Paediatrics (RCH) - Theses
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ItemModelling Inherited Kidney Diseases with Kidney Organoids Derived by Directed Differentiation of Patient Induced Pluripotent Stem Cells( 2019)Genetic kidney diseases are a heterogeneous group of disorders with varying phenotypes dependent on the affected nephron segment. Next generation sequencing has increased our appreciation of the breadth of gene variants associated with these diseases. It has also identified large numbers of variants of unknown significance (VUS), which require functional genomic validation. There is an unmet need for novel therapies for genetic kidney diseases as most invariably progress to dialysis or transplantation without any form of targeted treatment. Laboratory based research of genetic kidney disease requires the recapitulation of a disease phenotype in animal and/or in vitro cellular disease models. Interspecies variation in anatomy, physiology and gene function limits the translation of animal models to human disease and clinical care. Classical two dimensional cell cultures lack the complexity and intercellular cross-talk of in vivo three dimensional tissue. Kidney organoids are three dimensional, miniature, multicellular, human, in vitro micro-tissues, offering distinct disease modelling advantages over other models. Furthermore, kidney organoids can be regenerated from induced pluripotent stem cells (iPSC) reprogrammed from patients with genetic kidney disease, potentially providing outcomes with personalised clinical relevance. As a novel platform, the capabilities and limitations of kidney organoids as disease models are not well understood. By differentiating and characterising kidney organoids from the iPSC of patients with inherited kidney diseases, this thesis aims to explore the application of kidney organoids to disease modelling. As proof of concept, kidney organoids were first generated from iPSC reprogrammed from a patient with compound heterozygous variants in IFT140, an already validated nephronophthisis (NPHP) genotype. An isogenic control was generated by precision CRISPR-Cas9 gene editing. In this project, differential primary ciliary morphology within organoid tubules and transcriptional profiling of organoid epithelium validated the ability of the organoids to model genetic disease. Attempts were then made to validate novel, candidate variants for other pedigrees with unresolved trio whole exome sequencing. In a proband with clinically suspected NPHP, DNAH5 was selected as a candidate gene, despite previously association with a motile ciliary phenotype. In this project, kidney organoids were unable to validate the patient variant as pathogenic. In addition, a number of lessons were learned regarding the necessary variant curation process prior to making a commitment to modelling with kidney organoids. In the final chapter, kidney organoids validated a novel genotype for the glomerular disease steroid resistant nephrotic syndrome, via international collaboration with the laboratory of Prof Friedhelm Hildebrandt. Glomeruli within kidney organoids differentiated from iPSC expressing a patient-derived, homozygous variant in NOS1AP, displayed aberrant development, increased podocyte apoptosis and reduced expression of PAR polarity proteins. Together these projects demonstrate the strengths and challenges of using kidney organoids as models of inherited renal disease. Kidney organoids stand to complement animal and 2D unicellular disease models rather than replace them. We proposed that patient-derived kidney organoids are best placed to model paediatric onset kidney diseases with the future potential of providing personalised therapeutic screening.
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ItemInvestigating the biology of paediatric T cell acute lymphoblastic leukaemia to facilitate more effective individualised therapy( 2019)Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.
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ItemEnvironment versus genetics in the development of health-related problems in childhood obesity( 2017)Introduction: Childhood obesity is a major health concern affecting a quarter of all Australian youth. Obese youth are at risk of multi-systemic obesity-related comorbidities (ORC), such as Prediabetes and Type 2 Diabetes Mellitus (T2DM), Hypertension, Hyperlipidaemia, Non-alcoholic fatty liver disease (NAFLD), Polycystic ovarian syndrome (PCOS), Obstructive sleep apnoea (OSA), mental health and orthopaedic issues. However, within the obese population, the severity of obesity is a poor predictor of these comorbidities. As such, current guidelines advise screening obese youth for all comorbidities, which is both time consuming and costly. ORC, as with obesity itself, are complex polygenic disorders involving interaction between genetic and environmental risk factors. Furthermore, many of these interactions are likely mediated by epigenetic variation, linking environmental exposures with genetic predisposition. Therefore, clarification of the role of environmental exposures, genetic risk factors and epigenetic profile in obese youth may provide valuable insight into determining which obese youth are at risk of developing ORC. The aim of this study was to characterise and investigate the associations between environmental, genetic and epigenetic factors with obesity-related comorbidities in obese youth. This information obtained will allow clinicians to better predict and profile the comorbidity burden in obese youth for targeting interventions to those most likely to benefit. Materials and Methods: The Childhood Overweight BioRepository of Australia collected comprehensive clinical and anthropometric data, as well as blood samples for genetic, metabolic and hormonal profile testing from patients (age 1 to 18 years) attending the Weight Management Clinic at the Royal Children’s Hospital, Melbourne. Anthropometric measures included standard measurements of weight, height and waist circumference (converted into BMI z-score (BMI-Z) and waist-height ratio for analyses) as well as total percentage body fat (%FM) and percentage truncal body fat (%truncal) using bioimpedance. Dietary intake was assessed using the Australian Child and Adolescent Eating Survey and physical activity was measured objectively using accelerometry. Genetic typing and epigenetic profiling of single nucleotide polymorphisms (SNP) and measures of DNA methylation were performed using the SEQUENOM MassARRAY platform. Results: 349 participants were included in the study. Both metabolic and non-metabolic ORC are common in obese youth. The most commonly used measure of adiposity, BMI-Z, was the anthropometric marker least associated with ORC, while important ORCs such as T2DM and hyperlipidaemia were not significantly associated with any of the anthropometric measures. The presence of two or more significantly elevated anthropometric markers was associated with the presence of a higher number of ORCs. Almost all obese youth reported poor vegetable intake and high sedentary behaviour (TV-watching and total media) time. Although initial analyses observed associations between sedentary behaviour (TV-watching and total media use time) with prediabetes and hyperlipidaemia, unhealthy food options with mental health issues, and high fruit intake with NAFLD, these associations lost significance after correction for multiple testing by false discovery procedure (FDR). However, low vitamin D levels were significantly associated with hypertension, independent of adiposity and season. The Fat Mass and Obesity (FTO) gene has the largest influence on adiposity amongst obesity-risk genes to date and has been associated with ORCs. In our cohort, the FTO risk allele was associated with poorer longitudinal weight outcomes in pre-pubertal children, but not pubertal adolescents. The FTO SNP however was not associated with any ORCs. We also examined the association between the top 22 T2DM risk SNPs identified from previous studies and Prediabetes or T2DM status. The SLC30A8 rs13266634 SNP appeared to be protective of prediabetes/T2DM status in unadjusted analysis but lost significant on FDR testing. The addition of T2DM SNPs profile to TV-watching time improved the predictive model for Prediabetes/T2DM status. We were not able to replicate the previously reported association of FTO CpG hypomethylation with prediabetes/T2DM in obese children and adolescents. However FTO hypomethylation was seen in obese youth with NAFLD, independent of age, sex and total body adiposity. FTO methylation was independent of FTO expression, which was not associated with any ORC. Conclusion: Anthropometry is not associated with comorbidities in childhood obesity. Environmental factors do not appear to be associated with comorbidities. Low vitamin D level is associated with hypertension. Genetic factors impact on weight gain but not comorbidities, and epigenetic factors may contribute to NAFLD. Combining these environmental and genetic elements with established clinical risk factors in the assessment of each individual ORC may assist in enhancing current clinical models of care for obese children and adolescents.
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ItemA tool for risk profiling and accurate prognostication in paediatric glioma integrating clinical features with epigenetics: it is time to move on from the binary classification( 2016)Paediatric glioma, the most common group of brain tumours in children, encompasses a wide range of entities with highly variable prognoses. Gliomas are grouped by histopathological features into high and low grade glioma but this classification does not take into account many established and emerging risk factors in this disease. Research into the molecular features of these lesions has shown that histology does not always correlate with biology and, where they differ, molecular features are usually superior at predicting outcome. Risk classifications have been developed for other paediatric malignancies which combine clinical, radiological, pathological and molecular factors to predict disease risk and prognosis. A comprehensive risk classification has not been published for paediatric glioma despite many risk factors being established in this disease. Using a clinical cohort of all paediatric glioma treated at a single institution (Royal Children’s Hospital, Melbourne, Australia) over an 18 year period a database was developed incorporating clinical, radiological, pathological and treatment factors. Where sufficient tumour tissue was available genome-wide methylation analysis was performed. The results of this were processed and evaluated by an established cluster analysis algorithm. Breaking the cohort into clinically appropriate subgroups, risk factors for disease progression and death were determined and prognosis estimated for distinct groups. A tool for robust risk profiling and prognostication was established with 5 main risk groups and 10 subgroups. Patients in the very low risk group have a predicted 100% overall survival and the majority require no treatment. In contrast there was a group of patients with 100% mortality within a short space of time where attempts at curative therapy are futile and may be deleterious to quality of life. The intermediate risk group contained a number of entities with a prognosis genuinely falling between that of low grade glioma and that of high grade glioma. Histopathological diagnosis retained prognostic importance for some, but not all, groups and methylation analysis was shown to have a significant role to play, particularly in high grade lesions or where diagnosis was unclear. This is likely to become part of routine care in the future and its place is strongly supported by the analysis presented here. Risk profiling is possible in paediatric glioma with far more accuracy than the current binary classification provides. A combination of established clinical factors and emerging molecular features provides an accurate and nuanced assessment of risk and prognosis.
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ItemAppearance-altering facial surgery on children: An empirically informed ethical analysis( 2015)Using empirically informed ethical analysis, this thesis investigates the ethics of appearance-altering facial surgeries performed on children. These surgeries have attracted controversy in the media and ethics literature and are the subject of legislation and professional guidelines, which leave much to individual practitioners’ discretion. Despite their contentious nature, very little is known about surgeons’ practices and decision-making processes regarding these surgeries. This thesis is ‘empirically informed’ in two different ways: (1) by existing empirical data on psychosocial outcomes of children with facial differences and children who have undergone appearance-altering facial surgeries and (2) by new interview data from 22 surgeons who encounter requests for these surgeries. Using reflective equilibrium as my method of ethical analysis, these two kinds of empirical data are combined with established paediatric bioethical principles and concepts, including the zone of parental discretion and children’s assent and dissent, to arrive at a comprehensive position on the ethics of performing these surgeries. Using reflective equilibrium, I found some aspects of surgeons’ decision-making processes and practices diverged from existing ethical understandings about the relative roles of children and parents in medical decisions. One such area related to the ethical weight surgeons attach to children’s refusals. While most ethicists in the literature advocate including children in decisions, they do not necessarily recommend giving them decision-making authority. In contrast, most surgeons tended to give children a larger role in decisions about appearance-altering facial surgeries, viewing them as ultimate decision makers. In this thesis, I make normative claims about the relative levels of ethical weight surgeons should place on children’s and parents’ wishes when making decisions about these surgeries. Using the established ethical framework of the zone of parental discretion and related ethical concepts of harm and benefit, I claim that several morally relevant differences exist between these surgeries and other medical procedures requested for children. These include their uncertain risk-benefit ratio, their elective nature, uncertainty as to how the child will later perceive their facial difference and the acceptability of surgery, and the existence of alternative, less risky and invasive psychosocial interventions for alleviating appearance-related psychosocial harm. I argue these differences warrant placing greater (even absolute) ethical weight on children’s wishes, especially when children refuse these surgeries, and comparably less ethical weight on parents’ wishes, when making these decisions. Although my ethical position is similar to most of the surgeons’ judgments, I argue it would be worthwhile for surgeons to know about and use ethical principles and concepts more explicitly when making decisions and communicating with children and/or their parents. I also claim that surgeons ought to make more of a considered effort to ascertain children’s actual wishes and inform parents and/or children about non-surgical options for alleviating appearance-related psychosocial distress, and be taught strategies for saying no and how to articulate their ethical reasoning. These findings have significant implications for clinical practice, raise questions for further ethical analysis and contribute to refinement of existing understandings of children’s assent and dissent and parents’ role as proxy decision makers for their children.
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ItemAnosmia in paediatric traumatic brain injury( 2015)Objective: Paediatric traumatic brain injury (TBI) is a leading cause of childhood disability, and is associated with significant physical, sensory, psychosocial and neuropsychological sequelae. While our knowledge of outcomes after paediatric TBI has expanded over the past 20-30 years little is known about olfactory function outcomes. This is despite the fact that olfactory dysfunction (OD) is known to be common after adult TBI, has poor prognosis for recovery and is associated with significant functional implications in health, safety, and activities of daily living. In adults with TBI, OD has been linked to injury variables including severity, site of impact, neuropathology and skull fracture and to deficits in executive function (EF), with OD suggested as a potential marker of EF deficits following TBI, though little is known about these relationships in paediatric TBI. The overall aim of this thesis was to address the dearth of research in paediatric OD. The study investigated the frequency with which OD occurs following paediatric TBI, and the relationship between OD and injury variables such as severity, fracture, and injury impact. The relationship between olfactory function and EF was examined and recovery of OD documented up to 18 months post injury. Overall, it was hypothesised that children with moderate/severe TBI would demonstrate poorer olfactory function than those with mild TBI and that children with TBI and OD would perform more poorly on measures of EF than those without OD. Method: Thirty-seven children aged 8-16 years with TBI were recruited to our prospective longitudinal study. Olfactory assessment using the University of Pennsylvania Smell Identification Test was conducted at 0-3 months (T1), 8 months (T2) and 18 months (T3) post injury. Children completed EF tests at T2 and parents completed ratings of behavioural EF at T2 and T3. Results: At T1 19% of participants demonstrated impaired olfaction, with 5% classified as anosmic. OD was significantly related to injury severity at T1, with those with moderate/severe TBI showing poorer olfactory function than those with mild TBI. Longitudinal follow-up indicated evidence of recovery in olfactory function at T2, however, only 16% of those with the most severe OD showed recovery to normal olfactory function, with the remainder demonstrating ongoing OD at T3. Occipital site of impact was a significant predictor of olfactory performance at T3. Children with OD showed significantly poorer performance on a single EF measure of fluency at T2 compared to those with normal olfaction. Acute olfactory function did not significantly predict EF outcomes at either 8 or 18 months post injury. Conclusions: OD appears to be relatively common after paediatric TBI, with limited prospects for recovery, consistent with previous adult research. Acute OD was significantly related to injury severity, though those children at greatest risk of poor later olfactory function were those with an occipital site of impact. Evidence for a link between OD and EF was limited and in particular, acute olfactory function did not appear to be an accurate predictor of later EF deficits. Our findings indicate the need for a focus on OD in clinical practice with routine screening and implementation of education and management strategies recommended. There is a need for further longitudinal research in larger cohorts to elucidate further the links between OD and injury variables, identify clinical predictors of OD, and investigate the functional implications for children and adolescents with TBI.
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ItemMicroRNA expression and genome-wide epigenetic analysis of paediatric acute myeloid leukaemia( 2014)Introduction: Paediatric Acute Myeloid Leukaemia (AML) is the third most prevalent cancer in Australian children under the age of 14. Chromosomal and genetic lesions are commonly found within specific subtypes of paediatric AML and these help to direct treatments and prognostic predictions. However, there are no common genetic lesions across subtypes of paediatric AML and subtype-associated genetic changes may fail to induce leukaemogenesis. DNA methylation is the most commonly studied molecular alteration in cancer. Strong evidence indicates that methylation variations exist within paediatric cancer known to modify gene expression. Similarly, microRNA are small non-coding RNA that also regulate gene expression and whose dysregulation within paediatric cancer is now beginning to be appreciated. Therefore altered methylation and miRNA expression may contribute to malignancy through activating oncogenes or inactivating tumour suppressor genes, similar to genetic mutations. Furthermore, both DNA methylation and microRNA expression represent viable epigenetic mechanisms for application to clinical biomarkers of disease diagnosis, prognosis and disease tracking. At the beginning of this study, limited interrogations of genome-scale DNA methylation and microRNA expression had been conducted within paediatric AML with appropriate non-leukaemic controls. Furthermore, there has been few extensive studies evaluating the utility of current epigenetic techniques on primary clinical specimens. Therefore I chose to study genome-wide DNA methylation and large-scale miRNA expression changes in paediatric AML, as well as interrogating techniques for using archived primary patient samples. Materials and Methods: Two approaches were used to interrogate large-scale DNA methylation and microRNA expression; TaqMan OpenArray Human MicroRNA Panel (Life Technologies) was used to interrogate 5 primary paediatric AML patients with matched controls; and Illumina Infinium HumanMethylation450 BeadChip array (HM450) was used to interrogate 18 primary AML alongside matched non-leukaemic samples. Validation of AML DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER with an expanded cohort of 28 patients, in conjunction with validation of microRNA expression utilising quantitative real-time PCR (qRT-PCR) and TaqMan singleplex assays on 32 primary paediatric AML, together with matched control groups. This study also developed extensive extraction techniques for the analysis of microRNA expression within a range of samples, including archived bone marrow aspirate smear slides. Results: Genome-scale DNA methylation disruptions were characterised within paediatric AML, and identified hundreds of genes associated with disease compared to matched control samples. Interrogation of a refined subset of target genes also identified gene expression alterations within these regions, which were further associated with patient disease onset and predicted outcome. Conversely, large-scale microRNA expression disruptions were characterised within paediatric AML, whereby a small number of reliable targets were identified. Such microRNA disruption was found to be associated with DNA methylation regulation on the microRNA gene, and could also be used as reliable biomarkers to predict disease onset and patient outcome in connection with identified patient cytogenetic abnormalities. Integration of data from genome-scale DNA methylation and combined gene and microRNA expression analysis, identified common epigenetic disruptions within paediatric AML affecting known tumour suppressor genes, cytoskeletal organisation, cellular proliferation and immune function. Conclusions: The findings of this study reveal that DNA methylation alterations within paediatric AML can associate with gene disruptions with the potential to initiate and perpetuate malignant phenotypes. Likewise, microRNA deregulation can establish widespread gene disruptions as a catalyst for leukaemogenesis. Interestingly we establish that combined deregulation of epigenetic mechanisms, and the occurrence of one epigenetic mechanism working to deregulate another, may be a common feature of paediatric AML. Lastly this study identifies targets and techniques to extend such studies into the clinic and provides attractive targets for therapeutic intervention.
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ItemLong-term psychosocial outcomes following childhood traumatic brain injury( 2014)Introduction: Childhood traumatic brain injury (CTBI) is one of the most common causes of mortality and impairments in children, impacting on the development of neuropsychological, social and psychological functioning. A disruption in these areas often results in long-term impairments with interpersonal relationships, participation in leisure and social activities and quality of life. Background: Available literature has shown that children and adolescents with a CTBI demonstrate greater social and psychological dysfunction than typically developing children and adolescents. They often show lower self-esteem, have fewer friends, express more maladaptive, aggressive and antisocial behaviour in a social environment and have poorer emotional and behavioural regulation. These symptoms may become more severe and develop into a clinical disorder, with children and adolescents with a CTBI having a 49% risk of developing any kind of psychiatric disorder compared to 13% risk for healthy developing children and adolescents. Most research in this field has investigated these outcomes up to 5 years post-injury, although less is known about the long-term outcomes after CTBI into adulthood. Aim: The aim of this thesis was to investigate the development of long-term psychosocial outcomes over a period of 16 years following CTBI. The participants were recruited from a prospective, longitudinal sample ascertained between 1993-1997 and aged 1-7 years at time of injury. Children were included in the study if there was sufficient information regarding their CTBI to classify illness severity, if they were able to speak English and complete the protocol. They were excluded in cases of a non-accidental, penetrating or previous brain injury or if there was evidence of a pre-existing neurological or developmental disorder. A healthy control group was also recruited during the same period, matched for age, gender and socio-economic status (SES) with the CTBI sample. Both groups were followed up several times, with this thesis utilising data from the acute, 6 month, 5 year and 16 year post injury time points. Results: The results showed that young adult survivors of CTBI reported more depressive, anxious and withdrawn symptoms then their healthy peers. Ratings on psychosocial measures rated by the young adults with CTBI were compared with the ratings of their significant other, with analyses indicating fair to excellent agreement on the obvious and noticeable behaviours, such as communication, and alcohol and drug use. In contrast, there was poor agreement on the more internalising symptoms such as depressed/anxious and withdrawn symptoms. With regard to the developmental trajectory of social skills, and internalising and externalising symptoms, the CTBI and control group did not differ. However, there was a trend for the CTBI group to be rated higher on internalising symptoms by the parent at every time point. Conclusion: In summary, young adults with a CTBI experience more internalising symptoms than their healthy peers, and are at higher risk of developing more severe symptoms and even clinical disorders. Appropriate screening by health professionals and early intervention should be considered to prevent the development of any significant social or psychological symptoms later in life.
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ItemCharacterisation of immune differences in the development of food allergy in infants( 2013)Food allergy often develops early in life and has a major impact on the quality of life of the infant. It is characterised by a dysregulation of the immune response in which there is increased production of Th2 cytokines, resulting in B cell class-switching and production of food specific-IgE antibodies. Sensitisation to food allergens indicates the production of food specific IgE (sIgE) antibodies, but is not a definite indicator of a clinically allergic reaction upon ingestion. Although some individuals who produce food specific-IgE antibodies will react on consumption of the food in question (food allergic), others are sensitised but can consume the food without any adverse consequences (food sensitised). The mechanisms underpinning why some food-sensitised children are tolerant and some are allergic have not been fully elucidated, and it is postulated that tolerance to food antigens arises from regulatory T cells (Tregs) induced in the periphery, and that a diminished capacity to produce these cells results in the development of food allergy. Furthermore, due to the rapid rise in rates of food allergy combined with the lag in training new allergists, allergy services are currently overwhelmed, with patients having limited access to oral food challenge (OFC) tests. Although OFC remain the gold standard for diagnosing food allergy, these are time consuming, costly, and associated with a risk of anaphylaxis. While 95% positive predictive values thresholds for sIgE can assist with identifying increased likelihood of allergy among those who are sensitised, there are no specific biological markers that differentiate between allergic and sensitised individuals. Therefore, improved correlates of allergy status are required. This thesis aimed to investigate the key immunological differences that distinguish between the clinical phenotypes of food sensitisation and food allergy in one-year old infants, using samples from the HealthNuts study (a population-based cohort study of 5,276 infants recruited at 12 month old infants). In particular the role of Treg cells in the breakdown of tolerance in food allergics was studied. We explored whether there were any immune differences in the blood which could be utilised as biomarkers to distinguish allergic subjects from sensitised tolerant subjects. We determined that food allergic infants have a reduced capacity to regenerate their pool of Foxp3+ Treg cells following in vivo allergen stimulation in the form of an OFC compared to food sensitised tolerant infants. We also found a reduced level of circulating plasma IL-10 to further support the notion that the development of food allergy is the result from the failure to develop oral tolerance to the food antigen. Furthermore, we established that there were immunological differences between egg and peanut allergy, including different Treg and plasma cytokine profiles. Infants with egg allergy had significantly higher plasma levels of both IL-13 and IL-12, and lower levels of IL-10 compared to infants with peanut allergy. Unlike the peanut allergic infants, the kinetics of activated Tregs from egg allergics remained unchanged throughout the course of the 6 days in culture following allergen exposure. Together with the plasma cytokine profiles, this suggests that the development of egg allergy may be the result of a ‘temporary’ immunological response. The symptoms of egg allergy are generally not as severe as peanut allergy, which has the highest rate of food related anaphylaxis. Therefore, suggesting that the immune mechanisms involved may be dependent on the type of food allergy. In terms of using plasma cytokines biological markers that differentiate between allergic and sensitised individuals, we found that key Th1 and Th2 cytokines were unable to separate the clinical phenotypes of food allergy. In order for the plasma cytokine work to be useful as a tool for diagnosing food allergy, more work is needed to develop and establish relevant cytokine profiles for the various characteristics of food allergy. An example of developing a model by combining other immunological data points was demonstrated in chapter 3, using both peanut component sIgE (Ara h2) and whole peanut sIgE along with peanut SPT results to determine an improved algorithm of diagnosing peanut allergy which would reduce the number of current peanut OFCs by up to 3-4 fold. In summary, the results described in this thesis outline several immunological differences between the three clinical phenotypes of food allergy examined, and further work into understanding the mechanisms will help differentiate these phenotypes for i) diagnostic purposes; and more importantly ii) treatment of allergic diseases.
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ItemRemote ischaemic preconditioning-evoked intracellular signalling pathways in cardiac maturation and disease( 2013)Background & Aims: Children with congenital heart defects are burdened with additional stress on the myocardium, which is exacerbated by cardiopulmonary bypass (CPB) and cardiac surgery. Ischaemic preconditioning (IPC) induced by brief, intermittent periods of ischaemia and reperfusion (IR) on the coronary vasculature can activate intrinsic protective mechanisms. However, remote IPC (RIPC) induced by inflating and deflating a standard blood-pressure cuff attached to a limb is a practical, non-invasive and clinically applicable model for protection against sustained ischaemic injury. RIPC regulates phosphorylation of key intracellular proteins that are recruited by IR and propagate signalling for metabolic control of the heart. Agonism of G-protein coupled receptors promotes protein signalling via key kinases such as Akt, p38MAPK, GSK3β, and HSP27 amongst other important effectors for cell survival against IR injury. However, most RIPC studies have focussed on adult myocardium. There is a dearth of such studies in immature myocardium. Thus, the aims of this Thesis were to: • test the efficacy of RIPC in a double-blind randomised trial in patients undergoing cardiac surgery for tetralogy of Fallot (ToF) and to measure key signalling protein kinases; • determine whether these signalling pathways are developed in murine neonatal hearts, and to compare their activation and ability to functionally recover after sublethal IR compared to adults; • determine the efficacy of RIPC in immature (4 weeks) and adult (12 weeks) hearts to functionally recover and express kinase signalling proteins after sublethal IR; • examine the effects of chronic hypoxia and fentanyl on immature cardiomyocytes on modulating kinase signalling proteins; • characterise the ability for advanced age (72 weeks) murine hearts to functionally recover and express kinase signalling proteins after sublethal IR. Methods: Limb RIPC was induced by four 5 min periods of inflation (ischaemia) and deflation (reperfusion) of a pressure cuff. IR occurred in ToF neonates during cardiac surgery utilising CPB, and in murine hearts during Langendorff-mode isolated heart perfusion. Resected right ventricular outflow tract myocardium from neonates and murine heart protein homogenates were assayed for protein expression by western immunoblotting. Effects of chronic hypoxia and fentanyl were studied in immature cardiomyocytes differentiated from murine P19 cells. Results: The major findings from this Thesis are: • remotely preconditioned ToF neonates did not have different expression of pro-survival signalling proteins relative to sham controls, which had a high proportion of phospho-kinase activation masking the effects of RIPC; • complex proteomic changes and a greater ability to functionally recover from sublethal IR were evident in immature murine hearts relative to the adult; • remotely preconditioned immature murine hearts had improved left ventricular (LV) functional recovery and an increase in the total available pool of protein kinases available for phosphorylation; • immature cardiomyocytes exposed to chronic hypoxia had an increase in total abundance of protein kinases available for phosphorylation; hypoxia blunted the effect of the opioid receptor agonist fentanyl; • advanced age hearts had impaired LV functional recovery, with a greater propensity for apoptosis and equitable levels of necrosis and autophagy, relative to adults. Conclusions: A protective role exists for RIPC in immature murine hearts exposed to IR despite undetectable protection in RIPC neonates undergoing ToF surgery. An extrapolation of these findings indicates confounders related to cyanotic disease progression (adaptation to chronic hypoxia) and pharmacological intervention may limit protection by RIPC in the surgical setting.