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Unlabelled: 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., (© 2014 by the American Association for the Study of Liver Diseases.)

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.

Unlabelled: Isolation of hepatic stem cells from the adult liver (AL) has not yet been achieved due to the lack of specific cell surface markers. To identify new surface markers for hepatic stem cells, we analyzed differences in the gene expression profile of embryonic day (ED) 13.5 fetal liver stem/progenitor cells (FLSPC) versus AL by complementary DNA (cDNA) microarray technology. Using FLSPC purified to >90% by immunomagnetic selection for E-cadherin and high density (27k) mouse cDNA microarrays, we identified 474 genes that are more strongly expressed in FLSPC (FLSPC-up genes) and 818 genes that are more strongly expressed in AL (AL-up genes). The most highly overrepresented gene ontology (GO) categories for FLSPC-up genes are nucleus, cellular proliferation, and cell cycle control. AL-up genes are overrepresented for genes in metabolic pathways for specific hepatic functions. We identified 24 FLSPC-up gene surface markers and 69 AL-up gene surface markers. Western blot studies confirmed the expression of the FLSPC-up gene neighbor of Punc E11 (Nope) in fetal liver, but expression was not detectable in AL. Immunohistochemistry, confocal microscopy, and fluorescence-activated cell sorting (FACS) analysis of fetal liver demonstrated that Nope is specifically expressed on the surface of FLSPC within the fetal liver., Conclusion: This is the first microarray study to analyze the specific gene expression profile of purified murine FLSPC. Our analysis identified 24 new/potential cell surface markers for murine fetal hepatic stem cells, of which Nope may be particularly useful in future studies to identify, characterize and isolate hepatic stem cells from the AL.

Although it was proposed almost 60 years ago that the adult mammalian liver contains hepatic stem cells, this issue remains controversial. Part of the problem is that no specific marker gene unique to the adult hepatic stem cell has yet been identified, and regeneration of the liver after acute injury is achieved through proliferation of adult hepatocytes and does not require activation or proliferation of stem cells. Also, there are differences in the expected properties of stem versus progenitor cells, and we attempt to use specific criteria to distinguish between these cell types. We review the evidence for each of these cell types in the adult versus embryonic/fetal liver, where tissue-specific stem cells are known to exist and to be involved in organ development. This review is limited to studies directed toward identification of hepatic epithelial stem cells and does not address the controversial issue of whether stem cells derived from the bone marrow have hepatocytic potential, a topic that has been covered extensively in other recent reviews.

Epithelial cells in embryonic day (ED) 12.5 murine fetal liver were separated from hematopoietic cell populations using fluorescence-activated cell sorting (FACS) and were characterized by immunocytochemistry using a broad set of antibodies specific for epithelial cells (alpha-fetoprotein [AFP], albumin [ALB], pancytokeratin [PanCK], Liv2, E-cadherin, Dlk), hematopoietic/endothelial cells (Ter119, CD45, CD31), and stem/progenitor cells (c-Kit, CD34, Sca-1). AFP(+)/ALB(+) cells represented approximately 2.5% of total cells and were positive for the epithelial-specific surface markers Liv2, E-cadherin, and Dlk, but were clearly separated and distinct from hematopoietic cells (Ter119(+)/CD45(+)). Fetal liver epithelial cells (AFP(+)/E-cadherin(+)) were Sca-1(+) but showed no expression of hematopoietic stem cell markers c-Kit and CD34. These cells were enriched by FACS sorting for E-cadherin to a purity of 95% as defined by co-expression of AFP and PanCK. Purified fetal liver epithelial cells formed clusters in cell culture and differentiated along the hepatocytic lineage in the presence of dexamethasone, expressing glucose-6-phosphatase (G6P) and tyrosine amino transferase. Wild-type ED12.5 murine fetal liver cells were transplanted into adult dipeptidyl peptidase IV knockout mice and differentiated into mature hepatocytes expressing ALB, G6P, and glycogen, indicating normal biochemical function. Transplanted cells became fully incorporated into the hepatic parenchymal cords and showed up to 80% liver repopulation at 2 to 6 months after cell transplantation. In conclusion, we isolated and highly purified a population of epithelial cells from the ED12.5 mouse fetal liver that are clearly separate from hematopoietic cells and differentiate into mature, functional hepatocytes in vivo with the capacity for efficient liver repopulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).