Paediatrics (RCH) - Theses
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ItemThe innate immune mechanisms underlying the off-target effects of Bacillus Calmette-Guérin vaccine in infants( 2022-12)Bacillus Calmette Guerin vaccine (BCG) is administered to over 100 million newborn infants globally each year with the aim of protecting against tuberculosis. In addition to its specific anti tuberculous effects, BCG vaccine has off target effects that protect against a broad range of non mycobacterial infections in both children and adults. Immunological studies have implicated trained immunity in the mechanism underlying the off target effects of BCG vaccine in adults. Trained immunity is a de facto innate immune memory underpinned by the functional epigenetic and metabolic reprogramming of innate immune cells, such as monocytes. While much research has focused on elucidating the immunological mechanisms underlying the off target effects of BCG in adults, few studies to date have investigated the immunological mechanisms in children. Using samples from a well characterised sub cohort of 130 infants from MIS BAIR, a randomised trial of neonatal BCG vaccination in Australia, the projects in this thesis aim to characterise the phenotypic, functional, and epigenetic effects of neonatal BCG vaccination on the infant immune system in the first year of life. Chapter 2 of this thesis sought to better understand the role of vaccination and natural infection in shaping the human epigenome by a systematic review of the literature. This review identified a paucity of paediatric studies and highlighted a need for further research investigating the epigenetic mechanisms underlying the off target effects of BCG vaccine in children. This knowledge gap is addressed by the subsequent projects of this thesis. Immunological studies in infants are hampered by the ethical and logistical challenges of obtaining blood samples from this age group. Optimisation experiments detailed in Chapter 3 led to the refinement of laboratory methods that generate high dimensional immunological data from small volume infant blood samples containing as few as 3 million peripheral blood mononuclear cells (PBMC). Multi parameter flow cytometry was used to comprehensively profile and sort cell populations of interest for downstream analysis. The monocyte fraction was then used for parallel DNA methylation analysis and ex vivo heterologous stimulation assays. This analysis pipeline produced rich normative data on baseline immune cell profiles and monocyte cytokine responses in BCG naive infants. Subsequent studies in Chapters 4 and 5 found that BCG vaccinated infants have increased proportions of circulating classical and non classical monocytes, and activated regulatory CD4 T cells, compared to BCG naive infants at 13 months of age. Monocytes from BCG vaccinated infants also have an attenuated inflammatory response following heterologous stimulation and retain a DNA methylation signature of early life BCG exposure. Genes associated with this signature are involved in interferon signalling pathways and viral immunity. In summary, the studies in this thesis demonstrate that neonatal BCG vaccination induces durable changes in circulating immune cell profiles, monocyte cytokine responses and DNA methylation remodelling that persist over the first year of life. These changes are different to those observed in adults and strengthen evidence that immune responses to BCG vaccination are age dependent. Collectively, these findings contribute to a better understanding of the immunological mechanisms underlying the off target effects of BCG vaccine in infants that will inform the development of new immunomodulatory therapies and guide global vaccine policy.
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ItemInvestigation of the short- and long-term complications of anorexia nervosa/atypical anorexia nervosa and the impact of the coronavirus pandemic( 2022-10)Anorexia nervosa (AN) is an increasingly prevalent psychiatric disorder in which restricted energy intake leads to significantly low weight, a fear of weight gain, and a distorted perception of one’s body image. Individuals with this condition experience complications which affect the heart, muscles and bones, hormones, and mental health. The long-term prognosis for AN is difficult to predict; whilst many complications resolve with refeeding and weight gain, some damage may be permanent and disordered cognitions may persist. Furthermore, the impact of the coronavirus (COVID-19) pandemic on mental health and eating disorders is increasingly evident. However, reasons for declining mental health and the onset of disordered behaviours, as well as how the treatment of individuals is affected, remains unclear. The aim of this project was to (1) assess long-term cardiovascular and mental health following recovery from AN, and (2) determine the impact of the COVID-19 pandemic on both current and former AN patients. To achieve this, four separate but interrelated studies were conducted. The first study involved a four-year retrospective chart review of all patients presenting to the Royal Children’s Hospital Eating Disorder Service between 2017-2020. To assess the impact of the COVID-19 pandemic, the number and severity of presentations was compared between years via analyses of variance. The second study investigated long-term psychological outcomes and predictors of recovery and prognosis by administering online questionnaires to former AN patients from the Royal Children’s Hospital and Monash Children’s Hospital. The questionnaires assessed eating and exercising behaviours, mood, and COVID-19 impact. The third study involved non-invasive physical testing of the former patients to assess long-term cardiovascular health post AN recovery. Finally, further interrogation of carotid ultrasound images using wave analysis techniques provided valuable insight into the biomechanical interactions between the heart and vascular network. All outcome measures were compared to healthy controls and/or normal thresholds. Results revealed there was a surge in new AN cases under COVID-19 restrictions in 2020. Changes to routines, and feelings of isolation and loneliness were recognised as triggers for disordered behaviours. Mixed responses to telehealth and online learning were also reported. Former patient questionnaire responses indicated that psychological recovery from AN can be sustained long-term when individuals receive early and intense treatment. However, elevated levels of stress were exhibited by the former AN patients. This may reflect inherent personality features, such as perfectionism, which have been closely correlated with the condition. Despite increased stress and a history of an eating disorder, the former AN patients did not appear more susceptible to the impacts of the COVID-19 pandemic and faired comparably to controls. Cardiovascular assessments indicated that an increased propensity for cardiovascular disease may be associated with AN even after recovery. Regional changes in arterial stiffness, endothelial dysfunction, and abnormal autonomic regulation were identified in early adulthood. Overall, as the first research study to report on long-term cardiovascular and mental health following AN recovery, and the impact of the COVID-19 pandemic on an adolescent population within Melbourne (the most locked down city in the world), this thesis fills a critical gap in eating disorders literature. It identifies factors that contribute to patient course and outcome, reveals persisting cardiovascular adaptations, and informs of specific issues faced by both current and former AN patients during the pandemic. These findings have the potential to guide disease surveillance strategies and optimise clinical interventions.
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ItemNo Preview AvailableA Blood Based Study of COVID-19( 2022)Background: Coronavirus Disease 2019 (COVID-19), caused by the virus SARS-CoV-2, has had substantial impact on global health. In addition to the severe respiratory symptoms of COVID-19, approximately 20% of adult COVID-19 patients experience a clinically significant thrombosis. The relationship between COVID-19 and thrombosis is poorly understood, however the presence of thrombosis in even mild COVID-19 suggests that the haemostatic system is fundamental to COVID-19 pathogenesis. Children, while somewhat protected from COVID-19 coagulopathy, experience a severe, post-COVID-19 inflammatory condition known as multisystem inflammatory syndrome in children (MIS-C). Little is known about the underlying pathophysiology of MIS-C, and what differentiates it from typical presentation of severe COVID-19. Objectives: This research program explores the blood-based impacts of COVID-19 in adults and children, with a particular attention to understanding the differential thrombotic risk between adults and children, and MIS-C which seems to occur uniquely in children. This project investigated platelet phenotype and function in COVID-19 in vivo. SARS-CoV-2 infection induced fibrin-clot structure and characteristics were investigated in vitro. Additionally, the biological mechanisms underpinning MISC and severe COVID-19 were investigated in children. Methods: Platelet phenotype and function was investigated in 67 children and adults with mild COVID-19, by performing whole-blood platelet flow cytometry. These were compared to platelet flow cytometry samples collected prior to the COVID-19 pandemic. Fibrin clots were investigated in a novel in vitro model of endothelial involvement in Haemostasis. Human umbilical vein endothelial cells (HUVECs) were cultured and infected with SARS-CoV-2 and incubated with plasma from healthy children, healthy adults and adult vasculopaths. Experiments were repeated with the addition of anticoagulants bivalirudin, defibrotide, unfractionated heparin (UFH), and low molecular weight heparin (LMWH). Fibrin clot structure was determined using scanning electron microscopy. The plasma proteome of children who developed MIS-C or COVID-19 acute respiratory distress syndrome (ARDS) were compared to healthy children, by mass spectrometry. Abundance of proteins was compared to establish a protein signature of MIS-C and ARDS, and to determine underlying biological pathways in either syndrome. Results: Platelet activation increased in adults and children with mild COVID-19, which persisted at least four weeks after their initial infection. Additionally, close household contacts of the index case who did not test positive to COVID-19, also showed increase in platelet activation by PAC-1 and CD62P. Fibrin clots were induced in SARS-CoV- 2 infected cells. The fibrin network was denser and more composed of thinner fibrin fibres in the vasculopathic adults compared to healthy adults, and in healthy adults compared to healthy children. UFH and LMWH were effective at preventing fibrin clot formation, even at a half (LMWH) and quarter (UFH) prophylactic doses. 85 proteins were differentially expressed in MIS-C patients while 52 were differentially expressed in COVID-19 ARDS patients. Complement and coagulation pathways were impacted in both MIS-C and ARDS, however MIS-C showed specific FcGR and BCR activation while COVID-19 showed changes in haem scavenging and retinoid metabolism. Conclusion: COVID-19 induced changes to the coagulation system across three major aspects of haemostasis: platelets, a SARS-CoV-2 infected endothelium, and the plasma proteins. We show that there are prothrombotic platelet changes in the setting of mild COVID-19, and that vasculopathy patients may have increased risk of thrombosis. Our evidence that UFH is effective in reducing SARS CoV-2 induced coagulation in vitro sets the scene for future clinical research in establishing treatment of COVID-19 coagulopathy. We show that severe COVID-19 phenotypes (MIS-C and ARDS) are driven by coagulation and complement, showing the crucial interplay between immunity and coagulation in COVID-19.
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ItemTuberous sclerosis complex and autism spectrum disorder: phenotypic presentation and associated clinical factors( 2022)Background: Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem, genetic condition with variable disease severity, due to pathogenic variants in TSC1 or TSC2, which disrupt the mTOR pathway. Neurological morbidity, including seizures and structural brain changes, occur in most people. Seizures commonly present during the first three years of life and infantile spasms occur in around one third of children. Autism spectrum disorder (ASD) and intellectual disability (ID) are reported in half of TSC affected people. When this PhD commenced, it was unclear if ASD in TSC was similar to ASD without a co-existing medical condition, that is ‘idiopathic’ ASD, and if TSC clinical factors contributed to ASD like they impact on ID. Research had shown early seizure-onset, infantile spasms, TSC2 variant, and tubers to be potentially associated with ASD in TSC. However, findings were mixed, and many studies employed weak methods for ASD categorisation. A systematic and comprehensive investigation had not been undertaken. Aims: This thesis aimed to (A) define the autism phenotype in children with TSC and (B) investigate the major clinical factors: (1) seizures and associated factors, (2) genetic factors, (3) structural brain changes, and (4) individual child characteristics, associated with ASD outcome in TSC. Methods: Two systematic reviews with meta-analyses, methodically and comprehensively summarised the literature about (1) the TSC-ASD phenotype and (2) the TSC clinical, EEG, imaging and genetic factors reportedly associated with ASD. The systematic reviews informed development of a cohort study with 50 children which undertook robust ASD assessment and developmental phenotyping in children with TSC and investigated associations between ASD with TSC factors. Results: Systematic review and meta-analysis found 90 percent of children with TSC-ASD had ID, but little was reported about other phenotypic aspects. The cohort study found children with TSC-ASD had moderate ASD severity and typical characteristics, although most had severe ID. Co-occurring language disorder, ADHD, executive dysfunction and externalising behaviours occurred in most, and internalising behaviour in one-third. Systematic review and meta-analysis found significant associations between TSC-ASD and history of seizures (OR 3.79), seizure-onset in infancy (OR 2.65), infantile spasms (OR 3.04), and male sex, (OR 1.62). Seizure-related associations were not significant in the cohort study, but the trends were similar. Incorporation of cohort study data with the meta-analysis led to a significant association between TSC-ASD and TSC2 variant, (OR 1.95). Tuber burden was not associated with TSC-ASD in the systematic review meta-analysis or cohort study. Conclusion: Clarification of the TSC-ASD phenotype enables better counselling for families of affected children and improved early intervention planning. Early life seizures and TSC2 variant are likely important pathways to TSC-ASD. The role of cortical tubers is likely to be indirect, via seizures. Research following large TSC-ASD cohorts from infancy throughout childhood, measuring all relevant variables, will assist with disentangling the relationships. Studies investigating interventions targeting possible aetiological factors will be of significant value, aiding the understanding of causation and potentially improving developmental outcomes.
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ItemImmune Cell Profiling the Peripheral Blood of Heart and Lung Transplant Recipients( 2022)Protection against disease requires cells from both the innate and adaptive arms of the human immune system. The impact of disease is often significantly influenced by age. In humans, newborns have an immature immune system; thus, they are more susceptible to some infectious diseases such as influenza and respiratory syncytial virus (RSV). Whereas the immature immune system in infants below 14 months of age is advantageous in some settings such as heart transplants recipients, even if the donor and recipient are ABO mismatched. Later on in life, the immune system wanes and this renders elderly people more susceptible to diseases such as infections and cancers. This has recently been exemplified by COVID-19, which is more fatal in older age groups, particularly in individuals with other co-morbidities. Therefore, understanding the composition of the human immune system throughout life is crucial if we are to endeavor to manipulate the immune response for treating human disease. In this thesis, we used high-dimensional spectral flow cytometric analysis to generate an immune cell atlas encompassing more than 50 populations of immune cells from individuals aged below 1 month to 75 years of age and then we assessed changes to the immune system in adult lung transplant recipients (LTRs) and paediatric heart transplant recipients (HTRs). Specifically, the human immune landscape undergoes major changes early on in life and this likely influences how the body responds to certain diseases such as solid organ transplantation. Our results showed that naive T cells were replaced with different proportions of memory cell subsets in healthy people throughout life, while the trends were different among children who received heart transplantation. Moreover, the proportion of MAIT cells, gamma delta T cells and plasmablasts in healthy individuals were low in the first few years of life, peaking in teenagers, and declining in elderly. While in contrast, we found increased MAIT cells in peripheral blood of paediatric HTRs particularly infants below 2 years old who did not develop HTx rejection and increased gamma delta T cells in those who developed HTx rejection. Furthermore, we identified several immune cell biomarkers of HTx rejection. For example, CD16 on the surface of gamma delta T cells imbues these cells the ability to perform antibody-dependent cellular cytotoxicity (ADCC), a process which normally exhibit by natural killer (NK) cells. Our results revealed that gamma delta T cells from paediatric HTRs expressed lower levels of CD16 post-HTx, particularly in recipients that showed no sign of rejection, suggesting that gamma delta T cells could play a protective role via CD16-mediated activation. Moreover, we found that among those immunosuppressive drugs which organ transplant recipients routinely take, only calcineurin inhibitors blocked the activation of gamma delta T cells via CD16-mediated ADCC. These findings may lead to better detection of HTx rejection, modify the current treatments for disease and ultimately improved outcomes for solid organ transplant recipients. Altogether, this thesis provides the first comprehensive study of the immune system throughout ontogeny, which not only provides a foundation for understanding changes to the immune system in the context of heart and lung transplantation, but also for many other human diseases.
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ItemQuantifying the influence of arterial wave reflection on the heart and microvasculature( 2022)Hypertension is a leading risk factor for cardiovascular disease and accounts for significant healthcare expenditure. Furthermore, high systolic blood pressure (SBP) in childhood is associated with increased risk of cardiovascular events and mortality in adulthood. For more effective and targeted treatment, it is important to investigate the factors responsible for high SBP, both in the central arteries (causing elevated load on the left ventricle) and in the distal vasculature (causing end organ damage), and to better understand the effects of ageing on SBP. Wave reflection is thought to be a key factor modulating the pressure experienced by the microvasculature and the heart; however, controversies remain around its nature and effect on haemodynamics. The first controversy investigated in this thesis relates to whether an increase in microvascular pressure with advancing age arises from decreased wave reflection in conduit arteries, causing an increase in transmission of pressure waves from the heart to the periphery. This paradigm is controversial because the theory underpinning wave reflection dictates a decrease (rather than an increase) in pressure wave transmission with a decrease in wave reflection. Using transmission line modelling techniques, it is shown that the increase in microvascular pulse pressure with ageing is most likely linked to a decrease in conduit arterial compliance, and hence an increase in the forward pressure wave amplitude, rather than increased transmission of the forward pressure wave. The second controversy relates to the time when reflected waves arising in the arterial network return to the heart (‘return time’). Reflected waves are thought to arrive at the heart during diastole in youth (promoting coronary perfusion) and during systole with ageing (increasing SBP). However, current methods of estimating return time have only indicated systolic return times throughout life, which calls into question the traditional concept of wave reflection and/or the methodology used to estimate return time. To gain clarity on this, a computational model of the systemic vasculature of a young adult was developed. Given excellent agreement with human data, this model provides insights into the most likely origins of wave reflection and its effect on proximal and distal haemodynamics. In addition, a novel centroid method is developed whose higher accuracy (using the computational model) and sensitivity to diastolic and systolic return times (using in vivo experimental methods) than current methods is also demonstrated. Finally, the centroid method is applied to characterise return time across almost the entire life course, using data from various large cross-sectional studies in humans. Results provide the first quantitative evidence of a shift in return time from diastole towards late-to-mid-systole with age. These studies provide clarity on the changes in wave reflection with age and its effect on proximal and distal haemodynamics. Future studies should investigate the effectiveness of wave reflection metrics such as return time as a risk factor for cardiovascular events and as a monitor of treatment effectiveness. Following this, the widespread adoption of wave reflection indices in clinical settings via automated devices such as those used to measure blood pressure may hold promise for improving diagnosis and treatment of cardiovascular diseases.
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ItemDissecting human kidney morphogenesis at the cellular and molecular level using kidney organoids( 2022)Stem cell derived kidney organoids are complex tissues containing epithelial and mesenchymal populations reminiscent of in vivo kidneys. Kidney organoids provide a valuable resource to understand kidney development and disease. However, deficiencies including structural immaturity beyond an early trimester 2 equivalence and populations of non-renal cell types that arise during differentiation are known. For the promise of organoids to be realised in personalised medicine and clinically relevant translation, these deficiencies need to be addressed. Recent studies have highlighted differences at both the transcriptional and morphological level between pluripotent stem cell-derived and bona fide kidney tissue. Identifying specific cell-types transcriptionally using single cell sequencing is powerful but remains inexact, as many previously established marker genes are often broadly expressed and not restricted to a specific cell-type. Further, direct comparison of relative cellular composition between protocols has proved challenging. To improve the characterisation of cell types within kidney organoids, a novel human fetal kidney single-cell transcriptional dataset was annotated and sets of enriched genes associated with single identities extracted. These gene sets are used as comparisons to score transcriptional similarity to the reference data to illuminate cellular identity. This novel dataset was integrated with multiple single cell RNA-seq dataset and used to train classification models using the R package scPred. These models were organised into an hierarchical model that classifies cellular subtypes into nephron, stroma and ureteric epithelial elements. This model, provided in the R package DevKidCC, was then used to predict relative cell identity within published kidney organoid datasets generated using distinct cell lines and differentiation protocols, interrogating the impact of such variations. Finally, these tools were applied to a novel kidney organoid dataset. Kidney organoids with targeted perturbations to optimise mesoderm specification and downstream kidney organoid development were generated, covering 21 unique variations at four key time-points, generating a comprehensive single-cell dataset of organoid differentiation. The map identifies the heterogeneity within single time-points across kidney organoid differentiation. This comprehensive transcriptional map of kidney organoid differentiation across time provides a platform for advanced investigation of kidney organoid developmental dynamics. Recent studies have shown how crucial the presence of appropriate stromal cells are to the development of advanced nephron networks in kidneys, both in vivo and in vitro. However, the in vitro stromal populations that arise do not faithfully represent those in vivo, while the generation of unwanted off-target cell types may further impair appropriate nephron development. This screen identified conditions that limited the differentiation of off-target populations by improving on-target kidney lineage differentiation. Further, our investigation of stromal development in the literature identified the potential origin of these cells in the paraxial mesoderm, as opposed to the intermediate mesoderm. This dataset confirmed conditions in which populations expressing both paraxial mesoderm and stromal progenitor markers were present. This work provides tools to improve characterisation of kidney organoids and an improved understanding of key growth factor pathways influencing both on-target and off-target cell patterning. This data will further support advances for the kidney organoid field.
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ItemClinical and immunological features of severe RSV disease in preterm and term infants( 2022)Respiratory syncytial virus (RSV) is the leading viral pathogen causing acute lower respiratory tract infections in children under 5 years of age. Despite advancements in medical technology and in-hospital care, there is still no vaccine available for RSV, and the burden from RSV remains substantial, particularly in low- and middle-income countries (LMIC). Severe (hospitalised) RSV disease is also associated with the development of chronic respiratory conditions such as recurrent wheeze. One of the highest risk groups for severe RSV disease are preterm infants. While there are a number of complications associated with premature birth that may predispose preterm infants to severe RSV disease (e.g. bronchopulmonary dysplasia), their relatively immature immune system is thought to play a key role. The extent to which this immature immune system contributes to disease susceptibility remains an important question to be answered. Two cohorts were investigated in this thesis. The first cohort was used for results chapter 1 and the second cohort for results chapters 2 and 3. The first was a cohort of 970 children under the age of 2 years that were admitted to the Royal Children’s Hospital with RSV disease from 2017-2019 (RCH cohort). The aim of this cohort was to identify risk factors associated with severe disease by comparing demographic, clinical and laboratory data between moderate and severe groups defined using established criteria. The second cohort was based on cord blood mononuclear cell (CBMC) samples from 25 infants born preterm (30.4-34.1 weeks gestational age (wGA)) and 25 infants born term (37-40 wGA) from Tu Du Hospital, Ho Chi Minh City, Vietnam (Vietnam cohort). The aim was to explore differences in the immune response to RSV in preterm and term infants by comparing neutralising antibodies, immune profiles, cytokine response and immune gene signatures following ex vivo RSV stimulation. For the first results chapter, we analysed clinical data from hospitalised children with moderate versus severe RSV infection, as determined by the level of respiratory support received during their admission. In this chapter, we identified that young age (<2 months), parainfluenza virus type 3 (PIV3) co-detection and premature birth were significantly associated with severe RSV disease on multivariable analysis. The PIV3 co-detection as a predictor of severe disease is a unique finding that requires further investigation in the future. Additionally, we identified a reduced number of neutrophils in the peripheral blood of children with severe compared to those with moderate RSV disease, suggesting this may be associated with disease severity. In results chapter 2, we performed high-dimensional immune profiling on CBMCs to examine baseline differences between preterm and term infants. We identified reduced frequencies in classical monocytes, myeloid dendritic cells, gamma-delta T-cells and an increased frequency of regulatory T-cells (Treg) and transitional B-cells in preterm infants. Additionally, pro-inflammatory cytokines (IL-1b, IL-6 and IL-17A) and chemokines (IL-8, eotaxin, MIP-1a and MIP-1b) were reduced in the plasma of preterm infants. This suggested a reduced pro-inflammatory profile in preterm infants compared with term infants. In results chapter 3, we stimulated CBMBs with RSVA and RSVB to identify how their innate immune response differs. Stimulation of CBMCs with RSV resulted in similar cytokine and cellular profiles between preterm and term infants. The level of maternally derived RSV neutralising antibodies was also similar. However, key differences in their transcriptome were observed. Genes involved in pro-inflammation, immune regulation and cytotoxicity were expressed at higher levels in term infants compared to preterm infants (CSF2, IL-1b, IL-10, GZMB, SOCS1, SOCS3). Additionally, weighted gene co-expression network analysis (WGCNA) identified a unique module related to the pro-inflammatory response and immune regulation in term infants but not preterm infants. These results suggest that preterm infants may be less able to regulate the inflammatory response to RSV, thereby increasing their risk of severe RSV disease. In summary, this thesis found that premature birth was significantly associated with severe RSV infections which supports previous observations. This thesis conducted an in-depth look at severe RSV disease, immune differences in preterm and term infants and further explores these differences in cord blood in an RSV specific manner. Specifically, it was found that preterm infants have a reduced capacity to produce pro-inflammatory responses and for immune regulation in response to RSV. These results are crucial in our understanding of preterm susceptibility to severe RSV disease and will aid the development of strategies to reduce the burden of disease in this highly vulnerable group.
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ItemNo Preview AvailableHealthcare utilisation and comparative effectiveness of diagnostic strategies for the emergency department management of paediatric head injury in Australia( 2022)Head injuries are the most common cause of trauma-related emergency department (ED) presentations in children globally. There is limited information on ED presentations for paediatric head injuries in Australia. Traumatic brain injuries (TBIs) are associated with significant injury-related disabilities in children with substantial health and economic burden on families. Little is known about the healthcare utilisation and acute care costs for children who present to EDs with TBIs. This thesis aims to examine the health economic impact of paediatric head injuries in Australia. The first study aimed to estimate the trend of ED presentations for paediatric head injuries and determine the patient and population-level acute care costs. The patient-level costs were estimated for 17,841 Australian children enrolled in the prospective, multicentre Australasian Paediatric Head Injury Study (APHIRST) between 2011 and 2014. The population-level analysis utilised data on ED presentations for the financial years (FYs) 2014 to 2018 reported by the Independent Hospital Pricing Authority. Head injuries accounted for approximately 7% of the 2 million annual ED presentations for children in Australia, with acute care costs of AUD $154 million and a 13% increase over five years. While falls were associated with the highest total acute costs, transportation-related injuries from high speed were associated with the highest mean patient-level costs. The following two studies aimed to estimate the acute care costs of head injuries for the Australian cohort in APHIRST. Additionally, the combined effect of TBI severity with injury mechanisms on the total costs to the health system and mean patient-level costs were examined. Mild TBIs from falls, followed by moderate and severe TBIs from transportation-related injuries, accounted for the highest acute costs at the health system level. Transportation-related injuries across all TBI severity levels, particularly high-speed and pedestrians hit by motor vehicles, were associated with significantly higher patient-level costs compared to other mechanisms. Since sports activities are commonly associated with TBIs, the impact of TBI severity on sports-related head injuries was evaluated for children between 5 and 18 years. Mild TBIs from bicycle riding and severe TBIs from motorcycle riding were associated with the highest costs to the health system. The highest mean patient-level costs were motorcycle riding for boys and horse riding for girls. The last two studies examined resource utilisation for the diagnosis of TBIs in children with minor head trauma enrolled in APHIRST. While cranial computed tomography (CT) scans are the standard modality to diagnose TBIs with structural injury to the brain, the associated ionising radiation has increased lifetime risks of lethal malignancies. Planned observation in the ED is recommended as an alternative to obtaining an immediate CT scan to allow for symptom resolution or progression. Planned ED observation was an effective and cost-effective diagnostic management strategy to optimise CT use for most children with minor head trauma. In conclusion, this research informs on the economic impact of paediatric TBIs and has implications for safe sports participation and targeted injury prevention programmes. The cost-effectiveness evidence provides a system-level measure of comparative efficiency for resource allocation and optimisation.
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ItemEpigenetic dysregulation underpinning immune cell dysfunction in food allergy( 2022)Food allergy is a major health concern, particularly in Australia, where some of the highest prevalence has been recorded. Although food allergy is most common in younger age groups (between years 0 to 4), most of these allergies resolve within the first few years of life. However, allergies to peanuts and tree nuts tend to be lifelong, and are more likely to cause severe allergic reactions such as anaphylaxis. These severe allergic reactions occur most frequently in the adolescent age group, the highest risk group for fatalities from such reactions. Moreover, up to 30 percent of food allergic adolescents suffer from multiple food allergies, and there is no current effective treatment for this variable phenotype. Rates of food allergy have risen rapidly in the last few decades, at a pace that cannot be explained by genetics alone. While there is a genetic predisposition in food allergy, numerous studies have shown correlations between specific lifestyle and early life factors and food allergy development. Therefore, the origins of food allergy are multi-factorial, and the study of epigenetic factors may allow us to establish the interface between the role of these environmental exposures and our genomes. Indeed, past studies have shown that immune cells from food allergic individuals show aberrant phenotypes even in the absence of allergens, thereby indicating that these immune cells are fundamentally wired to respond differently upon non-specific activation, and these differences have been linked to epigenetic variation. This thesis aimed to identify immune pathways implicated in adolescent food allergy, using samples from individuals enrolled in the SchoolNuts study, a study profiling the prevalence of food allergy in adolescents aged 10 to 14 years in Melbourne, Australia. This was achieved in three parts, first by establishing the baseline immune response to activation in CD4 T cells from non-allergic adolescents. To do this, we compared the DNA methylation (epigenomic) profiles (DNAme) of purified naive CD4 (nCD4) T cells at quiescence (72 hour culture in media alone) or following activation (72hour culture in media with antiCD3 and antiCD28 beads) in healthy infants (aged 12 months, n 18) and adolescents (aged 10 to 15 years, n 15); using the Infinium MethylationEPIC array (EPIC array). This data was then integrated with transcriptomic (bulk RNAseq) and cytokine profiles obtained from culture media from the same samples (quiescent and activated), and publicly available naive CD4 open chromatin (ATACseq) signatures from unrelated infants. By showing that T cell activation induced widespread changes in DNA methylation, I revealed that T cell activation is intrinsically linked to DNA methylation reprogramming. Multi-omic data analysis of transcriptomic and open chromatin datasets validated these findings by showing that this level of reprogramming was reflected in gene expression patterns and altered chromatin conformation, with upregulated gene expression and increased DNA accessibility. While I found similar responses to activation in nCD4T cells from infants and adolescents, there were selected genomic sites showing age specific methylation patterns following activation. Many of these CpG sites were located within the T cell receptor gene locus, suggesting that age influences activation-associated remodelling of DNA methylation at this region. Analysis of cytokine production of these cells at quiescence and following activation indicated enhanced production of IFNgamma, IL2 and IL10 in adolescents, relative to infants. Correlation of these cytokine levels, and DNA methylation level of probes from the EPIC array located near genes encoding for them, highlighted probes (cg01720533, cg05646531, cg14284394, cg16247264) potentially associated with this altered response. I then characterised the epigenomes of naive CD4 T cells from adolescents with food allergy, by generating genome-wide DNAme data in purified nCD4 T cells at quiescence and following activation in adolescents (aged 10 to 15 years old) with peanut allergy (peanut only or peanut and one or more additional food allergy) (FA, n 29), and age-matched non-food allergic controls (NA, n 18). As with the previous study, I also analysed transcriptome-wide gene expression and cytokine production in these cells following activation. I showed that adolescents with FA exhibit unique DNA methylation signatures at quiescence and post-activation at key genes involved in Th1 and Th2 differentiation (RUNX3, RXRA, NFKB1A, IL4R). Combined analysis of DNA methylation, transcriptomic data and cytokine output in the same samples identified an attenuated interferon response in nCD4T cells from FA individuals following activation, with decreased expression of several interferon genes, including IFNgamma and a DMR at a key downstream gene, BST2. The third part of this thesis involved performing the first combined epigenetic and transcriptomic analysis of purified CD19 B cells from adolescents with FA, comparing single-food allergic (peanut only) (SA; n 10), multi-food allergic (peanut and one or more other food) (MA; n 7) and non-allergic (controls) (NA; n 9) individuals. Using bulk RNAseq data and genome wide DNA methylation data generated using the EPIC array, I identified 116 differentially expressed genes (DEGs) distinguishing B cells of FA (both SA and MA) adolescents from NA controls. Additionally, I showed higher methylation at PM20D1, a key regulator of several inflammatory processes, in FA individuals. This analysis also identified, for the first time, distinct epigenetic variation specific to the MA phenotype, which included differential methylation at a number of S100 genes (S100A1, S100A13, S100A14), which are involved in pathways such as inflammation, energy metabolism and cell cycle regulation. This thesis is the first to characterise nCD4 T cell responses across multiple age groups and highlight the levels of epigenetic regulation involved in T cell activation, which marks the first step in T cell differentiation to a range of downstream lineages, such as Th1 and Th2, previously implicated in FA. In doing so, this presents important findings in this field of research by identifying potential timepoints for intervention. Moreover, characterisation of age-specific responses to activation deepens our understanding of immune responses within each age group, which will help us determine timings for interventions relating to age-related decline in immune function. My thesis also presents novel findings in the field of adolescent food allergy, by showing epigenetic dysregulation at several genes implicated in T cell differentiation, as well as at a gene encoding an inflammatory regulator in B cells. Collectively, these results indicate that the immune dysfunction observed in adolescents with FA is likely mediated by epigenetic variations across multiple cell types, potentially in an age-specific manner. Moreover, I identified common epigenetic signature distinguishing MA adolescents from SA adolescents; an important finding given the heterogeneity of this MA clinical group, and point the way forward towards exploring targets for further research, which may allow us to identify potential therapeutic approaches to address peanut allergy in adolescents, and work towards establishing a molecular therapy for the multi food allergic phenotype.