Medicine (St Vincent's) - Research Publications
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ItemBronchial Smooth Muscle Cells of Asthmatics Promote Angiogenesis through Elevated Secretion of CXC-Chemokines (ENA-78, GRO-α, and IL-8)(PUBLIC LIBRARY SCIENCE, 2013-12-05)BACKGROUND: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors. OBJECTIVE: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors. METHODS: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay. RESULTS: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC. CONCLUSIONS: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC. IMPLICATIONS: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.
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ItemThe Expression and Activity of Cathepsins D, H and K in Asthmatic Airways(PUBLIC LIBRARY SCIENCE, 2013-03-06)Tumstatin is an anti-angiogenic collagen IV α3 fragment, levels of which are reduced in the airways of asthmatics. Its reduction may be due to the degradation by extracellular matrix (ECM) proteases. Cathepsins play a role in ECM remodelling, with cathepsin D, H and K (CTSD, CTSH and CTSK) being associated with lung diseases. CTSD modulates the NC1 domains of collagen molecules including tumstatin, while CTSH and CTSK are involved in ECM degradation. The role of these cathepsins in the regulation of tumstatin in the lung has not previously been examined. We demonstrated that CTSB, D, F, H, K, L and S mRNA was expressed in the airways. Quantification of immunohistochemistry showed that there is no difference in the global expression of CTSD, CTSH and CTSK between asthmatics and non-asthmatics. CTSD and CTSK, but not CTSH had the capacity to degrade tumstatin. No difference was observed in the activity of CTSD and H in bronchoalveolar lavage fluid of asthmatic and non-asthmatics, while CTSK was undetectable. This indicates that while CTSD possesses the potential to directly regulate tumstatin, and thus angiogenesis through this mechanism however, it is not likely to be involved in the dysregulation of tumstatin found in asthmatic airways.
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ItemInvestigation of herb-drug interactions with ginkgo biloba in women receiving hormonal treatment for early breast cancer(SPRINGER INTERNATIONAL PUBLISHING AG, 2013)Women receiving treatment for breast cancer commonly ingest herbal medicines. Little is known about the potential for herb-drug interactions in this population. The aim of this study is to investigate the effect of ginkgo biloba co-administration on the pharmacokinetics of tamoxifen, anastrozole and letrozole. This was a prospective open-label cross-over study in 60 women with early stage breast cancer taking either tamoxifen, anastrozole or letrozole (n=20/group). Participants received ginkgo biloba (EGb 761) for 3 weeks (120 mg twice daily). Trough concentrations of drugs were measured before and after ginkgo biloba treatment using LC-MS/MS. Toxicities were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events. Trough concentrations before and after treatment with ginkgo biloba were not significantly different for tamoxifen (93.5 ± 29.0, 86.5 ± 25.3 ng/mL; p=0.16), letrozole (91.1 ± 50.4, 89.6 ± 52.14 ng/mL; p=0.60) or anastrozole (29.1 ± 8.6, 29.1 ± 7.6 ng/mL; p=0.97). Ginkgo biloba was well tolerated, with no difference in toxicity during ginkgo biloba. Co-administration of ginkgo biloba does not significantly affect the pharmacokinetics of tamoxifen, anastrozole or letrozole. There was no difference in the toxicity profile of hormone therapy with ginkgo biloba use in women with early stage breast cancer.
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ItemLoss of TDAG51 results in mature-onset obesity, hepatic steatosis, and insulin resistance by regulating lipogenesis.(American Diabetes Association, 2013-01)Regulation of energy metabolism is critical for the prevention of obesity, diabetes, and hepatic steatosis. Here, we report an important role for the pleckstrin homology-related domain family member, T-cell death-associated gene 51 (TDAG51), in the regulation of energy metabolism. TDAG51 expression was examined during adipocyte differentiation. Adipogenic potential of preadipocytes with knockdown or absence of TDAG51 was assessed. Weight gain, insulin sensitivity, metabolic rate, and liver lipid content were also compared between TDAG51-deficient (TDAG51(-/-)) and wild-type mice. In addition to its relatively high expression in liver, TDAG51 was also present in white adipose tissue (WAT). TDAG51 was downregulated during adipogenesis, and TDAG51(-/-) preadipocytes exhibited greater lipogenic potential. TDAG51(-/-) mice fed a chow diet exhibited greater body and WAT mass, had reduced energy expenditure, displayed mature-onset insulin resistance (IR), and were predisposed to hepatic steatosis. TDAG51(-/-) mice had increased hepatic triglycerides and SREBP-1 target gene expression. Furthermore, TDAG51 expression was inversely correlated with fatty liver in multiple mouse models of hepatic steatosis. Taken together, our findings suggest that TDAG51 is involved in energy homeostasis at least in part by regulating lipogenesis in liver and WAT, and hence, may constitute a novel therapeutic target for the treatment of obesity and IR.
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ItemEnhanced lipid oxidation and maintenance of muscle insulin sensitivity despite glucose intolerance in a diet-induced obesity mouse model.(Public Library of Science (PLoS), 2013)BACKGROUND: Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity. METHODOLOGY: C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes. PRINCIPAL FINDINGS/CONCLUSIONS: A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle's insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.
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ItemMarkers of skeletal muscle mitochondrial function and lipid accumulation are moderately associated with the homeostasis model assessment index of insulin resistance in obese men.(Public Library of Science (PLoS), 2013)Lower skeletal muscle mitochondrial oxidative phosphorylation capacity (OXPHOS) and intramyocellular lipid (IMCL) accumulation have been implicated in the etiology of insulin resistance (IR) in obesity. The purpose of this study was to examine the impact of endurance exercise on biochemical and morphological measures of IMCL and mitochondrial content, and their relationship to IR in obese individuals. We examined mitochondrial content (subunit protein abundance and maximal activity of electron transport chain enzymes), IMCL/mitochondrial morphology in both subsarcolemmal (SS) and intermyofibrillar (IMF) regions by transmission electron microscopy, and intracellular lipid metabolites (diacylglycerol and ceramide) in vastus lateralis biopsies, as well as, the homeostasis model assessment index of IR (HOMA-IR) prior to and following twelve weeks of an endurance exercise regimen in healthy age- and physical activity-matched lean and obese men. Obese men did not show evidence of mitochondrial OXPHOS dysfunction, disproportionate IMCL content in sub-cellular regions, or diacylglycerol/ceramide accretion despite marked IR vs. lean controls. Endurance exercise increased OXPHOS and mitochondrial size and density, but not number of individual mitochondrial fragments, with moderate improvements in HOMA-IR. Exercise reduced SS IMCL content (size, number and density), increased IMF IMCL content, while increasing IMCL/mitochondrial juxtaposition in both regions. HOMA-IR was inversely associated with SS (r = -0.34; P = 0.051) and IMF mitochondrial density (r = -0.29; P = 0.096), IMF IMCL/mitochondrial juxtaposition (r = -0.30; P = 0.086), and COXII (r = -0.32; P = 0.095) and COXIV protein abundance (r = -0.35; P = 0.052); while positively associated with SS IMCL size (r = 0.28; P = 0.119) and SS IMCL density (r = 0.25; P = 0.152). Our findings suggest that once physical activity and cardiorespiratory fitness have been controlled for, skeletal muscle mitochondrial and IMCL profile in obesity may only partially contribute to the development of IR.
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ItemElevated mitochondrial oxidative stress impairs metabolic adaptations to exercise in skeletal muscle.(Public Library of Science (PLoS), 2013)Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 (+/-) mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 (+/-) mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 (+/-) mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.
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ItemA cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.(Public Library of Science (PLoS), 2013)BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
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ItemThe mesoscopic modeling of burst suppression during anesthesia(FRONTIERS MEDIA SA, 2013-04-30)The burst-suppression pattern is well recognized as a distinct feature of the mammalian electroencephalogram (EEG) waveform. Consisting of alternating periods of high amplitude oscillatory and isoelectric activity, it can be induced in health by deep anesthesia as well as being evoked by a range of pathophysiological processes that include coma and anoxia. While the electroencephalographic phenomenon and clinical implications of burst suppression have been studied extensively, the physiological mechanisms underlying its emergence remain unresolved and obscure. Because electroencephalographic bursting phenomenologically resembles the bursting observed in single neurons, it would be reasonable to assume that the theoretical insights developed to understand bursting at the cellular ("microscopic") level would enable insights into the dynamical genesis of bursting at the level of the whole brain ("macroscopic"). In general action potential bursting is the result of the interplay of two time scales: a fast time scale responsible for spiking, and a slow time scale that modulates such activity. We therefore hypothesize that such fast-slow systems dynamically underpin electroencephalographic bursting. Here we show that a well-known mean field dynamical model of the electroencephalogram, the Liley model, while unable to produce burst suppression unmodified, is able to give rise to a wide variety of burst-like activity by the addition of one or more slow systems modulating model parameters speculated to be major "targets" for anesthetic action. The development of a physiologically plausible theoretical framework to account for burst suppression will lead to a more complete physiological understanding of the EEG and the mechanisms that serve to modify ongoing brain activity necessary for purposeful behavior and consciousness.
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ItemKetamine, propofol, and the EEG: a neural field analysis of HCN1-mediated interactions(FRONTIERS MEDIA SA, 2013-04-05)Ketamine and propofol are two well-known, powerful anesthetic agents, yet at first sight this appears to be their only commonality. Ketamine is a dissociative anesthetic agent, whose main mechanism of action is considered to be N-methyl-d-aspartate (NMDA) antagonism; whereas propofol is a general anesthetic agent, which is assumed to primarily potentiate currents gated by γ-aminobutyric acid type A (GABAA) receptors. However, several experimental observations suggest a closer relationship. First, the effect of ketamine on the electroencephalogram (EEG) is markedly changed in the presence of propofol: on its own ketamine increases θ (4-8 Hz) and decreases α (8-13 Hz) oscillations, whereas ketamine induces a significant shift to beta band frequencies (13-30 Hz) in the presence of propofol. Second, both ketamine and propofol cause inhibition of the inward pacemaker current I h, by binding to the corresponding hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1) subunit. The resulting effect is a hyperpolarization of the neuron's resting membrane potential. Third, the ability of both ketamine and propofol to induce hypnosis is reduced in HCN1-knockout mice. Here we show that one can theoretically understand the observed spectral changes of the EEG based on HCN1-mediated hyperpolarizations alone, without involving the supposed main mechanisms of action of these drugs through NMDA and GABAA, respectively. On the basis of our successful EEG model we conclude that ketamine and propofol should be antagonistic to each other in their interaction at HCN1 subunits. Such a prediction is in accord with the results of clinical experiment in which it is found that ketamine and propofol interact in an infra-additive manner with respect to the endpoints of hypnosis and immobility.