Review Essays

These invited essays were written for the 2002 John Curtin School print Annual Review.

Dendritic Cells-a vital component of the immune response | Heparan Sulfate, a key molecule in cancer and other diseases | How changes in the brain affect emotions and memory | Emotions and the brain | Viral disease strategies | Molecular Biology and Pathogenesis of Flaviviruses like Murray Valley encephalitis and Japanese encephalitis virus | The science of hearing | What's in the histone code | A GST called Kappa | Arterial function and disease | High Blood Pressure and Glucocortoids | Asthma and the Immune Response | Breaking the code

 

Dendtitic Cells - a vital component of the immune response Joanne Banyer

The ability of the body to develop immunity against pathogenic infections is dependent on the activities of a particular type of immune cell called the dendritic cell. Progenitors of this cell type can be found migrating through tissues and circulating in the blood stream. They constitute a part of the body's first line of defense against invading microorganisms. The fate of this cell lies in the hands of the surrounding microenvironment where factors determine the types of properties these cells develop as they mature. Infections by invading microorganisms can cause massive changes to the components of the microenvironment. The progenitor dendritic cell is designed to detect these changes through special molecules on its cell surface. These molecules recognize features about pathogens that the cells of the body do not have, thus allowing a distinction between self and non-self. Recognition of the pathogen sets a cascade of events in motion that essentially rings an alarm bell for the body notifying it of an invading organism, and prepares the body to recognize and eliminate the pathogen.

As the properties of a dendritic cell reflect the nature of the microenvironment an immune response custom made for a particular pathogen can be generated. For example, the presence of a viral pathogen can stimulate production of cytokines such as IFN-? and IL-12 into the microenvironment that induce dendritic cells to develop properties that direct cytotoxic T cell immune responses against the virus. Whereas pathogens such as yeast induce the production of IL-4 that, instead, stimulate dendritic cells to develop properties that direct humoral antibody immune responses against yeast. The types of molecules and mechanisms that enable the dendritic cell to be so flexible at inducing different types of immune responses are the subject of intense investigation by many research groups around the world. This information will be highly important for improving the design of many vaccines and immunotherapeutics aimed at preventing and treating different diseases.

Our work aims to investigate the complex series of interactions between pathogens, cytokines and dendritic cells, which subsequently enables these cells to direct development of particular types of immune responses. To do this we utilize a broad range of genetic and biological analysis of both in vitro and in vivo cell systems. We have three areas of study, the first of these aims to identify genes that are induced by pathogenic and cytokine stimulus of dendritic cells. The significance of this area is in determining the mechanisms that enable dendritic cells to induce different types of immune responses. These genes may be useful as vaccine and immunotherapeutic adjuvants to support development of particular types of immunity. By utilising cDNA subtraction to identify gene profiles, we have established that different cytokine microenvironments stimulate expression of immunoregulatory genes that are tailored to induce suitable immune responses against pathogens. Through the development of a new in vivo model we hope to now demonstrate the time and location during an immune response at which dendritic cells are induced to express these immunoregulatory genes. By utilising the same model we also hope to examine the immunoregulatory phenotype that is developed by dendritic cells in response to different vaccination regimes, such as prime/boost, where the underlying mechanisms enabling this vaccine strategy to be highly effective are not resolved.

Our second area of study examines the effect of DNA and viral vaccine agents on dendritic cells. This work aims to establish the types of immunoregulatory properties induced by these agents and whether particular cytokines facilitate the uptake and processing of these agents. The significance of this work is two fold. 1. Determining optimal conditions for the generation of dendritic cells for use as immunotherapeutic or anti-tumor vaccines. 2. Determining how vaccine strategies, such as prime/boost are initiated through the interaction of vaccine agents with dendritic cells. These studies, as well as those performed by other groups, have found the choice of vaccine agent is highly important as some of these induce apoptosis (cell death) of dendritic cells whilst others contain immunostimulatory molecules that amplify immune responses. We have also found the maturation state and combination of cytokines used to stimulate dendritic cells have a significant affect on the immunoregulatory properties these cells develop, that subsequently also affects their ability to take up and process vaccine agents.

The third area of study examines the affect of pathogens on antigen-presenting dendritic and macrophage cells. We have a special interest in immune avoidance mechanisms elicited by persistent viral infections, such as Ross River virus and Hepatitis C. The significance of this work is in determining how these types of viruses avoid immune detection and establish low-level persistent infections in antigen presenting cells. Identifying associated molecules may provide useful targets for immunotherapy against persistent viral infections. In the pursuit of this area, in collaboration with Dr Lidbury from the University of Canberra, we have developed a novel in vitro cell line model that mimics Ross River virus persistent infection of macrophage cells. The virus in these cells undergoes periodic relapse in viral load and cytopathic effect. By utilising the genetic technique, cDNA subtraction, we hope to target genes that enable viral persistence and relapse in these cells.

Heparan Sulfate, a key molecule in cancer and other diseases Craig Freeman

We are investigating the roles that the sulfated carbohydrate heparan sulfate (HS), and the enzymes that degrade it, play in health and disease. Our major focus is how they are involved in the spread of cancer (tumor metastasis), inflammation and the growth of new blood vessels (angiogenesis).

HS plays a vital role in many biological processes, including cell growth and development, cell attachment to the surrounding matrix, the breakdown of triglycerides and the entry of viruses and other pathogens into cells. Many biologically important proteins, enzymes and growth factors bind to cell surface HS which can regulate their physiological actions. To ensure the specificity of their actions, many of these proteins recognise unique sugar sequences within the HS molecule, which is quite variable in its structure. We are therefore developing procedures to determine these particular sugar sequences which will allow us to design novel drugs (HS-mimetics) that mimic that particular HS sugar sequence. These drugs may then be used to specifically inhibit various physiological and pathological processes involving HS-protein interaction. For example, since tumor growth is critically dependent upon the development of a new blood supply, the selective blocking of angiogenic growth factor binding to cell surface HS has become a novel way to interfere with tumor development.

HS is also a key component of the extracellular matrix (ECM) and the vascular basement membrane that surrounds the blood vessels and acts as a barrier to cell invasion during tumor metastasis. Malignant tumor cells have elevated levels of the enzyme heparanase, which degrades HS, causing breakdown of the ECM structure and allowing tumor cell invasion. Heparanase is normally involved in embryonic development, angiogenesis, wound repair and inflammation permitting cell migration through the ECM and the release of growth factors stored within the ECM that stimulate cell growth. However, heparanase activity secreted by a growing tumor may release these growth factors, stimulating further tumor growth and new blood vessel growth that can allow subsequent tumor cell escape. Similarly, the uncontrolled invasion of leukocytes into the ECM can lead to inflammatory diseases such as inflammatory bowel disease and the progression of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis.

Previously, our group identified the HS-mimetic PI-88 which is both an effective inhibitor of heparanase activity and angiogenenic growth factor binding to HS. In animal models, PI-88 prevented the growth and spread of cancer as and it is currently undergoing clinical trials in cancer patients. In conjunction with Dr Martin Banwell at the Research School of Chemistry, ANU, we have developed a new series of HS-mimetics. Preliminary studies have shown that some of these compounds exhibit potent and selective inhibitory activity against heparanase activity and the binding of various growth factors, chemokines and proteins to HS. Such compounds may lead to the development of a new series of drugs to prevent cancer, inflammation, viral infection and to lower blood triglyceride levels.

Within the Cancer and Vascular Biology Group we are collaborating with Prof Chris Parish and Dr Mark Hulett to investigate the roles of heparanase in both health and disease, including the factors which regulate its normal activity. Using a novel enzyme assay, we were one of the first to purify and clone human heparanase, demonstrating that only one heparanase activity is expressed. Therefore heparanase represents an excellent target for the development of anti-cancer and anti-inflammatory drugs. We are currently characterising proteases that activate the enzyme as well searching for the presence of endogenous inhibitors to determine if control of these factors can also be used to inhibit heparanase activity. We are also studying the interaction between tumor cells and blood platelets which is occurs during tumor metastasis as well as identifying important ligands involved in this interaction. Our overall goal is to better understand both the biology and structure of heparanase to enable the development of inhibitors of the enzyme, which will hopefully lead to new drugs to prevent cancer spread, angiogenesis, and inflammation.

How changes in the brain affect emotions and memory John Power

As a member of the emotion laboratory my research has focused on understanding how changes in the brain mediate emotional learning and memory. We work on an area of the brain called the amygdala, which is the Latin word for almond, and is involved in emotional processing. Amygdala damage is associated with severe deficits in the expression of emotion and the recognition of emotion in others. My work is focused on the basolateral portion of the amygdala (BLA). The BLA is important in assigning emotional value to stimuli. This emotional associative learning can involve aversive stimuli or pleasurable stimuli, such as food, sex, and drugs. Emotional processing in the amygdala is thought to be involved in disorders such as phobias, post-traumatic stress disorder and addiction.

The long-term changes that underlie learning and memory are almost certainly mediated by rises in cytosolic calcium. Calcium activates a number of signaling pathways regulating nearly every cellular function including gene transcription. While much in known about amygdala dependent behavior, little is known about calcium handling in the amygdala. Over the last two years I have been examining the electrical and calcium responses in amygdalar neurons. To measure these signals glass electrodes are attached to individual neurons in rat brain slices. With this glass electrode we can record electrical activity and fill the neurons with a calcium sensitive fluorescent dye. Neurons in the BLA have an extensive network of fine branching processes called dendrites that can be less than 1 micron in diameter. The division's recent acquisition a two-photon scanning laser microscope has allowed us to monitor calcium in neurons at a sub micron resolution.

Using this technique we have found that stimulation of amygdalar neurons can evoke a rise in cytosolic calcium via either influx from the extracellular fluid, through voltage gated calcium channels and glutamate receptors, or via release from intracellular calcium stores. Sometimes these calcium rises are restricted to small areas of the dendrites. However, other times brief stimulation can evoke calcium rises over large areas of the neurons, including the nucleus where it is likely to initiated changes in gene transcription. We have also found that small stimuli that evoke little or no voltage response can produce large changes in calcium.

By understanding how the learning happens on a cellular level we hope to develop therapeutics for aberrant emotional learning thought to underlie addiction, phobias and post-traumatic stress disorder.

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Emotions and the brain Louise Faber

 

Our laboratory is interested in the part of the brain that mediates emotions, the amygdala, which is an almond shaped structure located in the medial temporal lobe. Of all the emotions, the amygdala has been shown to be particularly important for mediating fear. Lesions of the amygdala in monkeys cause an array of behavioural effects, including an apparent loss of fear. In humans the amygdala has been shown to be important for judging emotional expressions on faces, especially negative emotions such as fear and anger. In addition the amygdala has also been attributed with a role in subconscious processing of emotions, in effect being able to survey external surroundings for any potential threat. If a threat is perceived, the amygdala is then able to alert the person through activation of arousal systems and the "fight or flight" reflex. Malfunctions of amygdala circuitry are believed to underlie anxiety, phobias and post traumatic stress disorder.

The amygdala receives input from many different areas of the brain, including higher processing areas such as the cortex, and projects to many brain areas, including brainstem structures. The amygdala receives sensory information from the cortex and responds to this information by stimulating brainstem areas that initiate the appropriate behavioural response to the input, including changes in blood flow, heart rate and hormone release. Anatomically the amygdala is divided into 13 subdivisions. The basolateral complex, which is comprised of the basal and lateral nuclei, is considered the input zone because it receives most of the sensory input. In contrast the central nucleus is considered the output zone since it sends most projections to the brainstem. While a substantial amount of work has been carried out on these anatomical pathways, little is known about their physiology.

Our work concentrates on understanding the physiology of the basolateral complex of the amygdala. To do this we use the whole cell patch clamp technique, which is a technique that allows the electrical properties of single neurons to be recorded using glass microelectrodes. In particular we have been focusing on the properties of individual neurons within the lateral nucleus, in terms of the patterns of action potentials that they fire when they are stimulated. We found that cells display a continuum of firing patterns whereby some cells fire repetitively when stimulated while other cells fire in bursts. These different firing patterns will be important in determining how information is relayed through the amygdala. Following characterisation of a cell's physiological properties, the morphology (ie the size and shape of the cell's body and dendritic tree) can then be examined by injecting a dye into the cell. We found that the differences in firing patters were not due to differences in morphological properties.

In addition we have examined the mechanisms that control the firing patterns. There are a number of different proteins located on the cell membrane that can allow the flow of ions either into or out of the cell and thereby control the excitability of the cell. Potassium channels are a type of protein that open in response to changes in voltage or in intracellular calcium concentrations, allowing potassium ions to flow out of the cell. We have found that there are two main types of potassium channel, one voltage-activated and one calcium-activated, which control the firing patterns of neurons in the amygdala. These channels are susceptible to modulation by opioids or by the neurotransmitter acetylcholine. These findings point us in a new direction for understanding the cellular mechanisms underlying emotional processing in the brain, and may lead to future treatments for anxiety or post traumatic stress disorder.

Viral disease strategies Guna Karupiah

Viral diseases are a major social, health and economic problem worldwide. Effective vaccines have been successfully developed for only a small minority of disease-causing viruses and available antiviral agents are of limited efficacy and high cost. Furthermore, little effective intervention is currently available to minimize the often, serious immunopathology in established infections. The outcomes of our ongoing studies have the achievable goal of addressing some of these problems.

The immune response has several strategies to combat infectious disease. These include innate and adaptive components, all of which are regulated by a network of critically important cellular interactions and soluble mediators. Interestingly, however, the effector mechanisms that are generated to control and clear virus instead often cause immunopathology that has serious, sometimes lethal, consequences for the host. The objective is to modulate the response in order to direct the outcome towards efficient virus clearance and to minimize pathology. Our research efforts have been directed toward dissecting out the immunological parameters that allow the rapid resolution of virus infection with minimum pathology and also generates immunological memory. These studies are being carried out in parallel with others that attempt to reveal the many strategies that viruses have evolved to subvert the host immune response. Indeed, we have much to learn about our own immune system from these viruses.

In the last year, our group has made substantial progress in a number of key areas of our research. First, we now have a better understanding of the effector mechanisms employed by CD8+ lymphocytes (a key population of white blood cells necessary for virus control) to clear virus in vivo. Whether these lymphocytes employ their cytolytic potential or their ability to produce cytokines depends on both the type of virus and its virulence.

Second, in collaboration with Dr. Geeta Chaudhri, we have recently established an animal model for studying the consequences of 'reverse signalling" via membrane bound tumour necrosis factor (TNF), a key proinflammatory cytokine that is also involved in leukocyte recruitment. We have found that this cytokine is critical for recovery from some viral infections.

Third, we have some useful insights into how the antibody that is produced in response to a primary viral infection, helps control virus proliferation and spread. In addition, our studies on the role of antibody in providing protection against reinfection with the same virus challenge some widely held views on the role of CD8+ lymphocytes in this process.

Fourth, we have evidence that neutrophils are critical for helping to shape the protective immune response to viral infection. Neutrophils act as the first line of defense against invading pathogens but paradoxically, they are also involved in the pathology associated with various inflammatory conditions. There has been a widely held view that neutrophils do not play a significant role in the control of viral infections. This view stems from the observation that individuals with disorders of neutrophil function or numbers have an increased susceptibility to specific bacterial and fungal pathogens. Our data, however, clearly indicates an important role of this subset of leukocytes in the immune response to virus infection.

Finally, our group has been working for some time on the gas nitric oxide, an important molecule involved in normal physiology and an antiviral molecule. This soluble mediator is produced by the host, as a consequence of the immune response to an infection. We have shown that nitric oxide is also a mediator of pathology in influenza A virus infections and are in the process of identifying the effector mechanisms critical for the pathogenesis of influenza pneumonia.

Molecular Biology and Pathogenesis of Flaviviruses like Murray Valley encephalitis and Japanese encephalitis virus Mario Lobigs and Eva Lee

Of the dynamic host/pathogen interactions, those involving viruses are the most unpredictable. It has been repeatedly observed that viruses rapidly evolve new species- and tissue-tropisms, emerge in and adapt to new ecosystems, alter their virulence properties and pathogenic potential, and evolve mechanisms that allow viral evasion from the host immune response. A general aim in our group is to study the host/pathogen interaction of flaviviruses at the molecular, cellular, and whole system level and through this devise strategies for the prevention and treatment of flaviviral diseases. Flaviviruses are small RNA viruses transmitted to vertebrates by the bite of an arthropod vector (such as a mosquito); they include some of the most important viral pathogens for humans, which can cause life-threatening hemorrhagic fevers and encephalitis (e.g. yellow fever, dengue, Japanese encephalitis viruses).

Two of the medically important flaviviruses have recently emerged in geographic regions where they have previously not been encountered: West Nile virus in North America and Japanese encephalitis virus in Australia. This demonstrates the capacity of these viruses to alter or expand their geographic distribution, a process which may be aided by global warming (because it leads to the extension of the geographic range of the mosquito vectors) and rapid global movement of infected vertebrate hosts or mosquitoes. The altered flaviviral epidemiology raises urgent questions with respect to its impact on human health.

Receptors and virulence
Study of the fundamental processes of viral infection of a host cell or organism is crucial to our efforts to combat viral diseases. Viral infection begins with attachment of the virus to the cell surface and is followed by virus internalisation. For a great number of viruses, the availability of an appropriate cell surface receptor determines the ability of the virus to replicate in a particular cell type. Viral receptor usage can be altered by adapting a virus to grow in a particular cell type. This can impact on the virulence properties of a virus. We have demonstrated that virulence attenuated variants of encephalitic flaviviruses can be consistently generated by propagation of these viruses in a human adenocarcinoma cell line. These attenuated variants were altered in their entry properties, displaying an increased dependence on sulphated proteoglycans (long acidic sugar chains attached to the cell surface via a protein core). As a consequence of this altered receptor usage, the cell-adapted virus variants were rapidly removed from the circulation when inoculated into mice and prevented from invading the central nervous system. Knowledge of molecular determinants on viral variants that account for altered growth properties and of in vivo mechanism of virulence attenuation assists in the design of live-attenuated viral vaccines with multiple defects as well as in development of antiviral therapy.

Immune-escape
An intriguing phenomenon in the flavivirus host/pathogen interaction is the discovery by us that MHC class I molecules - the recognition elements for cytotoxic T cells critical in the elimination of the infected cells by 'killer' T cells - are presented in much larger number on the surface of infected cells. We have recently defined the molecular mechanism for this virus-induced effect. It involves the virus-mediated increase in peptide supply from the cytosol (liquid part of the cell) into the lumen (interior space) of the endoplasmic reticulum, an intracellular organelle where the assembly of peptides with MHC class I molecules takes place (see Figure 1). Peptide loading is critical for the MHC class I molecules to exit the intracellular compartment and travel to the cell surface. Thus an increased peptide supply into the endoplasmic reticulum up-regulates cell surface expression of the MHC class I restriction elements and peptide presentation to cytotoxic T cells. Flavivirus-induced up-regulation of MHC class I expression impinges on the cellular immune responses of the host against this virus. An important consequence of this modulation of the MHC class I pathway is the down-regulation of the antiviral activity of natural killer cells which serve in the 'early' control of viral infections. This mechanism constitutes a novel variation to the theme of viral immune-escape.

Immune-enhancement and vaccines
Japanese encephalitis virus (JEV) was first introduced into the Torres Strait and mainland Australia in 1995 and 1998, respectively, most likely due to the arrival of mosquitoes carrying the infectious agent and originating from neighboring countries north of Australia. Given the likelihood of recurrent outbreaks of JEV in northern Australia, vaccination of the 'at risk' population must be given critical consideration. A complicating factor is the co-circulation of the medically most important Australian flavivirus, Murray Valley encephalitis virus (MVE), with the newly introduced pathogen in this region. The two viruses are genetically closely related and elicit cross-reactive immune responses in humans and mice. However, these are not necessarily protective and may, in some circumstances, be deleterious to the disease outcome because of the phenomenon of antibody-mediated immune-enhancement of viral infection: it involves enhanced uptake into macrophage-like cells of a virus, when bound with antibodies that fail to neutralise. Thus, preexisting immunity to one virus can increase the severity of infection of a second, closely related virus.
We have investigated the potential occurrence of vaccine-induced immune-enhancement in the JEV-MVE pair. The only internationally approved vaccine against JEV is a 'killed' virus preparation grown in the brains of infant mice. We show for the first time in an animal model for flaviviral encephalitis that a 'killed' vaccine can dramatically increase the disease severity following challenge with the heterologous virus. In the light of this result a critical examination of the proposed use of the 'killed' JEV vaccine in Australia is warranted.

The science of hearing Sharon Oleskevich

How does hearing work?
Hearing is extremely important for language and environmental awareness. There are three distinct steps in the process of hearing. First, sounds enters the ear and is converted into electrical signals which travel along the auditory nerve towards the brain. Second, connection points (synapses) act as a bridge to transfer the electrical signals between nerve cells through a series of brain regions. Thirdly, there are the "hearing centres" in the brain where sound is finally perceived. The ear and auditory nerve are like a microphone and its wire, while the synapses act like an amplifier, regulating the gain of the electrical signals.

In the laboratory, we are concentrating on the synapse or connection points and how the synapse transfers sound information to the brain. There are billions of nerve cells in the brain and trillions of synapses, but technology allows us to focus in and concentrate on one connection point at a time. We are particularly interested in the synapses between the auditory nerve and the nerve cells in a brain region called the cochlear nucleus. We are working to find out how synapses are regulated in this region.

We have studied how the synapse functions both for normal hearing and for subjects who are deaf. In many forms of deafness, the problem is in the ear while the hearing centres in the brain remain functional. In some forms of congenital deafness, there is a loss in the number of connections, so it is very important to know how the remaining connections work.


How do we study synapses?
The team is working with a strain of mice which is profoundly deaf from birth, and whose deafness resembles certain forms of congenital deafness in humans. The deaf mice were originally discovered in a London hearing institute. Animals are deaf due to a naturally occurring defect in the inner ear. The mouse ear is similar to the human ear and is composed of an outer, middle and inner ear. Hearing in mice can be tested by measuring the electrical activity in the brain following the injection of sound into the ears. In contrast to normal mice, deaf mice have no electrical response to the sound. Slices of brain tissue from the normal and deaf mice are placed under a microscope so that we can see the nerve cells in the brain. We stimulate the auditory nerve and record the electrical response in a nerve cell. By monitoring the electrical signal, we can see how well the signal passes through the synapse.


What have we found in the deaf mice?
The research has shown that the connection points, or synapses, between the ear and the brain are almost three times stronger in deaf mice than in normal mice. The synapses seem to compensate for a lack of sound in the deaf animals. This agrees with other studies which show that the connection points in the brain are constantly changing.

Our research is discovering how the connections are different when they are made without any sensory experience (i.e. hearing). Our work in the auditory system may well prove valuable for patients with cochlear implants. The cochlear implant is a type of hearing aid used when mechanisms in the inner ear fail, but the auditory nerve to the brain is still functional (similar to the deaf mice). Our research may prove useful to cochlear implant technology, as well as providing valuable insights into how the connections in the brain are formed and how they can be modified.

What's in the histone code Sudha Rao

Genomic DNA is the ultimate template of our heredity. Despite the considerable excitement over the mapping of the human genome, many challenges remain in understanding the regulation and transduction of genetic information contained in genomic DNA. One of these is to explain the amazing discovery that the number of protein-coding genes in a human, estimated at approximately 35,000, is only double that of the comparatively simpler life form of Drosophila melanogaster, a fruit fly. The key question here, is whether DNA alone is responsible for generating the complete information necessary which ultimately results in a complex eukaryotic organism (one whose cells have a nucleus) such as a human being. It has been proposed that regulatory mechanisms at the level of histones, the DNA packaging proteins, may play a critical role and it is essential to explore the information potential held within the coding of histones.

In the nuclei of all eukaryotic cells, genes exist in the nucleus in a complex three dimensional structure that is referred to as chromatin. At its most fundamental level, chromatin consists of a repeated structure known as the nucleosome. Each nucleosome is made up of histone proteins (H2A, H2B, H3 & H4 organized as an octamer (eight-membered) structure) around which 145 base pairs of DNA are wrapped. This so-called "beads on a string" structure is further compacted into higher order chromatin structure known as the chromatin fibre and finally into chromosome structures.

Although chromatin was historically thought of as an inert repressive structure, we now know that the nucleus, the nuclear compartments, even the most fundamental units of the chromosome, the nucleosomes are all under continual transformation. Condensed chromatin is generally regarded as being repressive to transcription (the translation of the DNA code into proteins) and thus a key aim of our work has been to understand the mechanisms by which chromatin is modified or remodelled to allow gene transcription to occur. Several mechanisms have been identified that contribute to chromatin remodelling. They vary from those that alter histone-DNA interactions to those that physically dissociate the DNA from the histones. All of these processes are likely to act simultaneously and in a concerted manner regulate access to the DNA template. Recent studies clearly suggest that the sharp boundaries between active and inactive chromatin break down in cancerous tissues. Thus, the understanding of the mechanisms of chromatin mis-regulation in cancer cells is one of our key aims for the future.

A major challenge in our work has been to develop techniques to study chromatin structure on real genes in primary cell systems, where sampling material is limited. Towards this goal, we have successfully established a technique termed Chromatin Accessibility by Real-Time PCR (CHART-PCR) to monitor quantitatively for the first time chromatin alterations in vivo with a few thousand cells. Using such sensitive approaches, we have recently shown that the chromatin, within a precise region of a gene known as the promoter, becomes selectively altered for gene transcription to occur. In addition, we have identified key molecular switches that are co-ordinately recruited to the promoters of such genes, in response to external stimuli, that alter chromatin structure.

The post-sequencing era has been further nourished by the development of emerging technologies, such as the microarrays, which allows a snap shot of the behaviour of all genes expressed (that is, producing their proteins) in a cell in response to specific signals or at different stages of development. A host of recent studies illustrate elegantly how data obtained from expression profiling can be used to explore transcriptional regulatory networks in simple organisms such as yeast, providing a further insight into the mechanisms of gene regulation. We have been utilising a similar approach to decipher the role of specific molecular switches in more complex eukaryotic systems(Figure 1). There are two major issues here. Firstly, do large co-regulated groups of genes share common DNA binding domains that bind specific molecular switches? and secondly, do co-regulated groups of genes close to each other along linear DNA on the chromosome act, or are they simply co-localised in the nucleus? Hence, we hope, understanding the chromatin structural changes in response to external signals in normal cellular systems will allow us insight into potential dis-regulation in disease states. This will have a major impact on future therapeutic strategies.

Figure:1 The diagram illustrates a global approach to gene regulation, whereby expression profiles from different conditions or phenotypes are compared. By using cluster-analysis tools it is possible to identify groups of co-regulated genes, genes with related functions. Different samples can be grouped according to their expression profiles, which are used for molecular classification of cancer types.

A GST called Kappa Anna Robinson

Biological organisms are exposed to chemicals on a daily basis. These chemicals can be produced within an organism or externally, be naturally occurring, or synthesised by human beings as part of our complex lifestyle. Some chemicals are harmless and readily recycled in nature without causing problems. Others are very harmful to living things and can act as potential carcinogens, mutagens and teratogens. Pathologies caused by these chemical compounds can lead to many diseases and growth defects.

One of the mechanisms evolved by organisms to deal with such toxicological assault involves a family of enzymes called Glutathione Transferases (usually abbreviated to GSTs). GSTs are enzymes, proteins with biochemical and biophysical properties, that facilitate conversion of toxic compounds from one form to another. They are selectively produced in different tissues and cells. When toxic compounds enter the cells, GSTs bind the toxic compounds to a second or co-substrate, glutathione, which is naturally produced in all organisms. This changes the properties of the toxic compounds, making them either more soluble and readily excreted, or changed into a form that is suitable for further metabolism.

Different numbers of genes in different organisms encode the family of GST enzymes. The genes can also occur in different combinations within a species. This variability means some genes are continuously expressed (that is, turned on to make the protein they code for) and always produce the GST enzymes; others are expressed only in response to specific compounds. How the genes are regulated depends on many factors including variable exposure to different types of chemicals as well as genetic inheritance. Due to this genetic variation, certain individuals can be predisposed to specific diseases.

The family of GST enzymes has been classified according to similarities and differences between the enzymes' structure and function. Each class has been designated with a letter from the Greek alphabet such as Alpha, Mu, Psi… the most recent class to be identified being Kappa.

I have initiated studies into the human form of a Kappa class GST. There appears to be only one representative of this class in humans. Only one gene encoding the appropriate sequence exists and is located on chromosome 7. As yet, little is known about this enzyme. We think it is translocated to mitochondria, vital energy producing organelles that occur in most cells of all organisms and I am currently investigating how and why this occurs. In other work so far, I have identified the amino acid sequence of enzyme, characterised many of its biophysical and catalytic properties and initiated crystal structure analysis. This should show us how the protein folds into the three dimensional structure that catalyses reactions. In addition, I found that the GST Kappa Class has strong similarity to an enzyme in soil-borne bacteria involved in naphthalene degradation. It will be interesting to see whether in GST Kappa we have inherited this same function. It might explain why we can cope with all those mothball smells without fainting.

Arterial function and disease Shaun Sandow

Research in our laboratory aims to find new treatment targets for the production of novel drugs for the control of vascular disease, such as hypertension, atherosclerosis and stroke. This research concentrates on examining the structure and function of arteries in normal and disease states.

Arteries, which facilitate the supply of blood to the organs of the body, consist of two main cell types, endothelial cells and smooth muscle cells. Endothelial cells line the wall of arteries and are in contact with the blood. These cells are surrounded by multiple layers of smooth muscle cells. Contraction and relaxation of the smooth muscle cells results in changes in the diameter of arteries. Normal arterial function relies on a balance between factors causing relaxation of smooth muscle and those causing constriction of smooth muscle. Thus, the ability to control the balance between constriction and dilation represents a key to controlling diseases such as hypertension (high blood pressure) in which the balance between relaxation and constriction is disrupted.

The coordination of relaxation and constriction relies on the presence and maintenance of signals being passed within the walls of blood vessels via junctions in three ways; 1. between endothelial cells, 2. between smooth muscle cells and, 3. between endothelial cells and smooth muscle cells.

Studies in our laboratory aim to correlate the structural and functional relationships of endothelial cells and smooth muscle cells in a number of arteries to identify changes arising during disease. Currently, we have a number of projects underway.

Specifically, these concern the role of the endothelium in controlling smooth muscle relaxation. The endothelium of arteries produces factors that result in relaxation of the adjacent smooth muscle. Relaxation of the smooth muscle results in a reduction of blood pressure and thus represents a potential control target for disease. We are examining the dependence of one of the three main relaxing factors produced by the endothelium, endothelium-derived hyperpolarizing factor (EDHF), on the presence of junctions between smooth muscle and endothelial cells (myoendothelial gap junctions, MEGJs) in normal and pathological states.

Our studies are the first to show that EDHF activity in normotensive rats (that is those with normal blood pressure) is dependent on the presence of MEGJs. Furthermore, we have shown as artery size and the number of smooth muscle cell layers is reduced, EDHF activity and MEGJ incidence is increased. Thus, these studies show that there is a reciprocal relationship between the size of an artery and EDHF activity.

In further studies from our laboratory we have examined the relationship between MEGJs and EDHF under hypertensive conditions. In genetically hypertensive animals we have shown that the incidence of MEGJs and number of muscle cells is increased, whilst vessel and endothelial cell and is reduced. In arteries from hypertensive animals smooth muscle cell coupling is also reduced. Thus, the incidence of MEGJs is increased to compensate for other structural changes in the media in order to maintain a role for EDHF under hypertensive conditions.

High Blood Pressure and Glucocortoids Chris Schyvens

High blood pressure affects many Australians and is a major risk factor for heart attack and stroke and a range of other conditions. The High Blood Pressure Research Unit is investigating how high blood pressure develops, and the potential to prevent or reverse existing elevated blood pressure. In particular, we are interested in the development of high blood pressure caused by high levels of glucocorticoids - naturally occurring steroids released by the adrenal glands in response to stress or a glucocorticoid-secreting tumour; or when administered to patients in the treatment of various inflammatory or immune diseases.

Studies by this laboratory have identified that glucocorticoids alter the normal production and function of nitric oxide (NO). NO is a potent biochemical that has many actions throughout the body. It is its ability to dilate (open) blood vessels and thus lower blood pressure that is of prime interest to us. We are investigating this glucocorticoid-nitric oxide interaction in animal and human studies in collaboration with several investigators.

HOBAT is a chemical developed by Dr Bill Cowden (Division of Immunology and Genetics, JCSMR) that spontaneously releases NO when placed into solution. When mice that had normal blood pressure (normotensive) or high blood pressure (hypertensive) were administered HOBAT, there was a short-term but profound fall in blood pressure.

Oxygen is essential to life, but under certain conditions it changes its form to a superoxide and becomes toxic to cells. One of our hypotheses is that glucocorticoids increase the production of superoxide. We have become interested in superoxide and its ability to interact with NO and remove it from the circulation. To counter this effect, the body has set up certain defenses to scavenge the superoxide, which is further complemented by our diet which provides anti-oxidants. This year we have investigated the superoxide-scavenging ability of various anti-oxidants: Vitamins C and E, allopurinol and tempol. Tempol can prevent and reverse established glucocorticoid-induced high blood pressure indicating an important role for superoxide. Further work continues with the vitamins and in quantifying the production superoxide species in the body with Dr Kevin Croft (University of WA).

Genetic influences on the development of high blood pressure are of great interest in health and disease, and we are using three approaches to help us understand how glucocorticoids affect blood pressure. Firstly, in collaboration with Dr Tim Cole (University of Melbourne), we are investigating how mice deficient in the receptor for glucocorticoids maintain and control their blood pressure. We use implantable blood pressure devices that allow us to measure their blood pressure continuously over 24 hours. Secondly, in collaboration with Prof Brian Morris (Basic & Clinical Genomics Laboratory, University of Sydney) we have been investigating genetic links to high blood pressure. Thirdly, in collaboration with Dr Frances Shannon (Division of Molecular Bioscience, JCSMR) we are investigating changes in the gene expression in the kidney as a result of long-term glucocorticoid treatment. The latter uses innovative gene-chip technology to simultaneously account for the expression of several thousand genes.

Our laboratory has close links with the clinic. In collaboration with Dr George Mangos and A/Prof John Kelly (St George Hospital, Sydney) clinical studies were conducted to determine whether the route of administration of glucocorticoids affected how blood vessels dilate, and whether aspirin might also interact with nitric oxide and thus alter blood vessel function.

Asthma and the Immune Response Dianne Webb

Asthma is a complex inflammatory lung disease that arises from abnormal immune responses to environmental allergens such as pollens, moulds and house dust mite particles. Progressive thickening of the airway wall, the accumulation of sloughed airway lining cells and enhanced production of mucus develop during chronic asthma that, together with acute contracture of the muscle layer surrounding the conducting airways, can ultimately result in occlusion of the airway, wheezing and breathing difficulties. Large numbers of immune cells accumulate in the lungs of asthmatics and there appears a direct link between their numbers and disease severity. Therefore, a critical question in asthma research is how inflammation predisposes the development of abnormal tissue changes that result in respiratory impairment.

When the asthmatic lung is exposed to an allergen, a cascade of molecules is produced that causes activated immune cells, such as lymphocytes (a white blood cell) to flood into the lungs. Allergen-specific lymphocytes are termed T helper (Th)2 cells, which secrete factors that then induce the migration of another immune cell, the eosinophil, into the lung from the bone marrow and gut. Two of these key lymphocyte-derived factors are interleukin (IL)-13 and IL-5. Interleukin-13 stimulates epithelial cells that line the airway lumen to secrete eotaxin, a molecule that binds a receptor on the eosinophil to stimulate migration. Interleukin-5 is also important for the proliferation of eosinophils in the bone marrow and prolonging their survival. Eosinophils are commonly seen in parasite infections in the gut and contain a variety of highly toxic granules that may aid killing, and assisting the body to expel, these parasites. In asthma, the body develops an inappropriate immune response in the lung that is similar to that seen in parasite infections. The toxic products of eosinophils may contribute to lung tissue damage and induce contractile responses in the airway muscle layer in a similar manner to contractile mechanisms in the gut that help expel parasites from the body.

Although eosinophils are known to release toxic factors, respiratory dysfunction also develops if IL-13 is directly delivered to normal mouse lungs and this can occur before eosinophil infiltration. This observation suggested that IL-13 alone was sufficient to stimulate airways dysfunction and caused researchers to question if eosinophils were really the prime culprits in asthma pathology.

To address the relationship between eosinophils, IL-13 and IL-5 we have developed mouse models of allergic lung disease that produce asthma-like Th2 responses and eosinophilic inflammation, the hypersecretion of mucus, airway wall thickening and respiratory dysfunction. A powerful advantage of these models is the use of mice in which the DNA has been manipulated to prevent expression (that is, production and release under genetic control) of specific immune factors. Using genetically modified mice that were deficient in production of the eosinophil factors, IL-5 and eotaxin, we were able to show that eosinophils failed to be recruited to the lung in response to allergy-promoting treatment and that respiratory function was normal. This demonstrated that eosinophils play a crucial role in the tissue abnormalities associated with allergic lung disease. This study was ground-breaking because some researchers had considered eosinophils to be bystanders in the pathology associated with asthma. However, an interesting feature of mice deficient in eosinophils was that IL-13 was also reduced, suggesting that in fact eosinophils interact with lymphocytes to regulate the production of IL-13.

Our research for the first time has also identified and characterised a novel factor, called Ym2, that is secreted in the lungs of allergic mice in response to IL-13. Factors similar to Ym2 have been associated with the "remodelling" of tissue, for example in the reduction of mammary tissue following cessation of lactation in cows. We now believe that Ym2 is a down-stream factor induced by IL-13 that may be involved with aspects of airway wall thickening in asthma. This factor is now the focus of exciting ongoing research.

Our studies into the molecular mechanisms underlying asthma lay the groundwork for understanding precisely how the Th2 immune response generates abnormalities in the airway wall that ultimately results in respiratory dysfunction. This work is also critical in our bid to identify Th2 factors for which new treatments can be developed to specifically inhibit the interaction of these factors with the respiratory tract and aid in the treatment of asthmatics.

 

Breaking the code Adèle Yates

The genetic material of an individual is stored as a DNA code. The aim of our work is to unravel the code to understand how this information controls the activity and growth of cells in the body. In our lab we are using a new technique that utilizes a chemical to deliberately mutate the DNA code to cause medical defects in laboratory mice. The advantage of this approach is that it can find new genes that are responsible for causing disease. The mutation in the DNA can prevent cells developing, reducing the animal's resistance to infection, so the mice are more susceptible to becoming sick. Alternatively, a different error in the DNA in other mice can promote runaway cell growth and can lead to cancerous conditions.

Our research involves randomly mutating many mice, then finding the mice that are adversely affected by the DNA alteration and so develop cancer and other medical conditions. This is performed by analysing blood samples from the mice to detect any differences in the number of white blood cells (the cells responsible for fighting infection). Once a mutant mouse has been identified, we can then work out what went wrong with the development of their immune system. Where and what the DNA was changed to can then be discovered and may help identify the part of the DNA code that is likely to be involved in causing disease.

Of the mice I have been studying, the DNA alteration in four of these has been characterised. The Mr-T-Less mice do not produce mature T cells, white blood cells that are required to combat infection. The mutation in the DNA of these mice is within the ZAP-70 gene. Zap-70 is involved in activating T cells by passing signals from the T cell receptor located on the surface of the T cell to other molecules inside the T cell. Another mutant mouse strain, the Lochy mice, do not produce a molecule called CD45 as the DNA mutation has prevented production of this protein. CD45 has a similar role to Zap-70 in terms of passing signals within the cell, but is required for all white blood cells not just T cells. The Buffy mice have an altered version of the JAK-3 gene. JAK-3 is involved in the activation of white blood cells by passing signals on from the receptors that bind small proteins called cytokines. Similar mutations to these have been found in humans, and without activation of white blood cells, patients develop severe combined immunodeficiency. These new mouse strains will allow us to study the human conditions. A different type of mutant is the B-blast mouse that develops a variety of tumors. This is due to a variation in the p53 gene, and this is the most commonly altered gene in human cancers.

At this stage the mice strains characterised have alterations to known genes. However, research is currently being performed on mice that have a modification of a potentially new and unknown gene. As the DNA of mice and humans is very similar, identification of a new disease gene in mice should lead to the discovery of a comparable gene in humans.