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Wright K.T.,Keele University | Griffiths G.J.,Imagen Biotech | Johnson W.E.B.,Aston University
Journal of Biomolecular Screening | Year: 2010

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSCconditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ∼200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells. © 2010 Society for Biomolecular Sciences.


Grant
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2011-ITN | Award Amount: 4.31M | Year: 2012

The most prevalent chronic inflammatory diseases of humans are complex disorders of multifactorial aetiology influenced by genes, the environment and their interactions. Periodontitis (PD) and rheumatoid arthritis (RA) are two such chronic inflammatory diseases associated with significant morbidity and mortality, and have recently shown to have a bi-directional association. Moreover, the prevalence of both increases substantially with age, and given both the Europe-wide ageing population and the impact of both diseases upon the economy, health and quality of life, it is clear that novel and more cost effective approaches to management are urgently required. A key research goal of this project is to improve understanding of the pathogenesis of RA and PD and their inter-relationships, through the study of common risk factors linked to the activation of host and bacterial derived protein citrullination, which subsequently generates pro-inflammatory auto-antigens in the joints and periodontal tissues. Our vision is that enhanced biological understanding in this area will inform the future development of new approaches to disease prevention, early diagnosis and novel therapies. RAPID aims to provide a significant contribution to this by establishing a first class, dynamic training network of 12 partners and 5 associated partners for early career researchers who will be able to advance chronic inflammatory disease research by working across sectors and disciplines. The network is an interdisciplinary cooperative of medical and dental clinicians, epidemiologists, bio-scientists, industrial scientists, media and commercialization specialists. The aim of the Training Programme is to increase the knowledge base and experience of trainees in the different research areas and to develop their transferable skills for future careers in industry or academia, whilst advancing the field through new discovery.


PubMed | Imagen Biotech and University of Birmingham
Type: Journal Article | Journal: Journal of dental research | Year: 2015

Neutrophil extracellular traps (NETs) represent a novel paradigm in neutrophil-mediated immunity. NETs are believed to constitute a highly conserved antimicrobial strategy comprising decondensed nuclear DNA and associated histones that are extruded into the extracellular space. Associated with the web-like strands of DNA is an array of antimicrobial peptides (AMPs), which facilitate the extracellular destruction of microorganisms that become entrapped within the NETs. NETs can be released by cells that remain viable or following a unique form of programmed cell death known as NETosis, which is dependent on the production of reactive oxygen species (ROS) and the decondensing of the nuclear DNA catalyzed by peptidyl arginine deiminase-4. NETs are produced in response to a range of pathogens, including bacteria, viruses, fungi, and protozoa, as well as host-derived mediators. NET release is, however, not without cost, as the concomitant release of cytotoxic molecules can also cause host tissue damage. This is evidenced by a number of immune-mediated diseases, in which excess or dysfunctional NET production, bacterial NET evasion, and decreased NET removal are associated with disease pathogenesis. Periodontitis is the most prevalent infectious-inflammatory disease of humans, characterized by a dysregulated neutrophilic response to specific bacterial species within the subgingival plaque biofilm. Neutrophils are the predominant inflammatory cell involved in periodontitis and have previously been found to exhibit hyperactivity and hyperreactivity in terms of ROS production in chronic periodontitis patients. However, the contribution of ROS-dependent NET formation to periodontal health or disease remains unclear. In this focused review, we discuss the mechanisms, stimuli, and requirements for NET production; the ability of NET-DNA and NET-associated AMPs to entrap and kill pathogens; and the potential immunogenicity of NETs in disease. We also speculate on the potential role of NETs in the pathogenesis of periodontitis.


Bara J.J.,AO Research Institute Davos | Turner S.,Keele University | Roberts S.,Keele University | Griffiths G.,Imagen Biotech | And 3 more authors.
Experimental Cell Research | Year: 2015

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies. © 2015 Elsevier Inc.


PubMed | Keele University, AO Research Institute Davos and Imagen Biotech
Type: Journal Article | Journal: Experimental cell research | Year: 2015

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies.


Corfe B.M.,University of Sheffield | Kilner J.,University of Sheffield | Chowdry J.,University of Sheffield | Benson R.S.P.,Imagen Biotech | And 2 more authors.
Methods in Molecular Biology | Year: 2013

High content analysis (HCA; also referred to as high content biology) is a quantitative, automated, medium-throughput microscopy approach whereby cell images are segmented into relevant compartments (nuclei, cytoplasm) and the staining in each compartment quantified by computer algorithms. The extraction of quantitative information from the cell image generates a wealth of data which contributes significantly to the acceleration of drug discovery and biological research. Here we have adapted HCA to analyze protein acetylations in the cytoskeleton. This approach yields associative information on the link between acetylation and cytoskeletal organization. The protocol also describes optimization steps for cytoskeletal analysis and its application across different cell types, and HCA platforms. The methods described herein are readily adaptable to non-cytoskeletal acetylations and have been applied to the analysis of transcription factors. © Springer Science+Business Media, LLC 2013.


Waby J.S.,University of Sheffield | Waby J.S.,University of Hull | Chirakkal H.,University of Sheffield | Yu C.,University of Sheffield | And 4 more authors.
Molecular Cancer | Year: 2010

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC 50concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors. © 2010 Waby et al; licensee BioMed Central Ltd.


Grant
Agency: GTR | Branch: Innovate UK | Program: | Phase: Feasibility Study | Award Amount: 42.29K | Year: 2013

Beating cancer requires a treatment that kills cancer cells but leaves normal tissue unharmed. While this seems simple enough it is actually very difficult because cancer cells look so much like normal cells when it comes to treating with anti-cancer drugs. The drugs that are available to treat cancer all aim to target cancer cells in preference to normal cells but how good they are at doing this will depend on how toxic they are to the cancer of that particular patient. If a particular cancer is very sensitive to a drug then it can be used at lower doses thus minimizing nasty side effects. Similarly if the cancer is very resistant to a drug then using it will only damage normal tissue and make the situation worse. The currently funded project aims to develop a clever diagnostic assay that will tell the clincians which of the available drugs will be most effective for each patient thus personalising the treatment of cancer and so helping those who are afflicted with this terrible condition.


Imagen Biotech | Entity website

Investors Immunohistochemistry of normal mouse retina stained for vimentin (blue), synaptic vesicle marker SV2 (gold) and glycine transporter (red). Image courtesy of Duke University ...

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