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Scientific Newsletter 2006
Do Different, Think Different: Challenging Convention Dr Michael Wormstone, University of East Anglia
Work in the laboratory has progressed at a great rate. Through our use of human cell and tissue models we have made some potentially groundbreaking discoveries that could ultimately improve clinical approaches to deal with a number of conditions in the eye and throughout the body. Wound healing is a process that the body adopts to respond to physical insults. This response underpins events following cataract surgery, which can lead to secondary visual loss due to increased light scatter. This condition is known as posterior capsule opacification (PCO). One cellular event often implicated in wound healing is called transdifferentiation. In essence this means that a cell changes type to a supposedly more aggressive form and not dissimilar to the transformation of the Incredible Hulk when someone made his mild mannered alter ego mad. Certain proteins are powerful promoters of this change and are thus regarded as important factors in wound healing. One such protein is called Transforming Growth Factor beta (TGFb) and strong evidence supports its role in PCO formation. Light scatter is caused though matrix contraction, forming wrinkles, and this is associated with transdifferentiated cells. Current dogma suggests that it is the transdifferentiated cells that drive this process, however we now have a growing body of data, established using cutting-edge technology, to challenge this conventional view. We believe TGFb is important in generating light scattering wrinkles, but propose this arises from mechanisms other than transdifferentiation. The Universities motto is “Do different” and in this case we have also had to think different. There are many more discoveries we have made, but I have highlighted this finding because it could have massive implications to the wound-healing field and gives me confidence that the scientific approach we use will answer key questions that will ultimately drive the direction of future clinical therapies. Science is no longer a solitary pursuit and it is vital to establish a successful team and I am very lucky in this case. I am extremely proud and grateful to the efforts of Lucy Dawes (PhD Student) and Dr Julie Eldred (HRT funded Post-Doctoral Scientist) who have produced high quality data often at unearthly hours, which allows us to push the boundaries of convention and enter uncharted territories. Science is a great adventure and in our research we have a goal, which is to improve the quality of life of numerous individuals. I believe we can ultimately achieve this! Expression of Cataract-Related Genes in the Human Lens Lisa Hodgkinson, University of East Anglia My previous experience has been in the pharmaceutical industry with a
company in Nottingham and one in Cambridge. I worked on many projects
developing drugs for obesity and multiple sclerosis and also therapies to
treat blood clotting deficiencies. I then decided that I should take the
next step and begin a major study of my own. My interests lie in the
treatment of Human disease and so I was very excited and fortunate to find a
PhD position available with Professor George Duncan, the head of The Norwich
Design of in vitro human neurotoxicity tests using co-culture between human astrocytic and neural cells Elizabeth Woerhling, Aston University Neurotoxicity occurs when exposure to natural or manmade toxic substances disrupt or kills nerve cells (neurones). These are key cells that transmit and process signals in the brain and nervous system. Currently, new and potentially neurotoxic substances require animal testing before exposure to humans. However, the use of human cells in an in vitro neurotoxicity test system will prove more relevant to the human situation and thus reduce the number of animals used in neurotoxicological testing. In the human nervous system, nerve cells cannot exist without other cells known as astrocytes, which protect and nurture them. The aim of my research is to include both these cell types growing together in a co-culture, so the effects of toxins can be seen in the most realistic model possible. I have formed human neurones and astrocytes by treating the NT2.D1 human cell line with various chemical factors that operate within our own brains . I know that these cells are exactly like those in our brains, as I have measured several specific protein-based ‘markers’ which are substances that only authentic human nerve cells and astrocytes form. In my work I have been evaluating the use of this nerve/astrocyte co-culture with several known neurotoxins using a number of standard test systems, which can distinguish between ‘necrosis’ where cells die very quickly, due to major toxic effects and ‘apoptosis’, where cells continue for some time after the toxins’ effects, only to programme their own deaths several hours later. Essentially, this battery of tests will assess whether the co-culture will be a more realistic model than those currently used to show changes in human neural and astrocytic cells in the presence of the various neurotoxins. It is hoped that the model will be able to predict whether future drugs, food additives or toxins, might have damaging effects on the human nervous system, without the use of animal models. Tumour viruses can be used as signposts to proteins with an important role in human cancer Anthony Oliver, Lynne Hampson & Ian N. Hampson, St Mary’s Hospital, Manchester In the last 25 years revelations on the causes of cancer have arrived at an ever increasing pace and it is strange how most people do not know that many cancers are caused by viral infections. Up to 20% of all human cancers have so called “tumour viruses” associated with them, the most notable being cervical cancer in which > 95% of tumours contain the Human Papilloma Virus (HPV). Such viruses produce special “transforming proteins” that cause cells to become cancerous and we are studying this process to gain insights into how cancers form. HPV is approximately 140000 times smaller than the human genome and yet it can subvert the myriad of complex processes that are present in our cells to cause a cancer. This means that such viruses must be highly selective in their effects on cells and we are using this selectivity to identify which human proteins and their biological pathways are targeted by these small parasites. Basically we are using them as signposts to focus our efforts on human proteins with an important role in cancer. Clearly if the same human protein or pathway is targeted by different types of tumour virus it is going to be even more important and we are currently working on a protein that is targeted by two different tumour viruses- HPV and the adult T-cell leukaemia virus type 1 (HTLV1). Our studies have gained unique insights into how this protein may be involved in causing human cancer and we have expanded our search for similar proteins that are targeted by more than one tumour virus. We now have a unique collection of transforming proteins from virtually every virus with a known or suspected link to human cancer and we have already used this to identify several human proteins which are targeted in common by different viruses. Indeed we have found one such protein that is the target of five different tumour viruses. Future work will be aimed at investigating the function of these proteins to understand how they can participate in causing cancer.
Using Human Models to Study Diseases of the Human Retina Vicki Tovell, University of East Anglia The majority of my work over the last 2 years has focussed on the Retinal Pigment Epithelium (RPE) which is a layer of tissue that lies just behind the retina at the back of the eye. The function of this tissue is to help maintain a healthy retina by close interaction between the two layers.
Loss of contact between the retina and the RPE, for example in a patient with retinal detachme nt, can have a devastating effect on vision if left untreated. Detachment can occur when there is a build up of fluid between the retina and the RPE. The studies that I am involved in are looking at the characteristics of a healthy RPE and how external stimuli effect the calcium concentration inside these cells. It has been shown that the activation of a specific cell surface receptor in the RPE can help remove fluid that accumulates during retinal detachment via the increase of calcium. Part of my study has therefore been to characterise the receptor subtypes present in the RPE that may be involved in retinal detachment and glaucoma. None of this work would be possible of course with out the fantastic support from the Humane Research Trust and also without the location of the nearby hospital. The proximity of the Norfolk and Norwich University Hospital allows me to obtain fresh samples of Retinal Pigment Epithelium (RPE), sometimes less than 24hours old which is essential for certain experiments.
An Investigation into the Processes that Govern Renewal of the Human Colonic Epithelium Principal Investigator: Dr Mark Williams, School of Biological Sciences, University of East Anglia HRT-funded PhD student: Miss Alyson Parris The healthy status of the lining of the bowel is preserved by continuously replacing approximately ten billion ‘old’ cells each day with ‘new’ ones produced by intestinal stem cells. As a consequence, the lining of the bowel is almost entirely renewed every few days or so. The processes of tissue self-renewal take place in millions of pocket-like invaginations called colonic crypts. During the first year of Alyson’s PhD programme she has developed the methodology to isolate human colonic crypts from clinical biopsy samples and apply state-of-the-art imaging technologies. Alyson has demonstrated that isolated crypts maintain the exquisite hierarchy of cell production, cell migration and cell shedding along the crypt-axis and these observations represent the first demonstration of colonic crypt renewal in vitro. For the first time, the status of tissue renewal in healthy patients can now be compared to that in patients with a history of colon cancer or inflammatory bowel conditions. Moreover, the effects of different growth factors on tissue renewal can now be studied in order to elucidate the signalling pathways that underlie a predisposition to colorectal disease. Precise details by which growth factors and cellular signals modulate colonic crypt renewal will emerge over the final two years of the study. Dr Richard Phelps, University of Edinburgh Discovery of T cells epitopes is an important milestone on the path to creating the new types of ‘designer vaccine’. Such vaccines are widely expected to be powerful agents for the treatment and prevention of not only infectious diseases but also cancers and autoimmune diseases. We are seeking to demonstrate that a new technology we have invented permits T cell epitopes to be identified without recourse to experimental inoculation. The key to our approach is a novel labelling strategy that permits peptides with high potential to be useful T cell epitopes to be distinguished from the thousands of irrelevant peptides on the surface of human cells. Prior to our gaining support of the HRT we had taken our invention from an idea on the back of an envelope to an accepted labelling technology published in highly regarded scientific journal. With the support of the HRT our current aim is to demonstrate that the labelling technology works in the extremely challenging context of peptides washed from the surface of human cells. It has not been plain sailing but important advances have been made. The initial steps of generating labelled test proteins were accomplished as planed, but considerable difficulty was experienced obtaining cell washings that contained peptides without disruptive contaminants, such as cell wall lipids that gummed up the analysis instruments. After some effort we now have very good results with over 50 peptides identified as proof that our approach does allow peptide identification without inoculation. But to date none of the peptides we have identified are the ‘right’ peptides. The problem appears to be the fantastically greater complexity of the peptides mixtures that come off the surface of real cells as compared with the test systems used to validate the labelling technique. We anticipate that this problem will be overcome either by an exciting enhancement to our labelling strategy that we recently validated in another project in the lab, or by changing our analysis technique to utilise an ‘ion-trap’ mass spectrometer capable of ‘MSMS’. This type of mass spectrometer is considerably better than our own instrument for complex mixtures, but less good at seeing the label. Very likely a combined approach will turn out to be most effective. Development of a model of fat cell development using human cells Professor Keith Frayn, Oxford University We store fat in our bodies in specialised cells called adipocytes. As someone gets fatter, the adipocytes increase in size, thus storing more fat, and also new adipocytes are produced from ‘precursor’ cells sometimes called preadipocytes. It is widely recognised that excessive fat storage has adverse effects of health: people who are obese have increased risk of developing diabetes and coronary heart disease. This has led many people to believe that the fat cells play some role in these adverse effects. One interesting property of fat cells is that they can alter fatty acids (the building blocks of fat as it is found in the diet, and stored in our bodies). For instance, fat cells make an enzyme that converts saturated fats (as found in animal fats) into mono-unsaturated fats, as found typically in vegetable oils such as olive oil. This process is called ‘desaturation’ and the particular enzyme involved is known as stearoyl-CoA desaturase, or SCD. So far, the role of SCD in disease has been studied almost entirely in mice. In mice, if the gene for SCD is destroyed (so no SCD is produced) the animals are actually healthier than normal. We have some evidence from using a drug in humans that alters SCD activity, and also protects from diabetes, that in humans the situation is quite different. The Humane Research Trust have funded a studentship for Jenny Collins to develop a system whereby human preadipocytes can be grown in dishes
and encouraged to develop into adipocytes at the appropriate time. We can then use these cells to study the regulation and the role of SCD. As the preadipocytes develop into ‘mature’ adipocytes, we can see that a number of enzymes change their activity, and we can see fat storage increasing by staining the fat and looking at the cells down a microscope, that was bought for us by the Trust . This picture shows a human fat cell with the fat droplets stained with Oil Red O dye.
The use of a NASA-derived rotating cell culture system in the development of human cell neurotoxicity assays Ray Ransley, Aston University The objective of my particular research project is to utilise the NASA
developed Rotating Cell Culture System (RCCS) to facilitate the culture of cell types of the Central Nervous System (CNS) in 3D. It is our hope that the development of a three-dimensional in vitro model using human neuronal cell types in culture offers an opportunity to replicate the conditions within human neuronal tissues and potentially reduce and replace the number of animals used in neurotoxicity testing. A three-dimensional culture allows cells to form the interactions with each other that are representative of those which occur within actual human tissue. It also allows us to study the effects of neurotoxins on different cellular mechanisms. Initially I worked on establishing which cell types would be best suited for developing 3D aggregates within the RCCS. Cultures of the SK-N-SH and the U373MG human neural cell lines were successfully produced after being grown in rotary culture for up to 2 months. An analysis of the consumption of glucose supplied in the culture media showed these cell aggregates to be fully viable and functioning. My work has continued from that point, conducting trials to assess the response of our 3D neuronal cell clusters to neurotoxins. The initial studies in this have involved subjecting the SK-N-SH cell line in particular to different neurotoxins such as Hexandiones and
Trimethyltin (TMT). The most recent data obtained demonstrates that TMT in particular has a significant toxic effect on the SK-N-SH cell aggregates which is comparable to that seen in cells grown in more conventional two-dimensional cultures. Given these promising preliminary results it is our ultimate aim to establish long term trials to ascertain the chronic effects of such neurotoxins on neuronal cells and in three-dimensional culture. |
