Congenital intrahepatic portocaval shunts and hypoglycemia due to secondary hyperinsulinism: a case report and review of the literature.

BACKGROUND: Congenital portosystemic shunts present with various associated complications, such as other congenital malformations, hyperammonemia, or hepatopulmonary syndrome. Few cases of associated hypoglycemia have been reported so far and our case, to the best of our knowledge, describes the most severe extent of hypoglycemia. CASE PRESENTATION: We describe the case of a newborn Arab boy with two intrahepatic portosystemic shunts, resulting in severe and persistent hypoglycemia, due to which one of the shunts was closed by interventional radiology whereas the other shunt had already closed spontaneously. CONCLUSIONS: Because he showed elevated levels for insulin and prolonged high insulin levels in an oral glucose tolerance test, our case supports the theory that portocaval shunts cause a reduced hepatic insulin reduction due to the high blood volume bypassing the liver. This case provides further insights into glucose regulation mechanisms of the liver and we suggest a consistent screening for hypoglycemia in patients with congenital portosystemic shunts.

Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo.

Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis.

INTRODUCTION: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions. METHODS: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot. RESULTS: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated. CONCLUSIONS: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection.

BACKGROUND & AIMS: The limited availability of hepatitis Delta virus (HDV) infection models has hindered studies of interactions between HDV and infected hepatocytes. The aim was to investigate the antiviral state of HDV infected human hepatocytes in the setting of co-infection with hepatitis B virus (HBV) compared to HBV mono-infection using human liver chimeric mice. METHODS: Viral loads, human interferon stimulated genes (hISGs) and cytokines were determined in humanized uPA/SCID/beige (USB) mice by qRT-PCR, ELISA and immunofluorescence. RESULTS: Upon HBV/HDV inoculation, all mice developed viremia, which was accompanied by a significant induction of hISGs (i.e. hISG15, hSTATs, hHLA-E) compared to uninfected mice, while HBV mono-infection led to weaker hISG elevations. In the setting of chronic infection enhancement of innate defense mechanisms was significantly more prominent in HBV/HDV infected mice. Also the induction of human-specific cytokines (hIP10, hTGF-ß, hIFN-ß and hIFN-λ) was detected in HBV/HDV co-infected animals, while levels remained lower or below detection in uninfected and HBV mono-infected mice. Moreover, despite the average increase of hSTAT levels determined in HBV/HDV infected livers, we observed a weaker hSTAT accumulation in nuclei of hepatocytes displaying very high HDAg levels, suggesting that HDAg may in part limit hSTAT signaling. CONCLUSIONS: Establishment of HDV infection provoked a clear enhancement of the antiviral state of the human hepatocytes in chimeric mice. Elevated pre-treatment ISG and interferon levels may directly contribute to inflammation and liver damage, providing a rationale for the more severe course of HDV-associated liver disease. Such antiviral state induction might also contribute to the lower levels of HBV activity frequently found in co-infected hepatocytes.

Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism.

UNLABELLED: Chronic hepatitis B virus (HBV) infection has been associated with alterations in lipid metabolism. Moreover, the Na+-taurocholate cotransporting polypeptide (NTCP), responsible for bile acid (BA) uptake into hepatocytes, was identified as the functional cellular receptor mediating HBV entry. The aim of the study was to determine whether HBV alters the liver metabolic profile by employing HBV-infected and uninfected human liver chimeric mice. Humanized urokinase plasminogen activator/severe combined immunodeficiency mice were used to establish chronic HBV infection. Gene expression profiles were determined by real-time polymerase chain reaction using primers specifically recognizing transcripts of either human or murine origin. Liver biopsy samples obtained from HBV-chronic individuals were used to validate changes determined in mice. Besides modest changes in lipid metabolism, HBV-infected mice displayed a significant enhancement of human cholesterol 7α-hydroxylase (human [h]CYP7A1; median 12-fold induction; P<0.0001), the rate-limiting enzyme promoting the conversion of cholesterol to BAs, and of genes involved in transcriptional regulation, biosynthesis, and uptake of cholesterol (human sterol-regulatory element-binding protein 2, human 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and human low-density lipoprotein receptor), compared to uninfected controls. Significant hCYP7A1 induction and reduction of human small heterodimer partner, the corepressor of hCYP7A1 transcription, was also confirmed in liver biopsies from HBV-infected patients. Notably, administration of Myrcludex-B, an entry inhibitor derived from the pre-S1 domain of the HBV envelope, provoked a comparable murine CYP7A1 induction in uninfected mice, thus designating the pre-S1 domain as the viral component triggering such metabolic alterations. CONCLUSION: Binding of HBV to NTCP limits its function, thus promoting compensatory BA synthesis and cholesterol provision. The intimate link determined between HBV and liver metabolism underlines the importance to exploit further metabolic pathways, as well as possible NTCP-related viral-drug interactions.

Primary human hepatocytes from metabolic-disordered children recreate highly differentiated liver-tissue-like spheroids on alginate scaffolds.

Human hepatocyte transplantation has not been routinely established as an alternative to liver transplantation in liver disease due to low cell engraftment rates. Preimplantation in vitro engineering of liver tissue using primary human hepatocytes on three-dimensional scaffolds could be an alternative model. Alginate bioscaffolds were seeded with 1×10(6) hepatocytes freshly isolated from the livers of three children suffering from different metabolic disorders. During a culture period of 14 days only a marginal loss of hepatocytes was observed via measurement of DNA content per scaffold. Formation of hepatocyte spheroids was detected from day 3 onward using transmission light microscopy. Biochemical assays for albumin, α1-antitrypsin, and urea revealed excellent metabolic function with its maximum at day 7. Low lactate dehydrogenase enzyme release demonstrated minor cellular membrane damage. Hematoxylin and eosin and periodic acid Schiff staining displayed high cell viability and well-preserved glycogen storage until day 7. Immunofluorescent staining of hepatocyte nuclear factor 4, zonula occludens protein 1, and cytokeratin 18 revealed highly differentiated hepatocytes in spheroids with a tissue-like structure on scaffolds. Fluorescent labeling of cytochrome P450 and bile canaliculi demonstrated detoxification ability as well as a well-shaped bile canaliculi network. Almost constant expression levels in most target genes were detected by quantitative real-time polymerase chain reaction. The results of TUNEL reaction implicated a safe scaffold-dissolving procedure. Our results indicate that alginate scaffolds provide a favorable microenvironment for liver neo-tissue recreation and regeneration. Further, we demonstrate that livers from children with inherited metabolic disorders could serve as an alternative cell source for in vitro experiments.

Strong antiviral activity of the new l-hydroxycytidine derivative, l-Hyd4FC, in HBV-infected human chimeric uPA/SCID mice.

BACKGROUND: Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. ß-l-nucleoside analogues, especially ß-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these ß-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected ß-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivo investigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice. METHODS: Stably infected animals (median 6×10(7) HBV DNA/ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivo antiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR. RESULTS: Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes. CONCLUSIONS: Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent.

Primary human hepatocytes on biodegradable poly(l-lactic acid) matrices: a promising model for improving transplantation efficiency with tissue engineering.

Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end-stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix-based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l-lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver-like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte-specific cytoskeleton. Albumin, alpha-1-antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity.

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research.

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two-dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell-specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three-dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen-coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver-cell-specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver-specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real-time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well-preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF-4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re-established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO-1-positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes.