Research
All proceeds from '65km for Cystic Fibrosis' are directed to CF research or research that improves the quality of CF care in hospitals as this is known to be essential for good health outcomes.
To date we have funding the following projects:
2024 - Monash Foundation
Recombinant alpha-1 antitrypsin for the treatment of exacerbations of cystic fibrosis
Dr Paul King – Monash University
$49,000
Exacerbations of cystic fibrosis are episodes of acute clinical deterioration characterised by increased lung inflammation and have a major impact on patients with CF, both in terms of symptoms and deterioration in lung function. They are primarily treated with antibiotics, but there are no effective therapies available to target the inflammation that drives these episodes.
Alpha-1 antitrypsin (AAT) has a key role in reducing lung inflammation and patients with congenital deficiency may develop severe lung disease such as emphysema and bronchiectasis. AAT derived from human blood donors has been used for many years as a maintenance therapy for patients in the US with congenital deficiency; in this context it has been shown to be safe and well tolerated. However, as it is derived from blood donors it is very expensive and hard to obtain (not generally available in Australia).
We have developed a research program that has repurposed AAT to be used acutely to reduce lung inflammation (such as occurs in exacerbations of CF). We have extended this work to develop a unique form of recombinant AAT (rAAT) that can be made in a factory in large quantities at low cost, and our preliminary work has demonstrated this rAAT is highly effective in reducing acute lung inflammation.
In this study we will build on our established program to demonstrate that the rAAT reduces inflammation in clinical samples (e.g., blood and lung fluid) from patients with CF. We envisage this will lead to clinical trials and the first effective anti-inflammatory therapy for patients with CF.
NICHE Study
(Nutrition in Infants and Children exposed to early life highly effective modulator therapy)
Caitlin Miles
$35,560
Nutritional and gastrointestinal complications in infants and young children with CF are common and multifactorial in nature. Reduced pancreatic function and the inability to absorb nutrients from food further compounds nutritional status in 85% of infants and children with CF. Nutritional, gastrointestinal and pancreatic features of CF are known to impact both health and quality of life (QOL). Understanding the impact of CFTR modulator therapy on gastrointestinal and pancreatic function has been identified as a top translational research priority by people living with CF in 2023. The availability of highly effective modulator therapies (HEMTs) in infants and young children is an exciting development given the potential for early pancreatic and gastrointestinal tract preservation and the reversibility or prevention of complications such as pancreatic insufficiency, feeding difficulties, poor growth and malnutrition.
In Australia, CF dietitians and the wider multi-disciplinary team (MDT) have a unique opportunity to collaboratively and prospectively study the nutritional and gastrointestinal impacts of HEMT (ivacaftor (IVA) and elexacaftor/tezacaftor/ivacaftor (ETI)) in infants and young children ≤5 years of age. ‘Real world’ (i.e. non-clinical trial) studies are required to better understand the breadth, significance and longevity of nutritional, gastrointestinal and pancreatic changes in infants and young children who are exposed to early life HEMT. This research will ensure that we: have a better understanding of the short and long-term nutritional implications; are providing tailored nutritional advice based on genotype, phenotype and CFTR modulator therapy; and are responding to the priorities of the families and children we care for.
Pregnancy outcomes in people with cystic fibrosis taking Trikafta
Associate Professor Rasa Ruseckaite – Monash University
$49,000
Advances in the care of cystic fibrosis (CF) led to improved life expectancy. Thus, people with CF (pwCF) are able to reach adulthood, to get married and become pregnant. The CF transmembrane conductance regulator modulator (CFTRm) combination elexacaftor/tezacaftor/ivacaftor (ETI), also known as Trikafta, is a genetic mutation-targeted treatment in CF that results in profound improvements in clinical outcomes and increasing pregnancy rates. A few small studies suggest that ETI is safe in pregnancy, but currently there is little evidence regarding ETI outcomes in pregnant pwCF and their babies. Using a large multi-centre clinical registry, we will compare pregnancy outcomes in people who were on ETI versus those who stopped the treatment while pregnant. Secondary objectives of this study will include evaluation of pregnancy-related and obstetrical complications in pwCF and their infants, and developmental outcomes in infants and children for the first few years of life. A series of short surveys will be conducted regarding medication exposures during pregnancy, pregnancy outcome, neonatal/infant outcome and childhood health. Survey responses and data from the national CF registry will be analysed and compared across CF centres and internationally.
Preventing infection with Pseudomonas aeruginosa using novel non-antibiotic molecules
Professor Phil Robinson – Royal Children’s Hospital
$47,750
People with Cystic Fibrosis (pwCF) suffer from various bacterial infections in their airways, including from the notorious Pseudomonas aeruginosa bacteria, which is known to cause rapid lung function decline. Treating P. aeruginosa with antibiotics is difficult due to drug resistance and formation of a biofilm, a protective layer secreted by the bacteria which blocks antibiotic penetration into the bacterial colonies. Therefore, developing new therapies that will slow or prevent the initial infection are crucial.
Pseudomonas aeruginosa normally attaches itself to cilia, which are tiny hair-like structures found on the surface of respiratory epithelial cells. After attaching, P. aeruginosa neutralizes cilia function which normally act to remove bacteria from the lungs, thereby facilitating bacterial colonisation.
The primary attachment process of P. aeruginosa to the cilia can potentially be diminished using natural carbohydrate molecules, fucose and galactose, which are not classic antibiotics, but rather simple competitors for the binding sites. Preliminary data from our laboratory using healthy human cells supports this notion.
Our project aims to explore the potential protective effect of fucose and galactose against P. aeruginosa infection by administering these molecules to cultured respiratory cells taken from pwCF, and then infecting them with P. aeruginosa. The beneficial effect will be assessed with patients from Monash and RCH, using advanced cellular and molecular techniques available in CI-Robinson’s laboratory at MCRI.
The outcomes of this study will provide a basis for a novel preventative approach that may translate into clinical standard of care for pwCF, improving and extending their life.
2021 - Monash Foundation
Can exercise replace chest physiotherapy for people with cystic fibrosis? Partnering with consumers to address a research priority.
Professor Anne Holland – Monash University
$ 21,466
Background
Airway clearance techniques (ACTs), have been a key treatment for people with Cystic Fibrosis (CF). Many people with CF report that ACTs are the most taxing part of their treatment. Recent studies have indicated that people with CF support the need for a clinical trial to test whether exercise can be substituted for ACT’s without affecting health and wellbeing. The best way to conduct such a trial is not yet known.
Our project gathered the opinions and thoughts of people with Cystic Fibrosis (CF), their parents and their physiotherapists on whether exercise can be used to replace airway clearance for people with CF. Five discussion sessions were undertaken over Zoom to understand the perspective and preferences on the best way to conduct such a trial.
The keys findings included:
Importance
People with CF, their carers and their physiotherapists see a need and are supportive of a trial looking to explore if exercise can replace chest physiotherapy.
Trial Design and Safety
Choice for exercise and for airway clearance is essential.
Trial at least 6 months long.
People with good lung function should be included, but there was no clear understanding on who should be excluded.
Good adherence and commitment is essential.
Clear rules for modifying treatment when unwell will be important, and help to ensure the trial is safe.
Assessment
Lung function was considered the most important outcome.
Quality of life measures, treatment burden, school/work attendance and participation and social and emotional factors were equally important to review and monitor.
Keep assessments to a minimum and online where possible.
Incentive and Support
Incentives and support from the research team and treating health care team are important to encourage and sustain trial participation and to recognise time commitment.
These findings were directly relevant to patients, families and health care professionals and will help contribute towards the final design of this study.
Validating a potential new target for the treatment of cystic fibrosis
Professor Ray Norton – Monash Institute of Pharmaceutical Sciences
$ 49,000
This project sought to determine whether the ion channel Kv1.3 plays a role in airway inflammation in individuals with cystic fibrosis (CF). We aimed to examine broncho-alveolar lavage (BAL) fluid obtained from both young children with CF and adults with CF following lung transplantation.
Previous research has shown that KV1.3 is involved in other inflammatory diseases (such as rheumatoid arthritis and inflammatory bowel disease), but it was unknown if it is also important in CF lung disease or rejection in CF lung transplant recipients.
We also analysed BAL fluid from adults with CF post lung transplant, who are having BAL as part of their routine post-transplant care at the Alfred Hospital Lung Transplant Service. Children with CF having BAL as part of routine management at Monash Children’s Hospital were recruited. Lung inflammatory cells from BAL fluid were tested for the presence of Kv1.3.
Our preliminary results indicated that a substantial number of cells were Kv1.3-positive. Notably, Kv1.3 in cells from CF patients was present on the cell surface, suggesting that it may be possible to target Kv1.3 in these cells using inhibitors such as HsTX1[R14A], a potent peptide inhibitor developed at Monash University.
These highly promising results warrant further investigation of Kv1.3 expression in additional CF samples. As BAL samples comprise a heterogenous mixture of cells, we plan to stain with antibodies for markers of specific immune cell types
If our preliminary results are borne out in these additional samples, the next step would be to test whether blocking Kv1.3 with HsTX1[R14A] reduces inflammation and lung damage in animal models of CF lung disease and chronic rejection. In summary, this project has provided highly promising preliminary results that now need to be validated with further studies on additional samples. Although the period of funding has expired, these studies will be continued.
Motherhood and lung transplant
Dr. Miranda Paraskeva – Alfred Health
$ 22,856.33
Survival after lung transplantation continues to improve and many recipients (in particular those with cystic fibrosis) are of childbearing age. Pregnancy and parenthood after lung transplantation are important concerns for many but there is limited data regarding outcomes and no internationally accepted guidelines regarding the reproductive health of lung transplant recipients.
Though there are few studies reporting the outcomes of pregnancy post-transplant, most have shown that pregnancy in lung transplant recipients increases the risk of poor outcomes for both the mother and baby. For babies born to lung transplant recipients, this includes an increased risk of prematurity and low birth weight. Whilst for the mothers there is an increased risk of pre-eclampsia, gestational diabetes, acute and chronic rejection and death. Due to these risks some lung transplant recipients utilise surrogacy and other methods to have children.
A recent qualitative study undertaken in 2020 by our group (and funded by 65km for Cystic Fibrosis) explored the perspectives of young people on life after lung transplant. One key area identified, as an unmet clinical need by transplant recipients themselves was information about having children.
There is a lack of data on motherhood following transplant and the impacts on the recipient and their offspring. In particular, there are no studies looking at the experiences and beliefs of female transplant recipients and why they pursue parenthood. The aims of this study are to explore the experience, beliefs and motivations of women with cystic fibrosis and lung transplants who want to or have pursued motherhood. We plan to explore the lived experience of these individuals by undertaking semi-structured interviews examining their experiences and motivations, as well as whether they felt supported by their healthcare providers and had adequate information available to them.
2019 - Monash Foundation
A new diagnostic approach to guide treatment of non-tuberculous mycobacteria in cystic fibrosis
Dr. Paul King – Monash Health
$ 46,770
Infection with non-tuberculous mycobacteria (NTM) is a major issue for cystic fibrosis patients with more advanced disease. This bacteria is found in the normal environment and may also be present in people’s lungs, but not cause any inflammation/clinical disease. Alternatively, the NTM may cause severe inflammation and destroy the lung tissue. In patients with CF it is extremely difficult to determine when treatment should commence as there is no effective test available. This is important, as undertreatment may result in severe disease, however the antibiotics required need to be given for 12-18 months and have major side effects (that many subjects cannot tolerate).
The aim of this project was to assess whether a blood test can be used to measure the detection of NTM driven inflammation; there is no blood test currently available. There were significant technical challenges in the study, but we were able to demonstrate that our blood test could detect immune responses to NTM infection.
The results from this project are too preliminary to guide clinical practice but we have established the methods to get this test to work, which is a significant technical achievement. In the long-term we would like to build on this work by refining the technique and doing a larger patient study.
Does intensive glucose management, non invasively assessed by a flash continuous glucose monitoring system, improve clinical outcomes during acute pulmonary exacerbations?
Dr. Georgia Soldatos – Monash Health
$ 42,895
Preliminary analysis of the data from our previous 65km funded study entitled “Cystic Fibrosis Related Diabetes (CFRD) model of care: Optimising patient centred healthcare delivery in the inpatient and outpatient setting”, utilising a continuous glucose monitoring system (CGMS) have found a relationship between blood glucose levels during admission for a lung exacerbation and the duration of the intravenous antibiotics, time to recovery of lung function, length of hospital stay and risk for readmission within 6 months. The next logical step is to evaluate whether tight control of blood sugar levels during cystic fibrosis pulmonary exacerbations will improve outcomes such as recovery of baseline lung function, time spent in hospital and time spent on intravenous antibiotics.
CRFD becomes more prevalent with advancing age. With improved life expectancy in patients with CF, CFRD is likely to become a significant burden to the health of an ageing CF population. Its onset is associated with worsening health measures such as a decline in lung function and nutritional impairment. CFRD may also be associated with failure to improve lung function back to baseline after an exacerbation of lung disease.
We would therefore like to investigate whether an intensive insulin regimen, utilising continuous glucose monitoring to produce high control of blood sugar levels will improve these outcomes. Patients with cystic fibrosis admitted with exacerbations of their lung disease will be invited to participate and randomly assigned to “usual care” versus “tight glucose control”. Glucose levels will be measured every 5 minutes by utilising the Freestyle Libre Flash Glucose monitoring system which measures blood glucose levels without the need for a finger stick measurement. Insulin will be used to maintain blood glucose between between 5 and 10 mmol/L, consistent with current national and international guidelines. Patient satisfaction will also be measured to ensure that “tight glucose control” is not achieved at the expense of a higher burden of disease.
Adolescent and young adult perspectives on life after lung transplantation: a qualitative study
Dr. Miranda Paraskeva – Alfred Hospital
$ 29,858
Lung transplantation (LTx) is a treatment option for adolescents and young adults (aged 10-24) with end-stage lung disease. More than 70% of young people undergoing LTx have the underlying disease of cystic fibrosis (CF).
We believe that improved education and a patient-centred approach to care that is developmentally appropriate will benefit this group. By examining the lived experience and individual perceptions of young people themselves, we aim to understand the factors that influence their health and wellbeing. We plan to use the information obtained through this study to develop educational and clinical resources to optimise quality of life and longevity for those who live with both CF and LTx.
Sixteen interviews were undertaken with young people who had received lung transplants.
We wanted to know:
1. How did having a lung transplant effect their daily life?
2. What parts of their healthcare were they happy with and how could it be improved?
Most young people were happy with their care and life experiences after lung transplant.
In particular the positive things discussed were:
Having a second chance at life.
Achieving a sense of normality.
Increased independence.
Improved relationships with family and friends.
Patient centred care.
A number of challenges were identified including:
Adjusting to a life of medication.
Lack of youth-centred care and educational provision.
Interruptions in continuity of care.
Lack of focus on mental health.
The findings of these interviews will be presented to our project advisory group and the lung transplant service. The focus will now be on using these findings to develop a youth appropriate educational package, increase support around medication delivery and side effects and attempt to find solutions that improve continuity of care and allow a greater focus on mental health.
2017 & 2018 - Monash Foundation
Defining the presence of macrophage and neutrophil extracellular traps as a therapeutic target in early-stage cystic fibrosis
Dr. Paul King – Monash Health
$89,630
Cystic fibrosis lung disease arises from lung inflammation that causes the activation of lung white blood cells such as neutrophils and macrophages. This results in the development of bronchiectasis and over time, progressive lung destruction.
Bronchiectasis occurs in very young children with CF, but the processes responsible have not been well understood. In this study we explored the role of a white cell inflammatory pathway called extracellular trap formation in the development of lung damage.
We clearly found very strong involvement of extracellular traps from lung samples in young children both with CF and also in other children with chronic airway infection. We believe that these findings may help explain how young children develop lung disease.
Most importantly, we found that this inflammatory response (that causes lung disease) can be prevented by the currently available therapeutic medications, dornase alfa (DNase 1 or Pulmozyme) and by alpha-1 antitrypsin (AAT). These findings therefore have direct implications for the treatment of young children.
2018 - Monash Foundation
Combating Pseudomonas aeruginosa infections in paediatric patients with cystic fibrosis
Dr. Cornelia Langersdorfer – Monash University
$ 44,922
Paediatric patients with cystic fibrosis are substantially at risk of respiratory infections caused by Pseudomonas aeruginosa. Infections harbouring hypermutable Pseudomonas aeruginosa can be particularly critical because they are associated with accelerated development of antibiotic resistance.
We performed laboratory in vitro infection studies using three hypermutable Pseudomonas aeruginosa isolates from adolescents with cystic fibrosis. In these studies the combination of ceftolozane/tazobactam + tobramycin was superior to ceftazidime + tobramycin and piperacillin/tazobactam + tobramycin. We evaluated the effect of dosage regimens of ceftolozane/tazobactam and tobramycin that are clinically used or have been used in clinical pharmacokinetic studies, in monotherapies and combination regimens. The dynamic biofilm model was used to mimic the changing antibiotic concentrations over time, as would be observed in the lung fluid of adolescents with cystic fibrosis, and investigate the antibiotic effects on planktonic (free-floating) and biofilm bacteria. Tobramycin (intravenously or inhaled) and ceftolozane/tazobactam as monotherapies did not sufficiently suppress the development of resistance, although inhaled tobramycin was more effective than intravenous tobramycin. Mechanisms of bacterial resistance for these suboptimal regimens were identified. The combination regimens achieved substantial antibacterial effects and completely suppressed resistance in planktonic and biofilm bacteria. A novel mathematical model was developed to describe the antibiotic effects for different treatment regimens. The model can be used for translation of the regimens for future evaluation in animal studies or in patients.
Overall, our findings support the further investigation of ceftolozane/tazobactam in combination with tobramycin for the potential treatment of biofilm-associated P. aeruginosa infections in adolescents with cystic fibrosis.
The airway microbiota in advanced cystic fibrosis lung disease: its relationship to early life factors
Dr Katherine Frayman - PhD Candidate; Respiratory Paediatrician
$43,674.70
This study will describe the relationship between the bacterial community (microbiota) found in the sputum of adults with cystic fibrosis (CF) and early life factors, including the bacterial community in lower airway samples from infancy and early childhood. Infection and inflammation are intricately related to the development and progression of CF lung disease.
Historically, we believed that people with CF became infected with 1-2 of a small number of bacteria. More recently, studies using molecular techniques have demonstrated that there are complex bacterial communities in both healthy and CF airways, and that in CF, certain characteristics of these communities are associated with poorer outcomes. In the early 1990s, a birth cohort of Victorian infants with CF was established with the aim of exploring the role of infection in the development of CF lung disease. Participants underwent bronchoalveolar lavage (BAL; lung washings) under general anaesthetic approximately yearly during early childhood. Molecular analyses have been performed to describe the bacterial communities in their BAL fluid.
We are currently conducting a long term follow up study of this birth cohort, exploring the impact of early lower airway infection and inflammation of survival and on the development of lung disease over 25 years. This study will involve the molecular analysis of sputum samples collected from these adults, in order to describe the sputum microbiota. We will compare the early childhood and adult lower airway microbiota, and explore the relationship between early life factors and the sputum microbiota, therefore improving our understanding of the development of CF lung disease.
2017 - Monash Foundation
Cystic Fibrosis Related Diabetes (CFRD) Model of care: Optimising patient centred Healthcare Delivery in the Inpatient and Outpatient Setting
Georgia Soldatos, MBBS, FRACP, PhD
$ 50,000 grant
The development of Cystic fibrosis related diabetes (CFRD) is associated with poorer outcomes including impaired nutritional status, reduced body mass index (BMI), more frequent pulmonary exacerbations, increased prevalence of Pseudomonas aeruginosa colonisation, and lung function decline. Optimal management of CFRD is important to improve these outcomes.
This study showed that the Freestyle Libre Flash Glucose Monitor can be used in people with diabetes and cystic fibrosis to provide accurate measurements of their blood glucose level (BGL). The study also showed BGL are high especially in the initial phases of an exacerbation and improves after a few weeks. People with high BGL during exacerbation when compared to those with normal BGL were found to need antibiotics for a longer time, had more hospital admissions and poorer recovery of their lung function. These patients will hopefully benefit from closer attention to blood sugar control.
Most participants of an online survey felt diabetes was very important to their overall health. Despite this, less than half attended a hospital-based diabetes clinic for ongoing management and follow-up and almost a third stated they managed their diabetes by themselves. The majority wanted to receive diabetes management advice from an endocrinologist, who is a diabetes specialist.
Participants also preferred to see both their cystic fibrosis and diabetes specialist at the same clinic. Those who had used the Freestyle Libre Flash Glucose Monitor found it very satisfying however stated out-of-pocket costs were prohibitive. The majority of participants indicated burden of multiple clinics and conflicting advice from diabetes and CF specialists as some of the reasons they were poorly engaged with the diabetes services. They also hoped for more personalised approach to their diabetes care and more research into the area.
2016 - Monash Foundation
Is implementation of the 2006 Australasian Clinical Practice Guidelines for nutrition in cystic fibrosis associated with improvements in nutritional status? National data from the Australian Cystic Fibrosis Data Registry
Associate Professor David Armstrong - Monash Children’s Hospital
$ 22,200 grant
Nutrition in children with CF is important because poor nutrition is a risk factor for poor outcomes. However, poor nutrition remains a problem in CF because of increased energy requirements and problems with fat and protein absorption. The introduction of the 2006 Nutritional Guidelines for Cystic Fibrosis was the first step to optimising Nutritional care for people with CF, based on the best evidence available. This study looked at whether these guidelines led to improved nutrition in children. It also surveyed Cystic Fibrosis doctors and dieticians to find out what worked well with the guidelines and what still needed improvement.
The study found that after the introduction of the guidelines, weight and height increased in both younger and older children with CF. This increase was found even when accounting for other innovations which have improved health in children with CF since 2006. This suggests that the Nutrition Guidelines for CF have improved the health of children with CF.
The survey of doctors and dietitians found that the guidelines were in general easy to explain and follow but that more dietitians or more time with dietitians is recommended.
Cystic Fibrosis (CF) questionnaire assessing disease knowledge in adolescents with CF
Dr. Moya Vandeleur - Royal Children’s Hospital
$ 30,271 grant
Transition to adult care is a major life event for young people with cystic fibrosis. Many studies have reported that a successful transition partly relies on sufficient knowledge about CF; the disease itself and treatment plans. Historically CF education took place in early childhood with the parents being the primary recipient of the information. Today we recognise that transition must start in early childhood and involve education of both parents and their children by a multi-disciplinary CF team.
In this project we developed and piloted a new self-report questionnaire designed to assess CF disease knowledge and individual self-knowledge about health status and treatments in adolescents (11-18 years) and their parents. The questionnaire items addressed the same areas discussed in CF clinic as standard of care: respiratory health, gastrointestinal health and nutrition, reproduction and transition.
In general, adolescents had greater knowledge of respiratory and gastrointestinal than reproductive topics. Parents generally had higher levels of knowledge than adolescents. Young people and their parents indicated multiple topics they would like to know more about and a preference to receive this education either in person or via internet/web based resources.
The development of this questionnaire allows us to evaluate CF knowledge in adolescents and their parents. Such an assessment enables the CF multidisciplinary team to address any gaps in the young person’s knowledge about CF and to individualise their education and the transition process. The results of our pilot suggest that knowledge deficits in adolescents with CF are common-particularly regarding reproduction issues and disease self-knowledge and should be considered when developing CF education interventions in the future.
2015 - MCRI
How does activation of PAR1 (Protease-activated receptor-1) modify the response of lung immune cells in cystic fibrosis?
Associate Professor Phil Sutton
$ 45,339 Grant
Infection of the lung with bacteria is an important part of the disease-causing process in cystic fibrosis. In CF, these bacteria trigger an inflammatory response that recruit immune cells that cause lung damage and help drive progression to more serious lung disease. Control of these infections – or the inflammation that they produce – is an important feature of CF clinical management.
We have identified a factor, expressed by some immune cells, which may be able to be used to reduce the inflammatory response of these cells upon infection with disease-causing bacteria. However this effect is highly variable; targeting this factor reduces the inflammatory response of lung cells from only some people with CF. This project aims to increase our understanding of the conditions under which this factor is expressed by immune cells in CF, in order to help explain this variability. This will be valuable for determining if and how it may be developed as a possible treatment for lung inflammation in CF.
REPORT, NOVEMBER 2016: Protease Activated Receptor 1 (PAR1) is a molecule found on the surface of many different cell types, including those of the immune system, which has been shown to be able to change the severity of inflammation in a range of different diseases. We have been examining whether PAR1 might play a role in changing the severity of inflammation in the CF lung and, if so, whether it might therefore provide a novel therapeutic target.
The studies we have performed so far suggest that activation of PAR1 might modify the inflammatory response to pathogenic bacterial infection in lung immune cells some children with CF. Due to the high degree of variability of this effect, it does appear unlikely at this time that this approach would have widespread therapeutic benefit. However we are continuing our studies to get a better understanding of what PAR1 does in the CF lung in case there is still a potential approach we might be able to use.
Modelling complex variation in multiple breath washout (MBW) data to reveal unobserved characteristics of early childhood lung disease in cystic fibrosis
Professor John Carlin
$ 31,520 Grant
The multiple breath washout (MBW) technique has been widely adopted by respiratory researchers because of its usefulness in assessing lung function for various common and serious lung diseases, particularly cystic fibrosis (CF). Currently, MBW data are typically interpreted using simple summaries only, yet the data are rich in additional information that could be extracted using methods that have not previously been applied in respiratory research.
We will develop and apply sophisticated statistical models and techniques to MBW data using state-of-the-art software. We expect to reveal properties of the data that are clearly connected to the underlying lung biology. These advances will give researchers new perspectives on early childhood lung function, with a particular focus on identifying early markers of disease progression in CF.
A longer-term goal is the refinement of MBW and similar techniques to make them suitable for clinical use.
REPORT, NOVEMBER 2016: The MBW technique has been widely adopted by respiratory researchers because of its usefulness in assessing lung function of various common and serious lung diseases, particularly cystic fibrosis.
In this project we have developed and applied sophisticated statistical models and techniques to MBW data, and we expect in continuing work to reveal properties of the data that are clearly connected to the underlying lung biology. These advances will give researchers new perspectives on early childhood lung function, with a particular focus on identifying early markers of disease progression in CF.
A related goal is the refinement of MBW and similar techniques to make them suitable for clinical use. Improving the statistical methodology has taken us closer to this goal and we expect our findings from analysis of research data to demonstrate the clinical utility of these techniques. This research should make MBW testing more useful and cost-effective for both research and clinical purposes, thus providing a powerful tool for the study and management of CF.
We believe that we have generated new data and an interesting mathematical analysis that will be of interest to the CF community as new treatment trials use the MBW test. Our method means even when the testing time is short, as is often the case in young children, or relatively short as it may be in those who are sicker, useable results will be available in more children.
Role of an acute phase protein Alpha 1-acid glycoprotein as a biomarker of prediction of pulmonary exacerbation in young patients with cystic fibrosis
Rosemary Carzino - Progress Report
$ 41,367 Grant
SUMMARY: This project is to investigate if a specific blood protein can act as a “biomarker” to detect for the presence of a lung infection. Blood concentrations of these proteins are found to increase in response to the presence of inflammation and infection.
This protein has not yet been tested in children with CF and as part of the AREST CF program, we have an opportunity to study the blood samples that have already been collected and stored. These results will be studied alongside other information collected at the same time; including BAL, Chest CT, and lung function tests.
An early predictor of lung infection using a relatively simple blood test could help provide the information medical staff require to begin immediate treatment. Early intervention could help slow down lung disease leading to better long term health.
REPORT, NOVEMBER 2016: This project aims to investigate the role of an acute phase protein in blood as a biomarker for the prediction of a pulmonary exacerbation in young patients with CF.
To date we have not yet analysed any data for this project as the samples are currently being tested and results are yet to be received. Upon discussion, we have decided to change direction in methodology and send the samples off to a biomedical company in Finland. This means that besides analysing for the acute phase protein levels in question, a further 200 other biomarkers can also be tested simultaneously in a small volume of one sample by the use Nuclear Magnetic Resonance technology. This allows for a great opportunity to work alongside other groups in MCRI who are also utilising this service where results may be compared for further analysis.
Once results are received, they will be analysed alongside relative clinical information to ascertain the ability of the acute phase blood protein to predict a pulmonary exacerbation in young children with CF, with the added benefit of exploring other blood biomarkers in the meantime.
2014 - MCRI
Feasibility of human Epithelial Cell culture from Bronchial Brushings of cystic fibrosis patients for studies of airway modelling and research.
Paul Griffin, Cilial Function Scientist
$15,000 grant
SUMMARY: In this project we will use samples obtained from bronchial brushings obtained during broncho-alveolar lavage to establish a novel model and validate an in vitro model. This model can then be used to study a variety of conditions, including pathogenesis of, and recovery from various infectious organisms and the effects of environmental physiological changes on the respiratory epithelium. This will be done by growing the cells in an air-liquid interface culture using antibiotic media. The antibiotic clears infections in the sample allowing the cells to re-differentiate into “clean” new, airway epithelia. The cells are then challenged under different conditions and a comparison can be made to normal airway epithelia. Changes in the gross structure, function and ultrastructure of the cells will be analysed using high speed video and electron microscopy (and possibly fluorescence microscopy).
The model can further be used to test a wide range of pathogens in addition monitoring the time it takes to completely recover to normal function and the influence of pH on epithelial and ciliary function.
The aim is to adapt the cell culturing technique currently used on nasal epithelial brushings for bronchoscopic brushings from CF patients. Once validated, a functional and biologically representative cell model can be used to perform experiments. If successful, a second aim of this project would be to investigate the pathogenic sequence of infection of bacteria commonly isolated from CF specimens. The model can be used to check the time of clearance of the bacteria using antibiotics and how long recovery of normal function takes. Also, the effect on important aspects of the airway including cilia beat frequency and differences in ultrastructural components can be monitored. Thirdly, environmental conditions such as the pH of the media and Sodium Bicarbonate levels can be varied and again, the effects on functions and structure of the cells can be observed.
Very little detailed work has been reported on primary, differentiated CF airway epithelial cell culture. Most studies are done on explants or expensive commercial cell lines which are not as relevant. This project will provide an improved cell culture system for respiratory research that can provide novel experimental investigations. It also has the potential to allow for individual reports on patient samples. Our wealth of experience in cell culture, with the addition of world leading equipment and support (currently utilised in the RCH PCD diagnostic service), have shown images and videos produced here, are of the highest quality. Also, as it is thought that this method of culture should be more successful, it would therefore in theory, reduce the expense and time spent on culturing samples that fail to thrive.
A success model would be attractive to scientists and clinicians studying mucosal immunology, CF airway therapeutics and a myriad of other disciplines. To our knowledge, less than a handful of centres in the world possess this model for studying young children with CF.
REPORT: A clinical method for the brushing of Cystic Fibrosis patient’s airways was established. This was based on a method already in use at another hospital, altered to suit our project purposes and facilities.
Over 20 procedures have been performed, with no adverse effects. After overcoming a few problems, several individual samples have grown to the point where cilia function and mucus production was observed. To validate that a true CF airway cell environment had been generated, visualisation of this cell strata has been produced in two ways. A high speed video of the cilia beating was recorded and slowed down to a tenth of the actual speed, so the frequency and shape of the beat could be seen. These were both completely normal in all videos of the samples taken. The second method of visualisation was via electron microscope, where the structures of the cell organelles (including cilia) could be checked and confirmed to be as expected. This microscope allows magnification of the cells up to 30,000 times. Again, in all aspects of the cell formation, the structure was found to be as expected in a CF patient. There are still a few hurdles to overcome, and currently the correlation between health of patient at the time of brushing and the success rate of culture is being investigated. However, the successes so far have meant we can start to design and perform further experiments to characterise aspects of CF airways that likely could not be studied any other way.
Evaluating the role of elastase in the inflammatory response in cystic fibrosis
Associate Professor Phil Sutton, Group Leader, Mucosal Immunology
$45,000 Grant
SUMMARY: CF patients are highly susceptible to respiratory infections that result in life-threatening inflammation in their lungs. A/Prof Ranganathan has identified that a protease (an enzyme that cuts up proteins) called neutrophil elastase is significantly associated with CF pathogenesis. This elastase is probably produced in response to infection of the lung.
This project builds on a finding of potentially major significance that was published in one of the top medical journals.
Our findings indicate that the detection of free neutrophil elastase in lower respiratory secretions is associated with the development of early bronchiectasis in CF. The aim of this project is to investigate the potential use of this observation to identify suitable new targets for therapy in CF. This proposal will provide a proof-of-concept test of a theory regarding how we believe neutrophil elastase contributes to bronchiectasis in the airways. If correct this will identify new ways to prevent the pathway to destructive lung damage.
An important additional significant bonus of this project is that it will establish the CF mouse model at MCRI. Its introduction will increase the variety of techniques available for CF research.
This project is evaluating whether the protease activated receptors PAR1 and/or PAR2 play an important role in CF pathogenesis. The funded project aims to: 1) Establish a colony of Cftr mutant mice at MCRI. These would be used to provided cells and tissues for important tissue culture experiments. 2) Cross these Cftr mutant mice with mice lacking PAR1 or PAR2 in order to provide an animal model for examining what effect the PAR receptors have on CF pathology. 3) Examine the interaction of PAR1 and the CF mutation in human cells.
REPORT: We have had good success with examining the role of PARs and in particular PAR1 in CF.
We have started to examine the importance of PAR1 on the response of immune cells present in the lungs of children with CF. Importantly, we have found activation of PAR1 on these immune cells reduces or prevents the inflammatory response to stimulation with the important CF pathogenic bacterium, Pseudomonas aeruginosa. In other words, PAR1 activation may reduce the inflammatory response of CF immune cells.
We have also purchased neutrophil elastase and have started examining how this interacts with PARs using lung immune cells from CF children.
We have done considerable work on using a technology called “CRISPR” to be able to knock-out CFTR expression in any cell line we choose. CRISPR plasmids for targeting Cftr have been designed, made and tested successfully. We will soon start to make Cftr knock-out cell lines and use them in our studies.
We have very encouraging preliminary data suggesting activation of PAR1 may inhibit the inflammatory response of lung immune cells from some CF children; this may indicate a potential new therapeutic target. We are also developing powerful new tools to allow us to perform important tissue culture experiments that will help us evaluate this possibility.
2013 - MCRI
Exploring the early lung microbiota in cystic fibrosis
Sarath Ranganathan
$45,000 Grant
SUMMARY: New techniques have revealed that a much larger range of organisms than we previously believed colonise the human lung. These microbes are called the microbiota. We don't know which organisms reside in the lungs of people with CF. We believe it is important to study this as we have identified that the lungs can be damaged in CF before typical infections are detected. We hope that by turning our attention to different microbes that might be present in the lungs early in life and that may initiate the inflammation that causes damage to the structure of the lung.
The Royal Children's Hospital and MCRI previously (1992-1995) stored a unique resource of lower respiratory and saliva samples from infants and young children with CF, together with samples from controls without CF who underwent lung washings for investigation of other diseases. Together with our leading team of scientists from the Human Microbiome Project in the US, we want to analyse these samples in order to explore the range of microbes in early CF. This will enable us to investigate several important questions about the potential role of the microbiota in the development of CF lung disease.
REPORT (January 2015): Microbial diversity in lung fluid
Our analysis has now revealed the relative abundance of genera identified in the historic BAL samples from children with CF treated at RCH using 16s PCR sequencing. Deep sequencing is ongoing and will be used to identify individual bacterial species, virulence patterns and antibiotic resistance genes. Dr Katherine Frayman will be looking at long term clinical outcomes in relation to these early data. We are currently exploring how these sequencing data relate to conventional microbial culture and other aspects of disease pathogenesis and important clinical outcomes in preparation for publication. We will be the first group able to report on the microbiota in relation to inflammation and other clinical outcomes and so a manuscript is in preparation.
Differences between the left and right lung
We have assessed the number of reads and the relative abundance respectively of microbial genera in lung fluid samples obtained from the left and right lung. Where there were sufficient reads using 16s sequencing on BAL samples obtained from the left and right lung we identified that microbial genera were similar in most cases. In one case, there was a stark difference between left and right lung: the genus Staphylococcus was identified in the right lung but diverse flora in the left. We are investigating pulmonary inflammatory markers and evidence for regional structural lung damage in these individuals. This is a unique approach as few centres have recourse to such samples from the left and right lung.
Changes in the microbiota over time
In 15 subjects at least three BAL samples were analysed successfully so that changes in the microbiome over time can be analysed. Again, the data are unique because we are able to compare the microbiome with changes that occurred in pulmonary inflammation, standard culture and clinical progress. We are writing up the results and believe they will be of great interest to the CF and respiratory research community in helping to better understand the microbiota in the CF lung.
Prevalence of small-colony variant s.aureus among children with CF
Dr Emily Hart
$23,000 Grant
SUMMARY: Lung infections with the microorganism, Staphylococcus aureus, are common in young children with cystic fibrosis (CF). Many patients experience repeated infections during which time S. aureus adapts itself, becoming better suited to living in the human lung by forming what are known as small-colony variants (SCVs).
These changes make the bacteria more resistant against treatment with antibiotics and affect their ability to grow in the methods laboratories currently use to diagnose bacterial infections. Because this slower growth makes them more difficult to detect, at present we do not know whether SCVs in S. aureus are an important cause of infection in our patients.
This study will use updated diagnostic methods to specifically look for SCVs in respiratory specimens collected from patients attending the Royal Children’s Hospital (RCH) Melbourne, and we expect that SCV infections will be common in children with CF lung disease. Detection of these variants is particularly important, given their ability to resist treatment with certain antibiotics. Knowing the prevalence of this bacteria will help us to improve the way in which we diagnose respiratory infections in CF, and will also provide clinicians with more detailed information that can be used to optimise the treatment and management of their patients.
REPORT (January 2015): Twenty six children (mean age 11.35(5.05) years) from the AREST CF study (Melbourne) were identified with a positive S.aureus culture from lower respiratory tract specimens collected in 2014. All children were on at least one or multiple antibiotics prior to or at time of collection. The antibiotics were, namely Augmentin Duo (46%), Tobramycin (15%), Bactrim (11%), or other (27%). Fifty-seven percent of samples were visible on large MSA plates and subcultured onto wild-type HBA plates, but no SCVs of S.aureus were isolated.
Conclusion: SCV’s were not isolated from the lower airways specimens and therefore were not identified as contributing to lung infections in our cohort of young subjects with CF.
Establishing predictive indicators of amino glycoside nephrotoxicity and the incidence of toxicity in children aged less than 6 years with CF
Ms Courtney Munro
$42,000 Grant
SUMMARY: Cystic fibrosis (CF) is caused by a gene defect and affects the lungs and other body systems. People with CF have altered medicine metabolism. This is because CF affects how the body processes medicines.
Aminoglycosides are antibiotics commonly used in the treatment of CF. As CF is now mainly diagnosed by newborn screening, aminoglycosides are being used earlier than in the past. Thus people with CF are exposed to higher doses and to longer and repeated courses. This long and early exposure to aminoglycosides may pose risks that little is known about appropriate dosing or toxicity.
There are two important side effects from aminoglycosides. These are:
· Kidney damage and;
· Hearing loss or deafness.
The aim of this research project is to see if we can discover markers that show changes in kidney function, before and after treatment with aminoglycoside antibiotics. We will do this by testing blood and urine samples. The research project may help to find a marker of early kidney disease. This would help us to know when to reduce the dose of aminoglycoside or when to change the antibiotic to a different one, to prevent damage to the kidneys.
REPORT (January 2015): Patients under six years of age were recruited when commencing IV aminoglycosides (gentamcin 7.5 mg/kg or tobramycin 12 mg/kg) and had urinary and serum sampling of creatinine and magnesium. A total of seventeen patients aged 0.74-6.99 years, ten males, eight of them on gentamicin and nine of them on tobramycin, have been recruited to date. A total of 44 patients will be recruited.
Early results suggest that the metabolism and excretion of magnesium in CF warrants further study, and that aminoglycosides (gentamicin>tobramycin) considerably alter magnesium excretion.