Biliary Cells Research Articles

P2 B cell depletion therapy exacerbates murine primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of anti-mitochondrial antibodies (AMAS). Indeed, the presence of AMAS is the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Thus, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMA. The recent development of well characterized mAbs specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well defined xenobiotic induced model of autoimmune cholangitis, prompted us to utilize these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either antiCD20, anti-CD79, or isotype matched control mAb and followed for B cell development, the appearance of AMAs, liver pathology and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than control mice which is in contrast with results from a number of other autoimmune models which have documented an important therapeutic role of B cell specific depletion. The anti-CD20/CD79 treated mice have increased liver T cell infiltrates and higher levels of pro-inflammatory cytokines. In conclusion, our results

Bone Marrow Stem Cells Contribute to Alcohol Liver Fibrosis in Humans

Bone marrow-derived stem cell (BMSC) contribution to liver repair varies considerably and recent evidence suggests these cells may contribute to liver fibrosis. We investigated the mobilization and hepatic recruitment of bone marrow (BM) stem cells in patients with alcohol liver injury and their contribution to parenchymal/non-parenchymal liver cell lineages. Liver biopsies from alcoholic hepatitis (AH) patients and male patients, who received a female liver transplant and developed AH, were analyzed for BM stem cell content by fluorescence in situ hybridization and immunostaining. Y chromosome analysis was performed, along with co-staining for hepatocyte, biliary, myofibroblast, and Ki-67 markers. Blood CD34(+) levels were quantified in AH patients by flow cytometry. AH patients had increased CD34(+) cell counts in liver tissue (1.834% +/- 0.605%; P < 0.05) and in blood (0.195% +/- 0.063%; P < 0.05) as compared with matched controls (0.299% + 0.208% and 0.067% +/- 0.01%). A proportion of hepatic myofibroblasts were BM-derived (7.9%-26.8%) as deemed by the co-localization of Y chromosome/alpha-smooth muscle actin (alpha-SMA) staining. In the cross-sex liver grafts with AH, 5.025% of the myofibroblasts were co-staining for CD34, suggesting that a population of CD34(+) cells were contributing to the hepatic myofibroblast population. There was no evidence of BM contribution to hepatocyte or biliary cell differentiation, nor evidence of increased hepatocyte regeneration. Alcohol liver injury mobilizes CD34(+) stem cells into the circulation and recruits them into the liver. These BMSCs contribute to the hepatic myofibroblast population but not to parenchymal lineages and do not promote hepatocyte repair.

COP9 signalosome subunit 8 is required for postnatal hepatocyte survival and effective proliferation.

Studies using lower organisms and cultured mammalian cells have revealed that the COP9 signalosome (CSN) has important roles in multiple cellular processes. Conditional gene targeting was recently used to study CSN function in murine T-cell development and activation. Using the Cre-loxP system, here we have achieved postnatal hepatocyte-restricted knockout of the csn8 gene (HR-Csn8KO) in mice. The protein abundance of other seven CSN subunits was differentially downregulated by HR-Csn8KO and the deneddylation of all cullins examined was significantly impaired. Moreover, HR-Csn8KO-induced massive hepatocyte apoptosis and evoked extensive reparative responses in the liver, including marked intralobular proliferation of biliary lineage cells and trans-differentiation and proliferation of the oval cells. However, division of pre-existing hepatocytes was significantly diminished in HR-Csn8KO livers. These findings indicate that Csn8 is essential to the ability of mature hepatocytes to proliferate effectively in response to hepatic injury. The histopathological examinations revealed striking hepatocytomegaly in Csn8-deficient livers. The hepatocyte nuclei were dramatically enlarged and pleomorphic with hyperchromasia and prominent nucleoli, consistent with dysplasia or preneoplastic cellular alteration in HR-Csn8KO mice at 6 weeks. Pericellular and perisinusoid fibrosis with distorted architecture was also evident at 6 weeks. It is concluded that CSN8/CSN is essential to postnatal hepatocyte survival and effective proliferation.

Transcriptome atlas of aromatic amino acid family metabolism-related genes in eight liver cell types uncovers the corresponding metabolic pathways in rat liver regeneration

To explore gene expression of aromatic amino acid family metabolism and their metabolic pathways of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of percoll density gradient centrifugation and immunomagnetic bead methods. Rat Genome 230 2.0 Array was used to detect the expression changes of genes associated with aromatic amino acid family metabolism. The transcriptome atlas showed that the metabolic pathway of phenylalanine was mainly catalyzed into tyrosine in hepatic stellate cells in the initiation stage, tyrosine was oxidized into dopa and norepinephrine in biliary epithelia cells and dendritic cells, and norepinephrine was finally catalyzed into adrenaline in biliary epithelia cells and pit cells in the progress stage. Thyroid hormone of tyrosine catabolites was synthesized from tyrosine in almost all cells in different stage of LR, among which genes of T3 biosynthesis were increased in HCs, BECs, SECs and DCs in the progress stage. Tryptophan was decarboxylated to 5-hydroxytryptamine in dendritic cells in the progress stage. Based on the results as above, we concluded that phenylalanine is the major source of tyrosine, proliferation of biliary epithelia cells and dendritic cells maybe promote by tyrosine catabolites—dopa and norepinephrine, biliary epithelia cells and pit cells maybe promote by adrenaline. T3 maybe play a major role on proliferation of HCs, BECs, SECs and DCs in the progress stage. The proliferation of dendritic cells maybe promote by tryptophan catabolites—5-hydroxytryptamine.

Pathological changes and local defense reaction occurring in spontaneous hepatic coccidiosis in rabbits (Oryctolagus cuniculus).

Hepatic coccidiosis occurred in 56 rabbits belonging to different ages, sexes and breeds during the years from 2002 to 2005. Clinically, the severely affected rabbits showed decreased growth rate, anorexia, debilitation, listlessness, diarrhea, icterus and rough hair coat. Postmortem examinations revealed increased dirty dull straw colored peritoneal fluid. Hepatomegaly, with presence of discrete yellowish-white nodules of 1mm to 1 cm size on the surface and throughout the parenchyma, was seen. Smears from peritoneal fluid showed rafts of mesothelial cells. Impression smears from the liver nodules revealed presence of numerous developmental stages of Eimeria stiedae corresponding with the stage of the liver lesion. These were intermixed with sheats of hepatobiliary cells and inflammatory cells and the changes were reflective of the histological changes like biliary hyperplasia, cholangitis and vacuolar changes occurring in the liver. There was increase in the thickness of basement membrane with the advanced stage of the lesion. The basement membrane was up to 1-2 µm in thickness forming loop like structures over which resided hyperplastic biliary cells containing different stages of the parasite. Some thickened basement membranes were devoid of overlying cells which had desquamated and were present in the ductal lumens suggesting that the basement might have the local defensive role in warding of the coccidial infection. The basement membrane was positive for neutral mucopolysaccharides while as the inflammatory zones and biliary epithelia along with the gametogonic stages of the coccidium reacted positively to the increased acid mucopolysaccharides qualitatively. The results showed that the occurrence of hepatic coccidiosis in female rabbits coincided with the breeding and kindling periods. Direct wet mount smears from bile aid in diagnosis of the disease. Impression smears from the liver nodules might represent the histological changes in the liver. The basement membrane thickening might have a defensive role in warding off the coccidial infection.

Linking human beta retrovirus infection with primary biliary cirrhosis

Several environmental agents have been linked with primary biliary cirrhosis (PBC) that include bacteria, xenobiotics and viruses. A human beta retrovirus (HBRV) related to mouse mammary tumor virus has been cloned and characterized from patients with PBC. This agent can be detected in the majority of patients' perihepatic lymph nodes by immunochemistry and RT-PCR. The HBRV has recently been isolated in culture and integration sites have been identified in the genome of patients to provide convincing evidence of beta retrovirus infection in patients. Three lines of evidence support a role for the virus in PBC. First, the beta retrovirus is linked with aberrant expression of mitochondrial protein(s) on the biliary epithelium cell (BEC) surface, a disease specific phenotype. Second, the related agent, mouse mammary tumor virus has been linked with autoimmune biliary disease in the NOD.c3c4 mouse model for PBC. In this mouse model, the virus is localized to diseased biliary epithelium that also display aberrant expression of the mitochondrial autoantigens. In translational studies, both patients with PBC and NOD.c3c4 mice demonstrate significant improvement in biliary disease with combination antiviral therapy. An overview of the biological relevance of the beta retrovirus infection in PBC will be discussed in this review.

Interleukin-17 is involved in α-naphthylisothiocyanate-induced liver injury in mice

Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. The pathogenesis of DILI usually involves the participation of the parent drug or metabolites that either directly affect the cell biochemistry or elicit an immune response. However, in most cases the mechanisms are unknown. Alpha-naphthylisothiocyanate (ANIT) is known as a hepatotoxicant that causes biliary cell and hepatocyte damage and induces intense neutrophil infiltration in the liver. To investigate whether an immune-mediated mechanism is involved in ANIT-induced liver injury, we examined the plasma AST, ALT and T-Bil levels, hepatic expression of transcriptional factors, cytokines and CXC chemokine genes, plasma IL-17 level and histopathological changes in liver after ANIT administration in mice. Hepatic mRNA expression of retinoid related orphan receptor γt (RORγt) and macrophage inflammatory protein (MIP-2) and plasma IL-17 level was significantly increased in ANIT-administered mice as well as the plasma AST, ALT and T-Bil. Neutralization of IL-17 using anti-IL-17 antibody (100 μg/mouse, single i.p.) suppressed the hepatotoxic effect of ANIT. Co-administration of recombinant IL-17 (1 μg/mouse, single i.p.) to ANIT-administered mice resulted in a remarkable increase of the plasma AST, ALT and T-Bil levels. In conclusion, it was firstly demonstrated that IL-17 is involved in the ANIT-induced liver injury in mice.

Effect of Different Bile Duct Flush Solutions on Biliary Tract Preservation Injury of Donated Livers for Transplantation

Objectives The objective of this study was to explore the effect of various bile duct flush (BDF) solutions on biliary tract preservation of donated livers in rats. Methods We studied the effects of BDF solutions and cold ischemic times on biliary tract preservation, using 2 kinds of solutions: (1) BDFa with normal saline (NS), or hypertonic citrate-adenine kidney preservation in vivo (HCA), and (2) BDFb with University of Wisconsin solution (UW), or histidine-tryptophan-ketoglutarate solution (HTK). The cold ischemic times (CIT) were 4, 8, or 12 hours. Forty-five healthy male Wistar rats were randomly divided into 9 groups of 5 rats each using a [L 9(3 4)] orthogonal table. Biliary tract tissues were examined at the corresponding cold preservation times for the following: microscopic changes in bile duct cells; TUNEL (Transferase-mediated, dUTP-bitin nick end labeling) procedure assays apoptotic indices (AI) of endothelial cells in the biliary tract; ultrastructural changes; and average volumes (V) and density (Nd) of mitochondria in endothelial cells calculated using an image analysis system. The results were evaluated by analysis of variance (ANOVA) and direct analysis by an orthogonal design. Results AI of biliary tract endothelial cells showed significance ( P < .01) of cold preservation of the biliary tract of donor liver with BDFa or BDFb and CIT; furthermore, HCA, HTK, and 4-hour CIT were all ideal. V and Nd of mitochondrial endothelial cells were significantly increased ( P < .01) with BDFa, BDFb, and CIT; furthermore, factors HCA, HTK, and 4-hour CIT were all ideal. Conclusions Cold preservation injuries to the biliary tract of a donor liver may be greatly decreased by efficient and sufficient flushing of the bile tract. A suitable bile duct solution greatly decreases cold preservation injuries and protects endothelial cells.

Monolobar Hepatobiliary Fibropolycystic Disease

We herein report a case of monolobar hepatobiliary fibropolycystic disease. A 75-year-old woman presented with heartburn. Imaging modalities including US, CT, and MRI revealed marked atrophy and multiple biliary cysts of the hepatic left lobe. The hepatic right lobe was normal. ERCP and bile duct endoscopy revealed anomalous pancreaticobiliary union, choledochal dilation, dilation of left intrahepatic bile ducts, and small choledochal non-invasive adenocarcinoma. Polycystic kidney diseases were absent. The patient underwent pancreatico-duodenectomy and extended hepatic left lobectomy. Grossly, the hepatic left lobe was markedly atrophic, and studded with numerous biliary cysts. The left intrahepatic bile ducts were dilated (Caroli's disease) and the common bile duct showed type I choledochal dilation. The right hepatic lobe was normal. Histologically, the hepatic left lobe was replaced by fibroelastosis. The intrahepatic bile ducts showed ductal plate malformation such as irregular contours, invaginations, and protrusions. The numerous biliary cysts also showed ductal plate malformation. There were numerous persistent ductal plates and microhamartomas. Many hyalinized destructive biliary cysts and ductal plates were recognized. The liver parenchyma was scant and free of hepatocellular malformations. The portal veins showed old obliterative portal thrombosis. The right hepatic lobe was normal. Immunohistochemically, the biliary cells were positive for cytokeratin 7, 8, 18 and 19, and MUC6 and CD10, but negative for MUC2 and MUC5AC. The biliary cysts, persistent ductal plate, and microhamartomas were positive for fetal apomucin antigen MUC1.

Up-regulation of annexin A2 in cholangiocarcinoma caused by Opisthorchis viverrini and its implication as a prognostic marker

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is primarily associated with infection with the liver fluke Opisthorchis viverrini in northeast Thailand. The disease is associated with late presentation, poses challenges for diagnosis and has a high mortality rate – features that highlight the need for tumor markers. At present, there are no specific tumor markers that can indicate the early stages and status of CCA. Proteomic analysis of the proteins expressed on the surface of tumor cells is particularly difficult since proteome-wide analysis of surface membrane proteins has thus far been hampered by the lack of effective strategies to profile hydrophobic membrane proteins. In this study, a sequential protein extraction was utilized to overcome this problem. Membrane protein was extracted from four CCA cell lines with different tumor forming capabilities. The non-tumor H69 biliary cell line was used as a control. Two-dimensional-PAGE followed by MALDI-TOF-MS was used to identify differentially expressed proteins. Among 20 up-regulated membrane proteins identified in the CCA cell lines was ANXA2, a participant in tumor invasion and metastasis in other cancers. Accordingly, ANXA2 was verified in human subjects by probing, using a commercial anti-mouse monoclonal antibody and a tissue microarray of CCA (301 diagnosed cases), where it was found to associate with one of several tumor progression stages as reflected by lymphatic invasion ( P = 0.014) and metastasis ( P = 0.026). Patients with high expression of ANXA2 had a significantly shorter survival time ( P = 0.011). ANXA2 expression in tumors may be useful for predicting the poor outcome of CCA patients.

Monolobar ductal plate malformation disease of the liver

We herein report a unique monolobar hepatic disease composed of Caroli's disease, peribiliary cysts, ductal plate malformations, peribiliary gland proliferation, hepatolithiasis, and portal phlebosclerosis with thrombi. A 73-year-old man underwent abdominal imaging, which revealed multiple segmental dilations of the left intrahepatic bile ducts. Polycystic kidney diseases were absent. Intrahepatic cholangiocarcinoma was suspected, and extended left lobectomy of the liver was preformed. Grossly, the hepatic left lobe was atrophic, and partly replaced by fibrous tissue. The intrahepatic bile ducts were dilated (Caroli's disease), and showed small calcium bilirubinate hepatoliths. Microscopically, the intrahepatic bile duct showed non-obstructive segmental dilations (Caroli's disease), numerous peribiliary cysts, numerous ductal plate malformations, proliferation of intrahepatic peribiliary glands, and calcium bilirubinate hepatolithiasis. Portal veins showed phlebosclerosis with thrombi. Immunohistochemically, the various biliary epithelial cells were positive for cytokeratin (CK) 7, 8, 18, and 19, and for MUC6 and CD10. They were negative for MUC2 and MUC5AC. The ductal plate malformations were positive for fetal biliary antigen MUC1, but other biliary cell types were negative for MUC1. The present case resembles 'monolobar Caroli's disease'. We believe that the present monolobular liver disease was congenital in origin.

Characterisation of a stereotypical cellular and extracellular adult liver progenitor cell niche in rodents and diseased human liver

BackgroundStem/progenitor cell niches in tissues regulate stem/progenitor cell differentiation and proliferation through local signalling.ObjectiveTo examine the composition and formation of stem progenitor cell niches.MethodsThe composition of the hepatic progenitor cell...

S1870 Clinical Significance of Cd38-Positive Plasma Cells Surrounding Interlobular Bile Ducts in Primary Biliary Cirrhosis (PBC)

Background/Aims: There has been increased interest in the potential role of B cells and antimitochondrial antibodies in PBC. Indeed a new paradigm in PBC includes the potential of B cells to act as both regulatory elements and as components of the inflammasone induced by apoptosis of biliary cells. We submit that a rigorous dissection of the inflammatory infiltrate in PBC will provide further insight on these issues. Materials and Methods: We took advantage of well-characterized Mabs to CD3, CD4, CD8, CD20, CD38, CD56, CD68, and pan-keratin antigens, and immunohistochemistry (IHC), to study the distribution of liver infiltrating CD38-positive plasma cells in 26 consecutive patients with PBC (AMA positive in 20 and negative in 6), all of whom had detailed staged clinical data. All data was “blindly” evaluated. We simultaneously studied 10 ageand gender-matched patients with chronic hepatitis C as a control. Results: Noteworthy within the IHC data was the presence of an intense coronal arrangement (CR) of CD38-positive cells around interlobular bile ducts, generally in specimens with chronic non-suppurative destractive cholangitis (CNSDC). In contrast, CD20-positive B lymphocytes (precursor cells of plasma cells) were found scattered and/or aggregated within the lymphoplasmocytic infiltration. Such CD20positive B cells also occasionally formed follicle-like aggregations but importantly were not observed in the proximity of CNSDC. PBC patients with CR demonstrated significantly higher titers of AMA (119.5±16.1 vs. 59.7±17.1, p=0.018) and lower levels of total cholesterol (TC) (195.1±9.0 vs. 223.6±9.6, p=0.04) than those without CR. Interestingly the CR correlated with titer of AMA (r=0.46), IgM (r=0.32), the presence of CNSDC (r=0.32) and inversely with age (r=-0.37), γ-GTP (r=-0.38) and TC (r=-0.41). In contrast, CD4and CD8-positive T lymphocyte infiltration was noted either in proximity of, or within the degenerated cholangioepithelium, suggesting the participation of these cells in the destructive processes of interlobular bile ducts. No CR was found in control subjects. Conclusion: The presence of CD38+ plasma cells surrounding biliary epithelium has clinical and serologic significance; further it correlates with disease progression exemplified by TC decrease and the development of florid duct lesions. These data further highlight the functional significance of B cells/ plasma cells; it also reflects a multilineage loss of tolerance in PBC. We submit that study of B cells in PBC should go beyond simple measurement of AMA titer and include rigorous phenotypical and functional dissection of the liver specific B cell lineage populations.

S1873 The use of Liver Biopsy Evaluation in Determination of Autoimmune Hepatitis vs. Drug-Induced Liver Injury

Background/Aims: There has been increased interest in the potential role of B cells and antimitochondrial antibodies in PBC. Indeed a new paradigm in PBC includes the potential of B cells to act as both regulatory elements and as components of the inflammasone induced by apoptosis of biliary cells. We submit that a rigorous dissection of the inflammatory infiltrate in PBC will provide further insight on these issues. Materials and Methods: We took advantage of well-characterized Mabs to CD3, CD4, CD8, CD20, CD38, CD56, CD68, and pan-keratin antigens, and immunohistochemistry (IHC), to study the distribution of liver infiltrating CD38-positive plasma cells in 26 consecutive patients with PBC (AMA positive in 20 and negative in 6), all of whom had detailed staged clinical data. All data was “blindly” evaluated. We simultaneously studied 10 ageand gender-matched patients with chronic hepatitis C as a control. Results: Noteworthy within the IHC data was the presence of an intense coronal arrangement (CR) of CD38-positive cells around interlobular bile ducts, generally in specimens with chronic non-suppurative destractive cholangitis (CNSDC). In contrast, CD20-positive B lymphocytes (precursor cells of plasma cells) were found scattered and/or aggregated within the lymphoplasmocytic infiltration. Such CD20positive B cells also occasionally formed follicle-like aggregations but importantly were not observed in the proximity of CNSDC. PBC patients with CR demonstrated significantly higher titers of AMA (119.5±16.1 vs. 59.7±17.1, p=0.018) and lower levels of total cholesterol (TC) (195.1±9.0 vs. 223.6±9.6, p=0.04) than those without CR. Interestingly the CR correlated with titer of AMA (r=0.46), IgM (r=0.32), the presence of CNSDC (r=0.32) and inversely with age (r=-0.37), γ-GTP (r=-0.38) and TC (r=-0.41). In contrast, CD4and CD8-positive T lymphocyte infiltration was noted either in proximity of, or within the degenerated cholangioepithelium, suggesting the participation of these cells in the destructive processes of interlobular bile ducts. No CR was found in control subjects. Conclusion: The presence of CD38+ plasma cells surrounding biliary epithelium has clinical and serologic significance; further it correlates with disease progression exemplified by TC decrease and the development of florid duct lesions. These data further highlight the functional significance of B cells/ plasma cells; it also reflects a multilineage loss of tolerance in PBC. We submit that study of B cells in PBC should go beyond simple measurement of AMA titer and include rigorous phenotypical and functional dissection of the liver specific B cell lineage populations.

S1871 The Association of Low Level of Both Vitamin a and Retinol Binding Protein With the Liver Complications in Patients With Primary Sclerosing Cholangitis

Background/Aims: There has been increased interest in the potential role of B cells and antimitochondrial antibodies in PBC. Indeed a new paradigm in PBC includes the potential of B cells to act as both regulatory elements and as components of the inflammasone induced by apoptosis of biliary cells. We submit that a rigorous dissection of the inflammatory infiltrate in PBC will provide further insight on these issues. Materials and Methods: We took advantage of well-characterized Mabs to CD3, CD4, CD8, CD20, CD38, CD56, CD68, and pan-keratin antigens, and immunohistochemistry (IHC), to study the distribution of liver infiltrating CD38-positive plasma cells in 26 consecutive patients with PBC (AMA positive in 20 and negative in 6), all of whom had detailed staged clinical data. All data was “blindly” evaluated. We simultaneously studied 10 ageand gender-matched patients with chronic hepatitis C as a control. Results: Noteworthy within the IHC data was the presence of an intense coronal arrangement (CR) of CD38-positive cells around interlobular bile ducts, generally in specimens with chronic non-suppurative destractive cholangitis (CNSDC). In contrast, CD20-positive B lymphocytes (precursor cells of plasma cells) were found scattered and/or aggregated within the lymphoplasmocytic infiltration. Such CD20positive B cells also occasionally formed follicle-like aggregations but importantly were not observed in the proximity of CNSDC. PBC patients with CR demonstrated significantly higher titers of AMA (119.5±16.1 vs. 59.7±17.1, p=0.018) and lower levels of total cholesterol (TC) (195.1±9.0 vs. 223.6±9.6, p=0.04) than those without CR. Interestingly the CR correlated with titer of AMA (r=0.46), IgM (r=0.32), the presence of CNSDC (r=0.32) and inversely with age (r=-0.37), γ-GTP (r=-0.38) and TC (r=-0.41). In contrast, CD4and CD8-positive T lymphocyte infiltration was noted either in proximity of, or within the degenerated cholangioepithelium, suggesting the participation of these cells in the destructive processes of interlobular bile ducts. No CR was found in control subjects. Conclusion: The presence of CD38+ plasma cells surrounding biliary epithelium has clinical and serologic significance; further it correlates with disease progression exemplified by TC decrease and the development of florid duct lesions. These data further highlight the functional significance of B cells/ plasma cells; it also reflects a multilineage loss of tolerance in PBC. We submit that study of B cells in PBC should go beyond simple measurement of AMA titer and include rigorous phenotypical and functional dissection of the liver specific B cell lineage populations.

S1874 Drug-Induced Autoimmune Hepatitis: Clinical Characteristics and Prognosis

Background/Aims: There has been increased interest in the potential role of B cells and antimitochondrial antibodies in PBC. Indeed a new paradigm in PBC includes the potential of B cells to act as both regulatory elements and as components of the inflammasone induced by apoptosis of biliary cells. We submit that a rigorous dissection of the inflammatory infiltrate in PBC will provide further insight on these issues. Materials and Methods: We took advantage of well-characterized Mabs to CD3, CD4, CD8, CD20, CD38, CD56, CD68, and pan-keratin antigens, and immunohistochemistry (IHC), to study the distribution of liver infiltrating CD38-positive plasma cells in 26 consecutive patients with PBC (AMA positive in 20 and negative in 6), all of whom had detailed staged clinical data. All data was “blindly” evaluated. We simultaneously studied 10 ageand gender-matched patients with chronic hepatitis C as a control. Results: Noteworthy within the IHC data was the presence of an intense coronal arrangement (CR) of CD38-positive cells around interlobular bile ducts, generally in specimens with chronic non-suppurative destractive cholangitis (CNSDC). In contrast, CD20-positive B lymphocytes (precursor cells of plasma cells) were found scattered and/or aggregated within the lymphoplasmocytic infiltration. Such CD20positive B cells also occasionally formed follicle-like aggregations but importantly were not observed in the proximity of CNSDC. PBC patients with CR demonstrated significantly higher titers of AMA (119.5±16.1 vs. 59.7±17.1, p=0.018) and lower levels of total cholesterol (TC) (195.1±9.0 vs. 223.6±9.6, p=0.04) than those without CR. Interestingly the CR correlated with titer of AMA (r=0.46), IgM (r=0.32), the presence of CNSDC (r=0.32) and inversely with age (r=-0.37), γ-GTP (r=-0.38) and TC (r=-0.41). In contrast, CD4and CD8-positive T lymphocyte infiltration was noted either in proximity of, or within the degenerated cholangioepithelium, suggesting the participation of these cells in the destructive processes of interlobular bile ducts. No CR was found in control subjects. Conclusion: The presence of CD38+ plasma cells surrounding biliary epithelium has clinical and serologic significance; further it correlates with disease progression exemplified by TC decrease and the development of florid duct lesions. These data further highlight the functional significance of B cells/ plasma cells; it also reflects a multilineage loss of tolerance in PBC. We submit that study of B cells in PBC should go beyond simple measurement of AMA titer and include rigorous phenotypical and functional dissection of the liver specific B cell lineage populations.

Characterization of Volume-Activated Chloride Currents in Regulatory Volume Decrease of Human Cholangiocyte

Volume-activated chloride channel (VACC) plays vital roles in many physiological functions. In bile duct epithelium, VACC actively participates in biliary secretion and cell volume regulation, and it mediates regulatory volume decrease (RVD). Recently, we have shown that mouse cholangiocytes have an intact RVD via VACC and K(+) conductance. However, such cell volume regulation was not studied in the normal human cholangiocyte. Volume measurement by Coulter counter and whole-cell patch clamp technique were used to characterize the RVD and VACC in human cholangiocyte cell line (HBDC). When exposed to hypotonic solution, HBDC exhibited an intact RVD, which was inhibited by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), NPPB (5-nitro-2'- (3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanatostilbene-2-disulfonic acid), and tamoxifen, but was not affected by the removal of extracellular calcium. During RVD, HBDC exhibited large, outwardly rectifying currents and time-dependent inactivation at positive potential. The amplitude of the outward current was approximately 3 times of that of the inward current, and this volume-activated current returned to the baseline when switched to isotonic solution. The amplitude and reversal potential of the volume-activated current was dependent on Cl(-) concentration, and the VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, and acetate in the hypotonic solution. In addition, classical VACC inhibitors, such as NPPB or tamoxifen, inhibited the VACC. These inhibitory effects were reversible with washing out the inhibitors from the bath solution. The present study is the first to characterize and show that HBDC has an intact RVD, mediated by VACC, which has similar electrophysiological characteristics as that in mouse cholangiocytes.

Abstract 5205: EBP50, a PDZ-containing protein, regulates EGFR-induced cell scattering and migration in human cancer biliary epithelial cells

Abstract Background: Cholangiocarcinoma is the second most frequent primary malignant tumor of the liver, accounting for 10-15% of all primary liver cancers. Cholangiocarcinoma grows at the expense of epithelial cells that line intra- and extra-hepatic bile ducts. The adaptor protein EBP50 (Ezrin-radixin-moesin Binding Phosphoprotein 50) binds and regulates transmembrane transporters and receptors including Epidermal Growth Factor Receptor (EGFR). EBP50 and EGFR are both expressed in the normal biliary epithelium. While EGFR participates in biliary carcinogenesis, implication of EBP50 in this cancer is unknown. The aim of the study was to determine the potential links between EBP50 and EGFR in human biliary carcinoma. Methods: In vitro studies were performed in the human biliary epithelial cell line, Mz-ChA-1, that retains epithelial phenotype and endogenous expression of EBP50 and EGFR. The role of EBP50 was examined using siRNA. Cell scattering and migration were evaluated using E-cadherin immunostaining and Boyden chamber, respectively. For in vivo study, 112 patients (mean age: 59.5±11 years, 55 females and 57 males) who underwent surgical resection for hilar (n=53) and peripheral (n=59) cholangiocarcinoma in one single institution were analyzed retrospectively. Immunohistochemistry on tissue microarrays was performed to assess EBP50 and EGFR expression. Results: EGF activates EGFR and the three main signaling pathways, ERK1/2, STAT3 and Akt, in Mz-ChA-1 biliary cancer cells. Inhibition of EBP50 in EGF-treated cells increases and maintains activation of EGFR, and of ERK1/2 and STAT3 but not Akt. Enhanced EGFR activation in response to EBP50 depletion confers sensitivity to EGFR tyrosine kinase inhibitor, gefitinib. Functionally, upon EGF, EGFR drives cell scattering by inducing disruption of adherens junctions. In addition, EGF activates formation of lamellipodia structures and therefore cell migration. Interestingly, inhibition of EBP50 in non EGF-stimulated cell has the same effects on cell scattering and migration as those observed with EGF alone. Cell dispersion due to EBP50 inhibition is abolished in the presence of gefitinib. Moreover, impact on cell scattering and migration is even greater when both EGF addition and EBP50 siRNA are combined. In vivo, delocalization of EBP50 normally expressed beneath the apical membrane is associated with EGFR expression in human biliary carcinoma tissues (p=0.002). Conclusion: These data provide evidence for an essential role of EBP50 in repressing EGFR-induced signaling pathway and migration. EBP50 at the membrane may be critical to confine cancer progression by preventing migration of biliary cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5205.

Abstract 1473: Fra-2 and JunB are overexpressed in precancerous bile ducts and primary/transplanted hamster cholangiocarcinomas with a strong positive correlation

Abstract The transciption factor complex AP-1 plays important roles in cell proliferation, differentiation and apoptosis of which dysregulation is closely associated with cancer development. Fra-2 and JunB, components of AP-1 family proteins, had been found overexpressed in certain of tumors, suggesting their possible implication in carcinogenesis. In the present study, we investigated differential expression patterns of those two AP-1family proteins during hamster cholangiocarcinogenesis and aggressive transplanted cholangiocarcinomas (ChCs). First, we prepared appropriate liver tissue samples with precancerous lesions and ChCs from hamster ChC model as well as normal liver. The transplanted ChCs were produced by syngenic transplantation of the primary ChCs. We then performed immunohistochemistry (IHC) for Fra-2 and JunB using ABC methods. The typical nuclear staining for Fra-2 and JunB was determined as positive immunoreactivity and the ratio of positive cells was evaluated by counting 800-3000 cells, followed by statistical analysis. The correlation of Fra-2 and JunB expression was analyzed by scattergram. Our IHC indicated significantly increased frequency of Fra-2 and JunB positive cells according to the tumor progression, i.e., respectively in the early dysplatic bile ducts (10∼20%) and late dysplastic bile ducts (40∼50%), primary ChC (40∼60%) and transplanted ChC (higher than 70% positivity), while normal and simple hyperplastic bile duct epithelia were negative or indicated very low labeling index for both Fra-2 and JunB (under 1% positivity). The expression of Fra-2 and JunB showed a strong correlation (r=0.93) and the immunoreactive cells for Fra-2 and JunB were mainly localized in the invading areas adjacent to normal surrounding tissue. In summary, we found in the present study the followings 1) Expression of Fra-2 and JunB progressively increased as bile duct cells become carcinogenic, 2) Fra-2 and JunB have a high correlation in the expression patterns in the biliary cell populations including cancers, 3) In the transplanted biliary cancers, overexpression of Fra-2 and JunB (especially JunB) were localized in the neoplastic cells which are very aggressive to invade into adjacent normal tissue. Based on our results, Fra-2 and JunB could play significant roles in cholangiocarcinogenesis, possibly by collaboratively participating in dysplastic changes and acquisition of tumorigenicity of candidate bile duct cells for ChC. Also, those two AP-1 family proteins could contribute to invasiveness of biliary cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1473.

Uptake and phototoxicity of meso-tetrahydroxyphenyl chlorine are highly variable in human biliary tract cancer cell lines and correlate with markers of differentiation and proliferation

Photodynamic therapy (PDT) using Photofrin and, recently, Foscan has gained broad acceptance for palliative treatment of non-resectable cholangiocarcinoma (CC). No information, however, is available whether the phenotype of CC tumour cells has an effect on the efficiency of the treatment. Using a well-characterised set of n = 9 biliary tract cancer cell lines this study investigated the uptake, phototoxicity, and release of meso-tetrahydroxyphenyl chlorine (mTHPC, Foscan) after incubation with 200 or 400 ng ml(-1) mTHPC. For uptake of mTHPC we found great variations between the individual cell lines (up to a factor 2), resulting in even more pronounced differences in phototoxicity. Based on statistical classification by hierarchical cluster analysis, two groups of cell lines can be distinguished which are characterised by either high or low susceptibility towards mTHPC-based photodynamic treatment. Correlation analysis with previously established immunochemical parameters showed that cells with a low cytokeratin-19 (ductal differentiation), high vimentin (mesenchymal marker), and high proliferative phenotype preferentially show higher uptake of mTHPC and subsequent phototoxicity. These results demonstrate high variability of biliary tract cancer cells when subjected to mTHPC-based photodynamic treatment and identify possible markers that could be used in the clinical setting in order to predict the efficiency of PDT and adjust the dose for complete tumour elimination.