Previous studies from our group showed that HCV reduces the expression of ErbB3 in its host cell. As a result of this, the expression of EGFR, which is crucial for the viral entry, is enhanced, which suggests that HCV uses this rearrangement of the ErbB family members to improve its ability to enter the cell. Further investigations identified the ligand of ErbB3 receptor Neuregulin-1 to be responsible for its down-regulation and consequently triggering this circuit. The aim of the present study was to analyze whether down-regulation of ErbB3 by Nrg-1 also occurs in other human hepatoma cell lines and cells of other species like primary mouse hepatocytes and to assess whether this occurs at a transcriptional or post-transcriptional level.

The IL-1 (cid:2) induced activation of the p38 MAPK /MAPK-acti-vated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propa-gation in hepatocytes and the role of MK2. We established a mathematical model for IL-1 (cid:2) -induced activation of the p38 MAPK /MK2 pathway in hepatocytes that regeneration, cell and

Die TIPS Anlage stellt eine minimal-invasive Technik dar über die der portalvenöse Druck effizient gesenkt werden kann und die daher bei Versagen konservativer Therapien von Komplikationen der portalen Hypertension eine wichtige Therapieoption darstellt. Ziel der vorliegenden Untersuchungen war die retrospektive Erfassung des Therapieerfolgs, sowie die Identifikationen potentieller Prädiktoren für ein Therapieansprechen.

Übergewicht und Diabetes sind die Hauptrisikofaktoren für die Entwicklung einer nicht-alkoholischen Fettlebererkrankung (NAFLE). Die erhöhte Anhäufung von Fett in den Hepatozyten scheint in direktem Zusammenhang mit der Entwicklung von Insulinresistenz und pathologischer Glukosetoleranz zu stehen. Der Thrombinrezeptor PAR4 wird auf verschiedenen Zelltypen exprimiert. Eine erhöhte Thrombinaktivität im Fettgewebe wurde mit der Rekrutierung von inflammatorischen Makrophagen und der Entwicklung von Insulinresistenz bei Mäusen in Verbindung gebracht.

The Hepatitis C Virus influences the immune response by interaction of viral proteins with cellular signalling intermediates. Thereby, apart from others, it also affects the recruitment of several immune cells resulting in an alteration of the inflammatory microenvironment of its host cell. However, the underlying molecular mechasnisms are only incompletely understood. Aim of the present study was to further characterize the mechanisms by which HCV modifies the release of intercellular communication signals from its host cell.

The liver plays a pivotal role in innate and adaptive immunity and is critically involved in the acute phase response. Moreover, it has a high capacity of regeneration upon damage. The liver's resident tissue macrophages, which are an essential source of inflammatory cytokines, regulate the production of acute phase proteins by hepatocytes. Within macrophages the MAPKAP kinases (MK)2 and 3, which are downstream targets of the MAP kinase family member p38MAPK, coordinate the inflammatory outcome as they regulate cytokine and chemokine synthesis in response to pathogens or pathogen associated molecular patterns (PAMP) such as the bacterial component lipopolysaccharide (LPS). In case of cytokines like TNF-α MK2 and MK3 act cooperatively. It has been shown that serum levels of TNF-α are abrogated upon deletion of MK2 and further diminished by additional deletion of MK3.

The main risk factors for developing non alcoholic fatty liver disease (NAFLD) are overweight, diabetes, and metabolic syndrome. About 30 – 50% of type II diabetics suffer from NAFLD. Findings suggest that the development of NAFLD is not only an accompanying phenomenon of type II diabetes. Rather, the increased accumulation of fats in the hepatocytes appears to be directly related to the development of insulin resistance and pathological glucose tolerance. Accordingly, recent findings suggest that the NAFLD plays a pathogenically relevant role in the development of type II diabetes. The molecular mechanisms underlying this observation are poorly understood, some studies suggest that certain messengers (adipokines) are of particular importance in this context. The G protein-coupled thrombin receptor PAR4 is expressed on various cell types, e.g. thrombocytes, endothelial cells, smooth muscle cells, but also on adipocytes. Increased thrombin activity in adipose tissue has been associated with inflammatory macrophage recruitment and development of insulin resistance in mice. The contribution of PAR4 to metabolic and inflammatory changes in the course of diabetes and NAFLD development has not been reported to date.

Objective: The purpose of this study was to evaluate potential causes of Transjugular intrahepatic portosystemic shunt (TIPS) dysfunction. Material and Methods: We retrospectively evaluated 26 patients who required TIPS revision (group I) and 24 patients who did not require any further intervention (group II) within the first two years following TIPS implantation. The distance of the distal end of the stent to the hepatocaval junction was measured. Furthermore, the angle between the stent and the portal vein (inflow) and the angle between the stent and the hepatic vein (outflow) were measured. Furthermore, the following data were evaluated: pre- and postinterventional portal pressure gradients, maximal postinterventional flow and blood values [C-reactive protein (CRP), bilirubin, glutamicoxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT)]. Results: Compared with control subjects, patients who required TIPS revision showed a significantly longer distance from the distal end of the stent to the hepatocaval junction (I: 17.3 +/- 10 mm, II: 6.7 +/- 5.7mm, p < 0.001). There was a statistically significant correlation between the above named distance and the time to revision (Pearson's correlation coefficient, r = 0.5, p = 0.01). In addition, patients with TIPS revision had a significantly larger angle of portalvenous inflow (alpha angle) than the control group (I: 100.5 +/- 31.5 degrees, II: 64.5 +/- 31.6 degrees, p < 0.001). Conclusion: Our results show that the distance from the end of the stent to the hepatocaval junction and the angle of portalvenous inflow are technical factors that may influence the shunt's patency rate. Of these two, the distance to the hepatocaval junction can be influenced easily by the interventionalist.

TGR5 (Gpbar-1) is a G-protein coupled bile acid receptor, which is highly expressed in cholangiocytes, liver sinusoidal endothelial cells, liver macrophages and CD14 positive monocytes of peripheral blood. TGR5-/- mice show impaired liver regeneration after partial hepatectomy and the increased inflammation in liver disease models, thus emphasizing a protective role of TGR5 in liver. While TGR5 functions are being studied extensively, little is known about the transcriptional regulation of this receptor. Aim: To investigate the mechanism and transcriptional regulation of TGR5. Methods: The potential TGR5 promoter (-154/-79) was cloned into a pGL3 Luciferase expression vector. Luciferase gene expression was used to evaluate the effect of specificity protein 1 (Sp1) and Krüppel like factor 5 (KLF5) on TGR5 expression by co-transfection. Binding of the two transcription factors to the promoter was verified by Chromatin Immunoprecipitation (ChIP). Phosphorylation of Sp1 was shown by immunoprecipitation. Results: KLF5 increased the luciferase gene expression in a concentration dependent manner. The effect was lost when the binding site for KLF5 was mutated. High expression of Sp1 mediated a downregulation of luciferase activity, which was absent when mutated Sp1 with no DNA binding activity was co-transfected. KLF5 enhanced the phosphorylation of Sp1. ChIP analysis confirmed the binding of both transcription factors to the promoter region. Conclusion: This study demonstrates that the transcription factor Sp1 downregulates whereas KLF5 upregulates the gene expression of TGR5 in vitro. KLF5 increases the transcription of TGR5 either directly through interaction with the promoter and/or by decreasing the DNA binding activity of Sp1 via increased Sp1 phosphorylation.

Background: Bacterial lipopolysaccharide (LPS) efficiently stimulates the secretion of tumor necrosis factor (TNF)-α from non-parenchymal Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs). Paracrine-acting TNFα subsequently activates the canonical NF-kB pathway in hepatocytes, which is a central regulator of acute phase protein expression and the inflammatory response. However, the impact of individual liver cell types on this pro-inflammatory response has not been sufficiently analysed i.a. due to technical limitations. To establish a holistic view on this complex multi-scale process in a quantitative and time-resolved manner, systems biology serves as a valuable tool.

TGR5 (Gpbar-1) is a G-protein coupled bile acid receptor, which is highly expressed in cholangiocytes, liver sinusoidal endothelial cells, liver macrophages and CD14 positive monocytes of peripheral blood. TGR5-/- mice show impaired liver regeneration after partial hepatectomy and the increased inflammation in liver disease models, thus emphasizing a protective role of TGR5 in liver. While TGR5 functions are being studied extensively, little is known about the transcriptional regulation of this receptor. Aim: To investigate the mechanism and transcriptional regulation of TGR5. Methods: The potential TGR5 promoter (-154/-79) was cloned into a pGL3 Luciferase expression vector. Luciferase gene expression was used to evaluate the effect of Sp1 and KLF5 on TGR5 expression by co-transfection. Binding of the two transcription factors to the promoter was verified by Chromatin Immunoprecipitation (ChIP). Immunoprecipitation was used to confirm the interaction of HDAC1 and 2 with Sp1. Transfection of KLF5 siRNA was used to verify the TGR5 transcriptional regulation. Results: KLF5 increased the luciferase gene expression in a concentration dependent manner. The effect was lost when the binding site for KLF5 was mutated. High expression of SP1 mediated a downregulation of TGR5 promoter luciferase activity, which was absent when mutated Sp1 with a defective DNA binding domain was co-transfected. ChIP analysis confirmed the binding of both transcription factors to the predicted TGR5 promoter region. Sp1 forms an inhibitory complex with HDAC1 and 2 at the site of binding there by downregulating the transcription of TGR5. Inhibition of casein kinase 2 mediated phosphorylation of HDAC proteins by apigenin resulted in a decreased complex formation with Sp1 there by loss of Sp1 mediated downregulation. TGR5 is differentially regulated by Sp1 in different carcinoma cell lines (colon carcinoma and cholangiocyte carcinoma). KLF5 siRNA decreased the mRNA expression of TGR5 in colon and cholangiocyte carcinoma cell lines. Conclusion: This study demonstrates that the transcription factor Sp1 downregulates whereas KLF5 upregulates the gene expression of TGR5 in vitro. Sp1 decreases the transcription of TGR5 by binding to the promoter region and recruiting HDAC1 and 2 to forming an inhibitory complex thereby leading to the deacetylation of histones. Phosphorylation of HDAC proteins by casein kinase 2 is important in the formation of inhibitory complex. Differential regulation of TGR5 by Sp1 was confirmed in different carcinoma cell lines.

study demonstrates a role for mechano-sensitive signalling by the transcriptional regulators Yes associated protein (YAP) and WW domain-containing transcription regulator protein 1 (TAZ) during HSC activation. Methods: Expression was assayed by qPCR, immunoblot and immunocytochemistry of primary HSCs. Acrylamide gels were used to model altered matrix stiffness. Chromatin immunoprecipitation (ChIP) identified YAP binding sites. Results: During activation of HSCs, expression of Yap, Taz and their DNA binding partner Tead was increased. Yap was localised to the nucleus of HSCs positive for profibrotic markers alpha-smooth muscle actin, collagen 1 and Sox9. In contrast, HSCs cultured on soft substrates appeared quiescent-like (characterised by autofluorescence) with inactive Yap sequestered in the cytoplasm. Abrogation of Yap and Taz using siRNA reduced fibrotic gene expression. Hippo signalling (an alternative mechanism regulating Yap/Taz activation) via Lats1 and Lats2 has no effect on fibrotic gene expression in HSCs, confirming a role for mechano-transduction via Yap/Taz in HSC activation in vitro. Moreover, chemical inhibition of the Tead-Yap interaction caused reduced expression of Yap targets (Gli2, Ctgf ) and fibrotic markers (collagen, Sox9). ChIP identified Sox9 as a novel downstream Yap target. Conclusions: These data indicate a functional role for mechanotransduction by Yap/Taz during HSC activation. Capitalizing on this finding, particularly the ability to pharmacologically disrupt Yap target gene interaction may provide insight into novel therapeutic strategies.

The phospholipidfloppase MDR3 (gene symbol: ABCB4) is expressed in the canalicular membrane of hepatocytes and mediates the biliary excretion of phosphatidylcholine, which is required for the formation of mixed micelles in bile. Several mutations of ABCB4 have been identified, which cause cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 3 (PFIC-3), intrahepatic cholestasis of pregnancy (ICP) and the low phospholipid associated cholelithiasis syndrome (LPAC). Here, we report on four new (51076N; L23Hfs16X; c.286+ 1G > A; Q1181E) and one known (527G) MDR3 mutations in eight patients of three families. The patients presented with a wide spectrum of liver diseases. The clinical presentation and decisive laboratory findings or the association to a trendsetting family history led to the identification of the genetic background in these patients. Even the same mutation may be associated with varying disease progression.

April 23 liver.Insulin sensitivity was analysed by measuring fasting glucose and insulin levels (for calculation of HOMA) and by glucose-and insulin tolerance tests.We studied phosphorylation of Foxo1 at Ser256 by Western Blot and expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phophatase (G6Pase), two crucial gluconeogenetic key enzymes, and PGC1a, their major transcriptional coactivator, by quantitative RT-PCR.Results: Compared to C57BL6 mice, B6HCV mice have elevated fasting glucose (6.5 mmol/l vs. 4.9 mmol/l), fasting insulin (32.4 mIU/ml vs. 14.7 mIU/ml) and HOMA index (8.4 vs. 2.8).Glucose and insulin tolerance tests were pathologic.S256Foxo1 was hypophosphorylated in B6HCV mice and PGC1a expression was significantly upregulated.Also upregulation of PEPCK and G6Pase was observed. Conclusion:The transgenic expression of a HCV genotype 1b genome in hepatocytes results in a dysregulated expression of gluconeogenetic key enzymes and leads to systemic insulin resistance by a mechanism involving PP2A upregulation and interference with insulin signalling.

Ziele: Das hepatorenale Syndrom (HRS) ist eine bekannte Komplikation der Leberzirrhose. Diese retrospektive Studie wurde durchgeführt, um zu überprüfen, ob transjuguläre portosystemische Stentshunts (TIPSS) geeignet sind, ein HRS effektiv zu behandeln. Methode: Aus einer Gesamtgruppe von 229 Patienten (148Männer, 81 Frauen, mittleres Alter 56 +-12 Jahre), bei denen in einem Zeitraum von 10 Jahren (1995–2005) ein TIPSS implantiert wurde, lag bei 8 Patienten ein HRS mit einem mittleren Serum-Kreatinin von 4,2mg/dl als primäre Indikation zur TIPSS-Anlage vor. 6 dieser Patienten mussten zum Zeitpunkt der TIPSS-Anlage hämodialysiert werden. Eine zweite Untergruppe von 36 Patienten, bei denen ein TIPSS aufgrund eines therapierefraktären Aszites oder rezidivierender Varizenblutung implantiert werde musste, hatte klinisch zusätzlich ein HRS (mittleres S-Krea 2,5mg/dl). Nach Laboratoriumsdiagnostik hatten 30 weitere Patienten ein leicht erhöhtes S-Krea (>1,5mg/dl) und weitere 44 Patienten eine stark eingeschränkte Nierenfunktion (S-Krea >2,5mg/dl), jedoch klinisch kein HRS. Bei den übrigen Patienten lag eine normale Nierenfunktion vor. TIPSS-Shunts wurden in üblicher Technik unter Verwendung nicht-gecoverter selbstexpandierender Nitinol-Stents implantiert. Ergebnis: Das HRS war bei den 8 Patienten vollständig rückläufig, so dass innerhalb von drei Monaten bei allen Patienten die Hämodialyse beendet werden konnte, ihr S-Krea verbesserte sich auf 2,2 +-0,5mg/dl (n.s.). Bei den Patienten der zweiten Gruppe verbesserte sich das S-Krea signifikant auf 1,9mg/dl (+-0,3, p=0,04). 44 Patienten mit eingeschränkter Nierenfunktion zeigten einen deutlichen Abfall des S-Krea von 2,8 +-0,2mg/dl auf 1,9 +-0,2mg/dl (p=0,001) trotz der i.v.-Applikation von nicht-ionischem Kontrastmittel während der TIPSS-Anlage. Bei den übrigen Patienten blieb die Nierenfunktion stabil (1,2mg/dl +-0,1). Schlussfolgerung: TIPSS ist eine wirksame Therapie des hepatorenalen Syndroms bei Patienten mit Leberzirrhose. Die Reduktion des portalen Hochdrucks nach TIPSS kann eine verbesserte Nierenperfusion und damit bessere Nierenfunktion bedingen, so dass eine Nierenersatztherapie beendet werden kann.

Background: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. Aim: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. Methods: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl4), lipopolysaccaride (LPS), tumour necrosis factor α (TNFα), and anti-Fas antibody. Results: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl4, LPS, TNFα, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFα, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). Conclusions: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFα induced liver damage in vivo. The TNFα resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Einleitung: Hyperosmolarität (405 mOsm), CD95 Ligand (CD95L) und hydrophobe Gallensäuren induzieren in primären Hepatozyten eine EGF-R-vermittelte CD95-Tyrosin-Phosphorylierung mit folgendem CD95-Membrane trafficking und Formierung des Death inducing signaling complex (DISC).

AIM To define predictive parameters of a complicated clinical course after the TIPSS procedure. METHODS Blood cultures were drawn prospectively in 41 patients from a central line and from the portal venous blood before stent placement as well as from the central line 20 min after intervention. C-reactive protein (CRP) (mg/dl) and white blood cell count (WBC,/microl) on the day of TIPSS-procedure (d0), the first (d1) and seven (d7) days after TIPSS were compared in patients with a complicated clinical course (spontaneous bacterial peritonitis,pneumonia,sepsis; group I) to patients without clinical complications (group II) RESULTS: Group I showed a significant increase in CRP (d0: 1.8+/-1.0; d1: 3.2+/-1.5; d7: 4.3+/-3.2), and white blood cell count (d0: 7700+/-2600; d1: 10800+/-2800; d7: 7500+/-1800) on the first day after TIPSS-procedure in comparison to group II (CRP: d0: 1.6+/-0.6; d1: 1.8+/-1.0; d7: 1.9+/-0.6. WBC: d0: 6900+/-1500; d1: 8000+/-1600; d7: 7600+/-1400). Microbiological analysis showed in 12% skin or oral flora in the last sample. CONCLUSIONS The course of CRP and WBC-count during the first week after TIPSS procedure may indicate patients with a potential risk of a complicated clinical course.

Zusammenfassung Fragestellung. Definition prädiktiver Parameter infektiologischer Komplikationen bei Patienten nach TIPSS-Anlage. Methodik. Bei 41 Patienten wurden Blutproben prospektiv vor intrahepatischer Stentanlage zentralvenös und portalvenös sowie 20 min postinterventionell erneut zentralvenös entnommen und mikrobiologisch analysiert. C-reaktives Protein (CRP) (mg/dl) und Leukozytenzahl (/μl) wurden am Interventionstag (d0), am 1. (d1) sowie 7 Tage (d7) postinterventionell bestimmt. Patienten mit kompliziertem Verlauf (spontane bakterielle Peritonitis,Pneumonie, Sepsis; Gruppe 1) wurden von Patienten ohne klinische Komplikationen (Gruppe 2) unterschieden. Ergebnisse. Gruppe 1 wies einen signifikanten Anstieg des CRP (d0: 1,8±1,0; d1: 3,2±1,5; d7: 4,3±3,2) und der Leukozytenzahl (d0: 7700±2600; d1: 10800±2800; d7: 7500±1800) bereits am 1.Tag nach TIPSS im Vergleich zu Gruppe 2 auf (CRP: d0: 1,6±0,6; d1: 1,8±1,0; d7: 1,9±0,6.Leukozyten: d0: 6900±1500; d1: 8000±1600; d7: 7600±1400).Die mikrobiologische Analyse ergab in 12% der Fälle Haut- und Oberflächenkeime in der postinterventionellen Blutprobe. Schlussfolgerungen. Entwicklung von CRP und Leukozytenzahlen in der 1.Woche nach TIPSS können potenzielle Risikopatienten für einen komplizierten Verlauf anzeigen.