Most cells of the connective tissue have to cope with sizeable mechanical strains which also serve as cues to initiate cell responses like cell shape changes, cell reorientation and rearrangement of the cellular cytoskeleton. In our experiments we cultivated human umbilical cord fibroblasts in elastic chambers and exposed them to cyclic external strain of various amplitudes. We characterized both cell morphology and cytoskeletal structure simultaneously after various durations of straining by immunofluorescence microscopy. Digital image processing was employed to achieve high accuracy and high statistical significance of the results due to large numbers of evaluated cells. Moreover, regular microstructures micromolded into the elastomeric cell culture chamber were exploited to reliably quantify the amount of strain experienced by individual cells. Measured angular distributions of cell cytoskeleton orientations are in agreement with theoretical predictions in which a steady state is determined by a set point condition. The observed dependence of the results on the effective Poisson's ratio of the stretching chamber indicates that these cells regulate their mechanical homeostasis to control strain and not stress. Intriguingly, we observed a temporal delay between cytoskeleton and cell morphology reorientation with morphology lagging behind cytoskeleton orientation. First results on simultaneous molecular processes in focal adhesion complexes and cytoskeleton will be discussed.

With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappa B) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1 beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappa B controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1 alpha and HNF 4 alpha, two transcription factors essential for HBV gene expression and replication. Conclusion: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected. (HEPATOLOGY 2009;50:1773-1782.)

Hepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte-specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte-enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4alpha but not its potential cofactors or other hepatocyte-enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1alpha contributed to the control of HBV replication because it regulated the expression of HNF4alpha. Thus, a concerted action of HNF4alpha and HNF1alpha, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation.

4-hydroxyphenylpyruvate dioxygenase (HPD) (EC 1.13.11.27) is a key enzyme involved in tyrosine catabolism. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. The severe type I tyrosinemia, caused by a deficiency of fumarylacetoacetate hydrolase which functions downstream of HPD in the tyrosine degradation pathway, is often associated with decreased expression of HPD, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. The HPD gene was previously mapped to the chromosomal region 12q24→qter. In the present study high-resolution chromosome mapping localized the HPD gene to 12q24.31. DNase I footprinting, revealed that four regions of the HPD promoter were protected by rat liver nuclear proteins. Computer-assisted analyses suggested that these elements might bind Sp1/AP2, HNF4, HNF3/CREB, and C/EBP, respectively. In transient transfection experiments, the proximal 271 bp of the promoter conferred basal transcriptional activation in human Chang cells. Sequences in intron 1 were able to enhance the activity of this basal promoter. Finally, vaccinia virus-based expression provided evidence that HPD is subject to phosphorylation, and furthermore, allowed mapping of the HPD protein in the human keratinocyte 2D database.

Dendritic cells (DC) of hepatitis B virus (HBV) carriers have been reported to exhibit functional impairment. Possible explanations for this phenomenon are infection of HBV by DC or alteration of DC function by HBV. We therefore analyzed whether DC support the different steps of HBV infection and replication: uptake, deposition of the HBV genome in the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte-derived DC by adenoviral vectors, low-level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When monocyte-derived DC were subjected to wild-type HBV or a recombinant HBV expressing Renilla luciferase under a non-liver-specific promoter, intracellular HBV DNA was detected in a low percentage of cells. However, neither nuclear cccDNA was formed nor luciferase activity was detected, indicating that either uncoating or nucleocytoplasmic transport were blocked. To verify our observation in the in vivo situation, myeloid and plasmacytoid DC were isolated from blood of high viremic HBV carriers, and analyzed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DC had in vivo been exposed to more than 10(4) HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable as were viral capsids in the cytoplasm. In conclusion, circulating DC may take up HBV antigens, but neither support nucleocytoplasmic transport nor replication of HBV.

Aims: Hepatitis B virus (HBV) replication is restricted to hepatocytes. But not all hepatocytes containing HBV genomes support viral replication. We studied the cellular differentiation state and expression levels of liver specific transcription factors essential for efficient HBV replication. Freshly isolated primary human hepatocytes (PHH) were compared with hepatoma cell lines HepG2 and HuH7 and the hepatocyte cell line pop10, replicating HBV at high, medium and low levels, respectively.

In letzter Zeit wurde klarer, unter welchen Bedingungen sich hämatopoetische Stammzellen zu hepatozytenähnlichen Zellen ausdifferenzieren können. Hämatopoetische Zellen könnten z.B. als Reservoir für eine Reinfektion mit Hepatitis B Virus (HBV) nach Lebertransplantation dienen. Die zellulären Faktoren, die für eine Infektion mit HBV verantwortlich sind, sind bisher nur schlecht definiert. HBV kann sich sowohl in primären humanen Hepatozyten (PHH) als auch in Hepatozyten- (POP10) und Hepatom-Zellinien (HepG2, HuH7, Hep3B) replizieren, während eine Infektion mit diesem Virus nur in PHH möglich ist. Aus initialen Experimenten wissen wir, dass ein relativ hoher Grad der Differenzierung der Zelle für eine effektive Replikation des HBV notwendig ist. Für den Viruseintritt, der die Infizierbarkeit einer Zelle determiniert, ist das bisher nicht untersucht.