University of London, St. George's Hospital Medical School
The Chair of Cardiovascular Immunology
Held by: Prof. Christopher J.F. Spry MA DPhil FRCP FRCPath FESC
Annual lay report 1995
Our research group has had a very busy year in 1995. Not only have we finished a detailed study of two of the most important proteins from one of the blood cells that can cause a rare but fatal form of heart disease (eosinophilic endomyocardial fibrosis), but we have also started a new program of work to discover the basis of the most common form of heart muscle disease of unknown cause: dilated cardiomyopathy. We suspect that the development of dilated cardiomyopathy must be linked to the genetic makeup of the people who develop this serious disorder and the ways that their immune system reacts to infections. Two new Lecturers began work in the team with a technician (Mr. Robert Hollifield) in the Spring of 1995 . One Lecturer (Dr. Paul Nelson) is an Immunologist who is studying the immune responses in the heart and the other (Dr. Suzanne Ruddy) is a Molecular Biologist who is skilled in studying the genetic and molecular basis of disease. Together, we are beginning to set the ground work for the research that has to be done. We are also fortunate in being able to collaborate in our Medical School with Prof. Bill McKenna who is a leading authority on cardiomyopathies.
So far, after six months work, we have six projects under way and two are beginning to show useful results. We have discovered a protein on the surface of heart cells which may allow the persistence of some viruses there and we have shown that another protein is present in the hearts of fetuses but not adults, possibly accounting for a difference in the way that these proteins are involved in disease. It is too early to report on the other projects but they should take us close to the basis of dilated cardiomyopathy. These projects (a) deal with the genes that predispose to dilated cardiomyopathy, (b) examine antibodies to heart tissues, (c) measure the different ribonucleic acid molecules that form the proteins in cells to see if they are different in patients with dilated cardiomyopathy and ( d) provide a model of the disease. It looks as though 1996 will be even busier year than 1995 as we work to track down the elusive and complex processes that turn a normal healthy heart into one that begins to lose its capacity to pump properly so that the patient may die in heart failure. We acknowledge that it is a privilege to be allowed to work on such important topics and we take this opportunity to thank the British Heart Foundation, its loyal and generous supporters and our patients for enabling us to do this research in such an important area of cardiology.
Annual scientific report 1995
Last year we cloned, sequenced and defined the promoters of the principal granule proteins in eosinophils, the major basic protein (MBP) which is toxic to heart and other cells and the neurotoxin (EDN), which is particularly toxic to neurones and also damages heart cells. The work on ECP was published in the Biochemical Journal in February 1995 and Dr. Li’s Ph.D. thesis on this was accepted in September 1995. Dr. Sun’s work to clone and express EDN was also published in January 1996. Our clinical work to diagnose and treat patients with endomyocardial fibrosis has been less busy in 1995 as many of these patients can now be well looked after in their local hospitals, although we provide a tertiary referral service for the more complex clinical problems of patients with eosinophilia, with and without heart disease. Prof. Spry’s work in biomedical informatics has been noted in this rapidly developing field. Unfortunately the low band width and congestion on the UK cable and satellite data links to the States and pressure on Internet providers there has limited use of these systems. This will improve in 1996 as new resources are being installed both within the UK academic network, in the USA and on the Continent. Our facilities are among the best available today thanks to our considerable efforts in this field, and as a result of our collaboration with several of the principal commercial, academic and research teams implementing internet access and database provision internationally. The benefits of this will become more obvious as bandwidth and access times improve. In the Spring we began work on several important new area in cardiology research for our group. We recruited two Lecturer’s and a Technician to discover the nature of the molecular and immunological defects in patients with dilated cardiomyopathy. We have begun to characterize our many monoclonal antibodies to human heart antigens, the roles of Fas-ligand and adhesion molecules in this group of diseases, and define alterations in the mRNA profiles of myocytes in explanted hearts from patients undergoing heart transplantation. Two new projects are being done in close collaboration with Prof. Bill McKenna's group as outlined below, to map genetic defects leading to dilated cardiomyopathy and to learn more from an animal model of inflammatory heart disease leading to dilated cardiomyopathy.
Total annual support received in 1995:
Chair support, discretionary fund and equipment fund.
Lecturer salary for Dr. Paul Nelson for 6 months.
Private donation: £1,000
Investment income: £5,000
Summary of research in progress:
1. Fas ligand expression on myocytes in dilated cardiomyopathy. (Dr. Paul Nelson)
Some forms of dilated cardiomyopathy are associated with persistence of unusual varieties of Coxsackie and other viruses in the heart in the presence of a normal or exaggerated immune response that should eliminate it. As CD95 ligand (Fas-ligand) expression is associated with immune-privilege status, we have developed an in-situ RT-PCR hybridisation system to assess Fas ligand expression on cardiac myocytes in patients with dilated cardiomyopathy.
2. Adhesion molecules in myocarditis and dilated cardiomyopathy (Dr. Paul Nelson with Dr. T. Poulton, Dept. Immunology and Mr. Rod Tricker, University of Westminster)
We have shown by immunofluorescence and confocal microscopy that myocytes from cardiac tissues of some patients with dilated cardiomyopathy and some with myocarditis express the b1 integrin, but not the b2 and b4 forms. We have noted that adult human cardiac myocytes do not express ICAM-1 (CD54). In contrast, fetal human cardiac myocytes express do express this adhesion molecule. Although the significance of this is not yet clear, up-regulation of adhesion molecules may be of importance in virally induced myocarditis and enteroviruses associated with dilated cardiomyopathy in view of their importance in localizing and promoting acute and chronic inflammation.
3. Microsatellite mapping of dilated cardiomyopathy. (Dr. Suzanne Ruddy in collaboration with Prof. Bill McKenna)
Work has begun on mapping chromosome 1 using the Genethon markers most closely placed to the centromere on chromosome 1 (Kass, Nature Genetics 7 546 1994), studying family pedigrees collected under the direction of Prof. Bill McKenna and in conjunction with a Ph.D. student in his group. This initial mapping will allow resolution of 10 centimorgans. When an indication of linkage is established higher resolution markers will be used to focus on the relevant region. The first family to be studied has over 40 members with 8 definitely affected individuals. Some other individuals are still undergoing evaluation and may also have DCM.
Preliminary work has established the gel resolution of the markers being used: that they are sufficiently well separated on acrylamide gels to be able to distinguish polymorphisms of 2,4,8 etc. base pairs. This was done firstly non-isotopically and when these conditions were satisfactory, radioisotopes were used as this is generally accepted to improve the ease with which genuine bands can be determined over stutter bands. These are where the polymerase enzyme encounters a secondary structure in the template DNA, and stops, giving a false band.
Radioisotopic labelling has now been established as a satisfactory procedure for these primers and is currently being employed.
Advice has been sought locally from Dr. Steve Jefffries on strategies and recommended linkage programs and also from Dr. Jan Poleinecki on statistical requirements.
The work will initially centre on those chromosomes which appear in the published literature to have an indication of linkage in DCM cases. It will be possible with relative ease to exclude or establish linkage to these loci and in the event of linkage being found positional cloning will begin to isolate the relevant gene(s). If this is unsuccessful then a full genome scan can be undertaken if necessary. St. George’s Hospital Medical School is particularly well-appointed for this type of work as it has access to perhaps the largest group of patients with DCM.
4. Monoclonal antibodies (MAbs) to cardiac antigens. (Dr. Paul Nelson)
The antibodies, which were raised in our Group by Dr. Tai are being further characterized with regard to specificity and assay restriction . A dot-blot immunoassay for prognostic/diagnostic applications is being developed for use in myocarditis and DCM patients.
5. RNA fingerprinting and differential display polymerase chain reaction to illuminate changes in the expression pattern in dilated cardiomyopathy. (Dr. Suzanne Ruddy)
The technique of RNA fingerprinting and differential display (Liang and Pardee, Science (1992) 257, 967-971 and Liang and Pardee, Nucleic Acids Research (1994) 22 5763-5764), is currently being employed to investigate genes which are up or down regulated in the early stages of dilated cardiomyopathy.
This protocol involves the reverse transcription of mRNAs with oligo dTs anchored by one base at the beginning of the poly(A) tail. This allows the cDNA to be effectively divided into four sub-populations depending on whether the anchor at the reverse transcription stage was G, A, T or C. This is then followed by a polymerase chain amplification using an arbitrary primer which at low stringency will find sufficient sequence similarity to initiate an amplification reaction. If a radioactive isotope is incorporated at this stage the amplified population can be distributed and visualized on a sequencing gel. Messenger RNA samples analysed side by side can be compared and differentially expressed genes identified. Probes to the genes of interest can be recovered and used to clone their cDNAs or genomic DNA.
Work has begun on this technique with frozen samples which are readily available and it is hoped that the availability of fresh heart biopsy material which has been characterized with regard to the progression of the disease will facilitate the investigation.
6. Mouse model for dilated cardiomyopathy. (Dr. Suzanne Ruddy in collaboration with Prof. McKenna and Dr. Kamran Baig)
Research into dilated cardiomyopathy is restricted due to the limited availability of human tissue and the fact that the disease is often in an advanced stage of progression in these samples. There is a requirement for a satisfactory animal model of the condition.
Mice can be induced to develop an inflammation of the heart muscle known as myocarditis by injection with cardiac myosin, cardiac C protein, or by infection with virus such as Coxsackie B or encephalomyocarditis virus. Any of these initial challenges can result in progression to a condition similar to DCM in genetically susceptible mice. There is potential to develop a mouse model of DCM which would allow investigators to manipulate experimental conditions and investigate gene expression, both at the protein and RNA levels.
Cardiac myosin has been isolated from frozen mouse and human tissue. (Shiverick et al. 1974 'Purification of cardiac myosin', Elsevier Scientific Publishing Co. Amsterdam). An immunization program using cardiac myosin is currently underway. This was initiated by Dr. Kamran Baig in Prof. Bill McKenna’s group to establish myocarditis. It is hoped that when optimal conditions have been determined our samples of cardiac myosin can be used as an initiator.
Human patients with DCM have been shown to have elevated levels of IL-2 and IL-10, but there is no information available on interleukin levels in the mouse. It is intended that serum and RNA levels of interleukins would be measured from the time of immunization to determine if any of these were raised.
Antibody therapy has previously been proposed as a route to treating DCM and this could also be investigated using the mouse model. Antibodies to relevant interleukins in the mouse could be studied with the emphasis on whether or not they were capable of inhibiting disease progression.
7. Development of software for clinical and laboratory research. (Prof. C.J.F. Spry)
Last year we installed two workstations: a PC running Windows-NT 3.5 and a 24 bit Silicon Graphics ‘Indy’ workstation running IRIX 5.2. Software on both of these computers, which are the MS Windows servers and file servers for the Group respectively, has been extensively updated with improvements to their operating systems and range of programs. This has extended our outstanding suite of computer resources. Dr. Ruddy and Prof. Spry attended a Human Genome Mapping Project computer course for a week in September 1995 at Kings College, London to become more familiar with the many molecular biology and genetic resources being set up at Hinxton Hall, Cambridge by the MRC, Wellcome Trust and European Molecular Biology Laboratory. These are all accessible now from within our local network from any of the networked systems including two networked general purpose PCs running Windows95. We have set up PPP connections and enabled a number of video and sound resources on the Indy.
Conferences: Prof. Spry and Dr. Ruddy attended the International Symposium on The Role of Immune Mechanisms in Cardiovascular Disease in Berlin, September 1995. Prof. Spry gave an invited symposium lecture at the second Asian Pacific Congress of Allergology and Clinical Immunology in Taipei, Taiwan in November. Dr. Paul Nelson presented a poster on monoclonal antibodies with dual specificity corroborated by epitope mapping at the British Society for Immunology, Joint Congress of the British and Netherlands Societies for Immunology, Brighton. UK 6-8th December 1995.
Publications in 1995
Li M S, Sun L, Satoh T, Fisher L M, Spry C J. (1995) Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters. Biochem J 305, 921-7.
Spry C J. (1995) The hypereosinophilic syndrome. In: Eds. Weatherall D J. Ledingham J G G. Warrell D A. Oxford Textbook of Medicine. 3rd edition. Oxford University Press, Oxford., 3610-13.
Spry C J. (1995) The hypereosinophilic syndrome and the heart. In: Eds. Weatherall D J. Ledingham J G G. Warrell D A. Oxford Textbook of Medicine. 3rd edition. Oxford University Press, Oxford. 1995., 2396-8.
Wymann M P, Kernen P, Von Tscharner V, Tai P C, Spry C J, Baggiolini M. (1995) Activation of the respiratory burst in eosinophil leucocytes—a transduction sequence decoupled from cytosolic Ca2+ rise. Eur J Clin Invest 25, 25-31.
Nelson P N, Chambers T. (1995) Generation and characterization of putative monoclonal and polyclonal antibodies against tartrate-resistant acid phosphatase. Hybridoma 14, 91-4.
Nelson P N. (1995) Retroviruses in rheumatic diseases. Ann Rheum Dis 54, 441-2.
Blottiere H M, Daculsi G, Anegon I, Pouezat J A, Nelson P N, Passuti N. (1995) Utilization of activated U937 monocytic cells as a model to evaluate biocompatibility and biodegradation of synthetic calcium phosphate. Biomaterials 16, 497-503.
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