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Research

ARC has revised its research policy which has been in place since 1993. We shall continue to honour the memory of Harry Windsor and Ann Woolcock but in slightly different ways.

We have launched across Australia the ARC Harry Windsor Research Grants Scheme. These grants will be approximately $50,000 to support research in TB, respiratory diseases related to other infections and smoking-related respiratory diseases. There is an emphasis on community issues or the health of disadvantaged groups.

Applications will be linked to the National Health and Medical Research Council’s processes.

The ARC Ann Woolcock Fellowship is a 4-year full-time postdoctoral fellowship in biomedical, clinical or public health research anywhere in Australia. It is valued at approximately $100,000 and aims to encourage people of outstanding ability to develop research as a significant component of their career.

Further details about both schemes are here.

Research Updates

Update on ARC Fellow's research

Ingrid & Jacinta

ARC Ann Woolcock Research Fellow, Dr Ingrid Laing who is looking at the genetic influences on causal pathways of acute lower respiratory tract infections (ALRIs) in highly susceptible infants in PNG highlights the progress on her research at the at the Telethon Institute for Child Health Research in Perth.

Early in life, infants from the highlands of Papua New Guinea (PNG) experience dense bacterial infection of their nasal passages, leading to one of the highest childhood mortality rates due to pneumonia in the world. In the Asaro Valley, Papua New Guinea (PNG) where this study is based, Streptococcus pneumoniae (pneumococcus) is found in the nasal passages of all children before the age of 3 months and commonly by the age of 17 days. This early colonisation is associated with an increased risk of pneumonia and is the most common reason for children to be admitted to hospital or die in PNG, accounting for 34.7% of all deaths in children under 5yrs and 50% of all deaths in infants under1yr of age.

The PNG Institute of Medical Research, the Telethon Institute for Child Health Research and the School of Paediatrics and Child Health, University of Western Australia are conducting a trial of the 7 valent pneumococcal conjugate vaccine (7vPCV) in infants from the highlands of PNG. This fellowship project comprises a systematic investigation of the contribution of genetic factors to the development of immune responses and susceptibility to pneumonia in this population.

Between May 2005 and September 2006, 191 infants have been recruited into the study, 15 have dropped out and one infant has died. Preliminary microbiological studies of the weekly nasal swabs, collected from the infants in the first month of life, have shown that they are colonised with a wide variety of pneumococcal serotypes at a very young age (total of 34 different serotypes identified and most commonly by 20 days of age). Data collected on the incidence of acute respiratory infection has shown that there have been 165 episodes and 36 of these were moderate or severe.

In 2006 informed consent has been obtained from 152 mothers, for their infants and themselves to participate in the genetic studies. In July and August, ARC funded a trip to Goroka, PNG for the purpose of collecting the first batch of study DNA samples. Due to the limited resources at the PNGIMR, 32kg of laboratory reagents were taken to Goroka and included DNA extraction kits that had been tested and selected based on generating a high yield of DNA and their ease of use. As the blood samples collected from the study infants at 3 months of age have been small, limited cells were available for DNA extraction, necessitating the use of extraction kits.

The time in Goroka was spent extracting DNA and training Jacinta Kono, a technician at the PNGIMR, in the use of the DNA kits, so that she may continue extracting DNA from the study samples. DNA samples from 66 infants and 24 mothers were brought back to Perth for genetic analysis. Half of the extracted DNA sample remained in PNG. Due to the small volumes of DNA available, a method of quantitating DNA, using UV analysis in a 96-well plate, was tested. This was successful and showed that the samples from PNG ranged in concentration from 28.5 to 108.5ng/ul. In the next month preliminary genotyping of these samples, to determine the frequency of several immune gene polymorphisms in this population, will be completed.  

As part of this fellowship, two new projects that will investigate the immunogenetics of acute infection in the ears and lungs of Australian children have been developed in collaboration with Perth researchers. These projects will enable comparisons between those factors associated with susceptibility to pneumonia in PNG (a developing country) and Australia (a developed country).

A new non-toxic approach to controlling bacterial infection

Professor Robert Capon from the Institute for Molecular Bioscience, University of Queensland, Brisbane received a Harry Windsor Research Grant in 2006. He is working on a new non-toxic approach to controlling bacterial infection.

Traditional antibiotic discovery strategies focus on bacteriostatic and bactericidal agents - leading to antibiotics that are, in effect, selective toxins. The search for antibiotics that control rather than kill bacteria represents a valuable new discovery paradigm, able to yield drugs not compromised by emerging resistance. A wide variety of pathogenic bacteria utilise polar surface filaments called type IV pili (TFP) to attach to and colonise host epithelial surfaces. TFP extend and retract from the poles of the cell for surface translocation termed twitching motility (TM), which is critical for host infection. Type IV pili are a well-validated but totally under-utilised target for antibiotic discovery.

This project searches for new non-cytotoxic antibiotics that control infections by inhibiting the TM of a wide range of important bacterial pathogens, in particular, Pseudomonas aeruginosa. These new antibiotics will be well suited to treat immunologically compromised and seriously ill patients, such as those suffering cystic fibrosis, burns, AIDS or undergoing cancer chemotherapy, who are less able to tolerate cytotoxic side-effects. A TM assay capable of simultaneously assessing whether a test extract, fraction or pure compound is (a) non-bactericidal against P. aeruginosa, and (b) selectively inhibits the twitching phenomena has been developed successfully. The screen of a library from southern Australian and Antarctic marine extracts (~3000 invertebrates and algae) with this new assay resulted in fourteen extracts with promising TM inhibitory properties.

Using an optimised partitioning process the fourteen target extracts were converted into seventy-three fractions, with each subjected to the TM assay. Characteristic TM assay results are shown for sample A, which revealed non-cytotoxic TM inhibitors in wells B4-C2 and C5-C7. This conclusion is evident from the presence of a healthy bacterial colony on the agar surface (brown spot) but the absence of a bacterial halo indicative of twitching motility (a transparent zone). By contrast, wells C3 and C4 were cytotoxic (no bacterial growth or halo). Likewise, fractionation of sample B revealed two TM inhibitors in wells C2 and C3. To date 15 TM inhibitors have been isolated from the fourteen target marine extracts.

The discovery that marine metabolites can influence gram negative TM reveals a hitherto unrecognised ecological role. Marine sponges are well known for their ability to host symbiotic bacteria, and there ability to produce non-cytotoxic TM inhibitors represents a plausible ecological control strategy - to manage the density and localisation of bacterial colonisation with the sponge tissue.

Results to date confirm that marine extracts are a valid source of TM inhibitors, and that these are small "drug-like" molecules. Ongoing studies will define the structures of these metabolites, and will seek to determine whether such inhibitors have therapeutic potential.

Vaccine development

Professor David Jans of the Department of Biochemistry and Molecular Biology, Monash University, Melbourne received a Harry Windsor Research Grant in 2006 for the project Role of phosphorylation in regulating nuclear trafficking of the viral matrix protein during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in infants and young children throughout the world, with over 100,000 infants infected by RSV in Australia every year, (estimated annual cost of between $1 and 4 million in the State of Victoria alone), as well as being an important cause of pneumonia in the elderly, resulting in more deaths each winter than influenza. Currently there is no efficacious treatment for infection or vaccine to prevent RSV infection.

This study examined one of the RSV proteins, the matrix protein (M), which is central to virus propagation and resultant cell injury. It has been observed that M enters the cell nucleus (the site of cellular RNA synthesis) where it appears to inhibit RNA synthesis early in infection; later, it exits the nucleus in a step required for virus production. Understanding how M trafficks between nucleus and cytoplasm at precise times during infectious cycle could be the key to establishing its role in pathogenesis, and enable the development of new approaches to anti-viral therapy.

By examining the M protein both in isolation in living cells, and in infected cell systems, it has been possible firstly, to identify the key targeting signals in M that determine its ability to traffic, and the cellular transport molecules that mediate this. We have identified the signal conferring M nuclear entry through the action of the cellular transporter importin beta 1, as well as the signal mediating nuclear export through the exporter exportin 1. It had previously been observed that M was likely to contain a phosphate group at a specific site or sites on the protein in infected cells. Since M's phosphorylated state appeared to correlate with its cytoplasmic location later in infection, when it is actively exported from the nucleus, it was hypothesised that phosphorylation could be the switch between M nuclear import and export. A number of experiments were carried out to examine this in detail, in particular focussing on a protein kinase CK2 site close to the nuclear export signal that seemed likely to regulate M's export activity.

Live cell imaging experiments were first performed using quantitative microscopy to look at localisation of M in the presence and absence of a specific inhibitor of exportin 1, results showing that M nuclear localisation was increased. Importantly, the inhibitor of exportin 1 inhibits RSV virus production 25-fold, indicating that M nuclear export is important in the infectious cycle. Significantly, experiments using an inhibitor of CK2 revealed the same effect, implying that phosphorylation of M may be through CK2, and that this may be important for M nuclear export. Experiments looking at M's location in virus-infected cells in the presence of the CK2 inhibitor should confirm these observations. Parallel biochemical analysis indicates that the form of M in the nucleus and cytoplasm is different, and that the CK2 inhibitor changes the relative amounts of the respective forms of M. All of this is consistent with phosphorylation of M by CK2 playing a role in modulating M subcellular localisation.

Interestingly, the analysis of the CK2 site near the nuclear export signal, proved that this particular site is not critical in affecting M nuclear export. Currently other sites in M are being examined in an attempt to localise the key site(s) to then perform similar analysis.

In summary, although the work is ongoing, progress has been made in understanding the signals and cellular proteins including kinases responsible for M subcellular trafficking. When the work is complete, it will be possible to make viruses defective in M trafficking, both to test formally the importance of M trafficking in RSV infection, and as a possible first step to developing attenuated viruses for use in vaccine development.

Nutrition and tuberculosis

Kelly fig 3

Associate Professor Paul Kelly from the National Centre for Epidemiology & Population Health at the Australian National University and colleagues from Menzies School of Health Research in Darwin won an ARC Harry Windsor research grant in 2006 to study how Nutritional intervention might improve tuberculosis treatment outcomes in Timika, Indonesia: the NUTTS Study.

Background

The Australian Respiratory Council has provided funds to a team of Australian researchers working in collaboration with Indonesian counterparts to examine the effect of food on the outcomes of tuberculosis treatment in Timika, Papua Province, Indonesia. The Nutritional Upgrade for Tuberculosis Treatment Success (NUTTS) study is an extension of our previously ARC funded tuberculosis research program at the same site. Preliminary data show that patients have severe tuberculosis disease at diagnosis, low rates of HIV co-infection (4%) and MDR-TB (2%) and a high percentage (41%) are malnourished by WHO criteria (BMI<18.5kg/m2). Further, it has been demonstrated that these patients respond slowly to treatment: while there is clinical, radiological and anthropometric improvement after two months of treatment, average lung volumes measured by spirometry remain significantly lower than control values at both two and six months, and 31% of patients remain in the malnourished range at two months.

Despite the existence of effective medications and national tuberculosis control programs in most countries, tuberculosis remains a major cause of death due in part to poor compliance with treatment. A key issue in reducing tuberculosis morbidity and mortality is therefore: what are the most effective ways to improve tuberculosis treatment compliance? There is a well recognised link between adult malnutrition and tuberculosis, with malnourished adults more likely to develop active tuberculosis disease and active tuberculosis causing weight loss. Pulmonary nitric oxide (NO) production appears to be an important nutritionally-dependent mediator of myco-bacterial killing and amelioration of the immunopathology of tuberculosis in humans. No previous intervention studies have measured pulmonary immunological correlates of outcome to determine mechanisms of the effects seen.

The NUTTS Study

Within the context of a well functioning DOTS program we will randomise patients with pulmonary tuberculosis to intervention (daily food supplement for two months followed by weekly for four months) or a control group (nutritional advice alone). The food will consist of a relatively low-cost, locally available, culturally acceptable standardised food supplement which would be sustainable if this intervention is found to be effective. The following hypotheses arising from the work to date are being tested: food supplementation will improve compliance with tuberculosis treatment and given that malnutrition is associated with tuberculosis disease severity and delayed treatment response, correction of malnutrition with a food supplement will result in more rapid improvement in tuberculosis associated malnutrition and in measures of treatment outcome and lung inflammation.

Study progress

Preliminary results from a trial of arginine supplementation in uncomplicated malaria patients in Timika has resulted in substantial increases in exhaled NO (3g supplementation led to a mean increase of 40%). The immunohistopathological changes associated with tuberculosis suggest that NOS2 expression in tuberculosis which leads to the increase in endogenous NO production is at least as high as in malaria. Therefore, if the malaria result is replicated in our tuberculosis study, 1.2g supplemental arginine would be expected to increase exhaled NO by at least 16%. The proposed sample size is sufficient to demonstrate an effect of this size. Preliminary data from a related study in East Timor has shown that the study design is feasible and acceptable to the community, tuberculosis patients and clinic staff. We have been able to incorporate the lessons learnt in this study and apply them to a different setting in Timika where our well-established research collaboration will allow wider study of the food-tuberculosis interaction.

The principle investigator has made visits to Jakarta and Timika to finalise the study protocol in consultation with local stakeholders. Preliminary ethical approval has been obtained and the full ethics proposal is being prepared. A preliminary nutritional survey of Timika tuberculosis patients will be undertaken later this year to provide important baseline data and to test the acceptability of the proposed food intervention. The full proposal has been re-submitted to the National Health and Medical Research Council and the decision is awaited. An infectious diseases consultant will join the team in 2007. Two Indonesian research students with laboratory and nutritional expertise have been identified as field-based collaborators.

Biology of lung cancer

Associate Professor Kwun Fong and Dr Annalese Semmler of the Department of Thoracic Medicine at the Prince Charles Hospital, Brisbane received a Harry Windsor research grant in 2005 for their project Novel methylated genes in lung cancer

Lung cancer is a major public health problem. In Australia, lung cancer is the third leading cause of mortality and the fifth leading cause of burden of disease. Tobacco smoking is the major cause of lung cancer. Despite intensive efforts in primary prevention, screening, and therapy, long-term survival rates have not improved substantially since the 1960s. Therefore a more complete understanding of the biology of lung cancer is crucial to the development of effective new diagnostic and therapeutic strategies. One aspect of this has been the identification of genetic biomarkers associated with individual risk for developing the cell damage that leads to lung cancer.

In lung cancer, genes controlling cell growth are affected by numerous changes including changes to the structure of the genes or their protein product (mutational) or changes whereby certain genes are inappropriately switched off (non-mutational). Gene methylation is an example of the latter process and is the focus of this project. DNA methylation is a very effective mechanism to turn a gene off and if that gene is protective against cancer the loss of its function can leave cells more susceptible to malignant change. Research has shown that changes in the pattern of gene methylation are an early event in the development and progression of lung cancer. By detecting these methylation patterns, this disease can be better identified at an early stage, as well as potentially being able to develop new treatments based on reversing the process.

Identifying the key genes that have been switched off by methylation is therefore the first priority, and this is being investigated by using PCR based technology. Several putative regions of interest have so far identified within the genome. These regions include two known genes which play a key role in cell growth and development. A large number of lung cancers are now being screened to further pinpoint the importance of these two genes in lung cancer and to determine the suitability of using them as early diagnostic markers for lung cancer. The next challenge will be to work out ways of preserving the function of these genes and to evaluate the effect of restoring their function on tumour growth. An impact on tumour growth could indicate potential for manipulating gene methylation in developing new treatments for lung cancer.

Problems relating to lung transplantation

Dr Paul Reynolds and a team from the Royal Adelaide Hospital and Hanson Institute in Adelaide received a Harry Windsor research grant in 2005 for the project Infection versus rejection in lung transplant-related Bronchiolitis Obliterans Syndrome: can intracellular cytokines help?

Lung transplantation has become an established therapy for otherwise incurable lung diseases. Although the procedure leads to significant improvements in quality of life, patients often have many complex problems after the transplantation. All lung transplant patients must take powerful immunosuppressant drugs to prevent the body's natural tendency to reject the new lungs. These drugs suppress the immune system, which makes the patients susceptible to a wide range of infections, including those caused by viruses, bacteria and fungi.

In clinical practice a patient suffering from an episode of lung rejection can often have very similar symptoms to those with infection. Frequently, infection and rejection can occur at the same time.

There is an additional problem called Bronchiolitis Obliterans Syndrome or "BOS". BOS causes chronic lung failure due to narrowing and loss of the small airways, and is the major limiting factor in the long term survival of lung transplant patients. The cause of BOS is not clear but is likely related to a combination of infection and rejection processes.

This project involves the analysis of samples taken from the lungs (by bronchoscopy) and blood of lung transplant patients who are either stable, have infections or rejection, in order to define new cellular markers which will help us determine more accurately whether an individual patient has infection as the basis of their problems and to better understand the causes underlying BOS. So far, data from 39 bronchoscopy procedures (some being repeat procedures on the same patient) have been accumulated. At least 14 of these have had an identifiable infection, whereas mild rejection has also been seen in some. Data is being collated on many different inflammatory markers including IL-8, TNFa, IFNg, IL-6, IL-1b, IL-5, IL-10, IL-12.

In previous studies in neonatal sepsis these markers identified 89% of patients with infection. Important differences have also been noted between blood and lung derived inflammatory markers in stable lung transplant patients, and also differences between lung cells that are taken from the airway lining (using a small brush) and cells washed out from the air sacs (alveoli) of the lungs. In particular, it appears that current immunosuppressive regimens may not be adequately suppressing T-cell derived Interferon gamma (INFg), and monocyte derived IL8, MCP-1 and MCP-3 (these latter molecules stimulate the accumulation of neutrophils (white blood cells) and can stimulate fibrosis which may have a role in BOS). This may mean that the current practice of adjusting immunosuppressant drug doses based on monitoring of drug concentrations in the blood does not adequately assess the real impact of the drugs on the lungs. It is hoped to advance the ability to adjust immunosuppressive drug dosage, which will then help to reduce the incidence of infection and rejection.

By the end of the year further data will have been accumulated which will enable a determination which of the many markers that are currently being analysed will prove most useful. This assessment will be helped by further repeated studies done in the same patients when stable and when suffering an infection.

 

 
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