STAT3 inhibition reduces toxicity of oncolytic VSV and provides a potentially synergistic combination therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a refractory malignancy with a high mortality and increasing worldwide incidence rates, including the United States and central Europe. In this study, we demonstrate that a specific inhibitor of signal transducer and activator of transcription 3 (STAT3), NSC74859, efficiently reduces HCC cell proliferation and can be successfully combined with oncolytic virotherapy using vesicular stomatitis virus (VSV). The potential benefits of this combination treatment are strengthened by the ability of NSC74859 to protect primary hepatocytes and nervous system cells against virus-induced cytotoxicity, with an elevation of the VSV maximum tolerated dose in mice. Hereby we propose a strategy for improving the current regimen for HCC treatment and seek to further explore the molecular mechanisms underlying selective oncolytic specificity of VSV.

Randomized controlled multicenter trial on the effectiveness of the collagen hemostat Sangustop® compared with a carrier-bound fibrin sealant during liver resection (ESSCALIVER study, NCT00918619).

BACKGROUND: Despite improvements in liver surgery over the past decades, hemostasis during hepatic resections remains challenging. This multicenter randomized study compares the hemostatic effect of a collagen hemostat vs. a carrier-bound fibrin sealant after hepatic resection. METHODS: Patients scheduled for elective liver resection were randomized intraoperatively to receive either the collagen hemostat (COLL) or the carrier-bound fibrin sealant (CBFS) for secondary hemostasis. The primary endpoint was the proportion of patients with hemostasis after 3 min. Secondary parameters were the proportions of patients with hemostasis after 5 and 10 min, the total time to hemostasis, and the complication rates during a 3 months follow-up period. RESULTS: A total of 128 patients were included. In the COLL group, 53 out of 61 patients (86.9 %) achieved complete hemostasis within 3 min after application of the hemostat compared to 52 out of 65 patients (80.0 %) in the CBFS group. The 95 % confidence interval for this difference [-6.0 %, 19.8 %] does not include the lower noninferiority margin (-10 %). Thus, the COLL treatment can be regarded as noninferior to the comparator. The proportions of patients with hemostasis after 3, 5, and 10 min were not significantly different between the two study arms. Postoperative mortality and morbidity were similar in both treatment groups. CONCLUSION: The collagen hemostat is as effective as the carrier-bound fibrin sealant in obtaining secondary hemostasis during liver resection with a comparable complication rate.

Decrease of global methylation improves significantly hepatic differentiation of Ad-MSCs: possible future application for urea detoxification.

Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available "off the shelf." Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers α-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor.

BACKGROUND AND PURPOSE: Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACH: Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTS: All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONS: Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes.

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFalpha. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.

JNK inhibition sensitises hepatocellular carcinoma cells but not normal hepatocytes to the TNF-related apoptosis-inducing ligand.

BACKGROUND: cJun terminal kinase (JNK) is constitutively activated in most hepatocellular carcinomas (HCCs), yet its exact role in carcinogenesis remains controversial. While tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a major mediator of acquired immune tumour surveillance, and is currently being tested in clinical trials as a novel cancer therapy, the resistance of many tumours to TRAIL and concerns about its toxicity in vivo represent obstacles to its clinical application. In this study we investigated whether JNK activity in HCC could contribute to the resistance to apoptosis in these tumours. METHODS: The effect of JNK/Jun inhibition on receptor-mediated apoptosis was analysed by pharmacological inhibition or RNA interference in cancer cells and non-tumour cells isolated from human liver or transgenic mice lacking a phosphorylation site for Jun. RESULTS: JNK inhibition caused cell cycle arrest, enhanced caspase recruitment, and greatly sensitised HCC cells but not normal hepatocytes to TRAIL. TRAIL-induced activation of JNK could be effectively interrupted by administration of the JNK inhibitor SP600125. CONCLUSIONS: Expression and TRAIL-dependent feedback activation of JNK likely represent a mechanism by which cancer cells escape TRAIL-mediated tumour surveillance. JNK inhibition might represent a novel strategy for specifically sensitising HCC cells to TRAIL thus opening promising therapeutic perspectives for safe and effective use of TRAIL in cancer treatment.

[Acute pancreatitis: is there a need for surgery?]. / Akute Pankreatitis: Wann ist der Chirurg gefragt?

The treatment of acute pancreatitis is primarily non-surgical. An interdisciplinary approach as well as timely and aggressive intensive care has led to a significant improvement of the prognosis in severe necrotising pancreatitis. Early surgical procedures were associated with high morbidity and mortality and therefore were abandoned and replaced with forceful conservative treatment. However, there are still specific indications for surgery during the course of acute pancreatitis. These include cholecystectomy for biliary pancreatitis, surgical debridement of infected necrosis in septic patients and emergency operations for gastrointestinal perforations or haemorrhage. The following article focuses on surgical indications, optimal timing of surgery and competing surgical and non-surgical concepts like laparoscopic or endoscopic management. All mentioned procedures demand the cooperation of an experienced team of gastroenterologists, surgeons, radiologists and intensive care specialists, who are able to manage the potentially life-threatening complications of this disease. All patients with severe necrotising pancreatitis should be transferred to a specialised centre for interdisciplinary therapy.

Hepatic progenitor cells from adult human livers for cell transplantation.

OBJECTIVE: Liver regeneration is mainly based on cellular self-renewal including progenitor cells. Efforts have been made to harness this potential for cell transplantation, but shortage of hepatocytes and premature differentiated progenitor cells from extra-hepatic organs are limiting factors. Histological studies implied that resident cells in adult liver can proliferate, have bipotential character and may be a suitable source for cell transplantation. METHODS: Particular cell populations were isolated after adequate tissue dissociation. Single cell suspensions were purified by Thy-1 positivity selection, characterised in vitro and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Thy-1(+) cells that are mainly found in the portal tract and the surrounding parenchyma, were isolated from surgical liver tissue with high yields from specimens with histological signs of regeneration. Thy-1(+) cell populations were positive for progenitor (CD34, c-kit, CK14, M2PK, OV6), biliary (CK19) and hepatic (HepPar1) markers revealing their progenitor as well as hepatic and biliary nature. The potential of Thy-1(+) cells for differentiation in vitro was demonstrated by increased mRNA and protein expression for hepatic (CK18, HepPar1) and biliary (CK7) markers during culture while progenitor markers CK14, chromogranin A and nestin were reduced. After transplantation of Thy-1(+) cells into livers of immunodeficient mice, engraftment was predominantly seen in the periportal portion of the liver lobule. Analysis of in situ material revealed that transplanted cells express human hepatic markers HepPar1 and albumin, indicating functional engraftment. CONCLUSION: Bipotential progenitor cells from human adult livers can be isolated using Thy-1 and might be a potential candidate for cell treatment in liver diseases.

Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.

AIMS: To determine the expression of a protein termed augmenter of liver regeneration (ALR), recently found to have a specific and beneficial effect on the process of liver regeneration in normal and diseased human liver. METHODS AND RESULTS: ALR expression in normal and cirrhotic human livers with various underlying diseases as well as in tissue samples of hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) was analysed by immunohistochemistry and quantitative reverse transciptase-polymerase chain reaction (RT-PCR). Expression analysis of ALR in total liver protein extracts by Western blotting showed mainly dimeric ALR protein. Immunohistochemically, cytosolic and perinuclear immunosignals were found in hepatocytes and cholangiocytes in normal, cirrhotic or cancerous liver tissue and only weak signals in some endothelial cells in normal livers. Quantitative mRNA analysis revealed significantly increased ALR expression in cirrhosis compared with normal liver tissue. In HCC and CCC ALR mRNA expression was also significantly enhanced compared with normal liver tissue, but expression levels did not differ from the matching non-neoplastic tissue in the same patient. CONCLUSIONS: The findings suggest an important role for ALR in hepatocellular regeneration in liver cirrhosis as well as in hepatocarcinogenesis and therefore its potential value in the clinical diagnosis of hepatic cirrhosis and cancer.

Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes.

Extracellular matrices, like collagen layers, play an important role in preventing dedifferentiation of hepatocytes in long-term culture experiments. It has also been shown that polyamines are crucial for cell growth and liver differentiation - regeneration. Primary cultured hepatocytes with their low mitotic activity might be a valuable tool in studying the role of polyamines in differentiation. Here, our goal was to investigate whether an extracellular cell culture matrix can influence intracellular polyamine levels in human hepatocytes during long-term culture. Primary human hepatocytes were isolated from surgical tissue resections and were maintained either in single collagen (SG) or double collagen gel (DG) layer (sandwich) culture systems. Cell viability and function were examined and intracellular polyamine levels were measured using a highly sensitive high performance liquid chromatography (HPLC) method. Hepatocytes showed high viability in both culture systems used, but albumin secretion was diminished in SG cultured hepatocytes after 14 days. In general, total intracellular polyamine levels of hepatocytes decreased markedly in both SG and DG within the first days of culture, but remained constant until day 21 with a SG/DG ratio of about 1.4. Individual polyamines levels were dependent on the culture time and system, where spermine decreased and putrescine increased in both SG and DG over time (day 14), but spermidine increased only in DG. Our results suggest that polyamine levels, in particular putrescine, might be important regulators of hepatocyte specific function in vitro and therefore serve as a marker of differentiation for cultivated human hepatocytes.

High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices.

OBJECTIVE: Hepatocytes are increasingly used as functional units in bioartificial liver devices. The objective of the present study was to investigate the feasibility of culturing porcine hepatocytes in high density on a novel polyurethane-based nonwoven three-dimensional matrix. We investigated (1) the optimal cell density within this culture configuration, (2) the maintenance of liver-specific morphology and cell functions over long-term periods and (3) the necessity to apply an additional extracellular matrix component (collagen gel). METHODS: Nonwoven polyurethane matrices were manufactured by a specially developed fiber extrusion technology. Pig hepatocytes were cultured at various cell densities of 0.1, 0.25, 0.5, 0.75, 1 and 2 x 10(6) cells/cm(2) on three-dimensional networks of nonwoven polyurethane matrices and cell adhesion as well as functional parameters (DNA of nonattached/attached cells, lactate dehydrogenase release and cytochrome P450 activity) were determined. To assess the performance of cells within this configuration albumin and urea excretion was measured over 8 days. The potentially beneficial effect of an additional extracellular matrix configuration was evaluated by comparing the average albumin synthesis in groups of identical cell numbers. RESULTS: The optimal cell density in this three-dimensional culture configuration was 1 x 10(6) cells/cm(2). The functional capacity of hepatocytes was stable for 8 days at an average level of 53.7 +/- 5.6 ng/h/microg DNA and of 1.8 +/- 0.14 microg/h/microg DNA for albumin and urea excretion, respectively. The supplementation of an extracellular matrix configuration did not improve functional activity of cells. Average albumin synthesis was 35.6 ng/h/microg DNA (28.7, 42.8) and 32.7 ng/h/microg DNA (23.4, 49.2) for collagen-immobilized and control cultures, respectively. CONCLUSION: The results of the study indicate that nonwoven polyurethane sheets supply a biocompatible support structure for functionally active high density cultures. Thus, nonwoven polyurethane matrices should be further investigated on with respect to their role in the development, optimization and design of bioartificial liver systems.

[Hepatocellular transport of bile acids and organic anions in infection and SIRS--evidence for different mechanisms for regulating membrane transport proteins]. / Hepatozellulärer Transport von Gallensäuren und organischen Anionen bei Sepsis und SIRS--Anhalt für unterschiedliche Regulationsmechanismen der Membrantransportproteine.

The alteration of proinflammatory mediators during sepsis and SIRS results in a large variety of adaptive changes of metabolic and physiologic variables. This study investigated the alterations of hepatocellular transport in a rat sepsis model (LPS i.p.) as well as in a model inducing SIRS by sterile abscess formation (turpentine i.m.). Two bile acids (Cholyltaurine and Chemodeoxycholyltaurine) and one organic anion (Sulfolithocholyltaurine) were used as marker substrates to investigate the time course of hepatocellular transport function. Experiments were performed in isolated perfused rat livers and plasma membrane vesicles. During sepsis, both, the transport of bile acids and that of the organic anion was markedly reduced. In contrast no alteration of transport was detected during SIRS. However, biliary secretion of glutathione (+90%) and bile acid independent bile flow (%) were increased. mRNA levels of bile acid and organic anion transport proteins were reduced. The lowest values were noted 12 h after injection of LPS or turpentine. Almost unchanged kinetic parameters during SIRS pointed to a normal population of transporters with regard to quantity and substrate affinity. Therefore it seems that transcriptional regulation plays an important role for the expression of transport proteins during sepsis, whereas posttranscriptional regulation may be of importance during SIRS. The clinical phenomenon of septic cholestasis including jaundice implies endotoxemia and differenciates against SIRS.

Porcine hepatocytes for biohybrid artificial liver devices: a comparison of hypothermic storage techniques.

Two hypothermic preservation techniques were investigated to assess their possible role in on-demand cell supply for bioartificial liver support devices. Porcine hepatocytes from slaughterhouse organs were isolated and either cold stored in a modified University of Wisconsin solution for up to 72 h or directly cultured in a sandwich configuration, frozen at Day 3 of culture, and stored for up to 30 days with subsequent long-term culture (14 days) in both groups. Cold storage for 72 h resulted in a decreased viability of cells (58.7 +/- 7.9%) with well preserved ultrastructures in the remainder of cells. In subsequent culture, albumin secretion was slightly increased, and cytochrome P450 IA1 dependent 7-ethoxy-coumarine deethylation activity was reduced to about 40% of control values. After cryopreservation, hepatocyte cultures revealed no severe damage to ultrastructures of cells, and functional parameters (albumin, 7-ethoxycoumarine deethylation) were comparable with controls after an initial drop in activity directly after thawing. Length of storage time did not influence results. Both hypothermic preservation protocols might eventually play an important role for bioartificial liver processing and on-demand cell supply, dependent on the individual reactor design.

Hypothernic storage of pig hepatocytes: influence of different storage solutions and cell density.

For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.

Porcine hepatocytes from slaughterhouse organs. An unlimited resource for bioartificial liver devices.

Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) viable cells per isolation (viability: 93 +/- 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long-term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 +/- 5 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms DNA) indicated long-term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested.

[An artificial liver on its way to clinical reality?]. / Die künstliche Leber auf dem Weg in die klinische Realität?

Temporary extracorporeal liver assist devices based on liver cell cultures represent a promising approach for the treatment of liver failure as well as for perioperative care in liver surgery and transplantation. Primary hepatocytes from donor animals as well as human cell lines have been discussed as cell sources. A viable cell mass of 110-220 g (= 2.5-5 x 10(10) liver cells) has to be functionally active in bioreactors over a time period of several days. This cell number correlates to 10-20% of liver cells in an adult human liver. Neither in animal experiments, nor in clinical trials a complete liver replacement could yet be shown for cell-based bioreactors, however, beneficial effects have been demonstrated: partial detoxification and specific synthetic functions have been reported in animal experiments. Two authors have shown first preliminary clinical data on different cell-based liver assist devices with influences on blood parameters and the neurological status of treated patients. Nevertheless, clinical improvements also have been achieved by using non-biological support, like charcoal-hemoperfusion and modified hemodialysis. In order to assess the influence of liver support devices on better results in therapy of liver failure, randomized clinical trials have to be performed. Prior to this, artificial liver support systems have to demonstrate their biocompatibility and biosafety for clinical use as well as their efficacy and availability.