Publications by authors named "Jonathan M Gleadle"

49 Publications

Arginine Metabolites as Biomarkers of Myocardial Ischaemia, Assessed with Cardiac Magnetic Resonance Imaging in Chronic Kidney Disease.

Biomolecules 2021 Mar 11;11(3). Epub 2021 Mar 11.

Department of Cardiovascular Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia.

(1) Background: Cardiovascular disease (CVD) is the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Myocardial oxygenation and perfusion response to stress, using oxygen-sensitive cardiovascular magnetic resonance (OS-CMR) and stress T1 mapping respectively, are impaired in CKD patients with and without known coronary artery disease (CAD). Endothelial dysfunction, assessed by circulating levels of asymmetric dimethylarginine (ADMA) and homoarginine (HMA), promotes atherosclerosis. We hypothesized that in CKD patients, worsening endothelial dysfunction is associated with worsening myocardial oxygenation and perfusion as assessed by change in OS-CMR signal intensity (Δ OS-CMR SI) and stress T1 (ΔT1) values. (2) Methods: 38 patients with advanced CKD underwent cardiovascular magnetic resonance (CMR) scanning at 3 Tesla. OS-CMR and T1 mapping images were acquired both at rest and after adenosine stress and analyzed semi-quantitatively. Serum ADMA and HMA concentrations were assessed using mass spectrometry. (3) Results: There was no significant correlation between Δ OS-CMR SI and ADMA or HMA. Interestingly, there was a significant negative correlation seen between Δ T1 and ADMA (r = -0.419, = 0.037, = 30) but not between Δ T1 and HMA. (4) Conclusions: Stress T1 response is impaired in CKD patients and is independently associated with higher circulating ADMA concentrations.
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http://dx.doi.org/10.3390/biom11030416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002086PMC
March 2021

Cancer cell detection device for the diagnosis of bladder cancer from urine.

Biosens Bioelectron 2021 Jan 7;171:112699. Epub 2020 Oct 7.

Future Industry Institute, University of South Australia, Mawson Lakes, SA, 5095, Australia; School of Engineering, University of South Australia, Mawson Lakes, SA, 5095, Australia.

Bladder cancer is common and has one of the highest recurrence rates. Cystoscopy, the current gold standard diagnosis approach, has recently benefited from the introduction of blue light assisted photodynamic diagnostic (PDD). While blue light cystoscopy improves diagnostic sensitivity, it remains a costly and invasive approach. Here, we present a microfluidic-based platform for non-invasive diagnosis which combines the principle of PDD with whole cell immunocapture technology to detect bladder cancer cells shed in patient urine ex vivo. Initially, we demonstrate with model cell lines that our non-invasive approach achieves highly specific capture rates of bladder cancer cells based on their Epithelial Cell Adhesion Molecule expression (>90%) and detection by the intensity levels of Hexaminolevulinic Acid-induced Protoporphyrin IX fluorescence. Then, we show in a pilot study that the biosensor platform successfully discriminates histopathologically diagnosed cancer patients (n = 10) from non-cancer controls (n = 25). Our platform can support the development of a novel non-invasive diagnostic device for post treatment surveillance in patients with bladder cancer and cancer detection in patients with suspected bladder cancer.
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http://dx.doi.org/10.1016/j.bios.2020.112699DOI Listing
January 2021

Plasma enabled devices for the selective capture and photodynamic identification of prostate cancer cells.

Biointerphases 2020 05 15;15(3):031002. Epub 2020 May 15.

School of Engineering, University of South Australia, Mawson Lakes, Adelaide, SA 5095, Australia.

Prostate cancer is the second most common cancer in men and the second leading cause of male cancer deaths. The current blood test for detecting prostate cancers measures prostate-specific antigen. It has many limitations including a very high rate of false positives. Herein, prostate-specific membrane antigen (PSMA) based immunocapture and hexaminolevulinate (HAL) based photodetection are integrated into a new diagnostic device designed to selectively identify whole prostate cancer cells from voided urine with the aim of providing an accurate noninvasive alternative to current diagnosis methods. Prestained, prostate cancer cells spiked in urine samples at concentrations ranging from 1500 to 2000 cells/ml were captured with 89% sensitivity and 95% specificity. HAL, a cancer specific photosensitizer, was then used to circumvent the need for prestaining. Optimum HAL incubation conditions were identified (50 μM at 37 °C for 2 h) where the mean HAL-induced fluorescence intensity of LNCaP cells was three times that of healthy PNT2 cells, thus providing an independent way to discriminate captured cancer cells from background metabolites. Combining anti-PSMA immunocapture with HAL-induced fluorescent detection, 86% sensitivity and 88% selectivity were achieved, thereby proving the validity of the dual-method for the selective photospecific detection of prostate cancer cells.
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http://dx.doi.org/10.1116/6.0000047DOI Listing
May 2020

MicroRNA-Related Genetic Variants Are Associated With Diabetic Retinopathy in Type 1 Diabetes Mellitus.

Invest Ophthalmol Vis Sci 2019 09;60(12):3937-3942

Department of Ophthalmology, Flinders University, College of Medicine and Public Health, Flinders Medical Centre, Adelaide, South Australia, Australia.

Purpose: Few studies have explored the association of genetic variants in microRNA genes and binding sites with diabetic retinopathy (DR) in type 1 diabetes. We conducted a genome-wide scan for single nucleotide polymorphisms (SNPs) in these genes by using data from a genome-wide association study (GWAS).

Methods: All known SNPs were imputed from our GWAS data (n = 325) of DR cases and diabetic controls (no DR). Relevant SNPS were extracted using miRNASNP and PolymiRTS (version 2) databases. χ2 tests and logistic regression (adjusting for age, sex, duration of diabetes, HbA1c, and hypertension) were used to test the association between the imputed SNPs and DR phenotypes (any DR, nonproliferative DR [NPDR], proliferative DR [PDR], diabetic macular edema [DME], and sight-threatening DR defined as PDR, severe NPDR, or clinically significant macula edema [CSME]) compared with diabetic controls. Top-ranking SNPs were genotyped in a larger cohort (N = 560) to confirm their association with DR.

Results: Three SNPs (rs10061133, rs1049835, rs9501255) were selected and genotyped in the final cohort. Rs10061133 in MIR449b was protective of sight-threatening DR (odds ratio [OR] = 0.32, P = 3.68 × 10-4) and PDR (OR = 0.30, P = 8.12 × 10-4), and the associations became more significant as the cohort increased in size.

Conclusions: Rs10061133 in MIR449b is significantly associated with a decreased risk of PDR and sight-threatening DR in Caucasian patients with type 1 diabetes. This can guide future studies on genetic risk profiling and on developing microRNA-related therapies for sight-threatening DR.
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http://dx.doi.org/10.1167/iovs.18-25570DOI Listing
September 2019

In order for the light to shine so brightly, the darkness must be present-why do cancers fluoresce with 5-aminolaevulinic acid?

Br J Cancer 2019 10 13;121(8):631-639. Epub 2019 Aug 13.

Department of Renal Medicine, Flinders Medical Centre, Flinders University, Bedford Park, SA, 5042, Australia.

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection and photodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IX phenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies.
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http://dx.doi.org/10.1038/s41416-019-0516-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889380PMC
October 2019

Compensatory renal hypertrophy following nephrectomy: When and how?

Nephrology (Carlton) 2019 Dec 5;24(12):1225-1232. Epub 2019 May 5.

College of Medicine and Public Health and Medicine, Flinders University, Adelaide, South Australia, Australia.

Following surgical removal of one kidney, the other enlarges and increases its function. The mechanism for the sensing of this change and the growth is incompletely understood but begins within days and compensatory renal hypertrophy (CRH) is the dominant contributor to the growth. In many individuals undergoing nephrectomy for cancer or kidney donation this produces a substantial and helpful increase in renal function. Two main mechanisms have been proposed, one in which increased activity by the remaining kidney leads to hypertrophy, the second in which there is release of a kidney specific factor in response to a unilateral nephrectomy that initiates CRH. Whilst multiple growth factors and pathways such as the mTORC pathway have been implicated in experimental studies, their roles and the precise mechanism of CRH are not defined. Unrestrained hypoxia inducible factor activation in renal cancer promotes growth and may play an important role in driving CRH.
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http://dx.doi.org/10.1111/nep.13578DOI Listing
December 2019

Mitochondrial haplogroups are not associated with diabetic retinopathy in a large Australian and British Caucasian sample.

Sci Rep 2019 01 24;9(1):612. Epub 2019 Jan 24.

Department of Ophthalmology, Flinders University, Flinders Medical Centre, Adelaide, South Australia, Australia.

Mitochondrial haplogroups H1, H2 and UK have previously been reported to be associated with proliferative diabetic retinopathy (PDR) in Caucasian patients with diabetes. We aimed to replicate this finding with a larger sample and expand the analysis to include different severities of DR, and diabetic macular edema (DME). Caucasian participants (n = 2935) with either type 1 or type 2 diabetes from the Australian Registry of Advanced Diabetic Retinopathy were enrolled in this study. Twenty-two mitochondrial single nucleotide polymorphisms were genotyped by MassArray and haplogroups reconstructed using Haplogrep. Chi square tests and logistic regressions were used to test associations between haplogroup and DR phenotypes including any DR, non-proliferative DR (NPDR), proliferative DR (PDR) and DME. After stratifying the samples in type 1 and type 2 diabetes groups, and adjusting for sex, age, diabetes duration, concurrent HbA1c and hypertension, neither haplogroups H1, H2, UK, K or JT were associated with any DR, NPDR, PDR or DME.
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http://dx.doi.org/10.1038/s41598-018-37388-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345891PMC
January 2019

Circulating and Urinary miR-210 and miR-16 Increase during Cardiac Surgery Using Cardiopulmonary Bypass - A Pilot Study.

J Extra Corpor Technol 2018 03;50(1):19-29

School of Medicine, Flinders University, Adelaide, Australia.

A pilot study to measure and compare blood and urine microRNAs miR-210 and miR-16 in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and off-pump coronary artery bypass grafting surgery. Frequent serial blood and urine samples were taken from patients undergoing cardiac surgery with CPB (n = 10) and undergoing off-pump cardiac surgery (n = 5) before, during, and after surgery. Circulating miR-210 and miR-16 levels were determined by relative quantification real-time polymerase chain reaction. Levels of plasma-free haemoglobin (fHb), troponin-T, creatine kinase, and creatinine were measured. Perioperative serum miR-210 and miR-16 were elevated significantly compared to preoperative levels in patients undergoing cardiac surgery with CPB (CPB vs. Pre Op and Rewarm vs. Pre Op; < .05 for both). There were increases of greater than 200% in miR-210 levels during rewarming and immediately postoperatively and a 3,000% increase in miR-16 levels immediately postoperatively in urine normalized to urinary creatinine concentration. Serum levels of miR-16 were relatively constant during off-pump surgery. miR-210 levels increased significantly in off-pump patients perioperatively ( < .05 Octopus on vs. Pre Op); however, the release was less marked when compared to cardiac surgery with CPB. A significant association was observed between both miR-16 and miR-210 and plasma fHb when CPB was used ( = -.549, < .0001 and = -.463, < .0001 respectively). Serum and urine concentrations of hypoxically regulated miR-210 and hemolysis-associated miR-16 increased in cardiac surgery using CPB compared to off-pump surgery. These molecules may have utility in indicating severity of cardiac, red cell, and renal injury during cardiac surgery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848080PMC
March 2018

A platform for selective immuno-capture of cancer cells from urine.

Biosens Bioelectron 2017 Oct 9;96:373-380. Epub 2017 Feb 9.

School of Engineering University of South Australia, Mawson Lakes, SA 5095, Australia. Electronic address:

Urothelial cancers are amongst the 10 most common types of cancer and represent a major health problem worldwide. Current urinary diagnostic tests for urothelial cancer are expensive and have limited sensitivity and specificity. In this work, proofs of concept for a selective cancer cell capture platform are presented with the aim to achieve the first generation of specific urinary tests for the detection of cancer cells in urine specimen. The unique reactivity of plasma deposited polyoxazoline was used to covalently bind cancer specific antibodies in microchannels. Cancer cells dispersed in patient urine were successfully captured with up to 99% selectivity and 100% sensitivity over a wide range of cell concentrations. The streamlined two steps preparation process of the capture platform represents an important advance in medical diagnostics, with broader potential applications.
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http://dx.doi.org/10.1016/j.bios.2017.02.011DOI Listing
October 2017

Lack of appropriate investigations in making a diagnosis of syndrome of inappropriate antidiuretic hormone.

Intern Med J 2017 Mar;47(3):336-338

Department of Renal Medicine, Flinders Medical Centre, Adelaide, South Australia, Australia.

The syndrome of inappropriate antidiuretic hormone (SIADH) is reported as the most common cause of hyponatraemia. This retrospective cross-sectional study evaluated the diagnosis of SIADH in 110 hospitalised patients in an Australian tertiary hospital with reference to recently published clinical guidelines. Investigation of SIADH was incomplete in all but 20% of cases. Adrenal insufficiency and hypothyroidism were not excluded in a significant number of cases.
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http://dx.doi.org/10.1111/imj.13364DOI Listing
March 2017

Remote ischaemic preconditioning: closer to the mechanism?

F1000Res 2016 13;5:2846. Epub 2016 Dec 13.

School of Medicine, Flinders University, Adelaide, Australia; Cardiac Surgery Research and Perfusion, Cardiac and Thoracic Surgical Unit, Flinders Medical Centre, Adelaide, Australia.

Brief periods of ischaemia followed by reperfusion of one tissue such as skeletal muscle can confer subsequent protection against ischaemia-induced injury in other organs such as the heart. Substantial evidence of this effect has been accrued in experimental animal models. However, the translation of this phenomenon to its use as a therapy in ischaemic disease has been largely disappointing without clear evidence of benefit in humans. Recently, innovative experimental observations have suggested that remote ischaemic preconditioning (RIPC) may be largely mediated through hypoxic inhibition of the oxygen-sensing enzyme PHD2, leading to enhanced levels of alpha-ketoglutarate and subsequent increases in circulating kynurenic acid (KYNA). These observations provide vital insights into the likely mechanisms of RIPC and a route to manipulating this mechanism towards therapeutic benefit by direct alteration of KYNA, alpha-ketoglutarate levels, PHD inhibition, or pharmacological targeting of the incompletely understood cardioprotective mechanism activated by KYNA.
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http://dx.doi.org/10.12688/f1000research.9633.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5271585PMC
December 2016

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

J Virol 2017 Jan 16;91(1). Epub 2016 Dec 16.

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family.

Importance: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.
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http://dx.doi.org/10.1128/JVI.01430-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165212PMC
January 2017

Albuminuria is not associated with elevated urinary vesicle concentration but can confound nanoparticle tracking analysis.

Nephrology (Carlton) 2017 Nov;22(11):854-863

Department of Renal Medicine, Flinders Medical Centre, Adelaide, South Australia, Australia.

Aim: Extracellular vesicles, such as exosomes, are present in urine with reports of roles in intercellular signalling and diagnostic utility. However, the extent to which the concentration and characteristics of urinary vesicles are altered in albuminuric renal disease has not been well characterized. In this study, we examined the number and characteristics of extracellular vesicles in albuminuric urine.

Methods: Vesicles were isolated from the urine of 32 patients with varying levels of albuminuria using ultracentrifugation and density gradient purification and were examined using nanoparticle tracking analysis, immunoblotting and transmission electron microscopy. The size profile of particles in these urine preparations was compared with albumin-containing solutions.

Results: Overall, there were no substantial differences in the number, or characteristics, of vesicles released into proteinuric urine. Analysis of albumin-containing solutions showed particles of exosome-like size, suggesting that such particles can mimic exosomes in standard nanoparticle tracking analysis. Albumin and IgG depletion of proteinuric urine resulted in a substantial reduction in the concentration of particles detected by nanoparticle tracking analysis.

Conclusion: There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.
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http://dx.doi.org/10.1111/nep.12867DOI Listing
November 2017

Mending a broken heart but breaking the kidney.

Nephrology (Carlton) 2016 Oct;21(10):812-20

School of Medicine, Flinders University, Adelaide, South Australia, Australia.

The incidence of acute kidney injury (AKI) is a frequent and serious complication of cardiac surgery. In 2013, 95% of cardiac surgical procedures performed in Australia and New Zealand used cardiopulmonary bypass (CPB). AKI following CPB is well known, yet the perioperative factors contributing to its development are incompletely understood. AKI following CPB has significant implications on both short-term and long-term outcomes. The techniques for conducting CPB have evolved, moving towards evidence-based practice; however, there is still no generally accepted definition of optimal perfusion and its conduct. This review examines the current incidence of AKI following cardiac surgery and the short-term and longer-term effects of AKI on morbidity and mortality. The purpose of this review is to discuss the perioperative risk factors related to CPB and their contribution to the development of AKI. This review will also discuss outcomes in regard to off-pump cardiac surgery, the role of remote ischaemic preconditioning on AKI and outcomes in patients with chronic renal failure undergoing cardiac surgery.
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http://dx.doi.org/10.1111/nep.12799DOI Listing
October 2016

A single-nucleotide polymorphism in the MicroRNA-146a gene is associated with diabetic nephropathy and sight-threatening diabetic retinopathy in Caucasian patients.

Acta Diabetol 2016 Aug 21;53(4):643-50. Epub 2016 Mar 21.

Department of Ophthalmology, Flinders Medical Centre, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia.

Aims: This study aimed to investigate whether the single-nucleotide polymorphism (SNP) rs2910164 residing within microRNA-146a (miR-146a) is associated with diabetic microvascular complications diabetic nephropathy (DN), proliferative diabetic retinopathy (PDR) or diabetic macular oedema (DME) in either Caucasian patients with type 1 (T1DM) or type 2 (T2DM) diabetes mellitus.

Methods: Caucasian patients with T1DM (n = 733) or T2DM (n = 2215) were recruited from ophthalmology, renal and endocrine clinics in Australia and the UK. Patients with T2DM were required to have diabetes mellitus (DM) for at least 5 years and be on treatment with oral hypoglycaemic drugs or insulin. In total, 890 participants had DN (168 with T1DM and 722 with T2DM), 731 had PDR (251 with T1DM and 480 with T2DM) and 1026 had DME (170 with T1DM and 856 with T2DM). Participants were genotyped for SNP rs2910164 in miR-146a. Analyses investigating association were adjusted for relevant clinical covariates including age, sex, DM duration, HbA1c and hypertension.

Results: A significant association was found between the C allele of rs2910164 and DN in the T1DM group (OR 1.93; CI 1.23-3.03; P = 0.004), but no association found in the T2DM group (OR 1.05; CI 0.83-1.32; P = 0.691). In the subset of T2DM patients, the C allele was specifically associated with DME (OR 1.25; CI 1.03-1.53; P = 0.025). No association with DME was found in the T1DM group (OR 0.87; CI 0.54-1.42); P = 0.583), or with PDR for either type of DM.

Conclusions: Rs2910164 is significantly associated with microvascular complications DN in patients with T1DM and DME in patients with T2DM.
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http://dx.doi.org/10.1007/s00592-016-0850-4DOI Listing
August 2016

Human cytomegalovirus encoded microRNAs: hitting targets.

Expert Rev Anti Infect Ther 2015 28;13(12):1469-79. Epub 2015 Oct 28.

a School of Medicine , Flinders University , Adelaide , Australia.

Human cytomegalovirus (HCMV) infection is of particular concern in immunodeficient individuals notably transplant recipients, leading to increased morbidity and mortality. HCMV is predicted to encode multiple microRNAs (miRNAs) and several have been characterized in vitro. Furthermore, these miRNAs have been shown to target human and viral mRNAs. Pathways involved in human cellular targets have key roles in vesicle trafficking, immune evasion and cell cycle control. This demonstration of viral miRNA targets provides novel insights into viral pathogenesis. This review details the evidence for the existence of HCMV-encoded miRNA and their targets. HCMV miRNA in blood and other tissues is a potential diagnostic tool and blocking the effects of specific HCMV-encoded miRNA with sequence specific antagomirs is a potential new therapy.
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http://dx.doi.org/10.1586/14787210.2015.1106939DOI Listing
September 2016

Impaired Myocardial Oxygenation Response to Stress in Patients With Chronic Kidney Disease.

J Am Heart Assoc 2015 Aug 10;4(8):e002249. Epub 2015 Aug 10.

Department of Cardiovascular Medicine, Flinders Medical Centre, Bedford Park, South Australia, Australia (S.P., S.G., R.P., C.G.D.P., J.B.S.) School of Medicine, Flinders University, Bedford Park, South Australia, Australia (S.P., J.M.G., R.P., C.G.D.P., J.B.S.) South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia (S.P., S.G., J.B.S.).

Background: Coronary artery disease and left ventricular hypertrophy are prevalent in the chronic kidney disease (CKD) and renal transplant (RT) population. Advances in cardiovascular magnetic resonance (CMR) with blood oxygen level-dependent (BOLD) technique provides capability to assess myocardial oxygenation as a measure of ischemia. We hypothesized that the myocardial oxygenation response to stress would be impaired in CKD and RT patients.

Methods And Results: Fifty-three subjects (23 subjects with CKD, 10 RT recipients, 10 hypertensive (HT) controls, and 10 normal controls without known coronary artery disease) underwent CMR scanning. All groups had cine and BOLD CMR at 3 T. The RT and HT groups also had late gadolinium CMR to assess infarction/replacement fibrosis. The CKD group underwent 2-dimensional echocardiography strain to assess fibrosis. Myocardial oxygenation was measured at rest and under stress with adenosine (140 μg/kg per minute) using BOLD signal intensity. A total of 2898 myocardial segments (1200 segments in CKD patients, 552 segments in RT, 480 segments in HT, and 666 segments in normal controls) were compared using linear mixed modeling. Diabetes mellitus (P=0.47) and hypertension (P=0.57) were similar between CKD, RT, and HT groups. The mean BOLD signal intensity change was significantly lower in the CKD and RT groups compared to HT controls and normal controls (-0.89±10.63% in CKD versus 5.66±7.87% in RT versus 15.54±9.58% in HT controls versus 16.19±11.11% in normal controls, P<0.0001). BOLD signal intensity change was associated with estimated glomerular filtration rate (β=0.16, 95% CI=0.10 to 0.22, P<0.0001). Left ventricular mass index and left ventricular septal wall diameter were similar between the CKD predialysis, RT, and HT groups. None of the CKD patients had impaired global longitudinal strain and none of the RT group had late gadolinium hyperenhancement.

Conclusions: Myocardial oxygenation response to stress is impaired in CKD patients and RT recipients without known coronary artery disease, and unlikely to be solely accounted for by the presence of diabetes mellitus, left ventricular hypertrophy, or myocardial scarring. The impaired myocardial oxygenation in CKD patients may be associated with declining renal function. Noncontrast BOLD CMR is a promising tool for detecting myocardial ischemia in the CKD population.
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http://dx.doi.org/10.1161/JAHA.115.002249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599475PMC
August 2015

Myocardial perfusion is impaired in asymptomatic renal and liver transplant recipients: a cardiovascular magnetic resonance study.

J Cardiovasc Magn Reson 2015 Jul 10;17:56. Epub 2015 Jul 10.

Department of Cardiovascular Medicine, Flinders Medical Centre, Bedford Park, Adelaide, SA, 5042, Australia.

Background: Myocardial ischemia is a major cause of death in chronic kidney disease (CKD) patients, which can be caused by either epicardial or microvascular coronary artery disease (CAD). Although renal transplantation improves survival, cardiovascular disease remains a major cause of mortality in post renal transplant recipients, including those with no significant epicardial CAD pre-transplant. We aim to utilize stress cardiovascular magnetic resonance (CMR) and MR coronary angiography (MRCA) to assess silent myocardial ischemia and epicardial CAD in renal transplant recipients.

Methods: Forty-five subjects: twenty renal transplant (RT) with no known CAD, fifteen liver transplant (LT) controls without prior CKD and no known CAD, and ten hypertensive (HT) controls underwent stress perfusion CMR and MRCA.

Results: A total of 1308 myocardial segments (576 of RT, 468 of LT, and 264 of HT) were compared using mixed linear modeling. Left ventricular mass index, septal diameter and presence of diabetes mellitus were similar between the groups. The mean transmural MPRI was significantly lower in the RT and LT groups compared to HT controls (1.19 ± 0.50 in RT versus 1.23 ± 0.36 in LT versus 2.04 ± 0.32 in HT controls, p < 0.0001), in the subepicardium (1.33 ± 0.57 in RT versus 1.30 ± 0.33 in LT versus 2.01 ± 0.30 in HT controls, p < 0.001), and in the subendocardium (1.19 ± 0.54 in RT versus 1.11 ± 0.31 in LT versus 1.85 ± 0.34 in HT controls, p < 0.0001). Seven (35%) RT and five (33%) LT had significant epicardial CAD compared to none in HT controls, p = 0.12. One RT and one LT had LGE suggesting sub-endocardial infarction.

Conclusions: RT recipients have impaired myocardial perfusion independent of LVH or diabetes mellitus. The impaired myocardial perfusion in RT is similar to LT without prior renal disease, thus unlikely related to previous CKD. It is not fully explained by the presence of significant epicardial CAD, and therefore most likely represents microvascular CAD.
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http://dx.doi.org/10.1186/s12968-015-0166-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702380PMC
July 2015

Common Sequence Variation in the VEGFC Gene Is Associated with Diabetic Retinopathy and Diabetic Macular Edema.

Ophthalmology 2015 Sep 11;122(9):1828-36. Epub 2015 Jun 11.

Department of Ophthalmology, Flinders University, Flinders Medical Centre, Adelaide, South Australia, Australia.

Purpose: To investigate associations between single nucleotide polymorphisms (SNPs) in the VEGFC gene and the development of diabetic retinopathy (DR) in white patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM).

Design: Cross-sectional, case control study.

Participants: White patients with T1DM or T2DM (n = 2899) were recruited from ophthalmology and endocrine clinics in Australia and the United Kingdom. Patients with T2DM were required to have diabetes mellitus (DM) for at least 5 years and be receiving oral hypoglycemic treatment or insulin.

Methods: Participants were categorized according to their worst-ever DR grading, as having "no DR" (no history of nonproliferative DR [NPDR], proliferative DR [PDR], or diabetic macular edema [DME]) or "any DR" (further subclassified as NPDR or PDR, without or with DME). Clinical characteristics, glycemic control (hemoglobin A1c [HbA1c]), and presence of diabetic complications were determined at recruitment. Genotyping was performed for 13 VEGFC tag SNPs.

Main Outcome Measures: Odds ratios (ORs) were determined for associations with DR of VEGFC tag SNPs, individually and within haplotypes. Logistic regression was used to adjust for clinical covariates, including DM type, age, sex, DM duration, hypertension, nephropathy, HbA1c, and smoking.

Results: Participants with DM but "no DR" (n = 980) were compared with 1919 participants with DM and "any DR." Three VEGFC SNPs were associated with DR after logistic regression: rs17697419 (P = 0.001; OR, 0.67; confidence interval [CI], 0.52-0.85), rs17697515 (P = 0.001; OR, 0.62; CI, 0.47-0.81), and rs2333526 (P = 0.005; OR, 0.69; CI, 0.54-0.90). rs17697515 Was also specifically associated with DME in those with T2DM (P = 0.004; OR, 0.53; CI, 0.35-0.82). Haplotype analysis revealed 2 significantly associated haplotypes, both protective against DR development.

Conclusions: Significant associations were found between VEGFC tag SNPs (individually and within haplotypes) and the presence of any DR or DME in white participants with T1DM and T2DM.
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http://dx.doi.org/10.1016/j.ophtha.2015.05.004DOI Listing
September 2015

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

J Cell Sci 2015 Jan 20;128(2):225-31. Epub 2014 Nov 20.

School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, SA, Australia Centre for Molecular Pathology, University of Adelaide, Adelaide, SA 5005, Australia

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.
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http://dx.doi.org/10.1242/jcs.158451DOI Listing
January 2015

Hypoxia represses microRNA biogenesis proteins in breast cancer cells.

BMC Cancer 2014 Jul 22;14:533. Epub 2014 Jul 22.

Renal Department, Flinders Medical Centre, Flinders University School of Medicine, Bedford Park, Adelaide, South Australia 5042, Australia.

Background: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins.

Methods: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays.

Results: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production.

Conclusion: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs.
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http://dx.doi.org/10.1186/1471-2407-14-533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223767PMC
July 2014

The social determinants of health for people with type 1 diabetes that progress to end-stage renal disease.

Health Expect 2015 Dec 17;18(6):2513-21. Epub 2014 Jun 17.

Discipline of Public Health, School of Medicine, Flinders University, Adelaide, SA, Australia.

Objective: Self-management of type 1 diabetes over a lifetime is complex and challenging even in the best of circumstances, and the social environment can be a powerful determinant of health behaviours and outcomes. The aim of this study was to identify how social determinants of health can impact on the capacity of young people to manage their glycaemic control.

Methods: The findings emerged from a constructivist grounded theory approach through an in-depth examination of life course events that were recounted through qualitative interviews. The rich descriptive detail obtained from this enquiry locates common experiences and the context in which concordance with therapies occurs and health behaviours develop.

Results: This qualitative study of young people with type 1 diabetes who have developed end-stage renal disease demonstrates that there are many factors beyond individual control that can contribute to health outcomes. The social determinants of childhood environment, education, socio-economic status, gender and the culture of public health can contribute to disengagement from treatment regimens and the health-care system and to the development of microvascular complications at a comparatively young age.

Conclusion: These findings challenge the assumptions of health-care practitioners about individual responsibility and highlight the importance of considering how social determinants can shape lives, behaviours and health.
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http://dx.doi.org/10.1111/hex.12220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810664PMC
December 2015

Dipping your feet in the water: podocytes in urine.

Expert Rev Mol Diagn 2014 May 11;14(4):423-37. Epub 2014 Apr 11.

Department of Renal Medicine, Flinders Medical Centre, Adelaide, South Australia.

Podocyte injury and loss plays an important role in the pathogenesis and progression of many kidney diseases. Studies have shown that podocyte-related markers and products can be detected in the urine of patients with glomerular diseases such as focal segmental glomerulosclerosis, IgA nephropathy, lupus nephritis, diabetic nephropathy and pre-eclampsia. Therefore, detecting the loss of podocytes in the urine provides a useful noninvasive technique of gathering information about the disease type and/or activity of glomerular diseases. Currently, urine podocyte-related protein markers, mRNA, microRNA and exosomes have been used with varying degrees of success to study glomerular diseases. The determination of urinary podocyte loss may become an important noninvasive tool in the evaluation of glomerular diseases.
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http://dx.doi.org/10.1586/14737159.2014.908122DOI Listing
May 2014

Myocardial Ischemia Assessment in Chronic Kidney Disease: Challenges and Pitfalls.

Front Cardiovasc Med 2014 19;1:13. Epub 2014 Dec 19.

Department of Cardiovascular Medicine, Flinders Medical Centre , Bedford Park, SA , Australia ; School of Medicine, Flinders University , Bedford Park, SA , Australia.

Coronary artery disease is the leading cause of mortality and morbidity in the chronic kidney disease (CKD) population and often presents with atypical symptoms. Current diagnostic investigations of myocardial ischemia in CKD lack sensitivity and specificity or may have adverse effects. We present a case vignette and explore the challenges of diagnostic myocardial stress investigation in patients with CKD.
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http://dx.doi.org/10.3389/fcvm.2014.00013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668858PMC
December 2015

MicroRNAs: are they the missing link between hypoxia and pre-eclampsia?

Hypertens Pregnancy 2014 Feb 19;33(1):102-14. Epub 2013 Dec 19.

Department of Renal Medicine .

Pre-eclampsia is a multisystem disorder that occurs in the second half of pregnancy affecting 5% of pregnancies. It remains the leading cause of maternal and perinatal mortality and morbidity worldwide. Impaired placental implantation, hypoxia, endothelial dysfunction and systemic inflammation are thought to have a role in the pathogenesis of pre-eclampsia. MicroRNAs (miRNAs) are short non-coding RNAs. They are important regulators of gene expression and have been found to affect cell development, proliferation, differentiation and function. Specific patterns of miRNAs have been detected in the placenta and there is altered miRNA expression in the placenta of patients with pre-eclampsia to but their role in the pathogenesis remains unclear. Furthermore, deregulated miRNAs have also been reported in human villous trophoblasts during hypoxic stress. One of the more consistently elevated miRNAs by hypoxia and in the placenta of patients with pre-eclampsia is miR-210. Whether such miRNAs are bystander markers of hypoxia, or are directly involved in the pathogenesis of pre-eclampsia, needs to be clarified. There is potential for miRNAs to be used as predictors, markers or therapy in pre-eclampsia. This review provides current knowledge about miRNAs, particularly hypoxia-related miRNAs and the interaction of hypoxia, miRNAs and placenta in pre-eclampsia.
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http://dx.doi.org/10.3109/10641955.2013.832772DOI Listing
February 2014

Exosomes and the kidney: blaming the messenger.

Nephrology (Carlton) 2013 Jan;18(1):1-10

Department of Renal Medicine, Flinders Medical Centre, School of Medicine, Flinders University, Adelaide, South Australia, Australia.

Exosomes are membrane-bound vesicles of endosomal origin, present in a wide range of biological fluids, including blood and urine. They range between 30 and 100 nm in diameter, and consist of a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNA). Exosomes can act as extracellular vehicles by which cells communicate, through the delivery of their functional cargo to recipient cells, with many important biological, physiological and pathological implications. The exosome release pathway contributes towards protein secretion, antigen presentation, pathogen transfer and cancer progression. Exosomes and exosome-mediated signalling have been implicated in disease processes such as atherosclerosis, calcification and kidney diseases. Circulating levels of exosomes and extracellular vesicles can be influenced by the progression of renal disease. Advances in methods for purification and analysis of exosomes are leading to potential diagnostic and therapeutic avenues for kidney diseases. This review will focus on biophysical properties and biogenesis of exosomes, their pathophysiological roles and their potential as biomarkers and therapeutics in kidney diseases.
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http://dx.doi.org/10.1111/nep.12005DOI Listing
January 2013

Hypoxic enhancement of exosome release by breast cancer cells.

BMC Cancer 2012 Sep 24;12:421. Epub 2012 Sep 24.

Renal Department, Flinders Medical Centre, Flinders University School of Medicine, Bedford Park, South Australia, 5042, Australia.

Background: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.

Methods: Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.

Results: Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.

Conclusions: These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.
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http://dx.doi.org/10.1186/1471-2407-12-421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488584PMC
September 2012