E Van de Leur

Comparative analysis of adenoviral transgene delivery via tail or portal vein into rat liver.

Summary.The potential indications for gene therapy are expanding continuously. Currently, hepatotropic adenoviruses are useful vector systems for targeting liver in experimental animal models. Although this gene delivery technique is widely distributed, there is no common sense about how these viruses should be applied. In general, the local delivery into portal vein and the systemic application via tail vein induces above all substantial transgene expression. We here comparatively analysed both methods and found that the systemic administration of an adeno-virus expressing the green fluorescent protein resulted in a stronger infiltration, a more homogenous distribution, and a higher inter-individual reproducibility of reporter gene expression in rat liver than organ-specific administration via the portal vein.

Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes

STUDY QUESTION Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? SUMMARY ANSWER Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. WHAT IS KNOWN ALREADY Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. STUDY DESIGN, SIZE, DURATION We fertilized batches of 20–30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. PARTICIPANTS/MATERIALS, SETTING, METHODS Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. MAIN RESULTS AND THE ROLE OF CHANCE In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). LIMITATIONS, REASONS FOR CAUTION Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity. LARGE SCALE DATA None. STUDY FUNDING/COMPETING INTEREST(S) The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.

Lipocalin-2 (LCN2) in experimental liver injury and human liver diseases

Background: LCN2 is a member of the lipocalin family that is expressed under pernicious conditions such as intoxication, infection, inflammation and cellular stress. Sustained LCN2 expression is induced in experimental liver injury. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2. Methods: LCN2-/- mice were used in experimental models (CCl4, BDL, LPS and ConA) for both acute and chronic liver injury. Liver function analysis, FACS-based liver leukocyte characterization, qRT-PCR for chemokine and cytokine expression and Western Blot as well as histology for fibrotic markers was performed. In addition, serum LCN2 from 91 control and 194 liver diseased patients was analyzed in ELISA. Results: Acute single dose CCl4 intoxication showed lipid droplet accumulation in hepatocytes in LCN2-/- mice. Liver leukocyte isolation showed CD45+ and CD11b+F4–80+ to significantly decrease in LCN2-/- mice, while we found no difference after 5 day BDL or during chronic CCl4 application or BDL for 4 weeks. In ConA models however, LCN2-/- mice showed slightly more liver damage and increased values of CD11b+F4–80+, CD45+ and Tregs than wild type animals. Although serum LCN2 levels in diseased humans were higher than in control patients, we found no difference between cirrhotic and non-cirrhotic patients. Patients with Child A and Child C showed higher LCN2 levels compared to control patients that were independent from disease etiology and serum AST and ALT. By contrast, serum LCN2 showed significant correlation with glomerular filtration rate. Conclusion: LCN2 is a fatty acid transport protein that mobilizes fatty acids from hepatocytes. LCN2-/- mice are unable to appropriately respond to acute CCl4 intoxication. LCN2 expression during inflammatory responses in liver depends on the chosen model. It acts as an acute-phase response protein but the expression during chronic liver insult does not correlate to AST and ALT.