303-1310
21 Ishii M, Vroman B, LaRusso NF. Morphologic demonstration
of receptor-mediated endocytosis of epidermal growth factor
by isolated bile duct epithelial cells. Gastroenterology 1990; 98:
1284-1291
22 Elsing C, Hbner C, Fitscher BA, Kassner A, Stremmel W.
Muscarinic acetylcholine receptor stimulation of biliary epithe-
lial cells and its effect on bile secretion in the isolated perfused
liver [corrected]. Hepatology 1997; 25: 804-813
23 Alvaro D, Alpini G, Jezequel AM, Bassotti C, Francia C, Fraioli
F, Romeo R, Marucci L, Le Sage G, Glaser SS, Benedetti A.
Role and mechanisms of action of acetylcholine in the regula-
tion of rat cholangiocyte secretory functions. J Clin Invest 1997;
100: 1349-1362
24 Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD, Gla-
ser SS, Francis H, Mancino MG, Ueno Y, Barbaro B, Benedetti
A, Attili AF, Alvaro D. Nerve growth factor modulates the
proliferative capacity of the intrahepatic biliary epithelium in
experimental cholestasis. Gastroenterology 2004; 127: 1198-1209
25 Glaser S, Alvaro D, Roskams T, Phinizy JL, Stoica G, Francis H,
Ueno Y, Barbaro B, Marzioni M, Mauldin J, Rashid S, Mancino
MG, LeSage G, Alpini G. Dopaminergic inhibition of secretin-
stimulated choleresis by increased PKC-gamma expression
and decrease of PKA activity. Am J Physiol Gastrointest Liver
Physiol 2003; 284: G683-G694
26 LeSagE G, Alvaro D, Benedetti A, Glaser S, Marucci L, Baioc-
chi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis H,
Alpini G. Cholinergic system modulates growth, apoptosis,
and secretion of cholangiocytes from bile duct-ligated rats.
Gastroenterology 1999; 117: 191-199
27 Barbaro B, Glaser S, Francis H, Taffetani S, Marzioni M, LeS-
age G, Alpini GG. Nerve regulation of cholangiocyte func-
tions. In: Alpini G, Alvaro D, LeSage G, Marzioni M, LaRusso
NF, eds. Pathophysiology of the Bile Duct System. George-
town, Texas, USA: Landes Biosciences, 2004: 199-209
28 Reilly FD, McCuskey PA, McCuskey RS. Intrahepatic distri-
bution of nerves in the rat. Anat Rec 1978; 191: 55-67
29 Tsuneki K, Ichihara K. Electron microscope study of verte-
brate liver innervation. Arch Histol Jpn 1981; 44: 1-13
30 Gulbenkian S, Wharton J, Hacker GW, Varndell IM, Bloom
SR, Polak JM. Co-localization of neuropeptide tyrosine (NPY)
and its C-terminal flanking peptide (C-PON). Peptides 1985; 6:
1237-1243
31 Lundberg JM, Terenius L, Hkfelt T, Martling CR, Tatemoto
K, Mutt V, Polak J, Bloom S, Goldstein M. Neuropeptide Y
(NPY)-like immunoreactivity in peripheral noradrenergic neu-
rons and effects of NPY on sympathetic function. Acta Physiol
 Glaser S et al. Heterogeneity of bile ducts 3533
Scand 1982; 116: 477-480
32 Costa M, Furness JB. Somatostatin is present in a subpopula-
tion of noradrenergic nerve fibres supplying the intestine.
Neuroscience 1984; 13: 911-919
33 Gibbins IL, Furness JB, Costa M, MacIntyre I, Hillyard CJ,
Girgis S. Co-localization of calcitonin gene-related peptide-like
immunoreactivity with substance P in cutaneous, vascular and
visceral sensory neurons of guinea pigs. Neurosci Lett 1985; 57:
125-130
34 Jul Y, Clerc N, Niel JP, Condamin M. [Met]- and [Leu]enke-
phalin-like immunoreactive cell bodies and nerve fibres in the
coeliac ganglion of the cat. Neuroscience 1986; 18: 487-498
35 Schultzberg M, Dalsgaard CJ. Enteric origin of bombesin im-
munoreactive fibres in the rat coeliac-superior mesenteric gan-
glion. Brain Res 1983; 269: 190-195
36 Burt AD, Tiniakos D, MacSween RN, Griffiths MR, Wisse
E, Polak JM. Localization of adrenergic and neuropeptide
tyrosine-containing nerves in the mammalian liver. Hepatology
1989; 9: 839-845
37 el-Salhy M, Stenling R, Grimelius L. Peptidergic innervation
and endocrine cells in the human liver. Scand J Gastroenterol
1993; 28: 809-815
38 Fava G, Glaser S, Francis H, Phinizy JL, Venter J, Reichenbach
R, Taffetani S, Marzioni M, Marucci L, Benedetti A, Alpini G.
Neuropeptide Y (NPY) inhibits cholangiocarcinoma growth
by interaction with a G-protein coupled receptor by Ca 2+-
dependent modulation of Src/ERK1/2 phosphorylation. Gas-
troenterology 2004; 126: AT926
39 Inoue N, Sakai H, Magari S, Sakanaka M. Distribution and
possible origins of substance P-containing nerve fibers in the
rat liver. Ann Anat 1992; 174: 557-560
40 Goehler LE, Sternini C, Brecha NC. Calcitonin gene-related
peptide immunoreactivity in the biliary pathway and liver of
the guinea-pig: distribution and colocalization with substance
P. Cell Tissue Res 1988; 253: 145-150
41 Akiyoshi H, Gonda T, Terada T. A comparative histochemical
and immunohistochemical study of aminergic, cholinergic and
peptidergic innervation in rat, hamster, guinea pig, dog and
human livers. Liver 1998; 18: 352-359
42 Ohtani O, Kikuta A, Ohtsuka A, Taguchi T, Murakami T.
Microvasculature as studied by the microvascular corrosion
casting/scanning electron microscope method. I. Endocrine
and digestive system. Arch Histol Jpn 1983; 46: 1-42
43 Terada T, Ishida F, Nakanuma Y. Vascular plexus
around intrahepatic bile ducts in normal livers and portal
hypertension. J Hepatol 1989; 8: 139-149
44 Gaudio E, Onori P, Pannarale L, Alvaro D. Hepatic
microcirculation and peribiliary plexus in experimental biliary
cirrhosis: a morphological study. Gastroenterology 1996; 111:
1118-1124
45 Yamamoto K, Phillips MJ. A hitherto unrecognized bile
ductular plexus in normal rat liver. Hepatology 1984; 4: 381-385
46 Masyuk TV, Ritman EL, LaRusso NF. Hepatic artery and
portal vein remodeling in rat liver: vascular response to
selective cholangiocyte proliferation. Am J Pathol 2003; 162:
1175-1182
47 Lesage G, Glaser S, Ueno Y, Alvaro D, Baiocchi L, Kanno N,
Phinizy JL, Francis H, Alpini G. Regression of cholangiocyte
proliferation after cessation of ANIT feeding is coupled with
increased apoptosis. Am J Physiol Gastrointest Liver Physiol
2001; 281: G182-G190
48 Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri
A, LeSage G, LaRusso NF. Heterogeneity of the proliferative
capacity of rat cholangiocytes after bile duct ligation. Am J
Physiol 1998; 274: G767-G775
49 Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL, Francis H,
Lesage G. Bile acid feeding increased proliferative activity and
apical bile acid transporter expression in both small and large
rat cholangiocytes. Hepatology 2001; 34: 868-876
50 LeSage GD, Glaser SS, Marucci L, Benedetti A, Phinizy JL,
Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM,
Holcomb LA, Alpini G. Acute carbon tetrachloride feeding
induces damage of large but not small cholangiocytes from
BDL rat liver. Am J Physiol 1999; 276: G1289-G1301
51 LeSage GD, Benedetti A, Glaser S, Marucci L, Tretjak Z,
Caligiuri A, Rodgers R, Phinizy JL, Baiocchi L, Francis H,
Lasater J, Ugili L, Alpini G. Acute carbon tetrachloride feeding
selectively damages large, but not small, cholangiocytes from
normal rat liver. Hepatology 1999; 29: 307-319
52 LeSage G, Glaser S, Robertson W, Phinizy JL, Rodgers R,
Alpini G. Partial hepatectomy induces proliferative and
secretory events in small cholangiocytes. Gastroenterology 1996;
110: A1250
53 Alpini G, Ulrich CD 2nd, Phillips JO, Pham LD, Miller
LJ, LaRusso NF. Upregulation of secretin receptor gene
expression in rat cholangiocytes after bile duct ligation. Am J
Physiol 1994; 266: G922-G928
54 Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV,
Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and
functional heterogeneity of cholangiocytes from rat liver after
bile duct ligation. Am J Physiol 1997; 272: G289-G297
55 Tietz PS, Alpini G, Pham LD, Larusso NF. Somatostatin
inhibits secretin-induced ductal hypercholeresis and exocytosis
by cholangiocytes. Am J Physiol 1995; 269: G110-G118
56 Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin
stimulates bicarbonate secretion from rat cholangiocytes:
implications for neural regulation of bile secretion.
Gastroenterology 1997; 113: 311-321
57 Cho WK. Role of the neuropeptide, bombesin, in bile
secretion. Yale J Biol Med 1997; 70: 409-416
58 Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a
potent regulator of bile secretion from rat cholangiocytes.
Gastroenterology 1999; 117: 420-428
59 Caligiuri A, Glaser S, Rodgers RE, Phinizy JL, Robertson W,
Papa E, Pinzani M, Alpini G. Endothelin-1 inhibits secretin-
stimulated ductal secretion by interacting with ETA receptors
on large cholangiocytes. Am J Physiol 1998; 275: G835-G846
60 Alvaro D, Benedetti A, Marucci L, Delle Monache M,
Monterubbianesi R, Di Cosimo E, Perego L, Macarri G, Glaser
S, Le Sage G, Alpini G. The function of alkaline phosphatase
in the liver: regulation of intrahepatic biliary epithelium
secretory activities in the rat. Hepatology 2000; 32: 174-184
61 Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis
H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD.
Bile acid feeding induces cholangiocyte proliferation and
secretion: evidence for bile acid-regulated ductal secretion.
Gastroenterology 1999; 116: 179-186
62 Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Francis
H, Phinizy JL, Angelico M, Lesage G. Ursodeoxycholate
and tauroursodeoxycholate inhibit cholangiocyte growth
and secretion of BDL rats through activation of PKC alpha.
Hepatology 2002; 35: 1041-1052
63 Baiocchi L, Alpini G, Glaser S, Angelico M, Alvaro D,
Francis H, Marzioni M, Phinizy JL, Barbaro B, LeSage G.
Taurohyodeoxycholate- and tauroursodeoxycholate-induced
hypercholeresis is augmented in bile duct ligated rats. J
Hepatol 2003; 38: 136-147
64 Baiocchi L, LeSage G, Glaser S, Alpini G. Regulation of
cholangiocyte bile secretion. J Hepatol 1999; 31: 179-191
65 LeSage GD, Alvaro D, Glaser S, Francis H, Marucci L,
Roskams T, Phinizy JL, Marzioni M, Benedetti A, Taffetani
S, Barbaro B, Fava G, Ueno Y, Alpini G. Alpha-1 adrenergic
receptor agonists modulate ductal secretion of BDL rats via
Ca(2+)- and PKC-dependent stimulation of cAMP. Hepatology
2004; 40: 1116-1127
66 Francis H, Glaser S, Alvaro D, Taffetani S, Marucci L,
Benedetti A, Ueno Y, Marzioni M, LeSage G, Venter J,
Baumann B, Phinizy JL, Alpini G. The -2 adrenergic receptor
agonist, UK14,304, inhibits secretin-stimulated ductal secretion
of bile duct ligated (BDL) rats by activation of the G-protein
Gi. Hepatology 2003; 38: A1088
67 Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A,
Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J,
Fava G, Lesage GD, Alpini G. Autocrine/paracrine regulation
of the growth of the biliary tree by the neuroendocrine
hormone serotonin. Gastroenterology 2005; 128: 121-137
www.wjgnet.com
 3534 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
68 LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte
proliferation. Liver 2001; 21: 73-80
69 Alvaro D, Alpini G, Onori P, Franchitto A, Glaser S, Le Sage G,
Gigliozzi A, Vetuschi A, Morini S, Attili AF, Gaudio E. Effect
of ovariectomy on the proliferative capacity of intrahepatic rat
cholangiocytes. Gastroenterology 2002; 123: 336-344
70 Francis H, Glaser S, Ueno Y, Lesage G, Marucci L, Benedetti
A, Taffetani S, Marzioni M, Alvaro D, Venter J, Reichenbach R,
Fava G, Phinizy JL, Alpini G. cAMP stimulates the secretory
and proliferative capacity of the rat intrahepatic biliary
epithelium through changes in the PKA/Src/MEK/ERK1/2
pathway. J Hepatol 2004; 41: 528-537
71 Glaser S, Francis H, Marzioni M, Taffetani S, Phinizy
JL, LeSage G, Alpini G. Functional heterogeneity of the
intrahepatic biliary epithelium. In: Alpini G, Alvaro D, LeSage
G, Marzioni M, LaRusso NF, eds. Pathophysiology of the Bile
Duct System. Georgetown, Texas, USA: Landes Biosciences
2004: 245-254
72 Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno
N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G,
Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-
ligated rats by interaction with cholecystokinin-B/Gastrin
receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and
protein kinase C alpha-dependent mechanisms. Hepatology
2000; 32: 17-25
73 Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G,
Alpini G. Functional heterogeneity of cholangiocytes. Semin
Liver Dis 2002; 22: 227-240
74 Alvaro D, Mennone A, Boyer JL. Role of kinases and
phosphatases in the regulation of fluid secretion and Cl-/
HCO 3- exchange in cholangiocytes. Am J Physiol 1997; 273:
G303-G313
75 Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima
K, Glaser S, LeSage G, Shimosegawa T. Evaluation of
differential gene expression by microarray analysis in small
and large cholangiocytes isolated from normal mice. Liver Int
2003; 23: 449-459
76 Alpini G, Phillips JO, Vroman B, LaRusso NF. Recent
advances in the isolation of liver cells. Hepatology 1994; 20:
494-514
77 Lesage GD, Marucci L, Alvaro D, Glaser SS, Benedetti A,
Marzioni M, Patel T, Francis H, Phinizy JL, Alpini G. Insulin
inhibits secretin-induced ductal secretion by activation of
PKC alpha and inhibition of PKA activity. Hepatology 2002; 36:
641-651
78 Okada Y, Jinno K, Moriwaki S, Shimoe T, Tsuji T, Murakami
M, Thurin J, Koprowski H. Blood group antigens in the
intrahepatic biliary tree. I. Distribution in the normal liver. J
Hepatol 1988; 6: 63-70
79 Terada T, Kono N, Nakanuma Y. Immunohistochemical
and immunoelectron microscopic analyses of alpha-amylase
isozymes in human intrahepatic biliary epithelium and
hepatocytes. J Histochem Cytochem 1992; 40: 1627-1635
80 Terada T, Morita T, Hoso M, Nakanuma Y. Pancreatic
enzymes in the epithelium of intrahepatic large bile ducts
and in hepatic bile in patients with extrahepatic bile duct
obstruction. J Clin Pathol 1994; 47: 924-927
81 Mathis GA, Walls SA, DAmico P, Gengo TF, Sirica AE. En-
zyme profile of rat bile ductular epithelial cells in reference to
the resistance phenotype in hepatocarcinogenesis. Hepatology
1989; 9: 477-485
82 Martnez-Ans E, Castillo JE, Dez J, Medina JF, Prieto J. Im-
munohistochemical detection of chloride/bicarbonate anion
exchangers in human liver. Hepatology 1994; 19: 1400-1406
83 Hofmann AF. Current concepts of biliary secretion. Dig Dis
Sci 1989; 34: 16S-20S
84 Lamri Y, Erlinger S, Dumont M, Roda A, Feldmann G. Im-
munoperoxidase localization of ursodeoxycholic acid in rat
biliary epithelial cells. Evidence for a cholehepatic circulation.
Liver 1992; 12: 351-354
85 Gurantz D, Hofmann AF. Influence of bile acid structure on
bile flow and biliary lipid secretion in the hamster. Am J Physi-
ol 1984; 247: G736-G748
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
86 Aldini R, Roda A, Lenzi PL, Ussia G, Vaccari MC, Mazzella G,
Festi D, Bazzoli F, Galletti G, Casanova S. Bile acid active and
passive ileal transport in the rabbit: effect of luminal stirring.
Eur J Clin Invest 1992; 22: 744-750
87 Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC,
Levine S, Dawson PA, LaRusso NF. Rat cholangiocytes absorb
bile acids at their apical domain via the ileal sodium-depen-
dent bile acid transporter. J Clin Invest 1997; 100: 2714-2721
88 Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE,
Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional ex-
pression of the apical Na+-dependent bile acid transporter in
large but not small rat cholangiocytes. Gastroenterology 1997;
113: 1734-1740
89 Alpini G, Glaser S, Robertson W, Phinizy JL, Rodgers RE,
Caligiuri A, LeSage G. Bile acids stimulate proliferative and
secretory events in large but not small cholangiocytes. Am J
Physiol 1997; 273: G518-G529
90 Francis H, Glaser S, Ueno Y, Venter J, Reichenbach R, Sum-
mers R, Alpini G. Novel evidence for the activation of the
growth of small (but not large) murine cholangiocytes by
Interaction with H1 histamine Receptors. Hepatology 2005; 42:
A1145
91 Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H,
Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, Lesage
G. Bile acid depletion and repletion regulate cholangiocyte
growth and secretion by a phosphatidylinositol 3-kinase-de-
pendent pathway in rats. Gastroenterology 2002; 123: 1226-1237
92 Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Franchitto
A, Onori P, Ueno Y, Marzioni M, Fava G, Venter J, Reichenba-
ch R, Summers R, Alpini G. Administration of r-VEGF-A pre-
vents hepatic artery ligation-induced bile duct damage in bile
duct ligated rats. Am J Physiol Gastrointest Liver Physiol 2006;
291: G307-G317
93 Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Ueno Y,
Meininger CJ, Franchitto A, Onori P, Marzioni M, Taffetani
S, Fava G, Stoica G, Venter J, Reichenbach R, De Morrow S,
Summers R, Alpini G. Vascular endothelial growth factor
stimulates rat cholangiocyte proliferation via an autocrine
mechanism. Gastroenterology 2006; 130:1270-1282
94 Fava G, Glaser S, Phinizy JL, Francis H, Marucci L, Benedetti
A, Taffetani S, Venter J, Baumann B, Reichenbach R, Alpini G.
Thyroid hormone inhibits cAMP dependent proliferation of
cholangiocytes from bile duct ligated rats by a IP3/Ca2+/PKC-
dependent mechanism. Hepatology 2003; 38: A1097
95 Glaser S, Katki K, Supowit S, Francis H, Ueno Y, Venter J,
Reichenbach R, Dickerson I, Summers R, Chiasson V, DiPette
DJ, Alpini G. Alpha-calcitonin gene-related peptide (Alpha-
CGRP) stimulates the proliferation of large cholangiocytes
during obstructive cholestasis induced by bile duct ligation
(BDL) via cAMP-dependent activation of MAPK p38. Hepatol-
ogy 2005; 42: A13
96 Supowit SC, Zhao H, DiPette DJ. Nerve growth factor en-
hances calcitonin gene-related peptide expression in the spon-
taneously hypertensive rat. Hypertension 2001; 37: 728-732
97 Servillo G, Della Fazia MA, Sassone-Corsi P. Coupling cAMP
signaling to transcription in the liver: pivotal role of CREB and
CREM. Exp Cell Res 2002; 275: 143-154
98 Moriuchi A, Ido A, Nagata Y, Nagata K, Uto H, Hasuike S,
Hori T, Hirono S, Hayashi K, Tsubouchi H. A CRE and the re-
gion occupied by a protein induced by growth factors contrib-
ute to up-regulation of cyclin D1 expression in hepatocytes.
Biochem Biophys Res Commun 2003; 300: 415-421
99 Michalopoulos GK, DeFrances MC. Liver regeneration. Sci-
ence 1997; 276: 60-66
100 Choi EJ, Xia Z, Villacres EC, Storm DR. The regulatory diver-
sity of the mammalian adenylyl cyclases. Curr Opin Cell Biol
1993; 5: 269-273
101 Andrisani OM. CREB-mediated transcriptional control. Crit
Rev Eukaryot Gene Expr 1999; 9: 19-32
102 Alvaro D, Onori P, Metalli VD, Svegliati-Baroni G, Folli F,
Franchitto A, Alpini G, Mancino MG, Attili AF, Gaudio E.
Intracellular pathways mediating estrogen-induced cholangio-
cyte proliferation in the rat. Hepatology 2002; 36: 297-304
 Glaser S et al. Heterogeneity of bile ducts 3535
103 Seto-Young D, Zajac J, Liu HC, Rosenwaks Z, Poretsky L. The
role of mitogen-activated protein kinase in insulin and insulin-
like growth factor I (IGF-I) signaling cascades for progesterone
and IGF-binding protein-1 production in human granulosa
cells. J Clin Endocrinol Metab 2003; 88: 3385-3391
104 Clawson GA. Mechanisms of carbon tetrachloride hepatotox-
icity. Pathol Immunopathol Res 1989; 8: 104-112
105 Lakehal F, Wendum D, Barbu V, Becquemont L, Poupon R,
Balladur P, Hannoun L, Ballet F, Beaune PH, Housset C. Phase
I and phase II drug-metabolizing enzymes are expressed and
heterogeneously distributed in the biliary epithelium. Hepatol-
ogy 1999; 30: 1498-1506
106 Katayanagi K, Van de Water J, Kenny T, Nakanuma Y, An-
sari AA, Coppel R, Gershwin ME. Generation of monoclonal
antibodies to murine bile duct epithelial cells: identification
of annexin V as a new marker of small intrahepatic bile ducts.
Hepatology 1999; 29: 1019-1025
107 Diakonova M, Gerke V, Ernst J, Liautard JP, van der Vusse
G, Griffiths G. Localization of five annexins in J774 macro-
phages and on isolated phagosomes. J Cell Sci 1997; 110 (Pt 10):
1199-1213
108 Kurosawa H, Que FG, Roberts LR, Fesmier PJ, Gores GJ. He-
patocytes in the bile duct-ligated rat express Bcl-2. Am J Physiol
1997; 272: G1587-G1593
109 Charlotte F, LHermin A, Martin N, Geleyn Y, Nollet M, Gau-
lard P, Zafrani ES. Immunohistochemical detection of bcl-2
protein in normal and pathological human liver. Am J Pathol
1994; 144: 460-465
110 Nguyen T, Shapiro DA, George SR, Setola V, Lee DK, Cheng R,
Rauser L, Lee SP, Lynch KR, Roth BL, ODowd BF. Discovery
of a novel member of the histamine receptor family. Mol Phar-
macol 2001; 59: 427-433
111 Dickenson JM. Stimulation of protein kinase B and p70 S6
kinase by the histamine H1 receptor in DDT1MF-2 smooth
muscle cells. Br J Pharmacol 2002; 135: 1967-1976
112 Mitsuhashi M, Mitsuhashi T, Payan DG. Multiple signaling
pathways of histamine H2 receptors. Identification of an H2
receptor-dependent Ca 2+ mobilization pathway in human
HL-60 promyelocytic leukemia cells. J Biol Chem 1989; 264:
18356-18362
113 Garca-Sinz JA, Macas-Silva M, Olivares-Reyes A, Romero-
Avila MT. Histamine activates phosphorylase and inositol
phosphate production in guinea pig hepatocytes. Eur J Phar-
macol 1992; 227: 325-331
114 Francis H, Taffetani S, Glaser S, Venter J, Phinizy JL, Reichen-
bach R, Fava G, Alvaro D, Marucci L, Benedetti A, Marzioni
M, Alpini G. Histamine stimulates cholangiocyte proliferation
through transduction pathways involving the H1 and H2 his-
tamine receptor subtypes. Gastroenterology 2004; 126: AT925
115 Francis H, Glaser S, Venter J, Reichenbach R, Fava G, Alpini
G. H3/4 histamine receptor agonists inhibit cholangiocyte
growth in bile duct ligated (BDL) rats by negative regulation
of the cAMP-dependent PKA/ERK1/2 pathway. FASEB J
2005; 19: 480.417
116 Francis H, Glaser S, Venter J, Taffetani S, Reichenbach R, Fava
G, Marucci L, Benedetti A, Alvaro D, Summers R, Wyndham
M, Vaculin S, Alpini G. The specific H3/H4 histamine recep-
tor (HR) agonist, RAMH, inhibits cholangiocyte proliferation
in bile duct ligated (BDL) rats by negative regulation of the
cAMP- dependent PKA/ERK1/2 pathway. Hepatology 2004;
40: 468
117 Hughes K, Antonsson A, Grundstrm T. Calmodulin depen-
dence of NFkappaB activation. FEBS Lett 1998; 441: 132-136
118 Lipskaia L, Lompr AM. Alteration in temporal kinetics of
Ca2+ signaling and control of growth and proliferation. Biol
Cell 2004; 96: 55-68
119 Glaser S, Francis H, Venter J, Reichenbach R, Ueno Y, Sum-
mers R, Alpini G. De novo proliferation of small cholangiocytes
requires the activation of the calcium-dependent transcription
factor NFATc1/c4. Hepatology 2005; 42: A451
120 Cano E, Canellada A, Minami T, Iglesias T, Redondo JM. De-
polarization of neural cells induces transcription of the Down
syndrome critical region 1 isoform 4 via a calcineurin/nuclear
factor of activated T cells-dependent pathway. J Biol Chem
2005; 280: 29435-29443
121 Santini MP, Talora C, Seki T, Bolgan L, Dotto GP. Cross
talk among calcineurin, Sp1/Sp3, and NFAT in control of
p21(WAF1/CIP1) expression in keratinocyte differentiation.
Proc Natl Acad Sci U S A 2001; 98: 9575-9580
122 Marucci L, Alpini G, Glaser SS, Alvaro D, Benedetti A, Fran-
cis H, Phinizy JL, Marzioni M, Mauldin J, Venter J, Baumann
B, Ugili L, LeSage G. Taurocholate feeding prevents CCl4-
induced damage of large cholangiocytes through PI3-kinase-
dependent mechanism. Am J Physiol Gastrointest Liver Physiol
2003; 284: G290-G301
123 Marzioni M, LeSage GD, Glaser S, Patel T, Marienfeld C,
Ueno Y, Francis H, Alvaro D, Tadlock L, Benedetti A, Marucci
L, Baiocchi L, Phinizy JL, Alpini G. Taurocholate prevents the
loss of intrahepatic bile ducts due to vagotomy in bile duct-
ligated rats. Am J Physiol Gastrointest Liver Physiol 2003; 284:
G837-G852
124 Misra S, Ujhzy P, Gatmaitan Z, Varticovski L, Arias IM. The
role of phosphoinositide 3-kinase in taurocholate-induced traf-
ficking of ATP-dependent canalicular transporters in rat liver.
J Biol Chem 1998; 273: 26638-26644
125 Marzioni M, Glaser S, Francis H, Taffetani S, Marucci L, Bene-
detti A, Alvaro D, Phinizy JL, Baumann B, Venter J, Ueno Y,
Alpini G. Taurocholate feeding prevents the functional dam-
age of intrahepatic bile ducts induced by adrenergic denerva-
tion in a PI3K dependent manner. Hepatology 2003; 38: A28
126 Marzioni M, Francis H, Benedetti A, Ueno Y, Fava G, Venter J,
Reichenbach R, Mancino MG, Summers R, Alpini G, Glaser S.
Ca2+-dependent cytoprotective effects of ursodeoxycholic and
tauroursodeoxycholic acid on the biliary epithelium in a rat
model of cholestasis and loss of bile ducts. Am J Pathol 2006;
168:398-409
127 Strazzabosco M, Fabris L, Spirli C. Pathophysiology of chol-
angiopathies. J Clin Gastroenterol 2005; 39: S90-S102
128 Macdonald P, Palmer J, Kirby JA, Jones DE. Apoptosis as a
mechanism for cell surface expression of the autoantigen py-
ruvate dehydrogenase complex. Clin Exp Immunol 2004; 136:
559-567
129 Adams DH, Afford SC. Effector mechanisms of nonsuppura-
tive destructive cholangitis in graft-versus-host disease and
allograft rejection. Semin Liver Dis 2005; 25: 281-297
130 Xu WH, Ye QF, Xia SS. Apoptosis and proliferation of intrahe-
patic bile duct after ischemia-reperfusion injury. Hepatobiliary
Pancreat Dis Int 2004; 3: 428-432
131 Nakanuma Y. Necroinflammatory changes in hepatic lobules
in primary biliary cirrhosis with less well-defined cholestatic
changes. Hum Pathol 1993; 24: 378-383
132 Ishibashi H, Shimoda S, Gershwin ME. The immune response
to mitochondrial autoantigens. Semin Liver Dis 2005; 25:
337-346
133 Bjrnsson E, Boberg KM, Cullen S, Fleming K, Clausen OP,
Fausa O, Schrumpf E, Chapman RW. Patients with small duct
primary sclerosing cholangitis have a favourable long term
prognosis. Gut 2002; 51: 731-735
134 Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD.
Cholangiocarcinoma. Lancet 2005; 366: 1303-1314
135 Curry MP, Hegarty JE. The gallbladder and biliary tract in
cystic fibrosis. Curr Gastroenterol Rep 2005; 7: 147-153
136 Kinnman N, Lindblad A, Housset C, Buentke E, Scheynius A,
Strandvik B, Hultcrantz R. Expression of cystic fibrosis trans-
membrane conductance regulator in liver tissue from patients
with cystic fibrosis. Hepatology 2000; 32: 334-340
137 Schlenker T, Romac JM, Sharara AI, Roman RM, Kim SJ,
LaRusso N, Liddle RA, Fitz JG. Regulation of biliary secretion
through apical purinergic receptors in cultured rat cholangio-
cytes. Am J Physiol 1997; 273: G1108-G1117
138 Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG.
Endogenous ATP release regulates Cl- secretion in cultured
human and rat biliary epithelial cells. Am J Physiol 1999; 276:
G1391-G1400
139 Perrone RD, Grubman SA, Murray SL, Lee DW, Alper SL, Jef-
ferson DM. Autosomal dominant polycystic kidney disease
www.wjgnet.com
 3536 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

decreases anion exchanger activity. Am J Physiol 1997; 272: 10: 89-121

C1748-C1756
140 Arima T, Suita S, Shono T, Shono K, Kinugasa Y. The progres-
sive degeneration of interlobular bile ducts in biliary atresia:
an ultrastructural study. Fukuoka Igaku Zasshi 1995; 86: 58-64
141 Desmet VJ. Vanishing bile duct disorders. Prog Liver Dis 1992;
142 Poupon R, Chazouillres O, Poupon RE. Chronic cholestatic
diseases. J Hepatol 2000; 32: 129-140
143 Celli A, Que FG, Gores GJ, LaRusso NF. Glutathione depletion
is associated with decreased Bcl-2 expression and increased
apoptosis in cholangiocytes. Am J Physiol 1998; 275: G749-G757

L- Editor Pan BR E- Editor Liu WF

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 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Estrogens and the pathophysiology of the biliary tree
Domenico Alvaro, Maria Grazia Mancino, Paolo Onori, Antonio Franchitto, Gianfranco Alpini, Heather Francis,
Shannon Glaser, Eugenio Gaudio
Domenico Alvaro, Maria Grazia Mancino, Division of
Gastroenterology, University of Rome, La Sapienza, Rome,
Italy
Domenico Alvaro, University of Rome La Sapienza, Polo
Pontino, Latina, Italy
Gianfranco Alpini, Shannon Glaser, Department of Medicine,
The Texas A&M University System Health Science Center
College of Medicine, Scott & White Hospital, Temple, TX 76504,
United States
Gianfranco Alpini, Research, Central Texas Veterans Health Care
System, Scott & White Hospital, Temple, TX 76504,
United States
Gianfranco Alpini, Systems Biology and Translational Medicine,
The Texas A&M University System Health Science Center
College of Medicine, TX 76504, United States
Heather Francis, Shannon Glaser, Division of R&E, Scott &
White Hospital, Temple, TX 76504, United States
Paolo Onori, Department of Experimental Medicine, Section of
Human and Clinical Anatomy, State University of LAquila, L
Aquila, Italy
Antonio Franchitto, Eugenio Gaudio, Department of Anatomy,
University of Rome La Sapienza, Rome, Italy
Supported by MIUR grants PRIN, No.2003060498_002 and No.
2005067975_002 to Dr. Alvaro and by a grant award from Scott
& White Hospital and The Texas A&M University System Health
Science Center, a VA Merit Award, a VA Research Scholar Award
and the NIH grants DK58411 and DK062975 to Dr. Alpini
Correspondence to: Domenico Alvaro, MD, University of
Rome, La Sapienza, via R. Rossellini 51, 00137 Rome,
Italy. domenico.alvaro@uniroma1.it
Telephone: +39-06-49972023 Fax: +39-06-4453319
Received: 2006-01-25 Accepted: 2006-02-28
Abstract
The scientific framework concerning estrogen effects on
different tissues has expanded enormously during the
last decades, when estrogen receptor (ER) subtypes
were identified. Estrogens are not only essential for
the female reproductive system, but they also control
fundamental functions in other tissues including the
cardiovascular system, bone, brain and liver. Recently,
estrogens have been shown to target the biliary tree,
where they modulate the proliferative and secretory ac-
tivities of cholangiocytes, the epithelial cells lining bile
ducts. By acting on both estrogen receptors (ER-) and
(ER-) subtypes, and by activating either genomic or
non-genomic pathways, estrogens play a key role in the
complex loop of growth factors and cytokines, which
modulates the proliferative response of cholangiocytes
to damage. Specifically, estrogens activate intracellu-
World J Gastroenterol 2006 June 14; 12(22): 3537-3545
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
lar signalling cascades [ERK1/2 (extracellular regulated
kinases 1/2, PI3- kinase/AKT (phosphatidylinositol-3
kinase/AKT)] typical of growth factors such as insulin
like growth factor (IGF1), nerve growth factor (NGF)
and vascular endothelial growth factor (VEGF), thus po-
tentiating their action. In addition, estrogens stimulate
the secretion of different growth factors in proliferating
cholangiocytes. This review specifically deals with the
recent advances related to the role and mechanisms by
which estrogens modulate cholangiocyte functions in
normal and pathological conditions.
2006 The WJG Press. All rights reserved.
Key words: Estrogens; Cholangiocytes; IGF1; Prolifera-
tion; APDKD; PBC; Cholangiocarcinoma; SERMs
Alvaro D, Mancino MG, Onori P, Franchitto A, Alpini
G, Francis H, Glaser S, Gaudio E. Estrogens and the
pathophysiology of the biliary tree. World J Gastroenterol
2006; 12(22): 3537-3545
http://www.wjgnet.com/1007-9327/12/3537.asp
INTRODUCTION
The intrahepatic biliary tree is a complex three-dimension-
al network of interconnected ducts, which starts at the
level of canals of Hering, continues into intrahepatic ducts
of increasing diameter, and ends at the level of main extra-
hepatic bile ducts[1-3]. Recent studies demonstrated that the
intrahepatic biliary tree plays a critical role in many liver
functions including bile formation, regeneration, injury re-
pair, fibrosis, angiogenesis and regulation of blood flow[4,5].
Most of these events are regulated by a complex network
of neuropeptides, hormones, cytokines and growth fac-
tors, which target cholangiocytes[6,7], the epithelial cells lin-
ing the biliary tree. Cholangiocytes are the primary target
of damage in a group of chronic cholestatic liver diseases,
named cholangiopathies, with high social and economic
impact due to their high prevalence and morbidity[8]. Al-
together these pathologies represent a main indication for
liver transplantation and, in fact, in 2003, 10% of OLT in
the USA had as an indication PBC (primary biliary cirrho-
sis) and PSC (primary sclerosing cholangitis).
Although very heterogeneous in the etiology and
pathogenesis (autoimmune, infectious, vascular, drug-
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 3538 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
induced and cryptogenic)[8], the cholangiopathies share
common pathological features such as the injury of intra-
hepatic bile ducts, the proliferation of residual ducts and
the intralobular cholestasis[9]. In addition, most of these
pathologies evolve towards ductopenia that represents the
terminal stage of the disease[9]. For this reason, cholangio-
pathies have been also classified as vanishing bile duct syn-
dromes[8,9]. In the early stage of the diseases, the disappear-
ance of intrahepatic ducts is balanced by the proliferation
of the residual bile ducts[10], which tends to compensate for
the anatomical and functional loss of damaged ducts. In
fact, proliferating bile ducts display enhanced secretory ac-
tivities aimed to compensate for the impaired secretion of
injured ducts[11]. Thus, the course of these diseases is char-
acterized by a balance between damage (loss) of bile ducts
and compensatory proliferation of the residual ducts[10]. In
the terminal decompensated stages, the inefficacy of pro-
liferation to balance for the loss of intrahepatic bile ducts
leads to ductopenia, and thus to the clinical manifestations
of overt cholestasis[12]. Therefore, the design of a thera-
peutic strategy aimed at supporting an efficacious cholan-
giocyte proliferation could delay the progression toward
ductopenia and this represents a challenge for the future.
For these reasons, during the last few years, mechanisms
and agents involved in the modulation of cholangiocyte
proliferation have been extensively evaluated, especially at
the experimental level[5-7]. To this latter regard, the experi-
mental model of the common bile duct ligated (BDL) rat
was very helpful since the typical and selective cholangi-
ocyte proliferation that follows BDL induces a tremendous
expansion of cholangiocyte mass (30% of parenchymal
hepatic cells with respect to 2% in normal) thus facilitat-
ing the study of cholangiocyte pathophysiology. Thanks to
recent studies, we now know that cholangiocyte prolifera-
tion is regulated by several growth factors, hormones, neu-
ropeptides and bile salts[5-7]. One of the objectives of these
studies was related to estrogens[13-16] since, for many years,
we have become aware that the clinical appearance and
progression of different cholangiopathies are influenced
by gender and changes of estrogen status in the body[17].
Nevertheless, only in the last few years we have learned
that estrogens and their receptors influence the patho-
physiology of cholangiocytes and that this mainly occurs
during experimental and human conditions characterized
by cholangiocyte injury and proliferation[13-16]. Estrogens
have been considered for many years to play a role in the
development and progression of pathologies involving the
biliary tree[17,18]. This hypothesis was based on a number of
clinical observations including: (1) PBC, the most prevalent
acquired cholangiopathy, specifically affects females[17,19]
with a clinical presentation typically occurring during the
peri- and post-menopausal period[19-23]; (2) endocrine dys-
functions are frequent in PBC[17,19,21] including an increased
incidence of menstrual disturbance and hysterectomy[19,21]
and, the high incidence of post-menopausal osteoporosis
that is a sign of estrogenic deficiency[24] and that is correct-
ed by estrogen replacement therapy[25]; (3) chronic hepatic
rejection leading to ductopenia more frequently from male
donor into female recipient[26]; (4) in Turner syndrome, in
which liver morphology resembles that of a newborn liver,
bile duct pathology is frequent and is improved by estro-
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June 14, 2006 Volume 12 Number 22
gen replacement treatment[27]; (4) the progression of poly-
cystic liver disease is significantly influenced by female sex,
pregnancies and estrogen replacement treatment. In addi-
tion, marked alterations of estrogen hepatic metabolism
occur in cholestasis[28], which is one of the hallmarks of
cholangiopathies, leading to enhanced estradiol serum lev-
els[28], which could influence disease progression. In spite
of all these clinical considerations, the role and mechanism
by which estrogens modulate cholangiocyte functions have
been explored only in the last few years at both experimen-
tal and clinical levels[13-16].
EXPERIMENTAL STUDIES
In 1896, the British physician George Beatson, firstly
showed that oophorectomy induces the regression of
mammary tumors in a subset of premenopausal patients.
Since then, a variety of clinical and epidemiological obser-
vations have further substantiated the involvement of es-
trogens as inducers of growth and differentiation of target
cells expressing estrogen receptors (ERs)[29]. Estrogens ex-
ert their trophic action on many different target organs,
among which is the liver[30], where they modulate growth
and repair, contributing to neonatal liver growth and re-
generation after injury in adults[29]. In injured liver, ERs ex-
pression increases with respect to normal liver where ERs
are expressed at a very low level[30]. Furthermore, chronic
administration of estrogens for pharmacological purposes,
induces an enlargement of liver mass[31,32] and, after partial
hepatectomy, ERs expression in hepatocytes increases and
localizes predominantly into the nucleus with subsequent
transcription of genes involved in proliferation, thus fa-
voring the restoration of a normal liver mass[32]. Recent
findings indicate that estrogens also target the biliary tree
playing an important role in modulating cholangiocyte
proliferation[13]. Even if ER- and subtypes are also
expressed by normal rat cholangiocytes[14], their expres-
sion (especially ER-) markedly increases in proliferating
cholangiocytes of BDL rats, whereas hepatocytes, which
do not proliferate after BDL, display a ten-fold decrease
of ERs protein expression[13]. During cholestasis (after
BDL) estrogen serum levels are increased[20, 28] and this
could play a role in sustaining cholangiocyte proliferation
by a complex loop of agent among which estrogens may
act either directly or potentiating the effects of different
growth factors. In addition ER antagonists (tamoxifen, Ici
182,780) block the BDL-induced increase in intrahepatic
bile duct mass by impairing cholangiocyte proliferation
and enhancing apoptosis[13-16]. Similarly, in hepatocellular
carcinoma[33] and breast cancer cells, tamoxifen counteracts
estrogen mitogenic effect either by antagonizing estrogens
or by inducing apoptosis-related genes[33]. In cholangiocar-
cinoma cell lines[33-35], tamoxifen induces apoptosis mainly
by the activation of Fas receptor/Fas ligand pathway. In-
terestingly, the involvement of Fas antigen has been also
demonstrated in PBC and PSC[36,37], where the imbalance
between proliferation and apoptosis could play an im-
portant role in disease pathogenesis and progression. All
these findings suggest that, in BDL rats, estrogen antago-
nists could block cholangiocyte proliferation and activate
apoptosis by a Fas dependent mechanism, even if other
 Alvaro D et al. Estrogens and the biliary tree 3539
Src Shc
Raf
IGF1-R
IGF-1 Ras MAPKK
ERa
EE ERb
ERK 1/2 N
% Pos. Cholangiocytes
Figure 1 Additive effect of estrogens (EE) and insulin like growth factor-1 (IGF1)
in the modulation of cholangiocyte proliferation. EE and IGF1 play additive ef-
fects on cholangiocyte proliferation by acting at both receptor and post-receptor
levels IGF1 and EE induce, in cholangiocytes, additive increase of phosphorylated
ERK1/2.
mechanisms of activation, especially for tamoxifen, cannot
be excluded[32,33]. The concept that estrogens are positive
modulators of cholangiocyte proliferation is further sup-
ported by the fact that, in vitro[13] 17b-estradiol significantly
increases both proliferating cell nuclear antigen (PCNA)
protein expression and 3H-thymidine incorporation into
DNA and that two distinct ER antagonists (tamoxifen,
Ici 182,780) block 17b-estradiol effects[13]. In addition,
ovariectomy prevents cholangiocyte proliferation in BDL
rats[15] and causes a 3-fold decrease of bile duct mass with
respect to controls, in association with a 2.5-fold lower
expression of Er- and a 35-fold lower expression of
ER-[15]. When 17-estradiol was administered in BDL +
ovariectomy rats, bile duct proliferation was restored and
ER expression in cholangiocytes returned similar to con-
trols[15]. The proliferative effects of estrogens in target cells
expressing ER can involve both a direct genomic pathway
and an indirect nongenomic pathway where extracellular-
regulated kinases (ERK1/2 = p42/p44 MAP kinases) are
involved[38]. This has been also demonstrated in cholangi-
ocyte proliferation occurring after BDL where estrogen
administration increases the expression of phosphorylated
ERK1/2[39]. In contrast, tamoxifen decreases phosphor-
ylated ERK1/2 in association with impaired cholangiocyte
proliferation[39]. The Src/Shc/ERK1/2 cascade is typically
activated by several growth factors[40], thus suggesting that
estrogens could potentiate the effects of growth factors
by sharing similar intracellular signaling pathways. Consist-
ently, in cholangiocytes, estrogens exert additive effect with
NGF and IGF1[41,42] on modulating proliferation. As far as
IGF1 is concerned, the cooperation with estrogens (Figure
1) occurs at both receptor and post-receptor level, since
estrogens and IGF-1 increase phosphorylation of IGF1-R,
ERK and AKT, whereas the interplay between NGF and
estrogens occurs mainly at the level of PI3-kinase[42]. An-
other growth factor able to sustain cholangiocyte prolif-
eration is VEGF. Very recently, we showed that estrogens
induce VEGF synthesis and release in isolated cholangi-
ocytes and this was markedly amplified in proliferating
cholangiocytes[43]. This finding is very attractive since un-
der estrogen stimulation, proliferating cholangiocytes can
modulate, via VEGF release, their vascular supply whose
adaptive response is critical for sustaining the enhanced
functional and nutritional demands of the proliferating
biliary tree[43]. Moreover, VEGF released in the peribiliary
plexus by proliferating cholangiocytes following cholestasis
Immunohistochemistry of ER-a and ER-b in
cholangiocytes in different human pathologies
100%
ER-a d
b
0
100%
ER-b
0
n =5 n =9 n =5 n =5 n =8
Normal PBC PSC EtOH Cholangio-
Stage III Stage III Cirrhosis Carcinoma
Figure 2 Immunohistochemistry of ER- and ER- in cholangiocytes in different
human pathologies, bP < 0.01 , dP < 0.01 vs other columns.
could also favour the survival and repair of hepatocytes[43].
HUMAN STUDIES
Cholangiocytes of normal liver do not express ERs at the
immunohistochemical analysis[16] but they stain positive
for ER- and - in several pathological conditions includ-
ing PBC[16], polycystic liver disease[44] and cholangiocarci-
noma[45] (Figure 2). All these conditions are characterized
by reactive or neoplastic cholangiocyte proliferation, sug-
gesting that estrogens and their receptors may play a role
in modulating the proliferative activities of cholangiocytes
and therefore the course of these diseases.
ESTROGENS AND PBC
PBC is a chronic cholestatic liver disease, which represents
the most frequent acquired cholangiopathy[46]. Actually,
PBC is considered an autoimmune disease with a sexual
dimorphism[46]. The disease, in fact, predominantly affects
females (10:1 female/male ratio) with a typical clinical pres-
entation occurring during the peri- and post-menopausal
period[19,20,46]. The thought that estrogens can influence the
clinical course of PBC, as for most autoimmune disorders,
came from several studies showing that estrogens are able
to modulate the humoral and cellular immune response[47]
through the inhibition of Th1 pro-inflammatory cytokines
(IL-12, TNF-alpha and IFN-gamma)[48]. By switching the
immunological response toward the Th2 profile, estrogens
stimulate the production of Th2 anti-inflammatory cy-
tokines (such as IL-10, IL-4 and TGF-beta) thus potentiat-
ing the anti-inflammatory response[48]. In addition, estro-
gens can prevent oxidative stress in hepatocytes[49,50], which
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 3540 ISSN 1007-9327 CN 14-1219/ R
Primary biliary cirrhosis
ER- ER-b
Figure 3 Immunohistochemistry for ER- and ER-. Biopsies of human primary
biliary cirrhosis showing an intense positivity for both ER- and ER- in the prolif-
erating bile ducts. Orig. magn., x 20.
are injured by cholestasis[12]. In spite of all these considera-
tions, recent findings suggest that estrogens may influence
the course of PBC by directly modulating the pathophysi-
ology of cholangiocytes[13-16], which are the primary target
of damage in this disease.
In PBC, such as in other chronic cholestatic condi-
tions, estrogen serum levels are increased as a consequence
of impaired hepatic metabolism and biliary excretion of
estrogens and their metabolites[28]. Until recently, the high
serum concentration of estrogens occurring in PBC pa-
tients was considered to have a negative effect on disease
progression. For a long time, in fact, estrogens have been
used in rodents as an experimental model of intrahepatic
cholestasis[28]. Therefore, estrogen administration has been
avoided in PBC patients. Recently, however, estrogen re-
placement therapy, as osteoporosis treatment, has been
shown to be safe in PBC patients [51]. Thereafter, other
studies[25] with the same end-points (i.e. osteoporosis treat-
ment) indicate an improvement of liver serum enzymes
(including cholestasis enzymes) during estrogen adminis-
tration in PBC patients. These clinical studies allowed us
to rule out the concept that administration of estrogens in
PBC patients exerts deleterious effects on the liver but, on
the contrary, suggest that they can improve liver function
other than ameliorating bone mineral density.
Another old concept is the existence of a condition
of estrogenic dysfunction in PBC[19-21]. This concept was
based on the high frequency of menstrual abnormali-
ties and hysterectomy[21], on the increased risk of breast
cancer[22,23] and other malignancies[20], attributed to the in-
creased estrogen serum levels, and increased prevalence of
post-menopausal osteoporosis[24] which is corrected by es-
trogen replacement. However, these observations are still
the object of controversies[21-23,52,53]. The incidence of ext-
rahepatic malignancies[54], for example, in a cohort of Ital-
ian PBC patients, was shown to be lower with respect to
the general population and to what was shown for Ameri-
can and Northern European PBC patients. In the same
study, the incidence of breast cancer in PBC was compa-
rable to that expected in the general population[54]. Also
for metabolic bone disease associated with the progression
of PBC, definitive data are still lacking. Recent studies[55,56]
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
indicate that PBC is a risk condition per se for post-meno-
pausal bone disease (osteoporosis and osteopenia), which
occurs with the same incidence compared to other chronic
liver diseases. Therefore, advanced liver disease, rather that
PBC per se, represents a condition favouring the develop-
ment of osteoporosis[55,56].
Nevertheless, recent studies indicate that estrogens
play a role in the pathophysiology of PBC since both ex-
perimental and clinical studies indicate that they modulate
cholangiocyte survival and death[13-16]. Cholangiocytes lin-
ing interlobular bile ducts of PBC patients, but not normal
subjects, express both Er- and - subtypes, at the im-
munohistochemical analysis[16] (Figure 3). The ER expres-
sion varies according to different stages of disease and
correlates with markers of proliferation (PCNA) and death
(Tunel)[16]. ER- expression increases from 1% of cholan-
giocytes in PBC stage I to 12 % in stage III while, ER- is
stably high (50%-65%) in all histological stages of PBC[16].
Interestingly, in stages I-III of PBC, ER- expression cor-
relates with and co-localizes with PCNA indicating that the
expression of this receptor subtype is a typical feature of
proliferating cholangiocytes[16]. Furthermore, in stage IV of
PBC, when the maximal degree of ductopenia is reached,
cholangiocytes are negative for ER- and express the low-
est PCNA/TUNEL ratio[16]. This raises the speculation of
a relative proliferative deficiency of cholangiocytes in the
terminal ductopenic stages of PBC, which is associated
with the disappearance of ER-. In addition, ER- ex-
pression in cholangiocytes of PBC patients was markedly
lower when compared with primary sclerosing cholangitis
and alcoholic cirrhosis[16] (Figure 2), whose prevalence is
higher in male sex[9], suggesting that the female predomi-
nance of PBC and the typical post-menopausal presenta-
tion could be related to a defect in proliferative response
of cholangiocytes to estrogens. These findings could have
important therapeutic implication, since the modulation of
ERs could delay the progression of PBC toward ductope-
nia. During the last decade, estrogens have been involved
in the pathogenesis of different diseases including athero-
sclerosis, Alzheimer disease, multiple sclerosis, Parkinson
disease and obesity[57-61]. Estrogens, in fact, play biological
activities in several organs[62] including the cardiovascular
system, nervous system, digestive system (colon, liver) and
male organs such as prostate. In target tissues, estrogens
may exert opposite actions and heterogeneous effects,
promoting the resistance to apoptotic damage, modulat-
ing reparative processes and controlling inflammation[63-65].
The expression pattern of ER subtypes is fundamental
to evocate the different effects of estrogens[66]. ER-, for
example, is critical in estrogen induced protection against
brain injury during stroke[66]. On the contrary, overexpres-
sion of ER- has been linked with cancer development
and progression in different organs[67]. The functions of
ER- are less known although recent findings suggest a
protective effect against uncontrolled or neoplastic cell
proliferation[68]. In our setting, the modulation of cholangi-
ocyte proliferation exerted by estrogens and their receptors
could open new therapeutic scenario suggesting appro-
priate trials aimed to evaluate the effects of ER selective
modulators (SERMs) on PBC clinical expression. To this
latter regard, preliminary clinical observations by us and
 Alvaro D et al. Estrogens and the biliary tree 3541
others[69,70] indicate that tamoxifen (mixed agonist/antago-
nist for ER- and only antagonist for ER-) improves
biochemical parameters of cholestasis in PBC patients. We
described three cases of PBC[69] that underwent tamoxifen
treatment for breast cancer. In two patients, tamoxifen
administration for approximately 18 mo, caused a marked
decrease of alkaline phosphatase (70%-85%) and gamma-
GT (60%-75%) serum levels and, in the third case, where
tamoxifen was combined with UDCA, a strong reduction
of liver enzymes was observed[69]. Moreover, during the ob-
servational period, none of these three patients developed
complications of liver cirrhosis[69]. The positive effects of
tamoxifen on the serum levels of cholestasis enzymes in
PBC have been also described by Bassendine et al in two
PBC patients who developed breast cancer and received
tamoxifen, after surgical treatment[70]. However, other than
acting as ER modulator in cholangiocytes, tamoxifen could
improve cholestasis in PBC by additional mechanisms.
Recently, in fact, tamoxifen has been shown to activate
the human pregnane X receptor (PXR), a nuclear receptor
linked with cytochrome p 450 function and considered to
be relevant in the UDCA mechanism of action[71]. There-
fore, tamoxifen could theoretically activate these promis-
cuous nuclear receptors thus improving UDCA effects[71].
Nevertheless, tamoxifen could have important side effects
such as the increased risk of endometrial cancer when ad-
vised for disease lasting decades like PBC[72]. Second and
third generation SERMs do not have these side effects and
thus considered safer[73]. Our preliminary data (unpublished
observations) indicates that raloxifene (a second genera-
tion SERM avoids the proliferative effects on reproductive
tissues and which do not form adducts with DNA) causes
biochemical improvement of cholestasis enzymes in PBC
patients as shown for tamoxifen. Furthermore, it improves
bone mineral density in PBC[74]. Thus a therapeutic strat-
egy aimed to positively modulate estrogen proliferation
during the early stages of PBC represents a challenge for
the future, since cholangiocyte proliferation acts as a repair
and compensatory mechanism and its stimulation is always
associated with enhanced secretory activities[11]. Moreover,
very recent studies elucidated that ER- mediates many
other beneficial effects of estrogen including immuno-
protection in autoimmune disease[75]. ER- activation, in
fact, inhibits inflammatory gene expression by preventing
NF-kappa B nuclear translocation[76]. Interestingly, through
the ER-, estrogens can positively modulate the GH/
IGF-1 axis. This further supports a possible therapeutic
role of ER- positive modulators in cholangiopathies
since we demonstrated that IGF- 1 and estrogens play ad-
ditive effect on cholangiocyte proliferation (Figure 1)[42]
and that the GH/IGF-1 axis plays a pivotal role in liver
injury repair (unpublished observations). Consistently,
IGF-1 replacement therapy shows clinical benefit in PBC
and alcoholic cirrhotic patients[77].
ESTROGENS AND ADPKD
Autosomal dominant polycystic kidney disease (ADPKD)
is one of the most prevalent human genetic diseases with
an incidence of 1:800 individuals [78]. Hepatic cysts are
the most common extra-renal clinical manifestation of
ADPKD and a significant source of morbidity[79]. That
estrogens may have a role in the development and progres-
sion of hepatic cysts in ADPKD patients is suggested by
a number of different clinical observations[82]. In ADPKD
patients, the probability of developing hepatic cysts is
higher in women with respect to men and among patients
submitted to liver transplant for polycystic disease, more
than 92% are women[80]. Furthermore, the number and
size of hepatic cysts increase during post-menopausal
hormonal replacement therapy[81] and nulliparae display
less probability to develop hepatic cysts than pluriparae[82],
where number and size of hepatic cysts correlate with the
number of pregnancies[82]. As recently showed by us[44],
interestingly, hepatic cysts are lined by cholangiocytes[83],
that are different from normal biliary cells since they
lack of both microvilli and primary cilia[84]. It is currently
hypothesized that the lack of primary cilia, genetically
determined, generates a cell phenotype characterized by
hyperproliferating and hypersecretory properties. By act-
ing on this cell phenotype, estrogens could sustain the
enhanced proliferative and secretory activities, as experi-
mentally shown in BDL rats[15], by acting either directly
or potentiating the effects of growth factors. Recently, in
fact, a number of different cytokines and growth factors
(i.e. IL-8, IL6 and VEGF), markedly enriched in serum
or hepatic cystic fluid, have been shown to promote the
growth of hepatic cyst epithelium[85]. Immunohistochemi-
cal studies performed by our group (Alvaro, 2006, unpub-
lished observations) showed that the epithelial surface of
hepatic cysts of ADPKD patients display a marked and
diffuse staining for ERs (mainly-b), IGF1 and IGF1-R. In
addition, we demonstrated that[44] immortalized cell line
(LCDE), derived from hepatic cyst epithelium of patients
with ADPKD, also express ER- and ER- subtypes as
well as IGF1 and IGF-R and that estrogens and IGF1
markedly stimulate the proliferation of this cell line by act-
ing on their specific receptors[44]. Specific antagonists of
ER (Ici 182,780) and an IGF1-R blocking antibody (a-IR3),
in fact, inhibit the proliferating effect exerted by estrogens
and IGF1 respectively[44] These recent findings may explain
why estrogens induce the formation and worsen the pro-
gression of hepatic cysts in ADPKD patients. On the basis
of our findings, the hepatic cyst epithelium of ADPKD
patients could be considered a sort of estrogen responsive
tissue, where the interaction between female sex hormones
and growth factors, like IGF1 and VEGF, play a key role
in modulating the pathophysiology. This could open new
perspectives in pharmacological treatment of polycystic
liver disease based on estrogen antagonism or on the use
of selective SERMs.
ESTROGENS AND CHOLANGIOCARCINO-
MA
Cholangiocarcinoma is a tumor with increasing incidence
and prevalence, that still represents a diagnostic and thera-
peutic challenge. It is one of the cancers with the worst
prognosis, with a median 5-years survival rate of 7%-8%
from the diagnosis and a median survival time of only sev-
en months. The diagnosis is generally established late and
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 3542 ISSN 1007-9327 CN 14-1219/ R
Cholangiocarcinoma
ER- ER-b
Figure 4 Immunohistochemistry for ER- and ER-. Biopsies of human cholangi-
ocarcinoma showing an intense positivity for both ER- and ER-. Orig. magn., x
20.
surgery, when feasible, is the only effective treatment[86]
Cholangiocarcinoma, in fact, shows a strong resistance to
common chemotherapies and a treatment that slows the
progression of the disease doesnt exist[86]. As for other
cancers, an imbalance between proliferation and apoptosis
leads to uncontrolled cell growth and expansion[37] We have
recently shown, in human tissues and human cell lines,
that estrogens play a key role in development and progres-
sion of cholangiocarcinoma[45]. Liver samples obtained
from patients with intrahepatic cholangiocarcinoma were
intensely positive for ER- and ER- subtypes (> 80%
cholangiocarcinoma cells positive), while cholangiocytes
of the normal liver were negative[45] (Figure 4). Interest-
ingly, ER display both a cytoplasmic and nuclear staining,
the latter being indicative of activated receptors[45]. When
compared to benign cholangiocyte proliferation associated
with non-neoplastic biliary disease (i.e. PBC, PSC), cholan-
giocarcinoma cells showed a higher expression of ER-
and increased ER-/ER- ratio[54] (Figure 2). This ob-
servation is in keeping with many different reports show-
ing an increased ER-/ER- ratio in cancerous versus
normal tissues, including ovary, prostate, colon and breast
cancers[68]. In these tissues, primary events in neoplastic
transformation and progression have been correlated with
up-regulation ER- and down-regulation of ER- thus
associating the function of ER- subtype with a positive
modulation of cell proliferation[68]. The opposite was sug-
gested for ER-[68]. Human intrahepatic cholangiocarcino-
ma also overexpress IGF1 and IGF1-R[45], another growth
factor whose main source is liver and which is functionally
linked with estrogens and their receptors[87,88]. IGF1 has
been recently implicated in cancer development and pro-
gression since its serum concentrations correlate with an
increased risk of breast, prostate, colorectal, pancreas and
lung cancer[89-91] and, its receptor (IGF1R) is overexpressed
in many tumor cell lines and in some human tumors. By
studying a human intrahepatic cholangiocarcinoma cell
line (HuH-28), similar to intrahepatic cholangiocarcinoma
that over expresses ER-, IGF1 and IGF1-R, we showed
that IGF-1 and estrogens exert an additive proliferative
effect[45] blocked by both ER antagonists (Ici 182,780)
and IGF1-R blocking antibodies (a-IR3)[45]. Interestingly,
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
IGF1-R seems to play a primary role also in the estrogen
stimulation of neoplastic cell growth [45] since IGF1-R
blocking antibody (a-IR3) partially inhibited the effects
of 17-estradiol and transfection of HuH-28 cell with
IGF1-R antisense oligonucleotides caused a marked im-
pairment of HuH-28 cells proliferation (90% decrease of
PCNA expression)[45]. In estrogen sensitive tissues, a cross-
talk between IGF1 and estrogen play a major role in the
modulation of cell proliferation, where estrogens act at
several points of the IGF signal transduction pathway[87].
Estrogens not only may regulate the expression of IGF1-R
and IGF1-binding proteins but also that of crucial down-
stream proteins including IRS1, and the IGF1-R tyrosine
kinase main substrate[88]. Finally, the signaling activated by
estrogens and IGF-1 (Figure 1) may converge at different
common transduction pathways, including ERK and phos-
phatidylinositol-3 kinase/Akt pathway[92,93], and this was
observed in proliferating HuH-28 cells too. In addition,
ER-a and IGF1-R, once activated coprecipitate and, their
activation is potentiated by their coupling[45]. Neverthe-
less, estrogens can modulate other important mechanisms
involved in cholangiocarcinoma growth such as COX-2[94].
These new evidences highlight the role of estrogens and
IGF1 in regulating the growth of human cholangiocarci-
noma and suggests that a therapeutic strategy based on the
modulation of ERs and/or IGF1 system could be helpful
for the management of this cancer.
CONCLUSIONS AND FUTURE PERSPEC-
TIVES
In conclusion, recent studies support a relevant role of es-
trogens as modulators of benign (reactive) and neoplastic
cholangiocyte proliferation[16,45,69,70]. In our hypothesis, es-
trogens act in concert with growth factors in sustaining the
cholangiocyte proliferative machinery and in depressing
apoptosis[41-43]. This may have a number of clinical implica-
tions for diseases involving the biliary epithelium, where
cholangiocyte proliferation is a typical hallmark influencing
disease progression[10]. As far as PBC is concerned, we be-
lieve that an impaired cholangiocyte response to estrogens
characterizes the terminal ductopenic stages of the disease,
where cholangiocyte proliferation is unable to balance for
the enhanced apoptosis. On this basis, a SERM able to
selectively stimulate the ER- and conpemporarily depress
the ER- should be tested in controlled clinical trials.
Furthermore, it is of interest that new generation SERMs,
such as Idoxifene, play positive effects in controlling both
the oxidative stress and fibrosis, which are foundamental
processes in the progression of chronic cholestatic liver
diseases[95-97]. As far as cholangiocarcinoma and ADPKD
are concerned, pharmacological strategies aimed to inhibit
estrogens binding to their receptors, to decrease their syn-
thesis (by using aromatase inhibitors) and, finally, to down-
regulate ERs protein levels (by using pure anti-estrogens
such fulvestant), should be considered. Very recently an
ER-X putative membrane associated estrogen receptor
has been described in the brain and in the uterus[98]. Thus,
further studies aimed to check the presence of this recep-
tor subtype in cholangiocytes and to evaluate its role in the
pathophysiology of the biliary tree are needed.
 Alvaro D et al. Estrogens and the biliary tree 3543
REFERENCES
1 Roskams TA, Theise ND, Balabaud C, Bhagat G, Bhathal PS,
Bioulac-Sage P, Brunt EM, Crawford JM, Crosby HA, Desmet
V, Finegold MJ, Geller SA, Gouw AS, Hytiroglou P, Knisely
AS, Kojiro M, Lefkowitch JH, Nakanuma Y, Olynyk JK, Park
YN, Portmann B, Saxena R, Scheuer PJ, Strain AJ, Thung SN,
Wanless IR, West AB. Nomenclature of the finer branches of
the biliary tree: canals, ductules, and ductular reactions in
human livers. Hepatology 2004; 39: 1739-1745
2 Crawford JM. Normal and abnormal development of the
biliary tree. In: Alpini G, Alvaro D, Marzioni M, LeSage G,
LaRusso NF, eds. The Pathophysiology of Biliary Epithelia.
Georgetown, TX: Landes Bioscience, 2004: 1-13
3 Saxena R, Theise ND, Crawford JM. Microanatomy of the
human liver-exploring the hidden interfaces. Hepatology 1999;
30: 1339-1346
4 Wu CT, Davis PA, Luketic VA, Gershwin ME. A review of the
physiological and immunological functions of biliary epithelial
cells: targets for primary biliary cirrhosis, primary sclerosing
cholangitis and drug-induced ductopenias. Clin Dev Immunol
2004; 11: 205-213
5 Alvaro D. Biliary epithelium: a new chapter in cell biology.
Ital J Gastroenterol Hepatol 1999; 31: 78-83
6 Alvaro D, Gigliozzi A, Attili AF. Regulation and deregulation
of cholangiocyte proliferation. J Hepatol 2000; 33: 333-340
7 Demetris AJ. Participation of cytokines and growth factors in
biliary cell proliferation and mito-inhibition during ductular
reaction. In: Alpini G, Alvaro D, Marzioni M, LeSage G,
LaRusso NF, eds. The Pathophysiology of Biliary Epithelia.
Georgetown, TX: Landes Bioscience, 2004; 167-182
8 Boyer JL. Vanishing Bile Duct Syndrome-from bench to
bedside. In: Alvaro D, Benedetti A, Strazzabosco M, eds.
Vanishing Bile Duct Syndrome. London, UK: Kluwer
Academic Publishers, 1997: 240-246
9 Lazaridis KN, Strazzabosco M, Larusso NF. The cholangi-
opathies: disorders of biliary epithelia. Gastroenterology 2004;
127: 1565-1577
10 Desmet V, Roskams T, Van Eyken P. Pathology of the biliary
tree in cholestasis: ductular reaction. In: Manns MP, Boyer JL,
Jansen PLM, Reichen J, eds. Cholestatic liver diseases. London,
UK: Kluwer Academic Pubishers, 1988; 143-154
11 Lesage G, Glaser SS, Gubba S, Robertson WE, Phinizy JL,
Lasater J, Rodgers RE, Alpini G. Regrowth of the rat biliary
tree after 70% partial hepatectomy is coupled to increased
secretin-induced ductal secretion. Gastroenterology 1996; 111:
1633-1644
12 Desmet VJ, van Eyken P, Roskams T. Histopathology of
vanishing bile duct diseases. Adv Clin Path 1998; 2: 87-99
13 Alvaro D, Alpini G, Onori P, Perego L, Svegliata Baroni G,
Franchitto A, Baiocchi L, Glaser SS, Le Sage G, Folli F, Gaudio
E. Estrogens stimulate proliferation of intrahepatic biliary
epithelium in rats. Gastroenterology 2000; 119: 1681-1691
14 Alvaro D, Alpini G, Onori P, Franchitto A, Glaser SS, Le Sage G,
Folli F, Attili AF, Gaudio E. Alfa and beta estrogen receptors
and the biliary tree. Mol Cell Endocrinol 2002; 193: 105-108
15 Alvaro D, Alpini G, Onori P, Franchitto A, Glaser S, Le Sage G,
Gigliozzi A, Vetuschi A, Morini S, Attili AF, Gaudio E. Effect
of ovariectomy on the proliferative capacity of intrahepatic rat
cholangiocytes. Gastroenterology 2002; 123: 336-344
16 Alvaro D, Invernizzi P, Onori P, Franchitto A, De Santis
A, Crosignani A, Sferra R, Ginanni-Corradini S, Mancino
MG, Maggioni M, Attili AF, Podda M, Gaudio E. Estrogen
receptors in cholangiocytes and the progression of primary
biliary cirrhosis. J Hepatol 2004; 41: 905-912
17 Ahlqvist J. Can endocrine factors influence pathogenetic
mechanisms in chronic active hepatitis (CAH) and primary
biliary cirrhosis (PBC)? A hypothesis. Hepatogastroenterology
1980; 27: 64-67
18 Joplin RE, Neuberger JM. Antigen expression in bile duct
epithelia in cholestatic liver disease. In: Manns MO, Boyer JL,
Jansen PLM, Reichen J, Editors. Cholestatic Liver Diseases Falk
Symposium 102. Dordrecht: Kluwer Academic Publishers,
1998: 226-238
19 Floreani A, Titta M, Plebani M, Faggian D, Chiaramonte
M, Naccarato R. Sex hormone changes in post-menopausal
women with primary biliary cirrhosis (PBC) and with
cryptogenic chronic liver disease. Clin Exp Obstet Gynecol 1991;
18: 229-234
20 Floreani A, Paternoster D, Mega A, Farinati F, Plebani M,
Baldo V, Grella P. Sex hormone profile and endometrial cancer
risk in primary biliary cirrhosis: a case-control study. Eur J
Obstet Gynecol Reprod Biol 2002; 103: 154-157
21 Stellon AJ, Williams R. Increased incidence of menstrual
abnormalities and hysterectomy preceding primary biliary
cirrhosis. Br Med J (Clin Res Ed) 1986; 293: 297-298
22 Wolke AM, Schaffner F, Kapelman B, Sacks HS. Malignancy
in primary biliary cirrhosis. High incidence of breast cancer in
affected women. Am J Med 1984; 76: 1075-1078
23 Goudie BM, Burt AD, Boyle P, Macfarlane G, Birnie GG, Mills
PR, Gillis CR, MacSween RN, Watkinson G. Breast cancer in
women with primary biliary cirrhosis. Br Med J (Clin Res Ed)
1985; 291: 1597-1598
24 Hodgson SF, Dickson ER, Wahner HW, Johnson KA, Mann
KG, Riggs BL. Bone loss and reduced osteoblast function in
primary biliary cirrhosis. Ann Intern Med 1985; 103: 855-860
25 Olsson R, Mattsson LA, Obrant K, Mellstrm D. Estrogen-
progestogen therapy for low bone mineral density in primary
biliary cirrhosis. Liver 1999; 19: 188-192
26 Wiesner RH, Batts KP, Krom RA. Evolving concepts in the
diagnosis, pathogenesis and treatment of chronic hepatic
allograft rejection. Liver Transpl Surg 1999; 5: 388-400
27 Elsheikh M, Hodgson HJ, Wass JA, Conway GS. Hormone
replacement therapy may improve hepatic function in women
with Turners syndrome. Clin Endocrinol (Oxf) 2001; 55:
227-231
28 Chen J, Robertson G, Field J, Liddle C, Farrell GC. Effects
of bile duct ligation on hepatic expression of female-specific
CYP2C12 in male and female rats. Hepatology 1998; 28: 624-630
29 Diel P. Tissue-specific estrogenic response and molecular
mechanisms. Toxicol Lett 2002; 127: 217-224
30 Eagon PK, Porter LE, Francavilla A, DiLeo A, Van Thiel DH.
Estrogen and androgen receptors in liver: their role in liver
disease and regeneration. Semin Liver Dis 1985; 5: 59-69
31 Fisher B, Gunduz N, Saffer EA, Zheng S. Relation of estrogen
and its receptor to rat liver growth and regeneration. Cancer
Res 1984; 44: 2410-2415
32 Francavilla A, Polimeno L, DiLeo A, Barone M, Ove P, Coetzee
M, Eagon P, Makowka L, Ambrosino G, Mazzaferro V. The
effect of estrogen and tamoxifen on hepatocyte proliferation in
vivo and in vitro. Hepatology 1989; 9: 614-620
33 Cheng AL, Chuang SE, Fine RL, Yeh KH, Liao CM, Lay
JD, Chen DS. Inhibition of the membrane translocation and
activation of protein kinase C, and potentiation of doxoru-
bicin-induced apoptosis of hepatocellular carcinoma cells by
tamoxifen. Biochem Pharmacol 1998; 55: 523-531
34 Pickens A, Pan G, McDonald J, Vickers SM. Involvement of
FAS in tamoxifen-induced apoptosis of cholangiocarcinoma.
Gastroenterology 1998; 114: S0192
35 Sampson LK, Vickers SM, Ying W, Phillips JO. Tamoxifen-
mediated growth inhibition of human cholangiocarcinoma.
Cancer Res 1997; 57: 1743-1749
36 Kuroki T, Seki S, Kawakita N, Nakatani K, Hisa T, Kitada
T, Sakaguchi H. Expression of antigens related to apoptosis
and cell proliferation in chronic nonsuppurative destructive
cholangitis in primary biliary cirrhosis. Virchows Arch 1996;
429: 119-129
37 Celli A, Que FG. Dysregulation of apoptosis in the cholangio-
pathies and cholangiocarcinoma. Semin Liver Dis 1998; 18:
177-185
38 Migliaccio A, Di Domenico M, Castoria G, de Falco A,
Bontempo P, Nola E, Auricchio F. Tyrosine kinase/p21ras/
MAP-kinase pathway activation by estradiol-receptor complex
in MCF-7 cells. EMBO J 1996; 15: 1292-1300
39 Alvaro D, Onori P, Metalli VD, Svegliati-Baroni G, Folli
F, Franchitto A, Alpini G, Mancino MG, Attili AF, Gaudio
www.wjgnet.com
 3544 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
E. Intracellular pathways mediating estrogen-induced
cholangiocyte proliferation in the rat. Hepatology 2002; 36:
297-304
40 Peyssonnaux C, Eychne A. The Raf/MEK/ERK pathway:
new concepts of activation. Biol Cell 2001; 93: 53-62
41 Gigliozzi A, Alpini G, Baroni GS, Marucci L, Metalli VD,
Glaser SS, Francis H, Mancino MG, Ueno Y, Barbaro B,
Benedetti A, Attili AF, Alvaro D. Nerve growth factor
modulates the proliferative capacity of the intrahepatic biliary
epithelium in experimental cholestasis. Gastroenterology 2004;
127: 1198-1209
42 Alvaro D, Metalli VD, Alpini G, Onori P, Franchitto A,
Barbaro B, Glaser SS, Francis H, Cantafora A, Blotta I, Attili
AF, Gaudio E. The intrahepatic biliary epithelium is a target
of the growth hormone/insulin-like growth factor 1 axis. J
Hepatol 2005; 43: 875-883
43 Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Ueno Y,
Meininger CJ, Franchitto A, Onori P, Marzioni M, Taffetani
S, Fava G, Stoica G, Venter J, Reichenbach R, De Morrow S,
Summers R, Alpini G. Vascular endothelial growth factor
stimulates rat cholangiocyte proliferation via an autocrine
mechanism. Gastroenterology 2006; 130: 1270-1282
44 Alvaro D, Onori P, Torrice A, Cardinale V, Barbaro B, Alpini G,
Franchitto A, Battisti G, Jefferson DM, Strazzabosco M, Attili
AF, Gaudio E. Estrogens and Igf1 promote the proliferation
of hepatic cyst epithelium in autosomal dominant polycystic
kidney disease (ADPKD). Hepatology 2005; 42: 202A.
45 Alvaro D, Barbaro B, Franchitto A, Onori P, Glaser SS, Alpini
G, Francis H, Marucci L, Sterpetti P, Ginanni-Corradini S,
Onetti Muda A, Dostal DE, De Santis A, Attili AF, Benedetti A,
Gaudio E. Estrogens and insulin-like growth factor 1 modulate
neoplastic cell growth in human cholangiocarcinoma. Am J
Pathol 2006; 169: 877-888
46 Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J
Med 2005; 353: 1261-1273
47 Cutolo M, Sulli A, Seriolo B, Accardo S, Masi AT. Estrogens,
the immune response and autoimmunity. Clin Exp Rheumatol
1995; 13: 217-226
48 Salem ML. Estrogen, a double-edged sword: modulation
of TH1- and TH2-mediated inflammations by differential
regulation of TH1/TH2 cytokine production. Curr Drug
Targets Inflamm Allergy 2004; 3: 97-104
49 Crippin JS, Jorgensen RA, Dickson ER, Lindor KD. Hepatic
osteodystrophy in primary biliary cirrhosis: effects of medical
treatment. Am J Gastroenterol 1994; 89: 47-50
50 Mills PR, Boyle P, Quigley EM, Birnie GG, Jarrett F,
Watkinson G, MacSween RN. Primary biliary cirrhosis: an
increased incidence of extrahepatic malignancies? J Clin Pathol
1982; 35: 541-543
51 Lf L, Adami HO, Sparn P, Danielsson A, Eriksson LS, 73 Liu HB, Loo KK, Palaszynski K, Ashouri J, Lubahn DB,
Hultcrantz R, Lindgren S, Olsson R, Prytz H, Ryden BO.
Cancer risk in primary biliary cirrhosis: a population-based
study from Sweden. Hepatology 1994; 20: 101-104
52 Floreani A, Baragiotta A, Baldo V, Menegon T, Farinati
F, Naccarato R. Hepatic and extrahepatic malignancies in
primary biliary cirrhosis. Hepatology 1999; 29: 1425-1428
53 Floreani A, Mega A, Camozzi V, Baldo V, Plebani M, Burra P,
Luisetto G. Is osteoporosis a peculiar association with primary
biliary cirrhosis? World J Gastroenterol 2005; 11: 5347-5350
54 Floreani A. Osteoporosis is not a specific complication of
primary biliary cirrhosis (PBC). Gut 2002; 50: 898; author reply
898-899
55 Cid MC, Schnaper HW, Kleinman HK. Estrogens and the
vascular endothelium. Ann N Y Acad Sci 2002; 966: 143-157
56 Czlonkowska A, Ciesielska A, Gromadzka G, Kurkowska-
Jastrzebska I. Estrogen and cytokines production- the possible
cause of gender differences in neurological diseases. Curr
Pharm Des 2005; 11: 1017-1030
57 Toran-Allerand CD. Estrogen as a treatment for Alzheimer
disease. JAMA 2000; 284: 307-308
58 Liu X, Steffensen KR, Sanna A, Arru G, Fois ML, Rosati G,
Sotgiu S, Link H, Gustafsson JA, Huang YM. Anti-inflamma-
tory nuclear receptor superfamily in multiple sclerosis patients
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
from Sardinia and Sweden. Neurobiol Dis 2005; 20: 961-968
59 Lakoski SG, Herrington DM. Effects of oestrogen receptor-
active compounds on lipid metabolism. Diabetes Obes Metab
2005; 7: 471-477
60 Fritzemeier KH, Hillisch A, Elger W, Kaufmann U,
Kollenkirchen U, Kosemund D, Lindenthal B, Mller G, Muhn
P, Nubbemeyer R, Peters O, Siebel P, Hegele-Hartung C.
Biological effects of ERalpha- and ERbeta-selective estrogens.
Ernst Schering Res Found Workshop 2004; : 127-150
61 Asimiadou S, Bittigau P, Felderhoff-Mueser U, Manthey D,
Sifringer M, Pesditschek S, Dzietko M, Kaindl AM, Pytel M,
Studniarczyk D, Mozrzymas JW, Ikonomidou C. Protection
with estradiol in developmental models of apoptotic
neurodegeneration. Ann Neurol 2005; 58: 266-276
62 Tiidus PM. Influence of estrogen on skeletal muscle damage,
inflammation, and repair. Exerc Sport Sci Rev 2003; 31: 40-44
63 Koh KK. Effects of estrogen on the vascular wall: vasomotor
function and inflammation. Cardiovasc Res 2002; 55: 714-726
64 Merchenthaler I, Dellovade TL, Shughrue PJ. Neuroprotection
by estrogen in animal models of global and focal ischemia.
Ann N Y Acad Sci 2003; 1007: 89-100
65 Ikeda K, Inoue S. Estrogen receptors and their downstream
targets in cancer. Arch Histol Cytol 2004; 67: 435-442
66 Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of
ERbeta expression as a common step in estrogen-dependent
tumor progression. Endocr Relat Cancer 2004; 11: 537-551
67 Invernizzi P, Alvaro D, Crosignani A, Gaudio E, Podda M.
Tamoxifen in treatment of primary biliary cirrhosis. Hepatology
2004; 39: 1175-1176
68 Reddy A, Prince M, James OF, Jain S, Bassendine MF.
Tamoxifen: a novel treatment for primary biliary cirrhosis?
Liver Int 2004; 24: 194-197
69 Desai PB, Nallani SC, Sane RS, Moore LB, Goodwin BJ,
Buckley DJ, Buckley AR. Induction of cytochrome P450 3A4
in primary human hepatocytes and activation of the human
pregnane X receptor by tamoxifen and 4-hydroxytamoxifen.
Drug Metab Dispos 2002; 30: 608-612
70 Bergman L, Beelen ML, Gallee MP, Hollema H, Benraadt J,
van Leeuwen FE. Risk and prognosis of endometrial cancer
after tamoxifen for breast cancer. Comprehensive Cancer
Centres ALERT Group. Assessment of Liver and Endometrial
cancer Risk following Tamoxifen. Lancet 2000; 356: 881-887
71 Lewis JS, Jordan VC. Selective estrogen receptor modulators
(SERMs): mechanisms of anticarcinogenesis and drug
resistance. Mutat Res 2005; 591: 247-263
72 Levy C, Harnois DM, Angulo P, Jorgensen R, Lindor KD.
Raloxifene improves bone mass in osteopenic women with
primary biliary cirrhosis: results of a pilot study. Liver Int 2005;
25: 117-121
Voskuhl RR. Estrogen receptor alpha mediates estrogens
immune protection in autoimmune disease. J Immunol 2003;
171: 6936-6940
74 Ghisletti S, Meda C, Maggi A, Vegeto E. 17beta-estradiol
inhibits inflammatory gene expression by controlling NF-
kappaB intracellular localization. Mol Cell Biol 2005; 25:
2957-2968
75 Fournier B, Gutzwiller S, Dittmar T, Matthias G, Steenbergh
P, Matthias P. Estrogen receptor (ER)-alpha, but not ER-beta,
mediates regulation of the insulin-like growth factor I gene by
antiestrogens. J Biol Chem 2001; 276: 35444-35449
76 Conchillo M, de Knegt RJ, Payeras M, Quiroga J, Sangro
B, Herrero JI, Castilla-Cortazar I, Frystyk J, Flyvbjerg A,
Yoshizawa C, Jansen PL, Scharschmidt B, Prieto J. Insulin-like
growth factor I (IGF-I) replacement therapy increases albumin
concentration in liver cirrhosis: results of a pilot randomized
controlled clinical trial. J Hepatol 2005; 43: 630-636
77 Ramos A, Torres VE, Holley KE, Offord KP, Rakela J, Ludwig
J. The liver in autosomal dominant polycystic kidney disease.
Implications for pathogenesis. Arch Pathol Lab Med 1990; 114:
180-184
78 Everson GT. Hepatic cysts in autosomal dominant polycystic
kidney disease. Mayo Clin Proc 1990; 65: 1020-1025
 Alvaro D et al. Estrogens and the biliary tree 3545
79 Chapman AB. Cystic disease in women: clinical
characteristics and medical management. Adv Ren Replace
Ther 2003; 10: 24-30
80 Harris RA, Gray DW, Britton BJ, Toogood GJ, Morris PJ.
Hepatic cystic disease in an adult polycystic kidney disease
transplant population. Aust N Z J Surg 1996; 66: 166-168
81 Sherstha R, McKinley C, Russ P, Scherzinger A, Bronner T,
Showalter R, Everson GT. Postmenopausal estrogen therapy
selectively stimulates hepatic enlargement in women with
autosomal dominant polycystic kidney disease. Hepatology
1997; 26: 1282-1286
82 Gabow PA, Johnson AM, Kaehny WD, Manco-Johnson ML,
Duley IT, Everson GT. Risk factors for the development
of hepatic cysts in autosomal dominant polycystic kidney
disease. Hepatology 1990; 11: 1033-1037
83 Perrone RD, Grubman SA, Rogers LC, Lee DW, Moy E, Murray
SL, Torres VE, Jefferson DM. Continuous epithelial cell lines
from ADPKD liver cysts exhibit characteristics of intrahepatic
biliary epithelium. Am J Physiol 1995; 269: G335-G345
84 Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ,
LaRusso NF. Cholangiocyte cilia detect changes in luminal
fluid flow and transmit them into intracellular Ca2+ and
cAMP signaling. Gastroenterology 2006; 131: 911-920
85 Nichols MT, Gidey E, Matzakos T, Dahl R, Stiegmann G, Shah
RJ, Grantham JJ, Fitz JG, Doctor RB. Secretion of cytokines and
growth factors into autosomal dominant polycystic kidney
disease liver cyst fluid. Hepatology 2004; 40: 836-846
86 Gores GJ. Cholangiocarcinoma: current concepts and insights.
Hepatology 2003; 37: 961-969
87 Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R,
Grohe C. Estrogen receptor alpha rapidly activates the IGF-1
receptor pathway. J Biol Chem 2000; 275: 18447-18453
88 Adesanya OO, Zhou J, Samathanam C, Powell-Braxton L,
Bondy CA. Insulin-like growth factor 1 is required for G2
progression in the estradiol-induced mitotic cycle. Proc Natl
Acad Sci U S A 1999; 96: 3287-3291
89 Lin Y, Tamakoshi A, Kikuchi S, Yagyu K, Obata Y, Ishibashi
T, Kawamura T, Inaba Y, Kurosawa M, Motohashi Y, Ohno Y.
Serum insulin-like growth factor-I, insulin-like growth factor
binding protein-3, and the risk of pancreatic cancer death. Int J
Cancer 2004; 110: 584-588
90 Ma J, Giovannucci E, Pollak M, Stampfer M. RESPONSE:
Re: Prospective Study of Colorectal Cancer Risk in Men and
Plasma Levels of Insulin-Like Growth Factor (IGF)-I and IGF-
Binding Protein-3. J Natl Cancer Inst 1999; 91: 2052
91 Yu H, Spitz MR, Mistry J, Gu J, Hong WK, Wu X. Plasma
levels of insulin-like growth factor-I and lung cancer risk: a
case-control analysis. J Natl Cancer Inst 1999; 91: 151-156
92 Shelton JG, Steelman LS, White ER, McCubrey JA. Synergy
between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R
mediated cell cycle progression and prevention of apoptosis in
hematopoietic cells. Cell Cycle 2004; 3: 372-379
93 Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM. The
mitogen-activated protein kinase pathway mediates estrogen
neuroprotection after glutamate toxicity in primary cortical
neurons. J Neurosci 1999; 19: 2455-2463
94 Wu T, Leng J, Han C, Demetris AJ. The cyclooxygenase-2
inhibitor celecoxib blocks phosphorylation of Akt and induces
apoptosis in human cholangiocarcinoma cells. Mol Cancer Ther
2004; 3: 299-307
95 Pinzani M. Cholestasis and fibrogenesis. In: Alpini G, Alvaro
D, Marzioni M, LeSage G, LaRusso N. The Pathophysiology
of Biliary Epithelia. Georgetown, TX: Landes Bioscience, 2004:
210-218
96 Lu G, Shimizu I, Cui X, Itonaga M, Tamaki K, Fukuno H,
Inoue H, Honda H, Ito S. Antioxidant and antiapoptotic
activities of idoxifene and estradiol in hepatic fibrosis in rats.
Life Sci 2004; 74: 897-907
97 Zhou YJ, Yin DM, Chen HS, Shi JH, Sha BX, Wang X.
Inhibitory effects of idoxifene on hepatic fibrosis in rats. Acta
Pharmacol Sin 2005; 26: 581-586
98 Toran-Allerand CD. Estrogen and the brain: beyond ER-
alpha, ER-beta, and 17beta-estradiol. Ann N Y Acad Sci 2005;
1052: 136-144
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TOPIC HIGHLIGHT
Gianfranco D Alpini, PhD, Series Editor
Cholangiocytes and blood supply
Eugenio Gaudio, Antonio Franchitto, Luigi Pannarale, Guido Carpino, Gianfranco Alpini, Heather Francis,
Shannon Glaser, Domenico Alvaro, Paolo Onori
Eugenio Gaudio, Antonio Franchitto, Luigi Pannarale, Guido
Carpino, Department of Human Anatomy, University of Rome La
Sapienza, Rome, Italy
Domenico Alvaro, Division of Gastroenterology, University
of Rome La Sapienza, Rome, Italy, University of Rome La
Sapienza, Polo Pontino, Latina, Italy
Gianfranco Alpini, Central Texas Veterans Health Care
System, and Department of Medicine and Systems Biology and
Translational Medicine, College of Medicine, Temple, TX,
United States
Heather Francis, Division of Research and Education, Scott &
White Hospital and The Texas A&M University System Health
Science Center, College of Medicine, Temple, TX, United States
Shannon Glaser, Department of Medicine and Division of
Research and Education, Scott & White Hospital and The Texas
A&M University System Health Science Center, College of
Medicine, Temple, TX, United States
Domenico Alvaro, Division of Gastroenterology, University
of Rome La Sapienza, Rome, Italy; University of Rome La
Sapienza, Polo Pontino, Latina, Italy
Paolo Onori, Department of Experimental Medicine, Section
of Human and Clinical Anatomy, State University of L'Aquila,
L'Aquila, Italy
Supported by MIUR grants PRIN 2005 (prot. 2005067975_001)
to E. Gaudio and Biomedicina, Cluster C04, Progetto n. 5
to E.Gaudio-P.Onori; MIUR grants PRIN 2005 (prot.No:
2005067975_002) to D. Alvaro and a VA Research Scholar Award,
a VA Merit Award and the NIH grants DK58411 and DK062975
to Gianfranco Alpini
Correspondence to: Eugenio Gaudio, MD, University of Rome,
"La Sapienza", Department of Human Anatomy, Rome,
Italy. eugenio.gaudio@uniroma1.it
Telephone: +39-06-49918055 Fax: +39-06-49918062
Received: 2006-02-07 Accepted: 2006-02-28
Abstract
The microvascular supply of the biliary tree, the
peribiliary plexus (PBP), stems from the hepatic artery
branches and flows into the hepatic sinusoids. A
detailed three-dimensional study of the PBP has been
performed by using the Scanning Electron Microscopy
vascular corrosion casts (SEMvcc) technique. Considering
that the PBP plays a fundamental role in supporting
the secretory and absorptive functions of the biliary
epithelium, their organization in either normalcy and
pathology is explored. The normal liver shows the PBP
arranged around extra- and intrahepatic biliary tree.
In the small portal tract PBP was characterized by a
single layer of capillaries which progressively continued
with the extrahepatic PBP where it showed a more
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World J Gastroenterol 2006 June 14; 12(22): 3546-3552
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
complex vascular network. After common duct ligation
(BDL), progressive modifications of bile duct and PBP
proliferation are observed. The PBP presents a three-
dimensional network arranged around many bile ducts
and appears as bundles of vessels, composed by
capillaries of homogeneous diameter with a typical round
mesh structure. The PBP network is easily distinguishable
from the sinusoidal network which appears normal.
Considering the enormous extension of the PBP
during BDL, the possible role played by the Vascular
Endothelial Growth Factor (VEGF) is evaluated. VEGF-A,
VEGF-C and their related receptors appeared highly
immunopositive in proliferating cholangiocytes of BDL
rats. The administration of anti-VEGF-A or anti-VEGF-C
antibodies to BDL rats as well as hepatic artery ligation
induced a reduced bile duct mass. The administration
of rVEGF-A to BDL hepatic artery ligated rats prevented
the decrease of cholangiocyte proliferation and VEGF-A
expression as compared to BDL control rats. These data
suggest the role of arterial blood supply of the biliary
tree in conditions of cholangiocyte proliferation, such as
it occurs during chronic cholestasis. On the other hand,
the role played by VEGF as a tool of cross-talk between
cholangiocytes and PBP endothelial cells suggests
that manipulation of VEGF release and function could
represent a therapeutic strategy for human pathological
conditions characterized by damage of hepatic artery or
the biliary tree.
2006 The WJG Press. All rights reserved.
Key words: Peribiliary plexus; Periportal plexus;
Cholangiocytes
Gaudio E, Franchitto A, Pannarale L, Carpino G, Alpini
G, Francis H, Glaser S, Alvaro D, Onori P. Cholangiocytes
and blood supply. World J Gastroenterol 2006; 12(22):
3546-3552
http://www.wjgnet.com/1007-9327/12/3546.asp
Introduction
Cholangiocytes are currently the object of extensive
investigation since recent evidence demonstrates their
involvement in different processes (secretion, absorption,
proliferation, neoangiogenesis), which are fundamental for
 Gaudio et al. Bile duct vascularization 3547
the liver pathophysiology[1-4]. The metabolic and functional
demands of cholangiocytes are sustained by the hepatic
artery which forms a complex network of vessels, the
peribiliary plexus, around the biliary tree[5-9].
The central role of liver vascularization in hepatic
functions and the importance of microcirculation for
liver zonal organization were developed in the 17th
century by Marcello Malpighi[10] who first described the
"acinus", a hexagonal structure of the liver parenchyma
that was comparable with a glandular unit of the liver.
This description correlates with the current concept of
a liver morpho-functional unit, also termed "acinus",
described by Rappaport in 1952[11]. A number of studies
on liver vascularization clarified the typical microvascular
architecture that supplies the biliary tree. This typical
microvascular network r uns along the intra- and
extrahepatic biliary tree and, as previously mentioned, has
been termed peribiliary plexus (PBP)[12,13]. The structural
study of either the intrahepatic and extrahepatic PBP in
the liver under normal conditions has been the subject
of a number of studies using both two-dimensional and
three-dimensional approaches[5,12,14,15].
Although it is well known that the liver has a dual blood
supply from the hepatic artery (HA) and the portal vein
(PV), controversies still remain concerning the distribution
of hepatic arterial blood flow. It is generally accepted that
terminal branching of the HA opens into sinusoids, as
demonstrated by injection of dyes and vascular casting
[5,16-24]
. The question that remains is if the HA provides ar-
terial blood directly to sinusoids[16,20-22,23,25] or rather runs in
the portal tract stroma, thus feeding the PBP and the peri-
portal plexus (PPP) to finally arrive at the sinusoids[26-30].
The presence of a double feeding to the sinusoids from
the PV, at a pressure of 6-7 cm H2O, and from the HA, at
a pressure of 12-25 cm H2O, has generated many studies
in order to explain the prevalence of flow from the PV[31].
Arterio-portal anastomoses are mainly observed in termi-
nal portal tracts[23] but other studies deny the presence of
real arterio-portal venous anastomoses[16,27,32,33].
A number of reports indicate that structural changes
of the biliary tree, associated with experimental models
of liver damage or human pathologies, are followed
by substantial adaptive modifications of the PBP. At
the experimental level, the bile duct-ligated (BDL)
rat has been widely used as a model of typical and
selective cholangiocyte proliferation [9,34,35]. This model
is characterized by a dramatic increase of cholangiocyte
number which reaches 30% of the total parenchymal
hepatic cells compared with 2% in the normal liver [36].
In order to investigate the adaptive changes of hepatic
microvascular organization associated with an increased
mass of the biliary tree following BDL, our group has
used the method of vascular corrosion casts observed
with Scanning Electron Microscopy (SEM). This technique
provides the ability to follow the tiniest vessels in three
dimensions and to distinguish arterial, capillary and venous
vessels by surface characteristics [7,13,15,26,37]. As detailed
above, this technique demonstrates how the evolution
of PBP expansion follows the proliferation of biliary
tree suggesting a role of proliferating cholangiocytes in
sustaining, through the secretion of angiogenenic factors,
and the adaptive proliferation of their vascular supply. To
this regard, recent reports demonstrate that cholangiocytes
are an important source of vascular endothelial growth
factor (VEGF) in the damaged liver and that VEGF
secreted by cholangiocytes may play role in driving the
adaptive changes of the PBP, which accompany the
proliferation of the biliary tree. Therefore, cross talk
between cholangiocytes and endothelial cells could be
fundamental in determining the response of the liver to
cholestatic damage[38].
In this manuscript, we review recent studies related to
the physiopathology of hepatic microvasculature in experi-
mental models of liver damage and discuss the biological
and clinical implications.
Blood supply
The microvasculature organization was investigated in
normal or BDL rats by means of the Scanning Electron
Microscopy Vascular Cor rosion Casts (SEMvcc)
technique [9,15,35]. The casts of normal livers showed an
even distribution of sinusoids closely surrounding portal
spaces. In larger portal spaces the hepatic artery (HA)
branches were always evidently smaller than the portal
ones. In smaller portal spaces the HA was often difficult
to visualize[12,13,39,40]. The terminal branches of the HA are
always represented by either the extra- and intrahepatic
Peribiliary Plexus (PBP) or the periportal plexus (PPP). In
very small portal spaces a small capillary, representing the
terminal branch of a HA, can continue the course of the
arteriole and eventually run into the sinusoids. It must be
pointed out that it has all the characteristics of a capillary
as far as diameter and endothelial imprints are concerned.
This typical microvascular organization around the
extrahepatic and intrahepatic biliary tree takes origin from
the HA[15]. Hepatic artery branches paralleled the divisions
of the portal vein up to the interlobular vessels and always
showed regular profile and spindle shaped endothelial
nuclear imprints. The PBP and the PPP took their origin
from both the HA and from short collateral vessels of
larger arteries[13,39,42].
In larger portal spaces, the PBP is an anastomotic
network between short collateral arterioles from arising
from the same arterial branch. The network presents two
layers only next to the hilum, being generally mono-layered.
In smaller portal spaces the PBP is progressively reduced
up to one single capillary[13,39].Capillaries are recognized by
the poorly defined endothelial imprints and the irregular
profile of the cast[37,43]. Sometimes, capillaries both from
the PBP, the PPP as well as single capillaries, were more
dilated than normal but always exhibited a smaller diameter
than adjacent sinusoids[13,39]. The PPP can be recognized
as a true net only in very large portal spaces. However, in
other regions the PPP is represented by interstitial, isolated
capillaries. In smaller spaces it is not possible to discern
periportal from peribiliary capillaries[13,39].
The extrahepatic biliary tree shows a dense vascular
network arranged around the circumference of the
common bile duct [15]. Arterial and venous trunks are
very easily distinguished by observing their different
endothelial imprints on the cast surface. Two main layers
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 3548 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
A B
Figure 1 SEMvcc of normal rat liver (orig. magn., 20). A dense vascular
network is arranged around the circumference of the common bile duct. A:
transverse section of Extrahepatic peribiliary plexus. Observe the arterial and
venous vessels on the outer layer (B) and a dense capillary network in the inner
layer of the plexus (C).
were recognized in the cast. Closely meshed arterial and
venous vessels formed the outer layer of the plexus and a
dense capillary network in the inner layer (Figure 1). The
arterial network (30-40 m) ramifies into precapillaries. At
the origin of some of precapillary vessels, an indentation
produced by perivascular cells or the rearrangement of
smooth muscle fibers can be observed[40,44]. The capillaries
that originate from precapillary arterioles form a network
plane organized in the same plane of the arterial and
venous networks and in a deeper, denser and stratified
capillary network. This capillary plane, if observed in
its internal surface, exhibited well-delineated pits where
the capillaries are arranged around a vascular space that,
at LM observation, correspond to small acinar glands
surrounded by thin capillaries. Large venous vessels can be
observed at the same plane of the arterioles; these drained
blood arising from both the superficial and inner capillary
networks[15].
The extrahepatic PBP progressively continued with
the intrahepatic PBP. It was composed of afferent arterial
vessels and capillaries and was observed more easily in
large portal tracts. In small portal tracts, the PBP was
characterized by a single layer of capillaries that typically
defined the periphery of the hepatic lobule (Figure 2).
PBP supports the bile epithelial function: the extension
of the extrahepatic peribiliary tree underlies the ability
of the bile duct to absorb water and electrolytes during
the passage of bile through this collecting system [45].
Furthermore, the absence of the gallbladder in the rat
emphasizes the role of EPBP in water reabsorption during
the interdigestive phase[15]. Finally, the connection between
the inner capillary venular plexus, portal venules and
sinusoids, permits the transport of substances directly to
the liver parenchyma where they can exert their actions.
The reduction of complexity of the PBP inside the liver
is strictly correlated with the progressive decrease of the
diameter of the small bile duct, hence in the smaller portal
tract the PBP can be reduced to only few capillaries[15].
Cholangiocyte proliferation started after BDL at the
edge of the portal tract. During the first week from BDL
the hepatic microcirculation did not show any alterations
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June 14, 2006 Volume 12 Number 22
A B *
S
Figure 2 SEMvcc of normal rat liver (orig. magn., A: 40; B: 110). (A) small
portal tract: PBP is characterized by a single layer of capillaries. (B) * = terminal
hepatic artery, V = vena porta, arrows = peribiliary plexus, S = sinusoids.
A B
S
* PBP
Figure 3 SEMvcc of BDL rat liver (orig. magn., A: 40; B: 60). (A) proliferated
PBP run at the periphery of the lobule separated from the sinusoidal network (S)
by an empty space (*) which corresponds to proliferating connective tissue. (B) not
completely developed PBP with typical neovascular organization with dead end
vessels (arrows).
with respect to the normal liver. But after two weeks
from BDL SEMvcc showed extention of the PBP from
the portal tract. The proliferated PBP presented a three-
dimensional net that did not arrange around a single
lumen but delimited different troughs with a diameter of
20-30 m. An empty space separated proliferated PBP and
portal tract vessels from sinusoids. The sinusoids showed a
normal organization with the typical structure of a classic
lobule, in which sinusoids run evidently towards the central
vein[9,13,15,24-26].
Three to four weeks after common BDL, a typical
well-developed peribiliary microvascular plexus, originating
from arterioles derived from hepatic arterial branches, is
observed. The plexus runs at the periphery of the lobule
and appears hypertrophic, but otherwise normal in its
arrangement. The plexus is composed of many layers
and shows an intimate meshed network, characterized by
round loops, resembling the organization of the inner
vascular layer of the extrahepatic peribiliary plexus (Figure
3A). Between the PBP and the sinusoidal network there
is an empty space, which corresponds to proliferating
connective tissue, digested during the casting procedure
(Figure 3A). Infrequent vascular communications between
the PBP and sinusoids are also visible. The efferent vessels
that arise from the confluence of the capillaries form a
small vein that tends to drain into the interlobular vein. In
some areas of the liver the PBP does not seem to develop
completely. In these areas there is a typical neoangiogenetic
organization with interrupted vascular loops and dead-
end vessels (Figure 3B). These structures may represent
an attempt to increase metabolic exchange within the
ductular lumen and PBP[45,46]. In contrast to the profound
 Gaudio et al. Bile duct vascularization 3549
A B
Figure 4 Immunohistochemistry for VEGF-A (A) and VEGF-C (B). Proliferating
bile ducts in BDL rat show an intense positivity for both VEGF-A and VEGF-C (Orig.
magn., 20).
modification of the PBP, the sinusoidal organization
remains quite normal even in BDL rats.
After BDL, the intrahepatic biliar y epithelium
undergoes cholangiocyte proliferation[48,50], which leads
to bile duct mass expansion, followed by an adaptive
proliferation of the PBP[9]. Proliferating bile ducts are
characterized by enhanced cholangiocyte secretory and
proliferative activities [47]. Cholangiocyte proliferation
is modulated by a complex system of growth factors,
n e u r o p e p t i d e s a n d h o r m o n e s [51-54]. M o s t o f t h i s
information has been achieved in the BDL-rat model,
which is characterized by cholangiocyte proliferation[55-57]
followed by an adaptive proliferation of the PBP[9] that
occurs after that of the intrahepatic biliary epithelium.
Therefore, the adaptive proliferation of the PBP (and its
circulating factors including VEGF) seems to be adequate
for the increased mass of the bile ductal system.
VEGF regulation
Considering the enormous extension of the PBP during
BDL, it is interesting to evaluate the possible role played by
one of the most potent and well known angiogenic factors,
the VEGF. VEGF is a member of a family of related
growth factors that includes VEGF-A, -B, -C, -D, and -E,
and placenta growth factor[58-61]. VEGFs role in vascular
proliferation associated with tumour growth or wound
healing has been widely documented in different organs[62].
The expression of VEGF and its receptors is not restricted
to vascular endothelial cells, since their expression has
been detected in vascular smooth muscle cells, osteoblasts,
regenerating myotubes and hematopoietic stem cells[59-61,63].
Moreover, VEGF has also been secreted in a large number
of normal epithelial cells, such as keratinocytes, goblet
cells in nasal polyps, pulmonary cells, prostate cells, ductal
cells derived from normal pancreas and also in normal
hepatocytes[66-68]. Because cholangiocyte proliferation, as
described above, is regulated by growth hormones, we
investigated the expression of VEGF and its receptors in
normal and proliferating biliary epithelia.
After immunohistochemistr y of liver sections
from normal and BDL rats, we found that intrahepatic
cholangiocytes from both normal and BDL rats express
VEGFR-2 and VEGFR-3 but not VEGFR-1 [38]. This
data was confirmed by immunoblot analysis in a purified
cholangiocyte cell culture. Moreover, we found that
intrahepatic cholangiocytes from nor mal and BDL
Figure 5 Immunohistochemistry for VEGF-A. Normal rat liver. The hepatocyte of
pericentral zone shows positivity for VEGF-A (Orig. magn., 20).
rats express the protein for VEGF-A and VEGF-C [38].
Immunohistochemistry shows that the expression of
VEGF-A and VEGF-C was higher in bile ducts from
BDL rats (Figure 4) compared to bile ducts from normal
rats[38]. In addition, we found VEGF in the supernatant of
primary cultures of normal cholangiocytes, indicating that
the intrahepatic cholangiocytes secrete VEGF. Following
BDL, the amount of VEGF secreted by cholangiocytes
into the supernatant significantly increased compared to
the amount of VEGF secreted by normal cholangiocytes.
In normal rat liver, pericentral hepatocytes show positivity
for VEGF-A (Figure 5).
In order to confirm that VEGF plays an important
role in the regulation of cholangiocyte proliferation, we
also tested the effect of the administration of an anti-
VEGF-A or an anti-VEGFC antibody to BDL rats. The
administration of anti-VEGF to BDL rats induced an
increased number of apoptotic cholangiocytes compared
with BDL and a decrease in the number of PCNA and
CK-19 positive cholangiocytes. In order to demonstrate
that the proliferation of the PBP following BDL only
occurs after cholangiocyte proliferation, we measured the
number of PCNA-positive vascular endothelial cells and
cholangiocytes in liver sections from rats with BDL for 1
or 2 wk. After BDL for 1 wk, PCNA was expressed only
by proliferating cholangiocytes (and in rare hepatocytes),
whereas endothelial cells were negative. In contrast, after
BDL for 2 wk, both cholangiocytes and endothelial cells
became positive for PCNA. This was confirmed by the
immunohistochemical expression of Factor VIII-related
antigen (a marker of endothelial cells)[69] in liver sections
from normal, 1- and 2-wk BDL rats. The number of
Factor VIII- related antigen positive cells increase, in
comparison with normal rats, only after 2-wk BDL but
it was unchanged after 1-wk BDL, thus confirming that
the proliferation of PBP occurs after that of the biliary
epithelium.
In addition, to better define the role of VEGF-A in
the regulation of cholangiocyte and PBP proliferation
during BDL we performed in: (1) normal rats and normal
rats + hepatic artery ligation (HAL); (2) 1 wk BDL[4,5] rats;
and (3) rats that (immediately after BDL or BDI + HAL)
were treated by IP implanted with BSA or r-VEGF-A for
1 wk[70-73]. With the aim of evaluating PBP organization
and the cholangiocyte VEGF protein expression, SEMvcc
were performed. The peribiliary plexus, observed in BDL
rats[9], was not demonstrated in BDL + HAL rats by the
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 3550 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
7
6.25 1.90
6 5.61 1.91
Bile duct volume (% volume)
5
4 3.73 1.24
3
2
1
0.2 0.05
0 Gastroenterol 2005; 21: 337-343
N BDL BDL+HAL BDL+HAL+VEGF
Figure 6 Effect of hepatic artery ligation and chronic administration of VEGF on
bile duct volume.Bile Duct volume is measured as a volume fraction of the entire
liver tissue specimen. HAL induced in BDL rats a disappearance of PBP and an
impaired cholangiocyte proliferation. The effects of HAL on PBP and cholangiocyte
functions were prevented by r-VEGF by maintaining the integrity of the peribiliary
plexus and cholangiocyte proliferation.
SEMvcc technique. We did not observe PBP in BDL +
HAL rats because the PBP is nourished by the HA. In
normal rats, PBP was observed more easily in large portal
tracts[9]. In small portal tracts, the PBP was characterized
by single layer of capillaries or even by a single capillary;
in BDL + HAL we do not observed PBP. Administration
of r-VEGF-A to BDL + HAL rats prevented the HAL-
induced microvascular modification (absence of PBP).
The microvascular pattern observed after administration
of r-VEGF-A to BDL + HAL rats demonstrated the
presence of a PBP with similar characteristics previously
described in BDL rats[9,70].
Immunohistochemistr y in BDL liver sections
shows that bile ducts express VEGF-A, VEGFR-2 and
VEGFR-3. HAL induced a decrease in the number of
cholangiocytes positive for VEGF-A and VEGFR-2 and
VEGFR-3 receptors compared to liver sections from 1
wk BDL rats. Following the administration of r-VEGF-A
to BDL + HAL rats, the expression of VEGF-A and
VEGFR-2 and VEGFR-3 was similar or higher than
that of BDL rats. VEGFR-1 was not expressed by
cholangiocytes. These indicate that VEGF, predominantly
by an autocrine mechanism, plays an important role in
modulating cholangiocyte proliferation[70]. Furthermore, if
we drastically reduced the HA blood flow in BDL rats by
the HAL, we induced the absence of PBP, reduction of
both VEGF and VEGF receptors at the cholangiocyte level
and a reduction of bile duct mass with no induced effect
in the liver of normal rats (Figure 6). Evidently, when the
intrahepatic bile duct mass is expanded, as occurs after
BDL[47], blood supply through the hepatic artery becomes
fundamental in sustaining the enhanced nutritional and
functional demands of proliferating intrahepatic biliary
epithelium[9]. VEGF has been demonstrated to participate
in a complex that sustains cholangiocyte proliferation
after BDL[50]; therefore, concerning the effect of HAL
on bile duct mass, the fall of synthesis and release of
VEGF by cholangiocytes after HAL certainly has a role in
compromising cell proliferation.
In conclusion, recent data highlights the role of arterial
blood supply of biliary tree in conditions of cholangiocyte
proliferation, such occurs during chronic cholestasis. On
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June 14, 2006 Volume 12 Number 22
the other hand, the role played by VEGF as a tool of
cross-talk between cholangiocytes and PBP endothelial
cells suggests that manipulation of VEGF release and
function could represent a therapeutic strategy for human
pathological conditions characterized by damage of
hepatic artery or the biliary tree.
REFREENCES
1 Tietz PS, LaRusso NF. Cholangiocyte biology. Curr Opin
2 Alvaro D. Biliary epithelium: a new chapter in cell biology.
Ital J Gastroenterol Hepatol 1999; 31: 78-83
3 Demetris AJ. Participation of cytokines and growth factors in
biliary cell proliferation and mito-inhibition during ductular
reaction. In: Alpini G, Alvaro D, LeSage G, Marzioni M,
LaRusso NF, eds. Pathophysiology of the Bile Duct System.
Georgetown, Texas, USA: Landes Biosciences, 2004: 167-182
4 Strazzabosco M, Fabris L, Spirli C. Pathophysiology of
cholangiopathies. J Clin Gastroenterol 2005; 39: S90-S102
5 Ohtani O, Kikuta A, Ohtsuka A, Taguchi T, Murakami T.
Microvasculature as studied by the microvascular corrosion
casting/scanning electron microscope method. I. Endocrine
and digestive system. Arch Histol Jpn 1983; 46: 1-42
6 Yamamoto K, Phillips MJ. A hitherto unrecognized bile
ductular plexus in normal rat liver. Hepatology 1984; 4: 381-385
7 Gaudio E, Pannarale L, Ripani M, Onori P, Riggio O. The
hepatic microcirculation in experimental cirrhosis. A scanning
electron microscopy study of microcorrosion casts. Scanning
Microsc 1991; 5: 495-502; discussion 502-503
8 Gaudio E, Pannarale L, Onori P, Riggio O. A scanning electron
microscopic study of liver microcirculation disarrangement in
experimental rat cirrhosis. Hepatology 1993; 17: 477-485
9 Gaudio E, Onori P, Pannarale L, Alvaro D. Hepatic micro-
circulation and peribiliary plexus in experimental biliary
cirrhosis: a morphological study. Gastroenterology 1996; 111:
1118-1124
10 Malpighi M. De hepate. Bologna, 1666
11 RAPPAPORT AM, BOROWY ZJ, LOUGHEED WM, LOTTO
WN. Subdivision of hexagonal liver lobules into a structural
and functional unit role in hepatic physiology and pathology.
Anat Rec 1954; 119: 11-33
12 Ohtani O, Murakami T. Peribiliary portal system in the rat
liver as studied by the injection replica scanning electron
microscopic method. Scanning electron microscopy. Chicago:
SEM, 1978: 241244
13 Gaudio E, Pannarale L, Carpino F, Marinozzi G. Micro-
corrosion casting in normal and pathological biliary tree
morphology. Scanning Microsc 1988; 2: 471-475
14 Haratake J, Hisaoka M, Yamamoto O, Horie A. Morphological
changes of hepatic microcirculation in experimental rat
cirrhosis: a scanning electron microscopic study. Hepatology
1991; 13: 952-956
15 Gaudio E, Onori P, Pannarale L, Marinozzi G. Microcirculation
of the extrahepatic biliary tree: a scanning electron microscopy
study of corrosion casts. J Anat 1993; 182(Pt 1): 37-44
16 ELIAS H, PETTY D. Terminal distribution of the hepatic
artery. Anat Rec 1953; 116: 9-17
17 Hase T, Brim J. Observation on the microcirculatory archi-
tecture of the rat liver. Anat Rec 1966; 156: 157-173
18 Del rio Lozano I, Andrews WH. A study by means of vascular
casts of small vessels related to the mammalian portal vein. J
Anat 1966; 100: 665-673
19 Michels NA. Newer anatomy of the liver and its variant blood
supply and collateral circulation. Am J Surg 1966; 112: 337-347
20 Mitra SK. The terminal distribution of the hepatic artery with
special reference to arterio-portal anastomosis. J Anat 1966;
100: 651-663
21 Matsumoto T, Komori R, Magara T, Ui T, Kawakami M,
Tokuda T, Takasaki S. A study on the normal structure of
the human liver, with special reference to angioarchitecture.
 Gaudio et al. Bile duct vascularization 3551
Jikeikai Med J 1979; 26: 1-40
22 Kardon RH, Kessel RG. Three-dimensional organization of the
hepatic microcirculation in the rodent as observed by scanning
electron microscopy of corrosion casts. Gastroenterology 1980;
79: 72-81
23 Yamamoto K, Sherman I, Phillips MJ, Fisher MM. Three-
dimensional observations of the hepatic arterial terminations
in rat, hamster and human liver by scanning electron
microscopy of microvascular casts. Hepatology 1985; 5: 452-456
24 Gaudio E, Onori P, Bassi A, Feliciani E, Pannarale L. Liver
microcirculation and peribiliary plexus as seen by SEM in
normal liver and in bile duct ligated rats. In: Motta PM, editor.
Advances in Microscopy of Cells, Tissues and Organs. Roma,
Italy: Antonio Delfino Editore, 1997: 449-455
25 Ohtani O. The microvascularization of the liver, the bile
duct and the gallbladder. In: LJA DiDio, PM Motta, DJ Allen.
Biopathology of the liver. Amsterdam, the Netherlands:
Elsevier, 1988: 83-88
26 Murakami T, Itoshima T, Shimada Y. Peribiliary portal system
in the monkey liver as evidenced by the injection replica
scanning electron microscope method. Arch Histol Jpn 1974; 37:
245-260
27 Ohtani O. The peribiliary portal system in the rabbit liver.
Arch Histol Jpn 1979; 42: 153-167
28 Terada T, Ishida F, Nakanuma Y. Vascular plexus around
intrahepatic bile ducts in normal livers and portal
hypertension. J Hepatol 1989; 8: 139-149
29 Kono N, Nakanuma Y. Ultrastructural and immuno-
histochemical studies of the intrahepatic peribiliary capillary
plexus in normal livers and extrahepatic biliary obstruction in
human beings. Hepatology 1992; 15: 411-418
30 Ekataksin W, Wake K. New concepts in biliary and vascular
anatomy of the liver. Prog Liv Dis 1997; 15: 1-30
31 McCuskey RS. Morphological mechanisms for regulating
blood flow through hepatic sinusoids. Liver 2000; 20: 3-7
32 McCuskey RS. A dynamic and static study of hepatic
arterioles and hepatic sphincters. Am J Anat 1966; 119: 455-477
33 Nakata K, Kanbe A. The terminal distribution of the hepatic
artery and its relationship to the development of focal liver
necrosis following interruption of the portal blood supply.
Acta Pathol Jpn 1966; 16: 313-321
34 Carpino F, Gaudio E, Marinozzi G, Melis M, Motta PM. A
scanning and transmission electron microscopic study of
experimental extrahepatic cholestasis in the rat. J Submicrosc
Cytol 1981; 13: 581-598
35 Polimeno L, Azzarone A, Zeng QH, Panella C, Subbotin
V, Carr B, Bouzahzah B, Francavilla A, Starzl TE. Cell
proliferation and oncogene expression after bile duct ligation
in the rat: evidence of a specific growth effect on bile duct
cells. Hepatology 1995; 21: 1070-1078
36 Alvaro D, Alpini G, Onori P, Perego L, Svegliata Baroni G,
Franchitto A, Baiocchi L, Glaser SS, Le Sage G, Folli F, Gaudio
E. Estrogens stimulate proliferation of intrahepatic biliary
epithelium in rats. Gastroenterology 2000; 119: 1681-1691
37 Lametschwandtner A, Lametschwandtner U, Weiger T.
Scanning electron microscopy of vascular corrosion casts--
technique and applications: updated review. Scanning Microsc
1990; 4: 889-940; discussion 941
38 GaudioE, BarbaroB, AlvaroD, GlaserS, FrancisH, UenoY,
MeiningerCJ, FranchittoA, OnoriP, MarzioniM, TaffetaniS,
FavaG, StoicaG, VenterJ, ReichenbachR, De MorrowS, 57 Alpini G, Ulrich CD 2nd, Phillips JO, Pham LD, Miller
SummersR, AlpiniG. Vascular endothelial growth factor
stimulates rat cholangiocyte proliferation via an autocrine
mechanism. Gastroenterology 2006; 130: 1270-1282
39 Pannarale L, Borghese F, Conte D, Gaudio E. Terminal
Branches of the hepatic artery (where does the hepatic artery
flow into?). A scanning electron microscopic study of vascular
corrosion casts of rat liver. Ital J Anat Embryol 2004; 109: 157
40 Hodde KC, Steeber DA, Albrecht RM. Advances in corrosion
casting methods. Scanning Microsc 1990; 4: 693-704
41 Pannarale L, Onori P, Ripani M, Gaudio E. Precapillary
patterns and perivascular cells in the retinal microvasculature.
A scanning electron microscope study. J Anat 1996; 188(Pt 3):
693-703
42 Ekataksin W. The isolated artery: an intrahepatic arterial
pathway that can bypass the lobular parenchyma in
mammalian livers. Hepatology 2000; 31: 269-279
43 Miodonski AJ, Kuss J, Tyrankiewicz R. SEM blood vessels
cast analysis. In: LJA DiDio, PM Motta, DJ Allen, editors.
Three-dimensional microanatomy of cells and tissue surfaces.
Amsterdam, the Netherlands: Elsevier, 1981: 71-87
44 Konerding MA. Scanning electron microscopy of corrosion
casting in medicine. Scanning Microsc 1991; 5: 851-865
45 Schrschmidt BF. Bile formation and cholestasis, metabolism
and enterohepatic circulation of bile ducts and gallstone
formation. In: Zamin D, Boyer TD, eds. Hepatology. A
textbook of liver disease. Philadelphia: WB Saunders, 1982:
297-351
46 Luciano L, Reale E. The human gallbladder. In: Riva & Motta
PM, eds. Ultrastructure of the extraparietal glands of the
digestive tract. Boston: Kluwer Academic, 1990: 231-248
47 Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology
in rats with bile ductular cell hyperplasia. Evidence for a
secretory function of proliferated bile ductules. J Clin Invest
1988; 81: 569-578
48 Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri
A, LeSage G, LaRusso NF. Heterogeneity of the proliferative
capacity of rat cholangiocytes after bile duct ligation. Am J
Physiol 1998; 274: G767-G775
49 Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno
N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G,
Alpini G. Gastrin inhibits cholangiocyte growth in bile duct-
ligated rats by interaction with cholecystokinin-B/Gastrin
receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and
protein kinase C alpha-dependent mechanisms. Hepatology
2000; 32: 17-25
50 Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary
epithelia. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby
W, Schachter D, Shafritz DA, eds. The Liver; Biology &
Pathobiology, 4th Ed. Philadelphia, PA: Lippincott Williams &
Wilkins, 2001: 421-435I
51 Siegel EG, Schmidt WE, Flsch UR. Severe ischemic-type
biliary strictures due to hepatic artery occlusion seven years
after liver transplantation--a rare cause of late cholestatic graft
failure. Z Gastroenterol 1998; 36: 509-513
52 Alvaro D, Gigliozzi A, Attili AF. Regulation and deregulation
of cholangiocyte proliferation. J Hepatol 2000; 33: 333-340
53 Yamagiwa Y, Patel T. Cytokine regulation of cholangiocyte
growth. In: Alpini G, Alvaro D, LeSage G, Marzioni M,
LaRusso NF, eds. Pathophysiology of the Bile Duct System.
Georgetown, Texas, USA: Landes Biosciences, 2004: 227-234
54 Nozaki I, Lunz JG 3rd, Specht S, Stolz DB, Taguchi K,
Subbotin VM, Murase N, Demetris AJ. Small proline-rich
proteins 2 are noncoordinately upregulated by IL-6/STAT3
signaling after bile duct ligation. Lab Invest 2005; 85: 109-123
55 Castaing D, Houssin D, Bismuth H. Anatomy of the liver and
portal system. In: Castaing D, Houssin D, Bismuth H, eds.
Hepatic and portal surgery in the Rat. Paris: Masson, 1980:
27-45
56 Davis BH, Madri JA. Type I and type III procollagen peptides
during hepatic fibrogenesis. An immunohistochemical and
ELISA serum study in the CCl4 rat model. Am J Pathol 1987;
126: 137-147
LJ, LaRusso NF. Upregulation of secretin receptor gene
expression in rat cholangiocytes after bile duct ligation. Am J
Physiol 1994; 266: G922-G928
58 Clauss M. Molecular biology of the VEGF and the VEGF
receptor family. Semin Thromb Hemost 2000; 26: 561-569
59 Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and
its receptors. Nat Med 2003; 9: 669-676
60 Larrive B, Karsan A. Signaling pathways induced by vascular
endothelial growth factor (review). Int J Mol Med 2000; 5:
447-456
61 Zachary I. VEGF signalling: integration and multi-tasking in
endothelial cell biology. Biochem Soc Trans 2003; 31: 1171-1177
www.wjgnet.com
 3552 ISSN 1007-9327 CN 14-1219/ R
62 Folkman J. Seminars in Medicine of the Beth Israel Hospital,
Boston. Clinical applications of research on angiogenesis. N
Engl J Med 1995; 333: 1757-1763
63 Uchida S, Sakai A, Kudo H, Otomo H, Watanuki M, Tanaka
M, Nagashima M, Nakamura T. Vascular endothelial growth
factor is expressed along with its receptors during the healing
process of bone and bone marrow after drill-hole injury in
rats. Bone 2003; 32: 491-501
64 Pertovaara L, Kaipainen A, Mustonen T, Orpana A, Ferrara
N, Saksela O, Alitalo K. Vascular endothelial growth factor
is induced in response to transforming growth factor-beta in
fibroblastic and epithelial cells. J Biol Chem 1994; 269: 6271-6274
65 C o s t e A , B r u g e l L , M a t r e B , B o u s s a t S , P a p o n J F ,
Wingerstmann L, Peyngre R, Escudier E. Inflammatory cells
as well as epithelial cells in nasal polyps express vascular
endothelial growth factor. Eur Respir J 2000; 15: 367-372
66 Marszalek A, Daa T, Kashima K, Nakayama I, Yokoyama
S. Expression of vascular endothelial growth factor and its
receptors in the developing rat lung. Jpn J Physiol 2001; 51:
313-318
67 Kllermann J, Helpap B. Expression of vascular endothelial
growth factor (VEGF) and VEGF receptor Flk-1 in benign,
www.wjgnet.com
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premalignant, and malignant prostate tissue. Am J Clin Pathol
2001; 116: 115-121
68 Teraoka H, Sawada T, Nishihara T, Yashiro M, Ohira M,
Ishikawa T, Nishino H, Hirakawa K. Enhanced VEGF
production and decreased immunogenicity induced by TGF-
beta 1 promote liver metastasis of pancreatic cancer. Br J
Cancer 2001; 85: 612-617
69 Harach HR, Jasani B, Williams ED. Factor VIII as a marker of
endothelial cells in follicular carcinoma of the thyroid. J Clin
Pathol 1983; 36: 1050-1054
70 Gaudio E, Onori P, Franchitto A, Pannarale L, Alpini G,
Alvaro D. Hepatic microcirculation and cholangiocyte
physiopathology. Ital J Anat Embryol 2005; 110(2 Suppl 1): 71-75
71 Assy N, Paizi M, Gaitini D, Baruch Y, Spira G. Clinical impli-
cation of VEGF serum levels in cirrhotic patients with or
without portal hypertension. World J Gastroenterol 1999; 5:
296-300
72 Yin R, Feng J, Yao Z. Dynamic changes of serum vascular
endothelial growth factor levels in a rat myocardial infarction
model. Chin Med Sci J 2000; 15: 154-156
73 LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte
proliferation. Liver 2001; 21: 73-80
L- Editor Pan BR E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gianfranco D Alpini, PhD, Series Editor
Bile acid interactions with cholangiocytes
Xuefeng Xia, Heather Francis, Shannon Glaser, Gianfranco Alpini, Gene LeSage
Xuefeng Xia, Gene LeSage, University of Texas at Houston
Medical School, Houston, TX, United States
Heather Francis, Division of Research and Education, Scott &
White Hospital and The Texas A&M University System Health
Science Center, College of Medicine, Temple, TX, United States
Shannon Glaser, Department of Medicine, Division of Research
and Education, Scott & White Hospital and The Texas A&M
University System Health Science Center, College of Medicine,
Temple, TX, United States
Gianfranco Alpini, Central Texas Veterans Health Care System, and
Department of Medicine and Systems Biology and Translational
Medicine, United States
Supported by a NIH grant DK54208 to Gene LeSage, and a VA
Research Scholar Award, a VA Merit Award and the NIH grants
DK58411 and DK062975 to Gianfranco Alpini
Correspondence to: Gene LeSage, MD, Professor of Medicine,
University of Texas Houston Medical School, 6431 Fannin Street,
MSB 4.234, Houston TX 77030,
United States. gene.lesage@uth.tmc.edu
Telephone: +1-713-5006677 Fax: +1-713-5006699
Received: 2006-03-29 Accepted: 2006-04-26
Abstract
Cholangiocytes are exposed to high concentrations of bile
acids at their apical membrane. A selective transporter
for bile acids, the Apical Sodium Bile Acid Cotransporter
(ASBT) (also referred to as Ibat; gene name Slc10a2)
is localized on the cholangiocyte apical membrane. On
the basolateral membrane, four transport systems have
been identified (t -ASBT, multidrug resistance (MDR)3,
an unidentified anion exchanger system and organic
solute transporter (Ost) heteromeric transporter, Ost-
Ost. Together, these transporters unidirectionally move
bile acids from ductal bile to the circulation. Bile acids
absorbed by cholangiocytes recycle via the peribiliary
plexus back to hepatocytes for re-secretion into bile.
This recycling of bile acids between hepatocytes and
cholangiocytes is referred to as the cholehepatic shunt
pathway. Recent studies suggest that the cholehepatic
shunt pathway may contribute in overall hepatobiliary
transport of bile acids and to the adaptation to chronic
cholestasis due to extrahepatic obstruction. ASBT is
acutely regulated by an adenosine 3', 5-monophosphate
(cAMP)-dependent translocation to the apical membrane
and by phosphorylation-dependent ubiquitination and
proteasome degradation. ASBT is chronically regulated
by changes in gene expression in response to biliary
bile acid concentration and inflammatory cytokines.
Another potential function of cholangiocyte ASBT is to
allow cholangiocytes to sample biliary bile acids in order
World J Gastroenterol 2006 June 14; 12(22): 3553-3563
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
TOPIC HIGHLIGHT
to activate intracellular signaling pathways. Bile acids
trigger changes in intracellular calcium, protein kinase
C (PKC), phosphoinositide 3-kinase (PI3K), mitogen-
activated protein (MAP) kinase and extracellular signal-
regulated protein kinase (ERK) intracellular signals.
Bile acids significantly alter cholangiocyte secretion,
proliferation and survival. Different bile acids have
differential effects on cholangiocyte intracellular signals,
and in some instances trigger opposing effects on
cholangiocyte secretion, proliferation and survival. Based
upon these concepts and observations, the cholangiocyte
has been proposed to be the principle target cell for bile
acids in the liver.
2006 The WJG Press. All rights reserved.
Key words: Cholangiocytes; Bile acid; Liver
Xia X, Francis H, Glaser S, Alpini G, LeSage G. Bile acid
interactions with cholangiocytes. World J Gastroenterol
2006; 12(22): 3553-3563
http://www.wjgnet.com/1007-9327/12/3553.asp
INTRODUCTION
In earlier studies of hepatobiliary physiology, it was
generally concluded that biliary components do not
appreciably interact with bile ducts and after canalicular
secretion, bile acids and lipids pass through the biliary
system as if bile ducts were inactive conduits[1]. Twenty-five
years ago, the cholehepatic shunt pathway was proposed to
explain the hypercholeretic nature of certain bile acids[2,3].
This hypothesis suggested that bile acids, in a protonated,
uncharged form, undergo passive biliary absorption,
followed by transfer of bile acids back to hepatocytes
for re-secretion into bile (Figure 1)[2]. Later, other studies
suggested that in the presence of complete bile duct
obstruction, canalicular secretion of bile acids persists
and bile acids may pass back through cholangiocytes to
the circulation instead of the entering the intestine [4].
Subsequent to the discovery of the expression of a bile
acid transporter on the apical membrane of cholangiocytes
(apical sodium-dependent bile acid transporter or
ASBT)[5-8], there has been renewed interest in the potential
of cholehepatic shunting of bile acids. Cholangiocyte
ASBT adapts to chronic cholestasis induced by bile
duct obstr uction by upregulation of cholangiocyte
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 3554 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
transport capacity potentially leading to augmentation of
cholehepatic shunting of bile acids and protection from
cholestatic liver injury due to hepatocellular retention of
bile acids[9].
Bile acids also interact with cholangiocytes leading
to alteration of cholangiocyte secretion, proliferation,
apoptosis and differentiation[7,8,10-12]. In both hepatocytes
and stellate cells, bile acids when present intracellularly in
low concentrations (e.g. less than 10 mol/L) function
as intracellular signals triggering wide variety of protein
kinases, changes in intracellular Ca2+ and phosphorylation
of proteins. Since bile acids do not appreciably enter
cells in the absence of a membrane transporter, the
expression of bile acid transporter is required for bile
acid signaling, and the degree of bile acid transporter
expression may determine the sensitivity of cells to bile
acid signaling. Thus the bile acid transporter functions
in some respects like a membrane receptor, determining
both selective and sensitivity of the cell reactions to bile
acid agonists. Bile acids, alter Ca2+, cAMP, PKC and PI3K
intercellular signaling systems in cholangiocytes. ASBT
function was shown to be required for signaling and the
level of ASBT expression correlated with cholangiocyte
sensitivity to bile acids[7,8,10,13,14]. As a manifestation of the
signaling properties of bile acids in cholangiocytes, studies
show that bile acids may directly stimulate cholangiocyte
proliferation and secretion[11,12] and thus accumulating bile
acids due to chronic cholestasis may promote the ductal
hyperplasia that occurs in chronic cholestatic liver disease.
Changes in biliary bile acid composition or concentration
may also modulate cholangiocyte survival[15,16]. Increasing
biliary bile acid concentration, by bile acid feeding, reduces
cholangiocyte apoptosis induced by CCl 4 or vagotomy
in rats [15,16] . As another potential signaling function,
bile acids may alter cholangiocyte differentiation, since
small bile ducts following chronic exposure to bile acids
begin expressing proteins and functions normally only
present in large intrahepatic bile ducts [7]. Therapeutic
bile acids (ursodeoxycholate), perhaps surprisingly, have
opposing effects on cholangiocyte function compared
to endogenous bile acids. Ursodeoxycholate appears to
reduce cholangiocyte proliferation and reduce bile mass in
animal models of bile duct hyperplasia[8]. The implications
of these findings are discussed later.
The purpose of this review is familiarizing the reader
in the role of bile acids in cholangiocyte biology and
pathophysiology. Recent studies unexpectedly show that
cholangiocytes transport bile acids and adapt to changes in
biliary bile acids. Considering that one of the fundamental
events in cholestasis is the retention in the liver of com-
ponents that are normally secreted in bile, alternative
pathways for elimination of biliary components would be
an important adaptation of the liver to cholestasis. The
review will outline mechanisms for bile acid transport
in cholangiocytes, the role of bile acid regulation of
cholangiocyte proliferation, secretion and apoptosis in
animal models and human diseases. First the mechanisms
for bile acid uptake at the cholangiocyte apical membrane
will be reviewed. Second, basolateral bile acid efflux
mechanisms are outlined. Third, the current evidence
for cholehepatic shunting of bile acids is summarized.
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June 14, 2006 Volume 12 Number 22
Figure 1 Classic cholehepatic shunt
A B pathway (B) compared to direct biliary bile
acid secretion (A). Bile acids that are poor
substrates for coenzyme A (CoA) synthetase
and inefficiently conjugated by hepatocytes
are passively absorbed by biliary epithelium.
After canalicular secretion, the unconjugated
bile acid is absorbed by cholangiocytes
because of the high lipophilicity of the
protonated acid. The absorbed bile acid is
ASBT returned to the hepatocyte mass via the
periductular plexus. The osmotic effect of
multiple passages of the unconjugated bile
acid anions into canalicular bile results in
hypercholeresis. The presence of ASBT
on the apical membrane of cholangiocytes
provides a potential mechanism for absorption
of conjugated bile acids that then follows the
same shunt pathway as described above.
Next the mechanisms responsible for acute and chronic
regulation of ASBT activity in cholangiocytes are reviewed.
Finally, the concept of intracellular bile acid acting as
signaling molecules in cholangiocytes will be developed
and the evidence for bile acid regulation of cholangiocyte
secretion, proliferation and survival will be summarized.
CHOLANGIOCYTE BILE ACID UPTAKE
Although earlier studies have sug g ested bile acid
uptake mechanisms were present in cholangiocytes, the
identification of ASBT by Lazaridis et al [17] and Alpini
et al[5] brought to the forefront the interest in bile acid
transport in the biliary system. ASBT had previously
been identified in ileum and kidney tubules[18] and ASBT
has been proposed as the major transporter involved in
the reclamation of bile acids in the intestine and in the
nephron, respectively[18]. Studies by Alpini et al[5] showed
the presence of gene expression for both the ASBT and
ileal bile acid binding protein (IBABP) in cholangiocytes.
Immunof luorescence studies showed that ABAT
protein is expressed on the apical membrane of isolated
cholangiocytes and isolated bile duct units (IBDU)[5]. Our
studies showed the majority of [3H]-taurocholate uptake
is Na+-dependent with a Km of 43 mol/L and a Vmax of
190 pmol/min[5]. These values were lower compared to
measurements by Lazaridis et al[17], however studies by Alpini
et al[5] were performed in freshly isolated cholangiocytes
(which may be a more physiological model) whereas
those by Lazaridis were done in a rat cholangiocyte cell
line. In addition, the kinetics for taurocholate uptake in
freshly isolated cholangiocytes has a similar Km and Vmax
as reported by other investigators for ASBT-mediated
uptake in the ileum [19,20] . The K m for a transporter is
generally similar to the physiologic concentration of
the transported substrate. The markedly lower K m for
ASBT in cholangiocytes compared to biliary bile acid
concentration may be due to the effect of unstirred layer
adjacent bile duct lumen membrane that would reduce
the effective bile acid concentration immediately adjacent
to the cholangiocyte apical membrane[21]. Lazaridis et al[17]
demonstrated vectorial transport of bile acids from apical
to basolateral direction and an absence of transport in the
basolateral to apical direction in a normal rat cholangiocyte
 Xia X et al. Bile acid interactions 3555
cell line in a polarized culture system. No additional bile
acid uptake proteins have as of yet been identified in the
cholangiocyte apical membrane.
The natural substrate specificity of ASBT is narrow
and restricted to unconjugated bile acids as well as their
glycine- and taurine-conjugates[22]. ASBT appears to play
a major role in the enterohepatic circulation of bile acids
since dysfunctional mutations in the mouse or human
ASBT genes cause profound bile acid malabsorption in
humans [23]. In the renal tubule ASBT acts as a salvage
mechanism to prevent urinary excretion of bile acids that
undergo glomerular filtration. The role of ASBT in bile
ducts is not as obvious as in the intestine and kidney. We
have proposed that ASBT functions in a cholehepatic
shunt where bile acids are absorbed in the biliary tract,
secreted into the periductular capillary plexus, and carried
directly back to the hepatocyte for secretion, thereby
promoting bile flow. Alternatively, ASBT may function
to sense bile acid concentration in bile, a mechanism that
is dependent on bile acid uptake by ASBT and bile acid-
dependent activation of intracellular signaling systems in
cholangiocytes. The principles of cholehepatic shunting
and bile acid signaling are discussed in a later section.
Intracellular bile acid movement in cholangiocytes
In hepatocytes, cytosolic binding proteins have been
shown to sequester bile acids in a bound state that may
prevent the cytotoxicity of free intracellular bile acids[24,25].
The presence of high affinity binding sites would
significantly reduce the rate of transcellular transport of
bile acids. Indeed, previous studies have shown that the
transcellular transport comprises the greatest proportion
of time in the overall transcellular transport of conjugated
bile acids in hepatocytes[26].
The ileal bile acid binding protein (IBABP) is expressed
in cholangiocytes [5]. Very little is known as to whether
it functions to prevent intracellular toxicity, modulates
transcellular transport or it changes in expression in
response to increased bile acid flux in cholangiocytes
or in the presence of cholestasis. Recent studies show
expression of IBABP in the ileum is regulated by bile
acid concentrations through the effects of farnesoid X
receptor[27].
Bile acid efflux in cholangiocytes
Since Lazaridis et al [17] studies have shown that bile
acid transport in cholangiocytes is vectorial (e.g. apical-
to-basolateral), mechanisms are likely present in the
basolateral membrane that facilitates the efflux of bile
acids into peribiliary plexus circulation. Four mechanisms
have been identified in previous studies that together
account for bile acid efflux in cholangiocytes. The first
mechanism was identified employing bile duct fragments.
The studies showed that fluorescent bile acid analogs
can be taken up across the cholangiocyte basolateral
membrane [28] . The uptake process involved an anion
exchanger mechanism that was identified by inhibition
of bile acid uptake in the absence of Cl- or HCO3- or the
presence of 4, 4-diisothiocyanostilbene-2, 2-disulfonic
acid[28]. Although the authors studied uptake across the
basolateral membrane in their model, they proposed that
their studies reflected physiologically an anion exchanger
that effluxes bile acids out of cholangiocytes subsequent
to apical uptake. The protein responsible for this anion
exchanger was not identified.
The second mechanism for bile acid efflux was
identified by Lazaridis et al [6] as an exon-2 skipped,
alternatively spliced form of ASBT, designated t-ASBT.
Alternative splicing causes a frameshift that produces a
154-aa protein. T-ASBT is expressed in rat cholangiocytes,
ileum, and kidney and is localized to the basolateral
domain of cholangiocytes[6]. Transport studies in Xenopus
oocytes revealed that t-ASBT functions as a bile acid efflux
protein. Compared to ASBT, alternative splicing changes
the cellular targeting of ASBT provides a mechanism for
rat cholangiocytes to efflux bile acids at the basolateral
membrane [6]. The regulation of t-ASBT expression in
response to biliary or circulatory bile acid concentrations
or in the presence of cholestasis has not been determined.
The regional distribution of t-ASBT in large and small
ducts is not known. The third mechanism for bile acid
efflux in cholangiocytes is MDR3. Previous studies
have shown that MDR3 is expressed on the basolateral
membrane of cholangiocytes and MDR3 is upregulated
in chronic cholestasis associated with type 3 progressive
familial intrahepatic cholestasis[29]. It has been proposed
that up-regulation of MDR3 may promote cholehepatic
shunting in chronic cholestasis, thus preventing the toxic
effects of accumulating bile acids in cholangiocytes[29].
Similar upregulation of MDR3 in hepatocyte basolateral
membranes has been proposed to pump bile acids out of
hepatocytes with canalicular cholestasis[30] and MDR3 is
upregulated in patients with Dubin-Johnson syndrome[31].
There is no direct evidence that shows MDR3 effluxes
bile acids in cholangiocytes or provides a mechanism for
prevention of accumulation of bile acids in cholangiocytes
during cholestasis.
Recently the Ost-Ost heteromeric transporter was
initially identified in the liver, ileum, and kidney [32]. In
contrast to all other organic anion transporters identified to
date, transport activity requires the coexpression of both
Ost and Ost proteins. Substrates for this transporter
include the bile acid taurocholate, other steroids (estrone
3-sulfate and digoxin), and prostaglandin E2[33]. Ost and
Ost mRNA expression along the mouse gastrointestinal
tract mirrors that of ASBT, and both Ost and Ost
proteins are localized to the basolateral surface of ileal,
renal and bile duct cells[32]. Studies of bile acid transport in
Ost and Ost expressing Xenopus laevis oocytes showed
bile acid efflux and trans-stimulation, indicating that
transport occurs by facilitated diffusion. The selective
localization of Ost and Ost to the basolateral plasma
membrane of epithelial cells responsible for bile acid
and sterol reabsorption, the substrate selectivity of the
transporter, suggest that heteromeric Ost and Ost is an
important basolateral bile acid transporter in biliary, ileal
and renal epithelial cells.
Cholehepatic shunting of bile acids
Hoffman proposed bile acids may cycle between cho-
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 3556 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
langiocytes and hepatocytes through a cholehepatic shunt
pathway[3]. Unconjugated bile acids[34] or non-charged bile
acids (norursodeoxycholate)[34] were observed to induce a
greater degree of bile flow per bile acid molecule excreted
in bile. To account for this hypercholeretic effect, it was
proposed that unconjugated bile acids may be passively
absorbed by bile ducts; enter the peribiliary plexus
adjacent to intrahepatic bile ducts, then forwarded to the
hepatic sinusoids to be returned to cholangiocytes by
hepatocyte secretion[3]. Typically, cholehepatic shunting
initiated by passive absorption of non-ionized bile salt
results in the generation of bicarbonate molecules in
bile, which then increases biliary bicarbonate excretion.
Other criteria for cholehe patic shunting besides
hypercholeresis and alkalinization of bile have been
identified. With the hypercholeresis, the biliary transit
time for the unconjugated or non charged bile acids
was greater than expected which would be predicted by
longer retention time in the liver due to more than one
passage through the hepatobiliary axis[34]. Back perfusion
of the isolated perfused liver (infusion into the hepatic
vein), a route where the blood from the peribiliary plexus
does not appreciably enter the hepatic sinusoids, reduced
the hypercholeretic effect of ursodeoxycholic acid [35].
Increased number of bile ducts in animal models of
cirrhosis was associated with increased hypercholeresis
due to ursodeoxycholic acid infusion, an observation that
was attributed to increased cholehepatic shunting due to
increase bile duct mass[36]. Similarly, bile duct proliferation
in Mdr2(-/-) mice is associated with a disproportionably
high bile flow in response to tauroursocholate acid
infusion, a finding that was interpreted as due to enhanced
cholehepatic shunting of bile salts due to increased
number of bile ducts. The magnitude of absorption of
bile acids under physiologic or pathophysiologic conditions
in man is not known.
Identification of apical and basolateral bile acid
transport proteins in cholangiocytes, points to the
possibility that the cholehepatic shunt pathway functions
in bile secretion and may adapt in response to cholestasis.
From a functional point of view, the pathway provides a
mechanism to enhance bile acid-dependent bile flow and
biliary lipid excretion. With multiple passages of bile acids
through the canalicular membrane (as a result of recycling
through cholangiocytes), the cholehepatic shunt pathway
has the potential to increase the efficiency of bile acid-
induced biliary lipid excretion and bile acid-dependent
bile flow[3]. From the pathophysiologic point of view, the
pathway provides an alternative route for continuation
of hepato-cholangiocyte flux of bile acids despite the
presence of complete bile duct obstruction[37]. The latter
may well be an important pathophysiologic response of
the liver to bile duct obstruction.
In support of ASBT initiating cholehepatic shunting, our
studies[38] have shown that following the administration of
secretin to bile duct ligated rats, there is acute upregulation
of ASBT in cholangiocytes (as described in the next
section). With secretin stimulation of ASBT activity in
cholangiocytes, there is a marked increase in taurocholate-
induced choleresis (12 2 L per mol bile acid excreted cholangiocytes with the microtubule inhibitor colchicine
with basal cholangiocyte ASBT activity compared to 38
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June 14, 2006 Volume 12 Number 22
6 L per mol bile acid excreted during high cholangiocyte
ASBT activity). Similarly, with experimental augmentation
of cholangiocyte ASBT activity, taurocholate induces a
much greater increase in biliary phospholipid (1.5-fold
increase in mol phospholipid per mol bile acid excreted
compared to basal) and cholesterol secretion (2-fold
increase in mol cholesterol per mol bile acid excreted
compared to basal). Finally, following the administration
of secretion, the taurocholate transit time is increased
by 7 min. The taurocholate-induced hypercholeresis,
increased biliary lipid excretion and increased taurocholate
transit time are consistent with enhanced taurocholate
cholehepatic shunting due to up regulation of ASBT by
secretin. Additional studies will be needed to establish the
degree of cholehepatic shunting of conjugated bile acids
in normal rats.
OVERVIEW OF THE REGULATION OF ASBT
EXPRESSION IN CHOLANGIOCYTES
Alteration of bile acid transporter activity may occur
physiologically in response to local bile acid concentrations
to fine tune bile acid transport capacity. For instance,
ASBT has been shown by some studies to be upregulated
in the ileum with increased intestinal bile concentration[39],
so as to provide increased intestinal bile acid transport
activity in response to increased intestinal bile acid load.
Alternatively, bile acid transporter expression may be
chronically modified in pathologic conditions to prevent
intracellular accumulation of toxic bile acids due to
altered bile acid metabolism or retention[40]. For instance,
in chronic cholestasis due to bile duct obstruction, there
is downregulation of hepatocyte sinusoid transporters
and upregulation of hepatocyte canalicular transporters
that provides the combined effect to reduce hepatocyte
intracellular retention of bile acids that occurs with
cholestasis[41]. Regulation of bile acid transporters may also
occur regionally within anatomical confines of an organ.
For instance, ASBT expression is present exclusively in the
distal ileum (and not proximal small bowel)[42], restricted
to the mature enterocytes lining the villus, with little or
no detectable expression in the small intestinal crypt.
Sinusoidal transporters are present to a greater degree in
the periportal region compared to the pericentral region
of the hepatic lobule[41]. Temporally, regulation of bile acid
transporters has been shown to occur gradually through
changes in gene expression[39,40,43,44], or acutely by changes
in cell membrane transporter content by transporter
translocation [45,46] or by changes in the rate of protein
degradation.
Acute regulation of ASBT in cholangiocytes
Previous studies have shown that ASBT transport activity
acutely increases in cholangiocytes and ileal epithelial cells
by a cAMP-dependent mechanism[38,47]. In cholangiocytes,
increased cAMP induced by secretin doubles Na + -
dependent bile acid uptake in isolated cholangiocytes[38]
and in perfused bile duct fragments [48]. This effect is
likely due to protein translocation, since pretreatment of
prevents the cAMP-induced increase of Na+-dependent
 Xia X et al. Bile acid interactions 3557
bile acid transport[38]. The effects of secretin on protein
translocation of ASBT to the apical membrane of
cholangiocytes were studied employing isolated apical
membranes from cholangiocytes[38]. These studies showed
that cAMP increases apical membrane ASBT only in the
absence of colchicine. A model for cAMP-dependent
recycling of ASBT in the cholangiocyte apical membrane is
shown in Figure 2. The model shows that cAMP increases
apical ASBT membrane content, but also suggests that
ASBT recycles back to latent intracellular stores once the
secretin/cAMP stimulus has abated. This mechanism
for acute induction of ABAT activity in cholangiocytes
has been proposed to provide an accentuation of
cholehepatic bile acid shunting in the postprandial period,
thus accentuating bile flow and biliary lipid secretion (see
cholehepatic shunting section above)[38].
Chronic regulation of ASBT expression in cholangiocytes
ASBT expression in cholangiocytes changes chronically in
response to biliary bile acid concentrations, the presence
of cholestasis[7-9,15] or inflammation. With increase in biliary
bile acid concentration, due to feeding taurocholate to rats,
there is an increase in total liver ASBT[7]. In this model,
the increased ASBT is due to both increased number of
cholangiocytes in the liver and the maintenance of ASBT
expression per cell. In bile duct ligated rats depleted of
biliary bile acids for 12 h by external biliary drainage,
there is a marked decrease in cholangiocyte ASBT gene
and protein expression and transport activity[49]. ASBT
gene and protein expression and transport activity
can be restored in bile-depleted rats by infusion of
taurocholate to maintain biliary bile acid concentration[49].
These studies employing bile acid feeding and bile acid
depletion show that ASBT expression in cholangiocytes is
chronically regulated in a direct proportion to biliary bile
acid concentration. In contrast to taurocholate feeding
which increases cholangiocyte ASBT expression, feeding
ursodeoxycholic acid to bile duct ligated rats markedly
reduces cholangiocyte ASBT gene and protein expression
and taurocholate transport activity [8] . Although the
mechanism for differential effects of different bile acids on
ABAT expression in cholangiocytes has not been defined;
they are consistent with our studies showing differential
effects of different bile acids on cholangiocyte secretion
and proliferation (see below).
With chronic cholestasis due to bile duct ligation,
intrahepatic bile ducts markedly increase in number
(approximately 10 fold increase after 1 wk). In this model,
ASBT expression per cholangiocyte is maintained[9], so
that overall there is an effective increase in biliary bile
acid absorptive capacity in BDL rats. We propose that
the increased ASBT in BDL rats provides an alternative
excretory pathway in the presence of biliary obstruction
so as to prevent the bile acid stasis in the liver and
the subsequent accumulation of toxic bile acids in
hepatocytes[9].
Recently it was demonstrated that bile acids modulate
ASBT expression through activation of the peroxisome
proliferator-activated receptor alpha (PPARalpha) [18]
and activator protein 1 (AP-1) element regulates the
transcription of the rat ASBT gene[44].
Apical membrane
cAMP
Exocytosis
ASBT
Endocytosed ASBT
Endocytosis
Figure 2 Membrane recycling of ASBT. Increased cAMP enhances bile acid
uptake in cholangiocytes. Under basal conditions, intracellular ASBT resides
in an inactive position within the cytoplasm of cholangiocytes as well as on the
apical membrane. Increased cAMP results in translocation of ASBT to the apical
membrane where it inserts by exocytosis. Once on the apical membrane, ASBT
becomes active and mediates absorption of bile acids from bile. Membrane
recycling of ASBT is completed by removal of ASBT from the apical membrane by
endocytosis.
Previous studies have shown that the stress induced
alteration of ASBT genetic expression due to inflammation
and bile acids is a consequence of trans-activation of
the ASBT promoter by c-Jun/c-Fos and liver receptor
homologue-1, respectively[10,47] and that hepatocyte nuclear
factor-1 is critical for basal expression of ASBT.
Recently, the inflammatory cytokine IL-1 has been
shown to rapidly down-regulate ASBT in the terminal
ileum [50] . Dysregulation of the ASBT adaptation to
cholestasis (due to increased expression of IL-1) could
blunt the compensatory up-regulation of ASBT in response
to cholestasis and promote bile acid-induced liver damage.
Recent data demonstrates that the ubiquitin-proteasome
degradation system affects the activity of some membrane
transporters[51,52]. The system is responsible for the disposal
of many of the short-lived proteins in eukaryotic cells.
The ubiquitin-proteasome pathway targets proteins for
degradation via covalent tagging of the substrate protein
with a polyubiquitin chain[53]. This degradation pathway is
implicated in the regulation of many short-lived proteins
involved in essential cellular functions, including cell cycle
control, transcription regulation, signal transduction, and
protein translocation. We speculated that the initial ASBT
down-regulation due to ileal inflammation or due to IL-1
in vitro is caused by enhanced ASBT disposal by the
ubiquitin-proteasome pathway. Our studies showed that
ASBT is an unstable protein that is rapidly degraded with
a half-life of approximately 6 h. We showed that the rapid
IL-1 -dependent reduction of ASBT in cholangiocytes
is due to increased ASBT disposal via the ubiquitin-
proteasome pathway. IL-1 mediated down-regulation
of ASBT expression requires phosphorylation of ASBT
since mutation of two ASBT phosphorylation sites
reduces the rate of ASBT disposal under basal conditions
and markedly reduces IL-1 -dependent ubiquitination
and disposal of ASBT. These results indicate that the
proteasome plays an important role in the regulation of
ASBT protein level in cholangiocytes.
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 3558 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Regional ASBT expression in the biliary tree
Regionalization of bile duct function occurs in rat liver[5].
Large (greater than 20 m diameter bile ducts) that are
lined by large cholangiocytes contribute to hormone-
induced ductal secretion whereas small (smaller than 20
m diameter bile ducts) do not contribute to hormone-
induced ductal secretion [54,55]. Studies by Alpini et al [5]
showed that ASBT is expressed in large cholangiocytes but
not small cholangiocytes. The absence of ABST expression
in small intrahepatic bile ducts may lead to more efficient
hepatobiliary excretion of bile acids, since bile acid uptake
in small ducts, closely adjacent to the canalicular bile acid
secretion process, may hinder the post canalicular assembly
of polymolecular bile acid-lipid micelles and vesicles.
Recently, experimental models have been developed
where ASBT gene, and protein expression and transport
activity have been shown to extend into small bile ducts[7].
In taurocholate fed rats, a model where biliary bile acid
concentrate increases approximately two fold, Alpini
et al[7] found de novo ASBT expression in small ducts. The
authors suggested that with expansion of the bile acid pool
and increased biliary bile acid concentration, the extension
of ASBT expression into small ducts leads to enhanced
cholehepatic shunting of bile acids. Whether ASBT
expression in small ducts alters bile acid-lipid micelles or
vesicle formation has not been determined.
BILE ACID SIGNALING IN CHOLANGIOCYTES
Overview
In a variety of cells, bile acids have been shown to function
as intracellular signals and to profoundly alter cellular
functions such as proliferation, differentiation, secretion,
and apoptosis [56-61] . These studies have shown that
cellular uptake is required for bile acids to signal cellular
processes. In cells not expressing a bile acid transporter,
(which normally do not respond to the presence of bile
acids in the media) experimental expression of a bile acid
transporter activates de novo bile acid signaling[62]. Once
intracellular, bile acids at concentrations of less that 1
2+
mol/L, have been shown to alter intracellular Ca , PKC,
MEK, ERK and PI3K pathways in hepatocytes or chola
ngiocytes[16,59,61,63-66]. Through these downstream signals,
bile acids have been shown to alter cell proliferation,
secretion, apoptosis and gene expression [8,16,61,63-67]. In
addition, bile acids have been shown to induce activation (by
phosphorylation) of the EGF receptor in hepatocytes[64]
and cholangiocytes[68]. The signaling effects of bile acids
should be distinguished from the toxic effects of bile acids,
where bile acid in high concentration, through changes in
membrane lipids induces, in a nonspecific manner, cellular
damage[69].
Bile acid signaling of cholangiocyte secretion
It had previously been observed that ursodeoxycholic
acid increases biliary bicarbonate excretion into bile.
Shimokura et al[66] found that ursodeoxycholic acid directly
increases cholangiocyte secretion. They demonstrated
ursodeoxycholic acid increases intracellular Ca 2+ and
increases chloride channel activity in a malignant cho-
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June 14, 2006 Volume 12 Number 22
langiocyte cell line [66]. The ursodeoxycholate effect on
chloride channel activity was dependent on the increase
in intracellular Ca 2+. Our studies have shown that in
freshly isolated cholangiocytes, taurocholic acid or
taurolithocholic acid (1-20 mol/L) increases secretin-
stimulated cAMP levels and secretin-stimulated Cl-/HCO3
exchanger activity[12]. These effects were dependent on
taurocholate uptake by ASBT, since the stimulatory
effect of taurocholate was not present in the absence of
Na+[12]. Dependence of bile acid effects on cholangiocyte
secretion and ASBT transport activity, we found the
K m for ASBT in cholangiocytes to be close to the
concentration where in bile acids exert their maximum
response in cholangiocytes[12]. Similar to their effect in vitro,
taurocholate or taurolithocholate feeding to normal rats
for 7 d increased secretin-stimulated cAMP and Cl-/HCO3-
exchanger activity in cholangiocytes and resulted in an
increase of secretin-stimulated ductal bile flow in vitro[7].
The studies show that both in vitro and in vivo, bile acids
can augment secretin-stimulated ductal bile flow but the
intracellular signals responsible for this effect have not
been completely elucidated. Recently, cAMPindependent,
bile activation of CFTR was shown to be present in ileal
cells[70]. Like cholangiocytes, ASBT-dependent bile acid
uptake was required for CFTR activation but the molecular
mechanism for CFTR activation was not disclosed in this
study.
In contrast to the stimulatory effects of taurocholate
and taurolithocholate, ursodeoxycholate inhibits secretin-
stimulated cAMP synthesis and Cl -/HCO 3-exchanger
activity in isolated cholangiocytes and secretin-stimulated
ductal bile flow in vivo [8]. Inhibition of cholangiocyte
secretion by ursodeoxycholic acid was found to be
dependent on the ability of ursodeoxycholic acid to
increase intracellular calcium and activate PKCalpha
i n ch o l a n g i o c y t e s [ 8 ] . We h ave p r o p o s e d t h a t t h e
stimulatory and inhibitory effects of taurocholate and
ursodeoxycholate, respectively on cholangiocyte secretion
is due to their differing ability to activate intracellular Ca2+
and to activate different PKC isoforms[8].
Bile acid signaling of cholangiocyte proliferation
In vitro, taurocholate and taurolithocholate in 1-20 mol/L
concentrations increase H3-histone expression (a maker
of cholangiocyte proliferation)[12]. Feeding taurocholate or
taurolithocholate to rats increases 3H-thymindine uptake in
cholangiocytes, increases bile duct mass 2 to 3 fold[11] and
consistent with a ductal hyperplasia there is accentuation
of secretin-stimulated cAMP synthesis and ductal bile
flow. The effects of taurocholate and taurolithocholate on
bile duct proliferation in the bile acid feeding models occur
in the absence hepatic inflammation[11]. Recent studies[68]
show that taurocholate can induce phosphorylation of
EGF receptor in cholangiocytes, similar to that reported in
hepatocytes[64] and the transactivation of the EGF receptor
requires transforming growth factor alpha and matrix
metalloproteinase[71].
In contrast to taurocholate and taurolithocholate,
ursodeoxycholate inhibits cholangiocyte proliferation both
in vitro in isolated cholangiocytes and in vivo in bile duct
 Xia X et al. Bile acid interactions 3559
ligated rats[8]. The inhibition of cholangiocyte proliferation
was found to be dependent on activation of PKCalpha
and calcium-dependent pathways[8]. The inhibitory effect
of ursodeoxycholate on cholangiocyte proliferation may
be one mechanism for the histological and biochemical
improvement of diseases targeting the biliary tree (e.g.
primary biliary cirrhosis) with ursodeoxycholate treatment.
Inhibition of cholangiocyte proliferation may reduce
the number of proliferating cholangiocytes that release
proinflammatory cytokines [72] or profibrotic signaling
molecules such as platelet-derived growth factor[73]. The
lack of therapeutic effect for ursodeoxycholic acid in the
late stage primary biliary cirrhosis may be at least partially
related to lack of proliferating ducts (e.g. ductopenia) as
the disease progresses [74]. In a cholangiocarcinoma cell
line, we demonstrated that ursodeoxycholic acid inhibits
growth by inhibition of Raf through PKC-dependent
mechanism[13]. This study supports the need for clinical
trials examining the effect of ursodeoxycholic acid in the
promotion and progression of cholangiocarcinoma in
patients with primary sclerosing cholangitis.
Bile acid signaling of cholangiocyte death and survival
pathways
Previous studies in hepatocytes have shown bile acids
may be either cytotoxic[75-78] or cytoprotective[79]. Bile acid
cytotoxicity may be induced by abrupt permeability of
the inner mitochondrial membrane to ions leading to
mitochondrial membrane permeability transition (MMPT),
depolarization of the mitochondrial membrane potential
and uncoupling of oxidative phosphorylation [80]. The
uncoupling of oxidative phosphorylation, if extensive,
results in ATP depletion and cellular death by necrosis[80].
Furthermore, the associated mitochondrial swelling has
also been linked to redistribution of cytochrome c from
the intermembrane space to the cytosol. In the cytosol,
cytochrome c interacts with apoptotic protease-activating
factor 1 to activate caspase 9 and subsequently to cause
apoptosis[81]. Bile salt-induced hepatocyte apoptosis also
entails activation of the Fas death-receptor and subsequent
activation of caspase 8 followed by activation of Bid which
leads to mitochondrial dysfunction[75].
Alternatively, bile acids may provide cytoprotective
effects. Heuman et al[82] proposed that the protective effect
of ursodeoxycholic acid in opposing the hepatotoxicity
of bile acids was due to its direct interaction with plasma
membranes of hepatocytes. Ursodeoxycholic acid may
provide membrane stability via a physicochemical effect by
reducing the toxic bile salt disruption of cholesterol-rich
model membranes[82]. More recent studies, revealed that
ursodeoxycholic acid does not directly stabilize membranes
but rather prevents hydrophobic bile acid-induced
membrane disruption by alteration of the structure and
composition of mixed micelles[83].
In cholangiocytes, Benedetti et al [78] showed that in
vitro, unconjugated but not conjugated bile acids induce
ultrastructural evidence of cytotoxicity. These findings
were not observed in vivo. Even taurine depleted livers
did not show evidence of cytotoxicity despite having
high concentration of unconjugated bile acids in bile[78].
The authors concluded that bile acid toxicity, although
potentially present in biliary epithelium, is prevented in the
intact liver by the presence of active bile acid transport in
cholangiocytes.
Our studies have shown that taurocholate feeding is
protective against cholangiocyte apoptosis induced by
either CCl 4[16] or vagotomy [15]. In CCl 4 treated animals,
cholangiocyte apoptosis, demonstrated by the presence
of nuclear fragmentation, positive annexin staining and
loss of cholangiocyte function (secretin-stimulated cAMP
synthesis), was not observed in CCl4-treated rats that were
fed taurocholate[16]. Similarly, CCl4-induced apoptosis in
vitro was ablated by pretreating cholangiocytes with 20
mol/L taurocholate. The taurocholate inhibition of CCl4-
induced apoptosis required activation of PKCalpha [16].
Taurocholate feeding also prevents vagotomy induced
cholangiocyte apoptosis [15]. In the vagotomy-induced
model, apoptosis is associated with loss of PI3K activity
and activation of caspase activities[15]. Taurocholate feeding
prevented cholangiocyte apoptosis, loss of PI3K activity
and activation of caspase activity. Thus, taurocholate
is protective ag ainst CCl 4 - and vag otomy-induced
cholangiocyte apoptosis by activation of the PKC and
PI3K-dependent pathways, respectively.
Feeding ursodeoxycholate inhibits the ductal hyperplasia
in BDL rats, however these studies showed that the effect
of ursodeoxycholate was on inhibition of proliferation
without increasing cholangiocyte apoptosis[8]. In contrast,
Que et al [84] showed that ursodeoxycholate inhibits
beauvericin-induced apoptosis in a cholangiocarcinoma cell
line and that the inhibition was dependent on preventing
cytochrome C release from mitochondria and subsequent
activation of caspases.
BILE ACID SYNTHESIS AND CONJUGATION
IN CHOLANGIOCYTES
Bile acids, phospholipids and cholesterol are synthesized in
a cholangiocarcinoma cell line[85]. Cholangiocytes have also
been shown to conjugate bile acids[85]. The contribution
of cholangiocyte bile acid synthesis and conjugation
to the over all bile acid pool seems minimal since less
than 3 percent of the total liver mass is composed of
cholangiocytes[86].
Our preliminary data suggests that bile acid synthesis
through the alternative (mitochondrial) pathway by
cholesterol 27-hydroxylase (Cyp27) maybe important in the
regulation of cholesterol transport and prevent cholesterol
toxicity due to oxysterols in cholangiocytes[87]. Oxysterols
(which increase with a higher cell cholesterol pool) are
cytotoxic to smooth muscle and endothelial cells and are
potentially carcinogenic.
Oxysterols are present in bile, have been shown to
induce cholangiocyte apoptosis and may be carcinogenic
in biliary epithelium by increasing expression of COX-2[88].
The mechanisms for preventing oxysterol lipotoxicity
in cholangiocytes are unknown. Our preliminary studies
show normal rat cholangiocytes express the cholesterol
transporters ATP binding cassette subclass A (ABCA1)
on the basolateral membrane and Niemann-Pick C1 Like
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 3560 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
1 (NPC1L1) on the apical membrane [87,89]. The nuclear
receptors involved in sensing lipids liver X receptor
(LXR) and peroxisome proliferator-activated receptor
(PPAR ) and Cyp27 are abundantly expressed in
cholangiocytes[87]. Activation of LXR, PPAR, or both
in cholangiocytes induces ABCA1 gene and protein
expression and increases basolateral excretion of
cholesterol. Added oxysterols activate LXR expression
and increase basolateral membrane cholesterol efflux.
Elevated bile acid levels decrease CYP27 expression.
Cholangiocytes absorption of cholesterol at the apical
membrane is dependent on the expression of NPC1L1,
a cholesterol transporter recently shown to be required
for cholesterol absorption in the intestine. Like ABCA1,
NPC1L1 is upregulated by PPAR ligands. We propose
that upregulation of CYP27 leads to production of
oxysterols, which then activate LXR and cholesterol efflux
in cholangiocytes. These studies identify a potential reverse
cholesterol transport pathway in cholangiocytes regulated
by the cholangiocyte cholesterol and bile acids pools. We
propose, like the reverse cholesterol transport involving
macrophages, cholesterol or oxysterols taken up from bile
by cholangiocytes is released into the circulation return
to hepatocytes for elimination. The cholangiocyte reverse
cholesterol transport pathway may function to prevent
biliary damage due to oxysterols.
GALLBLADDER EPITHELIAL CELLS
Gallbladder epithelial cells and cholangiocytes share a
number of functions, however their primary role in the
liver and their reaction to disease are quite different. Similar
to cholangiocytes, gallbladder epithelial cells express ASBT,
and taurocholate have been shown to increase intracellular
calcium, activate chloride channels and stimulate mucin
secretion [90]. In contrast, ursodeoxycholate, through a
PKCalpha-dependent pathway inhibits gallbladder mucin
production[90]. The authors proposed that ursodeoxycholate
inhibition of gallbladder mucin could provide benefit to
biliary disorders such as cystic fibrosis.
BILE ACID EFFECTS ON CHOLANGIOCYTE
FUNCTION OR DYSFUNCTION IN HUMANS
Compared to the understanding of the effects of bile
acids on rodent cholangiocytes, very little is known
regarding bile acid signaling of cholangiocyte function
in health and disease in humans. Previous studies of
biliary bicarbonate secretion in humans, by employing
PET scanning, show that biliary bicarbonate secretion is
reduced in primary biliary cirrhosis, the prototypic disease
of bile duct damage in humans[91]. After treatment with
ursodeoxycholate, biliary bicarbonate secretion in primary
biliary cirrhosis patients is increased compared to the
pretreatment values[91]. It is not clear why ursodeoxycholic
acid inhibits cholangiocyte secretion in bile duct ligated
rats but increases cholangiocyte secretion in primary biliary
cirrhosis in humans. It is likely that the pathophysiology of
these two forms of biliary injury is different.
Cystic fibrosis also targets biliary epithelium in the
liver[92]. Clinical studies have shown that ursodeoxycholate
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June 14, 2006 Volume 12 Number 22
may improve liver tests in cystic fibrosis patients [93]. It
has been proposed that the mechanism for action of
ursodeoxycholate in cystic fibrosis is increased ductal
bile flow [as demonstrated by the opening of chloride
channels by Shimokura et al [66] ] that reduces the bile
plugs and obstruction due to thick biliary secretions[94].
Ursodeoxycholate has been found to have measurable
clinical effects in other diseases that target biliar y
epithelium (graft versus host disease involving the liver,
liver allograft rejection and bile-duct paucity syndromes)[95].
Finally, ursodeoxycholate has been used in other chronic
cholestatic liver disorders where biliary epithelium
is not the primary target (intrahepatic cholestasis of
pregnancy, progressive familial intrahepatic cholestasis,
non alcoholic steatohepatitis, alcoholic liver disease,
autoimmune hepatitis)[95]. The potential therapeutic effect
of ursodeoxycholate in human liver diseases is reviewed
elsewhere[95].
In summary, bile acids interact with cholangiocytes in
numerous ways. A specific bile acid transporter (ASBT)
is localized on the apical membrane, posed to absorb
biliary bile acids[5,17]. On the basolateral membrane, four
transport systems have been identified (t-ASBT, MDR3, an
anion exchanger system and the Ost-Ost heteromeric
transporter) [6,28,29]. Studies in cultured cholangiocytes
show that cholangiocytes transport bile acids from the
apical to the basolateral membrane[17]. Indirect evidence
for a cholehepatic shunt pathway initiated by bile acid
absorption from bile by ASBT that leads to bile acids
return via the peribiliary plexus to hepatocytes for secretion
into bile[7,34,36-38,41]. The contribution of the cholehepatic
shunt pathway in overall hepatobiliary transport of bile
acids and the role of the cholehepatic shunt pathway in
the adaptation to chronic cholestasis due to extrahepatic
obstruction remains to be determined. ASBT is both
acutely regulated by a cAMP-dependent translocation
to the apical membrane[38] and chronically regulated by
changes in gene expression in response to biliary bile acid
concentration and ubiquitination-dependent proteasome
disposal[9]. Biliary bile acid concentration and composition
may regulate cholangiocyte functions. After uptake by
ASBT, bile acids signal calcium, PKC, PI3K, MEK and
ERK intracellular signals in cholangiocytes with resultant
changes in cholangiocyte secretion, proliferation and
survival[11,12,59,61,66,96]. Different bile acids have differential
effects on cholangiocyte intracellular signals, resulting in
opposite effects on cholangiocyte secretion, proliferation
and survival[12,96,97].
In future studies, the mechanisms explaining how bile
acids with different structures can differentially regulate
different intracellular signals will be determined. To
address the question of how chronic cholestatic liver
disease may adapt by changes in cholehepatic shunting,
new experimental paradigms to directly quantify bile
acid absorption in bile ducts in experimental animals will
be developed. Since multiple transporters with varying
substrate specificity are present in the sinusoidal and
canalicular membrane of hepatocytes, additional bile
acid transporters may be found in cholangiocytes. When
the mechanisms for bile acid cytoprotective effects in
cholangiocytes are defined, a new therapeutic window
 Xia X et al. Bile acid interactions 3561
in human biliary disorders may open that operates
through modulation of biliary bile acid concentration and
composition. Finally, the role of cholangiocyte bile acid
transport in the promotion or adaptation to human liver
disease needs to be determined.
REFERENCES
1 Strange RC. Hepatic bile flow. Physiol Rev 1984; 64: 1055-1102
2 Kanai S, Kitani K, Sato Y. The nature of choleresis induced
by deoxycholate and its conjugates in the rabbit. Jpn J Physiol
1989; 39: 907-918
3 Hofmann AF. Current concepts of biliary secretion. Dig Dis
Sci 1989; 34: 16S-20S
4 Buscher HP, Miltenberger C, MacNelly S, Gerok W. The histo-
autoradiographic localization of taurocholate in rat liver after
bile duct ligation. Evidence for ongoing secretion and reab-
sorption processes. J Hepatol 1989; 8: 181-191
5 Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE,
Lasater J, Caligiuri A, Tretjak Z, LeSage GD. Functional ex-
pression of the apical Na+-dependent bile acid transporter in
large but not small rat cholangiocytes. Gastroenterology 1997;
113: 1734-1740
6 Lazaridis KN, Tietz P, Wu T, Kip S, Dawson PA, LaRusso NF.
Alternative splicing of the rat sodium/bile acid transporter
changes its cellular localization and transport properties. Proc
Natl Acad Sci U S A 2000; 97: 11092-11097
7 Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL, Francis H,
Lesage G. Bile acid feeding increased proliferative activity and
apical bile acid transporter expression in both small and large
rat cholangiocytes. Hepatology 2001; 34: 868-876
8 Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Francis H,
Phinizy JL, Angelico M, Lesage G. Ursodeoxycholate and tau-
roursodeoxycholate inhibit cholangiocyte growth and secre-
tion of BDL rats through activation of PKC alpha. Hepatology
2002; 35: 1041-1052
9 Alpini G, Glaser S, Phinizy JL, Kanno N, Francis H, Ludvik
M, and LeSage G.Regulation of cholangiocyte apical bile acid
transporter (ABAT) activity by biliary bileacids: different po-
tential compensatory changes for intrahepatic and extrahepat-
iccholestasis. Gastroenterology 2001; 120: A6
10 LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte
proliferation. Liver 2001; 21: 73-80
11 Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis
H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD. Bile acid
feeding induces cholangiocyte proliferation and secretion: evi-
dence for bile acid-regulated ductal secretion. Gastroenterology
1999; 116: 179-186
12 Alpini G, Glaser S, Robertson W, Phinizy JL, Rodgers RE,
Caligiuri A, LeSage G. Bile acids stimulate proliferative and
secretory events in large but not small cholangiocytes. Am J
Physiol 1997; 273: G518-G529
13 Alpini G, Kanno N, Phinizy JL, Glaser S, Francis H, Taffetani
S, LeSage G. Tauroursodeoxycholate inhibits human cholan-
giocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent
pathways. Am J Physiol Gastrointest Liver Physiol 2004; 286:
G973-G982
14 Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholan-
giocyte bicarbonate secretion. Am J Physiol Gastrointest Liver
Physiol 2001; 281: G612-G625
15 Marzioni M, LeSage GD, Glaser S, Patel T, Marienfeld C,
Ueno Y, Francis H, Alvaro D, Tadlock L, Benedetti A, Marucci
L, Baiocchi L, Phinizy JL, Alpini G. Taurocholate prevents the
loss of intrahepatic bile ducts due to vagotomy in bile duct-
ligated rats. Am J Physiol Gastrointest Liver Physiol 2003; 284:
G837-G852
16 Alpini G, Marucci L, Glaser S, LeSage G. Taurocholate (TC)
but not taurolithocholate (TLC) abrogates carbon tetrachloride
(CCl4)-induced cholangiocyte apoptosis by a phosphatidylino-
sitol 3-kinase (PI3K)-dependent pathway. Hepatology 1999; 30:
A897
17 Lazaridis KN, Pham L, Tietz P, Marinelli RA, deGroen PC,
Levine S, Dawson PA, LaRusso NF. Rat cholangiocytes absorb
bile acids at their apical domain via the ileal sodium-depen-
dent bile acid transporter. J Clin Invest 1997; 100: 2714-2721
18 Jung D, Fried M, Kullak-Ublick GA. Human apical sodium-
dependent bile salt transporter gene (SLC10A2) is regulated
by the peroxisome proliferator-activated receptor alpha. J Biol
Chem 2002; 277: 30559-30566
19 Marcus SN, Schteingart CD, Marquez ML, Hofmann AF,
Xia Y, Steinbach JH, Ton-Nu HT, Lillienau J, Angellotti MA,
Schmassmann A. Active absorption of conjugated bile acids in
vivo. Kinetic parameters and molecular specificity of the ileal
transport system in the rat. Gastroenterology 1991; 100: 212-221
20 Higgins JV, Paul JM, Dumaswala R, Heubi JE. Downregula-
tion of taurocholate transport by ileal BBM and liver BLM in
biliary-diverted rats. Am J Physiol 1994; 267: G501-G507
21 Aldini R, Roda A, Lenzi PL, Ussia G, Vaccari MC, Mazzella G,
Festi D, Bazzoli F, Galletti G, Casanova S. Bile acid active and
passive ileal transport in the rabbit: effect of luminal stirring.
Eur J Clin Invest 1992; 22: 744-750
22 Craddock AL, Love MW, Daniel RW, Kirby LC, Walters HC,
Wong MH, Dawson PA. Expression and transport proper-
ties of the human ileal and renal sodium-dependent bile acid
transporter. Am J Physiol 1998; 274: G157-G169
23 Oelkers P, Kirby LC, Heubi JE, Dawson PA. Primary bile
acid malabsorption caused by mutations in the ileal sodium-
dependent bile acid transporter gene (SLC10A2). J Clin Invest
1997; 99: 1880-1887
24 Stolz A, Sugiyama Y, Kuhlenkamp J, Osadchey B, Yamada
T, Belknap W, Balistreri W, Kaplowitz N. Cytosolic bile acid
binding protein in rat liver: radioimmunoassay, molecular
forms, developmental characteristics and organ distribution.
Hepatology 1986; 6: 433-439
25 Yamamuro W, Stolz A, Takikawa H, Sugimoto M, Kaplowitz
N. Distribution of 3 alpha-hydroxysteroid dehydrogenase (bile
acid binder) in rat small intestine: comparison with glutathi-
one S-transferase subunits. J Gastroenterol 1994; 29: 115-119
26 LeSage G, Hofmann AF. Effect of bile acid hydrophobicity on
biliary transit timeand intracellular mobility: A comparison of
four fluorescent bile acid analogues. Gastroenterology 1994; 106:
A929
27 Hwang ST, Urizar NL, Moore DD, Henning SJ. Bile acids
regulate the ontogenic expression of ileal bile acid binding
protein in the rat via the farnesoid X receptor. Gastroenterology
2002; 122: 1483-1492
28 Benedetti A, Di Sario A, Marucci L, Svegliati-Baroni G, Schtein-
gart CD, Ton-Nu HT, Hofmann AF. Carrier-mediated transport
of conjugated bile acids across the basolateral membrane of
biliary epithelial cells. Am J Physiol 1997; 272: G1416-G1424
29 Scheffer GL, Kool M, de Haas M, de Vree JM, Pijnenborg AC,
Bosman DK, Elferink RP, van der Valk P, Borst P, Scheper RJ.
Tissue distribution and induction of human multidrug resis-
tant protein 3. Lab Invest 2002; 82: 193-201
30 Hirohashi T, Suzuki H, Ito K, Ogawa K, Kume K, Shimizu
T, Sugiyama Y. Hepatic expression of multidrug resistance-
associated protein-like proteins maintained in eisai hyperbili-
rubinemic rats. Mol Pharmacol 1998; 53: 1068-1075
31 Knig J, Rost D, Cui Y, Keppler D. Characterization of the
human multidrug resistance protein isoform MRP3 localized
to the basolateral hepatocyte membrane. Hepatology 1999; 29:
1156-1163
32 Ballatori N, Christian WV, Lee JY, Dawson PA, Soroka CJ,
Boyer JL, Madejczyk MS, Li N. OSTalpha-OSTbeta: a major ba-
solateral bile acid and steroid transporter in human intestinal,
renal, and biliary epithelia. Hepatology 2005; 42: 1270-1279
33 Wang W, Seward DJ, Li L, Boyer JL, Ballatori N. Expression
cloning of two genes that together mediate organic solute and
steroid transport in the liver of a marine vertebrate. Proc Natl
Acad Sci U S A 2001; 98: 9431-9436
34 Gurantz D, Schteingart CD, Hagey LR, Steinbach JH, Grotmol
T, Hofmann AF. Hypercholeresis induced by unconjugated
bile acid infusion correlates with recovery in bile of unconju-
gated bile acids. Hepatology 1991; 13: 540-550
www.wjgnet.com
 3562 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
35 Perez Barriocanal F, Marin JJ, Dumont M, Erlinger S. Influence
of backward perfusion on ursodeoxycholate-induced cholere-
sis in isolated in situ rat liver. J Hepatol 1990; 11: 165-171
36 Elsing C, Sgesser H, Reichen J. Ursodeoxycholate-induced
hypercholeresis in cirrhotic rats: further evidence for cholehe-
patic shunting. Hepatology 1994; 20: 1048-1054
37 Lamri Y, Erlinger S, Dumont M, Roda A, Feldmann G. Im-
munoperoxidase localization of ursodeoxycholic acid in rat
biliary epithelial cells. Evidence for a cholehepatic circulation.
Liver 1992; 12: 351-354
38 Alpini G, Glaser S, Baiocchi L, Francis H, Xia X, Lesage G.
Secretin activation of the apical Na+-dependent bile acid
transporter is associated with cholehepatic shunting in rats.
Hepatology 2005; 41: 1037-1045
39 Shneider BL, Michaud GA, West AB, Suchy FJ. The effects of
bile acid feeding on the development of ileal bile acid trans-
port. Pediatr Res 1993; 33: 221-224
40 Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD,
Kramer W, Boyer JL. Adaptive regulation of bile salt trans-
porters in kidney and liver in obstructive cholestasis in the rat.
Gastroenterology 2001; 121: 1473-1484
41 Meier PJ, Stieger B. Bile salt transporters. Annu Rev Physiol
2002; 64: 635-661
42 Shneider BL, Setchell KD, Crossman MW. Fetal and neonatal
expression of the apical sodium-dependent bile acid transport-
er in the rat ileum and kidney. Pediatr Res 1997; 42: 189-194
43 Christie DM, Dawson PA, Thevananther S, Shneider BL.
Comparative analysis of the ontogeny of a sodium-dependent
bile acid transporter in rat kidney and ileum. Am J Physiol
1996; 271: G377-G385
44 Chen F, Ma L, Al-Ansari N, Shneider B. The role of AP-1 in the
transcriptional regulation of the rat apical sodium-dependent
bile acid transporter. J Biol Chem 2001; 276: 38703-38714
45 Webster CR, Blanch C, Anwer MS. Role of PP2B in cAMP-
induced dephosphorylation and translocation of NTCP. Am J
Physiol Gastrointest Liver Physiol 2002; 283: G44-G50
46 Sun AQ, Arrese MA, Zeng L, Swaby I, Zhou MM, Suchy FJ.
The rat liver Na(+)/bile acid cotransporter. Importance of the
cytoplasmic tail to function and plasma membrane targeting. J
Biol Chem 2001; 276: 6825-6833
47 Reymann A, Braun W, Drobik C, Woermann C. Stimulation
of bile acid active transport related to increased mucosal cyclic
AMP content in rat ileum in vitro. Biochim Biophys Acta 1989;
1011: 158-164
48 Alpini G, Glaser S, Chowdhury U, Francis H, Kanno N,
Phinizy JL, Eisel W, LeSage G. cAMP-dependent translocation
of the apical bile acid transporter (ABAT) to the cholangiocyte
apical membrane regulates ductal absorption of conjugated
bile acids. Hepatology 1999; 30: A1029
49 Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H,
Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, Lesage
G. Bile acid depletion and repletion regulate cholangiocyte
growth and secretion by a phosphatidylinositol 3-kinase-de-
pendent pathway in rats. Gastroenterology 2002; 123: 1226-1237
50 Chen F, Ma L, Sartor RB, Li F, Xiong H, Sun AQ, Shneider B.
Inflammatory-mediated repression of the rat ileal sodium-
dependent bile acid transporter by c-fos nuclear translocation.
Gastroenterology 2002; 123: 2005-2016
51 Skach WR. Defects in processing and trafficking of the cystic
fibrosis transmembrane conductance regulator. Kidney Int
2000; 57: 825-831
52 Hatakeyama S, Nakayama KI. Ubiquitylation as a quality con-
trol system for intracellular proteins. J Biochem 2003; 134: 1-8
53 Schwartz DC, Hochstrasser M. A superfamily of protein tags:
ubiquitin, SUMO and related modifiers. Trends Biochem Sci
2003; 28: 321-328
54 Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, La-
sater J, LeSage GD. Large but not small intrahepatic bile ducts
are involved in secretin-regulated ductal bile secretion. Am J
Physiol 1997; 272: G1064-G1074
55 Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV,
LeSage G, LaRusso NF. Morphological, molecular, and func-
tional heterogeneity of cholangiocytes from normal rat liver.
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
Gastroenterology 1996; 110: 1636-1643
56 Brady LM, Beno DW, Davis BH. Bile acid stimulation of early
growth response gene and mitogen-activated protein kinase is
protein kinase C-dependent. Biochem J 1996; 316 (Pt 3): 765-769
57 Podevin P, Rosmorduc O, Conti F, Calmus Y, Meier PJ, Pou-
pon R. Bile acids modulate the interferon signalling pathway.
Hepatology 1999; 29: 1840-1847
58 Di Toro R, Campana G, Murari G, Spampinato S. Effects of
specific bile acids on c-fos messenger RNA levels in human
colon carcinoma Caco-2 cells. Eur J Pharm Sci 2000; 11: 291-298
59 Nathanson MH, Burgstahler AD, Masyuk A, Larusso NF.
Stimulation of ATP secretion in the liver by therapeutic bile
acids. Biochem J 2001; 358: 1-5
60 Voronina S, Longbottom R, Sutton R, Petersen OH, Tepikin A.
Bile acids induce calcium signals in mouse pancreatic acinar
cells: implications for bile-induced pancreatic pathology. J
Physiol 2002; 540: 49-55
61 Marrero I, Sanchez-Bueno A, Cobbold PH, Dixon CJ. Tauro-
lithocholate and taurolithocholate 3-sulphate exert different
effects on cytosolic free Ca2+ concentration in rat hepatocytes.
Biochem J 1994; 300 (Pt 2): 383-386
62 Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG,
Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD,
Lehmann JM. Bile acids: natural ligands for an orphan nuclear
receptor. Science 1999; 284: 1365-1368
63 Kurz AK, Block C, Graf D, Dahl SV, Schliess F, Hussinger
D. Phosphoinositide 3-kinase-dependent Ras activation by
tauroursodesoxycholate in rat liver. Biochem J 2000; 350 (Pt 1):
207-213
64 Rao YP, Studer EJ, Stravitz RT, Gupta S, Qiao L, Dent P, Hy-
lemon PB. Activation of the Raf-1/MEK/ERK cascade by bile
acids occurs via the epidermal growth factor receptor in pri-
mary rat hepatocytes. Hepatology 2002; 35: 307-314
65 Kurz AK, Graf D, Schmitt M, Vom Dahl S, Hussinger D. Tau-
roursodesoxycholate-induced choleresis involves p38(MAPK)
activation and translocation of the bile salt export pump in
rats. Gastroenterology 2001; 121: 407-419
66 Shimokura GH, McGill JM, Schlenker T, Fitz JG. Ursodeoxy-
cholate increases cytosolic calcium concentration and activates
Cl- currents in a biliary cell line. Gastroenterology 1995; 109:
965-972
67 Spirl C, Nathanson MH, Fiorotto R, Duner E, Denson LA,
Sanz JM, Di Virgilio F, Okolicsanyi L, Casagrande F, Strazza-
bosco M. Proinflammatory cytokines inhibit secretion in rat
bile duct epithelium. Gastroenterology 2001; 121: 156-169
68 Yoon JH, Higuchi H, Werneburg NW, Kaufmann SH, Gores
GJ. Bile acids induce cyclooxygenase-2 expression via the epi-
dermal growth factor receptor in a human cholangiocarcinoma
cell line. Gastroenterology 2002; 122: 985-993
69 Puglielli L, Amigo L, Arrese M, Nez L, Rigotti A, Garrido
J, Gonzlez S, Mingrone G, Greco AV, Accatino L. Protective
role of biliary cholesterol and phospholipid lamellae against
bile acid-induced cell damage. Gastroenterology 1994; 107:
244-254
70 Bijvelds MJ, Jorna H, Verkade HJ, Bot AG, Hofmann F, Agel-
lon LB, Sinaasappel M, de Jonge HR. Activation of CFTR by
ASBT-mediated bile salt absorption. Am J Physiol Gastrointest
Liver Physiol 2005; 289: G870-G879
71 Werneburg NW, Yoon JH, Higuchi H, Gores GJ. Bile acids ac-
tivate EGF receptor via a TGF-alpha-dependent mechanism in
human cholangiocyte cell lines. Am J Physiol Gastrointest Liver
Physiol 2003; 285: G31-G36
72 Park J, Gores GJ, Patel T. Lipopolysaccharide induces cholan-
giocyte proliferation via an interleukin-6-mediated activation
of p44/p42 mitogen-activated protein kinase. Hepatology 1999;
29: 1037-1043
73 Grappone C, Pinzani M, Parola M, Pellegrini G, Caligiuri A,
DeFranco R, Marra F, Herbst H, Alpini G, Milani S. Expression
of platelet-derived growth factor in newly formed cholan-
giocytes during experimental biliary fibrosis in rats. J Hepatol
1999; 31: 100-109
74 Degott C, Zafrani ES, Callard P, Balkau B, Poupon RE, Poupon
R. Histopathological study of primary biliary cirrhosis and the
 Xia X et al. Bile acid interactions 3563
effect of ursodeoxycholic acid treatment on histology progres-
sion. Hepatology 1999; 29: 1007-1012
75 Higuchi H, Miyoshi H, Bronk SF, Zhang H, Dean N, Gores GJ.
Bid antisense attenuates bile acid-induced apoptosis and cho-
lestatic liver injury. J Pharmacol Exp Ther 2001; 299: 866-873
76 Danchenko E, Petermann H, Chirkin A, Dargel R. Effect of
bile acids on the proliferative activity and apoptosis of rat he-
patocytes. Exp Toxicol Pathol 2001; 53: 227-233
77 Meerman L, Koopen NR, Bloks V, Van Goor H, Havinga R,
Wolthers BG, Kramer W, Stengelin S, Mller M, Kuipers F,
Jansen PL. Biliary fibrosis associated with altered bile com-
position in a mouse model of erythropoietic protoporphyria.
Gastroenterology 1999; 117: 696-705
78 Benedetti A, Alvaro D, Bassotti C, Gigliozzi A, Ferretti G, La
Rosa T, Di Sario A, Baiocchi L, Jezequel AM. Cytotoxicity of
bile salts against biliary epithelium: a study in isolated bile
ductule fragments and isolated perfused rat liver. Hepatology
1997; 26: 9-21
79 Miyoshi H, Rust C, Guicciardi ME, Gores GJ. NF-kappaB is
activated in cholestasis and functions to reduce liver injury.
Am J Pathol 2001; 158: 967-975
80 Patel T, Steer CJ, Gores GJ. Apoptosis and the liver: A mecha-
nism of disease,growth regulation, and carcinogenesis. Hepa-
tology 1999; 30: 811-815
81 Ota K, Yakovlev AG, Itaya A, Kameoka M, Tanaka Y, Yoshi-
hara K. Alteration of apoptotic protease-activating factor-1
(APAF-1)-dependent apoptotic pathway during development
of rat brain and liver. J Biochem 2002; 131: 131-135
82 Heuman DM, Bajaj R. Ursodeoxycholate conjugates protect
against disruption of cholesterol-rich membranes by bile salts.
Gastroenterology 1994; 106: 1333-1341
83 Barrios JM, Lichtenberger LM. Role of biliary phosphatidyl-
choline in bile acid protection and NSAID injury of the ileal
mucosa in rats. Gastroenterology 2000; 118: 1179-1186
84 Que FG, Phan VA, Phan VH, LaRusso NF, Gores GJ. GUDC
inhibits cytochrome c release from human cholangiocyte mito-
chondria. J Surg Res 1999; 83: 100-105
85 Zoltowska M, Delvin EE, Paradis K, Seidman E, Levy E.
Bile duct cells: a novel in vitro model for the study of lipid
metabolism and bile acid production. Am J Physiol 1999; 276:
G407-G414
86 Alpini G, Prall RT, LaRusso NF. The pathobiology of biliary
epithelia. In: Arias IM, Boyer JL, Chisari FV, Fausto N, Ja-
koby W, Schachter D, Shafritz DA, eds. The Liver; Biology &
Pathobiology, 4th ed. Philadelphia, PA: Lippincott Williams &
Wilkins, 2001: 421-435
87 Jung D, Xia X, Moore DD, LeSage G. Reverse Cholesterol
Transport in Cholangiocytes is regulated by LXR. Hepatology
2004: 493A
88 Yoon JH, Canbay AE, Werneburg NW, Lee SP, Gores GJ. Oxy-
sterols induce cyclooxygenase-2 expression in cholangiocytes:
implications for biliary tract carcinogenesis. Hepatology 2004;
39: 732-738
89 Xia X, Zhang X, Xiao Y, Chukwunvere E, Gao D, Kone B, LeS-
age G. Niemann-Pick C1 Like 1 (NPC1L1) absorbs cholesterol
from bile and is regulated by PPARdelta incholangiocytes.
Hepatology 2005; 41: 41
90 Chignard N, Mergey M, Veissire D, Parc R, Capeau J, Pou-
pon R, Paul A, Housset C. Bile acid transport and regulating
functions in the human biliary epithelium. Hepatology 2001; 33:
496-503
91 Prieto J, Garca N, Mart-Climent JM, Peuelas I, Richter JA,
Medina JF. Assessment of biliary bicarbonate secretion in hu-
mans by positron emission tomography. Gastroenterology 1999;
117: 167-172
92 Diwakar V, Pearson L, Beath S. Liver disease in children with
cystic fibrosis. Paediatr Respir Rev 2001; 2: 340-349
93 Cotting J, Lentze MJ, Reichen J. Effects of ursodeoxycholic acid
treatment on nutrition and liver function in patients with cystic
fibrosis and longstanding cholestasis. Gut 1990; 31: 918-921
94 Lebensztejn DM. Application of ursodeoxycholic acid (UDCA)
in the therapy of liver and biliary duct diseases in children.
Med Sci Monit 2000; 6: 632-636
95 Lazaridis KN, Gores GJ, Lindor KD. Ursodeoxycholic acid
mechanisms of action and clinical use in hepatobiliary disor-
ders. J Hepatol 2001; 35: 134-146
96 Alpini G, Glaser S, Phinizy JL, Rodgers R, Robertson W, Ca-
ligiuri A, Lasater J, Tretjak Z, LeSage G. Bile acid depletion
decreases cholangiocyte proliferative capacity and secretin-
stimulated ductal bile secretion in bile duct ligated (BDL) rats.
Gastroenterology 1997; 112: A1210
97 Alpini G, Glaser S, Caligiuri A, Phinizy JL, Rodgers R, Francis
H, Robertson W,Papa E, Lasater J, LeSage G. Ursodeoxycholic
acid feeding inhibits secretin-inducedcholangiocyte secretory
processes in bile duct ligated rats. Gastroenterology 1998; 114:
AL0016
S- Editor Pan BR E- Editor Bi L
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REVIEW
Synchronous and metachronous occurrence of gastric
adenocarcinoma and gastric lymphoma: A review of the
literature
Erhan Hamaloglu, Serdar Topaloglu, Arif Ozdemir, Ahmet Ozenc
Erhan Hamaloglu, Arif Ozdemir, Ahmet Ozenc, Department
of Surgery, School of Medicine, Hacettepe University, 06100
Shhiye, Ankara, Turkey
Serdar Topaloglu, First Department of Surgery, Ankara Numune
Training and Research Hospital, 06100 Shhiye, Ankara, Turkey
Co-first-authors: Erhan Hamaloglu, Serdar Topaloglu
Co-correspondents: Erhan Hamaloglu
Correspondence to: Serdar Topaloglu, MD, Kl Apt. No 10/4, 6.
cadde, veler, 06450-1, ankaya, Ankara,
Turkey. serdartopaloglu@hotmail.com
Telephone: +90-31-24786109 Fax: +90-31-24182760
Received: 2006-02-14 Accepted: 2006-03-10
Abstract
The occurrence of both primary gastric lymphoma and
gastric adenocarcinoma in the same patient is a rare
entity. The possible causative factors of synchronous or
metachronous occurrence of both malignancies and vari-
eties in the treatment modalities are reviewed according
to published cases in English language medical literature.
2006 The WJG Press. All rights reserved.
Key words: Gastric adenocarcinoma; Gastric lymphoma;
Synchronous occurrence; Metachronous occurrence
Hamaloglu E, Topaloglu S, Ozdemir A, Ozenc A. Synchro-
nous and metachronous occurrence of gastric adenocarcino-
ma and gastric lymphoma: A review of the literature. World
J Gastroenterol 2006; 12(22): 3564-3574
http://www.wjgnet.com/1007-9327/12/3564.asp
INTRODUCTION
Secondary malignancies associated with Hodgkins or non-
Hodgkins lymphoma are commonly detected in relation
with the improvement in overall survival rate of these pa-
tients. The most common type of secondary malignancy
seen with lymphoma is acute myelogenous leukemia,
however, epithelial malignancies are also reported[1-3]. In-
tensive radiotherapy and chemotherapy administered to
these patients have raised questions about the long-term
effects of these therapeutic modalities on the development
of secondary malignancies. Primary gastric lymphoma
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World J Gastroenterol 2006 June 14; 12(22): 3564-3574
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
is a relatively uncommon malignancy, occurring in only
1%-7% of all malignant neoplasms of the stomach[4]. The
occurrence of both primary gastric lymphoma and gastric
adenocarcinoma in the same patient is a rare entity. We
review the possible causative factors of synchronous or
metachronous occurrence of both malignancies and va-
rieties in the treatment modalities according to published
cases in English language medical literature.
INCIDENCE
Since the first description of multiple gastric cancers by
Barth in 1855[5], case reports of benign and malignant mul-
tiple primary tumors have been appearing with an increas-
ing frequency. The incidence of multiple simultaneous
gastric cancers is high, ranging from 3.4% to 67.4%[6]. In
the first published case series in Germany, authors did not
mention the incidence of simultaneous occurrence of ma-
lignant lymphoma and adenocarcinoma in the stomach[7].
Thereafter, case series from Japan appeared in the litera-
ture. Kasahara et al[8] reviewed data of 35 Japanese patients
without giving incidence rate for synchronous occurrence.
Noda et al[9] found an association of both tumors in 2 of
2438 (0.08%) gastric carcinoma cases. The incidence of
simultaneous occurrence was 3.7% (9/247) in primary gas-
tric lymphomas and 0.098% (9/9150) in gastric carcinomas
in series by Nakamura et al[10]. Despite definite expression
of incidence rate for synchronous cases, the incidence of
metachronous occurrence of both tumors has not been
mentioned in the literature.
SYNCHRONOUS CASES
Since the first case reported by Rabinovitch et al[11], 56 cas-
es of synchronous occurrence of gastric adenocarcinoma
and lymphoma have been published in English language
medical literature[7-31]. Majority of these reports included
detailed analysis of histopathologic features of tumors in-
stead of clinical follow-up of patients. Basic characteristics
of the patients are summarized on Table 1. Twenty-three
cases were from the Eastern and 33 cases were from the
Western countries. The overall median age was 66 (range
27-85) years. The median age for the Eastern population
was 67 and for the Western population was 72 years. Male
predominance was observed in both the Western (1.75:1)
and the Eastern (3:1) populations. Overall, 62.5% of pa-
tients had early gastric carcinoma (EGC) and 30.4% of pa-
 Hamaloglu E et al. Gastric adenocarcinoma and lymphoma 3565

Table 1 Synchronous cases reported in the literature

Reference Age/sex Lymphoma type H pylori Adenocarcinoma type Therapy Survival

months
Rabinovitch[11] 64/m Lymphosarcoma NM EGC S. gastrectomy NM
Jernstrom[12] 72/f Lymphocytic lymphosarcoma NM AGC, I, W S. gastrectomy and 750 R radiotherapy 10
Manier[13] 65/m Histiocytic NM EGC, P T. gastrectomy and chemotherapy 24
72/m Histiocytic NM AGC, P T. gastrectomy 1.5
Lin[14] 56/m Diffuse lymphocytic NM EGC, W T. gastrectomy and 4500 cGy Co60 radiotherapy 24
Kane[15] 74/m Diffuse lymphocytic NM EGC, P S. gastrectomy, radiotherapy and chemotherapy 12
Planker[7] 65/m Immunocytoma NM EGC, I, M T. gastrectomy, splenectomy and 4000 cGy Co60 30
radiotherapy
Czerniak[16] 74/m Mix type NM EGC, W, S. gastrectomy, radiotherapy and chemotherapy 12
Kasahara[8] 77/m Small cleaved NM AGC, W S. gastrectomy 24
Noda[9] 61/m Diffuse large cell NM EGC, W T. gastrectomy and chemotherapy 9
Akosa[17] 79/m MALT, LG NM EGC, M No treatment NM
Kelly[18] 85/m MALT, LG + ?, I, M No treatment 2
Von Herbay[19] 79/f MALT, LG + HG + EGC, I, W T. gastrectomy
Wotherspoon[20] 55/f MALT, LG + AGC, P NM NM
55/f MALT, LG + HG + EGC, W NM NM
NM/m MALT, LG - AGC, W NM NM
60/f MALT, LG + AGC, W NM NM
34/f MALT, LG + AGC, P NM NM
67/f MALT, LG + EGC, W NM NM
55/f MALT, LG + EGC, W NM NM
55/f MALT, LG - EGC, P NM NM
69/f MALT, LG + AGC, W NM NM
Nishino[21] 71/m Diffuse, large cell type NM EGC, tubular type, W T. gastrectomy, splenectomy and 120
cyclophosphamide, oncovin, prednisolone
chemotherapy
Hardman[22] 56/m MALT, LG + AGC, Signet-ring cell, P T. gastrectomy and etoposide, cisplatin, adriamycin NM
chemotherapy
Nakamura[10] 27/m MALT, LG + EGC, I, W NM 57
38/m MALT, LG + EGC, D, P NM 45
70/m MALT, HG + EGC, I, W NM 81
72/m MALT, LG + AGC, I, W NM 1
75/f MALT, LG + EGC, I, W NM 13
53/m MALT, LG + EGC, I, W NM 67
67/f MALT,LG + EGC, I, W NM 31
42/m Immunoblastic + EGC, D, P NM 91
47/m MALT, LG + EGC, D, P NM 24
78/m T-cell pleomorphic + AGC, D, P NM 1
Ishihama[23] 68/m Diffuse, small cleaved + EGC, W T. gastrectomy NM
61/m Diffuse, large cell + EGC, P T. gastrectomy NM
61/f Diffuse, lymphocytic + EGC, P S. gastrectomy 132
77/m Diffuse, large cell + EGC, W Vincristine,endoxanpredonine, adriamicin NM
chemotherapy and endoscopic mucosectomy
Goteri[24] 51/M MALT, LG + HG + EGC, I, W NM 122
55/f MALT, LG + EGC, D, P NM 33
80/m MALT, LG - EGC, I, W NM 12
57/m MALT, LG + EGC, I, W NM 10
53/m MALT, LG + AGC, I, W NM 3
66/m MALT, LG - AGC, D, P NM 10
69/m MALT, LG - AGC, D, P NM 8
69/m MALT, LG - AGC, M, M NM 3
Kanamoto[25] 47/m MALT, LG + EGC, D, P T. gastrectomy 24
Montalban[26] 77/m MALT, LG NM GC NM NM
68/f MALT, HG NM GC NM NM
Cammarota[27] 47/m MALT, LG + AGC, I, P Etoposide, epirubicin, cisplatin chemotherapy and NM
T. gastrectomy
Chan[28] 71/m MALT, LG - EGC, P S. gastrectomy NM
58/f MALT, HG + EGC, P S. gastrectomy NM
75/f MALT, HG + EGC, P S. gastrectomy NM
Kafes[29] 78/m MALT, NM + EGC, I, P + stromal tumor T. gastrectomy 20
Sakai[30] 51/f MALT, LG + EGC, tubular, M T. gastrectomy NM
Suenaga[31] 73/m MALT, LG - AGC, I, W S. gastrectomy, D3 dissection 23

m: male, f: female, NM: not mentioned, EGC: early gastric carcinoma, AGC: advanced gastric carcinoma, GC: gastric carcinoma, I: intestinal type, D: diffuse
type, W: well differentiated, P: poor differentiated, M: moderately differentiated, MALT: mucosa-associated lymphoid tissue, HG: high grade, LG: low grade, S.
gastrectomy: subtotal gastrectomy, T. gastrectomy: total gastrectomy.
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 3566 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
tients had advanced gastric carcinoma (AGC), the type of
gastric carcinoma was undefined in the rest of the patients.
EGC constituted a majority of the Eastern cases (82%),
whereas 48% of cases were from the Western countries.
Before the report by Kelly et al[18] in 1994, the presence
of H pylori was not mentioned by the authors. Since then,
reported H pylori infection rate reached 79%. The infection
rate in the East (86%) was higher than that in the West
(72%). The prevalence of H pylori was higher in EGC (86%)
than that in AGC (64%). There was no significant correla-
tion between the prevalence of H pylori and the differentia-
tion of adenocarcinoma. The declared results of adenocar-
cinoma histopathology were not uniform in relation to the
variations in the classification systems used by the authors.
However, differentiation of the tumor was clearly revealed
by all authors. Poor and well differentiated adenocarcino-
mas showed an equal distribution in the Eastern (43.5%
and 52%, respectively) and the Western cases (42% and
42%, respectively).
Majority of the lymphomas were MALT (mucosa-
associated lymphoid tissue) type lymphoma (69.6%) and
low grade one (87.2%). The association of H pylori with
MALToma was 86% in the Eastern cases and 72% in the
Western cases. Six of 7 (86%) patients with other types of
gastric lymphoma associated with gastric adenocarcinoma
were H pylori-positive. There was no data about the size
of the tumor found in 32% of the cases. The size of the
lymphoma was larger than carcinoma in 57% of cases.
Overall, lymph node metastasis was found in 41% of the
cases. In patients with early carcinoma and positive lymph
node metastasis, the origin of metastatic lymph node was
usually lymphoma (89%). In the presence of advanced car-
cinoma, metastases originated from the adenocarcinoma,
either alone or in combination with the lymphoma. The
topographic interrelation of both tumors was also revealed
in the reports (n = 53/56). Majority of the cases (n = 29,
54.7%) had independent tumors. There was no significant
difference between the Eastern and the Western cases in
this respect. Collision of both tumors was reported in 14
(26.4%) cases. There were 4 cases with contiguous and 5
cases with intermingling tumors.
METACHRONOUS CASES
In contrast to synchronous occurrence of gastric ad-
enocarcinoma and lymphoma, majority of cases (90%,
27/30) with metachronous occurrence of both tumors
were reported from the Western countries[10,32-47]. In 1950,
McNeer et al[32] published the first case in the literature.
The features of the 30 patients are summarized in Table
2. The median age was 50 (8-82) years. Of these patients,
18 were male and 12 were female. Previously detected
malignancy in 28 patients was lymphoma. Only 2 cases
reported by Nakamura et al[10] were diagnosed as lymphoma
after the treatment of gastric adenocarcinoma. One of
these tumors developed in the remnant stomach in the 7th
year after subtotal gastrectomy and the other developed in
the 13th month after endoscopic mucosal resection. Rest
of the patients suffered from various types of lymphoma
previously. Leading type of lymphoma was MALT lym-
phoma (30%), followed by large cell lymphoma (20%).
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June 14, 2006 Volume 12 Number 22
The existence of H pylori was not mentioned in the reports
published before 1997. Thereafter, 9 of 10 reported cases
were found infected with H pylori. Median interval between
the treatment of lymphoma and the diagnosis of gastric
adenocarcinoma was 90 (6-408) mo. The reported inter-
val after the treatment of MALToma (median 27, range
6-108 mo) was shorter than the other types of lymphoma
(median 120, range 42-408 mo). Adequate description of
histopathology of adenocarcinoma was determined in 26
of 30 cases. After the latent period, majority of cases suf-
fered from advanced gastric adenocarcinomas (65%) and 9
patients had EGC.
PATHOLOGIC ASPECTS OF SYNCHRO-
NOUS OCCURRENCE
The relation of both tumors in the stomach was classified
in four categories: (1) separate tumors; (2) collision tumor;
(3) contagious tumor, without any intermingling between
malignant components; and (4) intermingling (admixture)
of both tumors. Histological classification of gastric lym-
phomas is based on systems originally designed for nodal
lymphomas as Musshoff modification of the Ann Arbor
staging system, Working formulation or Kiel classification.
However, these are not optimal for the documentation of
specific relevant features of primary extranodal lymphoma
in the gastrointestinal tract. Several modifications on TNM
staging system and alternatives have been proposed[48,49].
Recently, the European Gastro-Intestinal Lymphoma
Study Group (EGILS) proposed a new modification of
TNM staging system for the gastric lymphomas [50]. In
1983, the histologic relationship of extranodal B-cell lym-
phomas and MALToma was recognized by Isaacson and
Wright, and classification as a separate entity of MALT-
NHL was proposed - the MALT lymphoma concept[51,52].
However, the first report of synchronous occurrence of
MALToma with gastric adenocarcinoma appeared in the
literature in 1990. Metachronous occurrence of MALToma
was reported at the end of 1990s. Therefore, it is easily
speculated that authors are affected by the classification
systems for nodal lymphomas before 1990.
Histopathological disorders of gastric mucosa are not
specific for any neoplasm, but intestinal-type adenocarci-
nomas frequently showed atrophy, intestinal metaplasia,
and not uncommonly, dysplasia of the surrounding non-
neoplastic gastric mucosa. Diffuse-type adenocarcinomas
did not frequently show such lesions. Primary lymphomas
displayed expansive lymphoid follicles and also a high
percentage of atrophy and intestinal metaplasia of the sur-
rounding gastric mucosa[53]. In addition to the similar his-
topathological findings in the surrounding gastric mucosa
of intestinal-type adenocarcinoma and primary gastric
lymphoma, lymphocytic gastritis was found more fre-
quently in patients with gastric carcinoma and primary gas-
tric lymphoma than in unselected patients undergoing en-
doscopy[54]. This finding suggests that these two disparate
gastric tumors may share an immunological dysfunction
or a common pathogenesis. Zamboni et al[55] pointed out
that foveolar lymphoepithelial lesions or signet-ring cells
were found in 37% of MALT lymphomas. Like in MALT
 Hamaloglu E et al. Gastric adenocarcinoma and lymphoma 3567

Table 2 Metachronous cases reported in the literature

Reference Age/sex Lymphoma type Lymphoma therapy Interval Adenocarcinoma therapy

[32]
McNeer 27/f Reticulum cell sarcoma S. gastrectomy; radiotherapy for local recurrence 6.5 yr T. gastrectomy
Fleischer and 61/m Lymphosarcoma S. gastrectomy; 4000 cGy 5 yr Supportive treatment
Walker[33]
Bockus[34] 49/m Lymphosarcoma Radiotherapy 3.5 yr T. gastrectomy
Komarov[35] 40/m Round cell sarcoma S. gastrectomy 19 yr NM
Morgenstern[36] 46/f Reticulum cell sarcoma S. gastrectomy 25 yr Resection
Ettinger and 55/m Lymphosarcoma S. gastrectomy; 2000-2600 cGy 16 yr Palliative surgery
Carter[37]
Shani[38] 47/m Reticulum cell sarcoma S. gastrectomy 31 yr Resection
29/m Diffuse, poorly S. gastrectomy; orthovoltage for whole abdomen (6 34 yr Exploration
differentiated lymphocytic d)
51/f Diffuse, mixed S. gastrectomy; 1800 cGy X-R + 2000 cGy Co 60. 16 yr Exploration
1
lymphocytic histiocytic 2030 cGy for recurrence (16 yr later)
48/m Diffuse, poorly S. gastrectomy; 3000 cGy. 10 yr Exploration; combined chemotherapy
differentiated lymphocytic
Sellin[39] 45/m Diffuse, large cleaved cell 2300 cGy; S. gastrectomy; 4000 cGy (4 yr later) 13 yr Total gastrectomy
Brumback[40] 8/m Diffuse undifferentiated S. gastrectomy; cyclophosphamide, vincristine, 6 yr Gastrojejunal bypass;
(small noncleaved) prednisolone for 1 mo; 4075 cGy; 6-mercaptopurine, 5-FU, doxorubicine,
vincristine, prednisolone, methotrexate for 3 yr mitomycin chemotherapy

15/m Hodgkins disease, 3500 cGy to upper abdomen; 2000 cGy to left 10 yr NM
nodular sclerosis type axillary-cervical-supraclavicular; cyclophosphamide,
vincristine, procarbazine, prednisolone, adriamycin
for 2 yr

Baron[41] 58/m Diffuse large cell 3150 cGy to upper abdomen 10 yr S. gastrectomy
24/f Reticulum cell sarcoma S. gastrectomy; 4000 cGy 15 yr No treatment
60/m Diffuse large cell S. gastrectomy; cyclophosphamide, vincristine, 4 yr T. gastrectomy and S. esophagectomy
prednisolone, doxorubicin chemotherapy
56/m Well differentiated S. gastrectomy; 3700 cGy; oral cyclophosphamide 12 yr T. gastrectomy
lymphocytic chemotherapy
Zorlu[42] 43/m Diffuse, large cleaved cell S. gastrectomy; cyclophosphamide, vincristine, 8 yr Near total gastrectomy;
prednisolone ( 2 courses); 5-FU, mitomycin
4500 cGy; cyclophosphamide, vincristine,
prednisolone (4 courses)

35/f Diffuse, large cleaved cell S. gastrectomy; 4000 cGy; cyclophosphamide, 8 yr T. gastrectomy
vincristine, prednisolone (6 courses)
Nakamura[10]1 69/f Immunoblastic NM 7 yr S.gastrectomy
82/f MALT, HG NM 13 mo Endoscopic mucosal resection
Zauber[43] 78/f Large cleaved cell S. gastrectomy; 4500 cGy; i.v. cyclophosphamide 4 yr T. gastrectomy
chemotherapy (9 courses)
Montalban[26] 42/m MALT Cyclophosphamide, doxorubicin, 108 mo NM
vincristine, prednisone
Hasegawa[44] 72/f MALT Antibiotic therapy for H pylori 6 mo S. gastrectomy
Morgner[45] 74/m MALT, LG Antibiotic therapy for H pylori 4 yr Endoscopic mucosal resection
70/m MALT, LG Antibiotic therapy for H pylori 5 yr Endoscopic mucosal resection and
argon plasma coagulation
77/f MALT, LG Antibiotic therapy for H pylori 4 yr Endoscopic mucosal resection
Ghosdal[46] 32/m MALT, LG S. gastrectomy; Antibiotic therapy for H pylori (for 2 15 mo No treatment
wk);
Raderer[47] 67/f MALT Antibiotic therapy for H pylori ( for 9 mo) 9 mo S. gastrectomy
61/f MALT Antibiotic therapy for H pylori ( for 14 mo); 2CdA 27 mo S. gastrectomy
chemotherapy (4 courses)

m: male, f: female, NM: not mentioned, MALT: mucosa-associated lymphoid tissue, HG: high grade, LG: low grade, S. gastrectomy: subtotal gastrectomy, T.
gastrectomy: total gastrectomy, 1Only 2 cases reported by Nakamura et al were diagnosed as lymphoma after the treatment of gastric adenocarcinoma.

lymphomas, gastric carcinomas are invariably accompanied
by lymphoid proliferations ranging from reactive lymphoid
follicles to MALT lymphomas[56]. These observations also
supplied the theory of common pathogenesis of MALT-
type lymphoma and gastric carcinoma.
The incidence of early gastric carcinoma in syn-
chronous tumors was remarkably high. Especially in the
Eastern countries, routine screening for gastric carcinoma
is practiced, and up to about 50% of gastric carcinoma
diagnosed in the early stage[57]. This observation, together

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with the finding that most lymphomas were larger than ad- H pylori genotyping on archived gastric tissue revealed that
enocarcinomas, suggested that lymphomas might develop H pylori strains with certain combinations of virulence
before adenocarcinomas or that the presence of MALT subtypes are associated with gastric carcinoma or MALT
lymphoma might increase the risk of developing gastric lymphoma. Thus, the virulence subtype composition vacA
carcinoma. s1a and iceA1 occurs mainly in gastric carcinoma, whereas
the vacA s1t/m2 iceA1 combination is found in MALT
lymphoma[73]. Therefore, in the future studies, the compo-
ETIOLOGIC FACTORS OF SYNCHRONOUS sition of virulence subtypes might be taken into account
OCCURRENCE during the evaluation of possible sequelae of gastric mu-
cosal damage caused by H pylori infection.
H pylori
It is tempting to hypothesize that H pylori is a common
Epstein-Barr virus
etiological agent for synchronous occurrence of gastric
Studies of Epstein-Barr virus (EBV) suggest that an aber-
adenocarcinoma and primary gastric lymphoma. Since
rant antibody response to infection may occur years before
1994, the existence of H pylori has been investigated in
the appearance of a tumor [74]. The frequency of EBV
almost every report. In the present analysis, in areas with
detection has been reported to be 6%-18% in gastric lym-
high prevalence (Eastern) and low prevalence (Western) of
phomas[75-77] and 7%-16% in ordinary gastric adenocarcino-
H pylori, synchronous tumors are associated with H pylori
mas without lymphoid stroma[78-80]. Whether EBV plays a
infection rates of 86% and 72%, respectively, and are simi-
pathogenic role in either of these tumors is still unclear[81].
lar to or higher than the reported infection rates of isolat-
Although any mechanism related to EBV in tumorigen-
ed gastric adenocarcinoma and isolated MALT lymphoma
esis of gastric malignancies remains highly speculative, it
or other primary gastric non-hodgkin lymphoma[20,58-60].
has been demonstrated that there is a delay in apoptosis
Therefore, H pylori may have an important etiological role
in EBV-positive gastric carcinomas (associated with up-
for synchronous tumors in both high and low prevalence
regulation of BCL-2 and p53) and a decrease in cellular
areas.
differentiation (associated with decreased E-cadherin ex-
H pylori plays a key role in the natural history of gastric
pression)[82]. Nakamura et al[10] found no specific association
MALT lymphoma, representing an example of antigen-
between EBV infection and these double malignancies.
mediated tissue stimulation and lymphoproliferation, with
possible subsequent lymphomagenesis [58,61]. Antigenic
mimicry between H pylori and the host mucosa was held Exposure to atomic blast
It is controversial whether the incidence of gastric car-
responsible for inducing autoimmune responses which lead
cinoma is higher in persons exposed to an atomic blast
to development of the disease[62,63]. Kawahara et al[64] found
in comparison to non-exposed subjects[83-85], but Suehiro
that the increase of antibody titers to HCG-27 cells in
et al[83] suggested that the incidence is the highest in per-
H pylori-positive patients with MALT lymphoma compared
sons exposed within a 2.0-Km radius ground zero. The re-
to titers in patients with other gastroduodenal diseases
lation of primary gastric lymphoma and atomic bomb ex-
and in healthy subjects. Lymphoid follicles which are
posure is under debate. Recently, t (11; 18) (q21; q21) and
not present in the normal stomach show development
t (1;14) (p22; q32) translocations have been reported to be
in the setting of chronic gastritis. Genta et al[65] have ob-
associated with MALT-type lymphoma. The former trans-
served a strong correlation between follicular gastritis and
location results in the formation of fusion protein API2-
H pylori infection. Wotherspoon et al [58] suggested that
MALT1, and the latter in the fusion protein pf BCL10 and
H pylori might trigger the acquisition of MALT in the gas-
IgH. These fusion transcripts are thought to be molecular
tric mucosa, and this lymphoid tissue is thought to harbor
markers for MALT-type lymphoma not responding to
the precursor cells in MALT-NHL. These precursor cells
change gradually into malignant lymphoma cells with au- H pylori eradication and highly correlated with aberrant
nuclear BCL10 expression, which may serve as a screen-
tonomous and uncontrolled growth by accumulation of
ing tool for fusion[86-88]. These investigations support the
genetic alterations such as mutations, deletions, and am-
etiologic role of atomic bomb blast on the formation of
plifications. In vitro experiments have demonstrated that in
MALT-type lymphoma.
the earlier phases of development, the growth of geneti-
cally altered lymphoid cell is not yet fully autonomous, and
proliferation partly depends on H pylori-related proteins ETIOLOGIC FACTORS OF METACHRO-
and T cells[66-69]. On the other hand, H pylori infection has
been linked to the intestinal-type gastric adenocarcinoma
NOUS OCCURRENCE
through a chain of events that starts as acute gastritis and Epithelial malignancies, the common cancers of adult-
progresses to chronic gastritis, atrophic gastritis, intesti- hood, are detected rarely in the post-treatment course of
nal metaplasia, dysplasia, and eventually, adenocarcinoma Hodgkins disease and other lymphomas[2,3,26]. However,
formation[70,71]. A recent meta-analysis has revealed that Greene and Wilson[88,89] observed a significant increase in
H pylori infection is associated with a 2-fold increase in the incidence of gastric carcinoma in the period following
developing gastric adenocarcinoma[72]. These epidemio- the treatment of non-Hodgkin lymphomas.
logic and pathologic data about the relation of H pylori
infection with gastric adenocarcinoma and primary gas- Previous gastric surgery
tric lymphoma are supported by a recent genetic study[73]. Previous gastric surgery is a well known precancerous con-

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 Hamaloglu E et al. Gastric adenocarcinoma and lymphoma 3569
dition[90,91]. Gastroesophageal reflux and gastritis, formation
of nitrosamines due to gastric pH decrease and untreated
H pylori infection may play a role in carcinogenesis of the
gastric remnant[92]. So-called stump carcinoma develops
between 13-27 years after resection in patients treated with
gastrectomy for benign conditions[91].
Radiotherapy and chemotherapy
Though the stomach is considered relatively radio-resistant
compared to other parts of the gastrointestinal tract, side
effects and complications progressively increased when
the total dosage delivered was above 4500 cGy[93,94]. The
effects of radiotherapy on the risk of carcinogenesis are
well known[95]. Hirose et al[96] demonstrated that localized
radiation induced gastric adenocarcinoma development
in an experimental model. Brown et al[97] reported a slight
increase in the incidence of gastric adenocarcinoma in pa-
tients with ankylosing spondylitis under radiation therapy.
In contrast, when the relatively low dose (less than 3400
cGy) radiotherapy was used to decrease acid production in
2049 peptic ulcer patients, only two cases of leiomyosarco-
ma were reported. The elapsed time between radiotherapy
and the diagnosis of these cases were reported as 14 and
26 years[98].
Both series reported by Stanford and Milan concluded
that combined administration of chemotherapy and radio-
therapy increases the occurrence rate of secondary malig-
nancies[1,2]. In these series, the most common secondary
malignancy was especially acute myelogenous leukemia. Its
occurrence was associated with the administration of al-
kylating agents (nitrogen mustard, vincristine, prednisone,
and procarbazine in Milan series)[2]. Solid tumors were
commonly associated with radiation therapy[1,2]. Brumback
et al[40] concluded that secondary gastric adenocarcinoma
occurred in one of these cases probably due to oral ad-
ministration of procarbazine for Hodgkins disease. In
addition to these factors, H pylori infection was also held
accountable for metachronous occurrence of both tumors,
as discussed above.
DIFFICULTIES IN DIAGNOSIS
It may be difficult to diagnose the two different tumors in
the stomach before surgery. Because most of the patients
with gastric adenocarcinoma and coexisting primary gas-
tric lymphoma are associated with H pylori infection, the
presence of MALT-type or other types of primary gastric
lymphoma should be taken into consideration when gastric
adenocarcinoma with gastritis is detected on endoscopy. In
surgical specimens, not only the lesions of adenocarcino-
ma but also the background mucosa of cancerous lesions
must be examined with regard to infiltration of lympho-
cytes, and the presence of gastritis, as well as the presence
of H pylori. Signet-ring cells around MALT lymphomas or
lymphoid proliferations around gastric adenocarcinomas
should be kept in mind against over-diagnosis during the
histopathological examination. Considering the metachro-
nous occurrence of gastric adenocarcinoma and gastric
lymphoma, proper endoscopic follow-up should be per-
formed in the remnant stomach, especially when H pylori
infection is present.
TREATMENT
Treatment modalities for synchronous cases
Treatment of synchronous cases is generally applied ac-
cording to the presence of adenocarcinoma. Distal or
total gastrectomy is performed depending on the tumor
localization. Preoperative chemotherapy was preferred
only in two cases. Cammarota et al[27] decided to administer
etoposide, epirubicin, cisplatin chemotherapy according
to the presence of locally advanced tumor stage (T3 N1)
in laparoscopic exploration. Older patients in the series by
Ishihama et al[23] were exposed to 10 courses of vincristine,
endoxan, predonisdone, adriamicin chemotherapy and
endoscopic resection of the tumor. Postoperative chemo-
therapy was administered only to 6 (10.7%) patients.
Treatment modalities for metachronous cases
Eight of thirty patients underwent subtotal gastrectomy
and radiotherapy against lymphoma. Chemotherapy was
administered in different combinations with other treat-
ment modalities in 7 patients. Two patients were treated by
only chemotherapy. Radiotherapy was performed solely in
2 patients. Overall, 10 cases with lymphoma were treated
without surgical treatment. All patients with MALToma
were treated with antibiotic regimen against H pylori, except
cases reported by Nakamura et al[10] and Montalban et al[26],
the authors of both did not mention anything about the
treatment of H pylori infection in their reports. Every pa-
tient with H pylori infection was eradicated and no one was
re-infected or re-colonized during the follow-up period.
Except cases with widespread metastasis, total gastrectomy
or palliative resections were performed in patients with
AGC. Patients with EGC were exposed to more conserva-
tive surgery, such as endoscopic mucosal resection[10,45].
Surgery
Clear guidelines for management have not been designed
for synchronous adenocarcinoma and primary gastric
lymphoma. Traditionally, aggressive surgical resection has
been the mainstay of gastric lymphoma treatment because
by this treatment modality it would be possible to collect
definitive tissues for pathologic examination, allow ex-
ploration of the abdomen, reduce the tumor burden and
obviate the concern that gastric hemorrhage or perfora-
tion would complicate medical treatment of lymphomas.
Recently, radical gastrectomy is disputed and considered
unnecessary for gastric lymphomas. Lesser procedures are
now accepted where resection of the gross disease and
involved lymph nodes will provide adequate results[99-102].
Some authors advocate wide resection and extensive
lymph node dissection alone for adequate treatment of
stage 1E or pure MALT lymphomas with a survival rate of
> 95%[103,104]. In a retrospective study from Italy patients in
different stages of gastric lymphoma who underwent sur-
gical resection when feasible, the ten-year actuarial survival
rates were markedly higher (100% and 80%, respectively)
for stage IE and IIE as compared with stage IIIE and IVE
(21% and 0%, respectively)[105]. Surgical resection with clear
margins for lymphoma is advised in order to maximize the
chance of cure[100,106]. However, others have found no dif-
ference in survival, whether the margin of resection was
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 3570 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
clear or not, as long as post-operative chemotherapy was
given[107]. Operative mortality rates for gastric lymphoma
reached 25% in cases with advanced tumor stage [108].
Therefore, aggressive surgery for gastric lymphoma is not
indicated due to increased morbidity which outweighs the
benefit gained in terms of survival[107]. Surgery for gastric
lymphoma is now often reserved for patients with local-
ized disease, residual disease after non-surgical therapy or
for rare patients with complications[109]. If the coexisting
adenocarcinoma is diagnosed correctly, surgical treatment,
a first-line therapy, is preferred according to adenocarci-
noma treatment principles. Total or subtotal gastrectomy
with D1 or D2 dissection must be performed.
In metachronous cases, generally, the occurrence of
primary gastric lymphoma precedes gastric adenocarci-
noma, resection of the remnant stomach must be done
with or without a combination of other treatment modali-
ties. However, a close follow-up of the patients undergo-
ing gastric lymphoma treatment allows early detection of
carcinogenesis. Limited endoscopic resections might be
an alternative treatment for these cases. Similar to gastric
lymphoma as a previous malignancy, patients in whom
limited resection is performed for gastric adenocarci-
noma should undergo a close endoscopic follow-up. The
H pylori status with histopathologic alterations in the
resected stomach and in the remnant stomach must be
detected and followed up for early detection of metachro-
nous occurrence of gastric lymphoma.
Chemotherapy
The effect of chemotherapy as a sole treatment for
gastric lymphomas is still under debate. The needs behind
trying chemotherapy were the considerable morbidity
and mortality associated with resection[108]. Some authors
reported better survival rates in patients with primary
gastric lymphoma treated by chemotherapy alone or
combination with radiotherapy when compared to
surgical treatment alone[110,111]. Other reports showed no
apparent difference in survival between patients treated
by chemotherapy or surgery and chemotherapy with
survival rates of 67% and 60%, respectively. The fear
of chemotherapy-related complications, for instance,
bleeding and perforation, has been disputed, and less
significant compared with surgical resection [111,112] .
Therefore, chemotherapy has been suggested and adopted
as a primary mode of treatment for primary gastric
lymphomas. Whereas cyclophoshamide, vincristine and
prednisolone were adopted for low grade lymphomas, high
grade tumors were treated with doxurubicin, teniposide,
cyclophosphamide and prednisolone. The combination
of both regimens with surgical resection has increased
the survival rates up to 80% and 100%, respectively[113].
In contrast to primary gastric lymphomas, chemotherapy
regimens have not been used as a sole therapy in the
treatment of gastric adenocarcinoma, except in cases
with metastatic disease. Systemic chemotherapy for
metastatic disease has a marginal survival benefit when
compared with best supportive care, while no standard
worldwide regimen has been established yet. In Japan,
especially the number of patients treated with endoscopic
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June 14, 2006 Volume 12 Number 22
mucosal resection for early gastric adenocarcinoma is
increasing year by year. Chemotherapy may be a more
important part of treatment protocols in these patients
in near future, because of the increasing survival rates.
Cisplatin, 5-fluorouracil and mitomycin C were commonly
used in various combinations for patients with gastric
adenocarcinoma[114].
Radiotherapy
In most instances, radiotherapy is used as an adjuvant
to surgery, chemotherapy or both for primary gastric
lymphoma. It has rarely been tried as a single mode of
therapy[115,116]. However, limited trials have suggested that
radiotherapy can be utilized as a primary mode of treat-
ment with a reasonable outcome[116,117]. Radiotherapy has
been studied in comparison with other treatment modali-
ties for stage IE and IIE of primary gastric lymphomas
with a comparable outcome of 80%-89% survival[115,118,119].
Radiation was used post-operatively in high- and low-
grade lymphomas, for residual tumors in stages I and II to
improve the disease-free survival[120]. Combination of ra-
diotherapy with chemotherapy might improve the chance
of stomach conservation of these patients which may ap-
proach 100%[121]. Contradictory studies have found com-
bined radiotherapy with either resection or chemotherapy
no significant difference in both modalities, with a survival
rate of 82%-88%[116,119]. Radiation therapy has a limited
but well established role in the care of those afflicted with
gastric adenocarcinoma. Radiation therapy can provide
considerable relief of local gastric cancer symptoms. Ap-
proximately 50% to 75% of patients have had symptom-
atic improvement of problems, such as obstruction, bleed-
ing and pain in a variety of trials[122,123].
Antibiotic therapy
Because gastric MALT lymphomas have a high association
with H pylori infection, eradication of H pylori with antibi-
otics is very important[58,60,124,125]. Isaacson et al[125] suggested
that antibiotic eradication of H pylori removes the growth
stimulus from gastric MALT lymphoma without necessar-
ily eradicating the neoplastic B-cell clone. This clone may
re-expand, but in the absence of concomitant re-infection
with H pylori, this is likely to be a self-limiting event. Short
and long-term follow-up results of medical eradication
of H pylori in patients with gastric MALT lymphoma have
been published in the literature[124,125]. However, patients
still require periodic surveillance endoscopies and may
require more traditional treatment of their lymphoma.
Antibiotic therapy may fail to cure gastric lymphomas
when there is a bulky tumor with a high-grade compo-
nent or when the gastric lymphoma is associated with
carcinoma[126]. A combination of partial gastrectomy and
antibiotic therapy might be an alternative treatment for
primary gastric lymphomas. However, periodic surveillance
endoscopies are required after partial gastrectomy and
antibiotic therapy. There has been no defined role of an-
tibiotic therapy against H pylori in the treatment of gastric
adenocarcinoma. Eradication of H pylori infection can be
placed in preventive measures of gastric adenocarcinoma.
If H pylori infection is detected in the partially resected
 Hamaloglu E et al. Gastric adenocarcinoma and lymphoma 3571
stomach secondary to adenocarcinoma, the postoperative
antibiotic treatment against H pylori may be administered
for hindering metachronous occurrence of primary gastric
lymphoma.
PROGNOSIS
Reported period of follow-up for synchronous cases
ranged from 1 to 132 mo. Overall, data about surveillance
of patients were insufficient to perform analysis. Some
authors did not mention follow-up data whereas others did
not report treatment modalities. However, shorter post-
operative survey was observed in cases with AGC (up to
23 mo) when compared to cases with EGC. The follow-
up study conducted by Nakamura et al[10] revealed that the
survival rate of patients with synchronous occurrence of
gastric lymphoma and adenocarcinoma appeared to be
similar to that for previously reported patients with gastric
adenocarcinoma without lymphoma, and was significantly
worse than patients with primary gastric lymphoma with-
out adenocarcinoma. As discussed above, majority of
metachronous cases suffered from advanced gastric adeno-
carcinomas after the latent period. Eleven of 17 patients
with AGC died within 18 mo. Despite incomplete survival
data of cases with EGC, Zauber et al[42] reported 96 mo
disease-free survival after total gastrectomy. Nakamura et
al[10] did not reveal any survival data regarding primary gas-
tric lymphoma occurring after treatment of gastric adeno-
carcinoma.
In summary, with the help of case reports or series,
the synchronous or metachronous occurrence of primary
gastric lymphoma and gastric adenocarcinoma have
evolved in various aspects. Clinicians should be alert for
the possible synchronous or metachronous occurrence of
both tumors during diagnosis or follow-up of each other.
Authors suggest the application of principles for gastric
adenocarcinoma treatment to cases with synchronous
occurrence. The biologic behavior of the latest diagnosed
tumor deter mines the accurate management of
metachronous occurrence of primary gastric lymphoma
and gastric adenocarcinoma.
REFERENCES
1 Coleman CN, Williams CJ, Flint A, Glatstein EJ, Rosenberg
SA, Kaplan HS. Hematologic neoplasia in patients treated for
Hodgkins disease. N Engl J Med 1977; 297: 1249-1252
2 Valagussa P, Santoro A, Kenda R, Fossati Bellani F, Franchi
F, Banfi A, Rilke F, Bonadonna G. Second malignancies
in Hodgkins disease: a complication of certain forms of
treatment. Br Med J 1980; 280: 216-219
3 Neufeld H, Weinerman BH, Kemel S. Secondary malignant
neoplasms in patients with Hodgkins disease. JAMA 1978;
239: 2470-2471
4 Nakamura S, Akazawa K, Yao T, Tsuneyoshi M. A clinico-
pathologic study of 233 cases with special reference to
evaluation with the MIB-1 index. Cancer 1995; 76: 1313-1324
5 Barth. (Abstract of presentation). Bull Soc Anat Paris 1855; 30: 1
6 Ostertag H, Georgii A. Early gastric cancer: a morphological
study of 144 cases. Pathol Res Pract 1979; 164: 294-315
7 Planker M, Fischer JT, Peters U, Borchard F. Synchronous
double primary malignant lymphoma of low grade
malignancy and early cancer (collision tumor) of the stomach.
Hepatogastroenterology 1984; 31: 144-148
8 Kasahara Y, Takemoto M, Morishita A, Kuyama T, Takahashi
M, Tanji K. Coexisting adenocarcinoma and malignant
lymphoma of the stomach: case report and review of the
Japanese literature. Am J Gastroenterol 1988; 83: 190-193
9 Noda T, Akashi H, Matsueda S, Katsuki N, Shirahashi K,
Kojiro M. Collision of malignant lymphoma and multiple
early adenocarcinomas of the stomach. Arch Pathol Lab Med
1989; 113: 419-422
10 Nakamura S, Aoyagi K, Iwanaga S, Yao T, Tsuneyoshi M,
Fujishima M. Synchronous and metachronous primary gastric
lymphoma and adenocarcinoma: a clinicopathological study
of 12 patients. Cancer 1997; 79: 1077-1085
11 RABINOVITCH J, PINES B, GRAYZEL D. Coexisting
lymphosarcoma and ulcer-carcinoma of the stomach. AMA
Arch Surg 1952; 64: 185-191
12 Jernstrom P, Murray GC. Synchronous double primary
lymphosarcoma and adenosarcoma (collision tumor) of the
stomach with cancer to cancer metastasis. Cancer 1966; 19:
60-66
13 Manier JW, Reyes CN. Collision tumor of the stomach: Report
of two cases. Gastroenterology 1974; 67: 1011-1015
14 Lin JI, Tseng CH, Chow S, Weisberg J, Guzman L. Coexisting
malignant lymphoma and adenocarcinoma of the stomach.
South Med J 1979; 72: 619-622
15 Kane EP, Weingarten LA, Payson BA, Mori K, Sarlin JG.
Synchronous ulcerating adenocarcinoma and malignant
lymphoma of the stomach. Am J Gastroenterol 1982; 77: 461-463
16 Czerniak A, Lotan G, Engelberg IS, Rabau MY, Avigad I,
Schachter P, Wolfstein I. The simultaneous coexistence of
adenocarcinoma and primary malignant lymphoma in the
stomach. J Surg Oncol 1985; 30: 42-45
17 Akosa AB, Clark DM, Desa L. Synchronous adenocarcinoma
and primary malignant lymphoma of the stomach. Postgrad
Med J 1990; 66: 778-780
18 Kelly SM, Geraghty JM, Neale G. H pylori, gastric carcinoma,
and MALT lymphoma. Lancet 1994; 343: 418
19 von Herbay A, Schreiter H, Rudi J. Simultaneous gastric
adenocarcinoma and MALT-type lymphoma in Helicobacter
pylori infection. Virchows Arch 1995; 427: 445-450
20 Wotherspoon AC, Isaacson PG. Synchronous adenocarcinoma
and low grade B-cell lymphoma of mucosa associated
lymphoid tissue (MALT) of the stomach. Histopathology 1995;
27: 325-331
21 Nishino N, Konno H, Baba S, Aoki K, Nishimura T, Arai
T, Kino I. Synchronous lymphoma and adenocarcinoma
occurring as a collision tumor in the stomach: report of a case.
Surg Today 1996; 26: 508-512
22 Hardman WJ 3rd, Gal AA, Pascal RR. Gastric adenocarcinoma
and low-grade B-cell lymphoma of mucosa-associated
lymphoid tissue. South Med J 1997; 90: 426-430
23 Ishihama T, Kondo H, Saito D, Yamaguchi H, Shirao K,
Yokota T, Hosokawa K, Ono H, Iwabuchi M, Gotoda T,
Matsuno Y, Boku N, Ohtsu A, Yoshida S. Clinicopathological
studies on coexisting gastric malignant lymphoma and gastric
adenocarcinoma: report of four cases and review of the
Japanese literature. Jpn J Clin Oncol 1997; 27: 101-106
24 Goteri G, Ranaldi R, Rezai B, Baccarini MG, Bearzi I.
Synchronous mucosa-associated lymphoid tissue lymphoma
and adenocarcinoma of the stomach. Am J Surg Pathol 1997; 21:
505-509
25 Kanamoto K, Aoyagi K, Nakamura S, Hizawa K, Suekane H,
Sakamoto K, Fujishima M. Simultaneous coexistence of early
adenocarcinoma and low-grade MALT lymphoma of the
stomach associated with Helicobacter pylori infection: a case
report. Gastrointest Endosc 1998; 47: 73-75
26 Montalbn C, Castrillo JM, Lpez-Abente G, Abraira V,
Serrano M, Bellas C, Piris MA, Carrion R, Cruz MA, Garca-
Laraa J, Menarguez J, Rivas C. Other cancers in patients with
gastric MALT lymphoma. Leuk Lymphoma 1999; 33: 161-168
27 Cammarota G, Larocca LM, DUgo D, Persiani R, Cianci R,
Nocente R, Picciocchi A, Gasbarrini G. Synchronous gastric
adenocarcinoma and MALT lymphoma in a patient with H.
pylori infection. Could the two neoplasms share a common
www.wjgnet.com
 3572 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
pathogenesis? Hepatogastroenterology 2001; 48: 104-106
28 Chan AO, Chu KM, Yuen ST, Leung SY, Lam SK, Wong J.
Synchronous gastric adenocarcinoma and mucosa-associated
lymphoid tissue lymphoma in association with Helicobacter
pylori infection: comparing reported cases between the East
and West. Am J Gastroenterol 2001; 96: 1922-1924
29 Kaffes A, Hughes L, Hollinshead J, Katelaris P. Synchronous
primary adenocarcinoma, mucosa-associated lymphoid tissue
lymphoma and a stromal tumor in a Helicobacter pylori-infected
stomach. J Gastroenterol Hepatol 2002; 17: 1033-1036
30 Sakai T, Ogura Y, Narita J, Suto T, Kimura D, Ainai S, Fujita H,
Kamada M. Simultaneous early adenocarcinoma and mucosa-
associated lymphoid tissue (MALT) lymphoma of the stomach
associated with Helicobacter pylori infection. Gastric Cancer
2003; 6: 191-196
31 Suenaga M, Ohta K, Toguchi M, Sato T, Ohyama S, Yamaguchi
T, Muto T, Yanagisawa A, Kato Y. Colliding gastric and
intestinal phenotype well-differentiated adenocarcinoma of
the stomach developing in an area of MALT-type lymphoma.
Gastric Cancer 2003; 6: 270-276
32 McNEER G, BOOHER RJ, BOWDEN L. The resectability of
recurrent gastric carcinoma. Cancer 1950; 3: 43-55
33 FLEISCHER N, WALKER R. Gastric carcinoma following
successful therapy for primary gastric lymphosarcoma. South
Med J 1960; 53: 965-968
34 Bockus HL. Carcinoma of the stomach. In: Bockus H.
Gastroenterology, vol. 1, ed. 2 Philadelphia, W.B. Saunders
Company, 1963: 747
35 Komarov AS. [Cancer of the gastric stump 19 years after its
resection for sarcoma]. Khirurgiia (Mosk) 1969; 45: 131-133
36 Morgenstern L, Yamakawa T, Seltzer D. Carcinoma of the
gastric stump. Am J Surg 1973; 125: 29-38
37 Ettinger DS, Carter D. Gastric carcinoma 16 years after gastric
lymphoma irradiation. Am J Gastroenterol 1977; 68: 485-488
38 Shani A, Schutt AJ, Weiland LH. Primary gastric malignant
lymphoma followed by gastric adenocarcinoma: report of 4
cases and review of the literature. Cancer 1978; 42: 2039-2044
39 Sellin J, Levin B, Reckard C, Riddell R. Gastric adenocar-
cinoma followi n g g a st r ic l y mp h oma. R ol e of p ar t i a l
gastrectomy. Cancer 1980; 45: 996-1000
40 Brumback RA, Gerber JE, Hicks DG, Strauchen JA.
Adenocarcinoma of the stomach following irradiation and
chemotherapy for lymphoma in young patients. Cancer 1984;
54: 994-998
41 Baron BW, Bitter MA, Baron JM, Bostwick DG. Gastric
adenocarcinoma after gastric lymphoma. Cancer 1987; 60:
1876-1882
42 Zorlu AF, Atahan IL, Gedikoglu G, Ruacan S, Sayek I,
Tekuzman G. Does gastric adenocarcinoma develop after the
treatment of gastric lymphoma? J Surg Oncol 1993; 54: 126-131
43 Zauber NP, Berman EL. Synchronous and metachronous
primary gastric lymphoma and adenocarcinoma: a
clinicopathologic study of 12 patients. Cancer 1998; 82: 226-227
44 Hasegawa N, Kato K, Yamada K, Morita K, Kuroiwa M, Ito
H, Kamioka T, Matsuura T, Taguchi A, Ono K, Suzuki Y, Goto
H. Early-stage gastric adenocarcinoma: revealed after anti-
Helicobacter pylori therapy of MALT lymphoma. Gastrointest
Endosc 2001; 53: 495
45 Ghoshal UC, Guha D, Bandyopadhyay S, Pal C, Chakraborty
S, Ghoshal U, Ghosh TK, Pal BB, Banerjee PK. Gastric
adenocarcinoma in a patient re-infected with H. pylori after
regression of MALT lymphoma with successful anti-H.pylori
therapy and gastric resection: a case report. BMC Gastroenterol
2002; 2: 6
46 Raderer M, Pspk A, Stummvoll G, Lngle F, Chott A.
Early cancer of the stomach arising after successful treatment
of gastric MALT lymphoma in patients with autoimmune
disease. Scand J Gastroenterol 2003; 38: 294-297
47 Lim FE, Hartman AS, Tan EG, Cady B, Meissner WA. Factors
in the prognosis of gastric lymphoma. Cancer 1977; 39:
1715-1720
48 Shimodaira M, Tsukamoto Y, Niwa Y, Goto H, Hase S,
Hayakawa T, Nagasaka T. A proposed staging system for
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
primary gastric lymphoma. Cancer 1994; 73: 2709-2715
49 R u s k o n - F o u r m e s t r a u x A , D r a g o s i c s B , M orgner A,
Wotherspoon A, De Jong D. Paris staging system for primary
gastrointestinal lymphomas. Gut 2003; 52: 912-913
50 Isaacson P, Wright DH. Malignant lymphoma of mucosa-
associated lymphoid tissue. A distinctive type of B-cell
lymphoma. Cancer 1983; 52: 1410-1416
51 Isaacson P, Wright DH. Extranodal malignant lymphoma
arising from mucosa-associated lymphoid tissue. Cancer 1984;
53: 2515-2524
52 Arista-Nasr J, Jimnez-Rosas F, Uribe-Uribe N, Herrera-
Goepfert R, Lazos-Ochoa M. Pathological disorders of
the gastric mucosa surrounding carcinomas and primary
lymphomas. Am J Gastroenterol 2001; 96: 1746-1750
53 Griffiths AP, Wyatt J, Jack AS, Dixon MF. Lymphocytic
gastritis, gastric adenocarcinoma, and primary gastric
lymphoma. J Clin Pathol 1994; 47: 1123-1124
54 Zamboni G, Franzin G, Scarpa A, Bonetti F, Pea M, Mariuzzi
GM, Menestrina F. Carcinoma-like signet-ring cells in gastric
mucosa-associated lymphoid tissue (MALT) lymphoma. Am J
Surg Pathol 1996; 20: 588-598
55 Ioachim HL, Hajdu C, Giancotti FR, Dorsett B. Lymphoid
proliferations and lymphomas associated with gastric
metaplasia, dysplasia, and carcinoma. Hum Pathol 1999; 30:
833-842
56 Endo M, Habu H. Clinical studies of early gastric cancer.
Hepatogastroenterology 1990; 37: 408-410
57 Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson
PG. Helicobacter pylori-associated gastritis and primary B-cell
gastric lymphoma. Lancet 1991; 338: 1175-1176
58 Loffeld RJ, Willems I, Flendrig JA, Arends JW. Helicobacter
pylori and gastric carcinoma. Histopathology 1990; 17: 537-541
59 Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke
RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD.
Helicobacter pylori infection and gastric lymphoma. N Engl J
Med 1994; 330: 1267-1271
60 Isaacson PG. Mucosa-associated lymphoid tissue lymphoma.
Semin Hematol 1999; 36: 139-147
61 Negrini R, Savio A, Poiesi C, Appelmelk BJ, Buffoli F,
Paterlini A, Cesari P, Graffeo M, Vaira D, Franzin G. Antigenic
mimicry between Helicobacter pylori and gastric mucosa in the
pathogenesis of body atrophic gastritis. Gastroenterology 1996;
111: 655-665
62 Cammarota G, Gasbarrini G. Helicobacter pylori infection and
autoimmune pathogenesis of gastric neoplasias. Gut 2000; 46:
295
63 Kawahara Y, Yokota K, Mizuno M, Yunoki N, Uesu T, Okada
H, Kobayashi K, Hirai Y, Oguma K, Tsuji T. Antibodies to
human gastric epithelial cells and heat shock protein 60 in
Helicobacter pylori positive mucosa associated lymphoid tissue
lymphoma. Gut 1999; 45: 20-23
64 Genta RM, Hamner HW. The significance of lymphoid
follicles in the interpretation of gastric biopsy specimens. Arch
Pathol Lab Med 1994; 118: 740-743
65 Hussell T, Isaacson PG, Crabtree JE, Spencer J. The response
of cells from low-grade B-cell gastric lymphomas of mucosa-
associated lymphoid tissue to Helicobacter pylori. Lancet 1993;
342: 571-574
66 Hussell T, Isaacson PG, Crabtree JE, Spencer J. Helicobacter
pylori-specific tumour-infiltrating T cells provide contact
dependent help for the growth of malignant B cells in low
grade gastric lymphoma of mucosa-associated lymphoid
tissue. J Pathol 1996; 178: 122-127
67 Morgner A, Bayerdrffer E, Neubauer A, Stolte M. Malignant
tumors of the stomach. Gastric mucosa-associated lymphoid
tissue lymphoma and Helicobacter pylori. Gastroenterol Clin
North Am 2000; 29: 593-607
68 Zucca E, Bertoni F, Roggero E, Bosshard G, Cazzaniga G,
Pedrinis E, Biondi A, Cavalli F. Molecular analysis of the
progression from Helicobacter pylori-associated chronic gastritis
to mucosa-associated lymphoid-tissue lymphoma of the
stomach. N Engl J Med 1998; 338: 804-810
69 Correa P, Ruiz B. Helicobacter pylori and gastric cancer.
 Hamaloglu E et al. Gastric adenocarcinoma and lymphoma 3573
In: Rathbone BJ, Heatley RV. Helicobacter pylori and
gastroduodenal diseases. 2nd Ed. Oxford: Blackwell Scientific,
1992: 158-164
70 Asaka M, Takeda H, Sugiyama T, Kato M. What role does
Helicobacter pylori play in gastric cancer? Gastroenterology 1997;
113: S56-S60
71 Eslick GD, Lim LL, Byles JE, Xia HH, Talley NJ. Association
of Helicobacter pylori infection with gastric carcinoma: a meta-
analysis. Am J Gastroenterol 1999; 94: 2373-2379
72 Koehler CI, Mues MB, Dienes HP, Kriegsmann J, Schirmacher
P, Odenthal M. Helicobacter pylori genotyping in gastric
adenocarcinoma and MALT lymphoma by multiplex PCR
analyses of paraffin wax embedded tissues. Mol Pathol 2003;
56: 36-42
73 Mueller N, Mohar A, Evans A, Harris NL, Comstock GW,
Jellum E, Magnus K, Orentreich N, Polk BF, Vogelman J.
Epstein-Barr virus antibody patterns preceding the diagnosis
of non-Hodgkins lymphoma. Int J Cancer 1991; 49: 387-393
74 Ott G, Kirchner T, Seidl S, Mller-Hermelink HK. Primary
gastric lymphoma is rarely associated with Epstein-Barr virus.
Virchows Arch B Cell Pathol Incl Mol Pathol 1993; 64: 287-291
75 Hui PK, Tokunaga M, Chan WY, Ng CS, Chow J, Lee JC.
Epstein-Barr virus-associated gastric lymphoma in Hong Kong
Chinese. Hum Pathol 1994; 25: 947-952
76 Liu Q, Ohshima K, Masuda Y, Kikuchi M. Detection of the
Epstein-Barr virus in primary gastric lymphoma by in situ
hybridization. Pathol Int 1995; 45: 131-136
77 Shibata D, Weiss LM. Epstein-Barr virus-associated gastric
adenocarcinoma. Am J Pathol 1992; 140: 769-774
78 Tokunaga M, Land CE, Uemura Y, Tokudome T, Tanaka S,
Sato E. Epstein-Barr virus in gastric carcinoma. Am J Pathol
1993; 143: 1250-1254
79 Nakamura S, Ueki T, Yao T, Ueyama T, Tsuneyoshi M.
Epstein-Barr virus in gastric carcinoma with lymphoid stroma.
Special reference to its detection by the polymerase chain
reaction and in situ hybridization in 99 tumors, including a
morphologic analysis. Cancer 1994; 73: 2239-2249
80 Thompson MP, Kurzrock R. Epstein-Barr virus and cancer.
Clin Cancer Res 2004; 10: 803-821
81 Wu MS, Shun CT, Wu CC, Hsu TY, Lin MT, Chang MC, Wang
HP, Lin JT. Epstein-Barr virus-associated gastric carcinomas:
relation to H. pylori infection and genetic alterations.
Gastroenterology 2000; 118: 1031-1038
82 Suehiro S, Nagasue N, Abe S, Ogawa Y, Sasaki Y. Carcinoma
of the stomach in atomic bomb survivors. A comparison of
clinicopathologic features to the general population. Cancer
1986; 57: 1894-1898
83 Kai M, Luebeck EG, Moolgavkar SH. Analysis of the incidence
of solid cancer among atomic bomb survivors using a two-
stage model of carcinogenesis. Radiat Res 1997; 148: 348-358
84 Little MP, Muirhead CR, Charles MW. Describing time and
age variations in the risk of radiation-induced solid tumour
incidence in the Japanese atomic bomb survivors using
generalized relative and absolute risk models. Stat Med 1999;
18: 17-33
85 Maes B, Demunter A, Peeters B, De Wolf-Peeters C. BCL10
mutation does not represent an important pathogenic
mechanism in gastric MALT-type lymphoma, and the
presence of the API2-MLT fusion is associated with aberrant
nuclear BCL10 expression. Blood 2002; 99: 1398-1404
86 Nakamura T, Nakamura S, Yokoi T, Suzuki H, Ohashi K, Seto
M. Clinicopathologic comparison between the API2-MALT1
chimeric transcript-positive and negative gastric low-grade
B-cell lymphoma of mucosa-associated lymphoid tissue type.
Jpn J Cancer Res 2002; 93: 677-684
87 Okabe M, Inagaki H, Ohshima K, Yoshino T, Li C, Eimoto T,
Ueda R, Nakamura S. API2-MALT1 fusion defines a distinctive
clinicopathologic subtype in pulmonary extranodal marginal
zone B-cell lymphoma of mucosa-associated lymphoid tissue.
Am J Pathol 2003; 162: 1113-1122
88 Greene MH, Wilson J. Second cancer following lymphatic and
hematopoietic cancers in Connecticut, 1935-82. Natl Cancer Inst
Monogr 1985; 68: 191-217
89 Caygill CP, Hill MJ, Hall CN, Kirkham JS, Northfield TC.
Increased risk of cancer at multiple sites after gastric surgery
for peptic ulcer. Gut 1987; 28: 924-928
90 Stalnikowicz R, Benbassat J. Risk of gastric cancer after
gastric surgery for benign disorders. Arch Intern Med 1990; 150:
2022-2026
91 Pointner R, Schwab G, Knigsrainer A, Bodner E, Schmid KW.
Gastric stump cancer: etiopathological and clinical aspects.
Endoscopy 1989; 21: 115-119
92 Kellum JM, Jaffe BM, Calhoun TR, Ballinger WF 2nd. Gastric
complications after radiotherapy for Hodgkins disease and
other lymphomas. Am J Surg 1977; 134: 314-317
93 Roswit B, Malsky SJ, Reid CB. Severe radiation injuries of the
stomach, small intestine, colon and rectum. Am J Roentgenol
Radium Ther Nucl Med 1972; 114: 460-475
94 National Research Council: Committee on the biological
effects of ionising radiation. The Effects on Populations of
Exposure to Low Levels of Ionising Radiation. Washington,
DC: National Academy Press, 1980
95 Hirose F. Induction of gastric adenocarcinoma in mice by
localized x-irradiation. Gann 1969; 60: 253-260
96 Brown WM, Doll R. Mortality from cancer and other causes
after radiotherapy for ankylosing spondylitis. Br Med J 1965; 2:
1327-1332
97 Lieber MR, Winans CS, Griem ML, Moossa R, Elner VM,
Franklin WA. Sarcomas arising after radiotherapy for peptic
ulcer disease. Dig Dis Sci 1985; 30: 593-599
98 Al-Akwaa AM, Siddiqui N, Al-Mofleh IA. Primary gastric
lymphoma. World J Gastroenterol 2004; 10: 5-11
99 Rackner VL, Thirlby RC, Ryan JA Jr. Role of surgery in
multimodality therapy for gastrointestinal lymphoma. Am J
Surg1991; 161: 570-575
100 Maor MH, Velasquez WS, Fuller LM, Silvermintz KB. Stomach
conservation in stages IE and IIE gastric non-Hodgkins
lymphoma. J Clin Oncol 1990; 8: 266-271
101 Liang R, Todd D, Chan TK, Chiu E, Lie A, Kwong YL, Choy
D, Ho FC. Prognostic factors for primary gastrointestinal
lymphoma. Hematol Oncol 1995; 13: 153-163
102 Kitamura K, Yamaguchi T, Okamoto K, Ichikawa D, Hoshima
M, Taniguchi H, Takahashi T. Early gastric lymphoma: a
clinicopathologic study of ten patients, literature review, and
comparison with early gastric adenocarcinoma. Cancer 1996;
77: 850-857
103 Kodera Y, Yamamura Y, Nakamura S, Shimizu Y, Torii A,
Hirai T, Yasui K, Morimoto T, Kato T, Kito T. The role of
radical gastrectomy with systematic lymphadenectomy for the
diagnosis and treatment of primary gastric lymphoma.Ann
Surg 1998; 227: 45-50
104 Lucandri G, Stipa F, Mingazzini PL, Ferri M, Sapienza P,
Stipa S. The role of surgery in the treatment of primary gastric
lymphoma. Anticancer Res 1998; 18: 2089-2094
105 Vaillant JC, Ruskon-Fourmestraux A, Aegerter P, Gayet B,
Rambaud JC, Valleur P, Parc R. Management and long-term
results of surgery for localized gastric lymphomas. Am J Surg
2000; 179: 216-222
106 Popescu RA, Wotherspoon AC, Cunningham D, Norman
A, Prendiville J, Hill ME. Surgery plus chemotherapy or
chemotherapy alone for primary intermediate and high-
grade gastric non-Hodgkins lymphoma: the Royal Marsden
Hospital experience. Eur J Cancer 1999; 35: 928-934
107 Law MM, Williams SB, Wong JH. Role of surgery in the
management of primary lymphoma of the gastrointestinal
tract. J Surg Oncol 1996; 61: 199-204
108 Yoon SS, Coit DG, Portlock CS, Karpeh MS. The diminishing
role of surgery in the treatment of gastric lymphoma. Ann
Surg 2004; 240: 28-37
109 Salvagno L, Sorar M, Busetto M, Puccetti C, Sava C, Endrizzi
L, Giusto M, Aversa S, Chiarion Sileni V, Polico R, Bianco A,
Rupolo M, Nitti D, Doglioni C, Lise M. Gastric non-Hodgkins
lymphoma: analysis of 252 patients from a multicenter study.
Tumori 1999; 85: 113-121
110 Raderer M, Valencak J, Osterreicher C, Drach J, Hejna M,
Kornek G, Scheithauer W, Brodowicz T, Chott A, Dragosics B.
www.wjgnet.com
 3574 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Chemotherapy for the treatment of patients with primary high
grade gastric B-cell lymphoma of modified Ann Arbor Stages
IE and IIE. Cancer 2000; 88: 1979-1985
111 Liu HT, Hsu C, Chen CL, Chiang IP, Chen LT, Chen YC,
Cheng AL. Chemotherapy alone versus surgery followed
by chemotherapy for stage I/IIE large-cell lymphoma of the
stomach. Am J Hematol 2000; 64: 175-179
112 Ruskon-Fourmestraux A, Aegerter P, Delmer A, Brousse N,
Galian A, Rambaud JC. Primary digestive tract lymphoma:a
prospective multicentric study of 91 patients. Groupe d
Etude des Lymphomes Digestifs. Gastroenterology 1993; 105:
1662-1671
113 Ohtsu A. The latest advances in chemotherapy for gastro-
intestinal cancers. Int J Clin Oncol 2003; 8: 234-238
114 Sutherland AG, Kennedy M, Anderson DN, Park KG, Keenan
RA, Davidson AI. Gastric lymphoma in Grampian Region:
presentation, treatment and outcome. J R Coll Surg Edinb 1996;
41: 143-147
115 Kocher M, Mller RP, Ross D, Hoederath A, Sack H. Radio-
therapy for treatment of localized gastrointestinal non-
Hodgkins lymphoma. Radiother Oncol 1997; 42: 37-41
116 Pinotti G, Zucca E, Roggero E, Pascarella A, Bertoni F, Savio A,
Savio E, Capella C, Pedrinis E, Saletti P, Morandi E, Santandrea
G, Cavalli F. Clinical features, treatment and outcome in a
series of 93 patients with low-grade gastricMALT lymphoma.
Leuk Lymphoma 1997; 26: 527-537
117 Chandran RR, Raj EH, Chaturvedi HK. Primary gastro-
intestinal lymphoma: 30-year experience at the Cancer
Institute, Madras, India. J Surg Oncol 1995; 60: 41-49
118 Ferreri AJ, Cordio S, Paro S, Ponzoni M, Freschi M, Veglia
F, Villa E. Therapeutic management of stage I-II high-grade
primary gastric lymphomas. Oncology 1999; 56: 274-282
119 Fischbach W, Dragosics B, Kolve-Goebeler ME, Ohmann C,
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
Greiner A, Yang Q, Bhm S, Verreet P, Horstmann O, Busch
M, Dhmke E, Mller-Hermelink HK, Wilms K, Allinger S,
Bauer P, Bauer S, Bender A, Brandsttter G, Chott A, Dittrich
C, Erhart K, Eysselt D, Ellersdorfer H, Ferlitsch A, Fridrik
MA, Gartner A, Hausmaninger M, Hinterberger W, Hgel K,
Ilsinger P, Jonaus K, Judmaier G, Karner J, Kerstan E, Knoflach
P, Lenz K, Kandutsch A, Lobmeyer M, Michlmeier H, Mach H,
Marosi C, Ohlinger W, Oprean H, Pointer H, Pont J, Salabon
H, Samec HJ, Ulsperger A, Wimmer A, Wewalka F. Primary
gastric B-cell lymphoma: results of a prospective multicenter
study. The German-Austrian Gastrointestinal Lymphoma
Study Group. Gastroenterology 2000; 119: 1191-1202
120 Lin KM, Penney DG, Mahmoud A, Chae W, Kolachalam
RB, Young SC. Advantage of surgery and adjuvant
chemotherapyin the treatment of primary gastrointestinal
lymphoma. J Surg Oncol 1997; 64: 237-241
121 Mantell BS. Radiotherapy for dysphagia due to gastric
carcinoma. Br J Surg 1982; 69: 69-70
122 Henderson IW, Lipowska B, Lougheed MN. Clinical
evaluation of combined radiation and chemotherapy in
gastrointestinal malignancies. Am J Roentgenol Radium Ther
Nucl Med 1968; 102: 545-551
123 Bayerdrffer E, Neubauer A, Rudolph B, Thiede C, Lehn N,
Eidt S, Stolte M. Regression of primary gastric lymphoma
of mucosa-associated lymphoid tissue type after cure of
Helicobacter pylori infection. MALT Lymphoma Study Group.
Lancet 1995; 345: 1591-1594
124 Isaacson PG, Diss TC, Wotherspoon AC, Barbazza R, De
Boni M, Doglioni C. Long-term follow-up of gastric MALT
lymphoma treated by eradication of H. pylori with antibodies.
Gastroenterology 1999; 117: 750-751
125 Pescatore P, Heine M, Manegold BC. Cure of gastric lymphoma
with antibiotics. Gastroenterology 1995; 109: 334-335
S- Editor Pan BR E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Hepatocellular carcinoma in Lebanon: Etiology and prognostic
factors associated with short-term survival
Csar Yaghi, Ala l Sharara, Paul Rassam, Rami Moucari, Khalil Honein, Joseph BouJaoude, Rita Slim, Roger Noun,
Heitham Abdul-Baki, Mohamad Khalifeh, Sami Ramia, Raymond Sayegh
Csar Yaghi, Ala l Sharara, Paul Rassam, Rami Moucari,
Khalil Honein, Joseph BouJaoude, Rita Slim, Roger Noun,
Raymond Sayegh, Service de Gastroenterologie, Hotel Dieu de
France University Hospital, Beirut, Lebanon
Ala l Sharara, Heitham Abdul-Baki, Mohamad Khalifeh,
Departments of Internal Medicine and Surgery, American
University of Beirut Medical Center, Beirut, Lebanon
Paul Rassam, Service de Gastroenterologie, Hopital Saint
Georges, Beirut, Lebanon
Co-first-authors: Csar Yaghi and Ala l Sharara
Correspondence to: Ala I Sharara, MD, FACP, Head, Gastro-
enterology Division, American University of Beirut Medical
Center, PO Box 11-0236, Beirut 1107 2020,
Lebanon. as08@aub.edu.lb
Telephone: +961-1-350000 Fax:+961-1-366098
Received: 2006-01-24 Accepted: 2006-02-28
Abstract
AIM: To study the epidemiology of HCC in Lebanon and
prognostic factors predictive of early mortality.
METHODS: An observational follow-up cohort study of
HCC cases diagnosed over a five-year period was carried
out. Multivariate analysis was conducted to identify
prognostic factors in comparison to Cancer of the Liver
Italian Program (CLIP) score. Multiple variables including
the etiology of underlying liver disease, the demographic
characteristics of patients, and the severity of liver
disease evaluated by the Child-Pugh score were studied.
Tumor parameters included the time of diagnosis of HCC,
alpha-fetoprotein level, number and size of nodules,
presence of portal vein thrombosis, and treatment
modalities. Death or loss of follow-up was considered as
an end-point event.
RESULTS: Ninety-two patients (mean 60.5 22.3
years) were included. Etiology of underlying disease
was hepatitis B, C, and alcohol in 67%, 20%, and
23.5% respectively. Child-Pugh class at diagnosis was
A, B, and C in 34.8%, 39.3% and 25.8% respectively.
Overall survival was 44.8%, 32.8% and 17.6% at 1, 2
and 3 years respectively (mean F/U 40.2 23.5 mo).
Multivariate analysis identified three predictors of early
mortality (< 6 mo): bilirubin > 3.2 mg/dL (P < 0.01),
HCC as first presentation of liver disease (P = 0.035),
and creatinine > 1 mg/dL (P = 0.017). A score based
on these variables outperformed the CLIP score by Cox
proportional hazard. ROC curve showed both models to
be equivalent and moderately accurate.
World J Gastroenterol 2006 June 14; 12(22): 3575-3580
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
LIVER CANCER
CONCLUSION: HBV is the leading cause of HCC in
Lebanon. Independent predictors of early mortality
are elevated bilirubin, creatinine and HCC as first
manifestation of disease. Prospective validation of a
score based on these clinical parameters in predicting
short-term survival is needed.
2006 The WJG Press. All rights reserved.
Key words: Liver; Epidemiology; Hepatitis; Neoplasm;
Cancer; Cirrhosis
Yaghi C, Sharara Al, Rassam P, Moucari R, Honein K,
BouJaoude J, Slim R, Noun R, Abdul-Baki H, Khalifeh M,
Ramia S, Sayegh R. Hepatocellular carcinoma in Lebanon:
Etiology and prognostic factors associated with short-term
survival. World J Gastroenterol 2006; 12(22): 3575-3580
http://www.wjgnet.com/1007-9327/12/3575.asp
INTRODUCTION
Hepatocellular carcinoma (HCC) is a malignancy occurring
most often in the setting of liver cirrhosis. The causes
of the underlying liver disease differ according to the
geographical distribution. Common risk factors for HCC
include infection with hepatitis B virus (HBV), hepatitis C
virus (HCV), cirrhosis of any cause, alcoholic liver disease,
and inherited metabolic diseases such as hemochromatosis
and 1-antitr ypsin deficiency. HCC occurs most
commonly in sub-Saharan Africa and parts of the Far East
such as China, Taiwan, Korea, Japan, and Vietnam. HCV
infection with advanced fibrosis or cirrhosis is the main
cause of HCC in Japan, South Africa, Egypt, the United
States and Southern Europe[1-4]. In Northern and Central
European countries, ethanol is still the leading cause of
cirrhosis and is responsible for the majority of HCC
cases[5]. There has been no report on the epidemiology of
HCC in Lebanon. Epidemiologic data from neighboring
countries differ widely with the primary risk factor for
HCC attributable to HBV in Turkey [6], HBV/HDV in
Jordan[7], and HCV in Saudi Arabia[8,9].
The expected survival of patients with HCC is an
important parameter in predicting mortality on a liver
transplantation waiting list. The prognostic factors for
survival in patients with HCC have been identified in
previous studies and sub-classified into 3 groups: (1)
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 3576 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

demographic characteristics including age and gender; (2) Table 1 Characteristics of the 92 patients with hepatocellular
factors related to HCC such as tumor size, the number carcinoma n (%)
of nodules, vascular invasion, the presence of a tumor
capsule or metastasis; and (3) factors related to underlying Age(yr) At diagnosis of cirrhosis before 56.0 26.3
liver disease severity, including ascites, encephalopathy, development of HCC
At HCC diagnosis 60.5 22.3
or serum bilirubin. The Child-Pugh classification, used
Gender Male 78 (84.8)
in patients with cirrhosis, only considers liver disease and
Etiology HBV 62 (67.4)
not tumor characteristics. Orthotopic liver transplantation HCV 18 (19.6)
(OLT) is an excellent treatment for early hepatocellular HBV + HCV 77 (83.7)
carcinoma. There is, however, a paucity of data on survival Alcohol abuse 22 (23.9)
according to intention-to-treat analysis and on the rate Liver disease Child-Pugh A 32 (34.8)
of dropout from the waiting list for OLT among patients severity at time Platelet count < 75 000 16 (17.4)
with HCC due to shortage of grafts. This is compounded of diagnosis Bilirubin > 3.2 mg/dL 25 (27.2)
by the fact that up to 30% of eligible patients develop Albumin < 30 g/L 46 (50)
contraindications to the procedure while waiting for Mean MELD score 9.43 7.58

a donor. All of the above result in less than optimal Tumor Single nodule 41 (44.6)
characteristics 2-3 nodules 19 (20.7)
prediction of early mortality in HCC patients, particularly
Diffuse 33 (35.9)
in populations who present with advanced disease and Portal vein obstruction1 31 (34.8)
where therapeutic options may be limited. The aim of the Follow-up and HCC as first presentation of liver 53 (57.6)
present study was to describe the epidemiology of HCC survival disease
in Lebanon, and to identify factors predictive of early Mean F/U prior to HCC in those 40.2 mo
mortality in our patients who could seldom benefit from with known liver disease
Mean follow-up after HCC diagnosis 16.4 mo
liver transplantation.
Mean survival 14.9 mo

MATERIALS AND METHODS 1

Three patients had multiple nodules without details regarding vascular

Patients invasion or size of the nodules.

The study is a multicenter follow-up cohort study

consisting of 92 patients diagnosed with HCC between
1998 and 2003. The study involved three university
hospitals in Beirut: Hotel-Dieu de France (Universit Saint
Joseph), American University of Beirut Medical Center,
and Saint-Georges Hospital (Balamand University Medical
School). Patients were identified from the medical records
and from specialty liver clinics. Information regarding
deceased patients was obtained from medical records and
prospective follow-up was continued for new and existing
patients. Multiple variables including the etiology of
underlying liver disease, the demographic characteristics of
patients including age and gender, and the severity of liver
disease evaluated by the Child-Pugh score were studied.
Tumor parameters included the time of diagnosis of
HCC, alpha-fetoprotein level, number and size of nodules,
presence of portal vein thrombosis, and treatment
modalities. Death or loss of follow-up was considered as
an end-point event.
HCC was diagnosed according to any one of the
following criteria: (1) an abnormal liver morphology
with a tumor nodule that is enhanced during the arterial
phase following intravenous contrast injection on CT-
scan or MRI. (2) an elevated alpha fetoprotein level > 400
ng/mL in the presence of a liver nodule, and (3) biopsy-
proven HCC[10]. Patients who did not meet one or more
of these diagnostic criteria were not included in the study.
Cirrhosis was confirmed according to histology and/or
by clinical and biochemical signs of hepatic insufficiency,
ascites, esophageal varices, and ultrasonographic signs
of portal hypertension. Tumor eligibility-for-treatment
criteria included: a single nodule of less than 5 cm without
evidence of vascular invasion, or less than 3 nodules of
< 3 cm each with the absence of portal vein thrombosis.
Tumors more than 5 cm in diameter were considered
eligible for surgical resection when there was no evidence
of vascular invasion and when surgical resection was
technically possible. These patients were not considered
eligible for any other curative treatment namely liver
transplantation, percutaneous ethanol ablation, or radio
frequency ablation. Patients who had tumors eligible for
a curative treatment were further selected according to
transplantation indication with an upper age limit of 65
years.
Statistical analysis
Biochemical data were collected at the time of diagnosis
of hepatocellular carcinoma. Quantitative data were
expressed as mean standard deviation and range. Group
comparisons used the two-sample t-test or the Mann-
Whitney test for quantitative variables and the two-tailed
Fischers exact test was used for qualitative variables.
Comparison of survival used the Kaplan-Meier model, the
log-rank test and receiver operating characteristic (ROC)
curve.
RESULTS
Etiology
Characteristics of the 92 HCC patients are shown in
Table 1. The mean age was 60.5 22.3 years and the sex
ratio was 5.6:1 (M/F, 78/14). In 77 patients (83.7%), the
etiology of the underlying liver disease was viral. HBV
infection was found in 62 patients (67.4%) compared to 18
patients (19.6%) with HCV infection. Alcohol abuse was
found in 22 patients (23.9%) but was the only cause of

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 Yaghi C et al. HCC in Lebanon: Risk factors and prognosis 3577

liver disease in only 8.2% of our patients. Miscellaneous Table 2 Univariate analysis of overall survival in HCC patients
causes included autoimmune hepatitis, primary biliary
cir rhosis, hemochromatosis, and tyrosinemia and Criteria Median Mean Log
accounted for 5.9% of cases. survival survival rank P
(mo) (mo)
Age at diagnosis of < 55 18 40 0.008
Liver disease severity HCC (yr) > 55 8 14
HCC occurred in the setting of cirrhosis in all but one Child-Pugh score A 25 31.44 0.103
patient. The Child-Pugh class at time of diagnosis of HCC B 10 18.14
was A, B, and C in 32 (34.8%), 35 (39.3%), and 23 (25.8%) C 4 24.5
cases respectively. Model of end stage liver disease Bilirubin < 3.2 mg/dL 15 23 0.015
(MELD) score was calculated in 79 patients, and the > 3.2 mg/dL 3 14
mean was 9.4 7.6. MELD score was < 12, 12-16, 16-24, HCC diagnosis Known liver 25 33 0.003
and >24 in 68.4%, 12.7%, 12.7%, and 6.3% of patients disease
respectively. As first 6 12
manifestation of
liver disease
Tumor characteristics Eligibility for No 15 6 0.005
The diagnosis of HCC was established during follow- treatment Yes 27 33
up of liver cirrhosis in 36 patients (42.4%) with a mean INR <2 12 23 0.029
follow-up period of 40 mo (median = 24 mo). In 13 >2 2 9
patients (36.1%), HCC was diagnosed during the first Portal vein Absent 14 23 0.021
two years following the diagnosis of cirrhosis, and in thrombosis Present 3 12
26 patients (72.4%), within 5 years. The size of tumor
nodules ranged from 1 cm to 20 cm with a mean of 5.6
cm. The number of nodules was 1, 2, and 3 in 41 (46.1%),
reach statistical significance (P = 0.13).
14 (15.7%), and 5 (5.6%) patients respectively; 29 patients
(32.6%) had diffuse HCC. Portal vein thrombosis was
present in 29 patients (38.2%). Forty patients (43.5%) had Survival and follow-up
a single tumor of less than 5 cm or less than 3 nodules < Overall survival was 44.8%, 32.8%, and 17.6% at 1, 2,
3 cm of diameter; ten of those patients had evidence of and 3 years respectively. Individual prognostic factors
vascular invasion. The number of patients having a tumor were studied using the Log rank method. Survival was
considered eligible for a curative treatment was 32 (34.8%) studied with respect to multiple parameters including
versus 60 (65.2%) that were ineligible for curative resection prothrombin time, platelet count, bilirubin value, albumin,
on account of diffuse disease or tumor-associated with creatinine, alpha-fetoprotein, the diameter of the largest
portal vein thrombosis. nodule, the presence of portal vein thrombosis, eligibility
for curative treatment, Child-Pugh score, MELD score,
the presence of ascites, the etiology of liver disease, and
Eligibility for curative treatment
the grading of esophageal varices. Prognostic factors
Thirty-two patients had tumors considered eligible for
identified in univariate analysis included age > 55, bilirubin
curative treatment, but only 15 (46.9%) patients could
benefit from such therapy. This was primarily due to the < 3.2 mg/dL, HCC as the first manifestation of liver
inability to perform a curative treatment at first intent disease, eligibility for a curative treatment, International
because of age, tumor location for surgical resection, Normalized Ratio (INR) < 2, MELD score > 18, and the
the lack of regular organ allocation program, or general presence of portal vein thrombosis. Univariate analysis of
contraindications other than hepatic disease severity overall survival (Table 2) failed to demonstrate a significant
quantified by the Child-Pugh score. When patients older difference in survival with respect to Child-Pugh score
than 70 or 65 were excluded, eligibility for a curative in HCC patients. The mean survival was 31.4, 18.1, and
treatment decreased to 25.8% or 20.2% respectively. 24.4 months respectively in Child-Pugh class A, B, and
Other causes of failure to treat were the surveillance C patients (P = 0.10). In multivariate analysis using Cox
of small nodules of 1-2 cm of diameter, or patients regression model; survival in HCC patients was related to
awaiting OLT with no other therapeutic possibilities. age < 55 at diagnosis of HCC (P = 0.004), bilirubin value
Fifty-nine patients (64.1%) did not benefit from any < 3.2 mg/dL (P = 0.014), and HCC developing in the
treatment because of either decompensated liver disease setting of known liver disease (P = 0.016).
or advanced tumor cases; in the remaining cases, treatment Factors associated with six month survival are shown in
consisted of percutaneous ethanol injection (7 patients), Table 3. Death occurred within six months in 31 patients
chemoembolization (17 patients), OLT (8 patients) and (38.3%). Survival beyond six months was associated
surgical resection (7 patients). Eight patients had more with INR < 2, bilirubin < 3.2 mg/dL, tumor less than
than one treatment modality. There was a trend towards 5 cm of diameter or three nodules of less than 3 cm of
a difference in eligibility for treatment between patients diameter without portal vein thrombosis, MELD score
presenting with HCC as first manifestation of liver disease < 16, lower Child-Pugh class, and the absence of portal
(21% eligible for a curative treatment), compared to those vein thrombosis. Mean creatinine level, INR, and MELD
discovered on routine screening for HCC (41% eligible score were lower in patients with > 6 mo survival. In
for a curative treatment), although this difference did not multivariate analysis using Cox regression model, bilirubin

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 3578 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

100
Table 3 Factors associated with six months survival n (%) CLIP Score (Area = 0.689)
CBT (Area = 0.670)
1
Survival
< 6 mo > 6 mo P 75
Child-Pugh score A 4 (14.3) 14 (35.9) 0.0322
INR < 2 22 (70.9) 43 (93.5) 0.0122
Bilirubin < 3.2 mg/dL 17 (58.6) 35 (83.3) 0.0332

Sensitivity (%)
50
MELD score < 16 37 (90.2) 18 (62.1) 0.0062
Portal vein thrombosis 17 (54.8) 13 (30.9) 0.0352
Tumor < 5 cm or 3 nodules < 3 cm 3 (10.3) 11 (31.4) 0.0422
without portal vein thrombosis 25
Mean creatinine (mg/dL) 1.57 0.89 1.19 0.49 0.0213
Mean INR 1.68 0.47 1.47 0.42 0.0413
Mean MELD score 13.26 8.12 8.12 6.54 0.0053 0
0 25 50 75 100
1 Specificity (%)
Patients treated curatively by OLT or surgical resection were excluded from
this analysis. 2Fischer exact test. 3Mean comparisons by independent sample
t-test. Figure 2 Receiver Operating Characteristic (ROC) curve for early mortality using
the CBT (Creatinine, Bilirubin, and Tumor as primary manifestation) and CLIP
scores.

A CBT score:
100 0 (n = 13)
1 (n = 25) both prognostic models to be equivalent and of moderate
80 2 (n = 24) accuracy (Figure 2).
3 (n = 4)
Survival (%)

60 DISCUSSION
40
HCC is a relatively rare cancer in Lebanon, ranking 14th
among both males and females, with an age standardized
20 rate of 3.5 and 2.2 per 100 000 respectively[11]. Under-
standing the risk factors for HCC and predictors of poor
0 survival are important in prevention and treatment. The
6 12 18 24 mo
results of our study show that most HCC patients in
Lebanon have HBV-related liver cirrhosis, accounting for
B
100 CLIP score: nearly two thirds of our patients. This is concordant with
0-1 (n = 16)
2-3 (n = 26)
previous findings regarding the etiology of liver cirrhosis
80
4-6 (n = 31) in Lebanon where HBV was the most important cause
accounting for more than 50% of cirrhosis, followed by
60
HCV, and alcohol abuse[12]. Of note, the HBV and HCV
carrier state in Lebanese blood donors is 1%-2% and
Survival (%)

40
20
0
0 6 12 18 24 mo
Figure 1 A: Kaplan-Meier survival curves for 68 patients with hepatocellular
carcinoma based on 3 clinical criteria CBT (Creatinine, Bilirubin, and Tumor as
primary manifestation); B: Kaplan-Meier estimated survival curves according to the
Cancer of the Liver Italian Programme (CLIP) classification.
> 3.2 mg/dL (P = 0.001), creatinine > 1 mg/dL (P = 0.017),
and HCC as the first manifestation of liver disease (P =
0.035) were independent predictors of survival less than
six months (P = 0.006). Short-term survival (> 6 mo) was
69.2%, 56%, 45.8%, and 0% when none, one, two, or all
of these 3 independent factors were present (P = 0.0002)
(Figure 1). In comparison, the CLIP score predicted short-
term survival in our patient population was 69.3%, 68%,
and 29.6% when the score was respectively 0-1, 2-3, or
4-6 (Log rank P = 0.047) (Figure 1). A score based on
the 3 clinical variables identified above (CBT score for
creatinine, bilirubin, and tumor) outperformed the CLIP
score by Cox proportional hazard. ROC curve showed
0.1%-0.6% respectively[13-15]. Interestingly, there were no
cases of non-alcoholic steatohepatitis- or cryptogenic
cirrhosis-related HCC in this study population. Studies
from neighboring Middle Eastern countries showed wide
variation in the etiology of HCC. In Saudi Arabia, HCV
plays a major role in the epidemiology of HCC where
39.5% of patients were anti-HCV antibody positive [8].
This is in contrast to an overall HCV seroprevalence of
1.1%-2.9% among Saudi- blood donors [9] versus 0.6%
among Lebanese-blood donors[13]. In Jordan, an association
between HDV-positive status and HBs-antigen positive
primary HCC was found. The prevalence of hepatitis D
viral infection in Jordan was 23% in patients with chronic
liver disease and in 67% of patients with HCC [7] . In
contrast, none of our HCC patients was seropositive for
HDV. Lastly, HBV infection was found to be the leading
cause of HCC in Turkey, followed by HCV infection, and
alcoholic liver disease[6]. Hepatitis B, hepatitis C, excessive
alcohol intake were detected in 56%, 23.2%, and 15.9% of
Turkish HCC patients respectively.
Previous studies have identified multiple prognostic
factors in HCC [16-19]. Gender differences were studied
showing significantly longer median survival in females
(14 mo) than in male patients (4 mo)[20]. This does not

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 Yaghi C et al. HCC in Lebanon: Risk factors and prognosis 3579
seem to be the case in our patients. Previous multivariate
analysis demonstrated that high serum alpha-fetoprotein
levels, venous invasion, extrahepatic metastasis, and lack
of therapy were independent factors related to unfavorable
prognosis [17]. In another study, only type of estrogen
receptors and bilirubin showed independent predictive
value for mortality[21]. In Child B and C cirrhosis patients,
factors predictive of prolonged survival were albumin
level ( 30 g/L), absence of esophageal varices, tumor
size ( 3.0cm), tumor number (solitary), and alpha-
fetoprotein (AFP) level (< 400 ng/mL)[18]. In our study,
factors predictive of prolonged survival were bilirubin
(< 3.2 mg/dL) at diagnosis, age (less than 55), and HCC
developing in the setting of known liver disease. Serum
creatinine and INR values were not independent factors
associated with long-term survival, nor was the etiology
of the underlying liver disease. This explains somehow
that the MELD score was associated with the outcome in
univariate analysis, whereas this score has limited value for
HCC since prognosis is not only related to liver failure, but
also to tumor evolution which is not taken into account in
this score. Furthermore, the etiology of underlying liver
disease does not seem to play a role in prognosis either in
univariate or multivariate analysis. The finding that HCC
developing in the setting of known liver disease is an
independent predictor of outcome further supports the
role of routine screening for HCC in cirrhosis since some
of those patients may be amenable to curative treatment.
Data on short-term survival, particularly from the
Middle East, is scarce in the literature. Such data is
important for the evaluation of early mortality on liver
transplantation waiting lists. In a study from Kuwait
involving 145 HCC cases, the CLIP score was found to be
superior to the Okuda stage as a prognostic indicator[22].
However, the results from a study by Siddique et al seem
to reflect the natural history of the disease (91% did not
receive any specific therapy for HCC) [22]. In our study,
factors predictive of six month survival were bilirubin,
creatinine, and HCC developing in the setting of known
liver disease. Even for short-ter m sur vival, MELD
score does not seem to be fit for the evaluation of early
mortality in HCC patients. MELD score did, however,
show a significant difference in Kaplan-Meier survival
analysis when the chosen cut-off was 16, but no difference
in survival was found when the cut-off was 24. Again,
the etiology of liver disease does not seem to play a role
in the early mortality of HCC neither in univariate nor in
multivariate analysis.
Our study is limited by the fact that the number of
patients was relatively small which restricts the number
of variables that can be examined with confidence by
multivariate analysis. HCC is however, an uncommon
cancer in Lebanon, ranking 14th among both males and
females, with an age standardized rate of 3.5 and 2.2 per
100 000 respectively[11]. With a population of just over 3
million, the number of HCC in the whole of Lebanon
over a one-year period is estimated at less than 90 cases.
It is estimated that a third to half of these cases are
referred to the three major university hospitals involved
in this study for evaluation and management. We believe,
therefore, that our study cohort is relatively large for
Lebanon and is likely to be well representative of the
population at large particularly as it relates to etiology and
outcome.
In conclusion, our study shows that HCC in Lebanon is
primarily related to HBV cirrhosis with HCV and alcohol
abuse ranking as distant second and third risk factors.
The probability for a curative treatment is still low in
Lebanon because of advanced tumor stage at presentation
and the lack of a regular liver donation program. Thus
determination of independent prognostic factors for early
mortality is necessary, which include bilirubin (> 3.2 mg/
dL), creatinine (> 1 mg/dL), and HCC as first presentation
of liver disease. Further studies are needed to validate the
above identified prognostic factors and to evaluate drop-
out and mortality on the waiting list taking into account
these factors.
ACKNOWLEDGMENTS
The authors thank the Lebanese National Research
Council for partial funding of this project, the staff and
patients of the three university hospitals for their excellent
co-operation.
REFERENCES
1 Kew MC, Yu MC, Kedda MA, Coppin A, Sarkin A, Hodkinson
J. The relative roles of hepatitis B and C viruses in the etiology
of hepatocellular carcinoma in southern African blacks.
Gastroenterology 1997; 112: 184-187
2 Hassan MM, Zaghloul AS, El-Serag HB, Soliman O, Patt YZ,
Chappell CL, Beasley RP, Hwang LY. The role of hepatitis
C in hepatocellular carcinoma: a case control study among
Egyptian patients. J Clin Gastroenterol 2001; 33: 123-126
3 Caselmann WH, Alt M. Hepatitis C virus infection as a major
risk factor for hepatocellular carcinoma. J Hepatol 1996; 24:
61-66
4 Di Bisceglie AM, Order SE, Klein JL, Waggoner JG, Sjogren
MH, Kuo G, Houghton M, Choo QL, Hoofnagle JH. The role
of chronic viral hepatitis in hepatocellular carcinoma in the
United States. Am J Gastroenterol 1991; 86: 335-338
5 Hellerbrand C, Hartmann A, Richter G, Knll A, Wiest
R, Schlmerich J, Lock G. Hepatocellular carcinoma in
southern Germany: epidemiological and clinicopathological
characteristics and risk factors. Dig Dis 2001; 19: 345-351
6 Uzunalimolu O, Yurdaydin C, Cetinkaya H, Bozkaya H,
Sahin T, Colakolu S, Tankurt E, Sariolu M, Ozenirler S,
Akkiz H, Tzn N, Deertekin H, Okten A. Risk factors for
hepatocellular carcinoma in Turkey. Dig Dis Sci 2001; 46:
1022-1028
7 Toukan AU, Abu-el-Rub OA, Abu-Laban SA, Tarawneh MS,
Kamal MF, Hadler SC, Krawczynski K, Margolis HS, Maynard
JE. The epidemiology and clinical outcome of hepatitis D virus
(delta) infection in Jordan. Hepatology 1987; 7: 1340-1345
8 Shobokshi O, Al-Saffi Y, Zaharna J, Mohammad A. The
prevalence of hepatitis C virus in patients with established
primary hepatocellular carcinoma in the Western region of
Saudi Arabia. Saudi Med J 2003; 24 Suppl 2: S130
9 Shobokshi OA, Serebour FE, Al-Drees AZ, Mitwalli AH,
Qahtani A, Skakni LI. Hepatitis C virus seroprevalence rate
among Saudis. Saudi Med J 2003; 24 Suppl 2: S81-S86
10 Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R,
Burroughs AK, Christensen E, Pagliaro L, Colombo M, Rods J.
Clinical management of hepatocellular carcinoma. Conclusions
of the Barcelona-2000 EASL conference. European Association
for the Study of the Liver. J Hepatol 2001; 35: 421-430
11 Shamseddine A, Sibai AM, Gehchan N, Rahal B, El-Saghir
N, Ghosn M, Aftimos G, Chamsuddine N, Seoud M. Cancer
incidence in postwar Lebanon: findings from the first national
www.wjgnet.com
 3580 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
population-based registry, 1998. Ann Epidemiol 2004; 14:
663-668
12 Yaghi C, Sayegh R, Fayad N, Honein K, Bou Jaoude J, Khouri K.
[Cirrhosis and renal failure: the influence of creatinine value
on prognosis]. J Med Liban 2003; 51: 15-23
13 Irani-Hakime N, Tamim H, Samaha H, Almawi WY.
Prevalence of antibodies against hepatitis C virus among
blood donors in Lebanon, 1997-2000. Clin Lab Haematol 2001;
23: 317-323
14 Araj GF, Kfoury-Baz EE, Barada KA, Nassif RE, Alami SY.
Hepatitis C virus: prevalence in Lebanese blood donors and
brief overview of the disease. J Med Liban 1995; 43: 11-16
15 Nabulsi MM, El Saleeby CM, Araj GF. The current status of
hepatitis B in Lebanon. J Med Liban 2003; 51: 64-70
16 Sangro B, Herriz M, Martnez-Gonzlez MA, Bilbao I,
Herrero I, Beloqui O, Bets M, de-la-Pea A, Cienfuegos JA,
Quiroga J, Prieto J. Prognosis of hepatocellular carcinoma in
relation to treatment: a multivariate analysis of 178 patients
from a single European institution. Surgery 1998; 124: 575-583
17 Tangkijvanich P, Anukulkarnkusol N, Suwangool P,
Lertmaharit S, Hanvivatvong O, Kullavanijaya P, Poovorawan
Y. Clinical characteristics and prognosis of hepatocellular
carcinoma: analysis based on serum alpha-fetoprotein levels. J
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
Clin Gastroenterol 2000; 31: 302-308
18 Ueno S, Tanabe G, Nuruki K, Oketani M, Komorizono Y,
Hokotate H, Fukukura Y, Baba Y, Imamura Y, Aikou T.
Prognosis of hepatocellular carcinoma associated with Child
class B and C cirrhosis in relation to treatment: a multivariate
analysis of 411 patients at a single center. J Hepatobiliary
Pancreat Surg 2002; 9: 469-477
19 A new prognostic system for hepatocellular carcinoma: a
retrospective study of 435 patients: the Cancer of the Liver
Italian Program (CLIP) investigators. Hepatology 1998; 28:
751-755
20 Tangkijvanich P, Mahachai V, Suwangool P, Poovorawan Y.
Gender difference in clinicopathologic features and survival of
patients with hepatocellular carcinoma. World J Gastroenterol
2004; 10: 1547-1550
21 Villa E, Moles A, Ferretti I, Buttafoco P, Grottola A, Del Buono
M, De Santis M, Manenti F. Natural history of inoperable
hepatocellular carcinoma: estrogen receptors status in
the tumor is the strongest prognostic factor for survival.
Hepatology 2000; 32: 233-238
22 Siddique I, El-Naga HA, Memon A, Thalib L, Hasan
F, Al-Nakib B. CLIP score as a prognostic indicator for
hepatocellular carcinoma: experience with patients in the
Middle East. Eur J Gastroenterol Hepatol 2004; 16: 675-680
S- Editor Wang J L- Editor Zhu LH E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Apoptosis of human colon carcinoma HT-29 cells induced by
ceramide
Xiao-Feng Zhang, Bai-Xiang Li, Chun-Yan Dong, Rui Ren
Xiao-Feng Zhang, Bai-Xiang Li, Rui Ren, Department of
toxicology, Public Health College, Harbin Medical University,
Harbin 150081, Heilongjiang Province, China
Chun-Yan Dong, Department of Digestive Medicine, Changhai
Hospital, Shanghai 200433, China
Supported by the National Natural Science Foundation of China,
No. 30471447
Correspondence to: Bai-Xiang Li, Public Health College,
Harbin Medical University, 157 Baojian Road, Nangang District,
Harbin 150081, Heilongjiang Province,
China. libaix@ems.hrbmu.edu.cn
Telephone: +86-451-87502830 Fax: +86-451-87502885
Received: 2006-01-13 Accepted: 2006-02-18
Abstract
AIM: To investigate the effect of exogenous ceramide-
induced apoptosis on human colon carcinoma HT-29
cells.
METHODS: Light microscope, transmission electron
microscope and fluorescence microscope were used to
observe the morphology change of apoptosis in HT-29
cells. Agarose gel electrophoresis was performed to
detect the DNA fragment. Mitochondrial function was
detected by MTT assay. mRNA expression of Bcl-2 family
gene members was determined by reverse transcription
polymerase chain reaction (RT-PCR) assay.
RESULTS: A f t e r C 2 -ceramide treatment, typical
characteristics of apoptosis, such as nuclear chromatin
breakage, apoptotic body and DNA ladder, could be
observed. After exposure to 50 mmol/L C 2-ceramide
for 12 and 24 h, cell apoptosis was 64.1% and
81.3% respectively, which had a time-and dose-effect
relationship. Mitochondrial function started to decrease
from 6 h after exposure to ceramide. Meanwhile,
ceramide up-regulated or down-regulated the mRNA
expression of Bcl-2 family gene members.
CONCLUSION: Ceramide induces apoptosis of human
colon carcinoma HT-29 cells by affecting the expression
of Bcl-2 family gene members and impacting the
mitochondrial function.
2006 The WJG Press. All rights reserved.
Key words: Ceramide; Apoptosis; Human colon carcinoma
cells; Bcl-2 family gene member; Mitochondrial function
Zhang XF, Li BX, Dong CY, Ren R. Apoptosis of human colon
World J Gastroenterol 2006 June 14; 12(22): 3581-3584
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
COLORECTAL CANCER
carcinoma HT-29 cells induced by ceramide. World J Gastro-
enterol 2006; 12(22): 3581-3584
http://www.wjgnet.com/1007-9327/12/3581.asp
INTRODUCTION
Ceramide (N-acetyl-sphingosine) is a fundamental unit
of sphingomyelin which is a moiety of cytoplasmic
membrane. Ceramide, also an important bioactive
compound in vivo, can conduct specific signal transduction
from cell surface receptor and environmental stress to
nuclei, and then provoke multiple cytobiologic effects[1].
Meanwhile, ceramide is a common second messenger
molecule participating in sphingomyelin cycle, apoptosis
and differentiation of many cell types[2,3]. Apoptosis can
be induced by surrounding stimulating factors, such
as tumor necrosis factor a (TNF- a ), endotoxin, FAS
ligand, radiation, chemotherapeutics and heat shock,
and is associated with ceramide. Recent studies indicate
that ceramide has a close relationship with genesis and
progression of digestive tract tumor. But little information
about Bcl-2 family gene member and mitochondrial
function is available concerning the effect of ceramide-
induced apoptosis in colon carcinoma which is one of
the most aggressive forms of cancer. Even ceramide
could induce apoptosis of HT-29 cells. This study was
to determine how ceramide induces apoptosis of human
colon carcinoma cells and to discuss its mechanism.
MATERIALS AND METHODS
Materials
C 2-ceramide (N-acetyl-D-sphingosine) was purchased
from Sigma, dissolved to 5 mmol/L in DMSO as a stock
solution. Hoechest 33258 and MTT were purchased from
Sigma, dissolved in PBS solution. The two solutions were
preserved at 4 from light. Apoptosis DNA extract kit
was from Shanghai Huashun Biotechnology Company.
RPMI-1640 media, DMSO and fetal bovine serum (FBS)
were bought from GIBCO. Trypsin and EDTA were
obtained from Amresco.
Cell culture
HT-29 cells were provided by the Institute of Tumor
Prevention and Cure in Beijing, China. Cells were
cultured in RPMI-1640 media supplemented with 10%
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 3582 ISSN 1007-9327 CN 14-1219/ R
Table 1 Primer sequences of Bcl-2 family gene members
Size Tempera-
Gene Primer sequence
(bp) ture ()
Sense: 5-CCAGCTGCCTTGGACTGT-3
Bax 135
Antisense: 5-ACCCCCTCAAGACCACTCTT-3
Sense: 5-GTAAACTGGGGTCGCATTGT-3
Bcl-xl 146
Antisense: 5-TGGATCCAAGGCTCTAGGTG-3
Sense: 5-GGCAATGTGACTTTTTCCAA-3
Bcl-2 137
Antisense: 5-GGCTGATATTCTGCAACACTG-3
Sense: 5-AGGGCTGACCCAGATTCC-3
Bad 178
Antisense: 5-GTGACGCAACGGTTAAACCT-3
Sense: 5-GCTTCCAGTGTAGACGGAGC-3
Bid 116
Antisense: 5-GTGCAGATTCATGTGTGGATG-3
Sense: 5-GTGGACATCCGCAAAGAC-3
b-actin 303
Antisense: 5-TCAACGCAATGTGGGAAG-3
FBS and 2 mmol/L glutamine. Antibiotics added to
the media were 100 U/mL penicillin and 100 U/mL
streptomycin. Cells maintained in a humidified atmosphere
of 95 mL/L air and 50 mL/L CO 2 at 37 were then
passed at pre-confluent densities in a solution containing
0.25% trypsin and 0.02% EDTA. Exponentially growing
cells were used throughout all experiments.
Light microscopy
HT-29 cells were treated with C 2 -ceramide at final
concentrations of 12.5, 25 and 50 m mol/L for 24 and
48 h respectively. DMSO served as negative control.
During the procedure, cell morphology was observed
under light microscope for different time.
Transmission electron microscopy
Cells were collected and fixed with 4% glutaraldehyde in
phosphate buffer overnight at 4. After post-fixation with
1% OsO4 in cacodylate buffer for 1h at 4, the pellet was
dehydrated in graded ethanol solutions and embedded in
Epon. Ultrathin sections of pellet were counterstained
with uranyl acetate and lead citrate and observed under
transmission electron microscope.
Fluorescence microscopy
Cells (4 10 4) were treated as above, suspended with
FBS and incubated with Hoechest 33258 for 30 min
at 37 from light. Then the cells were observed under
fluorescence microscope. Three hundred cells were
counted and apoptotic cell rate was calculated.
Detection of DNA fragmentation
Cells (2 10 6 ) were treated as above and collected.
Fragmented DNA was extracted according to the
manufacturers instructions of apoptosis DNA extract
kit and underwent electrophoresis on 2 g/L agarose gel
containing 0.5 g/L of ethidium bromide and visulized by
UV transillumination.
Detection of mitochondrial function (MTT assay)
Cells (1.5 104) were cultured in 96-well plates and treated
with C2-ceramide for 1, 3, 6, 9, 12 and 24 h as above. Then
MTT and DMSO were added by turns. Absorbance was
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
Table 2 Apoptosis rate of HT-29 cells induced by C2-ceramide
(mean SD, %)
C2-ceramide Cell count (n ) 12 h 24 h
0 mmol/L 300 5.33 1.53 8.67 2.52
61
12.5 mmol/L 300 14.7 2.52a 28.0 9.85a
25 mmol/L 300 33.3 8.37a
43.3 12.3a
60
50 mmol/L 300 64.7 7.51a
81.3 9.07a
55 a
P < 0.05 vs control.
60
60 determined with a multi-well plate reader at wavelength
570 nm.
58
RT-PCR
Cells (1 10 6) were treated for 24 h as above. Total
RNA was isolated from the treated cells with Trizol and
reversely transcribed into cDNA with human specific
primers for Bax, Bad, Bid, Bcl-2, Bcl-xl and b -actin.
Sequences of primers are shown in Table 1. Briefly, 35
cycles of PCR amplification were performed at 94 for
30 s, annealing at 60 for 30 s and extension at 72
for 30 s in a 25 mL reaction system. PCR products were
confirmed by 1.5 g/L agarose gel electrophoresis and
visualized by UV transillumination. mRNA expression
of genes was assessed by correcting housekeeping gene
b-actin, which served as an internal control.
Statistical analysis
All statistical analyses were performed with SPSS 10.0
statistical package for Microsoft Windows. Data were
expressed as mean SD for all measurements. P < 0.05
was considered statistically significant.
RESULTS
C2-ceramide induced morphology changes of apoptosis in
HT-29 cells
With increasing concentration of C 2 -ceramide and
exposure time, HT-29 cells became round, atrophic
and poorly adherent and floating cells increased under
light microscope. Cells from control showed normal
distribution and morphology in all cellular organelles
except for a few necrotic cells under transmission electron
microscope. The main ultra-microstructural changes
seen in all treated groups were chromatin aggregation,
mitochondrial denaturation and apoptotic body, as well as
cytoplasmic compartments, swelling and disappearance of
mitochondrial cristae, etc (Figure 1). The necrotic changes
were more pronounced in cells treated with 50 mmol/L C2-
ceramide.
Apoptotic cells determined by Hoechest 33258 assay
increased in a time- and dose-dependent manner after
treatment with C 2-ceramide (Table 2). Normal cellular
chromatin did not change and uniformly spread over the
whole nuclei displaying diffusion uniformity fluorescence.
However, apoptotic chromatin was identifiable by its
scattered drop-like structure locating on the area of the
original nuclei which displayed high brightness lump or
punctiform fluorescence. The total size of apoptotic nuclei
 Zhang XF et al . Apoptosis of HT-29 cells 3583

Figure 1 Morphology change of apoptosis

A B C in HT-29 cells induced by C 2-ceramide at
different concentrations for different time
under transmission electron microscope. A:
0 mmol/L, 24 h, 5000; B: 50 mmol/L, 24 h,
4000; C: 50 mmol/L, 48 h, 3000.

Figure 2 Morphology change of

A B C apoptosis in HT-29 cells induced
b y C 2 - c e r a m i d e a t d i ff e r e n t
concentrations for different time
under fluorescence microscope.
A: 0 mmol/L, 24 h, 100; B: 50
mmol/L, 12 h, 100; C: 50 mmol/L,
24 h, 100).

Table 3 C2-ceramide damaged mitochondria function of HT-29 cells (mean SD)

C2-ceramide 1 h 3 h 6 h 9 h 12 h 24 h
(mmol/L)
0 0.505 0.061 0.596 0.050 0.528 0.083 1.088 0.134 1.371 0.066 1.770 0.161
12.5 0.478 0.044 0.609 0.048 0.502 0.029 0.833 0.149a 1.036 0.122 1.560 0.220
25 0.487 0.051 0.568 0.048 0.404 0.033a 0.646 0.158a 0.760 0.086a 1.484 0.346a
a a a
50 0.490 0.054 0.541 0.049 0.345 0.050 0.456 0.071 0.708 0.073 1.342 0.061a

a
P < 0.05 vs control.

Figure 3 DNA fragmentation Maker C L M H Maker C L M H

A B induced by C 2-ceramide in
HT-29 cells for 12 h (A) and Bcl-2
Bcl-xl
24 h (B). 1: 50 mmol/L; 2: 25
mmol/L; 3: 12.5 mmol/L; 4: 0
mmol/L.
1 2 3 4 1 2 3 4 Bad Bid

Bax b-Actin

Figure 4 Expression of apoptosis-related genes in HT-29 cells induced by C2-

ceramide for 24 h (C: 0 mmol/L; L: 12.5 mmol/L; M: 25 mmol/L; H: 50 mmol/L).
appeared smaller and more shrunken than the intact cells
(Figure 2).

C2-ceramide induced DNA fragmentation of HT-29 cells
DNA ladder was seen through DNA ag arose g el
electrophoresis, especially pronounced in the cells treated
with 50 and 25 mmol/L C2-ceramide for 12 and 24 h. Cells
treated with 12.5 mmol/L C2-ceramide and DMSO kept
their integrity, having no ladder appearance (Figure 3).
C2-ceramide damaged mitochondrial function of HT-29
cells
The absorbance value increased gradually with increased
concentration of C 2 -ceramide and prolongation of
exposure time. But the absorbance value after treatment
with 50 and 25 mmol/L C2-ceramide was obviously lower
than that in the control, showing statistical significance
from 6 to 24 h, indicating that C2-ceramide could decrease
mitochondrial function (Table 3).
C 2-ceramide affected mRNA expression of apoptosis-
related genes
After cells were treated with C2-ceramide for 24 h, the
mRNA expressions of Bax, Bad and Bid genes were up-
regulated. However, the expressions of Bcl-2 and Bcl-
xl were down-regulated (Figure 4). Moreover, the ratio
of Bcl-2 to Bax in 50 mmol/L C2-ceramid group was less
than 1.
DISCUSSION
Recently, the importance of ceramide in cell metabolism
has been broadly investigated. The biological effect of
ceramide on different cell lines is different. The role
of ceramide-induced apoptosis has been confirmed [4].
Recent studies indicate that ceramide is a common second

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 3584 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
messenger molecule of apoptosis. Apoptosis induced
by stimulating factors is mediated by ceramide from
sphingomyelin circulation way[5,6]. The effect of ceramide
on apoptosis has been studied extensively in neoplastic
cells but rarely in solid tumor cells. Ceramide is closely
related with genesis and progression of digestive tract
tumor. Decreased ceramide content increases the risk of
developing digestive tract tumor since ceramide content in
human colon carcinoma cells is lower than that in normal
colon mucosa cells[7].
To some extent, tumor chemotherapy is to induce
apoptosis. In this experiment, human colon cancer cells
were exposed to exogenous C2-ceramide. Results indicated
that C 2-ceramide could induce typical characteristics
of apoptosis, such as nuclear chromatin break and
apoptotic body as well as DNA ladder in a time-and dose-
dependent manner. Succinate dehydrogenase (SDH) in
the mitochondria is an index of cellular respiration and
energy, which reflects mitochondrial function. MTT assay
suggested that C2-ceramide could decrease mitochondrial
function. By studying Bcl-2 family gene members, we also
found that C2-ceramide could up-regulate or down-regulate
the mRNA expression of these genes, suggesting that
exogenous C2-ceramide induces apoptosis of human colon
carcinoma cells in vitro by affecting the expression of Bcl-2
family gene members and damaging the mitochondrial
functions.
C2-, C6 and C8-ceramides could induce apoptosis of cell
lines, but dihydroxy-ceramide lacking C4-C5 trans-double
bond located in basal framework of sphingolipid could
not induce apoptosis[8].
Studies also indicate that ceramide has many target sites
inducing apoptosis, such as ceramide-activated protein
kinase (CAPK)[9], ceramide-activated protein phosphatase
(CAPP) [10] , protein kinase C (PKC) family [11] , stress-
activated protein kinase/c-JUN N-terminal kinase (SAPK/
JNK)[12] and caspase cascade reaction[13]. There is a body
of evidence that mitochondria are involved in apoptosis,
for example, release of cytochrome C from mitochondria
triggers apoptosis[14-16].
In conclusion, mitochondria is the target of ceramide.
The importance and mechanism of mitochondria in
ceramide-induced apoptosis need to be further studied.
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June 14, 2006 Volume 12 Number 22
References
1 Perry DK, Hannun YA. The role of ceramide in cell signaling.
Biochim Biophys Acta 1998; 1436: 233-243
2 Hannun YA. The sphingomyelin cycle and the second mes-
senger function of ceramide. J Biol Chem 1994; 269: 3125-3128
3 Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks
Z, Kolesnick R. Ceramide synthase mediates daunorubicin-
induced apoptosis: an alternative mechanism for generating
death signals. Cell 1995; 82: 405-414
4 Obeid LM, Linardic CM, Karolak LA, Hannun YA. Pro-
grammed cell death induced by ceramide. Science 1993; 259:
1769-1771
5 Haimovitz-Friedman A, Kolesnick RN, Fuks Z. Ceramide sig-
naling in apoptosis. Br Med Bull 1997; 53: 539-553
6 Kolesnick RN, Haimovitz-Friedman A, Fuks Z. The sphingo-
myelin signal transduction pathway mediates apoptosis for
tumor necrosis factor, Fas, and ionizing radiation. Biochem Cell
Biol 1994; 72: 471-474
7 Selzner M, Bielawska A, Morse MA, Rdiger HA, Sindram
D, Hannun YA, Clavien PA. Induction of apoptotic cell death
and prevention of tumor growth by ceramide analogues in
metastatic human colon cancer. Cancer Res 2001; 61: 1233-1240
8 Ahn EH, Schroeder JJ. Sphingoid bases and ceramide induce
apoptosis in HT-29 and HCT-116 human colon cancer cells.
Exp Biol Med (Maywood) 2002; 227: 345-353
9 Stoica BA, Movsesyan VA, Knoblach SM, Faden AI. Ceramide
induces neuronal apoptosis through mitogen-activated protein
kinases and causes release of multiple mitochondrial proteins.
Mol Cell Neurosci 2005; 29: 355-371
10 Fishbein JD, Dobrowsky RT, Bielawska A, Garrett S, Han-
nun YA. Ceramide-mediated growth inhibition and CAPP are
conserved in Saccharomyces cerevisiae. J Biol Chem 1993; 268:
9255-9261
11 Signorelli P, Luberto C, Hannun YA. Ceramide inhibition of
NF-kappaB activation involves reverse translocation of classi-
cal protein kinase C (PKC) isoenzymes: requirement for kinase
activity and carboxyl-terminal phosphorylation of PKC for the
ceramide response. FASEB J 2001; 15: 2401-2414
12 Kurinna SM, Tsao CC, Nica AF, Jiffar T, Ruvolo PP. Ceramide
promotes apoptosis in lung cancer-derived A549 cells by a
mechanism involving c-Jun NH2-terminal kinase. Cancer Res
2004; 64: 7852-7856
13 Zhao S, Yang YN, Song JG. Ceramide induces caspase-depen-
dent and -independent apoptosis in A-431 cells. J Cell Physiol
2004; 199: 47-56
14 van Gurp M, Festjens N, van Loo G, Saelens X, Vandenabeele P.
Mitochondrial intermembrane proteins in cell death. Biochem
Biophys Res Commun 2003; 304: 487-497
15 Jiang X, Wang X. Cytochrome C-mediated apoptosis. Annu
Rev Biochem 2004; 73: 87-106
16 Li JM, Zhou H, Cai Q, Xiao GX. Role of mitochondrial dys-
function in hydrogen peroxide-induced apoptosis of intestinal
epithelial cells. World J Gastroenterol 2003; 9: 562-567
S- Editor Wang J L- Editor Wang XL E- Editor Bai SH
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Gene expression profiling defined pathways correlated with
fibroblast cell proliferation induced by Opisthorchis viverrini
excretory/secretory product
Chanitra Thuwajit, Peti Thuwajit, Kazuhiko Uchida, Daoyot Daorueang, Sasithorn Kaewkes, Sopit Wongkham,
Masanao Miwa
Chanitra Thuwajit, Peti Thuwajit, Daoyot Daorueang, Sopit
Wongkham, Department of Biochemistry, Liver Fluke and
Cholangiocarcinoma Research Center, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand
Sasithorn Kaewkes, Department of Parasitology, Liver Fluke and
Cholangiocarcinoma Research Center, Faculty of Medicine, Khon
Kaen University, Khon Kaen 40002, Thailand
Kazuhiko Uchida, Masanao Miwa, Institute of Basic Medical
Science, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
S u p p o r t e d b y the Thailand Research Fund, Grant No.
TRG4880004 and the Grants of Khon Kaen University 2004 and
2006; Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture of Japan; the Grant for
Student of Liver Fluke and Cholangiocarcinoma Research Center,
Faculty of Medicine, Khon Kaen University, 2003-2005
Co-first authors: Peti Thuwajit
Correspondence to: Chanitra Thuwajit, PhD, MD, Department
of Biochemistry, Faculty of Medicine, Khon Kaen University,
Khon Kaen 40002, Thailand. tchani@kku.ac.th
Telephone: +66-43-348386 Fax: +66-43-348386
Received: 2006-02-01 Accepted: 2006-03-01
Abstract
AIM: To investigate the mechanism of fibroblast cell
proliferation stimulated by the Opisthorchis viverrini
excretory/secretory (ES) product.
METHODS: NIH-3T3, mouse fibroblast cells were
treated with O. viverrini ES product by non-contact
co-cultured with the adult parasites. Total RNA from
NIH-3T3 treated and untreated with O. viverrini was
extracted, reverse transcribed and hybridized with the
mouse 15K complementary DNA (cDNA) array. The result
was analyzed by ArrayVision version 5 and GeneSpring
version 5 softwares. After normalization, the ratios of
gene expression of parasite treated to untreated NIH-
3T3 cells of 2-and more-fold upregulated was defined as
the differentially expressed genes. The expression levels
of the signal transduction genes were validated by semi-
quantitative SYBR-based real-time RT-PCR.
RESULTS: Among a total of 15000 genes/ESTs, 239
genes with established cell proliferation-related function
were 2 fold-and more-up-regulated by O. viverrini ES
product compared to those in cells without exposure
to the parasitic product. These genes were classified
into groups including energy and metabolism, signal
World J Gastroenterol 2006 June 14; 12(22): 3585-3592
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
BASIC RESEARCH
transduction, protein synthesis and translation, matrix
and structural protein, transcription control, cell cycle
and DNA replication. Moreover, the expressions of serine-
threonine kinase receptor, receptor tyrosine kinase and
collagen production-related genes were up-regulated by
O. viverrini ES product. The expression level of signal
transduction genes; pk C, pdgfra, jak 1, eps 8, tgfb 1i 4,
strap and h ras measured by real-time RT-PCR confirmed
their expression levels to those obtained from cDNA
array. However, only the up-regulated expression of pkC,
eps 8 and tgfb 1i 4 which are the downstream signaling
molecules of either epidermal growth factor (EGF) or
transforming growth factor-b (TGF-b) showed statistical
significance (P < 0.05).
CONCLUSION: O. viverrini ES product stimulates
the significant changes of gene expression in several
functional categories and these mainly include transcripts
related to cell proliferation. The TGF-b and EGF signal
transduction pathways are indicated as the possible
pathways of O. viverrini -driven cell proliferation.
2006 The WJG Press. All rights reserved.
Key words: Gene expression profile; Opisthorchis vi-
verrini ; Excretory/secretory product; cDNA array;
Fibroblast; Cell proliferation; Signal transduction;
Cholangiocarcinogenesis
Thuwajit C, Thuwajit P, Uchida K, Daorueang D, Kaewkes
S, Wongkham S, Miwa M. Gene expression profiling defined
pathways correlated with fibroblast cell proliferation induced
by Opisthorchis viverrini excretory/secretory product. World
J Gastroenterol 2006; 12(22): 3585-3592
http://www.wjgnet.com/1007-9327/12/3585.asp
INTRODUCTION
An Opisthorchis viverrini infection or opisthorchiasis is
the most important health problem in Southern Asia,
including Northeastern Thailand, Loas, Vietnam and
southern China [1] . It is a definite cause of bile duct
cancer or cholangiocarcinoma (CC) in humans and has
been classified as a carcinogen. Parkin et al[2] performed
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 3586 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
a case-control study of patients and estimated that two-
thirds of CC cases in Thailand were caused by O. viverrini
infestation. Nowadays, CC remains a major public health
problem in many parts of Southeast Asia. The incidence
rate of CC around the world is different with increasing
tendency[3,4]. The highest incidence is in the Northeastern
part of Thailand with the rate of 188 per 100000 [5].
The pathogenesis of O. viverrini-associated CC may be a
complex process, involving several mechanisms. The effect
of parasites on the bile duct epithelium is classified, as
both mechanical and chemical irritations. The mechanical
irritation is the direct contact of parasites to the bile duct
epithelium; the chemical irritation is believed to be caused
by the excretory/secretory (ES) product released from the
flukes. These chronic irritations caused by the parasite later
result in the proliferation of bile duct epithelium and leads
to epithelial hyperplasia[6]. This is an important step in the
genesis of cancer because hyperplastic cells are vulnerable
to a carcinogen and can easily turn to adenomatous and
finally cancerous cells [7]. Moreover, the alterations of
immune response and the stromal cells which are mainly
composed of fibroblasts were observed in opisthorchiasis
and CC in both animal models and human. The fibrosis
and mononuclear cell infiltration with lymphocyte
aggregation and, additionally, ductal dilatation were
observed in the gall bladders and extrahepatic bile ducts
of the hamsters infected with O. viverrini[8]. In addition,
the chronic inflammation and fibrosis of the bile ducts
may contribute to the strikingly enhanced susceptibility
to CC among people with heavy liver fluke infection[9].
The malignant transformation of bile duct epithelium
via dysplasia was detected in the abnormal intrahepatic
biliary tree of the patients with congenital hepatic
fibrosis[10]. There is the important relationship between
the gradual decreases of inflammation with a concomitant
increase in fibrosis after O. viverrini re-infection in the
experiment hamsters[11]. All of these data provide evidence
of the importance of fibrosis in the for mation of
cholangiocarcinoma.
Fibrosis is the excessive accumulation of extracellular
matrix proteins produced from the active and accumulated
fibroblasts. It occurs in most types of chronic diseases
including chronic liver disease. Liver fibrosis is considered
a model of the wound-healing response of the liver to
repeated injury[12]. Activated hepatic stellate cells, portal
fibroblasts, and myofibroblasts have been identified as the
major collagen-producing cells in the repeatedly injured
liver[13]. In the fibrogenesis, the increased numbers and
then the accumulation of fibrogenic cells including the
fibroblasts and myofibroblasts are the main observations.
As aforementioned, there is the correlation of fibrosis
with the development of CC. The interaction of O. viverrini
and the host immune cells has been proven to be the main
causative issue for chronic inflammation in opisthorchiasis.
The cytokines released from the immune cells will lead to
the formation of fibrosis later. Moreover, direct contact of
O. viverrini which have been strongly proven to induce bile
duct epithelium erosion and cell proliferation replacement
that occurs later on, may indirectly induce the surrounding
fibroblasts around the affected area to proliferate[14]. The
importance of hyperproliferative fibroblasts regarding
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June 14, 2006 Volume 12 Number 22
the induction of epithelial cancer has been increasingly
reported [15,16]. Though this phenomenon has not been
investigated in CC, the direct effect of O. viverrini ES
product to induce fibroblast cell proliferation has recently
been reported in vitro[14,17]. So far, there are no data for the
explanation of the fibroblast cell proliferation induced by
the O. viverrini ES product.
From all the evidence described above, it would appear
to be of great interest to study the mechanism of how O.
viverrini ES product induces fibroblast cell proliferation.
In order to know the response of cell to O. viverrini
ES product, the gene expression analysis of fibroblast
cell non-contact co-cultured with adult O. viverrini was
performed. The expression level of genes was compared
to those without O. viverrini ES product treatment. The cell
proliferation-associated gene expression was discussed in
relation to their roles in O. viverrini ES product-induced
fibroblast cell proliferation. The expression levels of
some signal transduction genes were also analyzed and
validated by real-time RT-PCR to indicate the possible
signal transduction pathway(s) utilized by O. viverrini in the
induction of fibroblast cell proliferation.
MATERIALS AND METHODS
Parasite preparation
Opisthorchis metacercariae were obtained from naturally
infected cyprinoid fish captured from fresh-water
reservoirs in the endemic area of Khon Kaen province,
Thailand. Pepsin-HCl was used to digest the flesh to
obtain metacercariae, which were then introduced to the
6-8-wk-old hamsters ad libitum. After 1 mo, the adult O.
viverrini were collected from the bile ducts of the hamsters
and were washed several times with PBS containing
100 mg/L penicillin and 100 kU/L streptomycin. To
prevent microbial contamination, the adult O. viverrini
were then incubated for 30 min in PBS containing
antibiotics before being used in the non-contact co-
culture technique. The animal holding protocol was
approved by the Animal Ethics Committee of Khon Kaen
University (AEKKU011/04), based on the Ethics of
Animal Experimentation of National Research Council of
Thailand.
Cell culture and non-contact co-culture
To prepare the fibroblasts stimulated with O. viverrini
ES product, the non-contact co-culture between the
fibroblasts and the adult parasites was performed. The
mouse fibroblast cell line, NIH-3T3, was bought from the
American Type Culture Collection (ATCC; Manassas, VA,
USA). It was maintained in DMEM (Gibco, Gaithersburg,
MD, USA) containing 100 mL/L calf serum (CS) (Gibco,
Gaithersburg, MD, USA), designated as the complete
medium, followed by incubation in a 50 mL/L CO 2
incubator at 37 and passaging twice a week. Trypan
blue staining was used to measure the viability of cells.
The cells with more than 95% viability were collected for
performing non-contact co-culture.
The NIH-3T3 cells and the intact viable O. viverrini
were non-contact co-cultured in a 24-well transwell (Costar,
Corning Incorporated, Cambridge, NY, USA). The
 Thuwajit C et al . Gene expression profile induced by O. viverrini 3587
parasites were incubated in the upper chamber containing
the 8-mm porous plate, which allowed the ES product to
diffuse to the lower chamber where the NIH-3T3 cells
adhered. The cell proliferation induction of ES product to
the NIH-3T3 cells was confirmed by the viable cell count
using trypan blue staining. Briefly, 10000 NIH-3T3 cells
were plated onto the lower chamber of a 24-well double-
chamber culture plate for 2 d with complete medium to
let most of cells adhere to the well. Then the media were
changed to DMEM without CS, serum-free medium. Five
viable adult O. viverrini were added to the upper chamber
of each well. The NIH-3T3 cells cultured in complete
medium and serum-free medium were used as positive
and negative controls, respectively. The cell numbers
obtained from the O. viverrini treatment cultured for 2 d was
compared to those of the negative and positive controls
with the same culturing time.
Hybridization and array data analysis
The in-house prepared mouse cDNA array was prepared
by Dr. Kazuhiko Uchida, Department of Biochemistry
and Molecular Oncology, Comprehensive Human Science,
Graduate School, University of Tsukuba, Japan. It consists
of 15 K of either mouse or human genes/ESTs. Firstly,
the total RNA extraction from NIH-3T3 cells treated and
untreated with adult O. viverrini ES product was performed.
Briefly, total of 2-5 106 cells were collected in RNAlater
solution (Ambion , Hilden, Germany) and processed
for total RNA with a commercial kit (ISOGEN; Nippon
Gene, Tokyo, Japan) according to the manufacturers
protocol. The RNA quality was confirmed by subjecting
the samples to electrophoresis on for maldehyde-
agarose gel and staining the 18S and 28S RNA bands for
visualization. Then, a 2-mg portion of the intact total RNA
was dissolved in 8 mL of RNase-free water, and 2 mL of
oligo (dT) primer (1 g/L; Invitrogen, Carlsbad, CA, USA)
was then added. The sample was heated for 10 min at 70
and immediately chilled on ice. The following reagents
were then added to the sample: 5 m L of first-strand
cDNA synthesis buffer, 1 mL of DTT (0.1 mol/L final
concentration), 1.5 mL of deoxynucleotide triphosphates
(20 mmol/L dATP, dGTP, and dTTP), 1.5 m L of
SuperScript (200 MU/L; Invitrogen), and [a-33P]dCTP (111
PBq/mol; Amersham). The reaction was incubated at 37
for 90 min. Labeled cDNA was then denatured for 3 min
at 95. The array was prehybridized at 42 for 2 h in
MicroHyb solution (Invitrogen, Carlsbad, CA, USA) with
0.5 mg/L poly(dA) and 1 mg/L COT1 DNA (Invitrogen,
Carlsbad, CA, USA). The probes were hybridized at 42
overnight in the same solution. The membrane array was
washed twice at 50 with 2 SSC-10 g/L SDS for 20
min and at room temperature with 0.5 SSC-10 g/L
SDS for 15 min. The membrane array was exposed to
an imaging plate which was scanned with a BAS 5000
Imaging Analyzer (Fuji Film, Tokyo, Japan). Spot intensity
was quantified with ArrayVision version 5.0 software
(Imaging Research, Ontario, Canada). GeneSpring version
5.0 software (Silicon Genetics, Redwood, CA) was used to
normalize values for each gene for data analysis.
Real-time RT-PCR
The mRNA was isolated using a Total RNA Extraction
Minipre p System (Viog ene, Feg ersheim, France)
according to the manufacturers instructions. The DNA
contaminated in the extracted RNA was destroyed by
DNaseI (Promega Corporation, Madison, WI, USA) at
37 for 30 min. The DNaseI inactivation reaction was
performed at 75 for 10 min. The first-strand cDNA
was synthesized from 1 mg of mRNA using the Reverse
Transcription System (Promega Corporation, Madison,
WI, USA). Semi-quantitative PCR was performed with
the SYBR-based method in 12.5 m L reaction in Rotor
Gene RG-3000 (Corbett Research). After the denaturing
of cDNA at 95 for 10 min, the reaction profile was
subjected to 50 amplification cycles, each cycle consisted
of denaturation at 95 for 30 s, annealing at different
temperatures for each gene (52, 55, 58, 58, 54, 52, 52, 52,
65, and 60 for abl 1, pkC, pdgfr , jak 1, eps 8, tgfb 1i4,
strap, csnk 1a1, h ras, and b 2m, respectively) for 30 s, and
extension at 72 for 45 s. After the PCR, a melting curve
was constructed by increasing the temperature from 50
to 99. b2-microglobulin (b2m), whose expression was
found to be directly proportional to the amount of mRNA
that presented in the sample stimulated by any growth
factors[18], was used as the internal control. The standard
curve between the CT of each gene expression and the
amount of the starting total RNA was performed. The CT
of the samples was then compared to the corresponding
standard curve and quantitated the amount of the starting
mRNA in that sample. The RT-PCR was performed three
times for each gene.
Table 1 shows the PCR primers designed by the Gene
Fisher Program according to the mRNA sequences of
each gene in the GenBank database. The primer sequences
can then be categorized into groups of growth factors they
belong to, in this case, the signal transduction molecules.
The result was analyzed for the statistical significance by
STATA version 8 (StataCorp LP, Texas, USA).
RESULTS
Gene responding to O. viverrini ES product
Using a mouse cDNA array, we examined the gene
expression profile of cell proliferation stimulated by O.
viverrini ES product. A total of 15000 genes and ESTs
were tested. O. viverrini caused widespread alteration in
gene expression. The ratios of gene expression between
treated and untreated with the parasite ES product
equal and greater than 2 were focused on because of
their striking changes. Among all genes/ESTs in 15K
cDNA array, 885 genes fitted to this criterion. Among
these genes, only 536 genes had a variety of molecular
functions and only 239 genes within these 536 genes had
cell proliferation-related functions. These 239 genes were
categorized in groups as proteins playing roles in energy
and metabolism, signal transduction, protein synthesis and
translation, matrix and structural protein, transcription
control, cell cycle and DNA replication. The percentage
distribution of genes in each group is 25.2, 21, 18.9, 17,
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 3588 ISSN 1007-9327 CN 14-1219/ R
1
Table 1 List of signal transduction genes validated the
expression by real-time RT-PCR, their corresponding PCR
2
primers , annealing temperature and product sizes
Group of
stimulating Gene Primer sequence temperature ()/
growth product size (bp)
factor
PDGF Forward: 5-TGAAGTTGGTGGGCTGCA-3
abl 1
/EGF Reverse: 5-TTTTCACTGGGCCCGCA-3
pkC
Forward: 5-GGCATGCCTTGTCCTGGAGA-3
Reverse: 5-TGTAGCCCTGCCTCGAGAGA-3
Forward: 5-CAGCCTCGCTCGTCCTT-3
jak 1
Reverse: 5-CCAAGCCCTTCAGAGCT-3
Forward: 5-TTTCCCGGATGCCTGCTA-3
raf 1
Reverse: 5-TCCTGCTGTCCCATGCA-3
h ras
Forward: 5-TGGTGGGCAACAAGTGTGA-3
Reverse: 5-CCGCAATTTATGCTGCCGAA
PDGF Forward: 5-TGCGGGGAAGGACTGGA-3
pdgfra
Reverse: 5-GTGAGGAGACAGCTGAGGA-3
EGF Forward: 5-CCGCTCCGTGGGTATGGA-3
eps 8
Reverse: 5-ACGTCCGACACACTGCTGA-3
TGF-b
tgfb 1i4
Forward: 5-GCAAGTGTGGTAGCTATCGA-3
Reverse: 5-CAGATTGTTCTCCTGCTCC-3
Forward: 5-GCCGGGAGATACAGGAGA-3
strap
Reverse: 5-TGCTTATGAGCCGGGTCA-3
1
They are classified based on their functions as the downstream signaling
molecules of the specific growth factor. 2 Primers for the internal
control (b 2 m) are: 5-CATGGCTCGCTCGGTGA-3 (forward) and
5-AATGTGAGGCGGGTGGAA-3 (reverse). The annealing temperature
and the product size of RT-PCR for b2m are 60 and 148 bp, respectively.
PDGF: platelet-derived growth factor; EGF: epidermal growth factor; TGF-b:
transforming growth factor-b.
12.6, 3.3, and 2, respectively. The examples of genes in
each group are summarized in Table 2.
Response of signal transduction-related genes to O.
viverrini ES product
A total of 59 genes/ESTs categorized by their molecular
function as the signal transduction genes showed 2-fold
and more up-regulation after O. viverrini ES product
stimulation. They are the downstream signal transduction
molecules for a variety of growth factors. The signal
transduction genes that corresponded to the stimulation
of either PDGF or EGF, PDGF, EGF and TGF-b were
categorized and summarized in Table 3. The most up-
regulated expressions were tgfb 1i4 and abl 1 which are the
representatives of TGF-b- and either PDGF- or EGF-
stimulated signal transduction pathways, respectively.
In addition to the different signal transduction
molecules responded to different growth factors, the
receptors for PDGF/EGF and TFF-b are classified in
different types as tyrosine kinase and serine-threonine
kinase receptors, respectively. The cDNA array data
represented the different up-regulated expression levels
between these 2 types of receptors (Table 4).
Nine signal transduction genes, which responded to
the specific growth factor as mentioned in Table 3, were
selected and tested for their expression levels by semi-
quantitative RT-PCR. The single peak of the melting curve
for each gene confirmed the appropriate PCR condition
(data not shown). The result indicated similar responses
in both cDNA array and RT-PCR analyses of pkC, jak 1, h
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World J Gastroenterol June 14, 2006 Volume 12 Number 22
ras, pdgfr , eps 8, tgfb 1i4, and strap. However, the significant
(P < 0.05) up-regulation was only found for those of
pkC, eps 8, and tgfb 1i4. The opposite result of mRNA
expression level was detected in abl 1 and raf 1 (Figure 1).
Annealing
DISCUSSION
Cell proliferation is regulated by the intricacy of cell
52/148
growth regulators, signaling cascades, and mediators of
55/149
cell cycle progression including cyclins, cyclin-dependent
kinases (cdks). Anti-proliferative genes coding for the
58/147 apoptotic protein, cyclin/CDK inhibitors are important in
controlling cell proliferation. Addition of growth factors
62/147
induces cell proliferation including the fibroblasts [19].
65/150 The changes in gene expression that accompany this
proliferative response have been the subject of many
58/122 studies, and the responses of dozens of genes have been
characterized[19]. A recent study indicated the effect of
54/146
O. viverrini ES product in induction of fibroblast cell
52/136 proliferation in vitro[17]. We have examined the effect of
this parasitic product in the gene expression profile using a
52/153 cDNA array. The NIH-3T3 cells were used because it not
only had the marked response to O. viverrini ES product[17],
but also being classified as the mesenchymal cells as the
real target of O. viverrini ES product; the stellate cells and
myofibroblasts. Among all genes/ESTs in 15K cDNA
array, 885 genes showed 2-fold and more up-regulation
after stimulation by O. viver rini ES product. Among
these genes, only 536 genes had a variety of established
molecular functions. Moreover, 239 of these 536 genes
had cell proliferation-related functions and were primarily
focused into groups based on their specific functions
because of their striking increased changes ( 2-fold).
Most of the genes which increased their expression
by O. viverrini ES product stimulation were the proteins
controlling the enzymatic metabolism and biosynthesis.
T he expressions of glyceraldehyde-3-phosphate
dehydrogenase, alpha-enolase and aldehyde dehydrogenase
involve the glycolysis pathway. Cytochrome C oxidase
and ATP synthase involve energy production from the
electron transport chain. These phenomena could be easily
understood because high levels of energy production and
synthesis of biological macromolecules are required for
the stimulation of quiescent fibroblasts by O. viverrini ES
product into the cell cycle progression. These data suggest
the possibility that ES product acts like a growth factor to
stimulate cell proliferation basically by the stimulation of
energy production-related gene expression[20].
In cell proliferation, the DNA replication is needed.
Many proteins, including RPII215 polymerase and
topoisomerase II, required for chromosome segregation at
mitosis; and DNA primase, required for DNA replication,
all increased their expressions in O. viverrini ES product-
treated cells. These increased expressions will help cells
undergo into cell cycle and consequently cell division.
Ribonucleotide reductase is associated with the anabolic
pathway of deoxynucleotide. The increased expression
of this enzyme causes an increase in the deoxynucleotide
triphosphate (dNTP) pool in the nucleus, which facilitates
DNA synthesis and repair[21]. In addition, when cells are
activated by the growth factors and then commit to enter
 Thuwajit C et al . Gene expression profile induced by O. viverrini 3589

Microarray
9 Real-time RT-PCR
Table 3 Up-regulation of signal transduction-related genes
in NIH-3T3 treated with O. viverrini , categorized in groups
a according to the response to the specific growth factor
Folding of expression

Related growth factor Gene name/production Folding

5 a
(Accession number)
PDGF/EGF
3 AW544655 Tyrosine kinase (v abl) 7.12
a C80388 Protein kinase C (pkC) 4.23
1 AU042564 Casein kinase I-alpha isoform (csnk 1a 1)1 3.81
C87299 Casein kinase I-epsilon (csnk 1e ) 2.88
abl 1 pkC jak 1 h ras raf 1 pdgfra eps 8 tgfb 1i 4 strap AU046274 Ras-GTPase activating protein SH3-domain 2.73
PDGF/EGF PDGF EGF TFG-b binding protein (c3bp-pending)
C87788 JAK1 protein tyrosine kinase 2.56
AU017366 H ras 2.26
Figure 1 RT-PCR analysis of signal transduction genes up-regulation by AW552623 N ras 2.01
O. viverrini ES product. The results of DNA array and RT-PCR are shown in
comparison for each gene (mean SD, aP < 0.05). PDGF
AW537708 PDGF receptor, alpha-polypeptide (pdgfr a ) 2.64

EGF
1 C88280 Epidermal growth factor receptor 2.32
Table 2 Proliferation-related genes with 2-fold and more
pathway substrate (eps 8)
up-regulation in fibroblast cell proliferation activated by O.
viverrini ES product compared to the negative control (not
TGF-b
exhaustive)
AW546174 Transforming growth factor beta-1-induced 8.24
transcript 4 (tgfb 1i4)
A Energy and metabolism Glyceraldehyde-3-phosphate
dehydrogenase (gapd), cyto- AU016757 c myc 3.70
chrome C oxidase, alpha- C79202 Serine/threonine kinase receptor-associated 3.40
enolase, ATP synthase beta sub- protein (strap)
u n i t 2, t h i o r e d o x i n , a l d e h y d e
1
dehydrogenase Represents human gene.
B Signal transduction Transforming growth factor beta-
1-induced transcript 4 (tgfb 1i4),
tyrosine kinase (abl 1), interleukin
1 receptor-associated kinase, MAP Table 4 cDNA array analysis showing up-regulation of genes
kinase phosphatase (mkp 6), colony encoded for the kinase receptors
stimulating factor 3 receptor,
protein kinase C, Janus kinase 2 (Jak Receptor kinases Gene names (accession number) Folding
2), casein kinase I alpha (csnk 1a1), Serine-threonine kinase Serine/threonine kinase 11 4.42
c-myc (C85710)
C Protein synthesis and translation Polyubiquitin C (Ubc) gene, Serine/threonine kinase 19 3.28
ribosomal protein S27a2, ribosomal (AW557191)
protein L27, elongation factor Tu, Serine/threonine kinase 4 2.88
RNA polymerase 1-2, translation (AU020804)
initiation factor 4 gamma2
D Matrix and structural protein Lysophospholipase 1 (lypla 1) Tyrosine kinase TYRO3 protein tyrosine kinase 2.60
E Transcription control Transformation/transcription 3 (AW556118)
domain-associated protein (trrap)2, JAK1 protein tyrosine kinase 2.56
telomeric repeat binding factor (C87788)
1 (terf 1), transcription factor EB Protein tyrosine kinase 9 1.83
(tcfeb), DEAD-box RNA helicase (AW544421)
(ddx 21)2 Downstream of tyrosine kinase 1.59
F Cell cycle Kinesin family member 3a (kif 3a), 1 (AW557123)
mitotic kinesin-linked protein 12,
cyclin-dependent kinase 4 (cdk 4),
cdk 5, cyclin B1
G DNA replication RPII215 polymerase II large sub-
later ones. This is the action of protein classified as the
unit, topoisomerase II alpha, DNA
primase p58 subunit (prim 2), transcription factor. In cells treated with O. viverrini ES
ribonucleotide reductase products, a variety of transcription factors are activated.
Cyclins and CDKs are the important proteins in cell
1

2
Sequence in each group is ordered from high to low expression increases; cycle progression[22]. The response of cell to O. viverrini ES
represents human gene. product indicated the increased expression of many CDKs
and cyclins. CDK4 and 5 are important at the G1 phase,
whereas cyclin B1 is crucial in G2 phase. The increased
the cell cycle, the cascade of gene expression within the expression of phosphorylated cyclin D1 was reported
cells is needed. This means it is important that the initial in fibroblast proliferation induced by O. viverrini ES
gene expressed to be protein for the expression of the product[17], thereby supporting the increased expression of

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 3590 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
CDK4 in this study. This result supports the importance
of cyclin D1-CDK4 expression in the re-entry of the
serum-stimulated fibroblasts into the cell-division cycle[23].
Moreover, O. viverrini ES product induced the expression
of kinesins which are a family of microtubles and are
involved in many crucial cellular processes including cell
division[24]. The kinesin family member 3a (kif 3a) and
mitotic kinesin-like protein are crucial for spindle assembly
and function, chromosome segregation, mitotic checkpoint
control, and cytokinesis in higher eukaryotes [25]. Cells
cannot divide without the appropriate functions of these
genes.
Rapid and efficient production of macromolecule
in protein synthesis and degradation of superfluous
proteins play important role in cell cycle progression[26].
The increased RNA polymerase which determines the
efficiency of mRNA production supports the capability of
other genes to increase their expressions. The ribosomal
proteins as the component of ribosomes, translation
initiation factors and elongation factors increased their
expressions which normally occur to promote proteins
synthesized for cell division. In addition, the expression
of polyubiquitin C (ubc) increased to get rid of the
superfluous proteins already synthesized and that have
reached their expired period. The increased expression of
these genes is crucial for the turnover of the appropriate
proteins for the right stimuli at the right time.
Matrix and str uctural proteins are important in
supporting cell proliferation[27]. In this study, some matrix
and structural proteins increased their expression level by
O. viverrini ES product stimulation. Lysophospholipase D
(lysoPLD) catalyses the production of lysophosphatidic
acid (LPA) from the lysophosphatidylcholine (LPC). LPA
has been demonstrated to be a potent inducer of cell
proliferation of multiple cell lineages[28]. Taken together,
all the data presented in this study confirm that O. viverrini
ES product can activate cell proliferation resulting in the
response of gene expression profile like a serum or growth
factor[29].
Array analysis is not only crucial in understanding
the effect of the ES product on cells but also to predict
what should be the growth factor available in O. viverrini
ES product. In this work, we took the advantage of this
technology to decipher the possible genome expression
circuit induced by O. viverrini ES product. The comparison
of O. viverrini ES product-induced gene expression profile
with that of established growth factor profiles may be
useful to know the cell signaling pathway utilized by O.
viverrini ES product in induction of cell proliferation.
The O. viverrini ES product stimulated cell proliferation
like the response of cells to the calf serum [19]. There
are many types of growth factors contained in the calf
serum including platelet-derived growth factor (PDGF),
epidermal growth factor (EGF), transforming growth
factor-b, and insulin-liked growth factor (IGF). Each of
them stimulates a different signal transduction profile
which is the most important feature used to distinguish
the types of growth factor-stimulated cell responses[30,31].
In this study, the signal transduction genes selected from
the array data can be categorized based on their functions
as the downstream signal transduction molecules of both
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June 14, 2006 Volume 12 Number 22
PDGF and EGF (PDGF/EGF), only PDGF, only EGF,
and only TGF-b, according to the previous reports[30,31].
Though PDGF and EGF are different growth factors
using different cell surface receptors, they used, in part, the
same signal transduction pathway[32] including abl 1, pkC,
csnk 1a1, ras and jak 1. The results of this study showed
that O. viverrini ES product stimulated the expression
of these genes. It may be possible that O. viverrini ES
product activated cell proliferation via PDGF/EGF-driven
signaling pathway. Moreover, pdgfra , eps 8, and tgfb 1i4, the
specific genes for PDGF, EGF, and TGF-b, respectively,
were found to be up-regulated as well. Based on the
increased expression, tgfb 1i4 was ranked in the uppermost
expression level. It is supposed that O. viverrini ES product
stimulated cell proliferation via TGF-b signaling pathway
as well.
The difference between signal transduction pathways
activated by TGF-b and PDGF/EGF is mainly focused
on their different types of receptors. The receptors of
TGF-b are membrane-bound receptors exhibiting intrinsic
serine-threonine kinase activities, whereas the tyrosine
kinase receptor is activated by EGF or PDGF[30,31]. From
cDNA array data, the activation of serine-threonine kinase
and tyrosine kinase receptors encoded gene expression
showed the involvement of both types of receptors in the
stimulation by O. viverrini ES product. This evidence still
supports the possibility that TGF-b and EGF/PDGF can
be the candidate signal transduction pathway induced by O.
viverrini ES product.
Since the uncertain mRNA expression level was
detected by cDNA array analysis, RT-PCR was used to
measure the exact expression. From the list of up-regulated
signal transduction-related genes, we selected 9 genes for
additional analysis to validate the array expression data.
The result showed marked increase of tgfb 1i4 expression.
It has been proven that tgfb 1i4 is a direct target of
TGF- b [33,34] and also is the transcriptional modulator
stimulated by TGF-b. The tgfb 1i4 gene expression was
transcriptionally activated by TGF-b, phorbol 12-myristate
13-acetate, and serum but not appreciably by EGF [34].
The up-regulation of tgfb 1i4 expression induced by O.
viverrini ES products may indicate the stimulation of the
TGF-b-activated signal transduction pathway. The normal
expression of strap, the inhibitor of the TGF- b signal
transduction cascade[35] , supports this conclusion.
For the EGF and PDGF-stimulated signal transduction
pathways, the expressions of the common genes, including
abl 1, jak 1, h ras and raf 1, were not significantly increased.
Moreover, the specific gene related to PDGF-stimulated
signal transduction, pdgfra , was expressed at the same level
as in cells without O. viverrini ES product treatment. This
result excludes the potential of O. viverrini ES product in
stimulating cell proliferation through the PDGF-mediated
signal transduction pathway. For the EGF-stimulated
signal transduction pathway, eps 8 is the transcription
factor required in the EGF-stimulated cell proliferation
and is confirmed to be the gene product that represents a
novel substrate for tyrosine kinase receptors[36]. Adoptive
expression of eps 8 cDNA in fibroblastic or hematopoietic
target cells expressing the EGFR resulted in an increased
mitogenic response to EGF, implicating the eps 8 product
 Thuwajit C et al . Gene expression profile induced by O. viverrini 3591
in control of mitogenic signals activated by EGF [36] .
That the O. viverrini ES product markedly stimulated the
expression of this gene represents the possibility that this
parasitic product stimulates cell proliferation via EGF-
mediated signal transduction cascade. There are 4 main
pathways induced by EGF, including the activations of
Ras/Raf/ERK, phosphatidylinositol 3-kinase (PI3K),
phospholipase C-g (PLC-g)/protein kinase C (PKC) and
c jun-N-terminal kinase (JNK). These pathways lead to
different cellular actions[37]. Only the Ras/Raf/ERK and
JNK-transduced signal transduction pathways involve
cell proliferation. In this study, the expression of h ras
and raf 1 was slightly up-regulated by O. viverrini ES
product, indicating O. viverrini ES product stimulates cell
proliferation via other pathways rather than the common
pathway of Ras/Raf/ERK. The up-regulated expression
of pkC represents the possibility of cell proliferation
activated by O. viverrini ES product via the EGF-stimulated
PKC signal transduction pathway. This is opposed to
the data that pkC-mediated signal transduction pathway
stimulated by EGF does not involve in control of cell
proliferation [37]. Moreover, the array data showed the
increased expression of JNK (2.78-fold, data not shown)
represents the possibility that EGF-associated JNK
pathway may be the pathway activated by O. viverrini ES
product.
Taken all together, O. viverrini ES product activates
cell proliferation via either TGF- b - or EGF-mediated
signal transduction pathways. These two signal pathways
have been proved to be strongly cor related with
cancer development [37,38]. TGF- b also mediates tumor-
promoting effects, through differential effects either on
tumor or stromal cells[38]. Its signaling pathway is being
evaluated as a prognostic or predictive marker for cancer
patients. Over-expression of EGF has been found in
many cancer types [37]. The roles of TGF- b and EGF
in cholangiocarcinogenesis need further investigations.
Regardless of the exact signal transduction pathway
stimulated by O. viverrini ES product, the induction of
fibroblast cell proliferation and the accumulation of
extracellular matrix (i.e. collagen) have been proven to be
associated with the fibrogenetic process[13]. The increased
expression of collagen type I, III, and IV (between
2-3-fold increase) was observed in cDNA array analysis
(data not shown). This supports the hypothesis that O.
viverrini ES product can stimulate cell proliferation and
collagen production leading to the fibrosis formation.
Since the fibrosis has been detected in cholangiocarcinoma
in both animal models and human [9-11] , it is possible
that O. viverrini ES product-induced fibrosis may be one
component in cholangiocarcinogenesis. Moreover, it may
be proposed that the ES product-induced fibrosis may act
synergistically with the immunological response against the
parasites to promote CC.
In summar y, this study demonstrates the g ene
expression profile obtained from cDNA array analysis to
determine the possibility of O. viverrini ES product as the
growth factor in induction of fibroblast cell proliferation.
Signal transduction-related genes may have a significant
expression increment in cells exposed to this parasitic
product. Among this set of genes, tgfb 1i4 and eps 8
were found to have the strongest expression levels with
corroboration data obtained by both cDNA array and RT-
PCR. These two genes are the specific signal transduction
molecules for TGF-b- and EGF-stimulated pathways. It
may therefore be proposed that O. viverrini ES product
activated cellular proliferation via the pathways of these
growth factors. The issues of whether one type of growth
factor is capable to cross-stimulate the two pathways
or two types of growth factors are available in the O.
viverrini ES product, needs future experiments to be clearly
elucidated. This report is an important resource to indicate
the mechanism of O. viverrini ES product-stimulated
fibroblast cell proliferation via TGF-b- or EGF-stimulated
signal transduction pathways. Since the activated fibroblast
has been shown to initiate non-tumorigenic epithelium to
carcinoma[16], O. viverrini ES product-stimulated fibroblast
may play a part to change bile duct epithelium into CC.
The ongoing research in understanding the signaling
pathways stimulated by O. viverrini ES product and roles of
fibroblasts in the development of CC will provide a novel
target for chemoprevention and treatment of fibrosis in
this cancer which may delay the formation of CC.
ACKNOWLEDGMENTS
The authors thank Professor James A Will, University of
Wisconsin-Madison, WI, USA for his assistance with the
English-language presentation of the manuscript and also
for the valuable comments and suggestions.
References
1 Sithithaworn P, Haswell-Elkins M. Epidemiology of
Opisthorchis viverrini. Acta Trop 2003; 88: 187-194
2 Parkin DM, Srivatanakul P, Khlat M, Chenvidhya D,
Chotiwan P, Insiripong S, LAbb KA, Wild CP. Liver cancer
in Thailand. I. A case-control study of cholangiocarcinoma. Int
J Cancer 1991; 48: 323-328
3 Khan SA, Taylor-Robinson SD, Toledano MB, Beck A, Elliott P,
Thomas HC. Changing international trends in mortality rates
for liver, biliary and pancreatic tumours. J Hepatol 2002; 37:
806-813
4 Taylor-Robinson SD, Toledano MB, Arora S, Keegan TJ,
Hargreaves S, Beck A, Khan SA, Elliott P, Thomas HC. Increase
in mortality rates from intrahepatic cholangiocarcinoma in
England and Wales 1968-1998. Gut 2001; 48: 816-820
5 Sriamporn S, Pisani P, Pipitgool V, Suwanrungruang K,
Kamsa-ard S, Parkin DM. Prevalence of Opisthorchis viverrini
infection and incidence of cholangiocarcinoma in Khon Kaen,
Northeast Thailand. Trop Med Int Health 2004; 9: 588-594
6 Riganti M, Pungpak S, Punpoowong B, Bunnag D, Harinasuta
T. Human pathology of Opisthorchis viverrini infection: a
comparison of adults and children. Southeast Asian J Trop Med
Public Health 1989; 20: 95-100
7 Watanapa P, Williamson RC. Experimental pancreatic
hyperplasia and neoplasia: effects of dietary and surgical
manipulation. Br J Cancer 1993; 67: 877-884
8 Sripa B, Kaewkes S. Gall bladder and extrahepatic bile duct
changes in Opisthorchis viverrini-infected hamsters. Acta Trop
2002; 83: 29-36
9 Elkins DB, Mairiang E, Sithithaworn P, Mairiang P, Chai-
yakum J, Chamadol N, Loapaiboon V, Haswell-Elkins MR.
Cross-sectional patterns of hepatobiliary abnormalities and
possible precursor conditions of cholangiocarcinoma associ-
ated with Opisthorchis viverrini infection in humans. Am J Trop
Med Hyg 1996; 55: 295-301
www.wjgnet.com
 3592 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
10 Yamato T, Sasaki M, Hoso M, Sakai J, Ohta H, Watanabe Y,
Nakanuma Y. Intrahepatic cholangiocarcinoma arising in
congenital hepatic fibrosis: report of an autopsy case. J Hepatol
1998; 28: 717-722
11 Pinlaor S, Sripa B, Sithithaworn P, Yongvanit P. Hepatobiliary
changes, antibody response, and alteration of liver enzymes in
hamsters re-infected with Opisthorchis viverrini. Exp Parasitol
2004; 108: 32-39
12 Friedman SL. Liver fibrosis -- from bench to bedside. J Hepatol
2003; 38 Suppl 1: S38-S53
13 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:
209-218
14 Sripa B. Pathobiology of opisthorchiasis: an update. Acta Trop
2003; 88: 209-220
15 Micke P, Ostman A. Tumour-stroma interaction: cancer-asso-
ciated fibroblasts as novel targets in anti-cancer therapy? Lung
Cancer 2004; 45 Suppl 2: S163-S175
16 Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD,
Cunha GR. Carcinoma-associated fibroblasts direct tumor pro-
gression of initiated human prostatic epithelium. Cancer Res
1999; 59: 5002-5011
17 Thuwajit C, Thuwajit P, Kaewkes S, Sripa B, Uchida K,
Miwa M, Wongkham S. Increased cell proliferation of mouse
fibroblast NIH-3T3 in vitro induced by excretory/secretory
product(s) from Opisthorchis viverrini. Parasitology 2004; 129:
455-464
18 Schmittgen TD, Zakrajsek BA. Effect of experimental treat-
ment on housekeeping gene expression: validation by real-
time, quantitative RT-PCR. J Biochem Biophys Methods 2000; 46:
69-81
19 Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JC,
Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D,
Shalon D, Botstein D, Brown PO. The transcriptional program
in the response of human fibroblasts to serum. Science 1999;
283: 83-87
20 Ma H, Zhang Z, Tong T. The effects of epidermal growth
factor on gene expression in human fibroblasts. In Vitro Cell
Dev Biol Anim 2002; 38: 481-486
21 Yamaguchi T, Matsuda K, Sagiya Y, Iwadate M, Fujino MA,
Nakamura Y, Arakawa H. p53R2-dependent pathway for
DNA synthesis in a p53-regulated cell cycle checkpoint. Cancer
Res 2001; 61: 8256-8262
22 Murray AW. Recycling the cell cycle: cyclins revisited. Cell
2004; 116: 221-234
23 Cook SJ, Balmanno K, Garner A, Millar T, Taverner C, Todd D.
Regulation of cell cycle re-entry by growth, survival and stress
signalling pathways. Biochem Soc Trans 2000; 28: 233-240
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
24 Vale RD. The molecular motor toolbox for intracellular
transport. Cell 2003; 112: 467-480
25 Walczak CE, Mitchison TJ. Kinesin-related proteins at mitotic
spindle poles: function and regulation. Cell 1996; 85: 943-946
26 Ciechanover A. The ubiquitin-proteasome proteolytic
pathway. Cell 1994; 79: 13-21
27 Boudreau NJ, Jones PL. Extracellular matrix and integrin
signalling: the shape of things to come. Biochem J 1999; 339 ( Pt
3): 481-488
28 van Corven EJ, Groenink A, Jalink K, Eichholtz T, Moolenaar
WH. Lysophosphatidate-induced cell proliferation:
identification and dissection of signaling pathways mediated
by G proteins. Cell 1989; 59: 45-54
29 Winkles JA. Serum- and polypeptide growth factor-inducible
gene expression in mouse fibroblasts. Prog Nucleic Acid Res
Mol Biol 1998; 58: 41-78
30 Claesson-Welsh L. Signal transduction by the PDGF receptors.
Prog Growth Factor Res 1994; 5: 37-54
31 D e r y n c k R , Z h a n g Y E . S m a d - d e p e n d e n t a n d S m a d -
independent pathways in TGF-beta family signalling. Nature
2003; 425: 577-584
32 He H, Levitzki A, Zhu HJ, Walker F, Burgess A, Maruta H.
Platelet-derived growth factor requires epidermal growth
factor receptor to activate p21-activated kinase family kinases.
J Biol Chem 2001; 276: 26741-26744
33 Kester HA, Ward-van Oostwaard TM, Goumans MJ, van
Rooijen MA, van Der Saag PT, van Der Burg B, Mummery CL.
Expression of TGF-beta stimulated clone-22 (TSC-22) in mouse
development and TGF-beta signalling. Dev Dyn 2000; 218:
563-572
34 Shibanuma M, Kuroki T, Nose K. Isolation of a gene encoding
a putative leucine zipper structure that is induced by
transforming growth factor beta 1 and other growth factors. J
Biol Chem 1992; 267: 10219-10224
35 Datta PK, Moses HL. STRAP and Smad7 synergize in the
inhibition of transforming growth factor beta signaling. Mol
Cell Biol 2000; 20: 3157-3167
36 Fazioli F, Minichiello L, Matoska V, Castagnino P, Miki T,
Wong WT, Di Fiore PP. Eps8, a substrate for the epidermal
growth factor receptor kinase, enhances EGF-dependent
mitogenic signals. EMBO J 1993; 12: 3799-3808
37 Pedersen MW, Poulsen HS. Epidermal growth factor receptor
in cancer therapy. Science Med 2002: 206-217
38 Wakefield LM, Roberts AB. TGF-beta signaling: positive and
negative effects on tumorigenesis. Curr Opin Genet Dev 2002;
12: 22-29
S- Editor Pan BR L- Editor Kumar M E- Editor Bai SH
 PO Box 2345, Beijing 100023, China
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Effect of abdominal trauma on hemorrhagic shock-induced
acute lung injury in rats
Bulent Kilicoglu, Erol Eroglu, Sibel Serin Kilicoglu, Kemal Kismet, Fusun Eroglu
Bulent Kilicoglu, Kemal Kismet, Department of Surgery, Ankara
Training and Research Hospital, Ulucanlar, Ankara,Turkey
Erol Eroglu, Department of Surgery, Suleyman Demirel
University School of Medicine, Isparta, Turkey
Sibel Serin Kilicoglu, Department of Histology and Embryology,
Ankara University School of Medicine, Ankara, Turkey
Fusun Eroglu, Department of Anesthesiology and Reanimation,
Suleyman Demirel University School of Medicine, Isparta, Turkey
Correspondence to: Dr. Bulent Kilicoglu, Ankara Egitim ve
Arastirma Hastanesi 4th, Genel Cerrahi Klinigi, 06340, Ankara,
Turkey. bulentkilicoglu@yahoo.com.tr
Telephone: +90-312-5953449 Fax: +90-312-3633396
Received: 2006-02-08 Accepted: 2006-02-28
Abstract
AIM: To evaluate the effects of abdominal trauma on
hemorrhagic shock-induced acute lung injury in rats.
METHODS: Five groups were allocated (n = 8) in the
study. Group I was taken as the control group, group
II as the hemorrhagic shock group, group III as
hemorrhagic shock + laparotomy, group IV as hemor-
rhagic shock + splenectomy and group V as splenec-
tomy + omentectomy + hemorrhagic shock group.
Hemorrhagic shock was induced by drawing blood
and reducing mean arterial pressure (MAP) to 40
mmHg within 10 min. After a hypotensive period of 1
h, animals were resuscitated. Bronchoalveolar lavage
(BAL) was performed to recover cells from the alveo-
lar space with 40 mL of BAL fluid after resuscitation
malondialdehyde (MDA) and L--glutamyl-L-cysteinyl-
glycine (GSH) levels were measured in serum, eryth-
rocytes and lung tissue.
RESULTS: Serum, erythrocyte, lung tissue MDA and
GSH levels were significantly increased in hemorrhagic
shock groups II-V (P < 0.05). Lymphocyte, neutrophil
and alveolar macrophage counts in BAL fluid indicated
a significant difference between control and shock
groups (P < 0.05).
CONCLUSION: The degree of trauma increases hem-
orrhagic shock-induced acute lung injury.
2006 The WJG Press. All rights reserved.
Key words: Hemorrhagic shock; Acute lung injury;
Abdominal trauma
World J Gastroenterol 2006 June 14; 12(22): 3593-3596
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
CLINICAL RESEARCH
Kilicoglu B, Eroglu E, Kilicoglu SS, Kismet K, Eroglu F.
Effect of abdominal trauma on hemorrhagic shock-induced
acute lung injury in rats. World J Gastroenterol 2006;
12(22): 3593-3596
http://www.wjgnet.com/1007-9327/12/3593.asp
INTRODUCTION
Acute respiratory distress syndrome (ARDS) and acute
lung injury (ALI) frequently occur after major trauma
with a high mortality[1,2]. Clinically, acute lung injury is
characterized by altered gas exchange, dyspnea, decreased
static compliance, and nonhydrostatic pulmonary edema[3].
Firmly implicated mediators are endotoxin, cytokines,
and products of arachidonic acid metabolism, leukocyte-
derived proteolytic enzymes, and toxic oxygen species.
The basic mechanisms of lung injury are focused on
oxidant-mediated damage[4]. Recent evidence supports that
leukocyte activation and the subsequent release of large
quantities of highly reactive oxygen radicals and hydrogen
peroxide are partially responsible for diffuse microvascular
and tissue injury in patients with hemorrhagic shock.
It has been shown that neutrophils in trauma patients
release greater amounts of superoxide anion, proteases,
and proinflamatory cytokine[5]. In addition to neutrophils,
alveolar macrophages and circulating macrophages
as well as endothelial cells aggravate lung injury and
alveolar neutrophil sequestration[6]. In response to various
inflammatory stimuli, lung endothelial cells, alveolar cells,
airway epithelial cells, and alveolar macrophages, produce
both nitric oxide and superoxide[7].
To address these issues we designed a trauma model
of controlled hemorrhagic shock. Free oxygen radical
metabolism of serum, erythrocyte, lung tissue and cellular
change in alveolar epithelium was investigated in this
model, but the main objective was to understand the role
of abdominal injury in hemorrhagic shock.
MATERIALS AND METHODS
Animals
Adult male Wistar Albino rats weighing 243-325 g were
used in experiments. Animals were housed and kept at
25 in a 12 h light/dark cycle and used in studies after an
acclimatization period of 7 d. Rats were allowed to have
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 3594 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
Table 1 Serum, erythrocyte, tissue MDA and GSH levels, and lymphocyte, neutrophil, alveolar macrophage counts in BAL fluid of
different groups (mean SD)
Group I Group II
Serum MDA (mol/L) 1.374 0.24a 2.095 0.246
Serum GSH (mol/L) 29.005 4.14 15.374 4.369
Erythrocyte MDA (mol/L) 411.048 31.61a 665.676 173.609
Erythrocyte GSH (mol/L) 74.145 7.35 52.702 10.725
Tissue MDA (mol/L) 11.721 1.79a 16.651 3.702
Tissue GSH (mol/L) 0.318 0.053 0.170 0.056
Lymphocyte/100 cells 0.0 0.0 0.5 1.07
Neutrophil/100 cells 2.88 0.35 1.88 1.36
Alveolar macrophage/100 cells 1.38 0.52 4.0 2.27
a
P < 0.05 vs hemorrhagic shock groups; cP < 0.05 vs other groups.
free access to rodent chow and water before experiment.
All animals were maintained in accordance with the
recommendations of the Guide for the Care and Use of
Laboratory Animals. The Sleyman Demirel University BAL fluid and its analysis
Medical School Animal Use and Care Committee approved
all experiments. In the experiments, forty rats were used
and allocated into five groups (n = 8). Group I was taken
as the control group, group II as the hemorrhagic shock
group, group III as hemorrhagic shock + laparotomy
group, group IV as splenectomy + hemorrhagic group, and
group V as splenectomy + omentectomy + hemorrhagic
shock group.
Methods
Animals were intraperitoneally anesthetized with 80
mg/kg ketamine and 8 mg/kg xylazine. The right carotid
artery was cannulated with a 24-gauge angiocathether
for monitoring the mean arterial pressure (MAP), blood
sampling, and resuscitation. Hemorrhagic shock was
induced by drawing blood and reducing MAP to 40 mmHg
for 10 min. Blood was collected into 0.1 mL citrate to
prevent clotting. After a hypotensive period of 1 h, animals
were resuscitated by transfusion of blood and Ringer
lactate in a volume equal to that of blood. After a period
of two hours blood samples were taken via carotid artery,
and the catheter was removed with the artery ligated. The
cervical incision was extended towards midline to isolate
trachea. Trachea was catheterized with a feeding tube
(No: 8) and stabilized with 4/0 silk suture to perform
bronchoalveolar lavage (BAL). BAL was performed to
recover cells from the alveolar space with 40 mL of
BAL fluid. Lung tissue was resected to measure tissue
malondialdehyde (MDA) and L--glutamyl-L-cysteinyl- Statistical analysis
glycine (GSH) levels after bronchoalveolar lavage. Control
animals underwent the same procedures, but hemorrhage
was not induced.
In groups III-V, additional surgical procedures were
added. In group III (hemorrhagic shock + laparotomy)
after hemorrhagic shock was maintained, a midline incision
in 2 cm length was performed on the abdominal wall. The
incision was sutured with 4/0 silk suture. In group IV
(hemorrhagic shock + laparotomy + splenectomy) after
laparotomy splenectomy was performed with its vessels
ligated, laparotomy incision was sutured as in group III. In
group V (hemorrhagic shock + laparotomy + splenectomy
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June 14, 2006 Volume 12 Number 22
Group III Group IV Group V
2.54 0.18 3.04 0.12 3.57 0.23c
11.149 1.71 8.240 2.65 5.055 0.86
723.287 89.0 792.28 235.6 900.28 254.9c
52.412 9.684 46.831 6.408 27.852 10.438
17.315 1.485 18.178 2.364 20.327 5.786c
0.101 0.020 0.082 0.001 0.019 0.001
1.0 0.0 2.75 0.46 3.5 1.07
2.63 0.74 3.38 0.74 4.75 0.89
4.57 1.58 3.25 0.71 3.63 0.52
+ omentectomy) omentectomy was added. All vessel
boundaries were ligated when the omentum was removed.
The lungs were lavaged using the feeding tube with BAL
fluid containing cold phosphate buffered saline (PBS),
8 mmol/L sodium phosphate, 2 mmol/L potassium
phosphate, 0.14 mol/L sodium chloride, and 0.01 mol/L
potassium chloride (pH7.4) and 0.1 mmol/L ethylenedia
minetetraacetic acid (EDTA). Forty mL of BAL solution
was used for lavage in each animal, and about 90% was
re-collected. The fluid was centrifuged at 700 r/min for
15 min. Supernatant was collected and re-suspended in
1 mL of the same solution. Two slides were prepared
from each sample, and stained with Wright-Giemsa stain.
A total number of 100 cells were counted in each slide
with immersion objective (100 ) and the number of
neutrophils, lymphocytes and alveolar macrophages was
counted respectively.
MDA and GSH levels in serum, erythrocyte and lung tissue
Hemolysate and serum of the blood specimens were pre-
pared and stored at -70 until assay. The lung specimens
were homogenized with a mechanic homogenizator (Ultra-
Turrax T25) and sonicated (Bandelin D.12207) in phos-
phate buffered saline (pH7.4). The homogenate was cen-
trifuged at 10000 r/min for 15 min at 4 and supernatant
was used for determination of the MDA and GSH con-
centrations. MDA, an end product of lipid peroxidation,
was assayed by the method of Drapper and Hadley[8]. GSH
levels were determinated by the method of Beutler et al[9].
All data were analyzed by one-way ANOVA with post hoc
test Bonferroni. Data were expressed as mean SD. P
0.05 was considered statistically significant.
RESULTS
There was no difference in MAP values before and after
hemorrhagic shock between the groups (P > 0.05). Serum,
erythrocyte and lung tissue MDA levels were significantly
higher in hemorrhagic shock groups (groups II-V) than
in control group (P < 0.05, Table 1). MDA levels were
also significantly different in groups II-V. Serum MDA
 Kilicoglu B et al. Abdominal trauma on hemorrhagic shock-induced lung injury
levels had a trend to increase with hemorrhagic shock and
additional surgical injury (P < 0.05). The same trend could
be seen in erythrocyte and lung tissue MDA levels (P < 0.05,
Table 1).
Serum, erythrocyte and lung tissue GSH values were
significantly lower in groups II-V than in control group (P
< 0.05, Table 1). All GSH levels were decreased especially
in group V (P < 0.05), but the difference in groups II-IV
was not significant (P > 0.05). The decreased serum GSH
levels were parallel to the severity of trauma, but the dif-
ference in groups II-V was not statistically significant (P
> 0.05, Table 1). Similar GSH levels in lung tissue were
obtained. Tissue GSH level in group V was significantly
lower than that in other groups (P < 0.05).
Lymphocyte, neutrophil and alveolar macrophage
counts in BAL fluid indicated a significant difference be-
tween groups (P < 0.05, Table 1). The highest lymphocyte
and neutrophil counts were found in group V. Although
there was no significant difference in group II-V, lympho-
cyte, neutrophil and alveolar macrophage counts were sig-
nificantly higher in groups II-V than in control group.
DISCUSSION
Many mediators of acute lung injury in animal models and
ARDS in human beings have been implicated as causative
factors. Selection of agents that can abrogate these
mediators is based on the efficacy of these agents in whole
animal models, and their relative potency and potential
toxicity. Through examination of metabolic processes,
GSH has been shown to be important in host defenses
against oxidative stress[3]. Another important agent showing
oxidative stress is MDA, a marker of free radical activity[3].
After ischemia-reperfusion, lipid peroxides induce a
substantial drop in intracellular GSH levels[10,11]. In this
study, hemorrhagic shock and resuscitation significantly
elevated ser um, er ythrocyte and lung tissue MDA
levels (P < 0.05). GSH levels were significantly lower in
hemorrhagic shock groups than in control group (P < 0.05).
It was reported that oxidative stress significantly elevated
MDA levels and reduced GSH levels[12,13]. These significant
alterations were also seen in our study. MDA levels were
significantly higher in group V ( laparotomy + splenectomy
+ omentectomy) than in other groups (P < 0.05). Serum
GSH levels of erythrocyte and lung tissue were also altered
in hemorrhagic shock groups (P < 0.05). All these findings
support the hypothesis that ischemia-reperfusion affects
free oxygen radical metabolism and additional surgical
trauma increases this effect. Lipid peroxidation seems the
main mechanism of free radical toxicity and results in
alterations of structural integrity and function of cellular
membranes[14]. Intracellular overproduction is not the only
mechanism of free radical-induced lung injury[15]. Hypoxia
can damage alveolar macrophages, which may release
chemotactic and activating factors for polymorphonuclear
neutrophils[16]. The release of toxic oxygen may result in
endothelial injury, which is an early event in the course of
oxygen-induced lung damage.
Neutrophils sequestered in the lung play a central role
in the pathogenesis of ARDS. Lung polymorphonuclear
3595
neutrophile accumulation in ARDS occurs as a result of
a cascade of cellular events initiated by either infectious
or noninfectious inflammatory stimuli[17]. Neutrophil and
lymphocyte counts were significantly increased in BAL
fluid of hemorrhagic shock groups in this study (P <
0.05). There was also a significant difference between these
groups. The count of these cells increased significantly
with the degree of surgical trauma (P < 0.05). Alveolar
macrophage count was similarly increased in BAL fluid
in shock groups (P < 0.05), the difference between these
groups was not significant. There was no relation between
the degree of trauma and alveolar macrophage count
increase (P > 0.05). These observations can be explained
with the findings in the studies of Jorens et al [18] and
Bernard et al[19]. Acute lung injury in ARDS is largely due to
activated neutrophils, which not only aggravate oxidative
stress in the lungs but also release mediators, including
cytokines and lipids.
In conclusion, oxidative stress and surgery increase free
oxygen radicals in serum, erythrocytes and lung tissue and
lead to epithelial injury in the lungs. The observed injury
seems related with the degree of trauma.
REFERENCES
1 Sauaia A, Moore FA, Moore EE, Lezotte DC. Early risk factors
for postinjury multiple organ failure. World J Surg 1996; 20:
392-400
2 Faist E, Baue AE, Dittmer H, Heberer G. Multiple organ
failure in polytrauma patients. J Trauma 1983; 23: 775-787
3 Bernard GR. N-acetylcysteine in experimental and clinical
acute lung injury. Am J Med 1991; 91: 54S-59S
4 Riley DJ, Kerr JS. Oxidant injury of the extracellular matrix:
potential role in the pathogenesis of pulmonary emphysema.
Lung 1985; 163: 1-13
5 Zallen G, Moore EE, Johnson JL, Tamura DY, Aiboshi J, Biffl
WL, Silliman CC. Circulating postinjury neutrophils are
primed for the release of proinflammatory cytokines. J Trauma
1999; 46: 42-48
6 Fan J, Marshall JC, Jimenez M, Shek PN, Zagorski J, Rotstein
OD. Hemorrhagic shock primes for increased expression of
cytokine-induced neutrophil chemoattractant in the lung: role
in pulmonary inflammation following lipopolysaccharide. J
Immunol 1998; 161: 440-447
7 Lang JD, McArdle PJ, O'Reilly PJ, Matalon S. Oxidant-
antioxidant balance in acute lung injury. Chest 2002; 122:
314S-320S
8 Draper HH, Hadley M. Malondialdehyde determination as
index of lipid peroxidation. Methods Enzymol 1990; 186: 421-431
9 BEUTLER E, YEH MK. Erythrocyte glutathione reductase.
Blood 1963; 21: 573-585
10 Ishikawa T, Sies H. Cardiac transport of glutathione disulfide
and S-conjugate. Studies with isolated perfused rat heart
during hydroperoxide metabolism. J Biol Chem 1984; 259:
3838-3843
11 Tamura M, Oshino N, Chance B. Some characteristics of
hydrogen- and alkylhydroperoxides metabolizing systems in
cardiac tissue. J Biochem 1982; 92: 1019-1031
12 Jackson JH, White CW, McMurtry IF, Berger EM, Repine JE.
Dimethylthiourea decreases acute lung edema in phorbol
myristate acetate-treated rabbits. J Appl Physiol (1985) 1986; 61:
353-360
13 Johnson KJ, Fantone JC 3rd, Kaplan J, Ward PA. In vivo
damage of rat lungs by oxygen metabolites. J Clin Invest 1981;
67: 983-993
14 Halliwell B, Gutteridge JM. Lipid peroxidation, oxygen
radicals, cell damage, and antioxidant therapy. Lancet 1984; 1:
www.wjgnet.com
 3596 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
1396-1397
15 Housset B. Biochemical aspects of free radicals metabolism.
Bull Eur Physiopathol Respir 1987; 23: 287-290
16 Repine JE, Tate RM. Oxygen radicals and lung edema.
Physiologist 1983; 26: 177-181
17 Fan J, Shek PN, Suntres ZE, Li YH, Oreopoulos GD, Rotstein
OD. Liposomal antioxidants provide prolonged protection
against acute respiratory distress syndrome. Surgery 2000; 128:
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
332-338
18 Jorens PG, Bossaert LL. Cytokine-networks and ARDS.
In: Vincent JL. Yearbook of Intensive Care and Emergency
Medicine. Berlin: Springer-Verlag, 1997: 83-90
19 Bernard GR, Korley V, Chee P, Swindell B, Ford-Hutchinson
AW, Tagari P. Persistent generation of peptido leukotrienes in
patients with the adult respiratory distress syndrome. Am Rev
Respir Dis 1991; 144: 263-267
S- Editor Pan BR L- Editor Wang XL E- Editor Bi L
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
The vital threat of an upper gastrointestinal bleeding: Risk
factor analysis of 121 consecutive patients
Peter Schemmer, Frank Decker, Genevieve Dei-Anane, Volkmar Henschel, Klaus Buhl, Christian Herfarth,
Stefan Riedl
Peter Schemmer, Frank Decker, Genevieve Dei-Anane, Klaus
Buhl, Christian Herfarth, Stefan Riedl, Deptartment of General
Surgery, Ruprecht-Karls-University, 69120 Heidelberg, Germany
Volkmar Henschel, Institute for Statistics and Medical Biometry,
Ruprecht-Karls-University, 69120 Heidelberg, Germany
Correspondence to: Dr. Peter Schemmer, Department of
General Surgery, Ruprecht-Karls-University of Heidelberg, Im
Neuenheimer Feld 110, 69120 Heidelberg,
Germany. peter_schemmer@med.uni-heidelberg.de
Telephone: +49-6221-566110 Fax: +49-6221-564215
Received: 2006-01-04 Accepted: 2006-01-24
Abstract
AIM: To analyze the importance in predicting patients
risk of mortality due to upper gastrointestinal (UGI)
bleeding under today's therapeutic regimen.
METHODS: From 1998 to 2001, 121 patients with the
diagnosis of UGI bleeding were treated in our hospital.
Based on the patients data, a retrospective multivariate
data analysis with initially more than 270 single factors
was performed. Subsequently, the following potential
risk factors underwent a logistic regression analysis:
age, gender, initial hemoglobin, coumarines, liver
cirrhosis, prothrombin time (PT), gastric ulcer (small
curvature), duodenal ulcer (bulbus back wall), Forrest
classification, vascular stump, variceal bleeding, Mallory-
Weiss syndrome, RBC substitution, recurrent bleeding,
conservative and surgical therapy.
RESULTS: Seventy male (58%) and 51 female (42%)
patients with a median age of 70 (range: 21-96) years
were treated. Their in-hospital mortality was 14%. While
12% (11/91) of the patients died after conservative
therapy, 20% (6/30) died after undergoing surgical
therapy. UGI bleeding occurred due to duodenal ulcer
(n = 36; 30%), gastric ulcer (n = 35; 29%), esophageal
varicosis (n = 12; 10%), Mallory-Weiss syndrome (n
= 8; 7%), erosive lesions of the mucosa (n = 20;
17%), cancer (n = 5; 4%), coagulopathy (n = 4; 3%),
lymphoma (n = 2; 2%), benign tumor (n = 2; 2%)
and unknown reason (n = 1; 1%). A logistic regression
analysis of all aforementioned factors revealed that
liver cirrhosis and duodenal ulcer (bulbus back wall)
were associated risk factors for a fatal course after UGI
bleeding. Prior to endoscopy, only liver cirrhosis was
an assessable risk factor. Thereafter, liver cirrhosis,
the location of a bleeding ulcer (bulbus back wall) and
World J Gastroenterol 2006 June 14; 12(22): 3597-3601
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
RAPID COMMUNICATION
patients gender (male) were of prognostic importance
for the clinical outcome (mortality) of patients with a
bleeding ulcer.
CONCLUSION: Most prognostic parameters used in
clinical routine today are not reliable enough in predicting
a patients vital threat posed by an UGI bleeding.
Liver cirrhosis, on the other hand, is significantly more
frequently associated with an increased risk to die after
bleeding of an ulcer located at the posterior duodenal
wall.
2006 The WJG Press. All rights reserved.
Key words: UGI bleeding; Mortality; Risk factors
Schemmer P, Decker F, Dei-Anane G, Henschel V, Buhl
K, Herfarth C, Riedl S. The vital threat of an upper
gastrointestinal bleeding: Risk factor analysis of 121
consecutive patients. World J Gastroenterol 2006; 12(22):
3597-3601
http://www.wjgnet.com/1007-9327/12/3597.asp
INTRODUCTION
Acute bleeding from the upper gastrointestinal (UGI)
tract remains a major cause of morbidity and mortality.
Over the last decades, mortality after UGI bleeding has
been reported to range between 8% and 14% of cases[1-8].
Selected groups, i.e. patients who underwent surgical
therapy for UGI bleeding, die at a risk of about 21%[9].
Despite improved diagnostic measures, emergency
endoscopy [10] , improved conser vative and surgical
treatment [5,11] , mortality after UGI bleeding remains
high[1]. An increase in the number of high-risk patients
with reduced ability to compensate for the consequences
o f b l e e d i n g m ay b e o n e o f t h e r e a s o n s f o r t h i s
phenomenon [11,12]. Further, UGI bleeding is the most
common emergency in gastroenterology, with an incidence
of about 50 to 150 bleeding episodes per 100 000
inhabitants per year[4,11,13,14].
Numerous prognostic factors have been described
in literature to be associated with a lethal outcome;
however, to date, it remains unclear whether a single or a
combination of these parameters is valid in predicting the
vital threat of a patient with an UGI bleeding[1,13,15,16]. Some
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 3598 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
factors were assessed for decision making on early surgical
therapy in patients with bleeding ulcers [5,17]. However,
they have not become clinical routine due to their poor
reliability.
There are very few studies which report on risk factors
for an increased threat after UGI bleeding based on
multivariate analysis[1]. Thus, our retrospective study was
designed to evaluate published risk factors for their value
in predicting the threat to patients with UGI bleeding.
MATERIALS AND METHODS
Patients
A database was established retrospectively using infor-
mation on consecutive admitted patients, with evidence
of UGI bleeding treated in the Department of General
Surgery, Ruprecht-Karls-University, Heidelberg, Germany
during 4 consecutive years from January 1 st , 1998 to
December 31st, 2001.
The patients were identified with endoscopy, patients
admission (electronic patients chart SARA-Med), and
an operation protocol database. Within the evaluated 4
years, a total of 264 patients with the suspicion of UGI
bleeding were found (Figure 1). Thirty-six of these were
treated temporarily in the outpatient clinic. In these cases
no hospitalization was necessary. Further 38 patients
were excluded from analysis since their charts could not
show enough evidence of an UGI bleeding. Forty-two
patients charts were not available for analysis. Thus, these
patients were excluded from further analysis. Twenty-six
cases have not been documented in the way needed for
an appropriate analysis. Since a gap-free documentation is
needed for statistics, a total of 65 patients were excluded
from this study. Moreover, patients were excluded from
this study if UGI bleeding was due to previous surgery or
endoscopic intervention (n = 1).
Literature research
MedLine was searched for gastrointestinal hemorrhage,
gastrointestinal bleeding, bleeding peptic ulcer, peptic
ulcer hemorrhage, bleeding from the upper gastrointesti-
nal tract, stigmata of hemorrhage, prediction of mortal-
ity, emergency endoscopy, predictive factors, and further
hemorrhage. Fifty-five publications on prognostic factors
and risk factors of UGI bleeding were found, including
papers mentioned in their references. Subsequently, factors
(i.e., renal failure, heart failure, diabetes mellitus and
others; n = 79) were classified according to the frequency
of their appearance. Factors which fulfill at least one of
the following conditions have been included: Factors
which have been mentioned at least twice in the context of
risks and prognosis for mortality, recurrence of bleeding,
failed endoscopic therapy or necessity of surgery and a
significant correlation between the parameter and mortality
after univariate or multivariate analysis.
Analysis at different time points of therapy
During various stages of therapy, different information
was available. Thus, analysis was performed at the time
of admission, right after endoscopy and after completed
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June 14, 2006 Volume 12 Number 22
Endoscopy database, operation
reports, and patients' admission
database (SARA-Med)
264 patients (suspicious for upper
gastrointestinal bleeding)
42 patients' charts not available
36 patients treated temporarily as
outpatients
186 patients (suspicious for upper
gastrointestinal bleeding)
Evaluation of the patients' charts
38 patients: no upper gastrointestinal
bleeding
26 patients' charts incomplete
1 iatrogenic bleeding
121 patients with upper
gastrointestinal bleeding
Figure 1 Identification of patients with an UGI bleeding qualifying for statistical
analysis (Identification of all patients with an UGI bleeding who received clinical
treatment within a period of 4 years from January 1st, 1998 to December 31st,
2001).
therapy. The following information on the patients were
given and analyzed:
At admission: Age, gender, medication [i.e. coumarines,
acetyl salicylic acid (ASS), non-steroidal antirheumatics
(NSA)], liver disease, laboratory findings (i.e. hemoglobin,
PT) were recorded.
At endoscopy: In addition to information obtained at
admission, the localization, the Forrest classification and
the presence of a vascular stump were elucidated.
At the end of therapy: In addition to the aforemen-
tioned information, the number of blood units transfused,
frequency of re-bleeding and the number of operations
were recorded.
Statistical analysis
The in-hospital survival was chosen as the endpoint. It was
analyzed as to how the probability of survival depends on
covariates by means of stepwise logistic regression[18] where
the significance level for entry of a covariate was chosen as
0.2 and the significance level for staying was chosen as 0.15.
Only parameters which were documented for all of
our 121 patients with a special focus on clinical relevant
parameters published before were analyzed. The pos-
sible covariates which were considered are: gender, age
(dichotomised at 70 years), medication (ASS, NSA, and
coumarines), liver cirrhosis, gastric ulcer (small curvature),
duodenal ulcer (bulbus back wall), varices, Forrest classifi-
cation, vascular stump, prothrombin time (dichotomised at
50%), hemoglobin concentration (dichotomised at 60 g/L),
transfused blood units (dichotomised at 6 units), recur-
rence of bleeding and operations.
Three models were applied. The first considered the
knowledge of covariates before the first gastroduode-
noscopy. The second model takes only the patients with
 Schemmer P et al. Prediction of a fatal course after UGI bleeding 3599

Table 1 Reason for the occurrence of upper gastrointestinal (UGI) bleeding

Reason Duodenal Gastric Erosive Esophageal Mallory-Weiss- Unknown

Cancer Coagulopathy Lymphoma Benign tumor
for bleeding ulcer ulcer mucosal lesion varicosis syndrome reason
% 29.8 28.9 16.5 9.9 6.6 4.1 3.3 1.7 1.7 0.8
Number (n = 125)1 36 35 20 12 8 5 4 2 2 1

1
Four patients presented with both duodenal and gastric ulcers with each showing an endoscopic evidence of bleeding and thus each ulcer was counted as a
whole.

Table 2 Logistic regressions analysis of covariables recorded at

dictability of dying as a result of an UGI bleeding. Liver
admission of 121 patients considering their ability in predicting cirrhosis at admission increased the risk of mortality due
the patients lethality to UGI bleeding by 4.5 fold (P = 0.005, 95%-confidence
interval: 1.4-13.8). The other prognostic parameters re-
Covariable P
Statistical inclusion
Odds ratio
95% confidence corded at admission (Hb, PT, age, gender and medication)
in model interval
did not have a predictive value (Table 2).
Cirrhotic liver
disease 0.005 Yes 4.5 1.4-13.8
At endoscopy: Sixty-seven patients with duodenal or gas-
Hb 0.256 No
tric ulcers were analyzed; fourteen prognostic parameters
Medication 0.419 No
which were recorded during endosopic examination were
PT 0.672 No tested on how far they prove to be a threat to the patients
Age 0.822 No survival. Three of the tested parameters were taken up
Gender 0.879 No into the model, as their existence could help in predict-
ing the faith of an ulcer patient. Cirrhotic liver disease (P
= 0.029; 95%-confidence interval: 1.1-34.4) increased the
mortality risk 5.9-fold as a result of UGI bleeding. Fur-
bleeding in the ulcers where the Forrest classification is thermore, male patients had a 4.8-fold higher risk of not
valid into account, whereas the third model considers the surviving an UGI bleeding (P = 0.065; 95%-confidence
covariates which are known after the termination of bleed- interval: 1.0-34.8). An ulcer located at the bulbus back wall
ing[18]. increased a patients lethality 3.5-fold (P = 0.135; 95%-con-
fidence interval: 0.6-20.7). Other parameters, such as the
Forrest classification, played no substantial role in improv-
RESULTS ing the predictive strength of the statistic model (Table 3).
Patients At the end of therapy: Only 2 of the 12 different
The total of 121 patients with UGI bleeding included in prognostic parameters tested at the end of therapy were
our analysis comprised of 70 males (58%) and 51 females of significance; a patient with cirrhotic liver disease had
(42%), making a gender distribution of 1.4:1. The median a 5.7-fold higher risk of dying of an UGI bleeding (P =
age distribution of the patients was 70 years, the youngest 0.005; 95% confidence interval: 1.7-19.3) as compared
being 21 and the oldest 96 years. The most frequent with a patient with healthy liver. An ulcer located at the
cause for UGI bleeding was duodenal ulcer (n = 36; 30%) bulbus back wall increased the lethality 3.4-fold (P =
of which n = 13 were located at the bulbus back wall. 0.092; 95%-confidence interval: 0.7-14.7). All remaining
Other reasons for UGI bleeding in descending order of parameters did not improve the predictability and were not
occurrence were gastric ulcer (n = 35; 29%) of which n = taken up in model (Table 4).
12 were located at the lesser curvature, erosive lesions of
the mucosa (n = 20; 17%), esophageal varicosis (n = 12; Subgroup analysis of liver disease patients
10%), Mallory-Weiss syndrome (n = 8; 7%), cancer (n = The logistic regression analysis at the 3 different time
5; 4%), coagulopathy (n = 4; 3%), lymphoma (n = 2; 2%), points showed that the covariable cirrhotic liver disease
benign tumor (n = 2; 2%) and an unknown reason (n = 1; significantly increased the predictive value at all time
1%). Four patients had both duodenal and gastric ulcers points. In 21 of our evaluated patients liver disease
with each ulcer showing endoscopic evidence of bleeding, occurred, analysis of the correlation between these patients
and hence each ulcer was counted as one (Table 1). and 9 covariables showed following findings: an initial Hb
< 6 mg/dL increased the risk of dying of an UGI bleeding
9.75-folds (P = 0.077; 95%-confidence interval: 0.8-121.8).
Total lethality
When these patients experienced recurrence bleeding dur-
Out of the 121 patients analyzed, 17 died, resulting in a
ing their hospital stay, the risk was increased 4.5-folds (P =
total mortality of 14%.
0.135, 95% confidence interval: 0.6-32.3). Moreover, addi-
tional surgical treatment resulted in a 4.5-fold risk increase
Logistic regression analyzed at different time points of
(P = 0.164; 95% confidence interval: 0.5-37.4). On the
therapy contrary, the necessity to transfuse more that 6 units red
At admission: Of all the prognostic parameters which blood cells (RBCs) as well as the location of a gastric ulcer
were considered for logistic regression, only the prognostic at the small curvature did not increase the risk factor (Table
parameter of cirrhotic liver disease could improve the pre- 5).

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 3600 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

Table 3 A logistic regression analysis evaluating 14 prognostic Table 4 Logistic regressions analysis indicating the predictive
parameters in their ability to increase the predictability of value of 12 covariables in predicting the lethality of an UGI
lethality of 67 patients with UGI bleeding resulting from bleeding
duodenal or gastric ulcer
Statistical
95 % confidence
Statistical Covariable P inclusion Odds ratio
95% confidence interval
Covariable P inclusion in Odds ratio in model
interval
model Cirrhotic liver disease 0.005 Yes 5.7 1.8-19.3
Cirrhotic liver Duodenal ulcer
disease 0.029 Yes 5.9 1.1-34.4 (bulbus back wall) 0.092 Yes 3.4 0.7-14.7
Gender 0.065 Yes 4.8 1.0-34.8 Bleeding recurrance 0.271 No
Duodenal ulcer Hb 0.341 No
(bulbus back wall) 0.135 Yes 3.5 0.6-20.7 Medication 0.345 No
Forrest 1 b 0.175 No Esophageal varicosis 0.401 No
Forrest 2 b 0.272 No Number of operations 0.42 No
Hb 0.285 No Substitution of RBCs 0.436 No
PT 0.641 No PT 0.625 No
Medication 0.693 No Gastric ulcer
Forrest 3 0.721 No (small curvature) 0.799 No
Forrest 1 a 0.748 No Gender 0.809 No
Forrest 2 a 0.782 No Age 0.863 No
Vascular stump 0.852 No
Age 0.926 No
Gastric ulcer
(small curvature) 0.964 No
Table 5 Subgroup analysis of 21 patients with UGI bleeding
and liver disease (Evaluation of the influence of 9 covariables on
the lethality within the group)

DISCUSSION 95% confidence

Covariable Odds ratio P
interval
The lethality of our evaluated patient group of 14%
Hb 9.75 0.8-121.8 0.077
confirms with records of selected patient groups in other
Bleeding recurrence 4.5 0.6-32.3 0.135
surgical departments[9]. These findings compared to the
Number of operations 4.5 0.5-37.4 0.164
standards of todays medicine where a lethality of more Substitution of RBCs 1 0.1-7.5 1
than 10% is classified as high, depict that the lethality Gastric ulcer (small curvature) 1 0.1-13.4 1
of UGI bleeding patients has been underestimated PT 0.72 0.1-5.2 0.744
tremendously. Although many risk factors are listed Gender 0.68 0.1-5.5 0.718
in literature and many scores [5,17] as well as indices [13,16] Age 0.3 0.03-3.3 0.322
have been suggested, there is still ongoing uncertainty Esophageal varicosis 0.17 0.02-1.8 0.137
as to which parameters are threatening or which patient
group is to be classified as high-risk group. The many
univariate analysed records found in todays literature do
not fully make up for the multi-factorial situation of an located at the bulbus back wall, were found to be of weak-
UGI bleeding, which requires multivariate analysis. The er predictable strength. On the other hand, other studies
disadvantages of prospective study designs, when it comes reported an evidence of disease [20,21], recurrent bleed-
to long-term studies, made us choose the limited but ing[2,21,22], transfusion of more than 5 units of RBCs[20,22]
short-termed retrospective study design. and an ulcer size of more than 1 cm[20,22] as significant
The results of our multivariate analysis which showed prognostic parameters. However, the parameters including
that of all the possible risk factors considered at admission patients age above 60 years[2,22], evidence of gastric bleed-
of a patient with UGI bleeding, only the covariable liver ing[2], an initial Hb below 100 g/L and complications[21] as
cirrhosis proved to be a strong enough prognostic factor prognostic parameters applicable for a patient with ulcer
in predicting the lethality of such patients. The only multi- bleeding could not be confirmed in our multivariate analy-
variate study, which also evaluated patients at admission[19], sis.
reported further risk factors such as age above 75 years, Other multivariate analyses of prognostic parameters
diagnosed carcinoma, evidence of blood in gastric aspirate for patients after UGI bleeding therapy found in literature
and a systolic blood pressure below 90 mmHg. These find- confirm our result that the covariable cirrhotic liver dis-
ings could not be confirmed in our study. The covariable ease is significant in predicting the lethality of patients[13,23].
blood in gastric aspirate was considered by us as a diagnos- Other reports which noted factors such as elevated serum
tic criterion and hence not taken into consideration. transaminases[19,23], high albumin levels[23] and existence of
The analysis of data gained after endoscopy showed in life-threatening disease[24] tend to confirm our findings as
our study that only the evidence of cirrhotic liver disease well, as these listed factors could be interpreted as descrip-
as covariable could significantly increase the ability to pre- tive of liver disease.
dict the lethality of a patient being treated because of UGI The existing correlation between liver disease and the
bleeding. Other variables, such as male gender and an ulcer covariables; Hb, the number of operations and recurrent

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 Schemmer P et al. Prediction of a fatal course after UGI bleeding 3601
bleedings during treatment of UGI bleeding suggests their
probable influence on the lethality of this patient group.
The relationship could be due to the fact that these pa-
rameters tend to outline the risk profile of liver disease pa-
tients. A study which reports that the lethality of patients
with liver disease correlates with the number of bleeding
occurrences and the extent of renal failure[25], confirms our
results.
In summary, most prognostic parameters used in
clinical routine today are not reliable enough in predicting
the patients vital threat due to UGI bleeding. Liver
cirrhosis is the only risk factor which shows a significantly
more frequent association with a fatal course after UGI
bleeding in our patients.
REFERENCES
1 Gilbert DA. Epidemiology of upper gastrointestinal bleeding.
Gastrointest Endosc 1990; 36: S8-13
2 Katschinski B, Logan R, Davies J, Faulkner G, Pearson J,
Langman M. Prognostic factors in upper gastrointestinal
bleeding. Dig Dis Sci 1994; 39: 706-712
3 Morgan AG, Clamp SE. OMGE international upper
gastrointestina l b l e e d i n g s u r v e y , 1 9 7 8 - 1 9 8 6 . S c a n d J
Gastroenterol Suppl 1988; 144: 51-58
4 Rockall TA, Logan RF, Devlin HB, Northfield TC. Incidence of
and mortality from acute upper gastrointestinal haemorrhage
in the United Kingdom. Steering Committee and members
of the National Audit of Acute Upper Gastrointestinal
Haemorrhage. BMJ 1995; 311: 222-226
5 Siewert JR, Bumm R, Hlscher AH, Dittler HJ. [Upper
gastrointestinal bleeding from ulcer: reduction of mortality by
early elective surgical therapy of patients at risk]. Dtsch Med
Wochenschr 1989; 114: 447-452
6 Silverstein FE, Gilbert DA, Tedesco FJ, Buenger NK, Persing J.
The national ASGE survey on upper gastrointestinal bleeding.
I. Study design and baseline data. Gastrointest Endosc 1981; 27:
73-79
7 Silverstein FE, Gilbert DA, Tedesco FJ, Buenger NK, Persing J.
The national ASGE survey on upper gastrointestinal bleeding.
II. Clinical prognostic factors. Gastrointest Endosc 1981; 27:
80-93
8 Sugawa C, Steffes CP, Nakamura R, Sferra JJ, Sferra CS,
Sugimura Y, Fromm D. Upper GI bleeding in an urban
hospital. Etiology, recurrence, and prognosis. Ann Surg 1990;
212: 521-526; discussion 526-527
9 Merkle NM, Hupfauf G, Herfarth C. [Criteria in the prognosis
of upper gastrointestinal hemorrhage. A retrospective
analysis]. Med Welt 1981; 32: 1431-1433
10 Kohler B, Riemann JF. Acute ulcer hemorrhage. [Evaluation
of endoscopic therapy methods]. Dtsch Med Wochenschr 1994;
119: 69-73
11 Sarkar MR, Kienzle HF, Bhr R. [Can endoscopic methods
decrease the mortality and complication rate of bleeding
stomach and duodenal ulcer? A study of the literature]. Leber
Magen Darm 1992; 22: 10-12, 15-18
12 Morris DL, Hawker PC, Brearley S, Simms M, Dykes PW,
Keighley MR. Optimal timing of operation for bleeding peptic
ulcer: prospective randomised trial. Br Med J (Clin Res Ed)
1984; 288: 1277-1280
13 Brsch G, Matuk ZE, Leverkus F. [Prognosis of acute upper
gastrointestinal hemorrhage. Univariate and multivariate
analysis of 477 episodes of hemorrhage]. Med Klin (Munich)
1987; 82: 774-780
14 Kohler B, Riemann JF. [Diagnosis of gastrointestinal bleeding].
Dtsch Med Wochenschr 1989; 114: 548-551
15 Guglielmi A, Ruzzenente A, Sandri M, Kind R, Lombardo
F, Rodella L, Catalano F, de Manzoni G, Cordiano C.
Risk assessment and prediction of rebleeding in bleeding
gastroduodenal ulcer. Endoscopy 2002; 34: 778-786
16 Kalula SZ, Swingler GH, Louw JA. Clinical predictors of
outcome in acute upper gastrointestinal bleeding. S Afr Med J
2003; 93: 286-290
17 Troidl H, Vestweber KH, Kusche J, Bouillon B. Hemorrhage
in peptic gastroduodenal ulcer: data as a deciding aid in the
concept of surgical therapy. Chirurg 1986; 57: 372-380
18 Agresti A. Categorical Data Analysis. New York: John Wiley
& Sons, Inc., 1990
19 Zimmerman J, Siguencia J, Tsvang E, Beeri R, Arnon R.
Predictors of mortality in patients admitted to hospital for
acute upper gastrointestinal hemorrhage. Scand J Gastroenterol
1995; 30: 327-331
20 Branicki FJ, Boey J, Fok PJ, Pritchett CJ, Fan ST, Lai EC, Mok
FP, Wong WS, Lam SK, Hui WM. Bleeding duodenal ulcer. A
prospective evaluation of risk factors for rebleeding and death.
Ann Surg 1990; 211: 411-418
21 Chow LW, Gertsch P, Poon RT, Branicki FJ. Risk factors for
rebleeding and death from peptic ulcer in the very elderly. Br J
Surg 1998; 85: 121-124
22 Branicki FJ, Coleman SY, Fok PJ, Pritchett CJ, Fan ST, Lai EC,
Mok FP, Cheung WL, Lau PW, Tuen HH. Bleeding peptic
ulcer: a prospective evaluation of risk factors for rebleeding
and mortality. World J Surg 1990; 14: 262-269; discussion
269-270
23 Zimmerman J, Meroz Y, Arnon R, Tsvang E, Siguencia J.
Predictors of mortality in hospitalized patients with secondary
upper gastrointestinal haemorrhage. J Intern Med 1995; 237:
331-337
24 Chojkier M, Laine L, Conn HO, Lerner E. Predictors of
outcome in massive upper gastrointestinal hemorrhage. J Clin
Gastroenterol 1986; 8: 16-22
25 del Olmo JA, Pea A, Serra MA, Wassel AH, Benages A,
Rodrigo JM. Predictors of morbidity and mortality after
the first episode of upper gastrointestinal bleeding in liver
cirrhosis. J Hepatol 2000; 32: 19-24
S- Editor Wang J L- Editor Kumar M E- Editor Liu WF
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RAPID COMMUNICATION
Roles of syndecan-1, bcl6 and p53 in diagnosis and
prognostication of immunoproliferative small intestinal
disease
Kim Vaiphei, Neeraj Kumari, Saroj Kant Sinha, Usha Dutta, Birinder Nagi, Kusum Joshi, Kartar Singh
Kim Vaiphei, Neeraj Kumari, Kusum Joshi, Department of
Histopathology, PGIMER, Chandigarh, India
Saroj Kant Sinha, Usha Dutta, Birinder Nagi, Kartar Singh,
Department of Gastroenterology, PGIMER, Chandigarh, India
Co-first-authors: Neeraj Kumari
Correspondence to: Dr. Kim Vaiphei, No. 127/C, Sector- 24/A,
Chandigarh, Pin-160023, India. kvaiphei@gmail.com
Telephone: +91-172-2755140 Fax: +91-172-2744401
Received: 2005-12-13 Accepted: 2006-02-18
Abstract
AIM: To evaluate roles of syndecan-1, bcl6 and p53 in
diagnosis and prognostication of immunoproliferative
small intestinal disease (IPSID) and to study profiles of
kappa () and lambda () light chains and IgA heavy
chain.
METHODS: The study consisted of 11 cases of IPSID
and similar number of controls which included 11 of
normal intestinal mucosa and 11 of high grade B cell
lymphoma of ileum. The parameters analyzed included
clinical profiles, biochemical and other laboratory
investigations, radiologic and histological findings
including immunohistochemistry.
RESULTS: All IPSID cases had demonstrable serum IgA
heavy chain and heavy mucosal plasma cell infiltration.
According to Galians histological staging, there were
4 patients with stage A and 7 with stage B. and
light chains were over-expressed in 7 patients; 1
stage A patient had H pylori -positive active gastritis
and eradication of H pylori led to disease remission.
Stage A biopsies had higher expression for syndecan-1,
while stage B had higher expression for bcl6 and p53.
Syndecan-1, and light chains and IgA heavy chain
showed inverse relationship with bcl6 and p53. All
patients were treated with doxycycline. CHOP regime
was added in 5 patients who developed frank lymphoma.
Three died of the disease due to extensive organ
infiltration.
CONCLUSION: Certain immunomarkers like syndecan-1,
and light chains and IgA heavy chain could be of
much help in identifying early stage IPSID. Stage B
IPSID showed higher expression for bcl6 and p53 than
stage A IPSID. bcl6 and p53 expressions correlated with
a more advanced disease stage and aggressive tumour
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World J Gastroenterol 2006 June 14; 12(22): 3602-3608
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
behavior.
2006 The WJG Press. All rights reserved.
Key words: IPSID; Syndecan-1; bcl6; p53 protein; and
light chains; Alpha heavy chain; H pylori
Vaiphei K, Kumari N, Sinha SK, Dutta U, Negi B, Joshi K,
Singh K. Roles of syndecan-1, bcl6 and p53 in diagnosis
and prognostication of immunoproliferative small intestinal
disease. World J Gastroenterol 2006; 12(22): 3602-3608
http://www.wjgnet.com/1007-9327/12/3602.asp
INTRODUCTION
Immunoproliferative small intestinal disease (IPSID)
or alpha heavy chain disease is quite frequently seen
in Mediterranean countries [1-7] compared to rest of the
world [8-12] . It usually affects young individual of low
socio-economic status. Reduction in incidence has been
observed with improvement of living conditions [13-15].
Plasma cells of IPSID secrete bio-chemically abnormal
IgA heavy chain in all body fluids[16-18]. Monoclonality in
plasma cell of early stage IPSID has been demonstrated by
immunohistochemistry and polymerase chain reaction[19-23].
IPSID is known to have an indolent course. Complete
remission could be obtained with long-term antibiotic
therapy in stage A IPSID, though the ultimate outcome
of the disease is unpredictable [22,23] . Syndecan-1 is a
member of cell surface trans-membrane heparan sulphate
proteoglycans [24] and is expressed by pre-B, mature
plasma cell and myeloma cell line[25-27]. In in vitro studies,
syndecan-1 has inhibitory effect on growth of myeloma
cell line and B cell lymphoma[27,28]. Reduction in syndecan-1
expression has been observed with dedifferentiation of
non-plasma cell malignant tumors[29] and down-regulation
of p53 and Ki67 expression [30] . bcl6 gene product, a
nuclear phospho-protein, is expressed independently of
bcl6 gene rearrangement[31]. bcl6 expression is restricted
to germinal center B cell and certain percentage of intra-
follicular T cell [32]. It is over-expressed in high grade
B-cell lymphoma, marginal zone B cell lymphoma and
in some cases of follicular lymphoma and nodular
lymphocytic predominance Hodgkins lymphoma[31-35]. In
good number of gastric MALTomas and intestinal diffuse
 Vaiphei K et al. Immunoprofiles in IPSID 3603
large B-cell lymphoma, expression patterns of bcl6 have
been evaluated[36]. p53 gene plays an important role in cell
cycle by checking and controlling apoptotic pathways and
abnormal cell proliferation. In absence of a functional p53
gene, apoptotic pathway does not become activated and
cell cycle progresses without the repair of the defective
DNA. Hence, it is known as gate-keeper gene [37] .
Mutation in p53 gene results in proliferation of abnormal
cell by blocking the apoptotic pathways and is seen in
various pre-malignant and malignant conditions [37-39].
H pylori-associated early stage MALToma are known to
regress following eradication of H pylori. This observation
suggests evolvement of MALToma from benign antigen-
driven B cell [40-42] . IPSID, like in H pylori-associated
MALTomas, likely represents follicle center and/or
marginal zone B-cell response to chronic infection by
bacteria or parasites and eventually acquiring uncontrolled
proliferation and probably monoclonality. Early staged
IPSID usually responds to antibiotic therapy alone[43,44] .
Histologically, Galian et al[23] has classified IPSID into
3 stages. Stage A is characterized by mucosal infiltrate by
mature plasma cell and lymphocytes. In stage B, mucosa
shows presence of atypical or immature plasma cells and
lymphocytes, which could extend to deeper portion of the
intestinal wall. Stage C is the frank lymphomatous stage
with involvement of draining lymph nodes. Stage A is
treated by antibiotics alone and stage B requires additional
cytotoxic drugs[45,46]. Differentiating between stage A and
B is of therapeutic and prognostic importance. With the
advancement in imaging techniques, clinical staging is
based on observations made on CT and MRI, without
subjecting the patient to laparatomy[7]. The protocol was
hence planned to study (1) histological profiles of IPSID
in endoscopic biopsies, (2) to evaluate roles of syndecan-1,
bcl6 and p53 in diagnosis and prognostication of IPSID,
and (3) expressions of kappa ( ) and lambda () light
chains, and IgA heavy chain.
MATERIALS AND METHODS
Materials
The study included 11 cases of IPSID who presented
with features of malabsorption. All had demonstrable
serum alpha heavy chain by immuno-electrophoresis.
Endoscopic biopsies of 11 patients showed heavy mucosal
infiltration predominantly by plasma cell with lymphocyte.
According to Galians et al [23], histological grading was
done as follows: stage A identified by heavy lamina propria
infiltration by mature plasma cell and lymphocyte; stage
B by lamina propria infiltration with extension into sub-
mucosa and/or with few scatter immature plasma cell; and
stage C by infiltrating immature plasma cell with variable
degree of intestinal wall infiltration and regional lymph
node involvement. In all patients studied, 3 to 5 mucosal
biopsy fragments were available from involved segments
of the intestine. Additional two tissue fragments from
pyloric antrum, one each from anterior and posterior
wall, were taken only when there was abnormality i.e.
either involvement by the ongoing disease or if there was
changes of gastritis.
Methods
Baseline assessment of all the biopsies was done on H&E-
stained sections for all the eleven patients. Peroxidase anti-
peroxidase technique was used for immunohistochemistry.
Primary antibodies used were: (1) Syndecan-1 (dilution 1:
50, Dako), (2) bcl6 (pre-diluted, Neomarker), (3) p53 (pre-
diluted, DO7, Dako), (4) and light chains (dilution
1:100, Dako), and (5) IgA specific for heavy chain (dilution
1:800, Dako). Antigen binding epitope site exposure was
done by heating in a microwave oven placing the slides
in a Coplin jar containing citrate buffer (pH 6) for 12
min in three divided cycles. Detection system used was
Envision (Dako). Di-aminobenzedine (DAB) was used
for labeling and samples were counterstained lightly with
haematoxylene.
The controls included positive control and normal
control: (1) CD20-positive non-maltomatous high grade
diffuse lymphoma of jejunum served as positive control;
and (2) normal intestinal mucosa from margin of surgically
resected small intestine served as normal control.
Immunostainings were interpreted with respect
to positively stained plasma cells and lymphocytes in
percentages of the total cells counted. Mean values were
calculated. Light chain over-expression was documented
when more than 75% of plasma cells and lymphocytes
showed positivity for one of the light chains.
Informed consent was obtained from all patients
before subjecting them to endoscopic procedure. Ethic
Committee of the Institute approved the protocol.
Statistical analysis
Chi-square test and paired Students t test were used
for comparison. P value less than 0.05 was considered
statistically significant.
RESULTS
The case collection ranged over thirteen years period. The
clinical details are depicted in Table 1. Besides anemia,
1 patient had low platelet count (37 000 /mL). Urinary
D-xylose excretion test (within 5 h after 5 g of oral
D-xylose administration) was abnormal in 10 patients
(normal > 1 g). Nine had increased fecal fat (7.67-30
g in 24 h, nor mal < 6 g/24 h). Abnor mal D-xylose
excretion test was recorded in 3 stage A and 7 stage B
IPSID patients. Fecal fat level was increased in 3 and 4
patients with stage A and stage B IPSID, respectively.
Stool examination for ova, cyst and parasite were negative
in all patients. All cases had increased level for serum
IgA. Pattern of intestinal involvement in radiology and
endoscopy are shown in Table 1. Diagnosis of IPSID
was based on demonstration of alpha heavy chain in
serum and increased IgA levels (range, -340 to 660 mg%).
Histology of all the 11 cases showed heavy plasma cell
infiltration of the lamina propria admixed with variable
amount of lymphocytes and scattered eosinophils. As
a result of heavy mucosal cellular infiltration, increased
mucosal thickness and wide spacing of the glands were
observed (Figure 1). The mucosal infiltrating cell showed
extension to sub-mucosa in 7 patients, and scattered
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 3604 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

Table 1 Clinical parameters, histological staging, treatment profiles and disease outcome

st nd rd th th th th th th th th
Parameters 1 2 3 4 5 6 7 8 9 10 11

Sex M M M M M M F M M F M
Age (yr) 32 20 48 28 51 45 48 21 34 27 39
Clinical features
Diarrhea + + + + - + + + + + +
Weight loss + + + + - + + + + + +
Pain abd + + - + + + - + + - +
Clubbing + - + + + - + + - - +
Anemia + + - - + - + + + + -
Fever + - - - + + - - - + -
Endoscopy
Stomach - - - + Infl - - - - - +N -
Duodenum + + +(U) + + +(U) - + +(U) +(U) +
Jejunum + + + + + + + + + +(U) +
Ileum - - - - - - + - + + -
Colon - - - - - - - - - + -
Radiology
Mesenteric LN - + + + - + - + + + -
Hepatomegaly - - - - - - - - + + -
Splenomegaly - - - - - - - - - + -
Histology
(S+D+J+C+R)
Number of tissue 4+4+0+0+0 4+4+0+0+0 5+3+0+0+0 2+4+4+0+0 3+4+0+0+0 4+5+0+0+0 4+5+0+0+0 3+5+0+0+0 3+4+3+0+0 2+3+3+3+4 4+4+0+0+0
Galians stage A A A A, HP B B B B B B B
Light chain -ive -ive -ive -ive
over-expression
Drugs received Dox Dox Dox Anti-HP Dox Dox Dox Dox Dox Dox Dox
Chm Chm Chm Chm Chm
Disease relapse - + + - - - + - + + -
Follow-up (mo) 120 36 29 40 165 93 No FU 46 No FU No FU 57

Abbreviations: Infl = inflammation, N = nodular, U = ulceration, + = disease present, - = disease absent, HP = H pylori, Dox = doxycycline, Chm = Chemotherapy
(CHOP regime), Anti-HP = anti-H pylori, S = stomach, D = duodenum, J = jejunum, C = colon, R = rectum.

Table 2 Immunoprofiles of the groups studied (mean SD)

Immuno-markers Stage A Stage B Lymphoma Normal

Syndecan-1 69 3.40 51 10.25 10.1 6.354 11 7.605

bcl6 1.75 1.63 3.64 3.541 4.25 4.152 0.55 0.51
p53 3.15 3.257 6.5 2.914 9.2 4.315 0.11 0.901
light chain 67.51 16.952 67.42 18.95 55 2.399 42 13.475
light chain 35 13.857 24 1.346 25.5 1.087 29 12.356
IgA heavy chain 72 4.570 62 24.521 22 2.972 36 6.513

Figure 1 Photomicrograph of mucosal biopsy showing sheets
of plasma cells infiltrating the lamina propria. Epithelial cell lining
the glands are well preserved (HE, 240).
immature plasma cells were also observed in 5 cases. One
patient had histological evidence of active gastritis with
H pylori in the gastric antral biopsy. In remaining cases,
antral biopsies showed mild chronic inflammation
of superficial mucosa and were negative for H pylori.
Histologically (Galians staging), stage A was found in 4
cases (1 case with H pylori infection) and stage B in 7 cases.
Bone marrow was examined in 7 patients of stage B, but
no atypical cells were detected in any biopsies.
Immunohistochemistry
Total number of positively stained cells was expressed in
percentages and a total of 1000 cells were counted for
each biopsy (Table 2).
Syndecan-1 expression: As shown in Figure 2, plasma
cells had strong cytoplasmic positivity. Epithelial,
endothelial and stromal cells showed faint cytoplasmic
positivity. Only the plasma cells were counted and
expressed in percentag es. T he mean values were
significantly higher in IPSID patients compared to normal
and lymphoma controls (P < 0.05). Stage A cases though
had higher expression of syndecan-1 compared to stage B
IPSID, the difference did not reach statistical significance.
bcl6 expression: As shown in Figure 3, bcl6 expression

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 Vaiphei K et al. Immunoprofiles in IPSID 3605
Figure 2 Photomicrograph showing strong syndecan-1-positive cells (PAP, 440).
Figure 3 Photomicrograph showing bcl6-positive cells (PAP, 440).
Figure 4 Photomicrograph showing p53 positivity in nuclei (PAP, 1000).
was low on the whole; higher number of positively stained
cells were seen in stage B IPSID and lymphoma control
than stage A IPSID and normal control. Four of normal
controls failed to stain. The differences between the mean
values were not significant.
p53 expression: As shown in Figure 4, p53 expression
was low and comparable amongst the different groups.
Lymphoma had maximum number of positive cells, while
lowest or negligible positive cells were observed in normal
control. Stage A IPSID and normal control showed
significantly lower mean values compared to lymphoma
control (P < 0.05). However, no significant difference was
observed between stage A, stage B and normal control.
Light chains
light chain: The expression of light chain was seen
both in plasma cell and lymphocyte. Highest value was
detected in stage A patients ranging from 12% to 80%;
two patients had 78% and 80%, respectively, one patient
had 12%, and the 4th patient had 58%. Stage B patients
had the values ranging from 46% to 87%, and 4 patients
showed more than 75%. Lymphoma had a range of 18%
to 82% and 5 had more than 75%. Normal control had
a range of 39% to 53%. Stage A and B IPSID patients
had significantly higher values compared to normal
and lymphoma controls (P < 0.05). light chain over-
expression (> 75%) was seen in 2 of stage A and 4 of
stage B patients. Few lymphocytes and plasma cells failed
to stain.
light chain: The expression of light chain was found
to be inversely correlated with light chain expression.
There was a wide range for light chain expression. One
stage A patient with light chain over-expression had
a value of 78%. Those patients with light chain over-
expression had values less than 25%. Two of stage A
patients with light chain over-expression had 9% and
15% for light chain. The 4th stage A patient had 38%.
Stage B patients had values ranging from 9% to 40%. The
four patients with light chain over-expression had 9%,
15%, 20% and 18% of light chain, respectively. In the
rest 3 patients, no over-expression of any of the two light
chains had 30%, 36% to 40% for light chain. Lymphoma
control had a range of 12% to 29% and one had 69%.
Normal control had a range of 26% to 37%. Seven IPSID
patients (3 stage A and 4 stage B patients) showed light
chain over-expression (i.e. light chain in 6 and light
chain in 1).
Heavy chain
IgA heavy chain expression was higher in IPSID patients
compared to other groups. Stage A patients had values
ranging from 58% to 81% and stage B had 40% to 91%.
No significant differences were observed between stage A
and B IPSID patients. Similarly, no significant differences
were found between the patients of stage A and B with
light chain over-expression and without light chain over-
expression. Lymphoma control had values ranging from
13% to 30% and 5 of the control cases failed to express
heavy chain. Normal control had values ranging from 32%
to 45%. Stage A and B patients had significantly higher
values than the two controls (P < 0.05).
Treatment and outcome
All cases were treated either with doxycycline (100 mg
BD for 4 wk, followed by 100 mg OD for the next 6 to
12 mo) or tetracycline (500 mg TDS for 6 to 12 mo).
Three patients initially received anti-tubercular drugs for
a month with no clinical response. Subsequently, all three
were diagnosed as IPSID (1 stage A and 2 stage B) based
on demonstration of serum alpha heavy chain and heavy
plasma cell infiltration in the endoscopic biopsies.
Stage A IPSID: The patient with light chain over-
expression received 8 mo of doxycycline and remained
disease-free at 120 mo of follow-up. Two patients with
light chain over-expression responded initially to 9
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 3606 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
mo of doxycycline and developed sub-acute intestinal
obstruction at 18 and 11 mo of follow-up. Laparotomy
revealed patches of proximal jejunal loops thickening.
Resected intestinal segments showed trans-mural plasma
cell infiltration mixed with lymphocyte and scattered
immature plasma cell. Both patients received CHOP
regime for six cycles and remained disease-free at 36
and 29 mo of follow-up, respectively. One patient with
duodenal and proximal jejunal involvement had associated
H pylori-positive active gastritis. Following treatment for
H pylori (Omeprazole 20 mg BD, amoxycilin 500 mg BD
and clarithromycin 250 mg TDS for 2 wk), he showed
marked clinical improvement. Two weeks latter, repeat
radiology and endoscopy showed no residual disease
and no evidence of gastritis on repeat follow-up biopsy.
He remained disease-free at 40 mo of follow-up. This
particular patient did not have positivity for bcl6. The
other 2 stage A patients failed show positive staining for
bcl6. The 2 patients who developed frank lymphoma on
follow-up had higher p53 and low syndecan-1 expression.
Stage B IPSID: Three patients received doxycycline for 9,
9 and 12 mo, respectively; all remained disease-free at 165,
93 and 46 mo of follow-up, respectively. One patient with
light chain over-expression with hepatosplenomegaly and
involvement of splenic flexure and stomach responded
to 6 courses of CHOP and remained in remission for 6
mo. She went into relapsed with no response to treatment.
At autopsy, terminal part of small intestine and proximal
half of colon showed diffuse thickening with mucosal
nodularity. Histology showed features of immunoblastic
lymphoma with hepatic and splenic infiltration. One case
with light chain over-expression was disease-free at 57
mo of follow-up. Three patients with light chain over-
expression responded to doxycycline therapy and remained
in remission at 35, 42, and 57 mo of follow-up. The first
two patients had recurrence of the symptoms after 35
and 42 mo and biopsies from proximal jejunum revealed
high-grade lymphoma. Subsequently, both were put on
CHOP regime but failed to respond and died of the
disease. Syndecan-1 expression was higher in four patients
(57%, 61%, 65%, and 69%) and all remained disease-free
at follow-up compared to the patients who had disease
recurrence or died of the disease (36%, 40%, and 42%)
(P < 0.05). Those 3 patients with unfavorable prognosis
also had higher values for bcl6 but the differences were
not significant (P > 0.05) (8%, 7%, and 5%) than those
in remission (0.5%, 2%, 3%, and 3%). Expression of p53
was also obviously higher in those who succumbed to their
illness (i.e. 11.5%, 9%, and 7%) than the ones who were in
remission (1%, 4%, 5%, and 6%) (P < 0.05).
DISCUSSION
Syndecan-1 expression was found to be higher in all
cases of IPSID compared to other groups. This would
be in relation to number of plasma cell present. Higher
mean values observed in stage A than stage B would
suggest a phenotypic alteration of the cells i.e. benign to
malignant clone. Syndecan-1-positive cell was significantly
lower in lymphoma control cases (P < 0.01). Patients of
stage A and B IPSID with unfavorable disease outcome
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June 14, 2006 Volume 12 Number 22
also had low syndecan-1 expression compared to the
ones with favorable outcome (P > 0.05). A higher
syndecan-1 expression (> 50% positive) would infer a
better prognosis. Similarly, such distinctive difference in
the number of positive cell has been recorded in certain
other types of tumors[26-30]. bcl6 expression was weak and
low. Stage B IPSID patients and control lymphoma had
comparatively higher values than stage A IPSID patients
and other groups (P > 0.05). These three stage B cases
subsequently developed high grade tumor had higher
bcl6 expression. In our study, bcl6 appears to be a poor
prognostic indicator. Similar observations have been made
in diffuse large and marginal zone B-cell lymphomas and
nodular variant of Hodgkins lymphoma[31,34-36]. Besides,
bcl6 expression would infer IPSID cells originating from
germinal center B cell. Ariatti et al[33] made such suggestion
with respect to bcl6-positive lymphoma. Mutant form of
p53 is over-expressed by many malignant tumors including
lymphomas. Similar to bcl6 expression, higher p53 values
were recorded in those cases with poor outcome, whereas
lower value was seen in those cases who were in remission
at follow-up (P < 0.05). Minimum value was expressed
by normal control followed by stage A and B IPSID and
highest by lymphoma control.
Over-expression of one type of light chain was
exhibited by our cases, as had been observed in few other
studies[19-21,23]. Number of cases with light chain over-
expression was more than that of light chain. Over-
expression of light chain had also been reported in
one case by Isaacson et al[19,20]. Monoclonality, exhibited
by our patients, further strengthens the view expressed
in previous studies, as an inherent character of IPSID as
in lymphoma. However, monoclonality appeared not to
influence the ultimate outcome of disease process. But
study in more number of cases would be required before
drawing such conclusion. IgA heavy chain over-expression
was diffuse and uniformly strong in IPSID cases. This
character of IPSID could be interpreted with important
clinical implication. And this would help to identify and
differentiate patients of IPSID, especially stage A, from
other similar clinical conditions and investigative findings
including histology.
Similar to our cases of H pylori-positive stage A
IPSID, Fishbach et al[44] reported one patient in whom the
underlying disease was controlled following eradication
of H pylori. As stage A IPSID could be successfully
treated with antibiotics, this effect of anti-H pylori could
be more generalized rather than specifically related to
H pylori eradication. Exposure to a long-standing cause
and effect relationship between H pylori infection and
IPSID localizing to foregut requires critical analysis and
documentation in more number of cases. Gastric biopsy
in the rest of cases did not show evidence of H pylori
infection, though 1 case had evidence of infiltration by
underlying disease process.
Colonic and hepatosplenic involvement in IPSID is
usually seen only with overt lymphomatous conversion or
in recurrent disease[47-50]. Three of our patients had gastric
and colonic involvement at presentation with no overt
lymphomatous conversion. But within one-year period, all
the three cases developed overt lymphoma and two of the
 Vaiphei K et al. Immunoprofiles in IPSID 3607
cases succumbed to their illness in spite of the treatment
In conclusion, over-expression of syndecan-1, IgA and
light chain would help in differentiating stage A IPSID
from normal and stage B IPSID. Expression of bcl6 and
p53 would indicate an aggressive behavior and a more
advanced disease stage. bcl6 and p53 exhibited an inverse
relationship with syndecan-1 expression. Gastric H pylori
infection, though was seen only in one case with the active
gastritis, would be advisable to look for in all cases of
IPSID by carrying out gastric or pyloric antral biopsies.
ACKNOWLEDGMENTS
We would like to thank Ms Anu Khuller and Monica Ahuja
for carrying out the immunostainings, Ms Surekha Sharma
for typographical help and Ms Tranum Kaur (Research
Scholar) for statistical calculation.
REFERENCES
1 Seligmann M, Danon F, Hurez D, Mihaesco E, Preudhomme
JL. Alpha-chain disease: a new immunoglobulin abnormality.
Science 1968; 162: 1396-1397
2 Doe WF, Henry K, Hobbs JR, Jones FA, Dent CE, Booth CC.
Five cases of alpha chain disease. Gut 1972; 13: 947-957
3 Seligmann M. Alpha chain disease: immunoglobulin
abnormalities, pathogenesis and current concepts. Br J
CancerSuppl 1975; 2: 356-361
4 Eidelman S. Abdominal lymphoma with malabsorption.
JAMA 1974; 229: 1103-1104
5 Selzer G, Sherman G, Callihan TR, Schwartz Y. Primary small
intestinal lymphomas and alpha-heavy-chain disease. A study
of 43 cases from a pathology department in Israel. Isr J Med Sci
1979; 15: 111-123
6 Seligmann M, Mihaesco E, Frangione B. Alpha chain disease.
Ann N Y Acad Sci 1971; 190: 487-500
7 Tabbane F, Mourali N, Cammoun M, Najjar T. Results of
laparotomy in immunoproliferative small intestinal disease.
Cancer 1988; 61: 1699-1706
8 Ghoshal UC, Chetri K, Banerjee PK, Choudhuri G, Pal BB,
Dabadghao S, Dhar K, Naik S, Naik SR. Is immunoproliferative
small intestinal disease uncommon in India? Trop Gastroenterol
2001; 22: 14-17
9 Puri AS, Kumar M, Khan EM, Pandey R, Aggarwal R, Naik S,
Choudhuri G, Naik SR. Immunoproliferative small intestinal
disease: a frequently missed diagnosis. Indian J Gastroenterol
1996; 15: 46-48
10 Dhir V, Mohandas KM. Immunoproliferative small intestinal
disease (IPSID): the Indian scenario. Indian J Gastroenterol 1997;
16: 38
11 Nair S, Mathan M, Ramakrishna BS, Mathan VI. Immuno-
proliferative small intestinal disease in South India: a clinical
and immunomorphological study. J Gastroenterol Hepatol 1998;
13: 1207-1211
12 Mohandas KM, Nagral A. Epidemiology of digestive tract
cancers in India. II. Stomach, and gastrointestinal lymphomas.
Indian J Gastroenterol 1998; 17: 24-27
13 Ramot B, Many A. Primary intestinal lymphoma: clinical
manifestations and possible effect of environmental factors.
Recent Results Cancer Res 1972; 39: 193-199
14 Alpha-chain disease and related small-intestinal lymphoma: a
memorandum. Bull World Health Organ 1976; 54: 615-624
15 C a m m o u n M , J a a f o u r a H , T a b b a n e F , H a l p h e n M .
Immunoproliferative small intestinal disease without alpha-
chain disease: a pathological study. Gastroenterology 1989; 96:
750-763
16 Seligmann M, Rambaud JC. IgA abnormalities in abdominal
lymphoma (alpha-chain disease). Isr J Med Sci 1969; 5: 151-157
17 Seligmann M, Mihaesco E, Hurez D, Mihaesco C, Preud
homme JL, Rambaud JC. Immunochemical studies in four
cases of alpha chain disease. J Clin Invest 1969; 48: 2374-2389
18 Seligmann M, Rambaud JC. Alpha-chain disease: an
immunoproliferative disease of the secretory immune system.
Ann N Y Acad Sci 1983; 409: 478-485
19 Isaacson PG, Price SK. Light chains in Mediterranean
lymphoma. J Clin Pathol 1985; 38: 601-607
20 Isaacson PG, Dogan A, Price SK, Spencer J. Immu-
noproliferative small-intestinal disease. An immu-
nohistochemical study. Am J Surg Pathol 1989; 13: 1023-1033
21 Smith WJ, Price SK, Isaacson PG. Immunoglobulin gene
rearrangement in immunoproliferative small intestinal disease
(IPSID). J Clin Pathol 1987; 40: 1291-1297
22 Khojasteh A, Haghshenass M, Haghighi P. Current concepts
immunoproliferative small intestinal disease. A Third-World
lesion. N Engl J Med 1983; 308: 1401-1405
23 Galian A, Lecestre MJ, Scotto J, Bognel C, Matuchansky C,
Rambaud JC. Pathological study of alpha-chain disease, with
special emphasis on evolution. Cancer 1977; 39: 2081-2101
24 Carey DJ. Syndecans: multifunctional cell-surface co-receptors.
Biochem J 1997; 327 ( Pt 1): 1-16
25 Sanderson RD, Lalor P, Bernfield M. B lymphocytes express
and lose syndecan at specific stages of differentiation. Cell
Regul 1989; 1: 27-35
26 Ridley RC, Xiao H, Hata H, Woodliff J, Epstein J, Sanderson
RD. Expression of syndecan regulates human myeloma
plasma cell adhesion to type I collagen. Blood 1993; 81: 767-774
27 Dhodapkar MV, Abe E, Theus A, Lacy M, Langford JK,
Barlogie B, Sanderson RD. Syndecan-1 is a multifunctional
regulator of myeloma pathobiology: control of tumor cell
survival, growth, and bone cell differentiation. Blood 1998; 91:
2679-2688
28 Snchez-Beato M, Snchez-Aguilera A, Piris MA. Cell cycle
deregulation in B-cell lymphomas. Blood 2003; 101: 1220-1235
29 Millet VG, Casado Prez S, Alvarez Grande J, Hernando
Avendao L. Cataracts after renal transplantation. Br Med J
1972; 4: 47
30 Kurokawa H, Matsumoto S, Murata T, Yamashita Y,
Tomoyose T, Zhang M, Fukuyama H, Takahashi T.
Immunohistochemical study of syndecan-1 down-regulation
and the expression of p53 protein or Ki-67 antigen in oral
leukoplakia with or without epithelial dysplasia. J Oral Pathol
Med 2003; 32: 513-521
31 Dierlamm J, Wlodarska I, Michaux L, Stefanova M, Hinz
K, Van Den Berghe H, Hagemeijer A, Hossfeld DK. Genetic
abnormalities in marginal zone B-cell lymphoma. Hematol
Oncol 2000; 18: 1-13
32 Ree HJ, Kadin ME, Kikuchi M, Ko YH, Suzumiya J, Go JH.
Bcl-6 expression in reactive follicular hyperplasia, follicular
lymphoma, and angioimmunoblastic T-cell lymphoma with
hyperplastic germinal centers: heterogeneity of intrafollicular
T-cells and their altered distribution in the pathogenesis of
angioimmunoblastic T-cell lymphoma. Hum Pathol 1999; 30:
403-411
33 Ariatti C, Vivenza D, Capello D, Migliazza A, Parvis G,
Fassone L, Buonaiuto D, Savinelli F, Rossi D, Saglio G,
Gaidano G. Common-variable immunodeficiency-related
lymphomas associate with mutations and rearrangements of
BCL-6: pathogenetic and histogenetic implications. Hum Pathol
2000; 31: 871-873
34 Akasaka T, Lossos IS, Levy R. BCL6 gene translocation in
follicular lymphoma: a harbinger of eventual transformation
to diffuse aggressive lymphoma. Blood 2003; 102: 1443-1448
35 Wlodarska I, Nooyen P, Maes B, Martin-Subero JI, Siebert
R, Pauwels P, De Wolf-Peeters C, Hagemeijer A. Frequent
occurrence of BCL6 rearrangements in nodular lymphocyte
predominance Hodgkin lymphoma but not in classical
Hodgkin lymphoma. Blood 2003; 101: 706-710
36 Kwon MS, Go JH, Choi JS, Lee SS, Ko YH, Rhee JC, Ree HJ.
Critical evaluation of Bcl-6 protein expression in diffuse large
B-cell lymphoma of the stomach and small intestine. Am J Surg
Pathol 2003; 27: 790-798
37 Levine AJ. p53, the cellular gatekeeper for growth and
www.wjgnet.com
 3608 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
division. Cell 1997; 88: 323-331
38 Nelson WG, Kastan MB. DNA strand breaks: the DNA
template alterations that trigger p53-dependent DNA damage
response pathways. Mol Cell Biol 1994; 14: 1815-1823
39 el-Deiry WS, Harper JW, OConnor PM, Velculescu VE,
Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill DE,
Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest
and apoptosis. Cancer Res 1994; 54: 1169-1174
40 Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke
RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD.
Helicobacter pylori infection and gastric lymphoma. N Engl J
Med 1994; 330: 1267-1271
41 Wotherspoon AC, Doglioni C, Diss TC, Pan L, Moschini A, de
Boni M, Isaacson PG. Regression of primary low-grade B-cell
gastric lymphoma of mucosa-associated lymphoid tissue type
after eradication of Helicobacter pylori. Lancet 1993; 342: 575-577
42 Isaacson PG, Spencer J. Is gastric lymphoma an infectious
disease? Hum Pathol 1993; 24: 569-570
43 Fine KD, Stone MJ. Alpha-heavy chain disease, Mediterranean
lymphoma, and immunoproliferative small intestinal disease:
a review of clinicopathological features, pathogenesis, and
www.wjgnet.com
June 14, 2006 Volume 12 Number 22
differential diagnosis. Am J Gastroenterol 1999; 94: 1139-1152
44 Fischbach W, Tacke W, Greiner A, Konrad H. Regression of
immunoproliferative small intestinal disease after eradication
of Helicobacter pylori. Lancet 1997; 349: 31-32
45 el Saghir NS, Jessen K, Mass RE, al-Mofleh I, Santhosh-Kumar
CR, Hall AD, Stuart AE. Combination chemotherapy for
primary small intestinal lymphoma in the Middle East. Eur J
Cancer Clin Oncol 1989; 25: 851-856
46 Ramakrishna BS. Malabsorption syndrome in India. Indian J
Gastroenterol 1996; 15: 135-141
47 Colombel JF, Rambaud JC, Vaerman JP, Galian A, Delacroix
DL, Nemeth J, Duprey F, Halphen M, Godeau P, Dive C.
Massive plasma cell infiltration of the digestive tract. Secretory
component as the rate-limiting factor of immunoglobulin
secretion in external fluids. Gastroenterology 1988; 95: 1106-1113
48 Tashiro T, Sato H, Takahashi T, Genda T, Sugitani S, Yoshida
T, Funakoshi K, Tukada Y, Narisawa R, Nomoto M. Non-
secretory alpha chain disease involving stomach, small
intestine and colon. Intern Med 1995; 34: 255-260
49 Rhodes JM, Jewell DP, Janossy G. Alpha-chain disease
diagnosed by rectal biopsy. Br Med J 1980; 280: 1043-1044
S- Editor Wang J L- Editor Kumar M E- Editor Liu WF
 PO Box 2345, Beijing 100023, China
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wjg@wjgnet.com
Expression of E-selectin, integrin 1 and immunoglobulin
superfamily member in human gastric carcinoma cells and its
clinicopathologic significance
Jin-Jing Ke, Qin-Shu Shao, Zhi-Qiang Ling
Jin-Jing Ke, Qin-Shu Shao, Zhejiang Provincial Peoples
Hospital, Hangzhou 310014, Zhejiang Province, China
Zhi-Qiang Ling, Zhejiang Academy of Medical Sciences,
Hangzhou 310013, Zhejiang Province, China
Supported by the Science and Technology Key Project of
Zhejiang Province, No. 2002c33015
Correspondence to: Dr. Jin-Jing Ke, Zhejiang Provincial
People's Hospital, Hangzhou 310014, Zhejiang Province,
China. kejinjing@hotmail.com
Telephone: +86-571-88226336 Fax: +86-571-85239988
Received: 2005-09-30 Accepted: 2005-11-10
Abstract
AIM: To study the expression levels of E- selectin, in-
tegrin 1 and immunoglobulin supperfamily member-
intercellular adhesion molecule-1 (ICAM-1) in human
gastric carcinoma cells, and to explore the relationship
between these three kinds of cell adhesion molecules
and gastric carcinoma.
METHODS: The serum contents of E-selectin, integrin
1 and ICAM-1 were detected by enzyme-linked immuno-
sorbent assay (ELISA), in 47 healthy individuals (control
group) and in 57 patients with gastric carcinoma (gastric
carcinoma group) respectively prior to operation and 7 d
after operation.
RESULTS: The serum E-selectin, ECAM-1 and integrin 1
were found to be expressed in both control and gastric
carcinoma groups. However, they were highly expressed
in patients with gastric carcinoma patients before
operation or with unresectable tumours. The expression
levels of ICAM-1 and integrin 1 were significantly higher
in gastric carcinoma patients than in controls (P < 0.01).
A comparison of the E-selectin levels between the two
groups showed statistically insignificant difference (P
= 0.64). In addition, the expression levels were all de-
creased substantially in the postoperative patients sub-
jected to radical resection of the tumours, indicating that
the high level expressions of these compounds might be
the important factor for predicting the prognosis of these
patients.
CONCLUSION: Serum E-selectin, ICAM-1 and integrin
1 expression levels are probably related to the metasta-
sis and relapse of gastric cancer.
2006 The WJG Press. All rights reserved.
World J Gastroenterol 2006 June 14; 12(22): 3609-3611
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
RAPID COMMUNICATION
Key words: Human gastric carcinoma cells; E-selectin;
Integrin 1; ICAM-1; ELISA
Ke JJ, Shao QS, Ling ZQ. Expression of E-selectin, integrin
1 and immunoglobulin superfamily member in human gas-
tric carcinoma cells and its clinicopathologic significance.
World J Gastroenterol 2006; 12(22): 3609-3611
http://www.wjgnet.com/1007-9327/12/3609.asp
INTRODUCTION
The adhesion between endothelial cells and cancer cells
is the essential intermediate link for tumor metastasis
mediated by specific cellular surface receptors including
selectin, integrin and cell adhesion molecule family[1]. Many
investigations indicate that adhesion family members are
involved in the progression of cancer[2]. The binding of
cancer cells to endothelial cells mediated by the E- selectin
is reportedly related to their metastatic potentials[3]. The
intercellular adhesion molecule-1 (ICAM-1) a member of
the immunoglobulin superfamily, its ligand is the adhesion
molecule (LFA-1) of the immunoglobulin superfamily.
Integrin- 1 is a member of the adhesion superfamily,
which not only mediates the adhesion between cells
and extracellular matrix, but also mediates the adhesion
between leucocytes and blood vessel endothelial cells.
The expression of E-selectin, ICAM-1 and integrin 1 on
small blood vessels in tumour-infiltrating area at a high
level is capable of suggesting the interaction between
endothelial cells[4]. In the present study, we detected the
serum E- selectin, ICAM-1 and integrin 1 contents in
patients with gastric carcinoma in order to evaluate their
clinicopathologic significance.
MATERIALS AND METHODS
Patients and healthy individuals
A total of 57 patients with gastric carcinoma, including
31 males and 26 females, at the age of 34-77 years ,
with a median age of 55 years, in Zhejiang Provincial
Peoples Hospital and First Peoples Hospital of Taizhou
were diagnosed as gastric carcinoma by pathological
examination from January 2000 to December 2003. Ac-
cording to the WHO staging standard, 13 patients were
classified into stage I, 5 patients stage II, 31 patients stage
III, and 8 patients stage IV. These patients could also be
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 3610 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
classified into intestine type and diffuse type based on
Laurens histological typing principle, and classified into
well-, moderately- and poorly- differentiated types based
on the predominant differentiation mode, respectively. All
patients did not receive radiotherapy, chemotherapy, blood
transfusion, steroid and opium drugs prior to operation.
All the patients were followed up for 9 mo (1-48 mo) and
their dates and causes of death were recorded.
Forty-seven healthy individuals, consisting of 31 males
and 16 females, (at the age of 32-68 years, with a medium
age of 51), were recruited as the control group. They were
selected as blood donors and did not have any disease.
Assay procedure
The healthy individuals and patients were all fasted
overnight. Blood samples were collected from their
peripheral veins at 8-9 h AM next day, and then the blood
samples were stored at -80 after the sera were separated
by centrifugation. The blood samples from the patients
with gastric carcinoma were collected both before and 7 d
after operation respectively.
Assays for serum E-selectin, ICAM-1 and integrin
1 were carried out using the solid phase ELISA test kit
provided by the Parameter R &D Systems (USA), following
the manufacturers instructions. The test sensitivities
were estimated to be 1 ng/mL, 2 ng/mL and 0.3 ng/mL
for E-selectin, integrin 1 and ICAM-1, respectively. Any
resulting values for the patients which were 95% higher
than those for the control were defined as the elevated
contents of serum adhesion molecules. The cut-off values
of E-selectin, ICAM-1 and integrin 1 were 50.4 ng/mL,
337 ng/mL and 5.2 g/mL, respectively. Therefore, if the
cut-off values were higher than the above values, the high
level expression should be considered as positive.
Statistical anaiysis
All data were analyzed by the SPSS 10.0 statistical soft-
ware package, and their abnormal distributions were
distinguished from the normal ones. At the same time,
the variability of the medium values and the distributive
ranges for the experimental data were evaluated using the
unvariate analysis. The non-matched and matched data
were evaluated using the variate Kruskal-Wallis analysis
(analysis of variance, ANOVA), Mann-Whitney U test and
Wilcoxon grade-related test. The multivariate analysis was
conducted using Cox Proportional Hazards Regression
Mode after the predicted variability was determined using
the single factor analysis.
RESULTS
Contents of E-selectin, integrin 1 and ICAM-1 and their
comparison
The E-selectin, integrin 1 and ICAM-1 were detected
in all serum samples. The content of serum integrin 1
in the preoperative gastric carcinoma group and control
group was 4.8 g/mL and 2.1 g/mL, respectively (P =
0.00002). The content of ICAM-1 in the preoperative
gastric carcinoma group and control group were estimated
to be 271 ng/mL and 193 ng/mL, respectively. (P = 0.0004).
The content of E-selectin in the preoperative gastric
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June 14, 2006 Volume 12 Number 22
Table 1 Diagnostic value of elevated E- selectin, ICAM-1 and
integrin 1 levels in prediction of gastric carcinoma
Diagnostic value
E- selectin ICAM-1 Integrin 1
(> 50.4 ng/mL) (> 337 ng/mL) (> 5.2 g/mL)
Sensitivity (%) 24.6 33.3 28.1
Specificity (%) 95.7 95.7 95.7
Positive prediction
value (%)
87.5 90.5 88.9
Negative prediction
51.3 54.2 52.3
value (%)
Predominance ratio 7.32 11.25 8.78
95% confidence region 1.57-34.15 2.46-51.42 1.9-40.73
carcinoma group and control group was 42.1 ng/mL and
39.6 ng/mL, respectively (P = 0.64).
Expression levels of E-selectin, ICAM-1 and integrin 1 in
gastric carcinoma patients
The positive rates of E-selectin, ICAM-1 and integrin 1
expression in gastric carcinoma group were 25.0%, 32.7%
and 28.8%, respectively. The accuracy for diagnosing
gastric carcinoma based on the expressions of these 3
adhesion molecules is shown in Table 1.
In gastric cacinoma group, 38 patients died in the pro-
gressive stage of gastric carcinoma, and tumours relapsed
in 3 of the 19 remaining survivors. The serum E-selec-
tin, ICAM-1 and integrin 1 levels were higher in the
progressive stage of tumour, and the elevated levels were
significantly correlated with the tumour staging (P < 0.05).
No cor relations were found between tumour T
staging and serum E-selectin (P = 0.053) and ICAM-1 (P
= 0.1)level. The serum E-selectin level [47.5 (36.8-58.3)
ng/mL] in the patients with T4 tumour, was significantly
higher than that [33.8 (28.4-41) ng/mL] of the patients
with T1-3 tumour (P = 0.006) . Furthermore, the serum
ICAM-1 level [292 (201-437) ng/mL] in the patients with
T3 and T4 tumour-infiltrating serous membrane layer was
significantly higher than that [231 (154-266) ng/mL] of
the patients with T1 and T2 tumours in mucous membrane,
submucous layer and proper muscular layer (P = 0.023).
The serum expression levels of E- selectin, ICAM-1
and integrin in patients with lymph node metastasis were
higher than those in patients with no lymph node metas-
tasis (the former vs the latter: P = 0.04, 0.004, and 0.0018,
respectively). As compared with the non-remote metastatic
group, the remote metastatic group had higher serum E-
selectin, ICAM-1 and integrin 1 levels (P < 0.05). The
serum E-seletin, ICAM-1 and integrin 1 levels were not
correlated with the tumour location (gastric antrum, gastric
body or gastric cardia), tumour typing and differentiation
degree (Table 2).
Effect of surgery on expression of E-selectin, ICAM-1 and
integrin 1
Among the 57 patients with gastric carcinoma, 41 were
subjected to radical gastric resection and regiongal
lymph node cleaning, 16 had unresectable tumours. All
the patients had no postoperative complications. The
postoperative serum E-selectin, ICAM-1 and integrin
 Ke JJ et al. E-selectin, integrin 1 and ICAM-1 in gastric carcinoma cells 3611

Table 2 Variability analysis of the prognoses and predictive

of their tumours. Moreover, sICAM-1 could not be
factors in patients with gastric carcinoma produced through different forms of mRNA splicing[8,10].
In the present study, all the preoperative ser um
Variables Risk ratio
95% confidence
P E-selectin, ICAM-1 and integrin 1 levels in the patients
region
with gastric carcinoma were found to be an important
Age 0.97 0.94-1.01 0.2 factor affecting the prognosis of the patients, suggesting
Sex 0.77 0.39-1.54 0.47 that the levels of E-selectin, ICA-1 and integrin 1 can be
Tumor location (gastric antrum, gastric used as supplementary markers to determine the disease
0.95 0.62-1.45 0.81
body or gastric cardia )
Tumour histology
condition, stage and prognosis, as well as the therapeutical
(intestine type and diffusion type)
1.32 0.70-2.52 0.38 efficacy in the patients. However, multiple statistical analy-
Tymour differentiaton ses of all the factors have revealed that tumor staging
1.19 0.71-1.96 0.49
degree (high,moderate, low) could be used to predict the survival of cancer patients as
Tumour staging (stages I-IV) 2.65 1.71-4.11 0.000 an independent factor. The discrepancy between effects
T (T1-4) 2.75 1.75-4.34 0.000 of the adhesion molecule levels in peripheral circulation
N (No, N1) 4.33 1.65-11.36 0.003
on the prognosis of patients with gastric carcinoma in
M (Mo, M1) 7.36 2.98-18.2 0.000
different reports is possibly attributed to the different
E-selectin (elevated, normal) 2.81 1.40-5.63 0.003
number of patients used and the different progression
ICAM-1 (elevated, normal) 2.71 1.37-5.36 0.004
stage of the disease[9-11].
Integrin 1 (elevated, normal) 2.1 1.05-4.18 0.035

1 contents decreased significantly (P = 0.04, 0.01, and
0.001, respectively). Conversely, the postoperative serum
E-selectin, ICAM-1 and integrin 1 levels in the16 patients
with nonresectable tumours were similar to those before
operation (P = 0.08, 0.09, and 0.2, respectively).
Variability analysis of prognosis and predictive factors in
patients with gastric carcinoma
The monovariate analysis revealed that the TNM staging,
tumour-infiltrating deepth in gastroparietes (T status),
lymph node metastasis, remote metastasis, as well as the
preoperative serum E-selectin, ICAM-1 and integrin
1 levels were the important factors affecting the total
survival rate of the patients (Table 2). The multiple
statistical analysis of all the factors was carried out, the
results indicated that tumour staging was the only indepen-
dent factor for predicting the survival of the patients.
DISCUSSION
The present study demonstrated that the preoperative high
levels of ICAM-1, integrin 1 and E-selectin in patients
with gastric carcinoma had higher specificity and lower
sensitivity for the diagnosis of gastric carcinoma. The se-
rum contents of the three kinds of adhesion molecules
significantly correlated with tumour staging, gastropariete
infiltrating, lymph node metastasis and remote metastasis.
Although the relationship between ser um ICAM-1
and metastasis in the present study further proved the
previous observations, the correlation between the serum
E-selectin and integrin 1 contents and remote metastasis
is reportedly contradictory sometimes[5-8].
Previous investigations showed that certain kinds of
cytokines are able to induce the expression of E-selectin,
ICAM-1 and integrin 1 [9,10] . Monocytes are the only
origin of sICAM-1. Large amounts of sICAM-1 in serum
could be found in the culture of certain tumour cell
strains indicating that tumour cells may also be the origin
of sICAM-1 which may explain the decreased serum
sICAM-1 in patients with gastric carcinoma after resecting
REFERENCES
1 Fidler IJ. Critical determinants of metastasis. Semin Cancer Biol
2002; 12: 89-96
2 Ohene-Abuakwa Y, Pignatelli M. Adhesion Molecules as
Diagnostic Tools in Tumor Pathology. Int J Surg Pathol 2000; 8:
191-200
3 Ramphal JY, Hiroshige M, Lou B, Gaudino JJ, Hayashi
M, Chen SM, Chiang LC, Gaeta FC, DeFrees SA. Ligand
interactions with E-selectin. Identification of a new binding site
for recognition of N-acyl aromatic glucosamine substituents of
sialyl Lewis X. J Med Chem 1996; 39: 1357-1360
4 Tzeren A, Kleinman HK, Grant DS, Morales D, Mercurio AM,
Byers SW. E-selectin-mediated dynamic interactions of breast-
and colon-cancer cells with endothelial-cell monolayers. Int J
Cancer 1995; 60: 426-431
5 Gearing AJ, Hemingway I, Pigott R, Hughes J, Rees AJ,
Cashman SJ. Soluble forms of vascular adhesion molecules,
E-selectin, ICAM-1, and VCAM-1: pathological significance.
Ann N Y Acad Sci 1992; 667: 324-331
6 Alexiou D, Karayiannakis AJ, Syrigos KN, Zbar A, Kremmyda
A, Bramis I, Tsigris C. Serum levels of E-selectin, ICAM-1
and VCAM-1 in colorectal cancer patients: correlations with
clinicopathological features, patient survival and tumour
surgery. Eur J Cancer 2001; 37: 2392-2397
7 Sawada R, Tsuboi S, Fukuda M. Differential E-selectin-
dependent adhesion efficiency in sublines of a human colon
cancer exhibiting distinct metastatic potentials. J Biol Chem
1994; 269: 1425-1431
8 Yoo NC, Chung HC, Chung HC, Park JO, Rha SY, Kim JH,
Roh JK, Min JS, Kim BS, Noh SH. Synchronous elevation
of soluble intercellular adhesion molecule-1 (ICAM-1) and
vascular cell adhesion molecule-1 (VCAM-1) correlates with
gastric cancer progression. Yonsei Med J 1998; 39: 27-36
9 Okada M, Matsuto T, Miida T, Inano K. Differences in the
effects of cytokines on the expression of adhesion molecules in
endothelial cells. Ann Med Interne (Paris) 1997; 148: 125-129
10 Morita M, Watanabe Y, Akaike T. Inflammatory cytokines
up-regulate intercellular adhesion molecule-1 expression
on primary cultured mouse hepatocytes and T-lymphocyte
adhesion. Hepatology 1994; 19: 426-431
11 Nasu R, Mizuno M, Kiso T, Shimo K, Uesu T, Nasu J, Tomoda
J, Okada H, Tsuji T. Immunohistochemical analysis of
intercellular adhesion molecule-1 expression in human gastric
adenoma and adenocarcinoma. Virchows Arch 1997; 430:
279-283
12 Snchez-Rovira P, Jimenez E, Carracedo J, Barneto IC,
Ramirez R, Aranda E. Serum levels of intercellular adhesion
molecule 1 (ICAM-1) in patients with colorectal cancer:
inhibitory effect on cytotoxicity. Eur J Cancer 1998; 34: 394-398

S- Editor Wang J L- Editor Wang XL E- Editor Liu WF

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CASE REPORT
Peliosis hepatis as an early histological finding in idiopathic
portal hypertension: A case report
Annalisa Berzigotti, Donatella Magalotti, Paola Zappoli, Cristina Rossi, Francesco Callea, Marco Zoli
Annalisa Berzigotti, Donatella Magalotti, Paola Zappoli,
Marco Zoli, Dipartimento di Medicina Interna, Cardioangiologia,
Epatologia, Policlinico S. Orsola-Malpighi, Universit di Bologna,
Italy
Cristina Rossi, Dipartimento di Scienze Radiologiche ed
Istocitopatologiche, Policlinico S. Orsola-Malpighi, Universit di
Bologna, Italy
Francesco Callea, Servizio di Anatomia Patologica, Ospedale
Pediatrico Bambino Ges, IRCCS, Roma, Italy
Supported by a grant on Vascular Disorders of the Liver from
Dipartimento di Medicina Interna, Cardioangiologia, Epatologia,
Universit di Bologna to Dr. A Berzigotti
Correspondence to: Professor Marco Zoli, Dipartimento di
Medicina Interna, Cardioangiologia, Epatologia, Policlinico
S.Orsola-Malpighi, Via Massarenti, 9; 40138-Bologna,
Italy. marco.zoli@unibo.it
Telephone: +39-051-6362211 Fax: +39-051-6362210
Received: 2005-10-04 Accepted: 2005-11-10
Abstract
Peliosis hepatis is a rare condition characterized by
dilatation of hepatic sinusoids and blood-filled spaces
in the liver mainly observed in subjects exposed to
toxic substances or estrogens, which is frequently
asymptomatic. Non-cirrhotic idiopathic portal
hypertension (NCIPH) is also a vascular disease of
the liver rarely observed in European countries, which
is usually diagnosed only when the hemorrhagic
complications of portal hypertension occur. We report
a case of NCIPH in a young Caucasian male who was
diagnosed with liver peliosis, showing ultrasonographic
and endoscopic signs of portal hypertension four years
after. A second biopsy was diagnostic for NCIPH. Even if
the pathogenesis remains obscure, peliosis hepatis can
be considered as an early sign of vascular disease of the
liver, which may progress to more definite conditions.
2006 The WJG Press. All rights reserved.
Key words: Peliosis hepatis; Idiopathic portal hyper-
tension; Hepatic veins catheterisation
Berzigotti A, Magalotti D, Zappoli P, Rossi C, Callea F, Zoli M.
Peliosis hepatis as an early histological finding in idiopathic
portal hypertension: A case report. World J Gastroenterol
2006; 12(22): 3612-3615
http://www.wjgnet.com/1007-9327/12/3612.asp
www.wjgnet.com
World J Gastroenterol 2006 June 14; 12(22): 3612-3615
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
INTRODUCTION
Portal hypertension in Europe is mainly due to intra-
hepatic causes, almost exclusively by liver cirrhosis, which
leads to an increase in sinusoidal pressure. While the
identification of pre-hepatic causes such as extra-hepatic
portal vein thrombosis is now feasible by non invasive
means such as eco-color-Doppler and CT-scan, rarer forms
of intra-hepatic pre-sinusoidal portal hypertension such as
non cirrhotic idiopathic portal hypertension (NCIPH) are
difficult, and rely on invasive techniques such as hepatic
vein catheterisation and hepatic biopsy. We describe a
case of NCIPH developed in a young Italian man who
was first diagnosed as peliosis hepatis carrier. Hepatic
vein catheterisation confirmed the clinical suspicion, and
the diagnosis was finally ascertained by histology. This is
the first reported case in which NCIPH follows a peliosis
hepatis. This may represent an early histological finding in
case of vascular diseases of the liver.
CASE REPORT
A 36-year-old Italian man was referred to our unit for a
sonographic evaluation of chronic liver disease. He was
in very good clinical condition, did not drink alcohol, and
was neither exposed to toxic substances nor to any drugs.
The patient also denied any exposure to anabolic steroids.
Family history indicated a grandmother died of digestive
bleeding in the setting of end-stage primary biliary
cirrhosis (PBC). Relevant findings in patients history
were a persistent slight elevation of gamma-glutamyl-
transferases and aminotransferases associated with leuco-
and thrombocytopenia noticed for the first time at the age
of 26 years. At the age of 28 years, the patient suffered
from spontaneous bilateral sublaxation of the shoulder,
for which he underwent surgical repair. A muscle biopsy
taken during the intervention was normal. Due to the
persistence of abnormal laboratory findings mentioned
above, at 32 year-old he was examined in another hospital
and screened for chronic liver disease. Laboratory tests
excluded viral, metabolic and autoimmune causes of liver
disease. Abdominal ultrasound scan showed only a slight
enlargement of the spleen (longitudinal diameter 130
mm), while liver and portal system were normal, no sign
of portal hypertension was evident. A liver biopsy was
performed, showing the typical aspects of microscopic
peliosis. The patient also underwent a bone marrow
 Berzigotti A et al. Peliosis in idiopathic portal hypertension 3613

A B
350 PLT/mmc WBC/mmc
AST 160 6
ALT
300 GGT 140
ALP 5
250 Platelets
120
WBC
4
U/I

200 100

150 80 3

60
100 2
40
50
1
20
0
91 95 97 99 01 03 Yr 0 0
19 19 19 19 20 20 9 1 95 97 99 01 03 Yr
19 19 19 19 20 20

Figure 1 Aminotransferases and cholestasis enzymes (A) and white blood cells (WBC) and platelets (B) during follow-up.

Table 1 Laboratory findings on admission and after ursodeox-

stature was 1.65 m, his weight 70 Kg, arterial blood
ycholic acid (UDCA) pressure was 115/70 mmHg, and heart rate was 48 bpm.
He showed hypertrophy of proximal muscles of upper
During UDCA and lower limbs (the patient made no significant physical
On admission treatment
(one year)
activity), multiple small telangiectasias on the trunk and
Haemoglobin (g/L) 146 148 lower limbs, splenomegaly, and white striae on the flanks.
White cells ( 103/mL) 3.12 3.5 Figure 1 shows laboratory findings in patients history,
Neutrophils (%) 59.2 60.3 and Table 1 lists those at the moment of first visit at
Platelets ( 103/mL) 93 107 our unit. Alanine aminotransferasis (ALT) and gamma-
Creatinine (mg/dL) 1.08 0.9 glutamyl transferases (GGT) were persistently elevated
Na+(mEq/L) 143 140 during the last 10 years, but no sign of liver failure was
K+ (mEq/L) 3.9 4.1 present. Autoantibodies for PBC, primary sclerosing
AST (IU/L) 44 31 cholangitis, connective tissue diseases and autoimmune
ALT (IU/L) 47 31 thyroiditis tested were negative. Inherited thrombogenic
ALP (IU/L) 241 235
disorders and hematologic diseases were also excluded.
GGT (IU/L) 95 67
An electrocardiogram showed sinus bradycardia (48 bpm)
CPK (IU/L) 182 160
but no other pathological aspects. An upper digestive tract
LDH (IU/L) 284 225
INR
endoscopy confirmed portal hypertension showing small
1.03 1.11
Albumin (g/L) 4.3 4.1
esophageal varices. The patient also underwent an invasive
Bilirubin (mg/dL) 1.04 0.6
hepatic hemodynamic study. By accessing via right brachial
Cholesterol (mg/dL) 212 228 vein under fluoroscopic control, a 5F balloon-catheter
Triglycerides (mg/dL) 81 89 was advanced in the main hepatic vein and hepatic venous
pressure gradient (HVPG) was obtained by the difference
between wedged (by inflating the balloon) and free
pressures. Figure 2 shows tracing of hepatic vein pressures.
biopsy in the same year, due to persistent leuco- and
thrombocytopenia, which revealed a normal pattern. Free pressure was normal, wedge pressure and HVPG
The Echo-color-Doppler perfor med at our unit were only slightly elevated (HVPG 7 mmHg, normal value
demonstrated a coarse liver echo-texture, splenomegaly 1-5 mmHg), suggesting idiopathic portal hypertension. A
(longitudinal diameter 155 mm), patency of portal vein needle liver biopsy was then obtained. Liver tissue samples
and intrahepatic branches, with normal portal blood flow were examined by the same experienced pathologist (F.C.)
velocity (15 cm/s, normal value > 14), patency of splenic who studied the first ones. Histology showed periportal
vein and superior mesenteric vein, and US signs of portal fibrosis, obliteration of small portal veins and some areas
hypertension, namely ectasia of left gastric vein with of nodular regenerative hyperplasia (NRH), which were
hepatofugal flow and a small spontaneous spleno-renal consistent with the diagnosis of NCIPH. No alteration of
shunt. Intraparenchymal branches of hepatic and splenic bile ducts was noted nor cholestasis. Peliosis hepatis was
artery showed high resistance and pulsatility indices. no more observable.
Hepatic veins were patent and showed a normal triphasic Due to the slightly cholestatic feature of liver disease,
flow. we empirically treated the patient with a high dosage (15
Since portal hypertension had not been detected mg/Kg of body weight) of ursodeoxycholic acid (UDCA).
before, the patient was hospitalized to perform a diagnostic After one year of treatment GGT decreased to near-
procedure. normal levels and aminotransferases became normal (Table
Physical examination demonstrated that the patient's 1 and Figure 1A).

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 3614 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
30
20
mmHg
Wedge P
10
-10
DISCUSSION
Peliosis hepatis is a rare disorder characterized by the
presence of cystic, blood-filled spaces of variable
size in the liver [1]. Endothelial cells cover the internal
surface of these cavities, and adjacent hepatocytes can
be normal or show atrophic or degenerative changes[2].
A microscopic for m and a macroscopic for m have
been described. In the microscopic form ultrasound
and CT do not show any macroscopic change in the
liver. It has been reported mainly in adult patients in
association with chronic diseases (AIDS, malnutrition,
tuberculosis, lepropsy, vasculitis, haematological neoplasias,
hepatocellular adenoma and hepatocarcinoma), various
pharmacological agents (androgenic anabolic steroids and
estrogens, immunosuppressive drugs, tamoxifene), toxic
substances (arsenic, vinyl chloride), infections (Bartonella
henselae)[3], and in renal transplant recipients receiving
immunosuppressive treatment [4,5]. The morphogenesis
of peliosis is controversial. It has been attributed to an
increased sinusoidal pressure because of difficulties in
blood outflow from the liver, the disappearance of normal
parenchyma by necrosis of liver cells, and sinusoid wall
weakness. Since the disease is very rare, data about its
natural history are scarce and clinical spectrum varies
from asymptomatic cases to severe complications, such as
hemoperitoneum[3].
Idiopathic non-cirrhotic intrahepatic portal hyper-
tension (NCIPH) was first identified by Banti in 1898[6].
An increased portal pressure with patent portal and
hepatic veins characterizes it, in the absence of cirrhosis[7].
The anatomic basis for these alterations is an increased
fibrous component in portal vessel wall and periportal and
perisinusoidal space, inducing a pre- and perisinusoidal
resistance to flow of portal venous blood. Therefore
wedge hepatic venous pressure does not reflect the
increased pre-sinusoidal portal pressure and HVPG is
usually normal or near-normal[8].
NCIPH is clinically characterized by the signs of portal
hypertension (varices and portosystemic collateral vessels),
overt splenomegaly with pancytopenia, and relatively mild
abnormalities in liver function tests[9]. Bilirubin is reported
to be normal in NCIPH series, but literature does not
mention GGT levels. To our knowledge this is the first
reported case in which GGT is elevated at presentation.
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June 14, 2006 Volume 12 Number 22
Figure 2 Hepatic hemodynamic
findings consistent with the
diagnosis (wedge pressure 12
mmHg, free pressure 5 mmHg,
hepatic venous pressure gradient
7 mmHg).
Free P
While NCIPH is quite common in India and Japan, it
is rarely observed in developed countries[10,11]. The clinical
course of the disease is usually silent until gastrointestinal
bleeding secondary to variceal rupture develops. If variceal
bleeding can be controlled, 5-year survival rate is about
95%[9].
The recognized causes are varied. Schistosomiasis[12]
and toxic substances (arsenic, vitamin A) are the most
frequent ones, interestingly the latter ones are the same
agents recognized as causes of peliosis hepatis. In many
cases the etiology remains unknown, and the disease
is defined idiopathic. In these cases immunological
disturbance (autoimmune diseases such as scleroderma,
mixed connective tissue disease, autoimmune thyroiditis),
thrombophilic status, latent myeloproliferative disorders,
infections and/or increased fibrogenesis in the portal tract
are suspected as being candidates for the primary agents[13].
Hillaire and colleagues[14] have recently analysed a series of
patients with NCIPH identifying a thrombophilic status
(overt or latent) in the majority, but this could be excluded
in the case we report.
As for biopsy features, many pathological entities such
as idiopathic portal hypertension, benign intrahepatic
portal hypertension, hepatoportal sclerosis, nodular
regenerative hyperplasia, and incomplete septal cirrhosis
can be grouped under the term idiopathic NCIPH, as they
frequently show a number of overlapping features[14,15].
The finding of peliosis hepatis in the first biopsy of our
patient suggests that this entity may represent a praecox
presentation of the vascular changes, which occur in
NCIPH. Our observation is supported by the paper of
Cavalcanti and colleagues[4], who performed serial liver
biopsies in a group of kidney-transplanted patients with
peliosis hepatis, showing that the disease can evolve,
leading to regenerative nodular hyperplasia, perisinusoidal
fibrosis and cirrhosis in some cases.
Another interesting finding in our patient is a second
grade relative affected by primary biliary cirrhosis (PBC).
Among autoimmune diseases, PBC has been shown
to have a strong heritability[16], and some familial cases
describing the involvement of second-degree relatives have
been reported[17,18]. PBC is a cause of non-cirrhotic portal
hypertension in the first stage of the disease, and some
cases of PBC resembling NCIPH have been previously
reported[19]. Even if bile ducts were found to be normal in
 Berzigotti A et al. Peliosis in idiopathic portal hypertension 3615
our patients biopsy and no clear evidence of an overlap
could be proved, interestingly an empiric treatment with
UDCA induced a normalization of aminotransferases and
a reduction of GGT (Table 1).
When all the known causes of NCIPH are excluded the
finding of previous bilateral glenohumeral instability, white
striae at flanks, hypertrophy of proximal muscles of upper
and lower limbs and peliosis hepatis in the same individual
make conceivable to hypothesize that some unrecognized
disturbances of connective tissue can be linked to all these
conditions, eventually leading to NCIPH.
REFERENCES
1 Spech HJ, Liehr H. Peliosis hepatis. [A clinical status
inventory]. Z Gastroenterol 1982; 20: 710-721
2 Nieves Cereceda C, Sols Herruzo JA, Muoz-Yage MT, De
Blas C. Hepatic peliosis. [Review of the literature]. Rev Esp
Enferm Apar Dig 1989; 75: 205-211
3 DeLeve LD. Vascular liver diseases. Curr Gastroenterol Rep
2003; 5: 63-70
4 Cavalcanti R, Pol S, Carnot F, Campos H, Degott C, Driss F,
Legendre C, Kreis H. Impact and evolution of peliosis hepatis
in renal transplant recipients. Transplantation 1994; 58: 315-316
5 Izumi S, Nishiuchi M, Kameda Y, Nagano S, Fukunishi T,
Kohro T, Shinji Y. Laparoscopic study of peliosis hepatis and
nodular transformation of the liver before and after renal
transplantation: natural history and aetiology in follow-up
cases. J Hepatol 1994; 20: 129-137
6 Banti G. Splenomegalie mit Leberzirrhose. Beitr Pathos Anat
1898; 24: 21-33
7 Okudaira M, Ohbu M, Okuda K. Idiopathic portal hyper-
tension and its pathology. Semin Liver Dis 2002; 22: 59-72
8 Sarin SK, Sethi KK, Nanda R. Measurement and correlation
of wedged hepatic, intrahepatic, intrasplenic and intravariceal
pressures in patients with cirrhosis of liver and non-cirrhotic
portal fibrosis. Gut 1987; 28: 260-266
9 Sarin SK. Non-cirrhotic portal fibrosis. J Gastroenterol Hepatol
2002; 17 Suppl 3: S214-S223
10 Nakanuma Y, Tsuneyama K, Ohbu M, Katayanagi K.
Pathology and pathogenesis of idiopathic portal hypertension
with an emphasis on the liver. Pathol Res Pract 2001; 197: 65-76
11 Dhiman RK, Chawla Y, Vasishta RK, Kakkar N, Dilawari
JB, Trehan MS, Puri P, Mitra SK, Suri S. Non-cirrhotic portal
fibrosis (idiopathic portal hypertension): experience with 151
patients and a review of the literature. J Gastroenterol Hepatol
2002; 17: 6-16
12 Da Silva LC, Carrilho FJ. Hepatosplenic schistosomiasis.
Pathophysiology and treatment. Gastroenterol Clin North Am
1992; 21: 163-177
13 Matsumoto T, Kobayashi S, Shimizu H, Nakajima M,
Watanabe S, Kitami N, Sato N, Abe H, Aoki Y, Hoshi T,
Hashimoto H. The liver in collagen diseases: pathologic study
of 160 cases with particular reference to hepatic arteritis,
primary biliary cirrhosis, autoimmune hepatitis and nodular
regenerative hyperplasia of the liver. Liver 2000; 20: 366-373
14 Hillaire S, Bonte E, Denninger MH, Casadevall N, Cadranel
JF, Lebrec D, Valla D, Degott C. Idiopathic non-cirrhotic
intrahepatic portal hypertension in the West: a re-evaluation
in 28 patients. Gut 2002; 51: 275-280
15 Ibarrola C, Colina F. Clinicopathological features of nine cases
of non-cirrhotic portal hypertension: current definitions and
criteria are inadequate. Histopathology 2003; 42: 251-264
16 Selmi C, Invernizzi P, Zuin M, Podda M, Gershwin ME.
Genetics and geoepidemiology of primary biliary cirrhosis:
following the footprints to disease etiology. Semin Liver Dis
2005; 25: 265-280
17 James SP, Jones EA, Schafer DF, Hoofnagle JH, Varma RR,
Strober W. Selective immunoglobulin A deficiency associated
with primary biliary cirrhosis in a family with liver disease.
Gastroenterology 1986; 90: 283-288
18 Bach N, Schaffner F. Familial primary biliary cirrhosis. J
Hepatol 1994; 20: 698-701
19 Kasuga Y, Kitajima S, Isobe H, Nakajima T, Kodama T,
Yamamoto M, Ohkubo I, Okano T, Yasunaga Y, Yoneshi-ma
M. [A case of primary biliary cirrhosis presenting histopatho-
logical similarity to idiopathic portal hypertension with
huge splenomegaly]. Nihon Shokakibyo Gakkai Zasshi 1995; 92:
1776-1781
S- Editor Wang J L- Editor Wang XL E- Editor Liu WF
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LETTERS TO THE EDITOR
Noni juice is not hepatotoxic
Brett J West, C Jarakae Jensen, Johannes Westendorf
Brett J West, C Jarakae Jensen, Research and Development
Department, Tahitian Noni International, American Fork, Utah,
United States
Johannes Westendorf, University Medical School of Hamburg,
Department of Toxicology, Hamburg, Germany
Correspondence to: Brett J West, Research and Development,
Tahitian Noni International, American Fork, UT 84003,
United States. brett_west@tni.com
Telephone: +1-801-2343621 Fax: +1-801-2341030
Received: 2006-02-24 Accepted: 2006-03-20
Abstract
Noni juice (Morinda citrifolia ) has been approved for use
as a safe food within the European Union, following a
review of safety. Since approval, three cases of acute
hepatitis in Austrian noni juice consumers have been
published, where a causal link is suggested between
the liver dysfunction and ingestion of anthraquinones
from the plant. Measurements of liver function in a
human clinical safety study of TAHITIAN NONI Juice,
as well as subacute and subchronic animal toxicity
tests revealed no evidence of adverse liver effects at
doses many times higher than those reported in the
case studies. Additionally, M. citrifolia anthraquinones
occur in the fruit in quantities too small to be of any
toxicological significance. Further, these do not have
chemical structures capable of being reduced to reactive
anthrone radicals, which were implicated in previous
cases of herbal hepototoxicity. The available data reveals
no evidence of liver toxicity.
2006 The WJG Press. All rights reserved.
Key words: Noni juice; Morinda citrifolia ; Novel food;
Human clinical safety study
West BJ, Jensen CJ, Westendorf J. Noni juice is not
hepatotoxic. World J Gastroenterol 2006; 12(22):
3616-3619
http://www.wjgnet.com/1007-9327/12/3616.asp
TO THE EDITOR
Morinda citrifolia (noni) fruit juice was approved as a novel
food ingredient in pasteurized fruit drinks by the European
Commission Decision of 05-06-2003. This approval was
based on the opinion of the EU Scientific Committee on
Food (SCF) of Tahitian Noni Juice, following a safety
review of this product[1].
www.wjgnet.com
World J Gastroenterol 2006 June 14; 12(22): 3616-3619
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
During 2004, an Austrian case report was published
in which it is suggested that noni fruit juice may be
responsible for acute hepatitis [2]. A second report was
published in the August 2005 issue of this journal in which
noni juice was again suggested to be the cause of two other
cases of acute hepatitis in Austria[3]. In both publications,
the authors suggest the presence of anthraquinones in the
juice may be responsible for the liver dysfunction observed
in their patients. However, experimental data fails to reveal
any direct liver toxicity from noni juice.
TAHITIAN NONI Juice, the brand associated with
the latest case reports, was investigated in a human clinical
study (unpublished data. Mugglestone C et al. A single
centre, double-blind, three dose level, parallel group,
placebo controlled safety study with TAHITIAN NONI
Juice in healthy subjects, BIBRA International Ltd. UK,
2003). Ninety-six subjects were randomly assigned to four
groups. These groups included a placebo group and three
test groups, receiving up to a dose of 750 mL TAHITIAN
NONI Juice per day. For 28 d, subjects drank 750 mL of
either the placebo or juice containing one of three doses
of TAHITIAN NONI Juice.
Several parameters were investigated with measure-
ments being made at study screening, d 0 (baseline), wk
2, wk 4, and wk 6. The measurements most applicable to
the evaluation of liver function and hepatocellular disease
were alkaline phosphatase (ALP), alanine aminotransferase
(ALT), aspartate aminotransferase (AST), total bilirubin
(BIL), gamma-glutamyl transferase (GGT), total protein,
and prothrombin time (which is useful for determining the
extent of hepatocellular disease).
Other study measurements included hemoglobin,
hematocrit, mean cell volume, red cell count, activated
partial thrombin time, total and differential white cell
count, platelet count, lipids (LDL, HDL, cholesterol,
triglycerides), creatine kinase, creatinine, gamma-
glutamyl transferase, glucose, total protein, and uric
acid. Additionally, urinalysis involved semi-quantitative
("dipstick") analysis for leucocytes, nitrite, urobilinogen,
protein, pH, blood, specific gravity, ketones, bilirubin and
glucose. Where necessary, a urine cyto-bacteriological
examination was performed to characterize or count
crystals, casts, epithelial cells, white blood cells, red blood
cells and bacteria. Vital signs were measured, including
systolic and diastolic blood pressure and heart rate. ECG
measurements (12 lead) were also made for each subject.
All adverse events were recorded. Selected mean laboratory
values for the various groups are presented in Table 1.
Differences between mean values were clinically
insignificant and well within the range of normal values.
Furthermore, there was no evidence of any dose-related
adverse events. The study results indicate that TAHITIAN
 West BJ et al. Noni juice is not hepatotoxic 3617

st
Table 1 Mean clinical laboratory values by week and dose Table 3 Serum values in male Sprague Dawley rats in 1 13-wk
groups study (Mean SD)

Parameter Value Placebo 30 mLTNJ 30 mL TNJ 750 mLTNJ Males

ALP (U/L) Mean wk 0 61.67 58.65 54.00 54.44 Dose ALT (kat/L) AST (kat/L) ALP (kat/L) BIL (mol/L) GGT (kat/L)
Mean wk 2 55.80 57.83 51.16 54.47 Control 2.47 0.42 2.11 0.58 2.97 0.33 1.59 0.32 0.01 0.01
Mean wk 4 63.56 59.48 54.99 53.07 0.4 mL/kg 2.27 0.52 1.8 0.56 2.82 0.39 1.5 0.41 00
Mean wk 6 62.69 61.50 58.22 55.04 4 mL/kg 2.35 0.44 1.86 0.32 2.77 0.36 1.5 0.36 00
ALT (U/L) Mean wk 0 17.80 18.00 17.88 21.98 8 mL/kg 2.4 0.61 2.06 0.76 3.18 0.44 1.6 0.49 0.01 0
Mean wk 2 18.53 19.53 17.80 24.11
Mean wk 4 18.99 20.10 17.75 22.71
Mean wk 6 19.21 16.90 17.63 20.92
AST (U/L) Mean wk 0 18.30 18.55 18.27 20.71 Table 4 Serum values in female Sprague Dawley rats in 2
nd

Mean wk 2 18.04 19.67 19.18 22.32 13-wk study (Mean SD)

Mean wk 4 18.93 19.55 19.19 20.90
Mean wk 6 18.42 18.95 18.64 19.87 Females
Total Mean wk 0 12.12 10.55 10.93 11.30 Dose ALT (kat/L) AST (kat/L) ALP (kat/L) BIL (mol/L) GGT (kat/L)
bilirubin Control 2.01 0.3 1.73 0.43 2.24 0.36 1.01 0.77 0.01 0.01
(mol/L) Mean wk 2 13.19 11.31 11.47 14.20 20 mL/kg 2.14 0.52 1.99 0.71 2.44 0.47 1.17 0.72 0.01 0.01
Mean wk 4 12.23 11.63 12.07 14.03 50 mL/kg 1.91 0.53 1.82 0.43 2.14 0.43 1.24 0.58 0.01 0.01
Mean wk 6 14.77 11.99 11.44 13.98 80 mL/kg 1.25 0.25 1.22 0.12 1.98 0.22 1.62 0.77 0.01 0.01
GGT (U/L) Mean wk 0 20.31 24.74 22.51 21.36
Mean wk 2 21.19 26.74 23.03 20.99
Mean wk 4 21.87 26.54 21.14 21.21
Mean wk 6 21.11 24.22 20.00 21.07 nd
Table 5 Serum values in male Sprague Dawley rats in 2 13-wk
study (Mean SD)

Males
Table 2 Serum values in female Sprague Dawley rats in 1st
Dose ALT (kat/L) AST (kat/L) ALP (kat/L) BIL (mol/L) GGT (kat/L)
13-wk study (Mean SD)
Control 2 0.26 1.83 0.21 3.51 0.42 1.23 0.25 00
Females 20 mL/kg 1.95 0.3 1.79 0.31 3.09 0.47 0.93 0.56 00
50 mL/kg 1.55 0.2 1.53 0.28 3.17 0.38 1.27 0.45 00
Dose ALT (kat/L) AST (kat/L) ALP (kat/L) BIL (mol/L) GGT (kat/L)
80 mL/kg 1.67 0.4 1.77 0.47 3.27 0.29 1.03 0.45 00
Control 2.1 0.5 2.26 0.46 2.07 0.27 1.21 0.61 0.02 0.01
0.4 mL/kg 1.89 0.62 2.15 0.81 2.22 0.29 1.43 0.52 0.01 0.01
4 mL/kg 1.98 0.34 1.99 0.39 2.08 0.41 1.36 0.54 0.01 0
8 mL/kg 1.94 0.33 1.74 0.18 2.3 0.45 1.22 0.63 0.01 0
TAHITIAN NONI Juice-A 13-wk oral (gavage) toxicity
study in rats. Scantox Biologisk Laboratorium, Lille
Skensved, Denmark, Lab no. 35 207, March 2000; and
NONI Juice is safe to consume, in quantities up to Glerup P. TAHITIAN NONI Juice-A 13-wk oral (gavage)
750 mL/d, confirming the EU SCF opinion that high toxicity study in rats. Scantox Biologisk Laboratorium, Lille
consumption quantities are appropriate. Skensved, Denmark, Lab no. 39 978, May 2001). In each
The aqueous extract of M. citrifolia fruit was evaluated study, 80 Sprague Dawley rats (40 males and 40 females)
in a repeat dose oral toxicity assay in rats [4]. For 28 d, were included. Six consecutively higher doses were
Sprague Dawley rats received 1000 mg/kg body weight. examined in each study, with a high dose equivalent to 80
Animals were observed for clinical symptoms, body weight mL TAHITIAN NONI Juice/kg per day (20 mL/kg of
was recorded, and hematology and serum chemistry 4-fold concentrated juice).
parameters were measured. Study measurements indicative of liver effects were
Among the serum chemistry measurements were AST, ALT, AST, ALP, GGT, total bilirubin, total protein, protein
ALT, and GGT. There were no significant differences by electrophoresis, globulins, albumin/globulin ratio,
between the males and females of the test and control and prothrombin time. Additionally, clinical observations
g roups, with the exception of a lower AST value included liver weights, and macroscopic and microscopic
observed in the females of the treatment group, and an (histological) examination of the livers. Laboratory data
exceptionally low ALT value for the males of the control for liver function for each dose group in each study are
group that resulted in a statistical difference for treated mentioned in Tables 2, 3, 4, and 5.
males. In either instance, however, the values observed in No treatment-related changes were observed in any of
the treatment groups were within the normal range for the dose groups for any of these measurements, including
these animals and did not correspond to a dose-response histological examinations, demonstrating a lack of toxicity
relationship. These results did not reveal any adverse liver to the liver. Consequently, the No Observable Adverse
effects. Effect Level (NOAEL) was determined to be greater than
Two 13-wk oral toxicity studies of TAHITIAN NONI 80 mL TAHITIAN NONI Juice/kg per day.

Juice in rats were performed (unpublished data, Glerup P. The NOAEL is much higher than the amounts ingested

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 3618 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol
by the patients described in the case studies. In the most
recent publication, the first patient consumed a daily dose
of 71.4 mL (1.5 L consumed over 3 wk). The second case
reported in this publication describes a daily consumption
rate of 23.8 mL (2 L consumed over 3 mo). The weight of
each patient is not given, but assuming an average weight
of 70 kg, the doses consumed would be 1.02 mL/kg per
day and 0.34 mL/kg per day. The NOAEL of the animal
studies is almost 80 times greater than these two doses.
The high dose in the clinical study was approximately 10
times higher.
The first published case report claims that the patient
drank a glass of noni juice every day for a few weeks. No
exact quantity is specified; no indication is given as to how
large the glass was or how much of it was filled. However,
assuming a full cup volume of 250 mL and a patient
weight of 70 kg, the dose would be 3.6 mL/kg. The
NOAEL of the animal studies is 22 times greater than the
ingestion rate of this patient, and the clinical study high
dose is three times more. Not only are all reported doses in
these cases well within the highest doses of the controlled
studies, where no liver effects were observed, but were also
sufficiently low to assume a large margin of safety. If there
is any true causal link, it is likely to be only idiosyncratic in
nature.
As a pretext for implicating noni juice in liver toxicity,
the authors of both reports cite publications containing
only limited information, including similar case reports for
the herbal laxative drugs Senna (Cassia augustifolia)[5] and
Cascara sagrada (Rhamnus purshianus)[6]. The laxative effects
of these herbal preparations are due to large quantities of
a specific class of anthraquinones[7] (different than those in
the noni plant).
In these cases, the patients had ingested large quantities
of the herbs for an extensive time. Consequently,
the investigators propose a potential involvement of
anthraquinones. A similar assumption is made in the few
case reports of other herbs cited by the authors of these
two publications [8-10]. In these instances, however, the
role of the anthraquinones directly isolated from these
herbs, and linked to the patients' liver dysfunction was
not supported by any additional experimental evidence.
In fact, support for these assumptions is based on limited
information from experimental studies of sennosides[11]
and luteoskyrin[12] , which do not occur in the noni plant.
The suggestion of an anthraquinone involvement in
the three Austrian cases is not based on solid data. The
first argument against such a causal link is a quantitative
one. Until very recently, investigators had been unable
to identify or isolate anthraquinones from noni fruit[13].
However, two recent publications have reported the
presence of anthraquinones in noni fruit, at very low
concentrations. The first report describes 48.3 mg of
isolated material, distributed amongst six anthraquinones,
from 1.3 kg of dried noni fruit from Indonesia[14]. On a
dry weight basis, the total concentration is 37 10-6. In the
juice form, the concentration would be approximately
310 -6 . The second publication describes isolating a
few micrograms of anthraquinones from 9 kg of dried
fruit[15]. The anthraquinone concentration in this study
was less than 1 10-6. Such low yields and concentrations
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June 14, 2006 Volume 12 Number 22
are responsible for the inability of earlier researchers to
identify anthraquinones in the fruit.
In comparison to Senna and Cascara sagrada, the
total anthraquinone content of noni fruit is insignificant.
The European Pharmacopoeia describes Senna leaf as
containing no less than 2.5% (25 10-3) anthraquinones;
the standardized extract is to contain no less than 5.5%.
Cascara sagrada is described as having a minimum anthra-
quinone content of 8% (80 10-3). These concentrations
are many thousands of times higher than that in noni juice.
Where a large quantity of Senna and Cascara is required to
induce liver toxicity, it is unreasonable to assume a barely
detectable quantity of anthraquinones in noni juice can
have the same effect.
Beyond the minute quantity of anthraquinones in
noni fruit juice is a second issue of chemical structure.
Anthraquinones do not have the same universal biological
effects. Differences in bioactivities, toxic or otherwise, are
seen amongst variants of other types of compounds as
well. Thus, it is not reasonable to assume that one category
of anthraquinones will have the same exact toxic action as
another. The anthraquinones present in the laxative herbs,
linked to the previous cases of hepatotoxicity, are 1, 8-di
hydroxyanthraquinones (1, 8-DHAs). On the other hand,
those that occur in M. citrifolia are substituted 9, 10-anthraq
uinones[16,17]. These structural differences result in different
modes of action. The 9, 10-anthraquinones can not be
reduced in biological systems to form anthrone radicals,
while 1, 8-DHAs can[18]. It is the formation of anthrone
radicals that is required to produce tissue damage[19].
In summar y, human and animal studies of high
doses of TAHITIAN NONI Juice reveal no adverse
liver effects. Further, anthraquinones in noni fruit are of
insufficient quantities and fail to possess the necessary
chemical structures to cause liver tissue damage. The
available data demonstrates that noni juice is not directly
toxic to the liver
REFERENCES
1 Scientific Committee on Food. Opinion of the Scientific
Committee on Food on Tahitian Noni Juice. SCF/CS/NF/
DOS/18 ADD 2 Final. 11 Dec. 2002. Available from: URL:
http://europa.eu.int/comm/food/fs/sc/scf/out151_en.pdf
2 Millonig G, Stadlmann S, Vogel W. Herbal hepatotoxicity:
acute hepatitis caused by a Noni preparation (Morinda
citrifolia). Eur J Gastroenterol Hepatol 2005; 17: 445-447
3 Stadlbauer V, Fickert P, Lackner C, Schmerlaib J, Krisper P,
Trauner M, Stauber RE. Hepatotoxicity of NONI juice: report
of two cases. World J Gastroenterol 2005; 11: 4758-4760
4 Mancebo A, Scull I, Gonzales Y, Arteaga ME, Gonzales BO,
Fuentes D, Hernandez O. Correa M. Ensayo de toxicidad a
dosis repetidas (28 dias) por via oral del extracto acuoso de
Morinda citrifolia en ratas Sprague Dawley. Rev Toxicol 2002;
19: 74-78
5 Beuers U, Spengler U, Pape GR. Hepatitis after chronic abuse
of senna. Lancet 1991; 337: 372-373
6 Nadir A, Reddy D, Van Thiel DH. Cascara sagrada-induced
intrahepatic cholestasis causing portal hypertension: case
report and review of herbal hepatotoxicity. Am J Gastroenterol
2000; 95: 3634-3637
7 De Smet PAGM, Keller K, Hnsel R, Chandler RF. Adverse
effects of herbal drugs. Berlin: Springer-Verlag, 1993:105-139
8 Li FK, Lai CK, Poon WT, Chan AY, Chan KW, Tse KC, Chan
TM, Lai KN. Aggravation of non-steroidal anti-inflammatory
 West BJ et al. Noni juice is not hepatotoxic 3619
drug-induced hepatitis and acute renal failure by slimming
drug containing anthraquinones. Nephrol Dial Transplant 2004;
19: 1916-1917
9 Park GJ, Mann SP, Ngu MC. Acute hepatitis induced by
Shou-Wu-Pian, a herbal product derived from Polygonum
multiflorum. J Gastroenterol Hepatol 2001; 16: 115-117
10 Itoh S, Marutani K, Nishijima T, Matsuo S, Itabashi M. Liver
injuries induced by herbal medicine, syo-saiko-to (xiao-chai-
hu-tang). Dig Dis Sci 1995; 40: 1845-1848
11 Mengs U. Toxic effects of sennosides in laboratory animals
and in vitro. Pharmacology 1988; 36 Suppl 1: 180-187
12 Ueno I, Sekijima M, Hoshino M, Ohya-Nishiguchi H, Ueno
Y. Spin-trapping and direct EPR investigations on the
hepatotoxic and hepatocarcinogenic actions of luteoskyrin, an
anthraquinoid mycotoxin produced by Penicillium islandicum
Sopp. Generations of superoxide anion and luteoskyrin
semiquinone radical in the redox systems consisted of luteo-
skyrin and liver NADPH- or NADH-dependent reductases.
Free Radic Res 1995; 23: 41-50
13 Zenk MH, el-Shagi H, Schulte U. Anthraquinone production
by cell suspension cultures of Morinda citrifolia. Planta Med
1975; Suppl: 79-101
14 Kamiya K, Tanaka Y, Endang H, Umar M, Satake T. New
anthraquinone and iridoid from the fruits of Morinda citrifolia.
Chem Pharm Bull (Tokyo) 2005; 53: 1597-1599
15 Pawlus AD, Su BN, Keller WJ, Kinghorn AD. An
anthraquinone with potent quinone reductase-inducing
activity and other constituents of the fruits of Morinda citrifolia
(noni). J Nat Prod 2005; 68: 1720-1722
16 Thomson RH. Naturally occurring quinones III, Recent ad-
vances. London: Chapman and Hall, 1987: 347-606
17 Leistner E, Zenk MH. Incorporation of shikimic acid into
1,2-dihydroxyanthraquinone (alizarin) by Rubia tinctorum
L.Tetrahedron Letters 1967; 6: 475-476
18 Westendorf J. Pharmakologische und toxikologische Bewer-
tung von Anthranoiden. Pharmazeutische Zeitung 1993; 48: 9-20
19 Cudlin J, Steinerova N, Sedmera P, Vokoun J. Microbial
analogy of Bayer-Villiger reaction with anthraquinone
derivative. Collect Czech Chem Comm 1978; 43: 1908-1910
S- Editor Wang J L- Editor Kumar M E- Editor Liu WF
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ACKNOWLEDGMENTS
Acknowledgments to Reviewers of World Journal of
Gastroenterology
Many reviewers have contributed their expertise and time
to the peer review, a critical process to ensure the quality
of World Journal of Gastroenterology. The editors and authors
of the articles submitted to the journal are grateful to the
following reviewers for evaluating the articles (including
those were published and those were rejected in this
issue) during the last editing period of time.
Gianfranco D Alpini, Professor
VA Research Scholar Award Recipient, Professor, Medicine and Systems
Biology and Translational Medicine, Dr. Nicholas C. Hightower Centennial
Chair of Gastroenterology, Central Texas Veterans Health Care System, The
Texas A & M University System Health Science Center College of Medicine,
Medical Research Building, 702 SW H.K. Dodgen Loop, Temple, TX, 76504,
United States
Domenico Alvaro, MD
Division of Gastroenterology, Department of Clinical Medicine, University
of Rome La Sapienza, Viale Universit 37, Rome 00185, Italy
Jasmohan Singh Bajaj, Assistant Professor
Division of Gastroenterology and Hepatology, Medical College of
Wisconsin, 9200 W Wisconsin Ave, Milwaukee WI 53212, United States
Yusuf Bayraktar, Professor
Department of Gastroenterology, School of Medicine, Hacettepe University,
Ankara 06100, Turkey
Josep M Bordas, MD
Department of Gastroenterology IMD, Hospital Clinic, Llusanes 11-13 at,
Barcelona 08022, Spain
Yogesh K Chawla, Dr, Professor
Department of Hepatology, Postgraduate Institute of Medical Education
and Research, Chandigarh 160012, India
Yoichi Chida, Assistant professor
Department of Psychosomatic Medicine, Graduate School of Medical
Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582,
Japan
Zong-Jie Cui, PhD, Professor
Institute of Cell Biology, Beijing Normal University, 19 XinJieKouWaiDaJie,
Beijing 100875, China
Kazuma Fujimoto, Professor
Department of Internal Medicine, Saga Medical School, Nabeshima, Saga,
Saga 849-8501, Japan
Kazuma Fujimoto, Professor
Department of Internal Medicine, Saga Medical School, Nabeshima, Saga,
Saga 849-8501, Japan
Xupeng Ge, MD, PhD
Division of Transplantation Surgery, CLINTEC, Karolinska Institute,
Karolinska University Hospital-Huddinge, Stockholm 14186, Sweden
Anna S Gukovskaya, Professor
VA Greater Los Angeles Health Care System ,University of California, Los
Angeles, 11301 Wilshire Blvd, Los Angeles 91301, United States
World J Gastroenterol 2006 June 14; 12(22): 3620
World Journal of Gastroenterology ISSN 1007-9327
2006 The WJG Press. All rights reserved.
Ming-Liang He, Associate Professsor
Faculty of Medicine , The Center for Emerging Infectious Diseases, The
Chinese University of Hong Kong, Hong Kong, China
Fu-Lian Hu, Professor
Department of Gastroenterology, Peking University First Hospital, 8 Xishiku
St, Xicheng District, Beijing 100034, China
Atif Iqbal, MD
Department of Surgery, Creighton University, c/o Dr Charles J Filipi, Suite
3728, 601 N 30th Street, Creighton University School of Medicine,
Omaha NE 68131, United States
Hartmut Jaeschke, Professor
Liver Research Institute, University of Arizona, College of Medicine, 1501 N
Campbell Ave, Room 6309, Tucson, Arizona 85724, United States
Gerd A Kullak-Ublick, Professor
Laboratory of Molecular Gastroenterology and Hepatology, Department of
Internal Medicine, University Hospital Zurich, Zurich CH-8091,
Switzerland
Shiu-Ming Kuo, MD
University at Buffalo, 15 Farber Hall, 3435 Main Street, Buffalo 14214,
United States
Louis Libbrecht, MD, PhD
Department of Pathology, University and University Hospitals of Leuven,
Minderbroedersstraat 12, Leuven 3000, Belgium
Hanns-Ulrich Marschall, Associate Professor
Karolinska Institutet, Department of Medicine , Division of Gastroenter-
ology and Hepatology, Karolinska University Hospital Huddinge, Stockholm
S-14188, Sweden
Kiyoshi Migita, Professor
Clinical Research Center, NHO Nagasaki Medical Center, Kubara 2-1001-1,
Omura 856-8562, Japan
James Neuberger, Professor
Liver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH,
United Kingdom
Wei Tang, MD, EngD, Assistant Professor
H-B-P Surgery Division, Artificial Organ and Transplantation Division,
Department of surgery, Graduate School of Medicine, The University of
Tokyo, Tokyo 113-8655, Japan
Hugo E Vargas, Associate Professor of Medicine
Division of Transplantation Medicine, Mayo Clinic, 5777 E. Mayo Blvd, 5E,
Scottsdale AZ 85054, United States
Jian Wu, Associate Professor of Medicine
Internal Medicine/Transplant Research Program, University of California,
Davis Medical Center, 4635 2nd Ave. Suite 1001, Sacramento CA 95817,
United States
Ming_shiang Wu, Dr, Associate Professor
Internal Medicine, National Taiwan University Hospital, No 7,Chung-Shan S.
Rd., Taipei 100, Taiwan, China
Eric M Yoshida, MD
Department of Medicine, University of British Columbia, 100-2647 Willow
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jbarnhart@continuingeducation.net EVENTS AND MEETINGS IN
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ILTS 12th Annual International Congress

Initiative 6th Annual Gastroenterology And 2007
22-24 February 2006 Hepatology
3-6 May 2006 9th World Congress on Gastrointestinal
Banff, Alberta 15-18 March 2006
Milan Cancer
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REFERENCES
Coding system
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[19,22-24 ], we know that...
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journal title (Journal title should be in its abbreviation form as shown in
PubMed), publication date, volume number (in black), start page, and end
page [PMID: 11819634]
Note: The author should test the references through reference testing
system (http://www.aushome.cn/cgi-bin/index.pl)
Style for book references
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of all authors should be typed with the initial letter capitalized and followed
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Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication
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Format
Journals
English journal article (list all authors and include the PMID where applicable)
1 Grover VP, Dresner MA, Forton DM, Counsell S, Larkman DJ, Patel N,
Thomas HC, Taylor-Robinson SD. Current and future applications of
magnetic resonance imaging and spectroscopy of the brain in hepatic
encephalopathy. World J Gastroenterol 2006; 12: 2969-2978 [PMID:
16718775]
Chinese journal article (list all authors and include the PMID where applicable)
2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of
Jianpi Yishen decoction in treatment of Pixu-diarrhoea. Shijie Huaren
Xiaohua Zazhi 1999; 7: 285-287
In press
3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signature of
balancing selection in Arabidopsis. Proc Natl Acad Sci U S A 2006; In
press
Organization as author
4 Diabetes Prevention Program Research Group. Hypertension,
insulin, and proinsulin in participants with impaired glucose tolerance.
Hypertension 2002; 40: 679-686 [ PMID: 12411462 ]
Both personal authors and an organization as author
5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One
Study Group. Sexual dysfunction in 1, 274 European men suffering
from lower urinary tract symptoms. J Urol 2003; 169: 2257-2261 [PMID:
12771764]
No author given
6 21st century heart solution may have a sting in the tail. BMJ 2002; 325:
184 [PMID: 12142303]
3623
Volume with supplement
7 Geraud G, Spierings EL, Keywood C. Tolerability and safety of
frovatriptan with short- and long-term use for treatment of migraine
and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99
[PMID: 12028325]
Issue with no volume
8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen section
analysis in revision total joint arthroplasty. Clin Orthop Relat Res 2002;
(401): 230-238 [ PMID: 12151900 ]
No volume or issue
9 Outreach: bringing HIV-positive individuals into care. HRSA Careaction
2002; 1-6 [PMID: 12154804 ]
Books
Personal author(s)
10 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed.
Oxford: Blackwell Sci Pub, 1993: 258-296
Chapter in a book (list all authors)
11 Lam SK. Academic investigators perspectives of medical treatment for
peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and
basis for therapy. New York: Marcel Dekker, 1991: 431-450
Author(s) and editor(s)
12 Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd ed.
Wieczorek RR, editor. White Plains (NY): March of Dimes Education
Services, 2001: 20-34
Conference proceedings
13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V.
Proceedings of the 5th Germ Cell Tumour Conference; 2001 Sep
13-15; Leeds, UK. New York: Springer, 2002: 30-56
Conference paper
14 Christensen S, Oppacher F. An analysis of Koza's computational effort
statistic for genetic programming. In: Foster JA, Lutton E, Miller J,
Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP
2002: Proceedings of the 5th European Conference on Genetic
Programming; 2002 Apr 3-5; Kinsdale, Ireland. Berlin: Springer, 2002:
182-191
Electronic journal (list all authors)
Morse SS. Factors in the emergence of infectious diseases. Emerg
Infect Dis serial online, 1995-01-03, cited 1996-06-05; 1(1): 24 screens.
Available from: URL: http//www.cdc.gov/ncidod/EID/eid.htm
Patent (list all authors)
16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible
endoscopic grasping and cutting device and positioning tool assembly.
United States patent US 20020103498. 2002 Aug 1
Inappropriate references
Authors should always cite references that are relevant to their article, and
avoid any inappropriate references. Inappropriate references include those
that are linked with a hyphen and the difference between the two numbers at
two sides of the hyphen is more than 5. For example, [1-6], [2-14] and [1, 3,
4-10, 22] are all considered as inappropriate references. Authors should not
cite their own unrelated published articles.
Statistical data
Present as mean SD or mean SE.
Statistical expression
Express t test as t (in italics), F test as F (in italics), chi square test as 2 (in
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sample number as n (in italics), and probability as P (in italics).
Units
Use SI units. For example: body mass, m (B) = 78 kg; blood pressure,
p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h, blood glucose
concentration, c (glucose) 6.4 2.1 mmol/L; blood CEA mass concentration,
p (CEA) = 8.6 24.5 g/L; CO2 volume fraction, 50 mL/L CO2 not 5% CO2;
likewise for 40 g/L formaldehyde, not 10% formalin; and mass fraction,
8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read 23 243 641.
The format about how to accurately write common units and quantum
is at: http://www.wjgnet.com/wjg/help/15.doc
Abbreviations
Standard abbreviations should be defined in the abstract and on first mention
in the text. In general, terms should not be abbreviated unless they are
used repeatedly and the abbreviation is helpful to the reader. Permissible
abbreviations are listed in Units, Symbols and Abbreviations: A Guide for
Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published
by The Royal Society of Medicine, London. Certain commonly used
 3624 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol June 14, 2006 Volume 12 Number 22

abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC,
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Quantities: t time or temperature, c concentration, A area, l length, m mass, Signed:
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