Bile Duct Epithelial Cells Research Articles

Effect of interleukin‐2 and tumor necrosis factor‐α on osteopontin: molecular function and biological process

Biliary atresia (BA) is the most common cause of neonatal cholestasis. Recently it was reported that osteopontin (OPN) expression by human bile duct epithelial cells in culture was responsive to interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha). This might be an important part in the pathogenesis of BA, but the function of IL-2 and TNF-alpha in OPN expression is not well understood. A new gene ontology technology was used to predict the molecular function and biological process of the effect of IL-2 and TNF-alpha on OPN. Using GoFigure server, the molecular function and biological process of the effect of IL-2 and TNF-alpha on OPN is predicted. Compared to IL-2, TNF-alpha and OPN, the IL-2-TNF-alpha-OPN combination has more functions. The IL-2-TNF-alpha-OPN combination presents many more functions including positive regulation of osteoclast and response to virus. IL-2 and TNF have a synergistic effect on OPN expression.

Obesity-Induced Lymphocyte Hyperresponsiveness to Chemokines: A New Mechanism of Fatty Liver Inflammation in Obese Mice

Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and "non-obese ob/ob" mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. Antibiotic treatment decreased liver infiltration with CD4(+) T, CD8(+) T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4(+) T, CD8(+) T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4(+) T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in "non-obese ob/ob" mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization.

Eimeria stiedae: Experimental infection in rabbits and the effect of treatment with toltrazuril and ivermectin

The aim of this study was to investigate the clinical, haematological, biochemical, lipid peroxidation, ultrasonographic and pathologic findings in hepatic coccidiosis induced by Eimeria stiedae in rabbits, and also to compare the treatment effects of both toltrazuril and ivermectin separately and in combination. In this study, 56 rabbits were divided into eight groups. The first group was designated as healthy control group. Rabbits were infected with 40.000 sporulated oocysts of E. stiedae. Groups 2, 3, 4, 5, 6, 7 and 8 were allocated as the infected control group, infected+toltrazuril-treated group, infected+ivermectin-treated group, infected+toltrazuril+ivermectin-treated group, non-infected+toltrazuril-treated group, non-infected+ivermectin-treated group, non-infected+toltrazuril+ivermectin-treated group, respectively. Haematocrit, Haemoglobin and MCV values as well as percentage of lymphocyte decreased in Groups 2 and 4 whereas leucocyte counts and percentage of granulocyte leucocyte increased. Serum GGT, ALT and AST activities increased but albumin value decreased. Plasma MDA concentrations increased whereas erythrocyte CAT, GSH-Px, and SOD activities decreased. Mean oocyst numbers in per gram faeces (epg values) increased in both groups during the study. Ultrasonographic examination revealed that the liver was enlarged and had hyperechogenic parenchyma. Bile ducts were dilated and hyperechogenic and the gall bladder was dilated. The livers of these animals were enlarged and typical macroscopic and microscopic findings of coccidiosis were present. Treatment with toltrazuril and toltrazuril+ivermectin combination were highly effective in reducing faecal oocyst output in infected rabbits. Haematological, biochemical and lipid peroxidation parameters and, ultrasonographic findings of the liver were close to control values for Groups 3 and 5. Necropsy of these animals showed no visible lesions related to hepatic coccidiosis although a few oocysts were detected in the bile duct epithelial cells.

Upregulation of AT1 Receptor in MDR2 Knockout Mice after low dose antifibrotic therapy with Telmisartan

Introduction: Angiotensin II stimulates liver fibrosis and cell proliferation via angiotensin II receptor type 1 (AT1R). In advanced liver disease, AT1R expression is increased on, among other cell types, bile duct epithelial cells, but in total AT1 R expression is downregulated. The biliary excretion of the AT1R blocker telmisartan may, therefore, argue for a positive impact of this substance on the course of chronic cholestatic liver disease. We examined the antifibrotic efficacy of telmisartan in MDR2 knockout mice, a well-established animal model of progressive cholestasis mimicking primary sclerosing cholangitis.

Physiology of bile secretion

The formation of bile depends on the structural and functional integrity of the bile-secretory apparatus and its impairment, in different situations, results in the syndrome of cholestasis. The structural bases that permit bile secretion as well as various aspects related with its composition and flow rate in physiological conditions will first be reviewed. Canalicular bile is produced by polarized hepatocytes that hold transporters in their basolateral (sinusoidal) and apical (canalicular) plasma membrane. This review summarizes recent data on the molecular determinants of this primary bile formation. The major function of the biliary tree is modification of canalicular bile by secretory and reabsorptive processes in bile-duct epithelial cells (cholangiocytes) as bile passes through bile ducts. The mechanisms of fluid and solute transport in cholangiocytes will also be discussed. In contrast to hepatocytes where secretion is constant and poorly controlled, cholangiocyte secretion is regulated by hormones and nerves. A short section dedicated to these regulatory mechanisms of bile secretion has been included. The aim of this revision was to set the bases for other reviews in this series that will be devoted to specific issues related with biliary physiology and pathology.

Hepatocyte growth factor improves viability after H 2O 2-induced toxicity in bile duct epithelial cells

Intracellular defence mechanisms against oxidative stress may play an important role in the progression of liver diseases, including cholangiopathies. The multifunctional anti-apoptotic hepatocyte growth factor (HGF) has been suggested to have antioxidant functions. The effect of HGF upon cell viability, the generation of ROS, the expression of genes that play a role in ROS defence, and the activation of caspase-3 were measured in bile duct epithelial (BDE) cells in the presence or absence of H 2O 2. HGF reduced H 2O 2-induced loss of viability, diminished H 2O 2-mediated ROS generation and abrogated H 2O 2-triggered changes in GSH/GSSG ratio. Furthermore, HGF increased the gene-expression of gamma-glutamylcysteine synthetase (GCLC) and glutathione reductase (GSR), while no effect was seen upon the gene-expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX1), and glutathione synthetase (GSR). Finally, HGF diminished the proteolytical activation of the key mediator of apoptosis (caspase-3) after H 2O 2 exposure. Together, HGF may improve viability in bile duct epithelia cells after H 2O 2 induced toxicity by proliferation, strengthening the intrinsic antioxidant defences, and/or by an attenuation of apoptosis. These in vitro results support the evaluation of HGF as antioxidative factor in hepatobiliary pathologies.

An Animal Model of Human Cytomegalovirus Infection

To develop a rat model that allowed in vivo progressive human cytomegalovirus (HCMV) infection, allogeneic liver transplantation was performed across a rat combination of Dark Agouti (DA) to Brown Norway (BN). AD169, a well-characterized laboratory strain of HCMV, was used to establish a rat model of HCMV infection by injection of 0.4 mL (30.0 logTCID50) supernate into the rat peritoneum. Histological and blood specimens were obtained from animals sacrificed at predetermined timepoints. We performed immunohistochemical staining in liver, heart, kidney, spleen, and lung for HCMV immediate-early antigen (IE), lower matrix protein (pp65) detection in peripheral blood leukocytes, and HCMV early antigen (EA) and late antigen (LA). We compared survival rates. Our results showed positive HCMV IE and pp65 antigenemia detected in peripheral blood leukocytes in transplanted recipients from day 1 to day 30. Positive HCMV EA and LA staining cells were only detected in sections 10 days after liver transplantation, namely, in hepatocytes, mononuclear cells, bile duct epithelial cells, and endothelial cells. Successful HCMV replication was due to the combination of liver transplantation and cyclosporine (CsA) immunosuppression. Survival analysis showed no significant differences between the HCMV-infected group and HCMV-uninfected group. This new rat model of HCMV infection may be helpful to understand immune system modulation of HCMV infection.

Anti-CD16 autoantibodies and delayed phagocytosis of apoptotic cells in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by chronic hepatic inflammation and immune mediated apoptosis of bile duct epithelial cells. Delayed macrophage phagocytosis of opsonized apoptotic cells, noted in other autoimmune diseases, may promote inflammation. Recent studies suggest serum anti-CD16 autoantibodies contribute to impaired macrophage phagocytosis by blocking complement receptor 3 (CR3) signaling via CD16. Therefore, serum anti-CD16 levels and the ability of monocyte derived macrophages from individuals with PBC to phagocytosis apoptotic cells were compared to controls. The mean level of anti-CD16 IgM autoantibodies (0.86 ± 0.62 v. 0.35 ± 0.22, respectively, p = 0.031) was increased in PBC compared to control sera, and mean PBC phagocytosis of opsonized apoptotic cells was significantly decreased compared to controls (23.9 ± 12.2% v. 43.9 ± 14.4%, respectively, p = 0.020). However, PBC phagocytosis of opsonized apoptotic cells was not significantly affected by the presence or absence of autologous serum (20.8 ± 13.5% v. 23.9 ± 12.2%, respectively, p = 0.560). PBC phagocytosis of opsonized apoptotic cells inversely correlated with CD16 (and CR3) expression levels on Day 5 after culture in the presence or absence of autologous serum ( r = − 0.546, p = 0.033 and r = − 0.519, p = 0.042, respectively). Phagocytosis of non-opsonized apoptotic cells did not correlate with CD16 or CR3 expression ( p > 0.050). In conclusion, PBC macrophage phagocytosis of opsonized apoptotic cells is impaired, irrespective of serum factors and may increase hepatic inflammation.

Proliferative effects of excretory/secretory products from Clonorchis sinensis on the human epithelial cell line HEK293 via regulation of the transcription factor E2F1

Clonorchis sinensis is one of the most prevalent parasitic helminths of humans in East Asia. Although several complications in bile duct epithelial cells are caused by C. sinensis infection, the mechanism is not clearly understood. To clarify the effects of C. sinensis excretory-secretory products (ES products) on bile duct epithelial cells, we investigated their effects on the human embryonic kidney epithelial cell line HEK293 in vitro. Our results show that ES products alter the proportion of cells in each stage of the cell cycle and induce HEK293 cell proliferation. Among cell cycle-related proteins, the expression of cyclin E increased markedly after treatment with ES products, indicating that the G1/S transition occurred. In addition, the expression of the transcription factor E2F1 was up-regulated by the addition of ES products. Small interfering RNA (siRNA) was used to demonstrate that the transcription factor E2F1 is a key factor in the control of cell proliferation in HEK293 cells. The present results demonstrate that ES products from C. sinensis stimulate cell proliferation by inducing E2F1 expression. We suggest that the ES products released from C. sinensis during infection may play an important role in the development of cholangiocarcinoma via the overgrowth of the bile duct epithelium.

Expression of microsomal prostaglandin E synthase‐1 in human hepatocelluar carcinoma

The objective of this study was to evaluate the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in hepatocellular carcinoma (HCC) tissues. Forty surgically resected HCC tissues with adjacent non-tumorous liver tissues and 14 surgically resected, histologically normal liver tissues were used. The immunohistochemical expressions of the mPGES-1 protein in these HCC tissues and normal control livers were analysed. mPGES-1 mRNA expression was also analysed by the real-time polymerase chain reaction method using the same tissues. Microsomal prostaglandin E synthase-1 was not expressed in hepatocytes but instead in vascular endothelial cells and bile duct epithelial cells in normal liver tissues. The mPGES-1 expression in HCC tissues was significantly greater than its expression in the non-tumorous tissues. All types of HCC expressed more mPGES-1 than normal or hepatitis livers, and the levels of mPGES-1 expression in poorly differentiated HCC were similar to the levels in well-differentiated HCC. The mPGES-1 mRNA expression paralleled its protein expression in these tumorous and non-tumorous tissues. The present study is the first to demonstrate a high expression of mPGES-1 in well-differentiated HCC as well as in poorly differentiated HCC. These findings suggest that mPGES-1 may play a role in the advanced as well as early stage of hepatocarcinogenesis.

Profibrogenic TGF-beta/ALK5 signaling via CTGF expression in hepatocytes

Connective Tissue Growth Factor (CTGF) plays an important role in TGF-β induced liver fibrogenesis. Hepatic stellate cells and bile duct epithelial cells were recognized as its cellular source in liver. Here, we demonstrate induction of CTGF expression in hepatocytes of damaged liver and identify a molecular mechanism responsible. Strong CTGF expression was found in bile duct epithelial cells, hepatic stellate cells and hepatocytes in fibrotic liver tissue from patients with chronic hepatitis B infection. Similarly, CTGF expression was induced in hepatocytes of CCl4 treated FVB mice. CTGF expression and secretion was detected spontaneously in medium of hepatocytes after 3 days of culture and was enhanced by stimulation with TGF-β. TGF-β-induced CTGF expression was mediated through the ALK5/Smad3 pathway, while ALK1 activitation antagonised this effect. CTGF expression in liver tissue of TGF-β transgenic mice correlated with serum TGF-β levels. Smad7 overexpression in cultured hepatocytes abrogated TGF-β-dependent and intrinsic CTGF expression. In line with these data, hepatocyte specific Smad7 expression reduced CTGF expression in CCl4 treated transgenic mice, whereas mice bearing a disrupted Smad7 gene displayed enhanced expression. Furthermore, IFN-g treatment of patients with chronic HBV infection induced Smad7 expression in hepatocytes leading to decreased CTGF expression and fibrogenesis. In conclusion, our data provide strong in vitro and in vivo evidence for pro-fibrogenic activity of TGF-β directed to hepatocytes mediated via upregulation of CTGF. Further, we identify ALK5 dependent Smad3 signaling as responsible pathway inducing CTGF expression, which can be interfered by an activated ALK1 pathway and completely inhibited by TGF-β antagonist Smad7.

A New Xenobiotic-Induced Mouse Model of Sclerosing Cholangitis and Biliary Fibrosis

Xenobiotics and drugs may lead to cholangiopathies and biliary fibrosis, but the underlying mechanisms are largely unknown. Therefore, we aimed to characterize the cause and consequences of hepatobiliary injury and biliary fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a novel model of xenobiotic-induced cholangiopathy. Liver morphology, markers of inflammation, cell proliferation, fibrosis, bile formation, biliary porphyrin secretion, and hepatobiliary transporter expression were studied longitudinally in DDC- and control diet-fed Swiss albino mice. DDC feeding led to increased biliary porphyrin secretion and induction of vascular cell adhesion molecule, osteopontin, and tumor necrosis factor-alpha expression in bile duct epithelial cells. This was associated with a pronounced pericholangitis with a significantly increased number of CD11b-positive cells, ductular reaction, and activation of periductal myofibroblasts, leading to large duct disease and a biliary type of liver fibrosis. After 4 weeks, we constantly observed intraductal porphyrin pigment plugs. Glutathione and phospholipid excretion significantly decreased over time. Expression of Ntcp, Oatp4, and Mrp2 was significantly reduced, whereas Bsep expression remained unchanged and adaptive Mrp3 and Mrp4 expression was significantly induced. We demonstrate that DDC feeding in mice leads to i) a reactive phenotype of cholangiocytes and bile duct injury, ii) pericholangitis, periductal fibrosis, ductular reaction, and consequently portal-portal bridging, iii) down-regulation of Mrp2 and impaired glutathione excretion, and iv) segmental bile duct obstruction. This model may be valuable to investigate the mechanisms of xenobiotic-induced chronic cholangiopathies and its sequels including biliary fibrosis.

Profibrogenic transforming growth factor-β/activin receptor–like kinase 5 signaling via connective tissue growth factor expression in hepatocytes

Connective tissue growth factor (CTGF) is important for transforming growth factor-beta (TGF-beta)-induced liver fibrogenesis. Hepatic stellate cells have been recognized as its major cellular source in the liver. Here we demonstrate the induction of CTGF expression in hepatocytes of damaged livers and identify a molecular mechanism responsible for it. CTGF expression was found by immunohistochemistry in bile duct epithelial cells, hepatic stellate cells, and hepatocytes in fibrotic liver tissue from patients with chronic hepatitis B infection. Similarly, CTGF expression was induced in hepatocytes of carbon tetrachloride-treated mice. CTGF expression and secretion were detected spontaneously in a medium of hepatocytes after 3 days of culture, which was enhanced by stimulation with TGF-beta. TGF-beta-induced CTGF expression was mediated through the activin receptor-like kinase 5 (ALK5)/Smad3 pathway, whereas activin receptor-like kinase 1 activation antagonized this effect. CTGF expression in the liver tissue of TGF-beta transgenic mice correlated with serum TGF-beta levels. Smad7 overexpression in cultured hepatocytes abrogated TGF-beta-dependent and intrinsic CTGF expression, indicating that TGF-beta signaling was required. In line with these data, hepatocyte-specific transgenic Smad7 reduced CTGF expression in carbon tetrachloride-treated animals, whereas in Smad7 knockout mice, it was enhanced. Furthermore, an interferon gamma treatment of patients with chronic hepatitis B virus infection induced Smad7 expression in hepatocytes, leading to decreased CTGF expression and fibrogenesis. Our data provide evidence for the profibrogenic activity of TGF-beta directed to hepatocytes and mediated via the up-regulation of CTGF. We identify ALK5-dependent Smad3 signaling as the responsible pathway inducing CTGF expression, which can be hindered by an activated activin receptor-like kinase 1 pathway and completely inhibited by TGF-beta antagonist Smad7.

Expression of Bax, Bcl‐xL and Bcl‐2 proteins in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C

To determine the expression of regulators of apoptosis in chronic hepatitis C. Expression of Bax, Bcl-xL and Bcl-2 proteins was assessed immunohistochemically in liver biopsy specimens obtained from 89 adults with chronic hepatitis C. Expression of Bax in hepatocytes correlated inversely with grade of inflammation (P < 0.001) and stage of fibrosis (P = 0.011), classified according to the Scheuer score; expression of Bcl-xL in hepatocytes did not correlate with grade of inflammation (P = 0.106) or stage of fibrosis (P = 0.078); maximum Bcl-xL expression was observed in grade 3 inflammation and stage 4 fibrosis. Expression of Bcl-2 protein in hepatocytes was present in only two cases (both with advanced disease); the expression of Bcl-2 protein in interlobular bile duct epithelial cells correlated with the grade of inflammation (P = 0.018), but not with stage of fibrosis (P = 0.154). The expression of Bcl-2 protein in lymphoid cells infiltrating portal zones and lobules did not correlate with grade of inflammation (P = 0.113) or stage of fibrosis (P = 0.815). Major differences in expression of studied proteins were observed in relation to grade of inflammation and stage of fibrosis in chronic hepatitis C.

Specific activation of the different fibrogenic cells in rat cultured liver slices mimicking in vivo situations

Due to the loss of cell-cell and cell-matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or beta-oestradiol. As in vivo, epidermal growth factor and beta-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, beta-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.

Response to Relationship between circulating levels of tumour necrosis factor-α and Child-Pugh scores in liver cirrhosis

To the Editor: Our study (tumour necrosis factor alpha levels in hepatitis B virus related chronic active hepatitis and liver cirrhosis and its relationship to Knodell and Child–Pugh scores) was aimed to investigate the possibility that circulating tumour necrosis factor alpha (TNF-α) levels might represent an effective indicator of the disease in patients with chronic active hepatitis B (CAH-B) and liver cirrhosis (LC). Plasma samples of 30 subjects from each group were taken and analysed using enzyme-linked immunosorbent assay technique. Results were compared with those of 30 healthy controls. As a result of our study, we concluded that plasma TNF-α levels are significantly elevated in patients with CAH-B proportionally with hepatitis activity index. This data is in agreement with those of previous studies. On the other hand, we found out an inverse relationship between Child–Pugh score and plasma TNF-α level in LC patients. This later result is in contradiction with the previously reported data as mentioned in the original paper. Tumour necrosis factor is a pleiotropic proinflammatory cytokine, mainly produced by activated macrophages and in smaller amounts by several other types of cells, with strong antitumoural capacities both in vitro and in vivo. TNF-α plays a main role in the extrinsic pathway of apoptosis. So, it serves the clearance of virus infected hepatocytes (1). As a matter of fact, TNF-α and other proinflammatory cytokines were reported to be elevated parallelly with transaminase levels (2,3). There are several reasons for elevated TNF-α level in cirrhotic patients. These are mostly related to concomitant factors such as exposure to bacterial lipopolysaccharids generated from apparent or unapparent bacterial infections, existence of hepatic encephalopathy or other complications associated with advanced cirrhosis or even poor psychological status (4–6). On the other hand, there are also a number of factors which may negatively influence the level of TNF in cirrhotic patients. In addition to reduced amounts of endogenous cells (Kupffer cells, endothelial cells, hepatocytes and bile duct epithelial cells), quantitative and functional abnormalities in other cellular source of TNF-α production such as natural killer cells has also been reported in cirrhosis (7–9). Furthermore, adipose tissue which is capable of TNF-α release, is commonly decreases in advanced cirrhosis (10). Similarly, peripheral blood neutrophilic granulocytes can also produce and release TNF-α (11). Hypersplenism because of the enlarged spleen as a result of portal hypertension may reduce neutrophil count and consequently TNF-α level. Increased plasma volume and dietary factors may also contribute to the alteration of the plasma level of TNF-α. We have carefully searched the presence of clinical or laboratory evidence of concomitant infectious and inflammatory diseases in our cases. All the cases were clinically stable. Finally, we believe that marked increase of TNF-α production in advanced cirrhosis is possibly determined by concomitant infections and several complications. Further studies on larger series of highly selected cases are necessary to confirm this hypothesis. Tumour necrosis factor plays an important role in host defence and tumour growth control. Therefore, its deficiency or anti-TNF antibody therapies may increase the risk of serious infections and malignancies (12,13). If reduction of TNF-α production in advanced cirrhosis is established, it will possibly be considered as one of the predisposing factors for increased risk of infection and carcinogenesis.

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). Three groups of mutant F344 dipeptidyl peptidase IV-deficient (DPPIV(-)) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV(+)) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. In group A, DPPIV(+) hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and alpha-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.

Comparison of membrane‐associated proteins in human cholangiocarcinoma and hepatocellular carcinoma cell lines

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. Cell line models, originating from Thai patients, are available for both diseases, including the human bile duct epithelial carcinoma cell line (HuCCA-1) and the HCC cell line HCC-S102. Here, we have prepared subproteomes enriched in membrane proteins or in cytosolic proteins from the HuCCA-1 and the HCC-S102 cell lines. Study of differential protein expression by 2-DE and LC/MS/MS showed 195 proteins expressed in the two cell lines, including both membrane-associated and cytosolic proteins. Eighteen proteins were found in both membrane and cytosolic fractions of HuCCA-1, but not in HCC-S102, while nine proteins were found in both membrane and cytosolic fractions of HCC-S102, but not in HuCCA-1. Ten membrane proteins were found in HuCCA-1 but not in HCC-S102, including integrin alpha-6 precursor, ezrin, hippocalcin-like protein 1, mitogen-activated protein kinase kinase kinase 2 (MAPK/ERK kinase kinase 2), and calgizzarin. Proteins showing increased expression in the membrane fraction of HuCCA-1 were mainly cytoskeletal proteins (40.9%), while proteins showing increased expression in the membrane fraction of HCC-S102 were mainly metabolic proteins (39.4%). The subproteomic approach used here facilitates detection of potential biomarkers undetected by regular proteomic methods.

Id3 is important for proliferation and differentiation of the hepatoblasts during the chick liver development

The specified hepatic endoderm (hepatoblasts), the bipotential progenitor for hepatocytes and bile duct epithelial cells, proliferates during the primordial stages of liver development. Despite extensive studies, the mechanism that regulates proliferation of bipotential hepatoblasts is not fully understood. Here we show that Id3, a negative regulator of helix–loop–helix transcription factors, is an important regulator of hepatoblast proliferation in the developing chick liver. Id3 was expressed in hepatoblasts at early developmental stages (stages 12–29) but not in hepatocytes at later developmental stages (stage 34 onwards). Depletion of Id3 in hepatoblasts by siRNA results in failure of cell proliferation, but is not associated with either cell death or failure of expression of Hhex and Fibrinogen, the earliest hepatoblast markers. These observations suggest that at early developmental stages, Id3 functions as a positive regulator of hepatoblast proliferation, independent of cell death or maintenance of the non-terminally differentiated state. Interestingly at later developmental stages, the expression pattern of Id3 is complementary to that of Albumin, a marker of mature hepatocytes. Overexpression of Id3 in liver explants delayed the initiation of Albumin expression. Taken together, our observations show that Id3 is not only a positive regulator of hepatoblast proliferation, but also an inhibitor of their differentiation into hepatocytes in the developing chick liver.

INOS‐dependent DNA damage via NF‐κB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation-mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535-42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV-infected hamsters. We also examined the expression of nuclear factor-kappaB (NF-kappaB), which functions as a tumor promoter in inflammation-associated cancer. Our results showed that although 1-week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV-induced 8-nitroguanine and 8-oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8-oxodG level in the liver of OV-infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF-kappaB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV-induced cholangiocarcinoma by inhibiting iNOS-dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.