Neuronal branching of sensory neurons is associated with BDNF-positive eosinophils in atopic dermatitis.

BACKGROUND: Pruritus is a major symptom of atopic dermatitis (AD) and is transmitted by a subpopulation of non-myelinated C-type free nerve endings in the epidermis and upper dermis. Stimulation of these nerve terminals is affected by histamine, neurotrophins and physical factors. Eosinophils of patients with AD are a source of neurotrophins, including brain-derived neurotrophic factor (BDNF), levels of which correlate with disease severity. OBJECTIVE: The purpose of this study was to determine the anatomical localization of eosinophils in the skin of patients with AD with regard to peripheral nerves and to investigate whether eosinophils induce sprouting and neurite outgrowth in murine sensory neurons. METHODS: Cryosections of skin derived from AD and control (NA) patients were subjected to immunofluorescence analysis with markers for eosinophils, BDNF and neuronal cells. Stimulated eosinophil supernatants were used for the treatment of cultured peripheral mouse dorsal root ganglia (DRG) neurons followed by morphometric analysis. RESULTS: Dermal axon density and the proximity of eosinophils to nerve fibres were significantly higher in AD patients vs NA. Both neuronal projections and eosinophils expressed BDNF. Furthermore, activated eosinophil supernatants induced BDNF-dependent mouse DRG neuron branching. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that BDNF-positive eosinophils are also localized in close proximity with nerve fibres in AD, suggesting a functional relationship between BDNF-expressing eosinophils and neuronal projections. These observations suggest that eosinophils may have considerable impact on pruritus by supporting sensory nerve branching.

Pre-osteoblast cell colonization of porous silicon substituted hydroxyapatite bioceramics: Influence of microporosity and macropore design.

Silicate-substituted hydroxyapatite scaffolds containing multiscale porosity are manufactured. Model parts containing macropores of five cross-sectional geometries (circle, square, rhombus, star and triangle) and two sizes are shaped by microstereolithography. Three open microporosity contents (0.5, 23 or 37 vol%) are introduced in the ceramic. MC3T3-E1 pre-osteoblasts are seeded onto these scaffolds. Analysis of cell colonization inside the macropores after 7 and 14 days of cultivation shows that the cellular filling is proportional to the macropore size and strongly influenced by macropore shape. Straight edges and convex surfaces are detrimental. High aspect ratios, the absence of reentrant angles and the presence of acute angles, by creating concavities and minimizing flat surfaces, facilitate cell colonization. Rhombus and triangle cross-sections are thus particularly favorable, while square and star geometries are the least favored. An increase in the microporosity content strongly impairs cell growth in the macropores. The data are statistically analyzed using a principal components analysis that shows that macro- and microtopographical parameters of scaffolds must be collectively considered with correlated interactions to understand cell behavior. The results indicate the important cell sensing of topography during the initial step of cell adhesion and proliferation and evidence the need for an optimized scaffold design.

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.

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.

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

UNLABELLED: 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/γ(c) -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.

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.

Gene expression analysis identifies novel genes participating in early murine liver development and adult liver regeneration.

Adult liver tissue regeneration may recapitulate molecular events of liver organogenesis. As gaps in our understanding of the fundamental processes that govern development and regeneration of the liver still exist, we studied gene expression in the developing liver at embryonic day 9.5 post coitum (E d9.5 p.c.). Microarray data from E d9.5 p.c. as well as previously published data from embryonic day 11.5 post coitum (E d11.5 p.c.) and embryonic day 13.5 post coitum (E d13.5 p.c.) were subjected to cluster analysis. This led to the identification of 130 genes which were characterized by continuous expression at all stages of liver development with peak expression of 44 genes at E d9.5 p.c. Five of these genes, previously not known to be associated with early liver development or with adult liver regeneration were selected for further analysis. The expression of the genes was studied by real-time polymerase chain reaction at 0, 2, 4, 6, 12, 24 and 48 hr after partial hepatectomy in the adult liver. Two of the genes, growth arrest protein 43 (GAP43) and paired-like homeodomain transcription factor 2 (Pitx2) were exclusively detected at 24 hr, whereas the genes Twist1, Midkine, and zinc finger protein of cerebellum 1 (Zic1) each showed a specific expression profile in the regenerating liver with peak expressions at 4, 24, and 6 hr, respectively. In summary, we were able to identify novel genes, that may act as regulators during liver formation as well as in the regeneration phase of adult liver. This information may contribute to the development of new targets for the treatment of liver diseases in the future.