Abstracts for all the poster presentation are below. To go directly to a particular abstract click on the Title & Name below.
P03 - Non-invasive measurement of renal tissue perfusion using arterial spin labelling (ASL) magnetic resonance imaging (MRI) - Cox, E.F., Buchanan, C.E., Selby, N.M., Taal, M.W., McIntyre, C.W., Francis, S.T.
P13 - Human in vitro model for Merlin-deficient tumours as a tool for mapping pathways involved in tumorigenesis and drug testing - Ammoun, S., Provenzano, L., Schmid, M.C., Zhou, L., Hilton, D.A., Ercolano, E., Barczyk, M.and Hanemann, C.O.
P14 - Lipid-loaded nanoparticle-delivery via an ‘all human’ blood brain barrier model: Importance in assessing novel therapies for brain tumours - Maherally, Z., Kumar, S., Alder, J., Fillmore, H.L., and Pilkington, G.J.
P01 - Behaviour of human brain vascular pericytes when seeded onto various proteins of the basal lamina
Willcock, A.P.1, Maherally. Z.1, Pilkington, G.J.1
1 University of Portsmouth, Portsmouth, UK
Knowledge on how the extracellular matrix (ECM) influences pericyte behaviour is modest. Our aim was to conduct adhesion and motility assays to assess the hypothesis that pericytes are highly motile but can be stabilised by basal lamina proteins. Results would also aid determination of the role of pericytes in human compared to rat/mouse models.
Human brain vascular pericytes (HBVP) were seeded onto agrin, collagen IV, fibronectin, laminin and perlecan substrates and cell adherence quantified via a colourimetric MTS assay. Motility assays were performed using live cell (time-lapse) imaging technology. Pericytes adhered equally to laminin and collagen IV, where reduced adhesion was seen in the absence of ECM, as well as on fibronectin, agrin and perlecan. Pericyte adhesiveness was not significantly different when cells on substrates were compared to cells only (p > 0.05). Highest to lowest distance travelled and speed of pericytes, when seeded onto each substrate, were: collagen IV, no ECM, laminin, agrin, fibronectin and perlecan respectively. A significant difference (p < 0.05) was seen where pericyte distance travelled and speed was compared between a few substrates (between collagen IV and perlecan and between fibronectin and collagen IV). Overall, findings were not suggestive of a major dependency of the pericytes on ECM in relation to cell anchorage and movement. This suggests we need to explore pericytes and their direct relationship with endothelial cells and astrocytes rather than the ECM.
P02 - The use of a human podocyte cell line to determine the implication of nets in lupus nephritis
Vernon, A.1, and Beresford, M.1,2
1 University of Liverpool, Liverpool, UK
2 Alder Hey Children’s Hospital, Liverpool, UK
INTRODUCTION: Monocyte chemoattractant protein-1 (MCP-1) has been identified as a biomarker of lupus nephritis (LN), involved in the early recruitment of pro-inflammatory cells to the kidney, resulting in further renal damage. Neutrophils undergo a process called NETosis, which results in the release of neutrophil extracellular traps (NETs). Impairment of NET degradation is associated with LN.
AIM: To use an in vitro model of LN to assess the involvement of NETs.
METHODS: Human podocyte cell-line with human-monocyte derived macrophages was used to create an in vitro model of (LN). Human monocytes were differentiated into macrophages using media containing monocyte colony stimulating factor. NETs were induced from isolated neutrophils using 600nm PMA. The macrophages were then either unstimulated (inactive cells) or incubated with IFN-γ at a concentration of 1ng/ml (active cells)+/- NETs. After 48 hours the supernatant from the macrophages +/- NETs was removed and added to mature podocytes. MCP-1 concentration (mean±SEM) was measured using ELISA. Podocytes following culture were visualised using light microscopy.
RESULTS: There was no difference in the concentration of MCP-1 from podocytes alone (1968±101pg/mL) compared to in the presence of inactive macrophages (1860±135pg/mL; p=0.373). However, there was significantly increased MCP-1 from the podocytes was produced following the addition of active macrophages (3874±292pg/mL; p=0.035) or NETs (4279±181pg/mL: p=0.011). When cultured together with active macrophages and NETs, the podocytes produced the highest concentration of MCP-1 (4716±420pg/mL; p=0.033).
P03 - Non-invasive measurement of renal tissue perfusion using arterial spin labelling (ASL) magnetic resonance imaging (MRI)
Cox, E.F.1, Buchanan, C.E.1,3, Selby, N.M.2,3, Taal, M.W.3, McIntyre, C.W.4, Francis, S.T.1
1 School of Physics & Astronomy, University of Nottingham, Nottingham, UK
2 Royal Derby Hospital, Derby, UK
3 School of Medicine, University of Nottingham, Nottingham, UK
4 University of Western Ontario, Canada
Chronic Kidney Disease (CKD) is a condition in which the kidneys are damaged in function and/or structure, and cannot filter blood as well as possible. Kidney damage can occur from a physical injury or a disease like diabetes, causing wastes to build up in the body leading to other health problems. Animal models are very commonly used to study CKD, and although most established animal models of chronic kidney disease bear some similarities to human chronic kidney failure, none is fully representative and ideal at replicating this in the human. Numerous animal studies use laser Doppler needle probes to assess renal tissue perfusion. However, MRI (magnetic resonance imaging) is a non-invasive technique which can be used to study both the structure and function of organs without the injection of contrast agents or exposure to radiation.
We aim to use MRI as an alternative to the invasive measures of laser Doppler needle probes to assess renal tissue perfusion. We will use arterial spin labelling (ASL), which uses the magnetic spins of freely diffusible water in blood magnetisation as an internal endogenous contrast agent (no exposure to gadolinium based compounds) to non-invasively label the magnetization of blood water and generate a perfusion image. We will also use MR relaxation time measures to assess structural changes in the tissue.
P04 - In vitro generation of human hepatocytes - a replacement for in vitro drug metabolism and toxicity screening
Fairhall, E.A.1, Lakey, A.F.1, and Wright, M.C.1
1 Newcastle University, Newcastle upon Tyne, UK
There is a need to generate an unlimited supply of functional human hepatocytes for cell-based approaches for drug and chemical toxicity testing in vitro. Recently, direct reprogramming of adult cells, by over-expression of defined transcription factors, has provided an alternative to stem-cell based studies. Using the expertise within our lab from studying pancreatic to hepatic transdifferentiation in a rat cell, a human pancreatic cell line has been developed with the aim of creating easily producible and functional human hepatocyte-like cells in vitro using serum-free culture conditions.
Human pancreatic cells were treated with dexamethasone (DEX); in addition a transcription factor central to liver development was over-expressed through a glucocorticoid receptor dependent mechanism. During culture the cells morphology altered to resemble a more hepatocyte-like cell. Over expression of the transcription factor resulted in the expression of a number of hepatocyte-specific genes, in both cell mRNA and protein, at levels comparable to human liver samples.
These early results have highlighted a quick and easy mechanism in which functional human hepatocyte-like cells can be produced, thus providing an in vitro model for drug and chemical toxicity testing. The replacement for both in vivo animal studies and animals as hepatocyte donors is therefore a real possibility.
P05 - Developing stem cell models to target self-renewal pathways in chronic myeloid leukaemia
Morrison, H.1, Toofan, P.1, Moka, H.1, Copland, M.1 and Wheadon, H.1
1 University of Glasgow, Glasgow, UK
Chronic myeloid leukaemia (CML) arises from a t(9;22) chromosomal rearrangement in a haemopoietic stem cell (HSC). This results in the expression of BCR-ABL, an oncogenic tyrosine kinase which confers a survival and proliferative advantage to the cancer stem cells (CSCs). The current tyrosine kinase inhibitor (TKI) therapies effectively target the progenitor and more mature leukaemic cells, but are unable to eliminate the CSC population responsible for minimal residual disease, relapse and disease progression. Patients therefore require long-term treatment to sustain remission, necessitating alternative strategies, either alone or in combination with TKI to eliminate the CSCs and provide a cure. One of the major hurdles in developing new treatments for CML is the ability to effectively test small molecule inhibitors. This project aims to develop and validate patient derived induced pluripotent stem cells (iPSC) as a fully humanised model system for pre-clinical drug-screening, replacing the need for animal testing early on in drug development. Our data has identified an important pathway for stem cell self-renewal and haemopoiesis, the TGFβ/Activin/BMP signalling pathway. This pathway is significantly differentially expressed in CML patients compared to normal donors and we are currently exploring this pathway as a potential target for developing CSC-directed therapies in CML in the future.
P06 - Will “Organ-on-a-chip” technology be a replacement for animal testing of drug or chemical toxicity?
Wilkinson, J.M.1, Phiri, K.1
1 Kirkstall Ltd., Rotherham, UK
There is currently great interest in applying microfluidics technology to the construction of increasingly complex in vitro models with the goal of simulating the behaviour of an organ, or even a whole organism – the so-called “Human-on-a-chip”. Enthusiasm for microtechnology has been generated by success in scaling cell culture from a petri dish down to 384 well plates and beyond. However the gold standard for clinical response is the human organism which is built out of microscopic cells, but incorporates larger functional units. 3D cell cultures are more representative of human physiology than 2D monolayers of cells on a plastic or glass surface. Correct organ function also requires gradients of oxygen and metabolites and removal of waste material, and co-culture of multiple cell types. Predictive models require both flow of media and 3D tissue-like cultures.
The goal for organ-on-a-chip is to demonstrate survival for a few days and that they mimic clinical behaviour. The models must demonstrate robustness and repeatability as well as ease of use and be economic compared with the alternative methods. This paper will present a study to identify the optimum size for each organ model. From that starting point, we then miniaturise the structure in a way that does not compromise the sensitivity of the assays used to monitor the outcomes of the experiments. Progress achieved to date with practical tools incorporating these principles will be presented.
P07 - Labelled IMS TAG proteins for profiling and quantitative mass spectrometry imaging of 3D tumour and skin models
Cole, L.M.1, Phillips K.L.E.1, Smith, D.1, Cross, N.A.1, Clench, M.R.1
1 Sheffield Hallam University, Sheffield, UK
The aim of this project is the development of methodology for quantitative Mass Spectrometry Imaging (MSI) of proteins in biological tissue. Whilst progress has been made in quantitative analysis of small molecules by MALDI-MS imaging, quantitative analysis of protein images has not been achieved.
“IMS-TAG” proteins are recombinant proteins produced to contain signature peptides from a variety of proteins of interest that are present in the tissue under study. We developed “IMS-TAG” proteins for use as positive controls for matching accurate mass, ms/ms spectra and ion mobility drift times in mass spectrometry imaging experiments. In order to progress the concept of quantitative imaging, 15N labelled IMS-TAG proteins are to be incorporated into tissue homogenates to create calibration standard arrays. Cut sections of the homogenate arrays (and the samples under study) will be homogenously deposited with 15N labelled IMS-TAG as an internal standard. Ratios of peptide m/z signals for corresponding labelled and unlabelled peptides will be used to generate quantitative data.
Preliminary data has been produced including mass spectrometry profiles of the IMS-TAG multi-peptide standard, tumour and skin models. Ongoing experimental studies include the study of apoptotic response to cytotoxic anti-tumour agents in tumours and protein induction of clinically relevant skin proteins involved in psoriasis and eczema in skin.
Successful development of the methodology will present an alternative to both in vivo and ex vivo testing using animal models.
P08 - Using an in vitro human airway model to study the toxic effects of components of e-cigarettes
Vasanthi Bathrinarayanan, P.1, Leslie, L.J.1, Marshall, L.J.2
1 School of Engineering and Applied Science, Aston University, Birmingham, UK
2 School of Health and Life Science, Aston University, Birmingham, UK
Cigarette smoke is a complex mixture of more than 4000 hazardous chemicals including the carcinogenic benzopyrenes. Nicotine, the most potent component of tobacco, is responsible for the addictive nature of cigarettes and is a major component of e-cigarette cartridges.
Our study aims to investigate the toxicity of nicotine with special emphasis on the replacement of animals. Furthermore, we intend to study the effect of nicotine, cigarette smoke and e-cigarette vapours on human airways. In our current work, the BEAS 2B human bronchial epithelial cell line was used to analyse the effect of nicotine in isolation, on cell viability. Concentrations of nicotine from 1.1µM to 75µM were added to 5x105 cells per well in a 96 well plate and incubated for 24 hours.
Cell titre blue results showed that all the nicotine treated cells were more metabolically active than the control wells (cells alone). These data indicate that, under these conditions, nicotine does not affect cell viability and in fact, suggests that there is a stimulatory effect of nicotine on metabolism. We are now furthering this finding by investigating the pro-inflammatory response of these cells to nicotine by measuring cytokine secretion via ELISA.
Further work includes analysing nicotine exposure at different time points and on other epithelial cells lines like Calu-3.
P09 - In silico simulation approach to study low dose fungal infections
Tanaka, R.J.1 and Bignell, E.2
1 Imperial College, London, UK
2 University of Manchester, Manchester, UK
Fungal infection is becoming a huge threat for human health. To combat this problem, there is an urgent need to understand the molecular basis of fungal host-pathogen interactions and how their system-level dynamics determines the outcome of disease in whole animal hosts. However, animal-based infection modelling cannot ethically or sustainably support these aims.
For example, some processes, such as localised host responses to low dose infections, are impossible to study or measure in whole animals due to the measurement sensitivity, although this is relevant to infections in human. An animal should not be the first stop for hypothesis testing. To replace animal experiments, computer simulation helps to expose flawed experimental designs and allows us to address hypotheses which are inaccessible in animals. We developed a mathematical model for the host-pathogen interaction in the human lung with Aspergillus fumigatus, a major opportunistic fungal pathogen of human. The model describes dominant biological processes with ordinary differential equations. A straightforward simulation of the mathematical model with an in silico titration of infectious dose (from 106 to 103 fungal spores) accurately captured information on pathogen burden, leukocyte populations and cytokine environment, which in reality would require ~ 2000 animals to perform. It successfully predicted clinically relevant features of the host response, such as cyclical exacerbation of infection in chronic disease at low dose infections and slower clearance of lower dose infection.
P10 - Electronic monitoring of a 3D in vitro model of the renal tubule with integrated microfluidics
Curto, V.1, Huerta, V.1, Ramuz, M.1, Rivnay, J.1, Serougne, R.1, Strakosas, X1 and Owens, R.M.1
1 Ecole des Mines de St. Etienne, Gardanne, France
There is an urgent need to move away from using animal models for toxicological testing as well as pharmacological evaluation of chemical substances, driven both by societal distaste and also the large cost of using animals. The alternative to testing on animal models is the use of cell-based models in vitro. However, the validity and predictive ability of in vitro based models has been shown to be poor and results in significant failures of drugs with a high associated price-tag. Limitations in technology are a major factor in the failure of the cell-based models. We take advantage of recent advances in materials research and tissue engineering to yield a highly performant 3D model of the kidney tubule integrated with microfluidics and in-line monitoring with on-board electronics. The system has been designed in a 12-well plate format, with simultaneous acquisition to allow medium-throughput screening. We demonstrate multi-parameter monitoring of relevant parameters to predict renal tubule toxicology, including TEER, and glucose levels, under fluidic conditions. Our system is compatible with high-resolution imaging and has potential for in situ immunofluorescence staining. In addition analysis of secreted biomarkers is possible by periodic sampling of efflux from the microfluidic chambers. We have monitored both canine and human kidney proximal tubule epithelial cells, in the presence or absence of endothelial cells to more closely mimic in vivo conditions.
P11 - Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells
Pallett, R.1, Leslie, L.J.2, and Marshall, L.J.1
1 School of Life and Health Sciences, Aston University, Birmingham, UK
2 School of Engineering and Applied Sciences, Aston University, Birmingham, UK
Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells.
Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA.
Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells.
Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.
P12 - The use of human (non-3D equivalent) skin assays (SkimuneTM) for detection of adverse reactions, sensitization, potency and efficacy
Ahmed, S.S.1, Dickinson A.M.1, Stebbings, R.2
1 Alcyomics Ltd, Newcastle upon Tyne, UK
2 National Institute of Biological Standards and Control (NIBSC), Potters Bar, UK
Alcyomics has developed human in vitro skin explant assays (Skimune™) as alternatives to the use of animal models as diagnostic tools for pharmaceutical, cosmetic and chemical industries for testing of drugs, novel compounds or monoclonal antibodies for potential allergic or hypersensitivity reactions. The current most favourable method to test compounds for allergenicity is the mouse local lymph node assay (LLNA). Recent EU legislation has banned animal testing on cosmetics. A number of alternative predictive test methods of skin sensitization testing are available but are inappropriate for assessment of relative potency. The Skimune™ assays have been evaluated against the LLNA, with 95% concordance (P<0.001 sensitivity 95%, specificity 95%) showing that it is a reliable tool for safety, potency and toxicity testing and also successfully identifies chemicals which have been shown to be negative in the LLNA but positive in man e.g. Nickel Sulphate.
The Skimune™ technologies can also test efficacy of novel immunomodulatory drugs or monoclonal antibodies, as well as potential allergic responses, before use in Phase I clinical trials. The Skimune™ assays could have predicted and prevented the TGN1412, Northwick Park incidence. The data demonstrate that the Skimune™ technologies provide novel and reliable approaches to the determination of skin sensitization, potency assessment, drug or monoclonal antibody evaluation and efficacy testing and can be used as a first step in the risk assessment process and as a replacement for animal models.
P13 - Human in vitro model for Merlin-deficient tumours as a tool for mapping pathways involved in tumorigenesis and drug testing
Ammoun, S.1, Provenzano, L.1, Schmid, M.C.1, Zhou, L.1, Hilton, D.A.2, Ercolano, E.1, Barczyk, M.1 and Hanemann, C.O.1.
1 Plymouth University, Plymouth, UK
2 Derriford Hospital, Plymouth, UK
Deficiency of the tumour suppressor Merlin causes development of schwannomas, meningiomas and ependymomas occurring spontaneously or as a part of a hereditary disease Neurofibromatosis type 2 (NF2). Merlin loss is also found in a proportion of other cancers e.g. mesothelioma, melanoma, breast cancer and glioblastoma. There are no effective therapies for these tumours and new drug treatments are needed. As schwannoma is a hallmark for NF2 and represents Merlin-deficient tumours we have developed an in vitro human schwannoma model, which replaces animals, to study tumour pathology and to test drugs. In contrast to animal models, the human in vitro model allows us to study actual diseased cells from patients and is an excellent tool for drug screening allowing faster from bench to bed transition by phase 0 clinical trials. Our strategy is to test drugs that are either already used clinically or drugs that have been successfully tested in Phase1/2 clinical trials for other diseases. Using our model, until now we have defined 13 potential targets such PDGFR, IGF-IR, Integrins, ErbB2/3, Axl, ERK1/2, AKT1/2, JNK, FAK, Src, NFκB, Wnt and p53/MDM2 complex. We have successfully tested seven drugs: Sorafenib, Nilotinib, Imatinib, Lapatinib, BEZ-NVP235, R1507 and AZD6244 (Selumetinib). Sorafenib and Nilotinib are currently being tested in phase 0 clinical trials on NF2 patients proving that our approach i.e. direct transfer from human in vitro studies into the clinics works.
P14 - Lipid-loaded nanoparticle-delivery via an ‘all human’ blood brain barrier model: Importance in assessing novel therapies for brain tumours.
Maherally, Z.1, Kumar, S.2, Alder, J.2, Fillmore, H.L.1, and Pilkington, G.J.1
1 University of Portsmouth, Portsmouth, UK
2 University of Central Lancashire, Preston, UK
The blood brain barrier (BBB) consists of endothelial cells, astrocytic endfeet and pericytes embedded in basal laminae. To evaluate the passage of novel brain tumour therapeutics across the BBB, an ‘all human’ model has been developed to assess the delivery of lipid-loaded nanoparticles (NPs).
A BBB model was constructed using human brain endothelial cells (hCMEC/D3), human cortical astrocytes (SC-1800) and human brain vascular pericytes (HBVP) in mono-, co- and tri- cultures. TEER values were measured using a Voltohmeter (EVOMTM). Western blotting and Immunocytochemistry revealed tight junction protein levels (ZO-1 and occludin). Response of cells post NP addition was assessed using cytotoxicity assay. Cellular uptake of lipid-loaded NPs tagged with rhodamine was analysed using confocal microscopy.
Co-cultures and tri-cultures resulted in significantly higher TEER values compared to endothelial cells alone (p<0.05). TEER values for tri-culture (E+A+P) were higher than co-culture (E+A) and monoculture (E). Western blot revealed higher expression of ZO-1 and occludin in co- and tri-cultures. Immunocytochemistry revealed localisation at cell-cell contacts compared to diffuse staining in monoculture. The barrier model showed complete recovery with no cytotoxic effects after addition of NPs. The NPs were seen to accumulate intracellularly. Astrocytes clearly contribute to BBB formation in this in vitro model. Preliminary in vitro tests have indicated efficient delivery of lipid-loaded NPs. Drug loaded NPs are now under investigation to provide data bridging the gap between drug discovery and clinical studies.