The Scleroderma Association of NSW Inc. cannot offer direct advice on treatment or available therapies. It is up to each patient to make all relevant health decisionsIn consultation with their own doctor or health professional.
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Australian Research between December 2002 and May 2004
Wendy Gold and Professor Keith Stanley from the Research Team
1. Report from Professor K Stanley, Scleroderma Laboratory, St Vincent's Hospital
- May 2004
2. Gene expression studies in Scleroderma and Morphoea
3. Scleroderma Gene Project Advances to Human Tissue Study
4. Human Genome Project Boosts Scleroderma Research
5. News from the Lab - August 2003
6. News from the Lab - May 2003
7. News from the Lab - Dec 2002
1. May 2004
Report from Professor K Stanley, Scleroderma Laboratory, St Vincent’s Hospital
In previous reports we have described our studies on the gene expression in different vascular cell types during inflammation with the aim of applying this knowledge to scleroderma and morphoea. The studies of these isolated cells have allowed us to identify markers of different vascular and skin types that might be involved in scleroderma, and also to identify for the first time a new family of genes involved in endothelial cell inflammation. This is particularly relevant as endothelial cells, which line all blood vessels, are known to be involved at early stages of disease. Therefore, blocking changes in endothelial cells associated with early sages of scleroderma may be a way to halt the progress of the disease.
We have very recently completed the analysis of the skin biopsies taken from our first morphoea volunteer who responded to an earlier appeal in the Scleroderma Association newsletter. We opted to start with a morphoea patient as the skin thickening advances with time so that there are always areas of unaffected skin, areas of currently changing skin (at the edge of the lesion) and skin that has been changed for some time. Our hypothesis has been that inflammation is likely to be an early event in connective tissue diseases affecting the skin, hence the first thing we have looked for is the level of inflammatory genes in different areas of skin from this volunteer. To our surprise the highest levels were found in the middle of the plaque, not the leading edge. Furthermore our novel inflammatory gene family is one of the genes that changes most. This immediately tells us that the inflammation is likely to be occurring in endothelial cells.
Currently we are examining the genes that directly affect collagen synthesis to see if these too are highest in the middle of the plaque, or whether they are elevated in the leading edge of the plaque. This might resolve the question of whether the inflammation is caused by the build up of collagen or vice versa.
Our major problem is that we cannot say whether the changes we are measuring are unique to one individual, or common to everyone with skin connective tissue disease until we have more biopsy samples from volunteers. We again would like to invite anyone with recently diagnosed morphoea or scleroderma to volunteer for our study so that we can confirm these results and get a clearer picture of what is happening at the cellular and molecular level of this disease. Please call Wendy on (02) 8382 2821 for more details.
2. Gene expression studies in Scleroderma and Morphoea
With the rapid advance in gene identification achieved in the human genome project it is now possible to investigate disease at the level of gene expression. This is potentially of special interest in diseases like scleroderma, where the cause of disease is unknown. The level of expression of many thousands of human genes can be measured simultaneously by measuring the amount of each gene in a piece of diseased tissue and comparing it to the gene expression in normal tissue. If a group of genes responsible for a particular function (e.g. inflammation) are all elevated (or depressed) this suggests that that pathway might be important in the initiation or progression of the disease. Our approach has been to define the groups of genes characteristic of different skin cell types under resting conditions and after stimulation with pro-inflammatory cytokines in vitro so that we can determine if inflammation in a particular cell type is a major contributor to the changes observed in scleroderma.
A recent publication has compared gene expression in skin from patients with diffuse scleroderma and normal controls (Whitfield et al., 2003: Systemic and cell type-specific gene expression patterns in scleroderma skin. Proc.Natl. Acad. Sci. 100,12319-12324). In this study the authors compare diseased areas of skin from scleroderma patients with clinically unaffected areas and also with skin from volunteers who were not suffering from scleroderma at all. The results of this study can be summarised as follows:
While this study could be criticised on the basis that only four subjects were studied, it shows for the first time that scleroderma can be identified by the pattern of gene expression and suggests, as previously suspected, that the endothelial cells that line blood vessels are of key importance.
We have now completed gene array analysis of one patient suffering from morphoea. Initial examination of our data suggests that some differences between unaffected and affected areas of skin do exist, although they are not as great as the differences between volunteer control skin samples and the patient samples. Our task now is to complete the gene array studies in morphoea by examining skin from at least two other patients and then to follow up the gene expression changes that we have observed. We would like to invite any volunteers with recently diagnosed morphoea or scleroderma who would be prepared to take part in this study to contact Wendy on (02) 83822821
3. Scleroderma Gene Project Advances to Human Tissue Study
Report from Prof. Keith Stanley, Scleroderma Laboratory, Centre for Immunology, St Vincent’s Hospital, Sydney
The power of the Human Genome Project has been harnessed for scleroderma research at the Centre for Immunology, St Vincent’s Hospital. Using state-of-the-art 'gene array's; technology the complete set of human genes has been screened for those responding to inflammation in blood vessel (endothelial) cells. This project is one of the most advanced of its kind in Australia and has already dramatically shown that endothelial cells, which are involved in early stages of scleroderma, produce a huge and broadly based response to inflammation. This work has been submitted for publication in an international scientific journal.
Our results show for the first time an overall picture of changes in gene expression during inflammation which allows us to place the response of individual genes into context. Among the genes that we have shown are involved in endothelial inflammation are many that are known to relate to connective tissue disease. A highlight of this study is the discovery of the involvement of over 50 unknown genes. These genes do not yet have a described function, but show large changes during endothelial cell inflammation. We are excited by the prospect that Some of these novel genes could be used as diagnostic markers of scleroderma, and potentially open up avenues for future therapy as the detailed function of these genes is determined.
Currently we are working on the development of a &’Custom array'; containing the most interesting known and unknown genes from our panel, to be used for screening of tissue obtained from volunteers with scleroderma. In this study the genes from small pieces of skin will be extracted and tested against our custom array to determine which genes are elevated in diseased and healthy areas of skin from a particular individual. This study will detail the local changes in gene expression that might be linked to disease and is the first step in addressing the molecular basis of scleroderma.
4. Human Genome Project Boosts Scleroderma Research
Report from Prof. Keith Stanley, Scleroderma Laboratory, Centre for Immunology, St Vincent’s Hospital, Sydney
Scleroderma research in Australia has now joined the genome age. Publication of the complete human genome in February 2001 heralded the beginning of the very exciting era for biomedical research. A group at the Centre for Immunology, St Vincent's Hospital Sydney is at the vanguard of this research, employing the latest 'Gene array'; technology to determine the patterns of gene expression that occur in scleroderma.
Gene arrays are like computer chips, but instead of circuits they have etched on their surface all of the human genes. By taking a sample of genes from any cell and building to the array the level of expression of genes in the tissue can be read. This gives a snap shot of what the cells in that piece of tissue are doing. This is very informative, indicating how happy’; the cells are, and if they are not, pointing to what the problem is. About 40% of human genes found by the human genome project currently have no known function. A particularly exciting aspect of this work is that some of these might turn out to be of importance in scleroderma.
The Scleroderma Research Group headed by Professor Stanley at the Centre for Immunology has devised a strategy for maximising the information obtained from this costly technology. In progress to date, the human genome has been screened for genes elevated during inflammation of endothelial cells, which line blood vessels and are believed to play an important part in the early stages of scleroderma. This set of genes, including many of unknown function, are now being used to create a customised "inflammation array" with which to screen tissue samples from scleroderma patients. Even though this project is at a very early stage, the results so far are fascinating. The inflammatory genes expressed specifically by endothelial cells include many expected genes, but also many surprises and a large number of genes which currently have no known function. The interesting question will be how the pattern of expression of these genes varies in skin from normal and scleroderma patients, and at different stages of disease. Once established we will go back with good samples of scleroderma affected tissue to the original human gene arrays to determine which genes other than inflammatory genes are expressed. This work will be the basis of a much more complete understanding of scleroderma than has been possible before.
5. News from the Lab - August 2003
Report from Prof. Keith Stanley, Scleroderma Laboratory, Centre for Immunology, St Vincent’s Hospital, Sydney
Gene profiling to start with Morphoea Progress has been made according to plan with the scleroderma research project at the Centre for Immunology. A major hurdle we had to surmount was how to handle the data obtained from analysis of gene expression in a skin biopsy if we did not know the cellular composition of the biopsy, or which cells were becoming activated. In the last quarter we have identified genes in all of the major skin cell types that can be used as reference points for gene expression. These genes do not change in expression under the conditions that we have tested, so we will be able to use them to measure the numbers of each cell type in the biopsy sample and to facilitate comparison between biopsy measurements. We have also identified genes that are highly activated in each cell type in response to inflammation. This will allow us to determine which cell types are becoming activated. This completes our preparation for the clinical study.
Our plan is to start the clinical study with two patients with recently diagnosed, or currently advancing morphoea. Our hypothesis is that the genes expressed at the advancing edge of the skin lesion will be important in the understanding of many forms of Scleroderma. The skin biopsies will be analysed using the latest techniques of ‘gene array’ profiling; and will identify any genes changing in concentration as the skin lesion occurs. This will tell us which cell types are involved and what changes are occurring. We would like to take three skin samples - one in an area of normal skin, one at the advancing edge of the morphoea, and one in a mature area of diseased skin. The biopsies will be performed by a medical registrar at the Skin & Cancer Foundation Australia under the supervision of Professor Steven Kossard. If you would like to volunteer for this arm of the study please contact Wendy Gold on (02) 8382 2821. Wendy can send you the information sheet that goes with the ethics permission for this study. We would like to thank all those who have already contacted Wendy. We intend to take volunteers with systemic scleroderma once we have completed the study on morphoea. As this is the first study of its kind we would like to progress in stages so as to maximise the information learnt and, in the long term, the benefit for sufferers of this disease.
6. News from the Lab - May 2003
Update: A recent report from the Scleroderma Laboratory Centre for Immunology, St Vincent’s Hospital, Sydney.
Progress is being made towards our goal of unravelling what is going on in tissue samples from patients with scleroderma by using gene expression studies. Our aim is to define the changes in cellular composition that occur, the cell types that are involved in disease and the changes that take place in those cells. To this end we have identified genes that are characteristic of several of the cell types that we might encounter and are currently setting up assays to measure their concentration. This will enable us to determine how the cellular composition of the skin samples is changing. In addition we will use a microscopic technique in which we can directly stain for the presence of key genes to determine in which cells these genes are being expressed. Using this technique we will be able to ask which genes are inflames or initiating fibrosis. We will also be able to determine the role of the unknown inflammatory genes that we are currently characterising. In another study we will examine the spectrum of gene expression changes using the new gene array techniques that will measure the concentration of over 8000 genes simultaneously. Since we want to be able to perform all of these studies on two small tissue biopsies, we are practising with control skin samples and hope to be ready to start a study of skin samples from patients with scleroderma by July. If you would like to take part in this study please please contact Wendy Gold on (02) 8382 - 2821 for more information.
This study will be the most thorough study of gene expression in Scleroderma conducted to date. By correlating gene expression data with clinical history and treatment record it may be possible to determine the role of individual cell types in disease progression and to determine the role of inflammation at different stages of the disease. This information should significantly improve treatment. In addition, large scale analysis of genes expressed in disease affected skin samples using gene arrays is likely to throw up novel genes that change expression in scleroderma leading to new hypotheses about disease initiation and progression. This study should also help to determine the role of our novel endothelial cell specific inflammatory genes; our hope is that they may eventually be used as new targets for therapeutic intervention in scleroderma.
7. News from the Lab - Dec 2002
A recent report from the Scleroderma Laboratory Centre for Immunology, St Vincent’s Hospital, Sydney.
Research at St Vincent’s Hospital is focused on defining the genes involved in blood vessel inflammation as a first step towards studying inflammatory genes as markers of disease progression in Scleroderma. It now appears that two previously uncharacterised genes might be involved in the inflammatory process. Professor Keith Stanley’s research group has identified these genes from a survey of the complete human genome conducted with the support of the Scleroderma Association of NSW. Although this work is still at a preliminary stage, it appears that both of the novel genes are highly unregulated, suggesting that they might play a fundamental role in the process of inflammation. Only 9 of the known genes in the genome survey showed similar levels of induction and these 9 genes are all critical components of the cellular response to inflammatory stimuli. Part of the current effort is to fully characterise the novel genes and determine their biological function.
In the second part of the project we will use the information learnt from our gene survey to study the role of inflammation in the progression and treatment of Scleroderma. The next step in this part of the project is to compare the response of each of the various types of skin cells to various inflammatory stimuli. We have preliminary evidence to suggest that we will be able to determine a characteristic response for each cell type. Thus when it comes to analysing skin samples from Scleroderma patients, which will contain signals arising from many different cell types, we should be able to identify the cell types responding by their characteristic signatures of gene expression. Ultimately this should enable us to understand to understand the inflammatory processes that are going on in Scleroderma and suggest better approaches to treatment.
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The Scleroderma Association of NSW Inc. cannot offer direct advice on treatment or available therapies. It is up to each patient to make all relevant health decisionsIn consultation with their own doctor or health professional.
Updated Updated Tuesday, 08. April 2008
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