Publications by authors named "Ann M Simpson"

26 Publications

  • Page 1 of 1

Studying the Oncosuppressive Functions of PTENP1 as a ceRNA.

Methods Mol Biol 2021 ;2324:165-185

School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia.

PTENP1 is a processed pseudogene of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). It functions posttranscriptionally to regulate PTEN by acting as a sponge for microRNAs that target PTEN. PTENP1 therefore functions as a competitive endogenous RNA (ceRNA), competing with PTEN for binding of microRNAs (miRNA) and thereby modulating PTEN cellular abundance. Studies of the overexpression of PTENP1 all confirm its oncosuppressive function to be mediated through the suppression of cell proliferation, induction of apoptosis, and inhibition of cell migration and invasion of cancer cells of differing types. These oncosuppressive functions are a direct consequence of miRNA binding by PTENP1 and the subsequent liberation of PTEN from miRNA induced suppression. In this chapter, we will focus initially on the description of a high efficiency transient transfection method to introduce and overexpress PTENP1 in the cell type of interest, followed by accurate methodologies to measure transfection efficiency by flow cytometry. We will then continue to describe two methods to analyze cell proliferation, namely the CCK-8 assay and Click-iT EdU assay. Due to commonalities in the manifestation of the oncosuppressive effects of PTENP1, mediated through its role as a ceRNA, the methods presented in this chapter will have wide applicability to a variety of different cell types.
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http://dx.doi.org/10.1007/978-1-0716-1503-4_11DOI Listing
January 2021

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues.

iScience 2021 Apr 31;24(4):102379. Epub 2021 Mar 31.

Centre for Transplant and Renal Research, Westmead Institute for Medical Research, University of Sydney, 176 Hawkesbury Road, Westmead, NSW 2145, Australia.

Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic β-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription.
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http://dx.doi.org/10.1016/j.isci.2021.102379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082091PMC
April 2021

Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice.

Cells 2020 10 2;9(10). Epub 2020 Oct 2.

School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia.

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin () to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver - ± the β-TF to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/ that results in transgene integration were used. Diabetic mice that received AAV8- became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8--FUR and AAV8- and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/-/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/ system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.
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http://dx.doi.org/10.3390/cells9102227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600325PMC
October 2020

Toward Systems Pathology for PTEN Diagnostics.

Cold Spring Harb Perspect Med 2020 05 1;10(5). Epub 2020 May 1.

School of Life Sciences, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.

Germline alterations of the tumor suppressor PTEN have been extensively characterized in patients with PTEN hamartoma tumor syndromes, encompassing subsets of Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus and Proteus-like syndromes, as well as autism spectrum disorder. Studies have shown an increase in the risk of developing specific cancer types in the presence of a germline mutation. Furthermore, outside of the familial setting, somatic variants of occur in numerous malignancies. Here we introduce and discuss the prospect of moving toward a systems pathology approach for PTEN diagnostics, incorporating clinical and molecular pathology data with the goal of improving the clinical management of patients with a mutation. Detection of a germline mutation can inform cancer surveillance and in the case of somatic mutation, have value in predicting disease course. Given that PTEN functions in the PI3K/AKT/mTOR pathway, identification of a mutation may highlight new therapeutic opportunities and/or inform therapeutic choices.
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http://dx.doi.org/10.1101/cshperspect.a037127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197423PMC
May 2020

High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.

Methods Mol Biol 2019 ;2029:197-214

The School of Life Sciences and Centre for Health Technologies, University of Technology Sydney, Sydney, NSW, Australia.

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.
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http://dx.doi.org/10.1007/978-1-4939-9631-5_16DOI Listing
April 2020

Peripheral Biomarker for Vascular Disorders.

Biomark Insights 2018 29;13:1177271918812467. Epub 2018 Nov 29.

School of Life Sciences and Centre for Health Technologies, University of Technology Sydney, Broadway, NSW, Australia.

Atherosclerosis is the underlying cause of most myocardial infarction (MI) and ischaemic stroke episodes. An early sign of atherosclerosis is hypertrophy of the arterial wall. It is known that increased intima media thickness (IMT) is a non-invasive marker of arterial wall alteration, which can easily be assessed in the carotid arteries by high-resolution B-mode ultrasound. Similarly, the other key element of MI and ischaemic strokes is the N-methyl-D-aspartate (NMDA) receptor which is an ionotropic glutamate receptor that mediates the vast majority of excitatory neurotransmission in the brain. NMDA activation requires the binding of both glutamate and a coagonist like D-serine to its glycine site. A special enzyme, serine racemase (SR), is required for the conversion of L-serine into D-serine, and alterations in SR activities lead to a variety of physiological and pathological conditions ranging from synaptic plasticity to ischemia, MI, and stroke. The amount of D-serine available for the activation of glutamatergic signalling is largely determined by SR and we have developed ways to estimate its levels in human blood samples and correlate it with the IMT. This research based short communication describes our pilot study, which clearly suggests that there is a direct relationship between the SR, D-serine, and IMT. In this article, we will discuss whether the activity of SR can determine the future consequences resulting from vascular pathologies such as MI and stroke.
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http://dx.doi.org/10.1177/1177271918812467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287300PMC
November 2018

Heart Rate Variability as a Biomarker for Predicting Stroke, Post-stroke Complications and Functionality.

Biomark Insights 2018 18;13:1177271918786931. Epub 2018 Jul 18.

Neuroscience Research Unit, School of Life Sciences, University of Technology Sydney, Broadway, NSW, Australia.

Background: Heart rate variability (HRV) is a non-invasive measure of the function of the autonomic nervous system, and its dynamic nature may provide a means through which stroke and its associated complications may be predicted, monitored, and managed.

Objective: The objective of this review is to identify and provide a critique on the most recent uses of HRV in stroke diagnosis/management and highlight areas that warrant further research.

Methods: The MEDLINE, CINAHL, and OVID MEDLINE databases were canvassed using a systematic search strategy, for articles investigating the use of HRV in stroke diagnosis and management. Initial paper selections were based on title alone, and final paper inclusion was informed by a full-text critical appraisal.

Results: The systematic search returned 98 records, of which 51 were unique. Following screening, 22 records were included in the final systematic review. The included papers provided some information regarding predicting incident stroke, which largely seems to be best predicted by time- and frequency-domain HRV parameters. Furthermore, post-stroke complications and functionality are similarly predicted by time- and frequency-domain parameters, as well as non-linear parameters in some instances.

Conclusions: Current research provides good evidence that HRV parameters may have utility as a biomarker for stroke and for post-stroke complications and/or functionality. Future research would benefit from the integration of non-linear, and novel parameters, the hybridisation of HRV parameters, and the expansion of the utilisation of predictive regression and hazard modelling.
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http://dx.doi.org/10.1177/1177271918786931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052496PMC
July 2018

Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.

J Gene Med 2018 05 16;20(5):e3017. Epub 2018 Apr 16.

School of Life Sciences, University of Technology Sydney, Sydney, Australia.

Background: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes.

Methods: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry.

Results: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and β-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes.

Conclusions: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.
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http://dx.doi.org/10.1002/jgm.3017DOI Listing
May 2018

PTEN/PTENP1: 'Regulating the regulator of RTK-dependent PI3K/Akt signalling', new targets for cancer therapy.

Mol Cancer 2018 02 19;17(1):37. Epub 2018 Feb 19.

Central Laboratory, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510080, China.

Regulation of the PI-3 kinase (PI3K)/Akt signalling pathway is essential for maintaining the integrity of fundamental cellular processes, cell growth, survival, death and metabolism, and dysregulation of this pathway is implicated in the development and progression of cancers. Receptor tyrosine kinases (RTKs) are major upstream regulators of PI3K/Akt signalling. The phosphatase and tensin homologue (PTEN), a well characterised tumour suppressor, is a prime antagonist of PI3K and therefore a negative regulator of this pathway. Loss or inactivation of PTEN, which occurs in many tumour types, leads to overactivation of RTK/PI3K/Akt signalling driving tumourigenesis. Cellular PTEN levels are tightly regulated by a number of transcriptional, post-transcriptional and post-translational regulatory mechanisms. Of particular interest, transcription of the PTEN pseudogene, PTENP1, produces sense and antisense transcripts that exhibit post-transcriptional and transcriptional modulation of PTEN expression respectively. These additional levels of regulatory complexity governing PTEN expression add to the overall intricacies of the regulation of RTK/PI-3 K/Akt signalling. This review will discuss the regulation of oncogenic PI3K signalling by PTEN (the regulator) with a focus on the modulatory effects of the sense and antisense transcripts of PTENP1 on PTEN expression, and will further explore the potential for new therapeutic opportunities in cancer treatment.
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http://dx.doi.org/10.1186/s12943-018-0803-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817727PMC
February 2018

"Dicing and Splicing" Sphingosine Kinase and Relevance to Cancer.

Int J Mol Sci 2017 Sep 2;18(9). Epub 2017 Sep 2.

School of Life Sciences, Faculty of Science, University of Technology Sydney, 15 Broadway, Ultimo, Sydney, NSW 2007, Australia.

Sphingosine kinase (SphK) is a lipid enzyme that maintains cellular lipid homeostasis. Two SphK isozymes, SphK1 and SphK2, are expressed from different chromosomes and several variant isoforms are expressed from each of the isozymes, allowing for the multi-faceted biological diversity of SphK activity. Historically, SphK1 is mainly associated with oncogenicity, however in reality, both SphK1 and SphK2 isozymes possess oncogenic properties and are recognized therapeutic targets. The absence of mutations of SphK in various cancer types has led to the theory that cancer cells develop a dependency on SphK signaling (hyper-SphK signaling) or "non-oncogenic addiction". Here we discuss additional theories of SphK cellular mislocation and aberrant "dicing and splicing" as contributors to cancer cell biology and as key determinants of the success or failure of SphK/S1P (sphingosine 1 phosphate) based therapeutics.
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http://dx.doi.org/10.3390/ijms18091891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618540PMC
September 2017

A parasite-derived 68-mer peptide ameliorates autoimmune disease in murine models of Type 1 diabetes and multiple sclerosis.

Sci Rep 2016 11 24;6:37789. Epub 2016 Nov 24.

The School of Life Sciences, University of Technology Sydney, New South Wales, Australia.

Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.
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http://dx.doi.org/10.1038/srep37789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121616PMC
November 2016

Role for the thromboxane A2 receptor β-isoform in the pathogenesis of intrauterine growth restriction.

Sci Rep 2016 07 1;6:28811. Epub 2016 Jul 1.

Division of Perinatal Research, Kolling Institute, Northern Sydney Local Health District, St Leonards, NSW, 2065, Australia.

Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPβ) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPβ attenuated these functions and inhibited migration. Expression of the TPβ transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPβ impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans.
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http://dx.doi.org/10.1038/srep28811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929481PMC
July 2016

Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line.

Int J Mol Sci 2016 Apr 8;17(4):534. Epub 2016 Apr 8.

School of Life Sciences and Centre for Health Technologies, University of Technology Sydney, P.O. Box 123, Broadway, 2007 Sydney, NSW, Australia.

Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 10⁶ cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.
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http://dx.doi.org/10.3390/ijms17040534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848990PMC
April 2016

Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

Mol Ther Methods Clin Dev 2015 8;2:15011. Epub 2015 Apr 8.

School of Medical and Molecular Biosciences and Centre for Health Technologies, University of Technology Sydney , Sydney, Australia.

As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D.
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http://dx.doi.org/10.1038/mtm.2015.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445011PMC
June 2015

Secreted proteins from the helminth Fasciola hepatica inhibit the initiation of autoreactive T cell responses and prevent diabetes in the NOD mouse.

PLoS One 2014 21;9(1):e86289. Epub 2014 Jan 21.

The i3 Institute, University of Technology Sydney, New South Wales, Australia.

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086289PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897667PMC
September 2014

The Inhibitory Effect of Haloxylon salicornicum on Contraction of the Mouse Uterus.

Evid Based Complement Alternat Med 2013 23;2013:714075. Epub 2013 Sep 23.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK.

Haloxylon salicornicum (H. salicornicum) is a plant that is frequently taken as a tea by Bedouin women in Egypt who are experiencing difficulties during pregnancy, as well as to provide relief from dysmenorrhoea. Despite its medical use, there has been no detailed evaluation of the effect of this plant on uterine tissue. Therefore, the initial aim of this study was to determine whether H. salicornicum affected the contraction of the mouse uterus in vitro. The crude aqueous extract of H. salicornicum was found to inhibit the spontaneous contractions of the uterus, with the effect being rapid in onset and completely reversible upon washout. Subsequent purification of the plant extract resulted in the identification of synephrine and N-methyltyramine, both of which were found to have inhibitory effects on the spontaneous contractions of the uterus. The EC50 for the purified constituent identified as synephrine was 0.82 ± 0.24  μ g/mL. The inhibitory activity of crude H. salicornicum, as well as the isolated constituents, could be prevented by pretreatment of the uterus with the β -adrenoceptor antagonist propranolol. In conclusion, the use of H. salicornicum during preterm labour appears to be justified, and its pharmacologic effect is consistent with it acting as a β -adrenoceptor agonist.
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http://dx.doi.org/10.1155/2013/714075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794631PMC
October 2013

Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy.

J Gene Med 2013 Jan;15(1):28-41

School of Medical & Molecular Biosciences, University of Technology Sydney, Sydney, Australia.

Background: Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing β-cells of the pancreas. The present study aimed to reverse T1D by gene therapy.

Methods: We used a novel surgical technique, which involves isolating the liver from the circulation before the 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 with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction 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 the expression of β-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-β, G6PC, AAT and GLUI was down-regulated 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, and 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, represents a novel therapeutic strategy for the treatment of T1D.
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http://dx.doi.org/10.1002/jgm.2692DOI Listing
January 2013

Isoforms of the heteropteran Nezara viridula ecdysone receptor: protein characterisation, RH5992 insecticide binding and homology modelling.

Pest Manag Sci 2011 Nov 18;67(11):1457-67. Epub 2011 May 18.

CSIRO Materials Science and Engineering and CSIRO Food and Nutritional Sciences, Sydney Laboratory, North Ryde, NSW, Australia.

Background: 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.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1002/ps.2200DOI Listing
November 2011

Gene expression profiling of HUH7-ins: lack of a granulogenic function for chromogranin A.

Islets 2009 Jul-Aug;1(1):62-74

Diabetes Transplant Unit, Prince of Wales Hospital, Sydney, New South Wales, Australia.

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.
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http://dx.doi.org/10.4161/isl.1.1.8994DOI Listing
March 2011

An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone.

Insect Biochem Mol Biol 2011 Feb 28;41(2):77-89. Epub 2010 Oct 28.

CSIRO Molecular and Health Technologies, and CSIRO Food and Nutritional Sciences, Sydney Laboratory, PO Box 184, North Ryde, NSW 1670, Australia.

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.
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http://dx.doi.org/10.1016/j.ibmb.2010.10.002DOI Listing
February 2011

Growth-promoting effect of Rh(D) antibody on human pancreatic islet cells.

J Clin Endocrinol Metab 2008 Sep 10;93(9):3560-7. Epub 2008 Jun 10.

Department of Newborn Care, Royal Hospital for Women, Randwick NSW 2031, Sydney, Australia.

Context/objective: Hyperinsulinism with islet cell hyperplasia is a frequent complication, of unknown cause, in hemolytic disease of the newborn, occurring in Rh(D)-positive infants of Rh-isoimmunized Rh(D)-negative mothers, but not in infants with other hemolytic disorders. We investigated the possibility that trans-placentally acquired anti-D Ig is the cause of both conditions.

Design: Monolayer cultures of human islet cells were exposed to sera from Rh-isoimmunized mothers and newborns, where jaundice, hyperinsulinism, and hypoglycemia in the infant had ensued. Parallel cultures with anti-D, specific anti-D monoclonal antibodies, normal human Ig (15 microg/ml), and serum controls were also undertaken. Islet cell proliferation was determined by [3H]thymidine incorporation. Insulin storage and chronic and acute insulin secretion to glucose were analyzed by RIA. Rh(D) surface antigen expression was determined on islet cells by flow cytometric analysis.

Results: Islet cell proliferation and insulin secretion were significantly greater in coculture with test sera (P < 0.01; n = 8) and with anti-D (P < 0.001; n = 8), compared with either controls or Ig. After 8 d of growth, the static incubation experiment showed a 3.5-fold response to glucose stimulus in all sera. Rh(D) antigen expression was detected on the islet cell surface by flow cytometry, and islet cell morphology was normal. Colocalization of the proliferation marker Ki67 with insulin by immunofluorescent staining further indicated that Rh(D) antibody promoted islet growth.

Conclusions: The anti-Rh(D) islet cell proliferative effect generates neonatal hyperinsulinism in Rh isoimmunization. Anti-Rh(D) may have application for islet cell proliferation in diabetes mellitus treatment for Rh(D)-positive subjects. Further analysis is required.
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http://dx.doi.org/10.1210/jc.2008-0510DOI Listing
September 2008

Association of SLC11A1 promoter polymorphisms with the incidence of autoimmune and inflammatory diseases: a meta-analysis.

J Autoimmun 2008 Aug 28;31(1):42-51. Epub 2008 Mar 28.

Department of Medical and Molecular Biosciences, University of Technology Sydney, PO Box 123, Broadway, NSW 2007, Australia.

Solute carrier family 11 member a1 (SLC11A1) exerts pleiotropic effects on macrophage function. Expression of SLC11A1 is regulated by a (GT)(n) microsatellite promoter repeat polymorphism of which nine alleles have been described. Enhanced activation of macrophages, associated with increased expression from allele 3, may be functionally linked to the development of autoimmune and inflammatory diseases. Conversely, low expression, driven by allele 2, may afford resistance. We have performed a meta-analysis to determine the association of SLC11A1 promoter alleles 2 and 3 with autoimmunity and inflammation. A random effects pooled odds ratio (OR) of 1.04 (95% confidence interval [CI]=0.20) for allele 3 suggested a weak association of this allele with an increased risk of disease. Calculation of the OR in the absence of asymmetry yielded a random effects pooled OR of 0.88 (95% CI=0.66), effectively reversing the above association. A fixed effects pooled OR of 0.90 (95% CI=0.24) was obtained for allele 2, suggesting a weak predominance of disease in the absence of this allele. Application of the trim-and-fill method resulted in a fixed effects OR of 0.80 (95% CI=0.22), thus strengthening this association. Associations of allele 3 with autoimmune and inflammatory diseases reported in several association studies may be attributable to some form of bias amongst published results.
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http://dx.doi.org/10.1016/j.jaut.2008.02.002DOI Listing
August 2008

Identification of vascular surface proteins by in vivo biotinylation: a method sufficiently sensitive to detect changes in rat liver 2 weeks after partial hepatectomy.

J Proteome Res 2007 Aug 30;6(8):3108-13. Epub 2007 Jun 30.

Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital and University of Sydney, St. Leonards NSW 2065, Australia.

We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to identify 32 proteins with changed expression in rat livers at 2 weeks or 5 weeks after partial hepatectomy, well after the 7 day tissue remodeling period. This method could be adapted to study other angiogenic tissues including tumors.
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http://dx.doi.org/10.1021/pr070032mDOI Listing
August 2007

ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimulated insulin secretion.

FASEB J 2003 Sep 18;17(12):1682-4. Epub 2003 Jul 18.

Department of Health Sciences, University of Technology, Sydney, Broadway, NSW, Australia.

As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a normal pancreatic beta cell.
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http://dx.doi.org/10.1096/fj.02-0051fjeDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2132862PMC
September 2003

Fetal pig beta cells are resistant to the toxic effects of human cytokines.

Transplantation 2002 Mar;73(5):714-22

Diabetes Transplant Unit, Prince of Wales Hospital and University of New South of Wales, Sydney, New South Wales, Australia.

Background: The cytokine tumour necrosis factor-alpha (TNF-alpha) is thought to be responsible for primary nonfunction of islets when transplanted. This, and two other cytokines, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) are also implicated in the autoimmune destruction of beta cells. It is unknown if the fetal pig beta cell, which is being transplanted as a treatment for type 1 diabetes, is affected by these cytokines.

Methods: We compared the effects of the cytokines on the function and viability of adult and fetal pig beta cells. The cells were cultured for up to 3 days in the presence of 2000 pg/ml of human IL-1beta, 1000 U/ml of TNF-alpha, and 1000 U/ml of IFN-gamma, as well as 1000 U/ml of porcine IFN-gamma. Cumulative insulin levels, insulin content, metabolic activity, and viability of these cells were examined. Additionally, nitric oxide production and the activity of antioxidant enzymes in these cells were also determined.

Results: TNF-alpha and the combination of the three human cytokines caused a transient increase in cumulative insulin levels. TNF-alpha alone enhanced insulin content on day 3. There was no effect of these human cytokines on mitochondrial function and viability. In contrast, porcine IFN-gamma inhibited fetal pig beta cell function and also caused their death. Adult pig islets are sensitive to the toxic effects of human TNF-alpha, IL-1beta, the combination of the three cytokines, and porcine IFN-gamma. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly higher in fetal pig beta cells than in adult islets, implying that this may be the reason for the lack of adverse effects of the cytokines on the fetal beta cell.

Conclusion: Fetal pig beta cells are resistant to the toxic effect of the human cytokines, TNF-alpha and IL-1beta, in vitro. This resistance suggests that fetal, but not adult beta cells, when transplanted into humans with type 1 diabetes may be protected from primary nonfunction and will be partially protected from autoimmune destruction.
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http://dx.doi.org/10.1097/00007890-200203150-00010DOI Listing
March 2002
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