Publications (62) View all
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Article: Long term reversal of diabetes in non obese diabetic mice by liver-directed gene therapy.
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ABSTRACT: BACKGROUND: Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing beta (β)-cells of the pancreas. The aim of this study was to reverse T1D by gene therapy. METHODS: We used a novel surgical technique, which involves isolating the liver from the circulation before delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR), or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared to the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests (IVGTT) were performed. Expression of insulin and pancreatic transcription factors was determined by reverse transcriptase PCR (RT-PCR) and immunohistochemistry and immunoelectron microscopy was used to localise insulin. RESULTS: Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed expression of β-cell transcription factors, murine insulin, glucagon and somatostatin, hepatic storage of insulin in granules. Expression of hepatic markers, C/EBP-β, G6PC, AAT, GLUI, were downregulated in INS-FUR-treated livers. Liver function tests remained normal with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, no pancreatic transdifferentiation or normal glucose tolerance was observed. CONCLUSIONS: This gene therapy protocol has for the first time permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, opens a novel therapeutic strategy for the cure of T1D. Copyright © 2013 John Wiley & Sons, Ltd.The Journal of Gene Medicine 01/2013; · 2.48 Impact Factor -
Article: Isoforms of the heteropteran Nezara viridula ecdysone receptor: protein characterisation, RH5992 insecticide binding and homology modelling.
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ABSTRACT: Certain bisacylhydrazine compounds such as tebufenozide (RH5992) have been shown to act as order-specific insecticides. Their compatibility with predatory Heteroptera, which are used as biological control agents, has also been demonstrated. However, the molecular mode of action of these ecdysone agonists has not been explored in a heteropteran, much less one that is a significant agricultural pest, such as Nezara viridula. Alternatively spliced ligand-binding regions of the N. viridula ecdysone receptor were expressed, purified and characterised by 2D gel analysis, mass spectrometry, homology modelling and competitive binding of a bisacylhydrazine insecticidal compound (RH5992) and various ecdysteroids. Ligand binding by the two splice isoforms was indistinguishable, and relative affinities were found to occur in the order muristerone A > ponasterone A > 20-hydroxyecdysone > inokosterone > RH5992 > α-ecdysone. The predicted difference in amino acid sequence between the ligand-binding domains of the N. viridula ecdysone receptor splice variants was verified by mass spectrometry. Both splice variant isoforms exhibit a greater affinity for the bisacylhydrazine insecticide RH5992 than do the other hemipteran ecdysone receptors characterised to date. Their affinities for a range of ecdysteroids also distinguish them from the ecdysone receptors of other Hemiptera characterised thus far. Homology models of both N. viridula receptor isoforms provide further insight into the bisacylhydrazine- and ecdysteroid-binding properties of these receptors, including their similar affinity for 20-hydroxyecdysone and the postulated pentatomomorphan moulting hormone makisterone A.Pest Management Science 05/2011; 67(11):1457-67. · 2.25 Impact Factor -
Article: Gene expression profiling of HUH7-ins: lack of a granulogenic function for chromogranin A.
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ABSTRACT: Previously, the insulin producing liver cell line HUH7-ins has been shown to synthesize, store and secrete insulin in response to glucose via secretory granules. The current study characterized the gene expression profile of HUH7-ins with the aim to identify changes possibly involved in the formation of granules. Additionally, experiments were conducted to determine the influence of chromogranin A (CgA) on secretory granule biogenesis (SGB) in HUH7-ins. Expression of 165 genes were significantly changed in HUH7-ins,though interestingly the majority of secretory granule associated genes, such as the chromogranins were unchanged. CgA was over-expressed in glucose unresponsive HUH7-ins cells to test whether CgA played a role in SGB and would restore the regulated secretory phenotype. Over-expression affected neither the storage nor regulated secretion of insulin. These data suggest that SGB may by regulated at a post-transcriptional level with no evidence to indicate that CgA regulates SGB in the cell line HUH7-ins.Islets 11/2010; 1(1):62-74. -
Article: An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone.
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ABSTRACT: It has been suggested that Pentatomomorpha utilise the C(28) ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C(27) hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His(6)-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His(6) tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [(3)H]ponasterone A (K(d) = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.Insect biochemistry and molecular biology 10/2010; 41(2):77-89. · 3.25 Impact Factor -
Article: Searching for the optimal living liver donor psychosocial evaluation.
American Journal of Transplantation 01/2012; 12(1):7-8. · 6.39 Impact Factor
