Dirk Nierhoff

Identification of Plateled-derived Growth Factor-BB as Cardiogenesis-Inducing Factor in Mouse Embryonic stem cells under Serum-free Conditions

Background/Aims: Embryonic stem (ES) cells may represent an alternative source of functionally mature cardiomyocytes for the treatment of heart diseases. ES cells spontaneously differentiate into spheroidal aggregates, also referred to as embryoid bodies (EBs). The identification of growth factors playing a decisive role in cardiogenesis is a crucial issue for the generation of mature cardiomyocytes. Methods: In order to identify growth factors promoting cardiac development, we established a new differentiation protocol using a defined serum-replacement medium (SRM) containing 5µg/ml insulin and 5µg/ml transferrin in combination with Dulbecco’s Modified Eagle Medium (DMEM). Furthermore, we added platelet-derived growth factor-BB (PDGF-BB) or sphingosine-1-phosphate (SPP) to promote cardiac differentiation. Results: Using SRM/DMEM, we obtained a 6-fold increase of cardiac specific myosin heavy chain α and β (cMHCα/β) in relation to 0,2% foetal calf serum (FCS)/DMEM (= 100%). Stimulation of EBs with PDGF-BB in the presence of SRM/DMEM resulted in a further 2,6-fold enhancement in comparison with the SRM/DMEM-induced increase of cMHCα/β (= 100%). A parallel increase in the number of beating EBs was observed. Similar results were obtained after stimulation of EBs with 5µg/ml SPP. Conclusion: We established a serum-free protocol and identify PDGF-BB and SPP as potent factors promoting cardiogenesis in ES cells.

NIKEI: a new inexpensive and non-invasive scoring system to exclude advanced fibrosis in patients with NAFLD.

Aims To develop, validate and compare a non-invasive fibrosis scoring system for non-alcoholic fatty liver disease (NAFLD) derived from routinely obtained clinical and biochemical parameters. Methods 267 consecutive patients with biopsy proven fatty liver or non-alcoholic steatohepatitis were randomly assigned to the estimation (2/3) or validation (1/3) group to develop a model for the prediction of advanced fibrosis. Univariate statistics were performed to compare patients with and without advanced fibrosis, and following a multivariate logistic regression analysis a new scoring system was constructed. This non-invasive Koeln-Essen-index (NIKEI) was validated and compared to the FIB-4 index by calculating the area under the receiver operating characteristic curve (AUC). We evaluated a stepwise combination of both scoring systems for the precise prediction of advanced fibrosis. To set in contrast, we additionally tested the diagnostic accuracy of the AST/ALT ratio, BARD score and the NAFLD fibrosis score in our cohort. Results Age, AST, AST/ALT ratio, and total bilirubin were identified as significant predictors of advanced fibrosis and used to construct the NIKEI with an AUC of 0.968 [0.937; 0.998] compared to 0.929 [0.869; 0.989] for the FIB-4 index. The absence of advanced fibrosis could be confirmed with excellent accuracy (99–100%). The positive predictive value of the FIB-4 index was higher (100% vs. 60%), however, the false negative rate was also high (33%). With a stepwise combination of both indices 82%–84% of biopsies would have been avoidable without a single misclassification. The AUROC for AST/ALT ratio, the NAFLD fibrosis score, and the BARD score were 0.81 (95% CI, 0.72–0.90), 0.96 (95% CI 0.92–0.99), and 0.67 (95% CI 0.55–0.78), respectively. Conclusion The NIKEI can reliably exclude advanced fibrosis in subjects with NAFLD. In combination with the FIB-4 index misclassification with inadequate clinical management can be avoided while the need for liver biopsies can be reduced.

Stepwise combination of simple noninvasive fibrosis scoring systems increases diagnostic accuracy in nonalcoholic fatty liver disease.

Objective: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease ranging from simple fatty liver to steatohepatitis, fibrosis, and cirrhosis. We aimed to analyze the diagnostic performance and clinical utility of simple noninvasive tests alone or in combination for the detection of advanced fibrosis in patients with NAFLD. Design and Subjects: Data from 323 patients with biopsy-proven NAFLD/NASH who presented to the Clinic for Gastroenterology and Hepatology, University Hospital of Cologne between July 1998 and November 2009, were analyzed retrospectively. Sensitivity, specificity, positive predictive values, and negative predictive values were determined along with the area under receiver operating characteristic curves (AUROC) using published formulas for NAFLD, FIB-4, and BARD fibrosis scores. Results: The area under receiver operating characteristic curves were as follows: NAFLD fibrosis score 0.96 [95% confidence interval (CI), 0.92-0.99], FIB-4 0.95 (95% CI, 0.91-1.00), BARD 0.82 (95% CI, 0.71-0.92) with negative predictive values for advanced fibrosis of 96%, 98%, and 96%, respectively. When applying the NAFLD, FIB-4, or BARD scoring systems 25%, 15%, or 26% of cases with advanced fibrosis would have been missed. Combining FIB-4 and BARD in a stepwise fashion, patients would have been correctly classified without biopsy in 67% of cases without missing a single case of advanced fibrosis. Conclusions: The FIB-4 and NAFLD fibrosis scores perform better than the BARD scoring system. Liver biopsy can securely be replaced only with a stepwise combination of simple noninvasive tests, otherwise the assessment of risk due to advanced fibrosis may be misleading in a clinically meaningful proportion of patients.

Hepatitis B virus infection enhances susceptibility toward adeno‐associated viral vector transduction in vitro and in vivo

Gene therapy has become an accepted concept for the treatment of a variety of different diseases. In contrast to preclinical models, subjects enrolled in clinical trials, including gene therapy, possess a history of infection with microbes that may influence its safety and efficacy. Especially, viruses that establish chronic infections in the liver, one of the main targets for in vivo gene therapy, raise important concerns. Among them is the hepatitis B virus (HBV), which has chronically infected more than 350 million people worldwide. Here, we investigated the effect of HBV on adeno‐associated viral (AAV) vectors, the most frequently applied gene transfer vehicles for in vivo gene therapy. Unexpectedly, we found that HBV greatly improved AAV transduction in cells replicating HBV and identified HBV protein x (HBx) as a key factor. Whereas HBV‐positive and ‐negative cells were indistinguishable with respect to cell‐entry efficiency, significantly higher numbers of AAV vector genomes were successfully delivered to the nucleus in the presence of HBV. The HBV‐promoting effect was abolished by inhibitors of phosphatidylinositol 3‐kinase (PI3K). PI3K was required for efficient trafficking of AAV to the nucleus and was enhanced in HBV‐replicating cells and upon HBx expression. Enhancement of AAV transduction was confirmed in vivo using HBV transgenic mice and could successfully be applied to inhibit HBV progeny release. Conclusion: Our results demonstrate that acute, as well as chronic, infections with unrelated viruses change the intracellular milieu, thereby likely influencing gene therapy outcomes. In the case of HBV, HBx‐mediated enhancement of AAV transduction is an advantage that could be exploited for development of novel treatments of HBV infection. (Hepatology 2014;59:2110‐2120)

Hepatocyte expression of angiotensin II type 1 receptor is downregulated in advanced human liver fibrosis.

Background: The renin–angiotensin system plays an important role in fibrosis. Angiotensin II regulates key steps in tissue remodelling processes through angiotensin II type 1 receptor (AT1R). In bile duct‐occluded rats, AT1R expression is significantly decreased in advanced liver fibrosis. Therefore, we studied the AT1R expression in human liver tissue during different stages of fibrosis caused by chronic hepatitis C.

Neighbor of Punc E 11: expression pattern of the new hepatic stem/progenitor cell marker during murine liver development.

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for “live” monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

Neighbor of punc E11, a novel oncofetal marker for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the 5th common malignancy worldwide, but established markers fail to detect up to one third of HCC. We have recently identified Neighbor of Punc E11 (Nope) as a surface marker for murine fetal liver stem cells. Similar to commonly used HCC markers such as α‐Fetoprotein (Afp) and Glypican‐3 (Gpc‐3), we here establish Nope as an oncofetal marker of murine and human HCC and investigate its specific expression in hepatoma cell lines and primary HCC. Murine and human hepatoma cell lines and Cre‐inducible SV40 T‐antigen transgenic mice (Alb‐SV40TAgind) were analyzed for Nope expression in comparison to common HCC markers by quantitative RT‐PCR, Western blot analyses and immunohistochemistry. Nope expression in primary human HCC was investigated using Oncomine Microarray database. Nope expression was elevated in 8 of 10 investigated murine and human hepatoma cell lines and in all tumors of our oncogenic mouse model but remained undetectable in normal liver and at preneoplastic stages of murine hepatocarcinogenesis. Furthermore, a significant induction of Nope was detected in primary human cancers compared to corresponding normal or cirrhotic tissue. Nope expression in tumor specimens and murine cell lines correlated closely with expression levels of Gpc‐3, whereas expression levels of Afp showed high variations. In conclusion, we identified Nope as a novel oncofetal surface marker for murine and human HCC. Nope is specifically expressed by epithelial tumor cells but not in preneoplastic stages and is a promising marker for clinical application because of its high detection rate in Afp‐positive and Afp‐negative tumors.

Insufficient evidence of benefit regarding mortality due to albumin substitution in HCC-free cirrhotic patients undergoing large volume paracentesis.

Current guidelines for clinical practice recommend the infusion of human albumin after large volume paracentesis. After inspecting the current evidence behind this recommendation, we decided to conduct a systematic review and meta‐analysis in order to address the effect of albumin on mortality and morbidity in the context of large volume paracentesis.

A Novel Easy-to-Use Prediction Scheme for Upper Gastrointestinal Bleeding: Cologne-WATCH (C-WATCH) Risk Score.

AbstractAcute upper gastrointestinal bleeding (UGIB) is the leading indication for emergency endoscopy. Scoring schemes have been developed for immediate risk stratification. However, most of these scores include endoscopic findings and are based on data from patients with nonvariceal bleeding. The aim of our study was to design a pre-endoscopic score for acute UGIB—including variceal bleeding—in order to identify high-risk patients requiring urgent clinical management.The scoring system was developed using a data set consisting of 586 patients with acute UGIB. These patients were identified from the emergency department as well as all inpatient services at the University Hospital of Cologne within a 2-year period (01/2007–12/2008). Further data from a cohort of 322 patients who presented to our endoscopy unit with acute UGIB in 2009 served for external/temporal validation.Clinical, laboratory, and endoscopic parameters, as well as further data on medical history and medication were retrospectively collected from the electronic clinical documentation system.A multivariable logistic regression was fitted to the development set to obtain a risk score using recurrent bleeding, need for intervention (angiography, surgery), or death within 30 days as a composite endpoint. Finally, the obtained risk score was evaluated on the validation set.Only C-reactive protein, white blood cells, alanine-aminotransferase, thrombocytes, creatinine, and hemoglobin were identified as significant predictors for the composite endpoint. Based on the regression coefficients of these variables, an easy-to-use point scoring scheme (C-WATCH) was derived to estimate the risk of complications from 3% to 86% with an area under the curve (AUC) of 0.723 in the development set and 0.704 in the validation set. In the validation set, no patient in the identified low-risk group (0–1 points), but 38.7% of patients in the high-risk group (≥ 2 points) reached the composite endpoint.Our easy-to-use scoring scheme is able to distinguish high-risk patients requiring urgent endoscopy, from low-risk cases who are suitable candidates for outpatient management or in whom endoscopy may be postponed. Based on our findings, a prospective validation of the C-WATCH score in different patient populations outside the university hospital setting seems warranted.