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ABSTRACT: Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.
The Journal of Immunology 06/2012; 189(2):551-7. · 5.79 Impact Factor
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ABSTRACT: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome.
To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes.
We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7).
Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF).
The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.
American Journal of Respiratory and Critical Care Medicine 03/2010; 181(12):1367-75. · 11.08 Impact Factor
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ABSTRACT: This chapter covers the breakdown of the process of angiogenesis into simple assays to measure discrete endothelial cell functions. The techniques described are suitable for studying stimulators or inhibitors of angiogenesis and determining which aspect of the process is modulated. The procedures outlined are robust and straightforward but cannot cover the complexity of the angiogenic process as a whole, incorporating as it does myriad positive and negative signals, three-dimensional interactions with host tissues and many accessory cells, including fibroblasts, macrophages, pericytes, and platelets. The extent to which in vitro assays predict responses in vivo (e.g., wound healing, tumor angiogenesis, or surrogate techniques such as Matrigel plugs, sponge implants, corneal assays, etc.) remains to be determined.
Methods in molecular biology (Clifton, N.J.) 02/2009; 467:159-81.
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ABSTRACT: We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2).
A full-length cDNA was isolated and predicted ECSM2 to be a putative 205-amino acid transmembrane protein that bears no homology to any known protein. Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown. Knockdown of ECSM2 expression in human umbilical vein endothelial cells using siRNA resulted in both reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane, both processes involved in angiogenesis. A yeast 2 hybrid analysis using the ECSM2 intracellular domain identified filamin A as an interacting protein. This interaction was confirmed by precipitation of filamin-A from endothelial cell lysates by a GST-tagged intracellular domain of ECSM2.
This study is the first to characterize a novel cell surface protein ECSM2 that regulates endothelial chemotaxis and tube formation, and interacts with filamin A. These studies implicate a role for ECSM2 in angiogenesis via modulation of the actin cytoskeleton.
Arteriosclerosis Thrombosis and Vascular Biology 07/2008; 28(9):1640-6. · 6.37 Impact Factor
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ABSTRACT: In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were in silico subtracted from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers.
From an endothelial cDNA library analysis, 431 genes were significantly up regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen.
This approach represents a significant advance on earlier work in its ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory. These methods are of value and available http://www.compbio.ox.ac.uk/data/diffex.html to researchers that are interested in the analysis of transcriptomic data.
BMC Genomics 02/2008; 9:153. · 4.07 Impact Factor
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Swee Y Sharp,
Kathy Boxall,
Martin Rowlands,
Chrisostomos Prodromou,
S Mark Roe,
Alison Maloney,
Marissa Powers,
Paul A Clarke,
Gary Box, Sharon Sanderson, [......],
Kwai-Ming J Cheung,
Karen Ball,
Angela Hayes,
Florence Raynaud,
Richard Marais,
Laurence Pearl,
Sue Eccles,
Wynne Aherne,
Edward McDonald,
Paul Workman
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ABSTRACT: The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.
Cancer Research 04/2007; 67(5):2206-16. · 7.86 Impact Factor
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ABSTRACT: Heat shock protein 90 (Hsp90) is a molecular chaperone involved in maintaining the correct conformation and stability of its client proteins. This study investigated the effects of Hsp90 inhibitors on client protein expression and key cellular functions required for tumor angiogenesis. The benzoquinone ansamycin Hsp90 inhibitors geldanamycin and/or its derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin inhibited production of vascular endothelial growth factor (VEGF)-A by tumor cells and blocked proliferative responses of human endothelial cells at nanomolar concentrations. 17-AAG also significantly reduced endothelial cell migration, tubular differentiation, invasion through Matrigel, and secretion of urokinase-type plasminogen activator at concentrations at or below those that inhibited proliferation. 17-AAG significantly reduced expression of VEGF receptor (VEGFR)-2 and established Hsp90 client proteins in human endothelial cells in vitro as well as in mouse vena cava, mesenteric vessels, and blood vessels within human tumor xenografts in vivo; this was associated with decreased tumor microvessel density. Finally, we showed for the first time that Hsp90 inhibitors also reduce expression of VEGFR-1 on human vascular endothelial cells, VEGFR-3 on lymphatic endothelial cells in vitro, and all three VEGFRs on mouse vasculature in vivo. Thus, we identify Hsp90 inhibitors as important regulators of many aspects of tumor angiogenesis (and potentially lymphangiogenesis) and suggest that they may provide therapeutic benefit not only via direct effects on tumor cells but also indirectly by inhibiting the production of angiogenic cytokines and responses of activated endothelial cells that contribute to tumor progression and metastasis.
Molecular Cancer Therapeutics 04/2006; 5(3):522-32. · 5.23 Impact Factor
