Ovarian hormones and pituitary responsiveness to gonadotrophin releasing hormone in mice

In the present study the role of progesterone and oestradiol in modulating the responsiveness at the anterior pituitary gland to gonadotrophin releasing hormone (GN-RH) was investigated by measuring the release of luteinizing hormone (LH) in ovariectomised mice which had been pretreated with these steroids.A significant release of LH was seen in the animals receiving GN-RH. Pretreatment with oestrogen and progesterone depressed LH levels in the animals which did not receive GN-RH, and resulted in a larger release of LH in the animals receiving GN-RH in comparison with the control group pretreated with oil

Determination of Odor Detection Threshold in the Göttingen Minipig

The aim of the study was to examine the ability of Göttingen minipigs to acquire an olfaction-based operant conditioning task and to determine the detection threshold for ethyl acetate and ethanol. We used an automated olfactometer developed for rodents to train and test 14 pigs. Odor sampling and reliable responding were obtained after three to fifteen 160-trial sessions. Successful transfer of the task from ethyl acetate to ethanol was achieved in 1–4 sessions. Detection threshold for ethyl acetate varied between 10−2% and 10−6% v/v and for ethanol between 0.1% and 5 × 10−6% v/v. The results provide evidence that minipigs can successfully acquire 2-odorant discrimination using a food-rewarded instrumental conditioning paradigm for testing olfactory function. This olfactory discrimination paradigm provides reliable measures of olfactory sensitivity and thereby enables detection of changes in olfaction in a porcine model of Alzheimer's disease currently being developed

Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection.

Hepatitis B virus (HBV) is a major human pathogen and about one third of the global population will be exposed to the virus in their life time. HBV infects hepatocytes where it replicates its DNA and infection can lead to acute and chronic hepatitis with high risk of liver cirrhosis and hepatocellular carcinoma. Despite this, there is limited understanding of how HBV establishes chronic infections. In recent years it has emerged that foreign DNA potently stimulates the innate immune response, particularly type I IFN production, and this occurs through a pathway dependent on the DNA sensor cGAS and the downstream adaptor protein STING. In this work we describe that human and murine hepatocytes do not express STING. Consequently, hepatocytes do not produce type I IFN in response to foreign DNA or HBV infection and mice lacking STING or cGAS exhibit unaltered ability to control infection in an adenovirus-HBV model. Stimulation of IFN production in the murine liver by administration of synthetic RNA decreases virus infection, thus demonstrating that IFN possess anti-HBV activity in the liver. Importantly, introduction of STING expression specifically in hepatocytes reconstitutes the DNA sensing pathway, which leads to improved control of HBV in vivo. In conclusion, the lack of a functional innate DNA sensing pathway in hepatocytes hampers efficient innate control of HBV infection. This may explain why HBV has adapted to specifically replicate in hepatocytes, and could contribute to the weak capacity of this cell type to clear HBV infection

Reduced choroidal neovascularization by AAV-anti-VEGF shRNA delivery

VEGF plays an essential role in ocular angiogenic diseases including the late-stage form of AMD, the primary cause of vision loss in the western world. Over-expression of VEGF leads to development of vasculature emanating from the choroid, invading the subretinal space through breaks in Bruch's membrane. Strategies leading to long-term suppression of inappropriate ocular angiogenesis are required. A panel of 10 shRNAs targeting the coding region of human VEGF165 was tested in HEK293 cells and in the human retinal pigment epithelial cell line, ARPE-19. VEGF knock-down up to 92% was achieved by co-transfecting shRNAexpressing constructs with plasmid encoding the Renilla luciferase gene fused to the VEGF165 sequence. For in vivo delivery of the most potent shRNA cassette, both single-stranded and self-complementary rAAV vectors were packaged in serotype 8 capsids. Intramuscular administration in mice led to localized expression and 96% knock-down of endogenous VEGF. Using eGFP as a marker, efficient gene transfer of retinal pigment epithelial cells, the cells thought to be responsible for the abnormal VEGF production, was obtained by subretinal delivery of rAAV2.8 vectors. The capacity of rAAV-encoded shRNAs to silence endogenous VEGF gene expression was evaluated in the laser-induced murine model of choroidal neovascularization (CNV). In this mouse model of AMD, sizes of the CNV were found to be significantly reduced following rAAV-shRNA subretinal delivery. Thus, our results indicate that gene transfer combining AAV-mediated delivery with triggering of the endogenous RNAi pathway can be used for anti-VEGF therapy and holds great promise for the treatment of AMD

Differences in Origin of Reactive Microglia in Bone Marrow Chimeric Mouse and Rat After Transient Global Ischemia.

Current understanding of microglial involvement in disease is influenced by the observation that recruited bone marrow (BM)-derived cells contribute to reactive microgliosis in BM-chimeric mice. In contrast, a similar phenomenon has not been reported for BM-chimeric rats. We investigated the recruitment and microglial transformation of BM-derived cells in radiation BM-chimeric mice and rats after transientglobal cerebral ischemia, which elicits a characteristic microglialreaction. Both species displayed microglial hyperplasia and rod cell transformation in the hippocampal CA1 region. In mice, a subpopulation of lesion-reactive microglia originated from transformed BM-derived cells. By contrast, no recruitment or microglial transformation of BM-derived cells was observed in BM-chimeric rats. These results suggest that reactive microglia in rats originate from resident microglia, whereas they have a mixed BM-derived and resident origin in mice, depending on the severity of ischemic tissue damage

Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC