Paediatrics (RCH) - Theses
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Item4.7 Tesla magnetic resonance imaging and spectroscopy of infants with neonatal encephalopathy(University of Melbourne, 2007)
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ItemSafety of Bacille Calmette-Guérin vaccination and revaccination( 2022-12)Bacille Calmette-Guerin (BCG) vaccine, derived from live-attenuated Mycobacterium bovis, is commonly given as a single dose in infancy to protect against tuberculosis (TB) in high TB-prevalence settings. There is growing interest in BCG revaccination for the protection of adolescents and adults against TB. Increasing recognition of potential novel applications for BCG to protect against diseases other than TB also mean that BCG vaccination (and revaccination) may become increasingly used in broader age groups and settings. For these reasons, a more comprehensive and detailed understanding of the safety of BCG vaccination and revaccination, particularly in adults, is necessary to help inform public health policy. My PhD research aimed to evaluate the incidence and predictors of reactions (normal, accelerated and adverse) to BCG vaccination, the impact of revaccination on BCG reactions, and the clinical management of adverse reactions to the vaccine. These aims were investigated using active safety surveillance of BCG vaccination reactions within an international randomised controlled trial of nearly 7000 adult participants – the BRACE Trial (BCG vaccination to reduce the impact of COVID-19 in healthcare workers), as well as through two systematic reviews and a meta-analysis of BCG vaccination safety in children. My thesis comprises three sections characterising reactions following BCG vaccination and revaccination: (i) the normal reaction; (ii) accelerated reactions; and (iii) adverse events. The first section reports a cohort study to assess the prevalence, factors influencing formation and vaccinee perception of BCG scarring (normal BCG reaction) following vaccination and revaccination in adults. The second section includes a systematic review and meta-analysis to investigate the clinical significance of an accelerated BCG reaction in children, reporting an association with prior mycobacterial exposure. A cohort study is then presented that evaluated the incidence and clinical implications of an accelerated BCG reaction in asymptomatic adults in low and high TB-prevalence settings. The third section includes a systematic review of the management strategies for BCG-associated lymphadenitis and injection site abscess in children. This is followed by a cohort study to assess the incidence and risk factors (host immune- and vaccination-related) for BCG-associated lymphadenitis and injection site abscess in adults. An additional cohort study in adults assesses the safety of co-administration of BCG with the influenza vaccine. The overall incidence of local adverse events and serious adverse events in the BRACE trial is then described, as well as the impact of previous BCG vaccination on local injection site reactions. Overall, BCG revaccination in adults was associated with increased frequency of normal (scar), accelerated and local adverse reactions (injection site abscess and lymphadenitis), compared to receiving BCG for the first time. Younger age was also associated with increased frequency of normal and local adverse reactions. Similarly, female sex was associated with an increased frequency of normal reactions and lymphadenitis following BCG vaccination, as well as increased frequency of accelerated BCG reactions. Latent TB infection was found to predispose to an accelerated BCG reaction, but was not associated with injection site abscess. Nonetheless, regardless of prior BCG vaccination status, age or sex, the majority of reactions were mild in nature, localised to the injection site and resolved without intervention. In summary, the studies in my thesis indicate that BCG vaccination and revaccination have an acceptable safety profile in children and adults.
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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.