ughlin Center for Molecular Medicine

Toronto, Canada

ughlin Center for Molecular Medicine

Toronto, Canada
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Nyende S.,Jinja Regional Referral Hospital | Conroy A.,University of Toronto | Opoka R.O.,Makerere University | Namasopo S.,Jinja Regional Referral Hospital | And 8 more authors.
Trials | Year: 2015

Background: Pneumonia is a leading cause of childhood mortality globally. Oxygen therapy improves survival in children with pneumonia, yet its availability remains limited in many resource-constrained settings where most deaths occur. Solar-powered oxygen delivery could be a sustainable method to improve oxygen delivery in remote areas with restricted access to a supply chain of compressed oxygen cylinders and reliable electrical power. Methods/Design: This study is a randomized controlled trial (RCT). Solar-powered oxygen delivery systems will be compared to a conventional method (oxygen from cylinders) in patients with hypoxemic respiratory illness. Enrollment will occur at two sites in Uganda: Jinja Regional Referral Hospital and Kambuga District Hospital. The primary outcome will be the length of hospital stay. Secondary study endpoints will be mortality, duration of supplemental oxygen therapy (time to wean oxygen), proportion of patients successfully oxygenated, delivery system failure, cost, system maintenance and convenience. Discussion: The RCT will provide useful data on the feasibility and noninferiority of solar-powered oxygen delivery. This technological innovation uses freely available inputs, the sun and the air, to oxygenate children with pneumonia, and can be applied "off the grid" in remote and/or resource-constrained settings where most pneumonia deaths occur. If proven successful, solar-powered oxygen delivery systems could be scaled up and widely implemented for impact on global child mortality. Trial registration: registration number NCT0210086. (date of registration: 27 March, 2014). © 2015 Nyende et al.

Jurasz P.,St Michaels Hospital | Jurasz P.,University of Alberta | Yurkova N.,St Boniface General Hospital Research Center | Kirshenbaum L.,St Boniface General Hospital Research Center | And 4 more authors.
Angiogenesis | Year: 2011

Hypoxia results in the apoptotic death of myocytes, neurons, and epithelial cells, through the actions of Bcl-2 and Nineteen kilodalton Interacting Protein-3 (BNIP3). On the contrary, endothelial cells are especially adept at surviving conditions of oxygen deprivation via up-regulation of vascular endothelial growth factor (VEGF) the most potent endothelial survival factor. Both VEGF and BNIP3 expression are transcriptionally regulated by hypoxia inducible factor and may antagonize each other's affects in endothelial cells (ECs). Since factors that promote and inhibit apoptosis may be expressed at the same time in endothelial cells under hypoxic conditions, we decided to investigate whether VEGF and BNIP3 have opposing actions in endothelial cells. Human microvascular endothelial cells were exposed to hypoxic conditions in a Billups-Rothenburg chamber. Under hypoxic conditions BNIP3 expression by endothelial cells increased as measured by real-time PCR and immunoblot. After 48 h of hypoxia, EC apoptosis was assessed by flow cytometry and was lower than in corresponding normoxia serum starved controls. The increase in EC survival under hypoxic conditions corresponded with an increase in the expression of VEGF. Under normoxic conditions adenoviral BNIP3 over-expression promoted apoptosis of ECs; however, recombinant VEGF (100 pg/ml) antagonized the BNIP3 apoptosis promoting affects. SiRNA knockdown of VEGF expression by hypoxic ECs resulted in increased apoptosis with a concomitant increase in BNIP3 expression. SiRNA knockdown of BNIP3 expression by hypoxic ECs reduced the increase in EC apoptosis as a result of VEGF knockdown. We conclude that under hypoxic conditions VEGF counteracts and masks the apoptosis promoting affects of BNIP3. © 2010 Springer Science+Business Media B.V.

Jurasz P.,St Michaels Hospital | Courtman D.,St Michaels Hospital | Courtman D.,University of Toronto | Babaie S.,St Michaels Hospital | And 5 more authors.
Pharmacology and Therapeutics | Year: 2010

Pulmonary arterial hypertension (PAH) is a progressive and lethal disease that has a strong female predominance, often affecting the young. Current therapies are mostly vasodilator agents, and while mainly addressing the endothelial dysfunction that has been widely reported in this disease, they may be less effective in treating arterial remodeling. The lung pathology of PAH is characterized by medial hypertrophy and intimal hyperplasia of muscular arteries as well as plexiform lesions, that lead to a widespread narrowing or obliteration of the pulmonary arteriolar bed. However, the pathogenesis of the functional and structural abnormalities of the lung microcirculation in PAH is poorly understood. Perhaps the greatest advancement in the last decade has been the discovery of the "PAH gene," bone morphogenetic receptor 2 (Bmpr2), however how the loss-of-function mutations in Bmpr2 lead to PAH is unclear. The BMPR2 pathway has recently been shown to mediate survival signaling in endothelial cells (EC), and thus the reduced activity will favor endothelial apoptosis, likely increasing the susceptibility to endothelial injury in response to various environmental triggers. EC apoptosis has been implicated as an initiating event in experimental PAH, leading either directly to the degeneration of pre-capillary arterioles or to the selection of hyperproliferative, apoptosis-resistant ECs that may contribute to "angioproliferative" plexiform lesions. The idea that EC apoptosis may play a central role in the initiation and progression of PAH suggests that therapeutic strategies aimed at endothelial repair and regeneration of ECs may be uniquely effective in the treatment of this disease. Preclinical evaluation and validation on the use of endothelial progenitor cells (EPCs) for the prevention and reversal of experimental PAH is reviewed and the design of a "first in man" clinical trial to assess the safety and efficacy of a combined EPC and endothelial NO-synthase gene therapy for patients that are refractory to standard therapies is discussed. © 2010 Elsevier Inc. All rights reserved.

Jin L.L.,ughlin Center for Molecular Medicine | Jin L.L.,University of Toronto | Jin L.L.,Hospital for Sick Children | Tong J.,ughlin Center for Molecular Medicine | And 12 more authors.
Journal of Proteome Research | Year: 2010

The stoichiometry of protein phosphorylation at specific amino acid sites may be used to infer on the significance of the modification, and its biological function in the cell. However, detection and quantification of phosphorylation stoichiometry in tissue remain a significant challenge. Here we describe a strategy for highly sensitive, label-free quantification of protein phosphorylation stoichiometry. Method development included the analysis of synthetic peptides in order to determine constants to relate the mass spectrometry signals of cognate peptide/phosphopeptide pairs, and the detection of the cognate peptides by using high resolution Fourier Transform mass spectrometry (FTMS) and selected reaction monitoring mass spectrometry (SRM). By analyzing extracted ion currents by FTMS, the phosphorylation stoichiometries of two tyrosine residues (tyrosine-194 and tyrosine-397) in the protein tyrosine kinase Lyn were determined in transfected human HEK293T cells and two cultured human multiple myeloma strains. To achieve high sensitivity to measure phosphorylation stoichiometry in tissue, SRM methods were developed and applied for the analysis of phosphorylation stoichiometries of Lyn phospho-sites in multiple myeloma xenograft tumors. Western immuno-blotting was used to verify mass spectrometry findings. The SRM method has potential applications in analyzing clinical samples wherein protein phosphorylation stoichiometries may represent important pharmacodynamic biomarkers. © 2010 American Chemical Society.

Wang C.,University of Toronto | Wang C.,Tianjin Institute of Urological Surgery | Tao W.,Harbin Medical University | Wang Y.,University of Toronto | And 9 more authors.
European Urology | Year: 2010

Background: Prostate cancer (PCa) is the most common malignancy in males in Western countries. Despite improvements in standard treatments such as surgery, radiotherapy, and chemotherapy, many patients still progress to advanced stages. Recent clinical trials have shown encouraging results regarding the application of angiogenic inhibitors in the treatment of angiogenesis-dependent diseases, paving the way for novel PCa therapies. Objective: To identify new antiangiogenic compounds and examine their therapeutic potential in models of PCa. Design, setting, and participants: We performed a chemical genetic screen in developing zebrafish embryos to identify small molecules inhibiting zebrafish angiogenesis. Transgenic Tg(flk1:EGFP) zebrafish embryos were used in the screening of the Spectrum Collection compound library. Subsequently, the antiangiogenic mechanism of an identified lead compound, rosuvastatin, was studied by conducting endothelial cell function assays and examining antitumor efficacy in a PCa xenograft mouse model. Measurements, results and limitations: Seven lead compounds, including isorotenone, dihydromunduletone, aristolochic acid, simvastatin, mevastatin, lovastatin, and rosuvastatin, were identified to inhibit the growth of the zebrafish intersegmental vessels. Of these seven leads, rosuvastatin was further evaluated for its antiangiogenic mechanism and anticancer efficacy. Rosuvastatin decreased the viability of the human umbilical endothelial cells (HUVECs) (one-half inhibitory concentration: 5.87 μM) by inducing G1 phase arrest and promoting apoptosis. Moreover, rosuvastatin remarkably inhibited the migration of HUVECs and dose-dependently inhibited the HUVEC capillary-like tube formation in vitro. Furthermore, we demonstrated that rosuvastatin suppressed xenografted PPC-1 prostate tumors in nonobese diabetic severe combined immunodeficiency (NOD-SCID) mice associated with decreased microvessel density (MVD) and tumor cell apoptosis. Conclusions: Collectively, our data suggest that rosuvastatin possesses antiangiogenic and antitumor activities and has therapeutic potential for the treatment of PCa. This study represents the first zebrafish antiangiogenic chemical genetic screen to identify a lead compound that targets cancer angiogenesis. © 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Turnbull H.,University of Alberta | Conroy A.,University of Toronto | Opoka R.O.,Makerere University | Namasopo S.,Jinja Regional Referral Hospital | And 6 more authors.
International Journal of Tuberculosis and Lung Disease | Year: 2016

Setting: A resource-limited paediatric hospital in Uganda. OBJECTIVE: Pneumonia is a leading cause of child mortality worldwide. Access to life-saving oxygen therapy is limited in many areas. We designed and implemented a solar-powered oxygen delivery system for the treatment of paediatric pneumonia. DESIGN: Proof-of-concept pilot study. A solar-powered oxygen delivery system was designed and piloted in a cohort of children with hypoxaemic illness. RESULT S: The system consisted of 25 × 80 W photovoltaic solar panels (daily output 7.5 kWh [range 3.8-9.7kWh]), × 3 220 Ah batteries and a 300 W oxygen concentrator (output up to 5 l/min oxygen at 88% [±2%] purity). A series of 28 patients with hypoxaemia were treated with solar-powered oxygen. Immediate improvement in peripheral blood oxygen saturation was documented (median change12% [range 5-15%], P < 0.0001). Tachypnoea, tachycardia and composite illness severity score improved over the first 24 h of hospitalisation (P < 0.01 for all comparisons). The case fatality rate was 6/28 (21%). The median recovery times to sit, eat, wean oxygen and hospital discharge were respectively 7.5 h, 9.8 h, 44 h and 4 days. CONCLUS ION: Solar energy can be used to concentrate oxygen from ambient air and oxygenate children with respiratory distress and hypoxaemia in a resourcelimited setting. © 2016 The Union.

Hawkes M.,University of Toronto | Hawkes M.,The Hospital for Sick Children | Opoka R.O.,Makerere University | Namasopo S.,Jinja Regional Referral Hospital | And 15 more authors.
Trials | Year: 2011

Background: Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.Methods/Design: This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.Discussion: Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.Trial Registration: Identifier: NCT01255215. © 2011 Hawkes et al; licensee BioMed Central Ltd.

Hawkes M.,University of Toronto | Li X.,University of Toronto | Crockett M.,University of Toronto | Diassiti A.,University of Toronto | And 9 more authors.
Microbes and Infection | Year: 2010

Tuberculosis (Mtb) and malaria are among the most important infectious causes of morbidity and mortality worldwide, causing an estimated 1.5 million and 1 million deaths every year, respectively. Here we demonstrate a biological interaction between malaria and mycobacteria in vitro and in vivo. Murine macrophages co-incubated with Plasmodium falciparum parasitized erythrocytes demonstrated impaired control of intracellular Mtb replication, and reduced production of reactive nitrogen species in response to mycobacteria. Infection of C57BL/6 mice with Plasmodium species exacerbated the course of acute mycobacterial infection (57% increase in peak splenic CFU, p = 0.043 for difference over time course of infection), induced disruption of the structural integrity of established granulomas, and caused reactivation of latent mycobacterial infection (2.6-fold increase in peak splenic CFU, p = 0.016 for difference over time course of reactivation). Malaria pigment deposition within the granulomas of co-infected mice suggested that the influx of dysfunctional hemozoin-laden monocytes into the locus of mycobacterial control may contribute to impaired containment of mycobacteria. Collectively, these results point to malaria-induced dysregulation of innate and adaptive anti-mycobacterial defences, and suggest that the interaction of these globally important pathogens may potentiate Mtb infection and transmission. © 2010 Institut Pasteur.

Hawkes M.,University of Toronto | Li X.,University of Toronto | Crockett M.,University of Toronto | Diassiti A.,University of Toronto | And 12 more authors.
BMC Infectious Diseases | Year: 2010

Background: Members of the CD36 scavenger receptor family have been implicated as sensors of microbial products that mediate phagocytosis and inflammation in response to a broad range of pathogens. We investigated the role of CD36 in host response to mycobacterial infection.Methods: Experimental Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection in Cd36+/+and Cd36-/-mice, and in vitro co-cultivation of M. tuberculosis, BCG and M. marinum with Cd36+/+and Cd36-/-murine macrophages.Results: Using an in vivo model of BCG infection in Cd36+/+and Cd36-/-mice, we found that mycobacterial burden in liver and spleen is reduced (83% lower peak splenic colony forming units, p < 0.001), as well as the density of granulomas, and circulating tumor necrosis factor (TNF) levels in Cd36-/-animals. Intracellular growth of all three mycobacterial species was reduced in Cd36-/-relative to wild type Cd36+/+macrophages in vitro. This difference was not attributable to alterations in mycobacterial uptake, macrophage viability, rate of macrophage apoptosis, production of reactive oxygen and/or nitrogen species, TNF or interleukin-10. Using an in vitro model designed to recapitulate cellular events implicated in mycobacterial infection and dissemination in vivo (i.e., phagocytosis of apoptotic macrophages containing mycobacteria), we demonstrated reduced recovery of viable mycobacteria within Cd36-/-macrophages.Conclusions: Together, these data indicate that CD36 deficiency confers resistance to mycobacterial infection. This observation is best explained by reduced intracellular survival of mycobacteria in the Cd36-/-macrophage and a role for CD36 in the cellular events involved in granuloma formation that promote early bacterial expansion and dissemination. © 2010 Hawkes et al; licensee BioMed Central Ltd.

PubMed | ughlin Center for Molecular Medicine and Princess Margaret Cancer Center
Type: | Journal: Cancer medicine | Year: 2016

Lenalidomide in combination with dexamethasone (Len-dex) represents a highly effective treatment in relapsed/refractory multiple myeloma (RRMM) patients. However, an increased risk of secondary primary malignancies (SPMs), including myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) has been described in patients receiving lenalidomide. In order to assess the incidence and features of this complication, we reviewed 195 patients with RRMM treated with Len-dex at our institution. The median follow-up time from diagnosis of MM was 73months (10-234months) and from initiation of Len-dex was 19months (1-104months). The median duration of Len-dex for all patients was 7.8months (range 1-90months). The incidence rate (IR) for all SPMs from start of Len-dex was 2.37 per 100 patient-years, which reflected an IR of 1.29 for MDS/AML and 1.08 for nonhematologic malignancies (NHM). MDS was the most common SPM noted. The cumulative IR of SPM at 5years was 1.54% from the time of MM diagnosis and 5.24% from starting Len-dex. Multivariable cumulative incidence of SPM analysis identified older age (P=0.005) and prior number of regimens (P=0.026) as adverse risk factors. We found more concomitant G-CSF use (P=0.029) in patients with MDS/AML, however, causal association is not clear. The progression-free survival after Len-dex was the longest for patients in MDS/AML group, and the 5-year overall survival did not differ among groups. Although the rate of SPM was relatively low with Len-dex, concomitant G-CSF should be used judiciously and patients receiving this regimen should be observed for the development of this complication.

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