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Introduction The majority (80%) of hepatocellular carcinomas (HCC) develop within the context of advanced liver fibrosis and cirrhosis. Recent studies with ultrasound elastography have demonstrated that increased liver stiffness is a strong predictor of HCC. Aim To establish whether alterations in matrix stiffness regulate the phenotype and chemotherapeutic response of HCC cells. Method Experiments were conducted using a system of ligand-coated polyacrylamide gels of variable stiffness. Matrix stiffness (expressed as shear modulus) was modelled across a physiologically-relevant range (1–12 kPa), corresponding to values encountered in normal and fibrotic livers. Experiments were conducted in two HCC cells lines (Huh7/ HepG2). Results In each cell type, there was a consistent morphological response to changes in matrix stiffness. There was increased cell spreading on stiff gels in association with both stress-fibre and mature focal adhesion formation. Increasing matrix stiffness promoted cellular proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold (p<0.001) and 12.2-fold (p<0.001) higher, respectively, when the cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Cells cultured on soft supports developed a quiescent (dormant) phenotype with marked reduction in cyclinD1/ D3 expression, without upregulation of p21/p27. We postulated that altered sensitivity to mitogenic growth factors mediates the stiffness-dependent regulation of proliferation. Matrix stiffness modulated both the magnitude and time-course of mitogenic signalling in response to HGF, with lower baseline and HGF-induced FAK, ERK and STAT3 pathway activation. Increasing stiffness results in upregulation of mesenchymal markers (including N-cadherin and vimentin), consistent with mesenchymal shift, and down-regulation of differentiated hepatocyte markers (including albumin, α-1-antitrypsin and HNF4). Following treatment with cisplatin, cells cultured on soft supports were more susceptible to apoptosis (PARP/ Caspase-3 cleavage). However, in both Huh7 and HepG2 cells, surviving cells from soft supports had 2.2-fold (p<0.05) and 2.4-fold (p<0.001) higher clonogenic capacity respectively, than surviving cells from stiff supports. This was associated with upregulation of cancer stem cell markers (Oct4, NANOG, CD44, CD133, c-kit and CXCR4). Conclusion HCC is a tumour that develops within an altered biomechanical niche. Increasing matrix stiffness regulates HCC mitogenic signalling, proliferation, differentiation and chemotherapeutic resistance. However, a soft microenvironment (as may be encountered by disseminated tumour cells) promotes stem cell characteristics following chemotherapy. This provides a possible explanation for the failure of systemic chemotherapy both in relation to treatment of primary HCCs and the eradication of disseminated tumour cells that give rise to metastases. The selective targeting of the cytoskeleton represents a potentially novel approach to the treatment of HCC. [ABSTRACT FROM PUBLISHER]

Background and Aims: Portal hypertension is the main pathophysiological driver of decompensation in patients with liver cirrhosis. Epithelial cell death markers, m30 and m65, correlate with hepatic injury and predict outcomes across various stages of liver disease. We aim (i) to evaluate whether portal hypertension itself contributes to liver outcome-relevant epithelial injury, and (ii) to analyse the capacity of m30/m65 to predict outcome in patients receiving a transjugular intrahepatic portosystemic shunt (TIPS) for refractory ascites., Methods: Sixty-six patients undergoing TIPS placement for refractory ascites and 20 patients with compensated cirrhosis as controls were prospectively enrolled in this monocentric cohort study. Epithelial cell death markers were analysed pre-TIPS, as well as 1-3 and 6-9 months post-TIPS. The capacity of baseline levels of m30/m65 in predicting six-month transplant-free survival rates was analysed by multivariable Cox proportional hazards regression., Results: Levels of m30 and m65 were higher in patients with decompensated cirrhosis (pre-TIPS) compared with compensated cirrhosis (controls). Following correction of portal hypertension by TIPS and recompensation, both markers decreased over time, reaching levels comparable to patients with compensated cirrhosis. On multivariable analysis, pre-TIPS baseline levels of m30 and m65 were not predictive for six-month survival., Conclusion: Correction of portal hypertension via TIPS reduces levels of epithelial cell death markers, indicating that portal hypertension is a driver of outcome-relevant, hepatic cell death in patients with decompensated cirrhosis. Baseline m30/m65 values do not affect six-month survival rates, which suggests that TIPS placement overcomes the unfavourable spontaneous prognosis otherwise indicated by elevated baseline m30/65 levels., (© 2024 The Author(s). Liver International published by John Wiley & Sons Ltd.)

Background & Aims: Cirrhosis is associated with an increased surgical morbidity and mortality. Portal hypertension and the surgery type have been established as critical determinants of postoperative outcome. We aim to evaluate the hypothesis that preoperative transjugular intrahepatic portosystemic shunt (TIPS) placement in patients with cirrhosis is associated with a lower incidence of in-house mortality/liver transplantation (LT) after surgery., Methods: A retrospective database search for the years 2010-2020 was carried out. We identified 64 patients with cirrhosis who underwent surgery within 3 months after TIPS placement and 131 patients with cirrhosis who underwent surgery without it (controls). Operations were categorised into low-risk and high-risk procedures. The primary endpoint was in-house mortality/LT. We analysed the influence of high-risk surgery, preoperative TIPS placement, age, sex, baseline creatinine, presence of ascites, Chronic Liver Failure Consortium Acute Decompensation (CLIF-C AD), American Society of Anesthesiologists (ASA), and model for end-stage liver disease (MELD) scores on in-house mortality/LT by multivariable Cox proportional hazards regression., Results: In both the TIPS and the control cohort, most patients presented with a Child-Pugh B stage (37/64, 58% vs. 70/131, 53%) at the time of surgery, but the median MELD score was higher in the TIPS cohort (14 vs. 11 points). Low-risk and high-risk procedures amounted to 47% and 53% in both cohorts. The incidence of in-house mortality/LT was lower in the TIPS cohort (12/64, 19% vs. 52/131, 40%), also when further subdivided into low-risk (0/30, 0% vs. 10/61, 16%) and high-risk surgery (12/34, 35% vs. 42/70, 60%). Preoperative TIPS placement was associated with a lower rate for postoperative in-house mortality/LT (hazard ratio 0.44, 95% CI 0.19-1.00) on multivariable analysis., Conclusions: A preoperative TIPS might be associated with reduced postoperative in-house mortality in selected patients with cirrhosis., Impact and Implications: Patients with cirrhosis are at risk for more complications and a higher mortality after surgical procedures. A transjugular intrahepatic portosystemic shunt (TIPS) is used to treat complications of cirrhosis, but it is unclear if it also helps to lower the risk of surgery. This study takes a look at complications and mortality of patients undergoing surgery with or without a TIPS, and we found that patients with a TIPS develop less complications and have an improved survival. Therefore, a preoperative TIPS should be considered in selected patients, especially if indicated by ascites., Competing Interests: The authors have no conflict of interest to declare regarding the work under consideration for publication. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Introduction: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET., Methods: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue., Results: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells., Conclusion: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET., (© 2022 S. Karger AG, Basel.)

This CIRSE Standards of Practice document is aimed at interventional radiologists and provides best practices for performing percutaneous transhepatic cholangiography, biliary drainage and stenting. It has been developed by an expert writing group established by the CIRSE Standards of Practice Committee., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature and the Cardiovascular and Interventional Radiological Society of Europe (CIRSE).)

Transjugular intrahepatic portosystemic shunt (TIPS) reduces portal hypertension in patients with liver cirrhosis. The exact cardiac consequences of subsequent increase of central blood volume are unknown. Cardiovascular magnetic resonance (CMR) imaging is the method of choice for quantifying cardiac volumes and ventricular function. The aim of this study was to investigate effects of TIPS on the heart using CMR, laboratory, and imaging cardiac biomarkers. 34 consecutive patients with liver cirrhosis were evaluated for TIPS. Comprehensive CMR with native T1 mapping, transthoracic echocardiography, and laboratory biomarkers were assessed before and after TIPS insertion. Follow-up (FU) CMR was obtained in 16 patients (47%) 207 (170-245) days after TIPS. From baseline (BL) to FU, a significant increase of all indexed cardiac chamber volumes was observed (all P < 0.05). Left ventricular (LV) end-diastolic mass index increased significantly from 45 (38-51) to 65 (51-73) g/m 2 (P =  < 0.01). Biventricular systolic function, NT-proBNP, high-sensitive troponin T, and native T1 time did not differ significantly from BL to FU. No patient experienced cardiac decompensation following TIPS. In conclusion, in patients without clinically significant prior heart disease, increased cardiac preload after TIPS resulted in increased volumes of all cardiac chambers and eccentric LV hypertrophy, without leading to cardiac impairment during follow-up in this selected patient population.

Background & Aims: Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. Therefore, we aimed to analyze the blood metabolome in patients with cirrhosis, with and without ACLF., Methods: We performed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 650 patients with AD (acute decompensation of cirrhosis, without ACLF), 181 with ACLF, 43 with compensated cirrhosis, and 29 healthy individuals., Results: Of the 137 annotated metabolites identified, 100 were increased in patients with ACLF of any grade, relative to those with AD, and 38 comprised a distinctive blood metabolite fingerprint for ACLF. Among patients with ACLF, the intensity of the fingerprint increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that the fingerprint reflected not only decreased kidney excretion but also altered cell metabolism. The higher the ACLF-associated fingerprint intensity, the higher the plasma levels of inflammatory markers, tumor necrosis factor α, soluble CD206, and soluble CD163. ACLF was characterized by intense proteolysis and lipolysis; amino acid catabolism; extra-mitochondrial glucose metabolism through glycolysis, pentose phosphate, and D-glucuronate pathways; depressed mitochondrial ATP-producing fatty acid β-oxidation; and extra-mitochondrial amino acid metabolism giving rise to metabotoxins., Conclusions: In ACLF, intense systemic inflammation is associated with blood metabolite accumulation and profound alterations in major metabolic pathways, in particular inhibition of mitochondrial energy production, which may contribute to the development of organ failures., Lay Summary: Acute-on-chronic liver failure (ACLF), which develops in patients with cirrhosis, is characterized by intense systemic inflammation and organ failure(s). Because systemic inflammation is energetically expensive, its metabolic costs may result in organ dysfunction/failure. We identified a 38-metabolite blood fingerprint specific for ACLF that revealed mitochondrial dysfunction in peripheral organs. This may contribute to organ failures., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Purpose: The standard of care treatment for patients with advanced pancreatic neuroendocrine tumors (pNET) is a combination of streptozotocin and 5-FU. Although widely used, little is known about the best long-term strategy with these substances., Methods: We here report our experience of 28 patients treated with streptozotocin/5-FU for advanced pNET with special consideration for long-term management using an extended cycle protocol., Results: Standard 6-weekly Moertel protocol resulted in a median progression-free survival of 21 months (range 3-128) and a median overall survival of 69 months (range 3-157+) in the whole cohort. Thirteen of the 28 patients were switched to an extended 3-month cycle protocol for maintenance therapy. Of these 13 patients, 2 achieved complete remission, 1 partial remission, and 8 stable disease as best response while 2 showed progressive disease following switch to the extended protocol, resulting in an additional median progression-free survival of 23 months. Median overall survival after the start of chemotherapy in this patient group was 69 months (21-157+). Patients benefitted from extended periods free of chemotherapy-associated side effects after switching to the extended cycle protocol., Conclusions: Switching to an extended cycle protocol of 3 months for maintenance therapy following initial standard cycles may achieve long-term disease stabilization in selected patients with advanced pNET with good patient acceptance.

Background & Aims: Refractory ascites is the main reason for the implantation of a transjugular intrahepatic portosystemic shunt (TIPS) in liver cirrhosis, but ascites control by TIPS fails in a relevant proportion of cases. Here, we investigated whether routine parameters pre-TIPS can predict persistent ascites after TIPS implantation and whether persistent ascites predicts long-term clinical outcome., Methods: A detailed retrospective analysis of 128 patients receiving expanded polytetrafluoroethylene-covered stents for the treatment of refractory ascites was performed. Persistent ascites post-TIPS was defined as the prolonged need for paracentesis >3 months after TIPS. The influence of demographics, laboratory results, pre-TIPS heart and liver ultrasound results, and invasive hemodynamic parameters on persistent ascites was evaluated by univariable and multivariable logistic regression. Predictors of the composite endpoint liver transplantation/death were analyzed using a multivariable Cox regression., Results: Ascites control post-TIPS was achieved in 95/128 patients (74%), whereas ascites remained persistent in 33/128 cases (26%). On multivariable analysis, a lower paracentesis frequency pre-TIPS (odds ratio 1.672; 95% CI 1.253-2.355) and lower baseline creatinine levels (odds ratio 2.640; CI 1.201-6.607) were associated with ascites control. Patients with persistent ascites post-TIPS had and impaired transplant-free survival (median 10.0 vs. 25.8 months), for which persistent ascites was the only independent predictor (hazard ratio 5.654; CI 3.019-10.59)., Conclusion: TIPS-placement in patients with lower paracentesis frequency and creatinine levels is associated with superior ascites control. Thus, TIPS implantation should be considered in moderate decompensation and not as a last resort. Persistent ascites post-TIPS seems to be the only predictor of liver transplantation and death., Lay Summary: The insertion of a transjugular intrahepatic portosystemic shunt (TIPS) in patients with refractory ascites should be considered in patients with moderate decompensation and not as a last resort, as lower paracentesis frequency and creatinine levels pre-TIPS are associated with superior ascites control. In turn, failure to control ascites seems to be the only predictor of liver transplantation and death., (© 2019 The Authors.)

Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl 4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl 4 -induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver.

Clinical options for systemic therapy of neuroendocrine tumors (NET) are limited. Development of new drugs requires suitable representative in vitro and in vivo model systems. So far, the unavailability of a human model with a well-differentiated phenotype and typical growth characteristics has impaired preclinical research in NET. Herein, we establish and characterize a lymph node-derived cell line (NT-3) from a male patient with well-differentiated pancreatic NET. Neuroendocrine differentiation and tumor biology was compared with existing NET cell lines BON and QGP-1. In vivo growth was assessed in a xenograft mouse model. The neuroendocrine identity of NT-3 was verified by expression of multiple NET-specific markers, which were highly expressed in NT-3 compared with BON and QGP-1. In addition, NT-3 expressed and secreted insulin. Until now, this well-differentiated phenotype is stable since 58 passages. The proliferative labeling index, measured by Ki-67, of 14.6% ± 1.0% in NT-3 is akin to the original tumor (15%-20%), and was lower than in BON (80.6% ± 3.3%) and QGP-1 (82.6% ± 1.0%). NT-3 highly expressed somatostatin receptors (SSTRs: 1, 2, 3, and 5). Upon subcutaneous transplantation of NT-3 cells, recipient mice developed tumors with an efficient tumor take rate (94%) and growth rate (139% ± 13%) by 4 weeks. Importantly, morphology and neuroendocrine marker expression of xenograft tumors resembled the original human tumor. Implications: High expression of somatostatin receptors and a well-differentiated phenotype as well as a slow growth rate qualify the new cell line as a relevant model to study neuroendocrine tumor biology and to develop new tumor treatments. Mol Cancer Res; 16(3); 496-507. ©2018 AACR ., (©2018 American Association for Cancer Research.)

In 2015, more than 13 000 people died due to the consequences of liver cirrhosis in Germany. Frequently, relevant liver fibrosis is diagnosed by non-invasive methods (e. g., ultrasound-based measurement of liver stiffness) already in the compensated stage. Following diagnosis of liver fibrosis, a thorough investigation of the underlying chronic liver disease and effective treatment are important to prevent progression to decompensated cirrhosis. Since morbidity and mortality dramatically increase in the decompensated stage (patients may present with jaundice, ascites, hepatic encephalopathy, gastrointestinal bleeding) with an upsurge in 1-year-mortality from 1 - 3.4 % to 20 - 57 %, prophylactic measures to prevent decompensation are indicated. Based on a risk stratification, these measures include propranolol or carvedilol as non-selective betablockers, as well as endoscopic band ligations as primary prophylaxis to prevent variceal bleeding. Because of the high risk for malignant transformation (2 - 8 % per year depending on the underlying etiology), surveillance by liver ultrasound every six months is essential to detect liver cancer in an early stage and to facilitate curative therapy. Currently under debate is the administration of antibiotics to prevent bacterial infections, which commonly trigger acute decompensation. To this regard, studies are not convincing and the risk to induce drug resistance has to be observed. However, health care providers should check the vaccination status and recommend missing vaccinations. The management of compensated liver cirrhosis also includes counseling and potentially also a drug therapy to prevent osteoporosis and muscle wasting. In this review, we will discuss specific prophylactic measures in the management of compensated liver cirrhosis based on the pathophysiological background and central clinical studies. If a patient decompensates despite these prophylactic measures (approximately 15 % of patients with liver cirrhosis per year), liver transplantation has to be discussed as definitive therapy (especially in patients with MELD > 15)., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interests.

Purpose: In order to assess the occurrence of blood congestion in the liver during liver resection, we aimed to evaluate the influence of a positive-end-expiratory-pressure (PEEP) and positioning of patients on central venous pressure (CVP) and venous hepatic blood flow parameters. We further analyzed correlations between CVP and venous hepatic blood flow parameters., Methods: In 20 patients scheduled for elective liver resection we measured CVP and quantified venous hepatic hemodynamics by ultrasound assessment of flow-velocity and diameter of the right hepatic vein and the portal vein after equilibration following these maneuvers: M1: 0° supine position, PEEP 0 cmH 2 O; M2: 0° supine position, PEEP 10 cmH 2 O; M3: 20° reverse-trendelenburg position; PEEP 10 cmH 2 O; M4: 20° reverse-trendelenburg position, PEEP 0cmH 2 O., Results: Changing from supine to reverse-trendelenburg position led to a significant decrease in CVP (M3 5.95 ± 2.06 vs. M1 7.35 ± 2.18 mmHg and M2 8.55 ± 1.79 mmHg). A PEEP of 10 cmH 2 O and reverse-trendelenburg position led to significant reduction of systolic (Vs HV ) and diastolic (Vd HV ) flow-velocities of the right hepatic vein (Vs HV M3 19.96 ± 6.47 vs. M1 27.81 ± 11.03 cm s -1 ;Vd HV M3 14.94 ± 6.22 vs. M1 20.15 ± 10.34 cm s -1 and M2 20.19 ± 13.19 cm s -1 ) whereas no significant changes of flow-velocity occurred in the portal vein. No correlations between CVP and diameters or flow-velocities of the right hepatic and the portal vein were found., Conclusions: Changes of central venous pressure due to changes of PEEP and positioning were not correlated with changes of venous hepatic blood flow parameters as measured after equilibration. Strategies aiming for low central venous pressure cannot be supported by these results. However, before ruling out low-CVP-strategies during liver resections these results should be confirmed by further studies.

Background. There are only few reports about travel-associated, imported tropical hepatitis E virus (HEV) genotype 1 infections within Western travellers. We describe the clinical course of a single outbreak of hepatitis E in a German travellers group returning from India and compare the results of two commercial HEV-seroassays., Material and Methods: After identifying hepatitis E in an index patient returning from a journey to India all 24 members of this journey were tested for anti-HEV-IgG and IgM using two commercial seroassays (Wantai and Mikrogen), for HEV-RNA by PCR and HEV-Ag by an antigen-assay (Wantai)., Results: 5/24 (21%) individuals were viraemic with viral loads between 580-4,800,000 IU/mL. Bilirubin and ALT levels in these patients ranged from 1.3-14.9 mg/dL (mean 7.3 mg/dL, SD 5.6 mg/dL) and 151-4,820 U/L (mean 1,832U/L, SD 1842U/L), respectively and showed significant correlations with viral loads (r = 0.863, p < 0.001; r = 0.890, p < 0.001). No risk factor for food-borne HEV-transmission was identified. All viraemic patients (5/5) tested positive for anti-HEV-IgG and IgM in the Wantai-assay but only 4/5 in the Mikrogen-assay. Wantai-HEV-antigen-assay was negative in all patients. Six months later all previously viraemic patients tested positive for anti-HEV-IgG and negative for IgM in both assays. However, two non-viremic individuals who initially tested Wantai-IgM-positive stayed positive indicating false positive results., Conclusions: Despite the exact number of exposed individuals could not be determined HEV genotype 1 infections have a high manifestation rate of more than 20%.The Wantai-antigen-test failed, the Wantai-IgMrapid- test and the Mikrogen-IgM-recomblot showed a better performance but still they cannot replace real-time PCR for diagnosing ongoing HEV-infections.

Background & Aims: Research on vasopressin (AVP) in cirrhosis and its role in the assessment of prognosis has been hindered by the difficulty of measuring AVP levels accurately. Copeptin, a 39-aminoacid glycopeptide, is released from the neurohypophysis together with AVP. Copeptin could have a role as biomarker of prognosis in cirrhosis as it may reflect circulatory dysfunction. The aim of this study is to investigate the role of copeptin as biomarker of disease progression and prognosis in cirrhosis., Methods: This prospective study is divided in 2 study protocols including 321 consecutive patients. Plasma copeptin levels were measured in all patients at study inclusion. Protocol 1: to investigate the relationship of copeptin with kidney and circulatory function (56 patients). Protocol 2: to investigate the relationship between copeptin and prognosis, as assessed by the development of complications of cirrhosis or mortality at 3months (265 patients admitted to hospital for complications of cirrhosis)., Results: Patients with decompensated cirrhosis showed significantly higher plasma copeptin levels compared to those of patients with compensated cirrhosis. Copeptin levels had a significant positive correlation with model for end-satge liver disease (MELD) score, AVP, endogenous vasoconstrictor systems, and kidney function parameters. Patients developing complications of cirrhosis or mortality had significantly higher plasma copeptin levels compared to those of the remaining patients. Plasma copeptin levels were an independent predictive factor of both the development of complications and mortality at 3months. This was confirmed in a validation series of 120 patients., Conclusions: Copeptin is a novel biomarker of disease progression and prognosis in cirrhosis., Lay Summary: Copeptin is a fragment of the vasopressin precursor, a hormone that is known to be increased in patients with cirrhosis and that plays a role in the development of complications of the disease. Vasopressin is difficult to measure, but copeptin is a more stable molecule and is easier to measure in blood. Solà and Kerbert and colleagues have shown in a series of 361 patients that copeptin is markedly increased in patients with cirrhosis who develop complications during the following 3months, compared to those patients who do not develop complications. Moreover, copeptin correlates with prognosis., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Intestinal inflammation can impair mucosal healing, thereby establishing a vicious cycle leading to chronic inflammatory bowel disease (IBD). However, the signaling networks driving chronic inflammation remain unclear. Here we report that CD4 + T cells isolated from patients with IBD produce high levels of interleukin-22 binding protein (IL-22BP), the endogenous inhibitor of the tissue-protective cytokine IL-22. Using mouse models, we demonstrate that IBD development requires T cell-derived IL-22BP. Lastly, intestinal CD4 + T cells isolated from IBD patients responsive to treatment with antibodies against tumor necrosis factor-α (anti-TNF-α), the most effective known IBD therapy, exhibited reduced amounts of IL-22BP expression but still expressed IL-22. Our findings suggest that anti-TNF-α therapy may act at least in part by suppressing IL-22BP and point toward a more specific potential therapy for IBD., (Copyright © 2016, American Association for the Advancement of Science.)

Background: Patients with acute alcoholic steatohepatitis are at a high risk for infections. To date, neither disease-specific pathogen patterns, nor typical sites of infection, nor antibiotic treatment strategies have been established for AH., Aims: To characterize incidence of infections, pathogen spectrum, sites of infection, and related mortality of patients with AH under steroid therapy., Methods: We retrospectively analyzed clinical data of 73 patients with severe alcoholic hepatitis (MELD ≥ 20)., Results: Infections were detected in 45 patients (73%). Patients who developed an infection after initiation of corticosteroid therapy had a higher 6-month mortality than patients without onset of infection after initiation of corticosteroid treatment (44% versus 24%, p = 0.116). The pathogen identified most frequently was Enterococcus species., Discussion: Infections frequently complicate severe alcoholic hepatitis and affect survival. The high rate of Enterococcus infections suggests that commonly used antibiotics, such as cephalosporins and quinolones, may represent an ineffective choice of empiric antibiotic treatment for complicated AH.

Background: Cirrhotic cardiomyopathy is a recently defined cardiac disorder in patients with end-stage liver disease. The frequency and exact manifestations of cardiac changes in liver cirrhosis is unknown., Goals: We aim to describe cardiac changes in a large autopsy study of patients with liver cirrhosis., Study: Postmortem data from 895 individuals with liver cirrhosis of different origin autopsied from 1995 to 2010 were analyzed. A total of 236 patients were excluded, mostly due to an advanced age above 70 years. The remaining 659 patients were assigned to 4 subgroups according to the etiology of cirrhosis: alcoholic cirrhosis (57.4%), nonalcoholic steatohepatitis (4.2%), viral hepatitis (9.3%), and cryptogenic cirrhosis (29.1%). Predefined clinical and cardiac parameters were assessed in these groups and compared by univariate and multivariate analyses to an age-matched and sex-matched control group including 40 deceased patients without evidence of chronic liver disease., Results: A critical heart weight (24%, P=0.024), hypertrophy of the right ventricle (24%, P<0.001), and dilatation of the right ventricle (36%, P=0.040) were significantly more frequent in the cirrhosis group compared with noncirrhotic controls. Cirrhosis patients had a greater risk for high-grade coronary sclerosis (30%, P=0.019). The etiology of cirrhosis was independently associated with hypertrophy and dilatation of the right ventricle, with nonalcoholic steatohepatitis patients being at the highest risk., Conclusion: Our results demonstrate a high rate of right-ventricular abnormalities and coronary sclerosis in individuals suffering from liver cirrhosis regardless of the etiology of cirrhosis.

Purpose: Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies., Experimental Design: Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo., Results: The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases., Conclusions: These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs., (©2012 AACR.)

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3-4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1-12 h). The initial efficiency of RGB marking can be assessed after 2-4 d by flow cytometry and/or fluorescence microscopy.

Background: Translocation of gut-derived bacterial products such as endotoxin is a major problem in liver cirrhosis., Methods: To assess the hepatic clearance of bacterial products in individuals with cirrhosis, we tested concentrations of Gram-negative bacterial lipopolysaccharide (LPS), LPS-binding protein (LBP), and the precursor of nitric oxide (NO), L-arginine, in a cohort of 8 stable patients with liver cirrhosis before and after elective transjugular portosystemic shunt (TIPS) implantation, including central venous, hepatic venous, and portal venous measurements., Results: Using an adapted LPS assay, we detected high portal venous LPS concentrations (mean 1743 ± 819 pg/mL). High concentrations of LPS were detectable in the central venous blood (931 ± 551 pg/mL), as expected in persons with cirrhosis. The transhepatic LPS gradient was found to be 438 ± 287 pg/mL, and 25 ± 12% of portal LPS was cleared by the cirrhotic liver. After TIPS, central venous LPS concentrations increased in the hepatic and central veins, indicating shunting of LPS with the portal blood through the stent. This paralleled a systemic increase of L-arginine, whereas the NO synthase inhibitor asymmetric dimethylarginine (ADMA) remained unchanged, suggesting that bacterial translocation may contribute to the pathogenesis of circulatory dysfunction post-TIPS., Conclusions: This study provides quantitative estimates of the role of the liver in the pathophysiology of bacterial translocation. The data indicate that the cirrhotic liver retains the capacity for clearance of bacterial endotoxin from the portal venous blood and that TIPS implantation attenuates this clearance. Thus, increased endotoxin concentrations in the systemic circulation provide a possible link to the increased encephalopathy in TIPS patients.

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.

Unlabelled: There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of "mechanically tunable" matrix-coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold and 12.2-fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness-dependent regulation of basal and hepatocyte growth factor-stimulated mitogenic signaling through extracellular signal-regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. β1-Integrin and focal adhesion kinase were found to modulate stiffness-dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c-kit, cysteine-X-cysteine receptor 4, octamer-4 (CXCR4), and NANOG., Conclusion: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;)., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC.

Unlabelled: Tracking stem/progenitor cells through noninvasive imaging is a helpful means of assessing the targeting of transplanted cells to specific organs. We performed in vitro and in vivo studies wherein adult human hepatocytes and human fetal liver stem/progenitor cells were labeled with indium-111 ((111)In)-oxine and technetium-99m ((99m)Tc)-Ultratag or (99m)Tc-Ceretec. The labeling efficiency and viability of cells was analyzed in vitro, and organ biodistribution of cells was analyzed in vivo after transplantation in xenotolerant nonobese diabetic/severe combined immunodeficiency mice through intrasplenic or intraportal routes. We found that adult hepatocytes and fetal liver stem/progenitor cells incorporated (111)In but not (99m)Tc labels. After radiolabeling, cell viability was unchanged. Transplanted adult hepatocytes or fetal liver stem/progenitor cells were targeted to the liver more effectively by the intraportal rather than the intrasplenic route. Transplanted cells were retained in the liver after intraportal injection and in the liver and spleen after intrasplenic injection, without translocations into pulmonary or systemic circulations. Compared with fetal liver stem/progenitor cells, fewer adult hepatocytes were retained in the spleen after intrasplenic transplantation. The distribution of transplanted cells in organs was substantiated by genetic assays, including polymerase chain reaction amplification of DNA sequences from a primate-specific Charcot-Marie-Tooth element, and in situ hybridization for primate alphoid satellite sequences ubiquitous in all centromeres., Conclusion: (111)In labeling of human fetal liver stem/progenitor cells and adult hepatocytes was effective for noninvasive localization of transplanted cells. This should facilitate continued development of cell therapies through further animal and clinical studies.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50 nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G(2)/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.

Improvements in surgical technique and the introduction of several new immunosuppressive medications mean that outcome after orthotopic liver transplantation (OLT) has improved continuously over the past 15 years. Given the increasing longevity of patients after OLT, the recognition and prevention of long-term complications after transplantation have become ever more important. With respect to graft function, physicians responsible for the everyday care of patients following transplantation should be particularly aware of the risk of late and chronic rejection episodes and of recurrence of the underlying liver disease. The major challenge of post-transplant care is, however, how best to prevent and manage the long-term adverse effects caused by the immunosuppressive medications prescribed. Screening investigations for early diagnosis of malignancy, strict control of cardiovascular risk factors, preservation of renal function, and prevention of infections are, therefore, fundamental. This Review suggests guidelines for the management of OLT recipients to improve long-term survival, overall outcome and health-related quality of life.

The incidence of de novo malignancies is increased in organ transplant recipients, and patients with hepatic carcinomas are at high risk for tumor recurrence after liver transplantation. Data about recurrent cancer after orthotopic liver transplantation (OLT) in patients with a history of nonhepatic malignancy are very limited. We retrospectively analyzed data from 606 adult OLT recipients and identified 37 patients (6.1%) with a preexisting extrahepatic malignancy. In the same group, 43 patients (7.0%) developed de novo cancer. Preexisting malignancies included 26 solid tumors and 11 hematological malignancies, including 7 patients with Budd-Chiari syndrome due to myeloproliferative disorders (MPDs). Patients had been selected for OLT because of the expected good prognosis of their preexisting malignancy. Except for 3 patients, recipients were tumor-free at OLT. The median interval from tumor diagnosis to OLT was 44 months (range, <1-321). After a median follow-up of 66 months post transplantation (range, 4-131), all but 1 recipient with incidental colon carcinoma were free of recurrence. No patient with MPD showed leukemic transformation, whereas a patient with neurofibromatosis experienced growth of skin fibromas. Our data and an included review of published OLT recipients with preexisting malignancies have enabled us to show that recurrence rates are comparable for nontransplanted patients and renal-transplant recipients. In conclusion, cancer recurrence is low if OLT recipients are carefully selected. Therefore, previous extrahepatic malignancy should not be considered a contraindication for OLT per se, but the oncologic/hematologic prognosis should be considered, particularly with respect to the current 5-year survival rate of OLT.

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL-positive and -negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34(+) cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.

Understanding the biological potential of fetal stem/progenitor cells will help define mechanisms in liver development and homeostasis. We isolated epithelial fetal human liver cells and established phenotype-specific changes in gene expression during continuous culture conditions. Fetal human liver epithelial cells displayed stem cell properties with multilineage gene expression, extensive proliferation and generation of mesenchymal lineage cells, although the initial epithelial phenotype was rapidly supplanted by meso-endodermal phenotype in culture. This meso-endodermal phenotype was genetically regulated through cytokine signaling, including transforming growth factor beta, bone morphogenetic protein, fibroblast growth factor and other signaling pathways. Reactivation of HNF3alpha (FOXA1) transcription factor, a driver of hepatic specification in the primitive endoderm, indicated that the meso-endodermal phenotype represented an earlier developmental stage of cells. We found that fetal liver epithelial cells formed mature hepatocytes in vivo, including after genetic manipulation using lentiviral vectors, offering convenient assays for analysis of further cell differentiation and fate. Taken together, these studies demonstrate plasticity in fetal liver epithelial stem cells, offer paradigms for defining mechanisms regulating lineage switching in stem cells, and provide potential avenues for regulating cell phenotypes for applications of stem cells, such as for cell therapy.

Transplantation of healthy cells to repair organ damage or replace deficient functions constitutes a major goal of cell therapy. However, the mechanisms by which transplanted cells engraft, proliferate, and function remain unknown. To investigate whether host liver sinusoidal endothelium could be replaced with transplanted liver sinusoidal endothelial cells, we developed an animal model of tissue replacement that utilized a genetic system to identify transplanted cells and induced host-cell perturbations to confer a proliferative advantage to transplanted cells. Under these experimental conditions, transplanted cells engrafted efficiently and proliferated to replace substantial portions of the liver endothelium. Tissue studies demonstrated that transplanted cells became integral to the liver structure and reacquired characteristic endothelial morphology. Characterization of transplanted endothelial cells by membrane markers and studies of cellular function, including synthesis and release of coagulation factor VIII, demonstrated that transplanted cells were functionally intact. Further analysis showed that repopulation of the livers of mice that model hemophilia A with healthy endothelial cells restored plasma factor VIII activity and corrected their bleeding phenotype. Our studies therefore suggest that transplantation of healthy endothelial cells should be considered for cell therapy of relevant disorders and that endothelial reconstitution with transplanted cells may offer an excellent paradigm for defining organ-specific pathophysiological mechanisms.

Purpose: Regulated expression of cell adhesion molecules could be critical in the proliferation, sequestration, and maintenance of stem/progenitor cells. Therefore, we determined fetal and adult stage-specific roles of cell adhesion in liver cell compartments., Methods: We performed immunostaining for the adhesion molecules, E-cadherin and Ep-CAM, associated proteins, beta-catenin and alpha-actinin, hepatobiliary markers, albumin, alpha-fetoprotein, and cytokeratin-19, and the proliferation marker, Ki-67. Expression of albumin was verified by in situ mRNA hybridization., Results: In the fetal liver, hepatoblasts showed extensive proliferation with wide expression of E-cadherin, beta-catenin, and alpha-actinin, although Ep-CAM was expressed in these cells less intensely and focally in the cell membrane to indicate weak cell adhesion. Hepatoblasts in ductal plate and bile ducts showed less proliferation and Ep-CAM was intensely expressed in these cells throughout the cell membrane, indicating strong adhesion. In some ductal plate cells, beta-catenin was additionally in the cytoplasm and nucleus, suggesting active cell signaling by adhesion molecules. In adult livers, cells were no longer proliferating and E-cadherin, beta-catenin, and alpha-actinin were expressed in hepatocytes throughout, whereas Ep-CAM was expressed in only bile duct cells. Some cells in ductal structures of the adult liver with Ep-CAM coexpressed albumin and cytokeratin-19, indicating persistence of fetal-like stem/progenitor cells., Conclusions: Regulated expression of Ep-CAM supported proliferation in fetal hepatoblasts through weak adhesion and helped in biliary morphogenesis by promoting stronger adhesion in hepatoblasts during this process. Restriction of Ep-CAM expression to bile ducts in the adult liver presumably facilitated sequestration of stem/progenitor cells. This stage-specific and cell compartment-related regulation of adhesion molecules should be relevant for defining how liver stem/progenitor cells enter, exit, and remain in hepatic niches during both health and disease.

The potential of cell and gene therapy has generated extensive interest over the past several years. More recently, identification of stem cells of various types, especially embryonic stem cells, reinforced this interest. Systematic studies are now being launched to define the biology of various stem cells, including after transplantation of cells in mmunodeficient animals. This requires robust and unequivocal means to identify transplanted cells. Ideally, it should be possible to screen animal tissues for human cells with relatively simpler methods, followed by more precise localization of transplanted cells. We describe the application of conserved primate Charcot-Marie-Tooth disease type 1A repeat element for polymerase chain reaction-based screening of animal tissues for human cells. Similarly, direct polymerase chain reaction labeling of pancentromeric human alphoid sequences with digoxigenin-UTP generates in situ hybridization probes for identifying transplanted human cells. This pancentromeric probe identifies human cells irrespective of the original tissue source and can be combined with additional in situ methods to analyze cell differentiation. Incorporation of these strategies will facilitate translational studies aimed at understanding mechanisms concerning the trafficking, engraftment, proliferation, differentiation and function of human stem cells in animals.

We investigated whether transplanted hepatocytes interact with hepatic stellate cells, as cell-cell interactions could modulate their engraftment in the liver. We transplanted Fischer 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Activation of hepatic stellate cells was analyzed by changes in gene expression, including desmin and alpha-smooth muscle actin, matrix proteases and their inhibitors, growth factors, and other stellate cell-associated genes with histological methods or polymerase chain reaction. Furthermore, the potential role of hepatic ischemia, Kupffer cells, and cytokine release in hepatic stellate cell activation was investigated. Hepatocyte transplantation activated desmin-positive hepatic stellate cells, as well as Kupffer cells, including in proximity with transplanted cells. Inhibition of Kupffer cells by gadolinium chloride, blockade of tumor necrosis factor alpha (TNF-alpha) activity with etanercept or attenuation of liver ischemia with nitroglycerin did not decrease this hepatic stellate cell perturbation. After cell transplantation, soluble signals capable of activating hepatic stellate cells were rapidly induced, along with early upregulated expression of matrix metalloproteinases-2, -3, -9, -13, -14, and their inhibitors. Moreover, prior depletion of activated hepatic stellate cells with gliotoxin decreased transplanted cell engraftment. In conclusion, cell transplantation activated hepatic stellate cells, which, in turn, contributed to transplanted cell engraftment in the liver. Manipulation of hepatic stellate cells might provide new strategies to improve liver repopulation after enhanced transplanted cell engraftment.

Background & Aims: Recognition and circumvention of the hepatic endothelial barrier is critical in the engraftment of transplanted cells. We examined whether interactions between integrin and extracellular matrix component receptors could be manipulated for improving transplanted cell engraftment and liver repopulation., Methods: Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV-deficient rats. Coating of cells or of liver sinusoids with natural collagen, natural laminin, or an engineered fibronectin-like polymer was studied with analysis of cell engraftment and liver repopulation using histologic and molecular assays. Focal adhesion complexes were identified by vinculin immunostaining. The role of integrin receptors in cell engraftment was analyzed with RGD peptide inhibition assays., Results: Coating of cells with extracellular matrix components before transplantation did not enhance cell engraftment. In contrast, intraportal infusion of collagen or fibronectin-like polymer in recipients prior to cell transplantation increased cell engraftment. Adherence of transplanted cells to the hepatic endothelium resulted in rapid activation of vinculin-containing focal adhesion complexes. Superior cell engraftment in animals treated with fibronectin-like polymer was RGD sensitive, verifying the integrin-dependent nature of this process. Moreover, studies in the retrorsine-partial hepatectomy rat model showed that intraportal infusion of the fibronectin-like polymer before cell transplantation significantly accelerated liver repopulation., Conclusions: Integrin-extracellular matrix component interactions can be manipulated for enhancing cell engraftment in the liver. Such mechanisms will be relevant for engraftment of other cell types and for strategies concerning liver-directed cell therapy.

Targeting of cells to specific tissues is critical for cell therapy. To study endothelial cell targeting, we isolated mouse liver sinusoidal endothelial cells (LSEC) and examined cell biodistributions in animals. To identify transplanted LSEC in tissues, we labeled cells metabolically with DiI-conjugated acetylated low density lipoprotein particles (DiI-Ac-LDL) or (111)Indium-oxine, used LSEC from Rosa26 donors expressing beta-galactosidase or Tie-2-GFP donors with green fluorescent protein (GFP) expression, and tranduced LSEC with a GFP-lentiviral vector. LSEC efficiently incorporated (111)Indium and DiI-Ac-LDL and expressed GFP introduced by the lentiviral vector. Use of radiolabeled LSEC showed differences in cell biodistributions in relation to the cell transplantation route. After intraportal injection, LSEC were largely in the liver (60 +/- 13%) and, after systemic intravenous injection, in lungs (67 +/- 9%); however, after intrasplenic injection, only some LSEC remained in the spleen (29 +/- 10%; P < .01), whereas most LSEC migrated to the liver or lungs. Transplanted LSEC were found in the liver, lungs, and spleen shortly after transplantation, whereas longer-term cell survival was observed only in the liver. Transplanted LSEC were distinct from Kupffer cells with expression of Tie-2 promoter-driven GFP and of CD31, without F4/80 reactivity. In further studies using radiolabeled LSEC, we established that the manipulation of receptor-mediated cell adhesion in liver sinusoids or the manipulation of blood flow-dependent cell exit from sinusoids improved intrahepatic retention of LSEC to 89 +/- 7% and 89 +/- 5%, respectively (P < .01). In conclusion, the targeting of LSEC to the liver and other organs is directed by vascular bed-specific mechanisms, including blood flow-related processes, and cell-specific factors. These findings may facilitate analysis of LSEC for cell and gene therapy applications.

ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression.

We determine here the functional integrity of auxiliary livers in containers fashioned from the small intestine. Liver microfragments from dipeptidyl peptidase 4 (DPP4)-deficient rats were transplanted into syngeneic normal animals with isolated intestinal segments characterized by mucosal denudation but intact vascular supply. Transplanted liver fragments were restored to confluent tissue with normal hepatic architecture and development of DPP4-positive vessels, indicating angiogenesis and revascularization. Auxiliary liver units expressed multiple hepatotrophic and angiogenic genes, and transplanted tissues remained intact for up to the 6-week duration of the studies with neither ischemic injury nor significant hepatocellular proliferation. Hepatic metabolic, transport and synthetic functions were preserved in auxiliary livers, including uptake and biliary excretion of (99m)Tc-mebrofenin in syngeneic recipients of liver from F344 rats, as well as secretion of albumin in allografted Nagase analbuminemic rats. This ability to produce functionally competent auxiliary livers in vascularized intestinal segments offers therapeutic potential for liver disease and genetic deficiency.