Urda Rüdrich

Induction of a mature hepatocyte phenotype in adult liver derived progenitor cells by ectopic expression of transcription factors

BACKGROUND/AIMS By ectopic expression of a distinct combination of transcription factors we aimed to induce a mature hepatocyte phenotype in an adult liver derived progenitor cell population (ALDPC). METHODS The open reading frames encoding murine Foxa2, Hnf4α and C/ebpα were cloned into lentivirus vectors and sequentially expressed in target cells. After seven days of culture, cells were analysed for expression of liver specific genes, and functional assays were performed. Fresh primary hepatocytes, twenty four hours in culture, served as positive controls. RESULTS Untransduced ALDPC under established differentiation conditions exhibited moderate signs of maturation, in particular in comparison with fresh hepatocyte controls. In transcription factor transduced cells, fifteen mRNA´s coding for secreted proteins, cytochrome p450 isoenzymes, liver metabolic enzymes were detected by RT-qPCR at levels close to controls. Albumin secretion increased incrementally in single (Foxa2), double (Foxa2, Hnf4α) and triple-transduced cells (Foxa2, Hnf4α, C/ebpα) and reached levels observed in primary hepatocytes. Glycogen storage as determined by PAS staining was detectable in double and triple transduced cells, comparable to controls. Ureagenesis was also induced in triple transduced cells, but at lower levels compared to primary hepatocytes. CONCLUSIONS Sequential expression of Foxa2, Hnf4α and C/ebpα induces a mature hepatocyte phenotype in an expandable liver derived progenitor cell line.

Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice

Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species‐specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47‐mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47‐negative counterparts. For the generation of human‐mouse chimeric livers in immunodeficient BALB‐ΔRAG/γc‐uPA (urokinase‐type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47‐positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. Conclusion: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization. (HEPATOLOGY 2012)

Human Basophils are Differentially Activated by and are a Source of IL-31

Basophils are important effector cells involved in the pathogenesis of inflammatory skin diseases including chronic urticaria which is associated by increased IL‐31 serum levels. So far the effects of IL‐31 on human basophils are unknown.

Substance P activates human eosinophils.

Substance P activates human eosinophils Mieke Raap, Urda R€ udrich, Sonja St€ ander, Manuela Gehring, Alexander Kapp and Ulrike Raap Department of Dermatology and Allergy, Hannover Medical School, Hannover, Germany; Department of Dermatology, University Hospital of M€ unster, Hannover, Germany Correspondence: Prof Dr med. Ulrike Raap, Department of Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany, Tel.: 49-511-532-7675, Fax: 49-511-532-18854, e-mail: raap.ulrike@mh-hannover.de

Increased Activity and Apoptosis of Eosinophils in Blister Fluids, Skin and Peripheral Blood of Patients with Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease that is more common in elderly individuals. The aim of this study was to determine the functional activity of eosinophils in patients with BP compared with healthy donors. Blood, skin and blister-derived eosinophils were strongly activated in patients with BP, seen by increased surface expression of CD69 compared with controls. CD11b was also increased in BP blood eosinophils, which may explain the striking accumulation of eosinophils in BP (1×106 per ml blister fluid). Furthermore, CCL26 was expressed by activated eosinophils in BP skin and in blister fluid. BP eosinophils also released IL-6, IL-8 and IL-1α in BP blister fluids. Apoptosis in cultivated BP eosinophils was increased and accompanied by enhanced surface externalization of CD95. Caspase 3 positive eosinophils in lesional BP skin and blister fluid also showed the initiation of apoptosis. These results reveal novel pathophysiological aspects of BP, with a strong activation pattern and increased apoptosis of eosinophils in the peripheral blood, skin and blister fluids.

An Extended ΔCT-Method Facilitating Normalisation with Multiple Reference Genes Suited for Quantitative RT-PCR Analyses of Human Hepatocyte-Like Cells

Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of in vitro cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies PSMB6, MDH1 and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells in vitro. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG - here identified suited for human hepatocyte-like cells - for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.

Childhood atopic dermatitis—Brain-derived neurotrophic factor correlates with serum eosinophil cationic protein and disease severity

Several studies have shown that neurotrophins including brain‐derived neurotrophic factor (BDNF) play a role in chronic inflammatory skin diseases such as atopic dermatitis (AD). BDNF is increased in the serum samples of adults with AD. Interestingly, eosinophils of these patients can release and produce BDNF. We analyzed BDNF serum levels with ELISA and their correlation with SCORAD score, eosinophil cationic protein (ECP), total IgE, IL‐4, IL‐13 and IL‐31 in children with AD (n = 56) compared to nonatopic healthy children (n = 25). In addition, we analyzed FLG loss‐of‐function mutations in 17 children with AD and their connection to BDNF. BDNF serum levels were significantly higher in children with AD. Further, BDNF correlated with disease activity, serum ECP, and total IgE serum levels in AD. There was no difference in BDNF levels of filaggrin‐positive or filaggrin‐negative children with AD, and there was no correlation of BDNF with IL‐31 and Th2 cytokines including IL‐4 and IL‐13. Together, our data add new insights into the pathophysiology of AD, suggesting that serum BDNF which correlates with disease severity contributes to the regulation of inflammation in an eosinophil‐, but not Th2‐dependent manner.

Eosinophils are a Major Source of Interleukin-31 in Bullous Pemphigoid

Bullous pemphigoid (BP) is characterized by substantial skin and blood eosinophilia as well as intensive pruritus. Since the pruritogenic cytokine interleukin (IL)-31 is increased in inflammatory skin diseases the aim of this study was to determine whether IL-31 plays a role in BP. Using immunofluorescence, IL-31 expression was analysed in eosinophils derived from blister fluids and skin from patients with BP and IL-31 levels in blister fluids, serum and culture supernatants were determined by enzyme-linked immunoassay (ELISA). High levels of IL-31 expression were observed in BP blister fluids, but they were only marginally elevated in BP serum compared with healthy controls. Eosinophils from either BP blister fluids or skin biopsies showed strong expression of IL-31. Furthermore, peripheral blood eosinophils from patients with BP, but not healthy controls, released high levels of IL-31, reflecting those in blister fluids. In conclusion, eosinophils are a major source of IL-31 in BP and this cytokine may contribute to itch in patients with BP.

Regulation of melanocortin 1 receptor in allergic rhinitis in vitro and in vivo

α‐melanocyte‐stimulating hormone (α‐MSH) was shown to inhibit allergic airway inflammation and exert suppressive effects on human basophils.