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Abstract Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. It is known that AIH originates not from the spleen but from the liver itself. Nonetheless, most details of the etiology and pathophysiology are unknown. We induced experimental murine AIH (emAIH) in NOD/Ltj mice by single administration of a replication-deficient adenovirus and performed splenectomy during late-stage disease. Biochemical disease remission occurred, which was characterized by improvement in transaminase levels. The causes of this remission included a shift in the transcriptomic signature of serum proteins toward regeneration. At the cellular level, there was a marked decrease in activated CD8+ T cells and an increase in intrahepatic regulatory T cells (Tregs). Here, intrahepatic Treg numbers correlated with biochemical remission. Notably, an imbalance in the T-cell/B-cell ratio was observed, with a disproportionate increase in total B cells. In summary, intrahepatic increases in Tregs, biochemical remission, and regeneration could be induced by splenectomy in the late stage of emAIH.

Background & Aims: EASL guidelines recommend 8 weeks of treatment with sofosbuvir plus velpatasvir (SOF/VEL) for the treatment of acute or recently acquired HCV infection, but only 6- and 12-week data are available. Therefore, the aim of this study was to evaluate the safety and efficacy of a shortened 8-week SOF/VEL treatment for acute HCV monoinfection. Methods: In this investigator-initiated, prospective, multicentre, single-arm study, we recruited 20 adult patients with acute HCV monoinfection from nine centers in Germany. Patients received SOF/VEL (400/100 mg) as a fixed-dose combination tablet once daily for 8 weeks. The primary efficacy endpoint was the proportion of patients with sustained virological response 12 weeks after the end of treatment (SVR12). Results: The median HCV RNA viral load at baseline was 104,307 IU/ml; the distribution of HCV genotypes was as follows: GT1a/1b/2/3/4: n = 12/1/1/3/3. Thirteen (65%) of the 20 patients were taking medication for HIV pre-exposure prophylaxis. SVR12 was achieved in all patients who complied with the study protocol (n = 18/18 [100%], per protocol analysis), but the primary endpoint was not met in the intention-to-treat analysis (n = 18/20 [90%]) because two patients were lost to follow-up. One serious adverse event (unrelated to study drug) occurred during 12 weeks of post-treatment follow-up. Conclusions: The 8-week treatment with SOF/VEL was well tolerated and highly effective in all adherent patients with acute HCV monoinfection. Early treatment of hepatitis C might effectively prevent the spread of HCV in high-risk groups. Clinical Trial Number: NCT03818308. Impact and implications: The HepNet acute HCV-V study (NCT03818308), an investigator-initiated, single-arm, multicenter pilot study, demonstrates the efficacy and safety of 8 weeks of daily treatment with the fixed-dose combination sofosbuvir/velpatasvir (400/100 mg) in patients with acute hepatitis C virus (HCV) infection. All patients who completed therapy and were followed-up achieved sustained virologic response. Thus, early treatment with SOF/VEL which might effectively prevent the spread of HCV in high-risk groups can be recommended for patients with acute HCV monoinfection.

Standard treatment of hepatitis delta virus (HDV) infection remains pegylated‐interferon alfa (peg‐IFNα) in most centers, which is not only associated with rather low efficacy but several adverse events. Hepatitis B core‐related antigen (HBcrAg) is linked to intrahepatic covalently closed circular DNA levels and has previously been suggested as response predictor in IFN‐based treatment of hepatitis B virus (HBV) mono‐infection. This study aimed to investigate the value of HBcrAg in the management of patients with HBV/HDV co‐infection undergoing peg‐IFNα treatment. The Hep‐Net‐International‐Delta‐Hepatitis‐Intervention Trial‐2 study included 120 patients co‐infected with HBV/HDV. Patients were treated for 96 weeks with peg‐IFNα and either tenofovir or placebo. Ninety‐nine patients with HDV‐RNA results 24 weeks after end of treatment (FU24) were included in this analysis, of whom 32 patients (32.3%) had undetectable HDV RNA at FU24. HBcrAg was measured at baseline, week 12, 24, 48, 96, and FU24. HBcrAg levels showed no significant correlation with HDV RNA but were significantly linked to treatment outcome. HBcrAg levels < 4.5 log IU/mL at baseline, week 24, and week 48 had high negative predictive value (NPV) for achieving undetectable HDV RNA at FU24 (81.8%, 87.1% and 95.0%, respectively). Similarly, HBcrAg levels at week 96 were significantly higher in patients with viral relapse until FU24 (3.0 vs. 3.63 log IU/mL; P = 0.0089). Baseline, week 24, and week 48 HBcrAg levels were also associated with the likelihood of achieving HBsAg level < 100 IU/mL at FU24 (HBcrAg < 3.0 log IU/mL: NPV 91.7%, 90.4% and 92.3%, respectively). Test statistics improved when combining HBcrAg with additional viral and clinical parameters. Conclusion: HBcrAg is linked to treatment response to peg‐IFNα in patients with HBV/HDV co‐infection and could be a promising marker to determine treatment futility.

Transjugular intrahepatic portosystemic shunt (TIPS) is an effective treatment for portal hypertension‐related complications. However, careful selection of patients is crucial. The aim of this study was to evaluate the prognostic value of serum cholinesterase (CHE) for outcomes and mortality after TIPS insertion. In this multicenter study, 389 consecutive patients with cirrhosis receiving a TIPS at Hannover Medical School, University Hospital Essen, or Medical University of Vienna were included. The Hannover cohort (n = 200) was used to initially explore the role of CHE, whereas patients from Essen and Vienna served as a validation cohort (n = 189). Median age of the patients was 58 years and median Model for End‐Stage Liver Disease (MELD) score was 12. Multivariable analysis identified MELD score (hazard ratio [HR]: 1.16; P

Abstract There are considerable differences between males and females regarding the etiology, progression and outcome of liver diseases. Infections are a frequent and severe complication in these patients. This study aimed to examine sex specific differences in the incidence and clinical course of nosocomial infections in patients with decompensated liver cirrhosis. A number of 556 consecutive hospitalized patients with decompensated liver cirrhosis and ascites were analyzed. The patients were followed up for the incidence of nosocomial infections, acute kidney injury (AKI), acute-on-chronic liver failure (ACLF) as well as liver transplantation and death (LTx-free survival). A number of 285 patients (111 women and 174 men) developed a nosocomial infection. Incidence was numerically lower in men (P = 0.076). While the frequency of a nosocomial spontaneous bacterial peritonitis was similar between males and females, the incidence of a nosocomial urinary tract infection was significantly higher in women (P

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High‐fat high‐carbohydrate (HF‐HC) diet feeding of NASH‐resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)‐deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF‐HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen‐driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1−/− mice was substantially worsened and accompanied by a sharp increase of M1‐like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti‐cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it.

In this study, we investigated compounds of plant and mushroom origin belonging to Traditional Chinese Medicine (TCM) and to Traditional Tibetan Medicine (TTM): a sandy beige mushroom Trametes robiniophila Murr, commonly known as Huaier/TCM as well as Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan, which both belong to TTM. We aimed to study the efficacy of TTM and TCM in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) in vitro. TCM and TTM were tested either as a monotherapy, or in combination with standard therapeutics: sorafenib for HCC treatment and gemcitabine for CCA. We also discovered a protective mechanism behind the most successful therapeutic combinations. The results demonstrated that TCM and TTM inhibited the proliferation of cancer cells in a time- and dose-dependent manner. The results were compared to classical chemotherapeutics currently used in the clinic: sorafenib for HCC and gemcitabine for CCA. In HCC settings, a combination of Huaier (16 mg/ml) with half of the human plasma concentration of sorafenib, Qiwei Honghua Shusheng Wan (1 mg/ml) monotherapy as well as its combination with half or even a quarter dose of the human plasma concentration of sorafenib represented the most efficient treatments, inhibiting the growth of HCC cells more effectively than the standard therapy. The inhibitory mechanism relied on a strong induction of apoptosis. In CCA settings, Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan as monotherapies or in combination with very low doses of gemcitabine inhibited the growth of CCA cells more efficiently than the standard therapy. Importantly, Ershiwuwei Songshi Wan at the 8 and 16 mg/ml concentrations and Qiwei Honghua Shusheng Wan at the 4 mg/ml concentration were efficacious with gemcitabine applied at massively reduced concentrations. The protective mechanism in CCA relied on a strong induction of early and late apoptosis. Cellular senescence and necroptosis were not associated with protection against HCC/CCA. Combination therapy with TCM or TTM allowed for a dose reduction of standard chemotherapeutics. This is especially important as both chemotherapeutic drugs show strong side effects in patients. The reduction of chemotherapeutics and the synergistic effect observed while applying them in combination with TCM and TTM has strong perspectives for the clinic and patients suffering from HCC and CCA.

The natural polymer, lignin, possesses unique biodegradable and biocompatible properties, making it highly attractive for the generation of nanoparticles for targeted cancer therapy. In this study, we investigated spruce and eucalyptus lignin nanoparticles (designated as S-and E-LNPs, respectively). Both LNP types were generated from high-molecular-weight (Mw) kraft lignin obtained as insoluble residues after a five-step solvent fractionation approach, which included ethyl acetate, ethanol, methanol, and acetone. The resulting S-and E-LNPs ranged in size from 16 to 60 nm with uniform spherical shape regardless of the type of lignin. The preparation of LNPs from an acetone-insoluble lignin fraction is attractive because of the use of high-Mw lignin that is otherwise not suitable for most polymeric applications, its potential scalability, and the consistent size of the LNPs, which was independent of increased lignin concentrations. Due to the potential of LNPs to serve as delivery platforms in liver cancer treatment, we tested, for the first time, the efficacy of newly generated E-LNPs and S-LNPs in two types of primary liver cancer, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), in vitro. Both S-LNPs and E-LNPs inhibited the proliferation of HCC cells in a dose-dependent manner and did not affect CCA cell line growth. The inhibitory effect toward HCC was more pronounced in the E-LNP-treated group and was comparable to the standard therapy, sorafenib. Also, E-LNPs induced late apoptosis and necroptosis while inhibiting the HCC cell line. This study demonstrated that an elevated number of carbohydrates on the surface of the LNPs, as shown by NMR, seem to play an important role in mediating the interaction between LNPs and eukaryotic cells. The latter effect was most pronounced in E-LNPs. The novel S- and E-LNPs generated in this work are promising materials for biomedicine with advantageous properties such as small particle size and tailored surface functionality, making them an attractive and potentially biodegradable delivery tool for combination therapy in liver cancer, which still has to be verified in vivo using HCC and CCA models.

Abstract Background Endoscopic placement of intestinal decompression tubes is a feasible technique for treatment of acute intestinal dilation. Given the heterogeneity of the underlying diseases leading to intestinal obstruction data on the significance of endoscopic procedures for treatment of these conditions are sparse. Methods In the study period from 2008 to 2019 all patients receiving a decompression tube were identified by retrospective chart review and analyzed. Results A total of 59 decompression tubes were placed in 50 patients. Technical success was achieved in 98% (58/59 tubes). As major complication one small bowel perforation occurred (1/59; 1.7%). Causes for impaired intestinal transit comprised tumor stenoses 22% (11/50), infections 18% (9/50), post-operative paralysis 14% (7/50), neurological diseases 8% (4/50), trauma 2% (1/50) and others 36% (18/50). Most patients (74%; 37/50) were critically ill and treated on intensive care unit. Treatment response after tube insertion was documented in 76% of patients (38/50) whereas 24% (12/50) did not fulfill response criteria. Patients with treatment response showed a significantly better outcome compared to non-responders. Responders had a median survival of 113 days (95% CI 41–186) compared to 15 days (95% CI 6–24) in non-responders (p = 0.002). Analysis of laboratory parameters after stratification in responders and non-responders to endoscopic therapy showed that non-responders had significantly higher levels of CRP and lower platelet count at baseline (CRP 262 mg/L (IQR 101–307) vs. 94 mg/L (IQR 26–153): p = 0.027; platelets 69 thsd/μL (IQR 33–161) vs. 199 thsd/μL (IQR 138–289): p = 0.009). Conclusions Endoscopic decompression is a safe procedure for acute management of impaired intestinal transit even in critically ill patients. Response to therapy is associated with improved outcome and markers of inflammation and organ function such as CRP, platelet count and serum lactate have to be taken into account for therapy monitoring and evaluation of prognosis.

Abstract Background Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. Methods Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. Results For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. Conclusions The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

Background & Aims: Treatment options remain limited for patients with autoimmune hepatitis (AIH), while there are still concerns over the consequences of long-term corticosteroid use. A few studies have suggested a role for B cell-driven autoimmune liver injury in AIH. This multicentre, international retrospective cohort study from the International Autoimmune Hepatitis Group aims to evaluate the clinical efficacy and safety of rituximab in difficult-to-manage AIH. Methods: Clinical data from 22 patients who received rituximab between 2007 and 2017 were collected from centres in the United Kingdom, Germany and Canada. Clinical response was assessed using changes in biochemical and immunological parameters up to 24 months post-rituximab infusion. In addition, we compared the doses of prednisolone used 3 months before and 12 months after treatment, and assessed freedom from AIH flares over the post-treatment period. Results: Twenty-two patients with type-1 AIH were included, with a median age of 40 years at diagnosis (range 19–79); 15/22 (68%) were female and 18/22 (82%) were Caucasian. The median period from diagnosis to the end of follow-up in these patients was 11 years (range 3–28). Values of alanine aminotransferase, aspartate aminotransferase and albumin improved significantly following rituximab therapy, and were sustained for up to 2 years (all p ≪0.001). Prednisolone doses were significantly reduced by 12 months post-treatment (p = 0.003), with 13/21 (62%) patients having a dose reduction. Over a median post-treatment follow-up period of 6 years (range 1–10), 5 patients developed AIH flares at a median of 22 months post-treatment, giving an estimated 71% freedom from AIH flare at 2 years. Four of these patients received a second course of treatment, of whom 2 had subsequent further flares. No serious adverse events attributable to rituximab were recorded. Conclusion: In patients with difficult-to-manage AIH, rituximab appears to be clinically effective and well tolerated. Rituximab was associated with sustained improvements in serum liver tests, an absence of clinical disease flares, and a reduction in prednisolone dose. Controlled trials are warranted to further evaluate B cell-targeting therapies in patients with AIH. Lay summary: Autoimmune hepatitis is an autoimmune condition of the liver, usually treated with medications that suppress the immune system, such as steroids. However, some patients do not respond to this treatment. We analysed the safety and efficacy of rituximab in patients who were not responding to first- or second-line therapies. Rituximab was safe and improved liver blood tests in 70% of patients over a 2-year follow-up period, while enabling steroid doses to be reduced in two-thirds of patients, which is a very positive clinical outcome. Keywords: Autoimmune hepatitis, Prednisolone, Difficult-to-manage, B cell depletion therapy, Rituximab

Abstract Chronic hepatitis B virus (HBV) infection may follow four different consecutive phases, which are defined by virology as well as biochemical markers and differ in terms of prognosis and need for antiviral treatment. Currently, host responses reflected by immune markers are not considered in this definition. We aimed to study soluble immune markers and their distribution in different phases of chronic HBV infection. In this cross-sectional retrospective study, we investigated a panel of 14 soluble immune markers (SIM) including CXCL10 in 333 patients with chronic HBV infection. In a small cohort of HBeAg positive patients we analyzed SIM before and after HBeAg seroconversion and compared seroconverters to patients with unknown outcome. Significant differences were documented in the levels of several SIM between the four phases of chronic HBV infection. The most pronounced difference among all investigated SIM was observed for CXCL10 concentrations with highest levels in patients with hepatitis. TGF-β and IL-17 revealed different levels between HBeAg negative patients. HBeAg positive patients with HBeAg seroconversion presented higher amounts of IL-12 before seroconversion compared to HBeAg positive patients with unknown follow up. SIM such as CXCL10 but also IL-12, TGF-β and IL-17 may be useful markers to further characterize the phase of chronic HBV infection.

The efficacy of oncolytic adenoviruses is limited by strong immune responses being induced against the oncolytic virus itself. Here, the authors generate a bispecific molecule capable of redirecting the adenovirus-specific antibodies to tumour cells and show this induces immune mediated cancer growth inhibition and enhances the therapeutic efficacy of viral oncolysis.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very poor prognosis and constantly growing incidence. Among other primary risks of HCC, metabolic disorders and obesity have been extensively investigated over recent decades. The latter can promote nonalcoholic fatty liver disease (NAFLD) leading to the inflammatory form of nonalcoholic steatohepatitis (NASH), that, in turn, promotes HCC. Molecular determinants of this pathogenic progression, however, remain largely undefined. In this study, we have focussed on the investigation of α-dicarbonyl compounds (α-dC), highly reactive and tightly associated with overweight-induced metabolic disorders, and studied their potential role in NAFLD and progression toward HCC using murine models. NAFLD was induced using high-fat diet (HFD). Autochthonous HCC was induced using transposon-based stable intrahepatic overexpression of oncogenic NRASG12V in mice lacking p19Arf tumor suppressor. Our study demonstrates that the HFD regimen and HCC resulted in strong upregulation of α-dC in the liver, heart, and muscles. In addition, an increase in α-dC was confirmed in sera of NAFLD and NASH patients. Furthermore, higher expression of the receptor for advanced glycation products (RAGE) was detected exclusively on immune cells and not on stroma cells in livers of mice with liver cancer progression. Our work confirms astable interplay of liver inflammation, carbonyl stress mediated by α-dC, and upregulated RAGE expression on CD8+ Tand natural killer (NK) cells in situ in NAFLD and HCC, as key factors/determinants in liver disease progression. The obtained findings underline the role of α-dC and RAGE+CD8+ Tand RAGE+ NK cells as biomarkers and candidates for a local therapeutic intervention in NAFLD and malignant liver disease.

Background: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease. Methods: In a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis. Findings: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89–0.97, p

Background & Aims: A DNA methylation (DNAm) signature derived from 353 CpG sites (the Horvath clock) has been proposed as an epigenetic measure of chronological and biological age. This epigenetic signature is accelerated in diverse tissue types in various disorders, including non-alcoholic steatohepatitis, and is associated with mortality. Here, we assayed whole blood DNAm to explore age acceleration in patients with primary sclerosing cholangitis (PSC). Methods: Using the MethylationEPIC BeadChip (850K) array, DNAm signatures in whole blood were analyzed in 36 patients with PSC enrolled in a 96-week trial of simtuzumab (Ishak F0-1, n = 13; F5-6, n = 23). Age acceleration was calculated as the difference between DNAm age and chronological age. Comparisons between patients with high and low age acceleration (≥ vs. < the median) were made and Cox regression evaluated the association between age acceleration and PSC-related clinical events (e.g. decompensation, cholangitis, transplantation). Results: Age acceleration was significantly higher in patients with PSC compared to a healthy reference cohort (median, 11.1 years, p

Natural killer (NK) cells are important immune cells for mediating antiviral immunity. Here the authors show that chronic hepatitis C virus infection in human can imprint lasting functional phenotypes in NK cells to increase their inter-individual but decrease intra-individual diversity.

Based on the hypothesis that autoimmunological factors coregulate the pathomechanism in biliary atresia (BA), adjuvant therapy with steroids has become routine, although its efficacy has never been proven. In 2010, a study on the advantages of budesonide compared to prednisolone in autoimmune hepatitis gave rise to experimental therapy using budesonide as an adjuvant BA treatment. Ninety-five BA patients prospectively received a budesonide 2 mg/dose rectal foam daily for three months (SG). A case-matched control group (CG: 81) was retrospectively recruited. The outcome measures were survival with native liver (SNL), determined at six months and two years after the Kasai procedure. The follow-up rate was 100%. At six months, SNL was statistically not different but became so after two years (SG: 54%; CG: 32%; p < 0.001). No steroid-related side effects were observed, except for eight patients with finally caught-up growth retardation. This study demonstrates for the first time a significantly longer survival with native liver in patients with BA after adjuvant therapy. However, indication, dosage, and duration of any budesonide application is not given in neonates with BA. Hence, we suggest extending the postoperative use of budesonide in a multicenter observational study with a clearly defined follow-up protocol, particularly in terms of potentially underestimated side effects.

Chronic hepatitis C can be treated very effectively with direct-acting antivirals (DAA) with only minor side effects compared to an interferon-containing treatment regimen. The significance of metabolic comorbidities after HCV cure is not well defined. This study aims to investigate short- and long-term weight change of patients receiving interferon-free antiviral treatment for chronic hepatitis C. The German Hepatitis C-registry (DHC-R) is a national multicenter real-world cohort. A total of 5111 patients were followed prospectively after DAA treatment for up to 3 years. Weight change compared to baseline was analyzed at end of treatment and at years 1, 2, and 3 after completion of antiviral therapy. Regression analysis was performed to identify baseline predictors for weight change. While there was no relevant mean weight change (−0.2 kg, SD 4.3 kg) at the end of antiviral treatment, weight started to increase during long-term follow-up reaching +1.7 kg (SD 8.0 kg, p < 0.001) compared to baseline at 3 years (follow-up year 3, FU3) after completion of antiviral therapy. 48%, 31%, and 22% of patients had a weight gain greater than 1, 3, and 5 kg at FU3, respectively. During follow-up, a body mass index (BMI)

Hepatobiliary and pancreatic cancers along with other gastrointestinal malignancies remain the leading cause of cancer-related deaths worldwide. Strategies developed in the recent years on immunotherapy and cancer vaccines in the setting of primary liver cancer as well as in pancreatic cancer are the scope of this review. Significance of orthotopic and autochthonous animal models which mimic and/or closely reflect human malignancies allowing for a prompt and trustworthy analysis of new therapeutics is underlined. Combinational approaches that on one hand, specifically target a defined cancer-driving pathway, and on the other hand, restore the functions of immune cells, which effector functions are often suppressed by a tumor milieu, are shown to have the strongest perspectives and future directions. Among combinational immunotherapeutic approaches a personalized- and individual cancer case-based therapy is of special importance.

Hepatocellular carcinoma (HCC) is known to be associated with protein alterations and extracellular fibrous deposition. We investigated the urinary proteomic profiles of HCC patients in this prospective cross sectional multicentre study. 195 patients were recruited from the UK (Coventry) and Germany (Hannover) between 1 January 2013 and 30 June 2019. Out of these, 57 were HCC patients with a background of liver cirrhosis (LC) and 138 were non-HCC controls; 72 patients with LC, 57 with non-cirrhotic liver disease and 9 with normal liver function. Analysis of the urine samples was performed by capillary electrophoresis (CE) coupled to mass spectrometry (MS). Peptide sequences were obtained and 31 specific peptide markers for HCC were identified and further integrated into a multivariate classification model. The peptide model demonstrated 79.5% sensitivity and 85.1% specificity (95% CI: 0.81–0.93, p < 0.0001) for HCC and 4.1-fold increased risk of death (95% CI: 1.7–9.8, p = 0.0005). Proteases potentially involved in HCC progression were mapped to the N- and C-terminal sequence motifs of the CE-MS peptide markers. In silico protease prediction revealed that kallikrein-6 (KLK6) elicits increased activity, whilst Meprin A subunit α (MEP1A) has reduced activity in HCC compared to the controls. Tissue expression of KLK6 and MEP1A was subsequently verified by immunohistochemistry.

Background: Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires lifelong immunosuppression. Frequent recurrences after the discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current treatments. Studies of other autoimmune diseases suggest that temporary depletion of B cells can improve disease progression in the long term. Methods: We tested a single administration of anti-CD20 antibodies to reduce B cells and the amount of IgG to induce intrahepatic immune tolerance. We used our experimental murine AIH (emAIH) model and treated the mice with anti-CD20 during the late stage of the disease. Results: After treatment, the mice showed the expected reductions in B cells and serum IgGs, but no improvements in pathology. However, all treated animals showed a highly altered serum protein expression pattern, which was a balance between inflammation and regeneration. Conclusions: In conclusion, anti-CD20 therapy did not produce clinically measurable results because it triggered inflammation, as well as regeneration, at the proteomic level. This finding suggests that anti-CD20 is ineffective as a sole treatment for AIH or emAIH.

Background and Study Aims. Patients undergoing therapeutic endoscopic retrograde cholangiography (ERC) may require different amounts of sedative agents depending on demographic characteristics, indication of ERC, and/or endoscopic intervention. Patients and Methods. We retrospectively analyzed all patients undergoing therapeutic ERC from 2008 – 2014 who received deep sedation with propofol ± midazolam. Results. A total of 2448 ERC procedures were performed in 781 patients. The cumulative per procedure propofol dose in the different groups was as follows: PSC 479 mg (±256), bile duct stones 356 mg (±187), benign stenosis/cholestasis 395 mg (±228), malignant stenosis 401 mg (±283), and postliver transplant complications 391 mg (±223) (p < 0.05). Multivariable analysis showed that dilatation therapy (p = 0.001), age (p = 0.001), duration of the intervention (p = 0.001), BMI (p = 0.001), gender (p = 0.001), platelet count (p = 0.003), and bilirubin (p = 0.043) influence independently the propofol consumption. Conclusions. Demographic characteristics and endoscopic interventions have a distinct influence on the amount of sedation required for therapeutic ERC. Although the sedation-associated complication rate is low optimization of sedative regimens is a prime goal to further reduce adverse events of therapeutic ERC.

Dysregulation of glucose homeostasis plays a major role in the pathogenesis of non-alcoholic steatohepatitis (NASH) as it activates proinflammatory and profibrotic processes. Beneficial effects of antiglycemic treatments such as GLP-1 agonist or SGLT-2 inhibitor on NASH in patients with diabetes have already been investigated. However, their effect on NASH in a non-diabetic setting remains unclear. With this aim, we investigated the effect of long-acting GLP1-agonist dulaglutide and SGLT-2 inhibitor empagliflozin and their combination in a non-diabetic mouse model of NASH. C57BL/6 mice received a high-fat-high-fructose (HFHC) diet with a surplus of cholesterol for 16 weeks. After 12 weeks of diet, mice were treated with either dulaglutide, empagliflozin or their combination. Dulaglutide alone and in combination with empagliflozin led to significant weight loss, improved glucose homeostasis and diminished anti-inflammatory and anti-fibrotic pathways. Combination of dulaglutide and empagliflozin further decreased MoMFLy6CHigh and CD4+Foxp3+ T cells. No beneficial effects for treatment with empagliflozin alone could be shown. While no effect of dulaglutide or its combination with empaglifozin on hepatic steatosis was evident, these data demonstrate distinct anti-inflammatory effects of dulaglutide and their combination with empagliflozin in a non-diabetic background, which could have important implications for further treatment of NASH.

miR125b-5p has been associated with acute liver failure. Here the authors show that this miRNA targets Keap1 and activates Nrf2 to inhibit liver cell death after APAP or FAS toxicity, plus they show that a miR125b-5p mimic can inhibit ALF in mice.

Background HBV DNA is the most important molecular marker in hepatitis B, used to determine treatment indication and monitoring. Most patients require lifelong hepatitis B virus (HBV) management, thus viral load (VL) monitoring may be performed at different laboratories, with different HBV assays, which may result in different VL results. This multicenter study compares the commutability and concordance of results from four different HBV DNA assays: CAP/CTM HBVv2, HPS/CTM HBVv2 and the new cobas 6800/8800 HBV and cobas 4800 HBV assays. Methods Across all four assays, HBV limit of detection (LoD) and linearity at lower concentrations were assessed using panels traceable to the World Health Organization international standard, and concordance was investigated at the important medical decision cutoffs 2000 and 20,000 IU/ml, using specimens from HBV-positive patients. Results The calculated LoD via a probit curve was 2.7 IU/ml for cobas 6800/8800 HBV, 2.8 IU/ml for cobas 4800 HBV, 9.6 IU/ml for CAP/CTM HBVv2, and 6.2 IU/ml for HPS/CTM HBVv2. The average accuracy was comparable between cobas 6800/8800 HBV, cobas 4800 HBV and CAP/CTM HBVv2 (0.04–0.05 log 10 IU/ml), while a slightly lower accuracy was documented for HPS/CTM HBVv2 (−0.16 log 10 IU/ml). A total of 211–245 clinical samples were used for a pairwise comparison. Mean paired log differences ranged from −0.17 log 10 IU/ml to −0.01 log 10 IU/ml. Coefficient of determination was over 98% for all pairs with high overall percent agreement at the 2000 and 20,000 IU/ml cutoffs (from 91.7% to 96.3%). In a subset of samples with VL±0.5 log 10 to the 2000 and 20,000 IU/ml thresholds, concordance was still 72% and 82%, respectively. Conclusions The new cobas 6800/8800 HBV and 4800 HBV assays show high accuracy in samples with low-level viremia and a high concordance with the established HBV tests, CAP/CTM HBVv2 and HPS/CTM HBVv2, at 2000 and 20,000 IU/ml. Thus, all four HBV assays have high commutability and may be used interchangeably in routine clinical practice.

Adaptive-like expansions of natural killer (NK) cell subsets are known to occur in response to human cytomegalovirus (CMV) infection. These expansions are typically made up of NKG2C+ NK cells with particular killer-cell immunoglobulin-like receptor (KIR) expression patterns. Such NK cell expansion patterns are also seen in patients with viral hepatitis infection. Yet, it is not known if the viral hepatitis infection promotes the appearance of such expansions or if effects are solely attributed to underlying CMV infection. In sizeable cohorts of CMV seropositive hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infected patients, we analyzed NK cells for expression of NKG2A, NKG2C, CD57, and inhibitory KIRs to assess the appearance of NK cell expansions characteristic of what has been seen in CMV seropositive healthy individuals. Adaptive-like NK cell expansions observed in viral hepatitis patients were strongly associated with CMV seropositivity. The number of subjects with these expansions did not differ between CMV seropositive viral hepatitis patients and corresponding healthy controls. Hence, we conclude that adaptive-like NK cell expansions observed in HBV, HCV, and/or HDV infected individuals are not caused by the chronic hepatitis infections per se, but rather are a consequence of underlying CMV infection.

Background and study aims Patients with primary sclerosing cholangitis (PSC) require repeated endoscopic retrograde cholangiography (ERC). Our aim was to evaluate whether patients with PSC require higher doses of sedation during ERC. Patients and methods We retrospectively analyzed all patients undergoing ERC from 2006 to 2013 who received conscious sedation with propofol and midazolam. The duration of the intervention and a potential progression of propofol consumption or intervention time by visit number were analyzed. Univariable and multivariable analyses were performed to identify independent factors which influence propofol consumption. Results A total of 2962 ERC procedures were performed in 1211 patients. Patients with PSC (n = 157) underwent 461 ERC procedures whereas patients without PSC (n = 1054) had 2501 ERC examinations. The total median propofol dose was 450 mg (290 – 630 mg) for patients with PSC and 300 mg (200 – 450 mg) for the non-PSC group (P

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.

Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.

Background. Intestinal intussusception in adults is associated with chronic inflammatory bowel disease, coeliac disease, abdominal tumors or previous abdominal surgery but most often of unknown origin.Aim. The aim of our study was to evaluate circumstances and identify risk factors for intussusceptions.Methods. All 65,928 abdominal ultrasound examinations performed at our tertiary medical center between January 2001 and June 2008 were analyzed retrospectively for the diagnosis “intussusception”. After identifying individuals with sonographically proven intussusception we analyzed various patients’ characteristics including age, gender and underlying disease as well as sonographic findings such as localization of the intussusception, absence or presence of ascites and lymph nodes.Results. We identified 32 cases of intussusceptions [mean age 45 years (range 18 to 88); 18 patients were male]. Twelve patients (38%) had a history of abdominal surgery including 8 patients who had undergone liver transplantation (2 patients with primary sclerosing cholangitis, 1 patient with cystic fibrosis, 1 patient with sarcoidosis, 1 patient with hepatocellular carcinoma and HCV infection, 1 patient with autoimmune hepatitis, 1 patient with Crigler-Najar-syndrome and one patient with echinococcus). A hepaticojejunostomy had been performed in 4 of the patients after liver transplantation. Liver transplanted patients were significantly overrepresented in the intussusception group compared with the overall cohort of patients undergoing abdominal ultrasound examination (25% vs. 8%, Chi-Square-test, p = 0.0023).Conclusion. In our retrospective study liver transplantation, in particular with hepaticojejunostomy, was identified as a new major risk factor for intestinal intussusceptions.

Introduction. Liver transplantation (OLT) for primary biliary cirrhosis (PBC) is characterized by disease recurrence of up to one third of patients. The diagnosis of recurrence requires a cholestatic profile and a typical histology representing a challenge for transplant hepatologists. Antimitochondrial antibodies (AMA) establish the initial diagnosis, persist after OLT, and are thus of limited value for the diagnosis of recurrence. Aim of this analysis was to identify serological parameters associated with recurrent PBC.Patients and methods. OLT performed between 1992 and 2006 at Hannover Medical School were evaluated retrospectively including histology before and after OLT, autoimmune serological parameters and clinical characteristics.Results. Between 1992 and 2006 72 patients underwent OLT with histologically confirmed PBC. Median follow up was 123 months. AMA persisted in 55 (767) patients. Anti-parietal cell antibodies (PCA) were detectable in 41% of the patients before and 47% after OLT. Liver biopsies were obtained in 34 patients post OLT upon clinical suspicion, and recurrent PBC diagnosed in 28% after a mean of 71 months (range 13-161). Anti-PCA were detected in 100% of patients with recurrence before and following transplantation, 54% of patients with anti-PCA before OLT developed recurrence during follow-up. There were no differences in immunosuppressive regimen.Discussion. Although unspecific for the diagnosis of PBC, anti-PCA prevalence increased after OLT, and was 100% in patients with recurrent PBC. Recurrent PBC developed in 54% of patients with anti-PCA before OLT suggesting a diagnostic role of anti-PCA as a simple and cost effective marker of recurrence.

Treatment of chronic hepatitis C with type I interferons and ribavirin can be associated with exacerbation of hepatitis and sometimes liver decompensation. We report two patients with chronic hepatitis C virus infection who experienced a severe increase of bilirubin levels of up to 17 times upper the limit of normal value in the absence of deterioration of hepatic function during therapy with pegylated-interferon and ribavirin. A genetic disposition for Gilbert’s syndrome explained the adverse events and permitted a continuation of therapy leading to a sustained clearance of chronic hepatitis C infection. Since one patient jaundiced already during a lead-in treatment period with ribavirin monotherapy we suggest that hyperbilirubinaemia during combination therapy is primarily caused by ribavirin rather than by effects of interferon alpha on UDP-glucuronosyltransferase activities. Of note, both patients recovered from their initial unconjugated hyperbilirubinemia despite continuation of ribavirin therapy, which indicates that compensatory mechanisms leading to a normalization of UGT1A1 activity are likely.

Background & Aims: Hepatitis C virus (HCV) cell entry is mediated by several cell surface receptors, including scavenger receptor class B type I (SR-BI). Oxidized low density lipoprotein (oxLDL) inhibits the interaction between HCV and SR-BI in a noncompetitive manner. We tested whether serum oxLDL levels correlate with sustained virologic response (SVR) rates after interferon-based treatment of chronic hepatitis C. Methods: Baseline oxLDL was determined in 379 participants with chronic HCV genotype 1 infection from the INDIV-2 study using a commercial enzyme-linked immunosorbent assay. The mechanistic in vitro studies used full-length and subgenomic HCV genomes replicating in hepatoma cells. Results: In the multivariate analysis, oxLDL was found to be an independent predictor of SVR. Oxidized LDL did not correlate with markers of inflammation (alanine transaminase, ferritin), nor was serum oxLDL affected by exogenous interferon administration. Also, oxLDL did not alter the sensitivity of HCV replication to interferon. However, oxLDL was found to be a potent inhibitor of cell-to-cell spread of HCV between adjacent cells in vitro. It could thus reduce the rate at which new cells are infected by HCV through either the cell-free or cell-to-cell route. Finally, serum oxLDL was significantly associated with the estimated infected cell loss rate under treatment. Conclusions: Oxidized LDL is a novel predictor of SVR after interferon-based therapy and may explain the previously observed association of LDL with SVR. Rather than being a marker of activated antiviral defenses it may improve chances of SVR by limiting spread of infection to naive cells through the cell-to-cell route. Keywords: Cell-to-Cell Spread, oxLDL, SVR, SR-BI

A major challenge in the field of hepatology is the fight against hepatitis C that affects more than 150 million people world-wide. Despite the enormous improvement that has been achieved in the therapy of chronic hepatitis C over the last decade, there is an urgent medical need for new therapeutic approaches. This review focuses on the optimization of the current standard therapy of hepatitis C and future treatment directions beyond pegylated interferon alpha and ribavirin.

Background and aims: Antimitochondrial antibodies (AMA) which recognize pyruvate acetyltransferase (PDC-E2) represent a highly diagnostic feature of primary biliary cirrhosis (PBC). The analysis of immunofluorescence (IF)-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2) alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay.

Acute liver failure is characterized by a sudden and severe deterioration of liver function, typically mirrored by a marked increase of INR and hepatic encephalopathy. Due to various possible causes hepatocytes get damaged via either apoptotic or necrotic pathways. Anticipating the natural prognosis of a patient with acute liver failure is one of the most challenging tasks in hepatology critical care. Important factors that influence the chance of spontaneous recovery are the underlying etiology of acute liver failure, the acuity of disease, and the severity of hepatic encephalopathy. Once an estimation of the prognosis in the individual patient has been made, this quickly has to be integrated in the discussion whether high-urgency liver transplantation is necessary and justifiable. This decision has to cover several medical, social and organisational issues. Well organized liver transplantation programs around the world have achieved an impressive improvement of the one-year survival rate in acute liver failure from around 40% without transplantation up to nearly 80% with transplantation. The recent debate on whether severe acute alcoholic hepatitis could represent a new candidate eligible for high-urgency liver transplantation shows that the topic is still open for discussion.

Background. Transjugular liver biopsy (TJLB) is frequently used to obtain liver specimens in high-risk patients. However, TJLB sample size possibly limits their clinical relevance. Methods. 102 patients that underwent TJLB were included. Clinical parameters and outcome of TJLB were analyzed. Control samples consisted of 112 minilaparoscopic liver biopsies (mLLBs) and 100 percutaneous liver biopsies (PLBs). Results. Fewer portal tracts were detected in TJLB (4.3±0.3) in comparison with PLB (11.7±0.5) and mLLB (11.0±0.6). No difference regarding the specification of indeterminate liver disease and staging/grading of chronic hepatitis was observed. In acute liver failure (n=32), a proportion of hepatocellular necrosis beyond 25% was associated with a higher rate of death or liver transplantation. Conclusions. Despite smaller biopsy samples the impact on the clinical decision process was found to be comparable to PLB and mLLB. TJLB represents a helpful tool to determine hepatocellular necrosis rates in patients with acute liver failure.

Directed endodermal differentiation of murine embryonic stem (ES) cells gives rise to a subset of cells with a hepatic phenotype. Such ES cell-derived hepatic progenitor cells (ES-HPC) can acquire features of hepatocytes in vitro, but fail to form substantial hepatocyte clusters in vivo. In this study, we investigated whether this is due to inefficient engraftment or an immature phenotype of ES-HPC. ES cells engrafted into recipient livers of NOD/SCID mice with a similar efficacy as adult hepatocytes after 28 days. Because transplanted unpurified ES-HPC formed teratomas in the spleen and liver, we applied an albumin promoter/enhancer-driven reporter system to purify ES-HPC by cell sorting. RT-PCR analyses for hepatocyte-specific genes showed that the cells exhibited a hepatic phenotype, lacking the expression of the pluripotency marker Oct4, comparable to cells of day 11.5 embryos. Sorted ES-HPC derived from β-galactosidase transgenic ES cells were injected into fumaryl-acetoacetate-deficient (FAH-/-) SCID mice and analyzed after 8 to 12 weeks. Staining with X-gal solution revealed the presence of engrafted cells throughout the liver. However, immunostaining for the FAH protein indicated hepatocyte formation at a very low frequency, without evidence for large hepatocyte cluster formation. In conclusion, the limited repopulation capacity of ES-HPC is not caused by a failure of primary engraftment, but may be due to an immature hepatic phenotype of the transplanted ES-HPC.

During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 × 109 cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 × 106 total and 2.6 × 106 viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.

We have investigated the contribution of intrasplenic bone marrow transplants or in vivo mobilized hematopoietic stem cells to the formation of hepatocytes in normal and injured liver. Direct intrasplenic injections of bone marrow mononuclear cells (5 × 10 cells), Sca1+/lin- hematopoietic stem cells (5 × 103) cells, and highly purified “side population” hematopoietic stem cells (5 × 103) derived from enhanced green fluorescent protein (EGFP)-transgenic mice [C57Bl/6-TgN(ActbEGFP)1Osb] were performed in normal C57Bl/6 mice (n = 6) and in C57Bl/6 mice following two thirds hepatectomy (n = 8). To test the effect of mobilized stem cells on transdifferentiation, C57Bl/6 mice (n = 12) were lethally irradiated and reconstituted with EGFP-positive bone marrow mononuclear cells in a second series of experiments. Eight to 12 weeks after bone marrow transplantation a subset of mice (n = 3 in each group) received either rhG-CSF for hematopoietic stem cell mobilization, rhG-CSF combined with an intraperitoneal application of carbon tetrachloride (CCl 4 ) as hepatocyte regeneration stimulus, or CCl 4 alone. All mice were completely perfused with PBS to remove circulating nonorgan cells for analyses 4 weeks later. Liver as well as heart, intestine, spleen, and kidney tissue was analyzed for the presence of EGFP-transgenic cells. In 100 sections (2.3 × 107 cells) of any recipient mouse no EGFP-positive hepatocytes were detected either by analysis of native EGFP fluorescence or by immunofluorescence analysis with anti-EGFP and antidipeptidyl peptidase (DPP) IV antibodies. EGFP-transgenic cells resembling heart, kidney, or intestinal cells could also not be proven. The results demonstrate that there is little or no contribution of bone marrow-derived cells to the regeneration of normal and injured liver in the animal models used. Thus, potential therapeutic prospects of hematopoietic stem cell therapy for liver disease have to be critically reassessed.