Biliary Fibrosis Research Articles

Epithelial‐mesenchymal transition‐related protein expression in biliary epithelial cells associated with hepatolithiasis

Epithelial-mesenchymal transition (EMT) of biliary epithelial cells (BECs) plays major roles in many cholangiopathies. This study evaluated whether EMT of BECs has a role in hepatolithiasis-induced biliary fibrosis and types of BECs that are involved. The expression of EMT-related proteins and epidermal growth factor receptor was evaluated by immunohistochemistry of liver tissues from 102 patients with hepatolithiasis, 32 patients with post-hepatitis cirrhosis, and 48 normal livers. Antibodies against E-cadherin, β-catenin, and cytokeratin were used to identify epithelial cells and antibodies against vimentin, S100A4, podoplanin, and α-smooth muscle actin (α-SMA) were used to identify mesenchymal cells. The relationship between clinical and histological parameters and immunohistochemistry findings in BECs, and the surrounding stroma were evaluated. Loss of E-cadherin and acquisition of S100A4 and vimentin were observed in BECs. In all BECs, cytokeratin and β-catenin expression were unchanged, while podoplanin and α-SMA were not expressed. Although hepatic fibrosis was more severe in post-hepatitis cirrhosis, EMT of BECs was more widespread in hepatolithiasis. In hepatolithiasis, EMT-related proteins were more highly expressed in small bile ducts than in medium or large bile ducts. Their expression was associated with the severity of biliary fibrosis and the expressions of epidermal growth factor receptor. Expression of α-SMA in fibroblasts from the portal space was closely linked to pathological changes in small bile ducts and EMT-related protein expressions in BECs. Proliferating cholangiocytes that form small bile ducts may contribute to cholangiopathies in hepatolithiasis through an EMT-like phenomenon or through interactions with stromal myofibroblasts.

Fibrosing cholestatic hepatitis in HBV transgenic mice after bone marrow transplantation

Introduction: Chronic hepatitis B virus (HBV) infection is associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Fibrosing cholestatic hepatitis (FCH) is a severe form of HBV infection, usually seen in immunocompromised patients, including liver and bone marrow transplant recipients. The aim of this study was to investigate the impact of bone marrow transplantation (BMT) on hepatic injury and fibrosis in transgenic mice that express HBV surface (HBs) proteins in the liver. Methods:12-week-old BALB/c transgenic mice that express HBs proteinsin the liver were lethally irradiated and whole bone marrow cells from BALB/c-GFP mice were transplanted by tail vein injection. 26- and 52-week-old transgenic mice (Tg-Tx) and their non-transgenic littermates (Lm-Tx) were sacrificed. The level of hepatic and bile duct injury was estimated by serum alanine transaminase (ALT) and alkaline phosphatise (AP) activity. Histological, immunohistochemical, microarray, and Western blot analyses were used to study liver pathology. Results: BMT resulted in strong increase of serum ALT and AP activity in 26-week-old Tg-Tx compared to Lm-Tx and non-transplanted mice. H&E and Sirius Red staining confirmed enhanced liver injury and development of biliary fibrosis in Tg-Tx. Furthermore, comparison of gene expression profile of 26-week-old Tg-Tx and Abcb4-/- mice, a well-established model for chronic cholestatic liver disease, supported the idea that major outcome of BMT is the development of FCH in HBV transgenic mice. Moreover, BMT induced senescence of liver cells, especially periportal hepatocytes and ductular reaction in Tg-Tx liver. Conclusion: BMT in transgenic mice expressing HBs proteins in the liver could be an attractive model for studies of FCH pathogenesis.

Cardiomyopathy reverses with recovery of liver injury, cholestasis and cholanemia in mouse model of biliary fibrosis

Triggers and exacerbants of cirrhotic cardiomyopathy (CC) are poorly understood, limiting treatment options in patients with chronic liver diseases. Liver transplantation alone reverses some features of CC, but the physiology behind this effect has never been studied. We aimed to determine whether reversal of liver injury and fibrosis in mouse affects cardiac parameters. The second aim was to determine whether cardiomyopathy can be induced by specifically increasing systemic bile acid (BA) levels. 6-8 week old male C57BL6J mice were fed either chow (n = 5) or 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) (n = 10) for 3 weeks. At the end of 3 weeks, half the mice in the DDC fed group were randomized to chow (the reversed [REV] group). Serial ECHOs and electrocardiographic analysis was conducted weekly for 6 weeks followed by liver tissue and serum studies. Hearts were analysed for key components of function and cell signalling. Cardiac physiological and molecular parameters were similarly analysed in Abcb11(-/-) mice (n = 5/grp) fed 0.5% cholic acid supplemented diet for 1 week. Mice in the REV group showed normalization of biochemical markers of liver injury with resolution of electrocardiographic and ECHO aberrations. Catecholamine resistance seen in DDC group resolved in the REV group. Cardiac recovery was accompanied by normalization of cardiac troponin-T2 as well as resolution of cardiac stress response at RNA level. Cardiovascular physiological and molecular parameters correlated with degree of cholanemia. Cardiomyopathy was reproduced in cholanemic BA fed Abcb11(-/-) mice. Cardiomyopathy resolves with resolution of liver injury, is associated with cholanaemia, and can be induced by BA feeding.

Loss-of-Function Mutations in the IL-21 Receptor Gene Cause a Primary Immunodeficiency Syndrome

D Kotlarz, N Zietara, G Uzel. J Exp Med. 2013;210(3):433–443 This report of 2 unrelated kindreds with newly recognized mutations in the inteleukin-21 receptor gene (IL-21R) highlights the molecular basis and phenotype of a new form of immunodeficiency. Two sets of kindreds were studied. The first set included a 4-year-old boy and a 10-year-old sister born from consanguineous Lebanese parents with phenotypes characterized by recurrent respiratory infections and chronic cryptosporidial gastrointestinal infection and associated chronic cholangitis, biliary fibrosis, and cirrhosis. A second unrelated set included an 8-year-old boy and a 13-year-old …

Intrahepatic bile acid retention in BALB/c-Abcb4-/--mice activates FXR-dependent rescue transporters at the basolateral and canalicular membrane

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive obliteration of bile ducts, accumulation of bile acids and secondary biliary fibrosis. Abcb4 involves the transport of phospholipids from hepatocytes into bile across the canalicular membrane. Abcb4 knockout disturbes the excretion of phospholipids into bile leading to a more toxic bile composition and bile duct alterations resemble sclerosing cholangitis. Hence we analyzed hepatic bile acid transport in Abcb4–/– mice during the course of the disease.

Impaired hepatocyte maturation, abnormal expression of biliary transcription factors and liver fibrosis in C/EBPα(Cebpa)-knockout mice.

Inactivation of the C/EBPα gene (Cebpa) in the mouse not only causes impaired hepatocyte maturation, but also induces pseudoglandular structures in the liver parenchyma. The present study was undertaken to determine how the expression of other transcription factors controlling differentiation into hepatocytes and biliary epithelial cells is affected, and how the hepatic architecture, including the bile and vascular systems, is disordered in the fetal knockout liver. Histochemical analyses demonstrated that the expression of HNF1α and HNF4α was heterogeneous in the knockout liver, and that not all parenchymal cells (pseudoglandular) expressed these transcription factors, whereas parenchymal cells in the wild-type liver homogeneously expressed these transcription factors. SOX9, which was expressed only in biliary cells in the wild-type liver, was detectable in many pseudoglandular cells of the knockout liver. Although the pseudoglandular cells often coexpressed SOX9 and HNF1α/HNF4α, cells expressing SOX9 but not expressing HNF1α/HNF4α (biliary cells) were sometimes detectable in the parenchyma. Periportal biliary structures were abnormal in their segregation from the parenchyma and in their expression of the transcription factors and Ep-CAM, a biliary adhesion molecule. These results suggest that the inactivation of the Cebpa gene causes unstable expression of liver-enriched transcription factors or biliary transcription factors and elevated expression of Ep-CAM, which may lead to abnormal biliary morphogenesis in the knockout liver. The impaired maturation of the parenchyma caused elevated expression of PECAM-1, desmin and Foxf1, suggesting that the maturation of the parenchyma plays an important role in the normal histogenesis of nonparenchymal cells (stellate cells and sinusoidal endothelial cells).

Extrahepatic Cholangiocyte Cilia Are Abnormal in Biliary Atresia

Biliary atresia (BA) is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atresia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities. The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extrahepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated α tubulin to identify primary cilia. Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human BA samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peribiliary glands adjacent to extrahepatic ducts in the BA sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared with controls. Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in RRV-infected primary cholangiocytes and the extrahepatic ducts from human patients with BA. This suggests that ciliary abnormalities are part of the pathophysiology of BA.

Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation

The use of livers from donors after cardiac arrest (DCD) is increasing in many countries to overcome organ shortage. Due to additional warm ischemia before preservation, those grafts are at higher risk of failure and bile duct injury. Several competing rescue strategies by machine perfusion techniques have been developed with, however, unclear effects on biliary injury. We analyze the impact of an end-ischemic Hypothermic Oxygenated PErfusion (HOPE) approach applied only through the portal vein for 1h before graft implantation. Rat livers were subjected to 30-min in situ warm ischemia, followed by subsequent 4-h cold storage, mimicking DCD-organ procurement and conventional organ transport. Livers in the HOPE group underwent also passive cold storage for 4h, but were subsequently machine perfused for 1h before implantation. Outcome was tested by liver transplantation (LT) at 12h after implantation (n=10 each group) and after 4 weeks (n=10 each group), focusing on early reperfusion injury, immune response, and later intrahepatic biliary injury. All animals survived after LT. However, reperfusion injury was significantly decreased by HOPE treatment as tested by hepatocyte injury, Kupffer cell activation, and endothelial cell activation. Recipients receiving non-perfused DCD livers disclosed less body weight gain, increased bilirubin, and severe intrahepatic biliary fibrosis. In contrast, HOPE treated DCD livers were protected from biliary injury, as detected by cholestasis parameter and histology. We demonstrate in a DCD liver transplant model that end-ischemic hypothermic oxygenated perfusion is a powerful strategy for protection against biliary injury.

Liver Failure and the Need for Transplantation in 6 Patients With Hepatoportal Sclerosis

Hepatoportal sclerosis (HPS), first reported by Mikkelsen et al in 1965, is a pathologic condition that does not cause cirrhotic portal hypertension. The primary hepatic lesion in HPS is found in portal vein branches with preserved synthetic function. Rarely do patients with HPS need liver transplantation. The aim of this study was to describe the clinical and pathologic features of 6 HPS cases who underwent liver transplantation (OLT). From 2000 to 2008, 6 OLT candidates were diagnosed with HPS: 3 displayed bleeding varices and 4 ascites. Child-Pugh evaluation was class B (n = 4) or C (n = 2). The Model for End-stage Liver Disease scores were 18 (n = 2), 20 (n = 3), and 22 (n = 1). Cirrhosis resulted from presumed diagnoses of alcohol n = (1), autoimmune n = (2) or cryptogenic cirrhosis n = (3). On histologic examination, there was marked phlebosclerosis in all cases, including nonocclusive portal vein thrombosis (n = 3), intense portal fibrosis (n = 1), moderate portal fibrosis (n = 5), and uniform moderate sinusoidal dilatation without megasinusoid formation, but with ductal biliary proliferation and ductal biliary fibrosis in all cases. Cholestasis was observed in 1 and incomplete septal cirrhosis in 4 cases. None of the subjects showed histological features of the presumed underlying liver disease. The overall survival of this group was no different from that of other OLT patients. HPS causing hepatic failure may require liver transplantation. Fhlebosclerosis andportal fibrosis may contribute to the loss of hepatic synthesis leading to the need for hepatic transplant. Significant portal fibrosis and phlebosclerosis can contribute to hepatic parenchymal and posterior synthetic loss.

Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver

To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis. DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl₄) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC). All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl₄ induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers. These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

Role of Integrin αvβ6 in the Pathogenesis of Ischemia-Related Biliary Fibrosis After Liver Transplantation

Biliary fibrosis has been referred to as the "final common pathway" of acute and chronic bile duct injury after orthotopic liver transplantation (OLT). We studied the role of integrin αvβ6 in the pathogenesis of ischemia-related biliary fibrosis after OLT. The mouse nonarterialized OLT model with prolonged cold ischemia time was used in this study. A total of 54 FVB/N mice were divided into three groups: sham-operated group (sham, n=18), OLT group that was given the blocking antibody to integrin αvβ6 (OLT+antibody, n=18), and OLT group that was given the isotype control immunoglobulin G (OLT+vehicle, n=18). The expression of αvβ6 and major fibrosis-related genes were studied by real-time polymerase chain reaction and immunohistochemistry. Serum and bile were collected and analyzed biochemically. The histopathologic evaluation was performed to determine the severity of biliary fibrosis and bile duct injury. Integrin αvβ6 was highly expressed on newly formed bile ducts because of cholangiocyte proliferation and was gradually upregulated with the progression of biliary fibrosis after liver transplantation. αvβ6 transcripts closely correlated with fibrosis stages but not bile duct injury severity. Inhibition of αvβ6 attenuated peribiliary collagen deposition remarkably, induced significant downregulation of fibrogenic genes, and improved hepatic function. Integrin αvβ6 is strongly induced de novo in newly formed bile ducts because of cholangiocyte proliferation during ischemia-related biliary fibrogenesis after liver transplantation. Inhibition of αvβ6 could retard the progression of biliary fibrosis of liver allograft significantly, suggesting that αvβ6 is a potential target for the treatment of ischemic biliary complications.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Chronic nicotine exposure stimulates biliary growth and fibrosis in normal rats.

Epidemiological studies have indicated smoking to be a risk factor for the progression of liver diseases. Nicotine is the chief addictive substance in cigarette smoke and has powerful biological properties throughout the body. Nicotine has been implicated in a number of disease processes, including increased cell proliferation and fibrosis in several organ systems. The aim of this study was to evaluate the effects of chronic administration of nicotine on biliary proliferation and fibrosis in normal rats. In vivo, rats were treated with nicotine by osmotic minipumps for two weeks. Proliferation, α7-nicotinic receptor and profibrotic expression were evaluated in liver tissue, cholangiocytes and a polarized cholangiocyte cell line (normal rat intrahepatic cholangiocyte). Nicotine-dependent activation of the Ca(2+)/IP3/ERK 1/2 intracellular signalling pathway was also evaluated in normal rat intrahepatic cholangiocyte. Cholangiocytes express α7-nicotinic receptor. Chronic administration of nicotine to normal rats stimulated biliary proliferation and profibrotic gene and protein expression such as alpha-smooth muscle actin and fibronectin 1. Activation of α7-nicotinic receptor stimulated Ca(2+)/ERK1/2-dependent cholangiocyte proliferation. Chronic exposure to nicotine contributes to biliary fibrosis by activation of cholangiocyte proliferation and expression of profibrotic genes. Modulation of α7-nicotinic receptor signalling axis may be useful for the management of biliary proliferation and fibrosis during cholangiopathies.

Cholanemia induces skeletal muscle wasting despite stimulation of translation initiation, decreased autophagy, activation of Yes Associated Protein (YAP) and proteosomal signal activation in mice

Skeletal muscle wasting is a common co‐morbidity in cholestatic liver diseases. We hypothesize that cholestasis and bile acid (BA) excess (cholanemia) directly induces muscle atrophy by decreasing translation and stimulating protein degradation in muscle. Biliary fibrosis and cholestasis was induced in 6–8 week old male C57BL/6J mice by feeding a DDC supplemented diet for 3 weeks and compared with isocaloric chow fed mice (n=3/group). DEXA scan, exercise tolerance, and fasting circulating BA, glucose and insulin were analyzed, and quadricep muscle signaling was determined. Compared to controls, DDC had 1/3 less food intake, lower body weight (17±0.6 vs. 25±0.5g), lean (11.4±0.8 vs. 16.1±0.4g) and fat mass (2.3±0.1 vs. 3±0.2g) and demonstrated early fatigue on the treadmill. DDC had liver injury, cholestasis, cholanemia (BA: 1000±250 vs. 7±1 μmol/L), and lower glucose (60±5 vs. 150±15mg/dl) and insulin levels (0.15±0.01 vs. 0.53±0.01 ng/L) than controls. DDC had lower muscle mass (0.05±0.006 vs. 0.14±0.01g), higher phosphorylation of AKT, 4E‐BP1 and eIF4G, lower AMPK, eIF2α, YAP phosphorylation, and lower LC3‐II and MuRF1 abundance than controls. These findings suggest that cholanemia stimulates translation initiation, and represses autophagy and protein degradation signaling despite physical evidence of muscle atrophy. (Supported by Pediatric Critical Care Internal Funding)

575 SPECIFIC IN VIVO DELIVERY OF PROCOLLAGEN a1(I) siRNA IN LIPID-LIKE CARRIERS INHIBITS THE PROGRESSION OF BILIARY LIVER FIBROSIS

Introduction: Hepatic fibrosis is characterized by excess production and deposition of extracellular matrix. Small interfering RNA (siRNA) is a powerful tool for post-transcriptional gene silencing in vitro. However, the systemic delivery of naked siRNAs has many obstacles, such as interaction with plasma proteins and degradation by plasma and tissue nucleases. Novel lipid-like particles (LPs) permit efficient delivery of siRNA, minimizing interference with blood cells and plasma and being devoid of side effects. Methods: Mice deficient in the phospholipid flippase (Mdr2) develop spontaneous and progressive biliary fibrosis (Popov et al, J Hepatol 2005). Optimized siRNA directed against the transcripts of the major scar tissue protein, procollagen a1(I), was encapsulated in C12–200 LPs and specifically delivered to the liver of Mdr2 knockout mice. LPs with siRNA to GFP (green fluorescent protein) served as negative controls. DiR-labeled C12–200 LPs were used for in vivo and ex vivo tracking using near infrared (NIR) and fluorescent imaging. Groups (n =7–8) of 8 week old Mdr2KO mice and their nonfibrotic wild-type controls received 4 injections of procollagen a1(I) or control (GFP) siRNA-LPs for 2 weeks (doses of 0.4 and 0.8mg siRNA per kg BW). 24h after the last injection liver collagen was quantified biochemically as hydroxyproline (Hyp) and in liver sections using Sirius red morphometry. Fibrosis related transcript levels were determined by qRT-PCR. Results: 90% of DiR-labeled C12–200 LPs were found in the liver 30 min after injection, in colocalization with hepatocytes>macrophages>myofibroblastic cells. Compared to the GFP control, procollagen a1(I) siRNA-LPs significantly suppressed the expression of its target gene in the fibrotic liver by 80%, which was accompanied by a significant reduction of collagen deposition by 25%. Parameters of hepatic inflammation (ALT and AST) and kidney function (creatinine) remained unchanged after siRNA-LPs injection, indicating the absence of proinflammatory or renal toxic side effects. Conclusion: C12–200 LPs are a specific and safe vehicle to deliver siRNAs to the liver with high efficiency. C12–200 LPs loaded with siRNA to fibrogenic transcripts are a promising approach to efficiently inhibit liver fibrosis progression in vivo.

Absence of beta‐catenin in liver attenuates bile duct injury

While the pathology of biliary fibrosis is well described, the signaling pathways involved in the proliferation and activity of the cholangiocyte compartment during cholestatic liver injury are incompletely understood. β‐Catenin, the chief downstream effector of the Wnt signaling pathway, has been shown to play an important role during bile duct development but its role in adult bile duct homeostasis remains undetermined. Hepatocyte and cholangiocyte‐specific β‐catenin knockout (KO) when fed a diet containing 0.1% 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 1–2 weeks showed decreased atypical ductular reaction as compared to wildtype littermates (WT). KO and WT were next subjected to bile duct ligation (BDL), which led to an increase in bilirubin in both WT and KO mice after 14 days. However, levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and gamma glutamyl transpeptidase (GGTP) were significantly lower in KO when compared to WT. This was associated with a dramatic decrease in ductular reaction and fibrosis in β‐catenin KO livers. Further analysis yielded a notable decrease in total hepatic bile acids (BA) in the KO, which was associated with an increased FXR/SHP2 activation. This led to reduced BA synthesis and increased excretion. Thus, loss of β‐ catenin limits cholestatic injury in multiple models by modulating BA biosynthesis. These findings support an important role of Wnt/β‐catenin signaling in bile duct homeostasis and repair. This study was funded by NIH grant 1R01DK62277 to SPM.

Primary Biliary Cirrhosis is More Severe in Overweight Patients

We sought to determine whether features of metabolic syndrome (MS) and histologic features of nonalcoholic steatohepatitis (NASH) are associated with increased fibrosis in patients with primary biliary cirrhosis (PBC). PBC is a chronic, progressive cholestatic disease. MS is strongly associated with NASH and fibrosis progression in some liver diseases. Patients with PBC seen consecutively at the University of Miami between 1985 and 2008 who had antimitochondrial antibody positivity and a liver biopsy performed at this center at the time of diagnosis were identified. Demographics, clinical features, and biochemical parameters were collected. All liver biopsies were reviewed by a single blinded pathologist for features of NASH, PBC, and fibrosis. The impact of NASH and features of MS on liver biopsy findings were analyzed. Forty-nine patients [median age 51 (34 to 78) years, 98% females] were enrolled. Higher degree of steatosis, severe inflammatory grade, and severe biliary duct damage were each associated with advanced fibrosis (P<0.0001). Regarding MS, only overweight status [body mass index (BMI) ≥ 25] was associated with nonalcoholic fatty liver activity score (NAS) ≥ 5 (P<0.0001), biliary duct damage (P<0.0001), and advanced fibrosis (71% vs. 32%, P=0.007). Patients with NAS ≥ 5 had more severe fibrosis (14/15, 96% vs. 11/34, 44%; P=0.0001) and more severe biliary duct damage (13/15, 87% vs. 3/34, 9%; P=<0.0001). NASH and BMI ≥ 25 are associated with severe biliary duct damage and fibrosis in patients with PBC. BMI could become a useful noninvasive tool to predict advanced fibrosis in PBC.

Vitamin D modulates biliary fibrosis in ABCB4 deficient mice

Background: Vitamin D deficiency is a hallmark of many chronic liver diseases. Impaired vitamin D-VDR signalling might represent an aggravating factor during liver injury (Zuniga et al. 2011) and recent studies suggest that vitamin D exerts a protective role in biliary-type liver diseases (Chignard et al. 2012). To assess the effect of vitamin D on biliary fibrosis, we now treated ATP binding cassette transporter knockout (Abcb4-/-) mice as established model of chronic cholangitis and biliary fibrosis with 25-hydroxyvitamin D.

Slowly progressive cholangiofibrosis induced in rats by α-naphthylisothiocyanate (ANIT), with particular references to characteristics of macrophages and myofibroblasts

A progressive cholangiofibrosis was developed as an animal model in 6-week-old male F344 rats by repeated intraperitoneal injections of α-naphthylisothiocyanate (ANIT) for 19 weeks; liver samples were examined at post-first injection (PFI) weeks 3, 7, 10, 13, 16 and 19, focusing on characteristics of macrophages and myofibroblasts by immunohistochemical analyses. In the affected Glisson's sheath consisting of inflammatory cell infiltrates, bile duct proliferation and advancing fibrosis, the number of macrophages reacting to OX6 (recognizing MHC class II) increased consistently (PFI weeks 3-19), suggesting a central role of antigen presenting cells in the biliary fibrosis; macrophages reacting to ED1 (CD68, reflecting phagocytic activity) and ED2 (CD163, relating to proinflammatory factor production) showed a significantly increased number at PFI weeks 7-19 and PFI weeks 13-19, respectively. Interestingly, macrophages positive for SRA-E5 (CD204, reflecting lipid metabolism) increased at PFI weeks 7-19, and the appearance was limited in the sinusoids around the affected Glisson's sheath. Myofibroblasts appearing in the affected Glisson's sheath reacted to vimentin and desmin at early (PFI weeks 3-7) and mid (PFI weeks 10-13) stages, and then they came to strongly express α-smooth muscle actin at late stage (PFI weeks 16-19). This study shows that macrophages exhibit heterogeneous properties depending on stages and locations; in association with such macrophage populations, myofibroblasts expressing various cytoskeletons participate in cholangiofibrosis. These characteristics would be useful in evaluating the pathogenesis of possible cholangio-toxicants.

Hepatobiliary parasites

Although a number of human parasites can occasionally be found in the liver and/or biliary ducts, there are some that are more commonly found in these body sites including: <i>Leishmania</i> spp., micro-sporidia, <i>Cryptosporidium</i> spp., and several trematodes (<i>Clonorchis, Opisthorchis</i>, and <i>Fasciola</i> spp.), the most important group. The liver trematodes are food borne; it is estimated that >50 million people are infected with >750 million at risk. Public health concerns include cholangiocarcinoma (<i>Clonorchis, Opisthorchis</i>) and severe liver disease (<i>Fasciola</i>). Diagnosis is usually confirmed by fecal examinations, while cholangiography, ultrasonography, and liver scans may reveal lesions consistent with liver fluke infection. Pathological changes (<i>Clonorchis, Opisthorchis</i>) are confined mainly to the biliary tree and morbidity is associated with the worm burden of the host. Chronic inflammatory processes, generation of active oxygen radicals, altered cellular detoxification mechanisms, activation of oncogenes, functional loss of tumour-suppressor genes and dysregulation of cell proliferation and apop-totic mechanisms have been identified as important contributors in the development of cholangiocarcinomas. In <i>Fasciola</i> infections, pathology depends on the number of flukes penetrating the liver. There may be hyperplasia of the biliary epithelium and fibrosis of the ducts with portal or total biliary obstruction. The gallbladder undergoes the same types of changes.