School of Biomedical Sciences - Research Publications
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ItemWhole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana(PUBLIC LIBRARY SCIENCE, 2015-03-01)Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.
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ItemDose planning objectives in anal canal cancer IMRT: the TROG ANROTAT experience(WILEY-BLACKWELL, 2015-06-01)INTRODUCTION: Intensity modulated radiotherapy (IMRT) is ideal for anal canal cancer (ACC), delivering high doses to irregular tumour volumes whilst minimising dose to surrounding normal tissues. Establishing achievable dose objectives is a challenge. The purpose of this paper was to utilise data collected in the Assessment of New Radiation Oncology Treatments and Technologies (ANROTAT) project to evaluate the feasibility of ACC IMRT dose planning objectives employed in the Australian situation. METHODS: Ten Australian centres were randomly allocated three data sets from 15 non-identifiable computed tomography data sets representing a range of disease stages and gender. Each data set was planned by two different centres, producing 30 plans. All tumour and organ at risk (OAR) contours, prescription and dose constraint details were provided. Dose-volume histograms (DVHs) for each plan were analysed to evaluate the feasibility of dose planning objectives provided. RESULTS: All dose planning objectives for the bone marrow (BM) and femoral heads were achieved. Median planned doses exceeded one or more objectives for bowel, external genitalia and bladder. This reached statistical significance for bowel V30 (P = 0.04), V45 (P < 0.001), V50 (P < 0.001), external genitalia V20 (P < 0.001) and bladder V35 (P < 0.001), V40 (P = 0.01). Gender was found to be the only significant factor in the likelihood of achieving the bowel V50 (P = 0.03) and BM V30 constraints (P = 0.04). CONCLUSION: The dose planning objectives used in the ANROTAT project provide a good starting point for ACC IMRT planning. To facilitate clinical implementation, it is important to prioritise OAR objectives and recognise factors that affect the achievability of these objectives.
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ItemFT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy(PUBLIC LIBRARY SCIENCE, 2015-07-29)Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.
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ItemInhibition of NOX1/4 with GKT137831: a potential novel treatment to attenuate neuroglial cell inflammation in the retina(BIOMED CENTRAL LTD, 2015-07-30)BACKGROUND: Inflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons. METHODS: Ischemic retinopathy was induced in Sprague-Dawley rats by exposure to 80 % O2 cycled with 21 % O2 for 3 h per day from postnatal day (P) 0 to P11, followed by room air (P12 to P18). GKT137831 was administered P12 to P18 (60 mg/kg, subcutaneous) and comparisons were to room air controls. Retinal inflammation was examined by measuring leukocyte adherence to the retinal vasculature, ionized calcium-binding adaptor protein-1-positive microglia/macrophages, and the mRNA and protein levels of key inflammatory factors involved in retinal disease. Damage to Müller cells was evaluated by quantitating glial fibrillary acidic protein-positive cells and vascular leakage with an albumin ELISA. To verify the anti-inflammatory actions of GKT137831 on glia and neurons involved in ischemic retinopathy, primary cultures of rat retinal microglia, Müller cells, and ganglion cells were exposed to the in vitro counterpart of ischemia, hypoxia (0.5 %), and treated with GKT137831 for up to 72 h. ROS levels were evaluated with dihydroethidium and the protein and gene expression of inflammatory factors with quantitative PCR, ELISA, and a protein cytokine array. RESULTS: In the ischemic retina, GKT137831 reduced the increased leukocyte adherence to the vasculature, the pro-inflammatory phenotype of microglia and macroglia, the increased gene and protein expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and leukocyte adhesion molecules as well as vascular leakage. In all cultured cell types, GKT137831 reduced the hypoxia-induced increase in ROS levels and protein expression of various inflammatory mediators. CONCLUSIONS: NOX1/4 enzyme inhibition with GKT137831 has potent anti-inflammatory effects in the retina, indicating its potential as a treatment for a variety of vision-threatening retinopathies.
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ItemInhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin(IMPACT JOURNALS LLC, 2015-05-10)Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.
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ItemThe peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of the glutamate transporter excitatory amino acid transporter 2 (EAAT2) in human glioblastoma cells(IMPACT JOURNALS LLC, 2015-08-28)Glioma cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of the excitatory amino acid transporter 2 (EAAT2) expression maintains high extracellular glutamate levels in the glioma microenvironment, causing excitotoxicity to surrounding parenchyma. Not only does this contribute to the survival and proliferation of glioma cells, but is involved in the pathophysiology of tumour-associated epilepsy (TAE). We investigated the role of the peroxisome proliferator activated receptor gamma (PPARγ) agonist pioglitazone in modulating EAAT2 expression in glioma cells. We found that EAAT2 expression was increased in a dose dependent manner in both U87MG and U251MG glioma cells. Extracellular glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in both U87MG and U251MG cells at a concentration of ≥ 30 μM pioglitazone (p < 0.05). The PPARγ antagonist GW9662 inhibited the effect of pioglitazone on extracellular glutamate levels, indicating PPARγ dependence. In addition, pioglitazone significantly reduced cell viability of U87MG and U251MG cells at ≥ 30 μM and 100 μM (p < 0.05) respectively. GW9662 also significantly reduced viability of U87MG and U251MG cells with 10 μM and 30 μM (p < 0.05) respectively. The effect on viability was partially dependent on PPARγ activation in U87MG cells but not U251MG cells, whereby PPARγ blockade with GW9662 had a synergistic effect. We conclude that PPARγ agonists may be therapeutically beneficial in the treatment of gliomas and furthermore suggest a novel role for these agents in the treatment of tumour associated seizures through the reduction in extracellular glutamate.
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ItemIdentification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers(ROCKEFELLER UNIV PRESS, 2015-06-29)Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid-related orphan receptor γt (RORγt), whereas RORγt(lo) MAIT cells predominantly express T-bet and produce IFN-γ. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.
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ItemGeneration of adaptive immune responses following influenza virus challenge is not compromised by pre-treatment with the TLR-2 agonist Pam(2)Cys(FRONTIERS MEDIA SA, 2015-06-05)Immunostimulatory agents provide a new category of anti-microbial agents that activate the host's innate immune system allowing control of viral and/or bacterial infections. The TLR-2 agonist PEG-Pam2Cys has been shown to mediate potent anti-viral activity against influenza viruses when administered prophylactically (1). Here, we demonstrate that the treatment of mice with PEG-Pam2Cys does not compromise their ability to generate adaptive immune responses following subsequent challenge with influenza virus. The antibody induced in mice pre-treated with Pam2Cys possessed hemagglutination-inhibiting activities and the CD8(+) T-cell responses that were elicited provided protection against heterologous viral challenge. In the absence of an effective influenza vaccine, an agent that provides immediate protection against the virus and does not compromise the induction of influenza-specific immunity on exposure to infectious virus provides an opportunity for population immunity to be achieved through natural exposure to virus.
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ItemTargeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance(PUBLIC LIBRARY SCIENCE, 2015-04-01)Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.
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ItemNo Preview AvailableAlcohol consumption for different periods in life, intake pattern over time and all-cause mortality(OXFORD UNIV PRESS, 2015-12-01)BACKGROUND: Conventionally, cohort studies have assessed the association between alcohol and all-cause mortality by using alcohol intake at enrolment. METHODS: In the Melbourne Collaborative Cohort Study, participants were asked about usual frequency and quantity of beverage-specific alcohol intake for 10-year periods starting at age 20 from which current, past and lifetime intakes were calculated. We used Cox regression to estimate hazard ratios for mortality for 39 577 participants of the Melbourne Collaborative Cohort Study aged 40-69 at baseline. RESULTS: After a mean follow-up of 15 years/person, we identified 4639 deaths. Associations between all-cause mortality and lifetime, current (baseline) and past intake were J shaped, with lower mortality at low intake (e.g. <40 g/day for men and 10 g/day for women using lifetime intake) and elevated mortality at higher intake. For men, consistent light-to-moderate drinking (>0-39/>0-39 g/day) from age 20 to baseline age was associated with a 16% lower mortality, while heavy drinking at both ages (≥80/≥40 and ≥40/0 g/day) was associated with higher mortality compared with stable abstinence. CONCLUSIONS: Our findings support a reduced mortality risk associated with low-dose drinking but also highlight a higher mortality risk for consistent heavy drinking from a young age.