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Although normothermic machine perfusion (NMP) provides superior preservation of liver grafts compared to static cold storage and allows for viability testing of high-risk grafts, its effect on the liver immune compartment remains unclear. We investigated the innate immune response during 6 h of continuous NMP (cNMP) of livers that were directly procured (DP, n = 5) or procured after 60 min warm ischemia (WI, n = 5), followed by 12 h of whole blood (WB) reperfusion. WI livers showed elevated transaminase levels during cNMP but not after WB reperfusion. Perfusate concentrations of TNF-α were lower in WI livers during cNMP and WB reperfusion, whereas IL-8 concentrations did not differ significantly. TGF-β concentrations were higher in WI livers during NMP but not after WB reperfusion, whereas IL-10 concentrations were similar. Endoplasmic stress and apoptotic signaling were increased in WI livers during cNMP but not after WB reperfusion. Additionally, neutrophil mobilization increased to a significantly lesser extent in WI livers at the end of NMP. In conclusion, WI livers exhibit a distinct innate immune response during cNMP compared to DP livers. The cytokine profile shifted towards an anti-inflammatory phenotype during cNMP and WB reperfusion, and pro-apoptotic signaling was stronger during cNMP. During WB reperfusion, livers exhibited a blunted cytokine release, regardless of ischemic damage, supporting the potential reconditioning effect of cNMP.

Background: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites., Methods: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte., Results: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue., Conclusions: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

The wider use of livers from expanded criteria donors and donation after circulatory death donors may help to improve access to liver transplantation. A prerequisite for safely using these higher risk livers is the development of objective criteria for assessing their condition before transplantation. Compared to simple cold storage, hypothermic machine perfusion (HMP) provides a unique window for evaluating liver grafts between procurement and transplantation. In this proof-of-concept study, we tested basic parameters during HMP that may reflect the condition of human liver grafts, and we assessed their morphology after prolonged HMP. Seventeen discarded human livers were machine-perfused. Eleven livers were nontransplantable (major absolute contraindications and severe macrovesicular steatosis in the majority of the cases). Six livers were found in retrospect to be transplantable but could not be allocated and served as controls. Metabolic parameters (pH, lactate, partial pressure of oxygen, and partial pressure of carbon dioxide), enzyme release in the perfusate [aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)], and arterial/portal resistances were monitored during HMP. Nontransplantable livers released more AST and LDH than transplantable livers. In contrast, arterial/portal vascular resistances and metabolic profiles did not differ between the 2 groups. Morphologically, transplantable livers remained well preserved after 24 hours of HMP. In conclusion, HMP preserves the morphology of human livers for prolonged periods. A biochemical analysis of the perfusate provides information reflecting the extent of the injury endured., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background/aims: Meckel syndrome is an autosomal-recessive disease characterized by a combination of renal cysts, anomalies of the central nervous system, polydactyly and ductal plate malformations (DPM), which are hepatic anomalies consisting of excessive and abnormal foetal biliary structures. Among the genomic loci associated with Meckel syndrome, mutations in four genes were recently identified. These genes code for proteins associated with primary cilia and are possibly involved in cell differentiation. The aim of the present work was to investigate the formation of the primary cilia and the differentiation of the hepatic cells in foetuses with Meckel syndrome., Methods: Sections of livers from human foetuses with Meckel syndrome were analysed by immunofluorescence, immunohistochemistry and electron microscopy., Results: The primary cilia of the biliary cells were absent in some Meckel foetuses, but were present in others. In addition, defects in hepatic differentiation were observed in Meckel livers, as evidenced by the presence of hybrid cells co-expressing hepatocytic and biliary markers., Conclusions: Defects in cilia formation occur in some Meckel livers, and most cases show DPM associated with abnormal hepatic cell differentiation. Because differentiation precedes the formation of the cilia during liver development, we propose that defective differentiation may constitute the initial defect in the liver of Meckel syndrome foetuses.

Background & Aims: The Rho small guanosine triphosphatase Cdc42 is critical for diverse cellular functions, including regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell-cycle progression, and cell transformation. This implies that Cdc42 might be required for liver function., Methods: Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure., Results: Mice lacking Cdc42 in their hepatocytes were born at Mendelian ratios. They did not show increased mortality but showed chronic jaundice. They developed hepatomegaly soon after birth, and signs of liver transformation, such as formation of nodules and tumors, became visible macroscopically at age 6 months. Hepatocellular carcinoma was observed 8 months after birth. Tumors grew slowly and lacked expression of nuclear beta-catenin. Lung metastases were observed at the late stage of carcinogenesis. Immunofluorescent examination and electron microscopy revealed severe defects in the liver. At the age of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis., Conclusions: We describe a mouse model in which chronic liver disease leads to hepatocarcinogenesis.

Unlabelled: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis., Conclusion: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.

Background: We recently showed that human hepatic progenitor cells (HPCs) express muscarinic acetylcholine (Ach) receptor subtype 3 and that--following liver transplantation--HPC numbers are significantly reduced. To further elaborate on this, we examined whether HPC also express receptors for vasoactive intestinal peptide (VIP), which, besides Ach, also is an important parasympathetic neurotransmitter. VIP expressing nerves are known to be present in the liver., Methods: We performed immunohistochemistry for VIP receptor subtypes 1 and 2 (VIPR1 and 2), on sections of normal and diseased human liver (n=17), and double staining for VIPR2 and known HPC markers. We performed RT-PCR for VIPR1 and 2 on total RNA from purified rat HPC. To document the probability of direct interaction, we also performed double immunostaining for nerve markers and HPC markers on human liver sections., Results: VIPR2 immunostaining was clearly positive in HPC and reactive bile ductules on paraffin-embedded and frozen tissue sections. We could not demonstrate VIPR1 protein expression in the liver, with either of two VIPR1 antibodies tested. The presence of VIPR2 mRNA in HPC was confirmed by RT-PCR. Nerve endings were shown to abut on reactive bile ductules., Conclusion: We show here for the first time that HPC express VIPR2 and receive nerve endings. These features, and the fact that HPC numbers are influenced by the presence or absence of the autonomic innervation of the liver, suggest a direct interaction.

Diagnostic liver histopathology is currently based on the microscopic assessment of morphological and immunohistochemical features in a biopsy. In other fields of diagnostic pathology, it has been shown that the diagnostic process can be improved by the incorporation of ancillary molecular techniques and this will most likely also occur in liver pathology in the future. This article gives an overview of several liver diseases for which the diagnosis could be improved by the addition of a molecular technique to the classical histopathologic working method. For each of these diseases, the most promising molecular techniques are discussed. It is very likely that ancillary molecular techniques will be applied in routine practice to improve diagnosis and prognostication of liver tumors in the foreseeable future.

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette transport protein that is expressed in several organs including the liver. Previous studies have shown that ABC transport proteins play an important pathophysiological role in several liver diseases. However, to date, expression pattern and possible role of BCRP in human liver diseases and animal models have not been studied in detail. Here we investigated the expression pattern of BCRP in normal liver, chronic parenchymal and biliary human liver diseases, and parallel in different rat models of liver diseases. Expression was studied by immunohistochemistry and additionally by RT-PCR analysis in Thy-1-positive rat oval cells. Bile ducts, hepatic progenitor cells, reactive bile ductules, and blood vessel endothelium were immunoreactive for BCRP in normal liver and all types of human liver diseases and in rat models. BCRP was expressed by the canalicular membrane of hepatocytes in normal and diseased human liver, but never in rat liver. Remarkably, there was also expression of BCRP at the basolateral pole of human hepatocytes, and this was most pronounced in chronic biliary diseases. In conclusion, BCRP positivity in the progenitor cells/reactive ductules could contribute to the resistance of these cells to cytotoxic agents and xenotoxins. Basolateral hepatocytic expression in chronic biliary diseases may be an adaptive mechanism to pump bile constituents back into the sinusoidal blood. Strong differences between human and rat liver must be taken into account in future studies with animal models.

Background/aims: Hepatic stellate cells (HSC) have been hypothesised to derive from the neural crest, based on their expression of multiple neural/neuroendocrine features and their contacts with autonomic nerve endings., Methods: We studied the emergence of HSC in the liver during embryonic development in a transgenic mouse line expressing yellow fluorescent protein (YFP) in all neural crest cells and their derivatives. Cellular YFP expression in these mice was compared with desmin expression between embryonic day (E) 11.5 and adulthood., Results: YFP was abundantly expressed in neural crest cells delaminating from the neural tube and in all known neural crest-derived structures and cell populations. In particular, YFP expressing cells perfectly mimicked the spatial and temporal pattern of enteric nervous system development from neural crest cells migrating from the postotic region. Cells within the adrenal medulla were also YFP positive. Analysis of the liver showed that desmin-expressing, stellate-shaped, perisinusoidally located HSC were evident from E11.5 onwards. However, no detectable YFP expression was seen in the developing liver or in HSC, from E11.5 until adulthood., Conclusions: These findings suggest HSC do not descend from the neural crest, and therefore may derive from the septum transversum mesenchyme, from endoderm or from the mesothelial liver capsule.

Alagille syndrome (AGS) is a developmental, multiorgan disease caused by mutations of the Jagged1 gene. The liver is one of the major organs affected in AGS, and the hallmark of liver pathology in AGS is an age-related increase in the proportion of portal tracts that have no bile duct, but without evidence of prominent bile duct damage. The pathogenesis of this bile duct paucity is currently not well understood. (Immuno)histochemical and molecular analyses were performed on several liver biopsies that were taken during macroscopic examination of the explant liver of a 17-year-old AGS patient. The liver periphery was macroscopically pale and was microscopically characterized by complete absence of bile ducts and presence of severe cholestasis, but there was no ductular reaction. Conversely, the central, hilar portion contained normally developed bile ducts showing no or minimal damage and cholestasis. A missense mutation in the Jagged1 gene was present in both parts of the liver, indicating that mosaicism did not cause this peculiar picture. There was also a hypertrophy of the hepatic arterial branches in the liver periphery. Together with previous indirect findings, the current study of the explant liver of an AGS patient strongly suggests that a lack of branching and elongation of bile ducts during postnatal liver growth is the mechanism by which peripheral bile duct paucity and cholestasis develops in AGS. Our findings also suggest that anomalies of the intrahepatic arterial branches may be part of AGS in some patients.

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)-SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA(+/+)-SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated "mature" hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Liver progenitor cells as well as hepatic stellate cells have neuroendocrine features. Progenitor cells express chromogranin-A and neural cell adhesion molecule, parathyroid hormone-related peptide, S-100 protein, neurotrophins, and neurotrophin receptors, while hepatic stellate cells express synaptophysin, glial fibrillary acidic protein, neural cell adhesion molecule, nestin, neurotrophins, and their receptors. This phenotype suggests that these cell types form a neuroendocrine compartment of the liver, which could be under the control of the central nervous system. We recently showed that the parasympathetic nervous system promotes progenitor cell expansion after liver injury, since selective vagotomy reduces the number of progenitor cells after chemical injury in the rat. Similarly, after transplantation, which surgically denervates the liver, human livers that develop hepatitis have fewer progenitor cells than native, fully innervated livers with similar degrees of liver injury. There is also accumulating experimental evidence linking the autonomic system, in particular the sympathetic nervous system (SNS), with the pathogenesis of cirrhosis and its complications. Recently, it has been shown that hepatic stellate cells themselves respond to neurotransmitters. Moreover, inhibition of the SNS reduced fibrosis in carbon tetrachloride-induced liver injury. In view of the denervated state of transplanted livers, it is very important to unravel the neural control mechanisms of regeneration and fibrogenesis. Moreover, since there is a shortage of donor organs, a better understanding of the mechanisms of regeneration could have therapeutic possibilities, which could even obviate the need for orthotopic liver transplantation., (Copyright 2004 Wiley-Liss, Inc.)

Erythropoietic protoporphyria (EPP) is an inherited disease of haem synthesis caused by a mutation in one of the alleles of the enzyme ferrochelatase. This mutation leads to partial deficiency of the enzyme, resulting in increased concentrations of protoporphyrin (PP) in blood, liver, and faeces. Five to ten per cent of patients with EPP develop severe liver disease characterized by the presence of PP deposits. This study used histochemistry and immunohistochemistry to investigate the histopathological features present in the livers of 44 mice with a heterozygous or homozygous point mutation in the ferrochelatase gene (fch/+ and fch/fch mice, respectively). Some fch/+ mouse livers showed mixed steatosis and large cell dysplasia. The livers of fch/fch mice showed periportal or septal fibrosis accompanied by an atypical ductular reaction. These findings suggest that the obstruction and damage of a proportion of large and small bile ducts by PP deposits cause an accumulation of PP in the parenchyma, which leads to damage and loss of hepatocytes due to the toxic effects of PP. The classical stages of hepatocarcinogenesis were observed and hepatic progenitor cells appear to be involved in this process. PP acts as the promoting agent and is probably also the initiating agent., (Copyright 2002 John Wiley & Sons, Ltd.)

The canals of Hering and bile ductules in human liver contain hepatic progenitor cells that can differentiate towards the biliary and hepatocytic lineage. Proliferation and differentiation of hepatic progenitor cells is referred to as 'activation' and this process occurs to a variable degree in almost all human liver diseases. Several studies indicate that hepatic progenitor cell activation in diseased liver is regulated by neural and neuroendocrine factors such as the vagal innervation. Analogous to oval cells in animal liver, there is evidence that human hepatic progenitor cells may be able to give rise to hepatocellular carcinoma and other liver tumors.

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

Background/aims: The development of the intrahepatic bile ducts most likely requires interactions between epithelial and mesenchymal cells. In view of the epithelial-mesenchymal interactions between portal myofibroblasts (pMFs) and biliary epithelial cells in adult diseases of the bile ducts, we investigated the presence and function of pMFs during the development of intrahepatic bile ducts, as well as the development of intrahepatic branches of the hepatic artery., Methods: We performed haematoxylin-eosin-stainings and immunohistochemistry for alpha-smooth-muscle actin, cytokeratin 19 and vimentin on serial sections of 45 fetal and postnatal liver biopsies., Results: The mesenchyme of portal tracts in the ductal plate stage devoid of a hepatic artery branch, contained numerous and diffusely scattered pMFs. Portal tracts with a hepatic artery branch were always larger than those without and showed a decreasing number of pMFs. In the remodeling stage, all portal tracts contained a hepatic artery branch, and pMFs were restricted to the periductal mesenchyme. These periductal pMFs disappeared after full incorporation of the bile duct., Conclusion: Our findings strongly suggest interactions between pMFs and epithelial cells of the developing bile ducts. The development of the intrahepatic arterial branches always precedes the incorporation of the tubular segments of the ductal plate.

Background: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Mice lacking the tumor-suppressive protein phosphatase 2A subunit B56d (Ppp2r5d) spontaneously develop HCC, correlating with increased c-MYC oncogenicity. Materials and Methods: We used two-dimensional difference gel electrophoresis-coupled matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify differential proteomes of livers from wild-type, non-cancerous and HCC-affected B56d knockout mice. Results: A total of 23 proteins were differentially expressed/regulated in liver between wild-type and non-cancerous knockout mice, and 119 between noncancerous and HCC knockout mice ('cancer proteins'). Overlap with our reported differential transcriptome data was poor. Overall, 56% of cancer proteins were reported before in HCC proteomics studies; 44% were novel. Gene Ontology analysis revealed cancer proteins mainly associated with liver metabolism (18%) and mitochondria (15%). Ingenuity Pathway Analysis identified 'cancer' and 'gastrointestinal disease' as top hits. Conclusion: We identified several proteins for further exploration as novel potential HCC biomarkers, and independently underscored the relevance of Ppp2r5d knockout mice as a valuable hepatocarcinogenesis model. [ABSTRACT FROM AUTHOR]

Background/Aims: Focal nodular hyperplasias (FNHs) are benign liver lesions considered to be a hyperplastic response to increased blood flow in normal liver. In contrast, FNH-like lesions/nodules occur in cirrhotic liver but share similar histopathological features. We conducted a transcriptome analysis to identify biological pathways deregulated in FNH. Methods: Gene expression profiles obtained in FNH and normal livers were compared. Differentially-expressed genes were validated using quantitative-RT-PCR in 70 benign liver tumors including FNH-like lesions. Results: Among the deregulated genes in FNHs, 19 displayed physiological restricted distribution in the normal liver. All six perivenous genes were up-regulated in FNH, whereas 13 periportal genes were down-regulated. Almost all these genes are known to be regulated by β-catenin. Glutamine synthetase was markedly overexpressed in anastomosed areas usually centered on visible veins. Moreover, activated hypophosphorylated β-catenin protein accumulated in FNH in the absence of activating mutations. These results suggest the zonated activation of the β-catenin pathway in FNH, whereas the other benign hepatocellular tumors, including FNH-like lesions, demonstrated an entirely different pattern of β-catenin expression. Conclusions: In FNH, increased activation of the β-catenin pathway was found restricted to enlarged perivenous areas. FNH-like nodules may have a different pathogenetic origin. [Copyright &y& Elsevier]

Abstract: Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H2O2, the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H2O2 to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O2 − ) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H2O2 directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation. [Copyright &y& Elsevier]