Some Recently Funded Research Projects
Clinical translation of bioimpedance spectroscopy for monitoring the impact of interventions for acute burn swelling
Burn injury causes marked inflammation leading to swelling.
Swelling in the tissues increases pain and slows wound healing.
Removal of swelling is difficult and slow with traditional methods.
However, to improve treatments of this problem after acute burn, researchers must measure change in swelling without removing all the wound dressings.
In 2009, the research group showed that bioimpedance spectroscopy (BIS), a non-invasive measure of fluid shift and body composition, showed promise as a method that could provide this information in burn patients.
However, BIS required specific placement of electrodes on distal limbs and could not sensibly measure limb or localised wound swelling. This study showed that with new electrode configurations the BIS technique can measure localised wound swelling changes. Healthy and burn injured subjects took part in the research. A series of projects were completed in the burn unit and ambulatory clinic. The study, conducted with the support of the MRF, has been integral in breaking down the barriers to application of BIS to help acute burn fluid resuscitation (whole body fluid tracking) but more importantly, whole limb tissue swelling monitoring and localised, small wound swelling responses.
Dr Edgar’s research team now has a strong platform from which to develop new interventions for swelling after burns and more importantly, in all wounds. All wounds, trauma, stings and a number of disease states create localised swelling and thus this study has provided a real-time, non-invasive, sensitive method of tracking the change in tissue fluid of any cause, not only after burn injury.
The project has resulted in one submission for publication to date with two others now in preparation.
Impact of gene variants on plasma lipids in hepatitis C patients following antiviral treatment
Hepatitis C is an infectious disease primarily affecting the liver, caused by the hepatitis C virus (HCV). The infection is often asymptomatic, but can lead to scarring of the liver and ultimately to cirrhosis, and in some to liver failure or cancer. Treatments for HCV are available but are not effective in all patients.
There is a close link between HCV infection and human fat (lipoprotein) metabolism. Hepatic lipoprotein assembly and secretion requires the association of apolipoprotein B (apoB) and lipids, a process facilitated by the protein MTTP. The HCV lifecycle is dependent on this same process. There is also evidence that other proteins involved in lipoprotein metabolism can influence HCV infectivity. The project aimed to determine the effect of variation in genes involved in lipid/lipoprotein metabolism on plasma lipids in chronic HCV patients before and during/after treatment.
Plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, apoB and apoA-I concentrations were determined in 186 samples from 102 HCV patients taken before, during and/or after therapy. Variants were genotyped in genes known to influence lipoprotein metabolism, including MTTP, APOE and LPL.
Patients who responded to therapy had higher blood concentrations of total cholesterol, LDL-cholesterol and apoB compared to those patients who did not respond. The viral genotype influenced response to treatment; only 52% of patients with HCV genotype 1 responded to therapy, compared to 73% of patients with other HCV genotypes. Novel findings included lower baseline concentrations of LDL-cholesterol and apoB in patients carrying the MTTP I128T variant, which increased post-therapy, while lipid levels in patients not carrying this variant did not change.
This new study has revealed some interesting findings, providing a starting point for the future exploration of the mechanisms underlying these associations.
Long-term outcomes following an episode of acute kidney injury requiring renal replacement therapy in the Intensive Care Unit
The Intensive Care Unit (ICU) provides care to the most critically ill patients and those having undergone complex surgery. Almost 100,000 patients per year are treated in an ICU in Australia and an increasing proportion of these patients are at risk of, or develop, kidney damage known as acute kidney injury (AKI). AKI is associated with both short term and long term increased risk of death and need for dialysis, and is an issue of substantial public health importance.
The aim of this research project is to describe the long term outcomes of patients admitted to the ICU with severe AKI requiring dialysis. Using the Western Australian (WA) Department of Health Data Linkage Service and individual ICU clinical databases, data for all patients admitted to all adult ICUs in the state over a 10 year period will be linked to prior and subsequent health outcomes. This comprehensive database will allow an accurate estimation of the burden and trends of severe AKI in WA vital for health policy planning, as well as being a first step in identifying potential new interventions to prevent long term harm of this increasingly common and serious condition.
Identification of ADAM28 as a novel metalloproteinase involved in the shedding of TNF-α in obesity
Over nutrition promotes obesity, which greatly increases the risk of type 2 diabetes and cardiovascular disease. Recently gained data implicated a relatively newly discovered metalloproteinase known as ADAM28 in inflammation, obesity and type 2 diabetes.
Metalloproteinases may play a major role by cleaving numerous pro-inflammatory mediators from the cell surface. Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) have been associated with the metabolic syndrome, and prior reports have suggested that the metalloproteinase ADAM28 is able to cleave synthetic peptides possessing the TNF-α shedding site. This research aimed to ascertain whether human ADAM28 correlates with parameters of the metabolic syndrome and if ADAM28 is a novel sheddase of human TNF-α.
To identify novel metalloproteinases associated with the metabolic syndrome, investigators conducted micro-array studies on peripheral blood mononuclear cells from a well characterised human cohort. In cell culture experiments, human ADAM28 and TNF-α were over-expressed and ADAM28 expression or activity was reduced with small interfering RNA (siRNA) or pharmacological inhibition. TNF-α levels were then measured in cell culture supernatant. Investigators also conducted ADAM28 inhibition studies in human macrophages, and discovered that human ADAM28 expression levels were positively correlated with parameters of the metabolic syndrome. Importantly, when human ADAM28 and TNF-α were over-expressed in cells, both proteins co-localised, co-immunoprecipitated and promoted TNF-α shedding. The shedding was significantly reduced when ADAM28 activity was inhibited or ADAM28 expression was down-regulated. In human macrophages, endogenous ADAM28 and TNF-α were co-expressed and TNF-α shedding was significantly reduced when ADAM28 was inhibited by pharmacological inhibition or siRNA knockdown. These results have now been published in the journal “Immunology and Cell Biology”.
The project is most significant, as it has demonstrated for the first time the importance of ADAM28 in the metabolic syndrome, and further supports that metalloproteinase inhibition is a potential therapeutic target for anti-obesity agents. Importantly, there is the potential for ADAM28 mediated TNF-α cleavage to be implicated in numerous other immunopathological conditions.
CMV infection as a determinant of immune function in older HIV patients with a stable virological response to ART
This study used patients recruited from Outpatient Clinics at Royal Perth Hospital to answer a series of specific research questions regarding the role of chronic CMV infection in the accelerated immuno-senescence, which is characteristic of HIV patients who have maintained a stable response to ART for 10 years or more.
Peripheral blood mononuclear cells (PBMC) were collected from aviraemic HIV patients on long term (>10 years) ART and age-matched healthy controls; all were over 50 years old. Flow cytometric analysis was performed on T cells to phenotype for senescence markers. NK cells were phenotyped for the expression of inhibitory and activatory receptors, and NK function was assessed.
Patients had significantly higher CMV antibody levels, indicating ineffective less effective control of CMV, but CMV DNA was not detected. Patients and controls had similar levels of inhibitory and activatory receptors on their NK cells, where only LIR-1 expression in controls correlated with CMV antibody. This suggests that LIR-1 on NK cells could be a possible marker of previous CMV infection and disease in controls, but not in patients.
T-cell senescence was higher in patients than controls. Although CD8 T-cell senescence was associated positively with CMV antibody levels in patients, the inverse was true for controls. CD8 T-cell responses to CMV VLE peptide were also related to CD8 T cell senescence in patients but not controls. Together, these results may indicate that CMV drives T cell senescence in patients due to ineffective control of the virus.
The research also found that despite more than 10 years on ART, NK cells remain less functional in HIV patients compared to controls. This may facilitate more frequent or longer periods of CMV replication. This finding supports CMV therapy as a clinically useful strategy in a subset of patients.
Using synthetic biology to discover new antibiotics
The emergence of “superbugs”; bacteria that resist all current antibiotics, such as methicillin-resistant Staphylococcus aureu (MRSA), poses a significant risk to public health. Over prescription of current antibiotics, overuse of antibiotics in agriculture, and increased international travel are rapidly accelerating the rise of antibiotic resistant bacteria.
With all the pharmaceutical giants abandoning or severely curtailing their antibiotic discovery programs the need for publicly funded research laboratories to develop new approaches to address this problem is immediate. This project focuses on developing a radical new approach using synthetic biology to discover new antibiotics that cannot be evaded by current superbugs. In this approach the research group has built new genetic pathways in cells grown in the laboratory that enable it to generate millions of cells, each of which could produce a different antibiotic.
Funding from the Royal Perth Hospital Medical Research Foundation has enabled a PhD student to optimise this breakthrough new technology and begin screening cells for new and useful antibiotics. This technology will provide an opportunity to put the future of drug discovery in the hands of the wider scientific community and provide new tools for Australian industries.