Tumour necrosis factor is a cytokine made by specific leukocytes, in two stages. First, the cytokine is exposed on the surface of the cell and then it is clipped off and released as a soluble form. In either form it can interact with specific receptors on other cells and, in this way, change the cells’ activities. We have found that binding of tumour necrosis factor, while it is in its membrane form, to its receptor can also send a message ‘backwards’ into the cell bearing the tumour necrosis factor. This process, known as ‘reverse signalling’, then changes the activity of this cell and constitutes a major new route through which information transfer can occur. We are currently characterising the biological changes that result from reverse signalling in specific types of leukocytes.

We are also looking at the role of membrane tumour necrosis factor in two separate models of viral disease. The first is influenza pneumonia that is responsible for a great deal of morbidity and mortality worldwide. The second is a model of poxvirus infection (mousepox) that mimics the disease smallpox in humans. The incidence of human poxvirus infections, such as monkeypox, is on the rise and there is an increased threat of the use of smallpox as a weapon of bioterrorism. Mousepox is a good model for the study of generalised viral infections and is also an excellent example of a virus that encodes proteins specifically designed to interfere with host tumour necrosis factor. Our studies will focus on the role of this cytokine in host-virus interactions and the outcome of infection.

Immunopathology, nitric oxide and autoimmunity
Dr Bill Cowden, Immunopathology Group

Work in the Immunopathology Group is focused on developing novel treatments for and understanding key biochemical processes underlying debilitating cell-mediated disease processes. As the term ‘immunopathology’ suggests, we are interested in the pathological, or tissue damaging, changes that occur as a direct result of either normal or abnormal immunological processes. Autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis are examples of pathologies that occur as the result of an abnormal immunological process because in these cases cells of the immune system appear to ‘attack’ apparently healthy tissue rather than perform their intended role of dealing with invading pathogens. Such cell-mediated autoimmune diseases are characterised by an accumulation of leucocytes in the affected tissue. Leucocytes are white blood cells and are the mainstays of the immune system. In the case of MS, an abnormal accumulation of leucocytes is found in the brain and spinal cord, while in arthritis this build-up occurs in the diseased joint. The usual role for these cells is in fighting infection but, in the case of autoimmunity, the cells appear to ‘recognise’ normal tissue as being ‘foreign’ and as a consequence of this ‘misinterpretation’ these cells attack the affected tissue. This results in damage to the tissue and is reflected in clinical symptoms. Symptoms of MS range in type and severity depending upon the areas of the brain affected. Transient mild weakness or tingling in the limbs may be the only symptom in some MS patients while others may suffer paralysis or blindness. Arthritis symptoms are invariably directly associated with the inflamed joint and once again the clinical symptoms range in severity from mild discomfort to crippling disfiguration.

Dr Bill Cowden, Immunopathology Group

Since an essential component of these diseases is the accumulation of inflammatory cells, a potential approach to treating them is to reduce or prevent the accumulation of leucocytes in the affected tissue, the inflammatory site. Leucocytes normally circulate through the body, along with erythrocytes (red blood cells), inside the blood vessels. The mechanism by which leucocytes arrive at the inflammatory site is both complex and incompletely understood. There are, however, many potential points at which interference with this process may occur. Work carried out in collaboration with others within the School and by the Neurosciences Research Group at The Canberra Hospital has identified a promising biochemical target for preventing cell migration. Research in our group is currently targeting one of the final biochemical steps involved in the migration of the leucocytes from blood vessels into the adjacent tissue. The translation of basic findings into clinical outcomes is central to our efforts and since experimental results have been promising we hope to trial the potential treatment in the clinic. One of our goals over the next twelve months is to initiate and facilitate a multicentre clinical trial in the treatment of relapsing-remitting MS. Planning for these trials is underway.

Another major interest of the group is the role of nitric oxide in autoimmune processes. Nitric oxide is produced in large amounts by certain leucocytes that have been stimulated by invading pathogens — especially protozoan parasites, such as those that cause malaria, and bacteria — that live and replicate inside host cells. Infection with some viruses also elicits the production of nitric oxide. The nitric oxide molecule is chemically reactive and this attribute may be responsible for its ability to inhibit the growth and spread of certain infectious agents. This property may also contribute to its widely reported immunopathological properties in both infectious and autoimmune diseases. Another way of looking at this is that cells of the immune system produce nitric oxide in order to damage invading microorganisms but in the process normal tissue in the immediate vicinity can also be damaged by this chemically reactive molecule. On the other hand, nitric oxide is also produced in small quantities by cells other than those of the immune system. In this situation, nitric oxide has some roles that are directly related to normal, nonimmunological processes such as the control of high blood pressure and neurotransmission. Other research groups within the school study some aspects of these activities.

The Immunopathology Group has investigated the potential pathological role of nitric oxide in some autoimmune diseases such as type-1 or juvenile-onset insulin-dependent diabetes and in a multiple sclerosis-like disease. In contrast to many reports in the medical and scientific literature, our group has discovered that in some autoimmune disease settings, nitric oxide has a ‘down-regulating’ effect on the disease. In other words, in addition to possibly having a localised, tissue-damaging role in these diseases, it may, in contrast, actually slow down or reverse the disease process. In this regard our group has found that in the absence of nitric oxide production by the immune system, some autoimmune diseases are actually more severe and protracted than would otherwise be the case. This finding implies that nitric oxide may function in a feedback manner to signal the immune system to reduce or halt its attack. This was further supported by our discovery that by increasing systemic levels of nitric oxide, either through immunological manipulation or drug treatment, these diseases were less severe. This finding is perhaps important from a therapeutic perspective and because of this the group is actively studying potential means by which nitric oxide production can be elevated in the hope that such therapy may be of benefit in the treatment of autoimmune diseases.

Nature and nurture in human characteristics
Professor Simon Easteal, Human Genetics Group

Professor Simon Easteal, Human Genetics Group

Research in the Human Genetics Group is primarily aimed at understanding how genetic and environmental factors combine to affect people’s health. Vital to this work are collaborations with colleagues from different research centres here at the Australian National University and elsewhere. For our major research effort on the determinants of common forms of mental illness associated with anxiety and depression, we work with the National Health and Medical Research Council (NHMRC) Centre for Mental Health Research (CMHR) at the ANU and its Director Professor Tony Jorm. The Path Through Life project is built around a CMHR longitudinal study of three groups of people of different ages sampled from the general population. Variation in genes encoding proteins important in the central nervous system are being studied in these people in combination with life history and environmental information to look for patterns associated with measures of mental health and wellbeing.

The combined study of many genes and environmental factors in large samples of people requires new approaches to statistical analysis of results. We are working with statistical colleagues in the Centre for Bioinformation Science (CBiS) at ANU who are developing novel statistical methods that will feed into the Path Through Life project. Consistent with the results of similar studies elsewhere, to date the work has not produced any clear-cut examples of genetic associations with common mental health characteristics. This underscores the complexity of this form of human variation. In another collaboration, we are working with researchers at the University of Sydney and the Australian Institute of Sport to identify genetic variation that influences performance in elite athletes. Last year the team discovered a significant association between a common loss-offunction variant of a muscle protein and elite participation in both power and endurance sporting events. Our other big interest is in human evolution. In a study with Drs Turakulov and Isaev from CBiS, we used information about variation across the entire human genome in combination with the recently published chimpanzee genome to identify variation in human populations from Africa, Europe and Asia that have been affected by natural selection. This evolutionarily important variation represents a major part of the genetic responses of human groups exposed to different diets, climates and diseases.

On the hunt for genes that protect us from autoimmune disease
Dr Gerard Hoyne, Medical Genome Centre

Autoimmune diseases are widespread and can be life threatening. They include disorders such as juvenile type 1 diabetes, systemic lupus erythematosis (SLE)-like disease, rheumatoid arthritis and some types of anaemias and thyroid dysfunction. In individuals with autoimmune diseases, the immune system which normally plays a vital role in protecting them from infection with microbial pathogens has turned on itself. It directs responses against their own tissues or organs leading to their destruction and the development of diseases. Normally during their development, lymphocytes, a variety of white blood cell involved in the immune system, are educated not to respond against the body’s own proteins (known as self proteins or antigens), and the vast majority of self-reactive lymphocytes are removed from the immune repertoire through a process known as clonal deletion. Unfortunately clonal deletion is not foolproof and a small but significant number of self-reactive lymphocytes escape deletion and enter the peripheral circulation even in healthy individuals. However the immune system has additional mechanisms in place to control the activation of these self-reactive cells that keeps them silent. Collectively this process of removal or silencing of selfreactive lymphocytes is referred to as immunological tolerance.

Dr Gerard Hoyne, Medical Genome Centre

Autoimmune diseases occur due to a breakdown of 'self tolerance' and this results in the activation of self-reactive lymphocytes that display specificity for self proteins expressed in particular organs. Through their capacity to secrete insulin, pancreatic beta cells play a critical role in regulating the level of blood glucose. Juvenile type 1 diabetes results from a breakdown in tolerance to X-cell derived proteins such as insulin leading to the destruction of the beta cells and development of diabetes. The disease normally begins during adolescence and requires patients to receive daily injections of insulin in order to stabilise their blood glucose. (Type 2 diabetes is not an autoimmune disease and is a late onset disease occurring in patients in their late 50s or 60s. It is caused by environmental or dietary factors that lead to a decrease in insulin production by pancreatic beta cells.)

As immunologists we would like to understand how autoimmune diseases begin. To accomplish this we need to be able to track the fate of organ specific, self-reactive lymphocytes in the blood and lymphoid tissues. This is difficult in a normal individual since the frequency of self-reactive cells in the blood is vanishingly small.

To circumvent this problem we use an experimental transgenic animal model that requires two different strains of mice. Lymphocytes can be divided into two types known as T- and B-lymphocytes or T- and B-cells. One strain of mice produces a receptor specific for a model protein Hen Egg Lysozyme (HEL) on virtually all of its T-lymphocytes — a T-cell receptor (TCR) transgenic. A second strain of mice has been engineered to express the HEL protein in a specific organ using a tissue-specific promoter. Under these conditions the immune system will treat the transgenic protein as a self antigen and mice should become tolerant to the HEL protein as it would to any other self protein in the body. To study type 1 diabetes we need to create double transgenic mice by breeding a TCR transgenic with a transgenic mouse that expresses the HEL protein in pancreatic beta cells — an InsHel mouse. The double transgenic (TCR:InsHel) mice are predisposed to developing type 1 diabetes since the majority of their T-cell repertoire is focused to a single protein antigen. Thus if these mice display any breakdown in immune tolerance they will succumb to type 1 diabetes as the HEL-reactive lymphocytes will attack and destroy the pancreatic beta cells.

Previous studies in the laboratory have shown that introduction of mutations in the mouse genome using a powerful chemical mutagen ethyl-nitroso urea (ENU) can be used to identify genes that are critical for lymphocyte development and growth. The process of chemical mutagenesis gives rise not only to loss-of-function mutations, but it can generate interesting new alleles, or variants, of known genes that can provide additional information about the biological activity of a given protein. We are particularly interested in identifying genes that are inherited in a recessive manner — that only cause disease when an animal inherits two mutant copies of the same gene, one from each parent. This mode of inheritance is normally associated with the development of human autoimmune diseases.

To identify genes that play a critical role in protecting an individual from autoimmune disease we have set up a genetic screen using the TCR:InsHel double transgenic mouse model. So far we have screened a total of 180 different strains of mice from two different ENU mutant libraries and observed diabetes in approximately 40 strains (approximately 20 per cent). However, we have only been able to successfully propagate the diabetic trait in three different strains and we are waiting to confirm inheritance in a further 13 strains. Although the screen is designed primarily to identify genes that protect from type 1 diabetes, we have identified three different strains that seem to develop diabetes that is independent of immune function. We anticipate these three strains may define genes that protect against type 2 (late onset) diabetes. Since pancreatic beta cells have to produce large amounts of the transgenic HEL protein this will place these cells under a degree of cellular stress, and combined with the underlying genetic mutation this may be sufficient to give rise to diabetes.

Once we have demonstrated inheritance of the phenotype, the observable characteristics, in individual animals that show the disease, we need to identify the chromosomal region where the mutation resides. This requires further rounds of breeding and genetic analysis of individual mutant mice to localise the mutated gene down to a small interval on a single chromosome that will hopefully contain only a handful of candidate genes. Once the interval has been defined we can then use a combination of DNA sequencing and bioinformatics to finally reveal the identity of the mutated gene. Also we can study the biology of the disease process in the mutant off spring using standard immunological and biochemical techniques, and this should reveal how the gene mutation leads to the breakdown in immune tolerance in self-reactive lymphocytes.

We hope this novel approach of using genome-wide mutagenesis will provide some fundamental insights into the genetic control of immune responses in vivo. Since the mouse and human genomes have now been sequenced, we will be able to determine if any of the genes we identify have been previously identified as susceptibility genes in human patients. Although the focus of the study described here is towards type 1 diabetes, we have also implemented a number of other immunological screens that will lead to a better understanding for the genetic basis of other autoimmune disease states such as systemic lupus erythematosis (SLE)-like disease and vaccination responses.

Turning the immune system against cancer
Professor Chris Parish, Cancer and Vascular Biology Group

Professor Chris Parish, Cancer and Vascular Biology Group

For over 100 years immunologists have tried to harness the immune system to eliminate cancers. Usually there have been attempts to immunise cancer patients against their own cancer cells, a procedure that is called cancer immunotherapy. Such an approach has many advantages. One would predict that side effects would be few, if not nonexistent, the approach would be cheap and cancers that fail to respond to current treatments could be eliminated. Unfortunately, so far, cancer immunotherapy has yielded disappointing results. Originally it was thought that the failure to immunise patients against their own cancer is because cancer cells are very similar to normal cells and, therefore, cannot be recognised as foreign by the immune system. It was eventually realised that this is not the case. Cancer cells are genetically very unstable and produce many abnormal or mutant proteins that can be detected by the immune system as foreign. A study in 1999 highlighted this point, the researchers calculating that a single carcinoma can carry as many as 11,000 genetic alterations when compared with normal 'healthy' cells! In fact, there are many reports that show that killer T-lymphocytes, a variety of white blood cell that attacks and kills other cells, can be generated against cancer cells, even in patients that are carrying the same malignant tumour.

Why then has immunotherapy against cancer been relatively unsuccessful? As discussed above, there is no doubt that immune responses can be produced against cancer cells. It is now becoming clear, however, that due to their enormous genetic instability, a cancer can rapidly produce mutant cells that can evade the immune system. Many viruses use a similar approach, the most notable of these being HIV which is particularly adept at dodging the immune system of the infected host. In the case of cancer there is mounting evidence that at the time of diagnosis many cancers have already been selected so that they are resistant to elimination by the immune system. It is no wonder that cancer vaccination has been such a frustrating area of research.

My group, the Cancer and Vascular Biology Group, has been attempting to devise vaccination strategies against cancer that overcome the problem of immune evasion. The conventional cancer vaccination strategy is to produce killer T-lymphocytes, or T-cells, that directly recognise and kill individual cancer cells. Unfortunately, using this approach there is often very rapid selection for cancer cells that are resistant to killing by the T-cells. Our rationale has been to produce an immune response against the tumour that is much harder to evade. The immune response we have chosen is that mediated by what are called Th2 cells. These T-cells recognise molecules secreted by the tumour and produce a range of anti-cancer effects. First, upon recognising tumour-derived molecules, they secrete molecules called cytokines that inhibit blood vessel growth. Tumours lack a blood supply and must induce the growth of new blood vessels, a process called angiogenesis, or they are unable to grow beyond about 1 millimetre in diameter. Thus a Th2 immune response against a tumour can shut down tumour growth by starving it of a blood supply. Second, the Th2 cells can attract into a tumour a class of white blood cell called eosinophils that contain intracellular granules full of toxic proteins. The Th2 cells, by a process that is not fully understood, instruct the eosinophils to degranulate and release their toxic payload. The result is the non-specific killing of many tumour cells in the vicinity. Interestingly, Th2 cells are associated with chronic asthma and much of the lung damage seen in patients with chronic asthma is due to degranulating eosinophils. We predict that it will be difficult for cancers to escape the anti-angiogenic effect induced by Th2 cells and the toxic products produced by degranulating eosinophils.

Experiments in animals support this new approach to cancer immunotherapy. Thus we have shown that tumour-specific Th2 cells can produce spectacular regression of establish melanomas in mice. The melanomas that we have examined were already resistant to elimination by killer T-cells and, therefore, were a good test of our new approach. In ongoing studies we are examining how important eosinophils are in controlling the emergence of cancers in animals. Using a mutant mouse that is unable to recruit eosinophils into tissues we have found these mice to be much more cancer prone than their normal counterparts. Thus, eosinophils are not only good cells to recruit into and control established tumours but they also appear to play an important role in controlling the emergence of cancers. We are currently attempting to translate our findings into the clinic by developing a new cancer vaccination strategy that involves the generation of tumour-specific Th2 cells.

Natural killer cells: Keys to the innate immune response
Dr Hilary Warren, Cancer and Human Immunology Group

In the late 1970s, a type of white blood cell was identified as a spontaneous killer of various cultured tumour cells without prior exposure to them. These natural killer (NK) cells are part of the 'innate' immune response to infectious organisms and cancerous cells, and they function immediately to protect the host. This contrasts with lymphocytes, another type of white blood cell involved in the immune system, that need a period of time following encounter with diseased cells to mature and produce antibodies and killer T-lymphocytes. NK cells are essential in the early stages of the immune response, whilst the antigen-specific immune lymphocytes develop. Indeed the few patients described who lack NK cells had recurrent viral infections. The challenge in understanding NK cells is to identify just how they regulate their activity. It is now clear that NK cells must be activated to kill by interaction with diseased cells (virally infected cells or cancers), and that this activation must not occur when NK cells encounter normal cells in the body. NK cells are regulated from killing normal cells by knowing the normal level of the body's own antigens (called self tissue antigens or transplantation antigens). Disease processes often lead to a lower level of these self tissue antigens, and when this happens NK cells are permitted to kill. The function of NK cells is not only to kill diseased cellsproduce a range of chemical messengers (cytokines) that attract other cells of the immune system. This sets up inflammation which is necessary to 'start' the immune response resulting in the production of antibodies and killer T-lymphocytes specific for the initiating pathogen. Inflammation is also necessary to 'resolve' the immune response which clears destroyed cells. As part of this process NK cells proliferate and become more potent producers of cytokines. Once the destroyed cells are removed, the stimulus for NK cell activity is removed, and the NK cells return to a 'resting' state and remain silent until another diseased cell is encountered.

Dr Hilary Warren, Cancer and Human Immunology Group

Our laboratory is currently focusing on NK cell proteins that are responsible for NK cell activation, in particular what they encounter on the diseased cell that allows them to kill and to proliferate. This work uses molecular biology to 'engineer' the proteins so that they can be attached to fluoresceinated lipid vesicles. These vesicles can then be used to identify the proteins on the diseased cell with which they interact. The advantage of using liposome vesicles is that they simulate the cell surface by allowing lateral movement and focusing of the attached proteins. This work uses technology developed at the JCSMR by Professor Chris Parish and colleagues.

Part of our work is undertaken at research laboratories at The Canberra Hospital, allowing us to interact with clinical colleagues and to translate some of our research to clinical investigations. In recent years we have investigated patients with the rare disorder of chronic NK cell lymphocytosis. These patients are either asymptomatic or present with only mild symptoms, and appear to tolerate vast numbers of NK cells. Our studies suggest that NK cells in these patients are the result of extensive proliferation of mature NK cells. We have shown that NK cells in these patients can monitor normal levels of self tissue antigens, and are effectively silenced. Interestingly, some patients spontaneously resolve their NK lymphocytosis over a period of years. A current study is investigating whether the status of various NK cell proteins are measures of autoimmune or inflammatory disease. These studies are ongoing and involve an analysis of cord blood cells from full-term healthy infants, and from preterm infants where placental pathology is well documented.

Division of Molecular Bioscience

Viruses and Ion Channels: A potential Achilles Heel? | Immune Responses on a Genome-wide Scale

Dr Frances Shannon, Head of Division

The Division of Molecular Bioscience has a broad focus with the general aim of understanding the molecular mechanisms governing the function of cells and applying this information to the aberrant molecular events that lead to disease. Areas of research within the Division relate to many diseases such as diabetes, asthma, allergy, anaesthetic complications, cancer and viral diseases. The scientific highlights for 2003 include a demonstration by the Baker lab that the pathway in cells that degrades unwanted proteins is linked to a signalling pathway often perturbed in cancer. The Casarotto and Dulhunty labs (Membrane Proteins in Health and Disease Program) have used biochemical and structural studies to design a series of peptide analogues that can regulate calcium levels in muscle. These peptides may have therapeutic implications in areas such as heart failure, malignant hypothermia and muscular dystrophy. The discovery by the Gready lab of a new human gene that is related to prion protein and exclusively expressed in the brain demonstrates the power of combining experimental and computational approaches with the availability of whole genome sequences to discover important new genes and cellular processes. Once again the Division was highly successful in obtaining project grants from the National Health and Medical Research Council (NHMRC), the Australian Research Council (ARC) and other sources. The NHMRC program grant on the Molecular Mechanisms in the Regulation of Allergy and Inflammation commenced this year supporting a continuation of multidisciplinary basic research into the pathogenesis of allergic diseases of the lung and gut. A common link has been identified between the induction of gastrointestinal hypersensitivity and bronchial hyperresponsiveness in asthma with the identification of the cytokine IL-13 as a key therapeutic target. In addition, a novel cytokine binding interface has been identified in the human beta common receptor, an important receptor in eosinophilic inflammation.

Professor Young was part of a team that obtained ARC-LIEF funding for mass spectrometry equipment to be located at the ANU. Several younger members of our Division were also successful in obtaining funds for their research with Natalia Gousseva awarded an International Union against Cancer Fellowship to travel to France to pursue part of her PhD studies. A large number of PhD students successfully completed their studies in 2003 highlighting the input of the Division in postgraduate education in the University.

Viruses and ion channels: A potential Achilles heel
Professor Peter Gage, Membrane, Physiology and Biophysics Group

The search for novel antiviral agents continues and the need is desperate. Most viruses are widespread and the diseases they cause are without effective cures. In Australia in 2002, there were 16,000 new cases of infection by the hepatitis C virus (HCV). The majority of infected persons are between 20 and 49 years of age. The financial cost of HCV infection in the Australian community is very high. In 1996–97, the last year for which accurate figures were published, the direct health-related costs were approximately $75 million while the indirect costs, such as production loss, were estimated to be $32.5 million. HCV-related morbidity in Australia is predicted by the National Centre for HIV Epidemiology and Clinical Research to triple by 2020. Refined regimens now result in clinical recovery in about 50 per cent of patients. However, treatment is expensive and many patients experience unpleasant side effects. In addition, only around 9,000 patients have been treated in Australia, due to the cost and strain on the resources of liver clinics. Novel antiviral agents are desperately required. Since HCV infects only humans and chimpanzees, there are no experimental systems, other than the HCV-infected chimpanzee, in which to examine antiviral agents which target other steps (both early and late) in the virus replication cycle. As the cost of an individual chimpanzee is currently US$180,000, it is clear that other systems in which to examine the efficacy of the complete range of putative antiviral agents against HCV are also desperately required.

Professor Peter Gage, Membrane Physiology and Biophysics Group

The Membrane Physiology and Biophysics Group has been investigating ion channels made by viruses in the membranes of host cells as potential targets for drugs which may prevent viral replication. If we could find how to block any virus ion channels we discover, it might be possible to affect replication of the virus and to come up with a new kind of antiviral drug. Studying viral ion channels might also give us information about how more complicated channels work.

Ion channels are specialised proteins embedded in all cell membranes that let ions, such as sodium and potassium ions, through when they switch open. They are important portals that convey information between the outside and the inside compartments of cells. In the brain, heart and skeletal muscle, movements of ions across ion channels generate the electrical signals that are essential for normal function.

About 12 years ago, it was discovered that a protein (M2) of an influenza virus formed an ion channel that was needed for normal replication of the virus. This was a 'simple' channel: 4 to 6 identical proteins are the staves of an ion channel 'barrel'. This was a surprising and provocative discovery. Even prior to the discovery of the M2 ion channel activity, a number of studies had drawn attention to changes in permeability of membranes in cells infected with a range of animal viruses. These changes occur at two distinct stages of infection: either during entry of the viral genome (the viral genetic material) into the cytoplasm (the body of the cell) or later in infection after expression of the appropriate viral gene.Individual virus proteins responsible for altering the permeability of cellular membranes have been identified.

Conceivably, these proteins may affect changes in membrane permeability by non-specific damage, by activating the host cell's own membrane transporters or channels; or by assembling to form new channels in the membrane, like M2. Structural comparison of small proteins from other viruses with M2 reveals a group of similar, small, hydrophobic, virus proteins that could conceivably form ion channels. Experimental evidence that these proteins affect membrane permeability has been reported for relatively few of these virus proteins but many more people are now beginning to study proteins from viruses that might form ion channels.

We had been investigating ion channels in excitable cells, such as nerve cells, for many years. So, about 10 years ago, we decided to investigate whether other viruses made ion channels and, if so, whether and how they were involved in virus replication. The first viruses we looked at were a different kind of influenza virus and the virus responsible for AIDS: the human immunodeficiency virus HIV-1. We soon discovered that both viruses made proteins that formed ion channels. For example, a protein called Vpu from HIV-1 made beautiful ion channels — good, clear openings and closings — and we discovered some drugs that could block them. There was good evidence for a connection between ion channel activity and budding of the virus when it escaped from cells. We found that our drugs that blocked ion channel activity also blocked budding of the virus. In experiments on the actual virus, it was found that the drugs depressed HIV-1 replication in white blood cells, the blood cells essential for combating infection and disease. So it does look as though another virus ion channel is involved in virus replication in some way. These discoveries have been protected with patents and are currently being developed by Biotron, a start-up biotechnology company.

Over the past two years, we have been enlarging our repertoire by studying proteins from other viruses such as the alphavirus responsible for Ross River fever, the hepatitis C virus (HCV) and the virus responsible for subacute respiratory syndrome (SARS). We have found that all these viruses make proteins that form ion channels. Since it is such a problem and the need is so great, several groups have focused on a small protein from HCV that forms ion channels.

We, and two other groups in the UK, have shown that the protein p7 of HCV forms an ion channel. Each group has identified different ion channel blockers: amantadine, long-alkyl-chain iminosugar derivatives and hexamethylene amiloride (HMA). The next major step in this project and our other projects is to show that drugs that block the channels also affect replication of the viruses. If we are successful in this, we may have contributed to the discovery of a novel kind of anti-viral agent.

Immune responses on a genome-wide scale
Dr Frances Shannon, Cytokine Gene Expression Laboratory

Dr Frances Shannon, Cytokine Gene Expression Laboratory

We now know the sequence of the human genome and of the genomes of many other organisms. This new knowledge is changing the way we approach biological and medical problems. The genes contained within the genome are a code for the proteins that form the working components of the cells that make up the body. Some genes continuously produce proteins, those proteins usually required for the day-to-day workings of all cells. Other genes only produce proteins in specific cell types — the proteins that are needed for brain function are only produced in cells of the brain, for example. The establishment of the cell-specific pattern of gene expression occurs as the organism develops and inappropriate control leads to developmental defects.

An organism can also respond to its environment and does so very rapidly by altering the pattern of proteins produced from the genome. In humans, one of the most highly responsive systems in the body is the immune system which is triggered into action as soon as the body is invaded by a foreign organism. The cells of the immune system respond by rapidly switching on the production of an array of proteins that are required to fight the invasion. Each gene contains a highly tuned molecular switch that allows the production of protein to be switched on and off rapidly in response to the appropriate signals. Here in the Cytokine Gene Expression Laboratory, we are studying the function of these molecular switches in the cells of the immune system.

Until very recently, it was only possible to study the production of protein from one gene at a time. As a result of the Human Genome Project, microarray technology has been developed that allows us to study tens of thousands of genes at one time. Its use to study the expression of genes is referred to as 'gene expression profiling'. The use of microarray technology allows us to visualise the response of the genome as a whole and not just the response of individual genes. The advantages of such a view are that we can now define regulatory networks within the cell and compare the function of these networks in disease states. In this way, we can identify entire pathways within the cell that are malfunctioning in disease, opening up the possibility of targeting these pathways for disease treatment.

In our laboratory, we are using microarray technology and gene expression profiling to gain insights into the function of T-lymphocytes, a variety of white blood cell that is responsible for cell-mediated immunity, when they are activated by an immune signal. With the aid of computational analyses, we have discovered groups of genes that are co-expressed and identified common molecular switches. By using animal models where key control proteins are deleted, we can visualise the global impact of these key proteins on cell function.

Such projects require cooperation between experimentalists, computational biologists and statisticians and it is this type of teamwork that will allow us to tackle important biological questions using the power of these new technologies.

Division of Neuroscience

Why Study the Brain | Visual Neuroscience Laboratory | How the brain communicates with itself | Understanding Hearing and Congenital Deafness |

Professor Caryl Hill, Head of Division

The central and peripheral nervous systems are complex constellations of nerve cells intricately connected and ultimately controlling motor, sensory and cognitive functions. Research in the Neuroscience Division is concerned with the cellular and molecular mechanisms by which these networks are formed during development and how they function in maturity to integrate multiple inputs and transmit information along neural pathways in the brain and in the organ systems which they control. An increased understanding of the processes underlying normal physiological function is an essential basis for determining how systems are perturbed during disease states. In 2003, research into both the central and peripheral nervous systems was bolstered with the arrival of Professor Trevor Lamb to head the Visual Neuroscience Laboratory, Associate Professor Christian Stricker to head the Neuronal Network Laboratory and Professor David Hirst to head the Autonomic Neuroeff ector Transmission Laboratory. These groups add new dimensions in the areas of visual processing in the retina, synaptic dynamics and gastrointestinal motility. Calcium plays a crucial role in both pre and postsynaptic events and complex imaging techniques are being employed by many groups within the Division to investigate at high resolution the compartmentalization of intracellular signaling components. Success with major equipment grants in 2003 has facilitated the purchase of a digital imaging setup with deconvolution software and will further enable the purchase in 2004 of a confocal microsope for gastrointestinal and vascular studies.

VWhy study the brain?
Dr John Clements, Brain Modelling Laboratory

Very few people these days have seen a brain 'in the flesh', and even fewer have been awed by the experience. More likely they felt a sense of surprise or mild repulsion at the sight of the strangely convoluted lump of pinkish grey meat. What is it that makes the brain so fascinating and vitally important that more than 50,000 scientists worldwide dedicate their working lives to it? One clear justification is the broad clinical relevance of neuroscience research. Brain malfunctions produce a plethora of diseases: epilepsy, depression, schizophrenia, stroke, Parkinson's disease, Alzheimer's disease, attention deficit hyperactivity disorder (ADHD) and bipolar disease (manic depression), to name but a few. The extraordinary diversity of these problems highlights the complexity of the system we are struggling to understand. Medical outcomes are important, but there are other justifications for studying the brain. Neuroscience, for me, began with a deep curiosity about how animals work. What controls their movements? How do they see and hear? How do higher animals learn, and form strategies for survival? Much of contemporary neuroscience research is curiosity-based. In discussing this work the emphasise is on the intrinsic fascination that draws us to study the complex inner workings of a normal healthy brain.

Dr John Clements, Brain Modelling Laboratory

The building blocks of the brain are its nerve cells, or neurons, and my lab focuses on their dynamic properties. An example of a dynamic property is the way in which a neuron adapts when it is stimulated several times in quick succession. Generally it will respond most strongly to the first one or two stimuli, then the response will fade. But more subtle behaviours are sometimes seen. These properties are important because neurons typically communicate with one another by sending out a short, intense burst of signals. The frequency or length of the burst encodes information. In turn, the dynamic properties of the neuron that receives a burst of signals determine how the encoded information is processed. These bursts are loosely analogous to the 'packets' of information that carry information across the Internet.

Sophisticated proteins found in the outer membrane of a neuron determine its dynamic properties, the 'receptor' proteins, which receive signals from other neurons. Transmitter molecules are released from a nerve fibre and immediately switch on receptor proteins in the neuron to which it connects, much as a key opens a lock. The dynamic properties of a neuron are influenced by the speed with which the transmitter molecules can unlock the receptors, and how long the receptors then remain active. In collaboration with colleagues at the University of Freiburg in Germany, we have studied a receptor which plays a vital role in forming memories that link two events (associative memories). We have shown that when a signal reaches the receptor, there is a significant and unexpected delay before it becomes active — the key turns slowly in the lock. The net effect of this newly discovered property is to finetune the receptor as a coincidence detector. We now have a physical explanation of why an associative memory is more likely to be formed when two events occur very close together in time.

The most clinically relevant research undertaken in my lab this year focused on another receptor which is found in neurons that control voluntary movements. A mutation in this receptor underlies the rare, inherited neurological disease hyperekplexia, or startle disease. A sudden noise or unexpected physical contact can produce a nasty seizure in people who suffer from this disease. Students working in my lab tested a wide range of drugs to see whether they could improve the function of the defective receptor. Although we found drugs which boosted its activity, they did so only at toxic concentrations. Nonetheless, the results of our study will be useful in future efforts to find a cure for hyperekplexia. The good news for people suffering from this debilitating disease is that a German group has just reported the discovery of a drug that boosts the defective receptor without toxicity.

Visual Neuroscience Laboratory
Professor Trevor Lamb, Visual Neuroscience Laboratory

Professor Trevor Lamb, Visual Neuroscience Laboratory

The Visual Neuroscience Laboratory is a new laboratory which was established in 2003 upon my arrival at JCSMR. Our overall research aim is to provide a detailed understanding of the molecular steps involved in the first stage of vision: the conversion of light into a neural signal in the rod and cone photoreceptors of the retina. This is being tackled by recording the electroretinogram (ERG) from the human eye, either with full-field illumination or with spatiotemporal patterns of illumination, and also by modelling and theoretical approaches. Human electroretinogram ganzfeld recordings During the year, a major piece of equipment was designed and constructed by the School's mechanical and electronic workshops: an 'ERG ganzfeld'. This apparatus permits us to record the electrical activity of neurons in the retina, and we have been particularly interested in the responses both of the photoreceptors and of the bipolar cells that process their output. By presenting a brief flash of light uniformly over the entire visual field ('ganzfeld') and measuring the resultant voltage at the front of the eye using a very fine conductive fibre electrode, we are able to measure the electrical responses of the rods and cones, as well as the rod bipolar cells.

It is a common experience that after exposure of the eye to very intense illumination one, at first, cannot see very well in a dimly lit environment. For example, on entering a cave after being out on a bright sunny beach, one is at first almost blind, but then steadily over a period of minutes one's vision improves. This phenomenon, termed 'dark adaptation', occurs not because of the absence of visual pigment rhodopsin, but because of continued signalling by rhodopsin molecules that have recently absorbed light. We are investigating this phenomenon in the retina by tracking the recovery of the photoreceptors and bipolar cells following exposure of the eye to intense light. We find that the cone cells recover much more rapidly than the rod cells, which take tens of minutes to recover, and we are now measuring the speed of recovery at the next stage of processing: in the retinal bipolar cells.

Human electroretinogram multifocal recordings
In a parallel study, we are investigating the spatial distribution of neural responses across the retina by recording the ERG elicited by 'multifocal' stimuli (mfERGs). The basic idea is that if different regions in the visual field are stimulated with pseudo-random sequences of illumination, then we can extract the signals elicited by stimulation of the individual regions. We are particularly interested in the spatial distribution of sensitivity and circulating current in the rod and cone photoreceptors. We have shown that the first negative-going wave of the mfERG corresponds closely to the 'a-wave' of the conventional full-field photopic ERG, which is known to be elicited by the activity of the cone photoreceptors and the cone OFF bipolar cells. We have examined the spatial profile of these responses and have shown that they obey 'spatial superposition' — that is, stimulation of a pattern of regions elicits the same response as the sum of responses to stimulation of the individual regions.

Modelling of human dark adaptation
We have also investigated human dark adaptation through an approach of cellular modelling and theoretical analysis in a collaborative study with Professor EN Pugh Jr in Philadelphia. Our analysis shows that the time-course of psychophysical dark adaptation and of visual pigment regeneration appear to be set by the rate at which bleached rhodopsin ('opsin') can recombine with 11-cis retinal that is diffusing down a concentration gradient from the retinal pigment epithelium to the rod photoreceptors in the retina. One outcome of this research is that it is now possible to obtain an estimate of the relative concentration of the critical retinoid 11-cis retinal in the living eye through simple non-invasive measurements.

Our approaches are providing us with a better understanding of the first stage of vision: the mechanism by which rod and cone photoreceptors respond to light, and the way in which they recover after intense illumination. These studies will next be complemented with 'single-cell recordings' from rod and cone photoreceptors isolated from the retina, now that the next major piece of apparatus is nearly complete.

How the brain communicates with itself
Associate Professor Christian Stricker, Neuronal Network Laboratory

To understand the complex functions of the brain, it is necessary to understand how individual nerve cells communicate with each other and how their interactions shape the fl ow of information within networks of cells in the brain. However, the strength of communication between two neurons (nerve cells) is not static, but is changeable, depending on previous experience. ? e modulation of the strength of the signal is thought to be central to the brain’s capacity to learn, form memories and adapt to a continually changing environment. Neuronal communication is classically thought of as resulting from the entry of calcium ions (Ca2+) through channels in the membrane at the end of a nerve cell resulting in the release of a chemical called an additional source of calcium within terminals called intracellular reticulum, a cellular compartment into which calcium in the form of calcium ions is actively pumped. Calcium release from the intracellular stores is controlled in such a way as to play a crucial role in controlling the strength of signals between cells and so has significant consequences for the flow of information in the brain.

Associate Professor Christian Stricker, Neuronal Network Laboratory

During sustained neural activity, the time course of calcium release within the nerve terminal and, therefore, the efficiency of message transmission between cells is determined by control of calcium flow from within and outside the cell from the sources mentioned which together determine the calcium level at the cellular level.

The importance of studying the contribution of internal calcium stores to the release of the neurotransmitter stems from the observation that dysfunctional regulation of calcium levels has been implicated in the pathogenesis of Alzheimer's disease. One current hypothesis is that sustained disruption of the level of calcium causes the degeneration associated with Alzheimer's disease; that an increase in calcium concentration elicits the characteristic lesions, including accumulation of beta amyloid fibrils; that changes in calcium signalling occur during the initial phases of the disease (even before the onset of overt symptoms); and that disruption of calcium regulation in its storage compartment, the endoplasmic reticulum brings about the nerve signal cascades that lead to Alzheimer's disease.

We are examining the capacity of communication within the smallest network imaginable: that formed between two nerve cells connected with each other in a slice of brain from the cortex of a rat. Within this microcircuit and under well-defined conditions, we are able to quantify the biophysical properties determining the rate of transmitter release at different stimulus frequencies, the changes in efficacy observed within a burst of activity, the rate of recovery after the burst and the mechanisms involved in shaping these parameters using pharmacological tools. Our aim is to determine within an animal model the changes that alter the effectiveness of communication within the brain in conditions like Alzheimer's disease.

Understanding hearing and congenital deafness
Professor Bruce Walmsley, Synapse and Hearing Laboratory

Professor Bruce Walmsley, Synapse and Hearing Laboratory

Congenital deafness is often due to dysfunction of the cochlea: the organ in the ear which converts sound into electrical impulses. These electrical impulses are normally transmitted directly to the brainstem via the auditory nerve, where they are processed and sent to higher brain centres for interpretation. In peripheral congenital deafness, the connections between auditory neurons in the brain are still formed during development, but they may be abnormal since they do not experience normal auditory activity. As with all of our sensory systems, it is thought that auditory experience during development is important in shaping the properties of neurons and their synaptic connections. We are addressing this issue by studying the properties of neurons and synaptic connections in a mouse model of congenital deafness called dn/dn. Our results have revealed that the properties of auditory neurons and synapses are very different in the deaf mice. The most striking difference is in the response properties of the neurons to synaptic input. The cells in the deaf mice are much more excitable, and produce many nerve impulses in response to a single synaptic input, whereas the same types of cells in a normal hearing mouse respond with just a single nerve impulse. Our results are providing important new insights into the fundamental question of the role of nerve activity during development in regulating neuronal properties. Our results are also relevant to the functioning of cochlear implants, in which the auditory nerve is electrically stimulated, since our research shows that the response of the auditory neurons can be very different in congenital deafness.