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Grantholder Publications
Here are a selection of research articles published by our Grantholders in the past few months.
The Generation of Three-dimensional Tissue Structures with Mesenchymal Stem Cells
Genever P.G.
ATLA, 38(Suppl 1), 2010 Dec, 31-34
Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow moredetailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.
A Three-dimensional In Vitro Model of Breast Cancer: Toward Replacing the Need for Animal Experiments
Holliday D.L.
ATLA, 38(Suppl 1), 2010 Dec, 41-44
While the events leading to breast cancer development are not fully understood, a pre-invasive lesion, ductal carcinoma in situ (DCIS), is recognised as the main precursor of invasive disease. Understanding how pre-invasive lesions develop into invasive breast cancer is critical, since currently there is no way of predicting which tumours are likely to progress, leading to unnecessary surgical intervention or chemotherapy. With a lack of good animal models able to mimic DCIS progression in a laboratory setting, there has been a shift toward developing in vitro human models which more accurately represent human disease. By manipulating individual cell populations in these models, we can recapitulate the complex cellular interactions involved in disease progression, an essential step in understanding breast cancer behaviour.
Development and Application of Human Virtual Excitable Tissues and Organs: From Premature Birth to Sudden Cardiac Death
Holden A.V.
ATLA, 38(Suppl 1), 2010 Dec, 87-99
The electrical activity of cardiac and uterine tissues has been reconstructed by detailed computer models in the form of virtual tissues. Virtual tissues are biophysically and anatomically detailed, and represent quantitatively predictive models of the physiological and pathophysiological behaviours of tissue within an isolated organ. The cell excitation properties are quantitatively reproduced by equations that describe the kinetics of a few dozen proteins. These equations are derived from experimental measurements of membrane potentials, ionic currents, fluxes, and concentrations. Some of the measurements were taken from human cells and human ion channel proteins expressed in non-human cells, but they were mostly taken from cells of other animal species. Data on tissue geometry and architecture are obtained from the diffusion tensor magnetic resonance imaging of ex vivo or post mortem tissue, and are used to compute the spread of current in the tissue. Cardiac virtual tissues are well established and reproduce normal and pathological patterns of cardiac excitation within the atria or ventricles of the human heart. They have been applied to increase the understanding of normal cardiac electrophysiology, to evaluate the candidate mechanisms for re-entrant arrhythmias that lead to sudden cardiac death, and to predict the tissue level effects of mutant or pharmacologically-modified ion channels. The human full-term virtual uterus is still in development. This virtual tissue reproduces the in vitro behaviour of uterine tissue biopsies, and provides possible mechanisms for premature labour.
Development of a Cell-based Diabetic Wound Assay
Stephens P.
ATLA, 38(Suppl 1), 2010 Dec, 45-48
Chronic wounds require prolonged healthcare and adversely affect the quality of life of patients. They are particularly prominent in patients with diabetes, and their relative numbers are set to increase with the rise of diabetes within our population. Research is still needed to understand the factors leading to such wounds, to understand why they persist for such long periods of time, and also to develop new and efficacious treatment strategies. One problem facing this research is a lack of adequate animal models, as the current models do not truly reflect the human condition and often lead to much animal suffering. Hence, over the past four years, our group has been trying to develop a human-based in vitro diabetic wound model, which could be used as a high-throughput screening system to pre-screen potential chronic diabetic wound healing agents and to reduce unnecessary animal pain and suffering. To this end, we have isolated healthy and diseased skin fibroblasts from patient tissue biopsies. Crucially, to create a cell reporter system that can be widely used in the future, the cells were immortalised in order to escape senescence. By using microarray analysis, gene expression pattern differences have been identified between healthy and diseased cells, and disease-specific ‘reporter’ genes have been selected for further studies. The promoters of these reporter genes have been coupled to fluorescent reporter constructs and inserted back into the diseased fibroblasts, so that we now have proof-of-concept for a real-time diabetic reporter system for future exploitation.
Neuronal network pharmacodynamics of GABAergic modulation in the human cortex determined using pharmaco-magnetoencephalography.
Hall SD, Barnes GR, Furlong PL, Seri S, Hillebrand A.
Hum Brain Mapp. 2010 Apr;31(4):581-94.
Neuronal network oscillations are a unifying phenomenon in neuroscience research, with comparable measurements across scales and species. Cortical oscillations are of central importance in the characterization of neuronal network function in health and disease and are influential in effective drug development. Whilst animal in vitro and in vivo electrophysiology is able to characterize pharmacologically induced modulations in neuronal activity, present human counterparts have spatial and temporal limitations. Consequently, the potential applications for a human equivalent are extensive. Here, we demonstrate a novel implementation of contemporary neuroimaging methods called pharmaco-magnetoencephalography. This approach determines the spatial profile of neuronal network oscillatory power change across the cortex following drug administration and reconstructs the time course of these modulations at focal regions of interest. As a proof of concept, we characterize the nonspecific GABAergic modulator diazepam, which has a broad range of therapeutic applications. We demonstrate that diazepam variously modulates theta (4-7 Hz), alpha (7-14 Hz), beta (15-25 Hz), and gamma (30-80 Hz) frequency oscillations in specific regions of the cortex, with a pharmacodynamic profile consistent with that of drug uptake. We examine the relevance of these results with regard to the spatial and temporal observations from other modalities and the various therapeutic consequences of diazepam and discuss the potential applications of such an approach in terms of drug development and translational neuroscience.
The E2 protein of human papillomavirus type 8 increases the expression of matrix metalloproteinase-9 in human keratinocytes and organotypic skin cultures.
Akgül B, García-Escudero R, Ekechi C, Steger G, Navsaria H, Pfister H, Storey A.
Med Microbiol Immunol. 2011 Jan 28. [Epub ahead of print]
Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.