Paediatrics (RCH) - Theses
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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.
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ItemThe epidemiology and risk factors of anaphylaxis and food-induced anaphylaxis worldwide( 2019)Anaphylaxis is a severe allergic reaction that is rapid in onset and may cause death. There are increasing reports from individual countries and regions on anaphylaxis prevalence or incidence; however, there has been no systematic summary of the worldwide evidence among the paediatric population. An increasing rate of hospital admissions for food-induced anaphylaxis was observed in Australia from 1993 to 2012, especially among young children. Although rising rates of anaphylaxis have also been reported in other western countries, little is known about the time trends in Asian regions. Time trends of adrenaline auto-injectors (AAI) prescription is a good supplement surrogate for the time trends of anaphylaxis risk in the community. Some studies reported time trends of AAI prescription internationally, such as USA, UK and Canada; however, there is little information on the time trends of AAI prescription or dispensing in Australia in recent years. Previous international studies have reported that ethnicity is associated with the risk of anaphylaxis. Food allergy was found to be more common in children born in Australia with Asian parents than children born in Australia with Caucasian parents. However, it is not known whether ethnicity is also a risk factor for the development of anaphylaxis and food-induced anaphylaxis in Australia. Food allergy is an important cause of anaphylaxis. People with food allergy have a high risk of anaphylaxis, but not all of them will have an anaphylactic reaction. It is hence crucial to know the risk factors of having anaphylactic reactions in the food allergic population. Few studies have examined risk factors for food-induced anaphylaxis in food-allergic children. The characteristics of children with food allergy who are more likely to experience anaphylaxis are unknown. Therefore, this thesis aims to describe the worldwide incidence and prevalence of anaphylaxis and identify risk factors for anaphylaxis and food-induced anaphylaxis in both the general population and the food-allergic population. Firstly, I conducted a systematic review to describe the incidence and prevalence of anaphylaxis in children worldwide. I found a high heterogeneity between studies which limited the interpretation of an overall combined incidence and prevalence. I found increasing time trends of all-cause anaphylaxis and food-induced anaphylaxis in children from included studies and studies in developing areas were underrepresented. By using hospital admission data for anaphylaxis from the Hospital Authority of Hong Kong, I reported increasing time trends of both all-cause anaphylaxis and food-induced anaphylaxis in the paediatric population in Hong Kong between 2001 and 2015. By analysing AAI dispensing data from the Australian Pharmaceutical Benefits Scheme (PBS) database, I found an increasing incidence rate of patients with AAI in Australia from 2005 to 2014. Different trends were reported by sex, age and state. I found a shift towards more AAI prescriptions being provided by general practitioners (GPs) rather than specialists in most regions in Australia. By using the data from the School Entrant Health Questionnaire in Victoria, Australia, I investigated the risk factors of anaphylaxis in the general population. I found an association between Asian ethnicity and anaphylaxis risk in children living in Australia and identified the high-risk group (Australian-born children with Asian-born mothers) for anaphylaxis. Lastly, I used data from the HealthNuts study to explore the frequency and risk factors of anaphylaxis in food allergic children from a community setting. I found a high frequency of experiencing anaphylactic reactions (11.5%) in the preceding 12 months in children with food allergy. In summary, the results presented in this thesis have provided further knowledge on the epidemiology of anaphylaxis and food-induced anaphylaxis in the general population and identified important predictors of anaphylaxis in the general population and the food allergic population. The identification of these essential predictors has important implications for the management of anaphylaxis and will improve our understanding of the development of anaphylaxis.
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ItemEnvironmental and genetic risk factors for food allergy in children with Asian ancestry( 2019)Epidemiological and genetic studies on food allergy to date have focused primarily on the Caucasian population. This is despite emerging evidence that food allergy appears to be rising in Asian countries, alongside the increasing Westernisation and urbanisation in these countries. Even less is known about Asian migrants living in Western countries. A population-based study on food allergy found the risk of food allergy to be three times higher in infants of East Asian ancestry than infants of Caucasian ancestry. It is thought that infants growing up in Australia are exposed to environmental agents that have a differential effect on the immune system depending on their genetic background. However, the influence of specific genetic and environmental risk factors is currently unknown. It is also unclear if the increased risk of food allergy translates to higher risk of other allergic diseases later in childhood. The main objective of this thesis is to identify specific environmental and genetic factors on the risk of food allergy in the Asian population living in Australia. This thesis aims to quantify the prevalence of, and identify risk factors for food allergy and allergic comorbidities in the Asian population. An additional aim is to identify genetic variants that increase the risk of food allergy in the East Asian population and compare this to the Caucasian population living in Australia. This thesis primarily used data and samples from the longitudinal HealthNuts study where 5,276 1-year-old infants attending council run vaccination sessions across Melbourne were recruited. Skin prick tests to a range of food were carried out on infants and those with a wheal size less than 1 mm underwent an oral food challenge. The 1-year-old infants were followed up again at age 6 years and data collected at this follow-up visit were also used for analyses in this thesis. Additionally, data collected from a Growing Up in Singapore Towards Healthy Outcomes (GUSTO) birth cohort was also used to compare the risk factors and prevalence of allergic diseases between East Asians living in Melbourne and East Asians living in Singapore. This thesis reports that Australian-born children with East Asian parents have a higher burden of allergic rhinitis, eczema and aeroallergen sensitisation but not asthma, in the first six years of life compared to children of Caucasian ancestry. Moreover, children with IgE-mediated food allergy and eczema in infancy were 3 times more likely to have asthma and 2 times more likely to have allergic rhinitis at age 6 years, irrespective of ancestry. Additionally, East Asian children living in Melbourne have a higher risk of food allergy compared to East Asian children living in Singapore. Despite delayed introduction of allergens into the diet compared to the Asian population in Melbourne, Asian children in Singapore had less food allergy. While eczema rates were lower in Singapore than in Melbourne, early onset eczema was associated with an increased risk of food allergy in both Singapore and Melbourne. In terms of genetic risk factors, a systematic review conducted as part of this thesis identified several genes of interest known to be involved in immune regulation, cell function and epidermal barrier function. However, studies were of varied quality and the reproducibility of findings for the same SNPs were minimal. Some of the highly re-producible genes identified from the literature include HLA, FLG and IL13. Additionally, there was also a paucity of studies carried out in the Asian population that were able to elucidate underlying mechanisms for the differential food allergy risks observed in the population. This highlighted the need for genetic studies focused in this population. This thesis found that HLA rs7192 minor allele was associated with increased risk of peanut allergy in the Caucasian population but not East Asian population. Among sensitised children with two East Asian born parents, those with the minor allele for rs231735, rs231804 or rs11571291 (all CTLA4) have a reduced risk of egg allergy. The findings of this thesis identify Asian children living in Australia as a high risk allergic group not just in infancy but throughout early childhood. As a multifactorial disease, both environmental and genetic factors are known to contribute to the pathogenesis of food allergy. Therefore, it may be that the increased risk of food allergy observed in genetically predisposed East Asian children living in Melbourne unmasked upon exposure to environmental risk factors.
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ItemExploring the prevalence, natural history and adverse events related to tree nut allergy( 2018)Peanut and tree nut allergies are the most commonly reported trigger of food induced anaphylaxis and fatalities. Whilst most childhood allergies are outgrown by school age, peanut and tree nut allergy resolution has been reported to be as low as 10%. Despite the potential severity and lifelong nature of both peanut and tree nut allergy much of the literature to date has focused on peanut allergy epidemiology, and tree nut allergy prevalence, natural history while adverse events have been under studied. There is very limited data on the prevalence of allergies to the individual tree nuts nor the factors related to development of tree nut allergy. Therefore, this thesis explores several key areas regarding tree nut allergy including the prevalence, elements of the natural history and development of tree nut allergy, as well as the frequency of adverse food reactions to tree nuts. This PhD has utilised data from the population-based HealthNuts and SchoolNuts studies and the Royal Children’s Hospital allergy clinic populations all based in Melbourne, Australia. In a systematic review I published, I found limited tree nut allergy prevalence estimates based on challenge-confirmed outcomes with current estimates less than 2%, while probable tree nut allergy prevalence ranged from 0.05 to 4.9%. Prevalence of individual tree nut allergies varied significantly by region with hazelnut the most common tree nut allergy in Europe, walnut and cashew in the USA and Brazil nut, almond and walnut most commonly reported in the UK. There was no challenge-confirmed Australian tree nut allergy data. Among 6 year old children in the HealthNuts study, 3.3% had challenge confirmed tree nut allergy and 2.6% of 10 to 14 year olds self-reported one or more tree nut allergies in the SchoolNuts study. Cashew was the most common tree nut allergy both at 6 and 10 to 14 years of age. Among those with peanut or egg allergy at 12 months of age in the HealthNuts study, sensitisation rates to tree nut were as high as 48%, with 39% of those tree nut sensitised at 12 months tree nut allergic at 6 years of age. Confirming other reports of adverse food reactions, the work conducted as part of this PhD has found peanut and tree nut the most common triggers of adverse food reactions in the past year for children aged 10 to 14 years, with cashew the most common individual tree nut trigger reported. Finally, a cashew SPT wheal size of 10mm was found to have 95% PPV to challenge confirmed cashew allergy using the population based HealthNuts and SchoolNuts cohorts. In summary, the results presented in this thesis have reported the first challenge confirmed tree nut allergy prevalence rates in Australia and have highlighted one of the highest reported tree nut allergy rates in the world to date, with cashew the most common tree nut allergy. With up to half of those with food allergies already sensitised to tree nuts as early as one year of age, improved methods for identifying and targeting children at highest risk of tree nut allergy along with development of early prevention strategies are desperately needed, with cashew allergy a priority tree nut in Australia.
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ItemPredictors and phenotypes of IgE-mediated food allergy and the development of tolerance in children( 2014)The prevalence of IgE-mediated food allergies has increased dramatically in recent decades. In clinical cohorts, food allergy resolves in the majority of children with egg allergy, but only 20% of those with peanut allergy. The relevance of these findings to the wider population is unknown since the natural history of challenge-confirmed food allergy has not been assessed in a population-based setting. Finally, the role that food allergy plays in the atopic march is yet to be fully elucidated. Although food allergy, eczema and wheeze commonly co-occur in infancy, the interplay between these conditions is not well understood. It is unclear whether different phenotypes of allergy and food allergy exist in infancy, and what the consequences of these are. The increase in food allergy has placed an unprecedented demand on the Australian health system with waiting times to see an allergy specialist now exceeding 12 months. Oral food challenges (OFC) remain the gold standard of food allergy diagnosis, however they are resource consuming and potentially dangerous. Tests of sensitisation, the skin prick test (SPT) or serum specific-IgE (sIgE), offer a more accessible approach to assess the risk of food allergy, although a positive test only confirms sensitisation, not necessarily a clinical reaction. The utilisation of these tests can be improved by identifying decision points with 95% positive predictive value (PPV), reducing the need for OFC. However, the predictive value of SPT and sIgE has not been assessed in a population-based setting nor in a large number of infants, the age most likely to present for diagnosis of food allergy. Although SPT and sIgE evolve with progression of food allergy, reliable indicators of when and in whom tolerance will develop are lacking. Presently, the only way of confirming the development of tolerance is through repeat OFC. Therefore this PhD has focussed on three key areas of paediatric food allergy, i) to identify thresholds for SPT and sIgE that have 95% PPV to the diagnosis of food allergy, ii) to describe the natural history of food allergy and identify predictors of persistence or tolerance, and iii) to identify phenotypes of allergy and food allergy. The HealthNuts study is the first population-based, longitudinal food allergy study using the gold standard OFC to confirm food allergy and is the ideal platform to address these aims. Over 5000 12-month-old infants were recruited and underwent SPT to four common food allergens, egg, peanut sesame and cow’s milk or shrimp. Any infant showing objective evidence of sensitisation was invited to undergo OFC (egg, peanut and sesame only). A subgroup of infants with egg allergy participated in a sub-study which examined tolerance to baked egg and the early resolution of egg allergy at age 2. All participants were invited to undergo follow-up at 4 years of age, which included repeat OFC to detect the development of tolerance, amongst those who were challenge-confirmed food allergic at age 1. In 12-month old infants, SPT wheal size ≥ 8 mm for peanut and sesame and ≥ 4 mm for egg, and sIgE levels ≥ 34 kU/L for peanut and ≥ 1.7 kU/L for egg have 95% PPV to challenge-confirmed food allergy. Only 20% of children with challenge-confirmed peanut allergy at 12 months-of-age, developed tolerance to peanut at 4 years of age. SPT and sIgE measured at age 1 were predictive of persistent of peanut allergy at age 4; 95% PPVs were SPT ≥ 13mm and sIgE ≥ 5.0 kU/L. Thresholds at age 4 with 95% PPV to persistent peanut allergy were SPT ≥ 8mm and sIgE ≥ 2.1 kU/L, while SPT ≤ 3mm had 90% negative predictive value (NPV) and SPT ≤ 2mm and sIgE ≤ 0.35 kU/L had 50% NPV to the development of tolerance. Among infants who were challenge-confirmed allergic to raw egg at 12-months, 80% could safely tolerate baked egg on challenge and nearly 50% outgrew their egg allergy by 2 years of age. Infants who were able to tolerate baked egg at age 1 were more likely to outgrow their raw egg allergy, compared to those who reacted to baked egg (aOR 5.27, 95% CI 1.36-20.50, p=0.02). Frequent ingestion of baked egg (≥ 5 times per month) was also associated with the resolution of egg allergy, (aOR 3.52, 95% CI 1.38 – 8.98, p = 0.009.) Finally, five phenotypes of food allergy in infancy where identified using latent class analysis: no allergic disease, non-food-sensitised eczema, single egg allergy, multiple food allergies (predominantly egg) and multiple food allergies (predominantly peanut). Shared and differential risk factors for these phenotypes were identified, suggesting that different mechanisms are involved in the development of food allergy phenotypes. In summary, the results presented in this thesis have identified important predictors and phenotypes of food allergy and the development of tolerance. This has important implications for the management of food allergy in the clinical setting and will improve our understanding of the development of food allergy and the role it plays in the atopic march.
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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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ItemThe role of filaggrin in food allergy and eczema in the first year of life( 2012)Eczema and IgE-mediated food allergy often develop early in life and have a substantial impact on the quality of life of a child for many years. Family history is a strong risk factor for the development of allergic disease and the strongest genetic association with eczema to date is the filaggrin gene (FLG), which produces a key structural protein in the epidermis. Eczema, food sensitisation and food allergy commonly co-associate in early childhood. Although many studies have now identified the link between FLG and eczema, to date only one study has looked at the association between FLG and peanut allergy. No other study has investigated whether the presence of FLG mutations increases the risk of developing food allergy in early life. The recent rise in the prevalence of eczema and food allergy has occurred too rapidly to be explained by genetic factors alone, suggesting that environmental factors play a role. However, the rise might only be occurring amongst those genetically at risk. This warrants the investigation of the role of gene-environment interactions, in particular epigenetic regulation, which remains largely unexplored. Within this thesis, I investigated the association of FLG mutations with infantile eczema, food sensitisation and food allergy in one-year-old infants, using samples from the HealthNuts study. HealthNuts is a large population-based cohort study of 5276 infants recruited at 12 months of age in Melbourne, Australia. During recruitment, all infants were examined for eczema and underwent a skin prick test to four common food allergens. Those with a wheal size ≥ 1mm underwent a hospital-based oral food challenge (the gold standard for food allergy diagnosis) and bloods were taken. Non-sensitised infants were randomly invited to clinic to act as negative biological controls. I also investigated the role of DNA methylation in regulating FLG expression and the outcome of allergic disease using a small pilot study and the HealthNuts study. The association of FLG mutations with infantile eczema was replicated in this study, with an odds ratio [OR] of 4.2 (95% CI, 2.0-8.8; P <0.001) for broad definition of eczema; an OR of 2.4 (95% CI, 1.5-4.1; P=0.001) for diagnosed eczema; and OR of 2.0 (95% CI, 1.3-3.3; P=0.003) for current eczema. FLG mutations were associated with atopic eczema (eczema with IgE-mediated food sensitisation) with an OR of 4.4 (95% CI, 2.1-9.6; P< 0.001), but not non-atopic eczema (p=0.984). FLG mutations increased the risk of IgE-mediated food sensitisation in the first year of life, after adjusting for the presence of eczema (adjusted OR [aOR], 3.0; 95% CI, 1.0-8.7; p= 0.043). However, there was no significant difference in FLG mutations when comparing food-tolerant infants with those with food allergy (OR, 0.7; 95% CI, 0.3-1.7; P= 0.478). This indicates that FLG mutations do not further increase the risk of IgE-mediated food allergy over and above that of IgE-mediated food sensitisation. Together these results support the biologically plausible hypothesis that decreased skin barrier function increases the risk of food sensitisation in early life, but other as yet undetermined factors must be important in the conversion from food sensitisation to allergy. FLG was found to be expressed in adult buccal epithelia, ~19% relative to FLG expression in adult skin tissue. FLG expression in infant buccal epithelia was found to be twice as high as those of adult buccal epithelia. Methylation was measured at three CpG sites upstream of FLG transcription start site. However, there was no clear correlation between methylation and FLG expression. Therefore, although associations between FLG methylation and various allergic outcomes were identified, the clinical relevance of these findings is unclear. Nevertheless, this epigenetic study has contributed novel data to add to our understanding of the role of gene-environment interaction involving FLG in allergic disease. In summary, this study has generated evidence for an important role for FLG in the establishment of food sensitisation and food allergy in infants.
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ItemThe epidemiology of egg allergy and other IgE-mediated food allergies in infants( 2010)IgE-mediated food allergy often develops early in life and has a major impact on the quality of life of the child and its family. Recent increases in the prevalence of food allergy, which have occurred too rapidly to be explained by genetic factors alone, suggest that environmental factors may play an important role. Potential candidates include maternal and infant diet and infant microbial exposures. However, few studies to date have examined risk factors for food allergies. I investigated the prevalence and risk factors for egg allergy, the most common IgE-mediated food allergy in infants, within the HealthNuts study. HealthNuts is an ongoing population-based cross-sectional study of 12-month-old infants in Melbourne, Australia. As part of the study, parents reported on infant feeding and other environmental exposures. All infants subsequently received a skin prick test for four foods including egg white and those with a wheal size ≥ 1mm underwent an oral food challenge with raw egg white. This thesis includes data from the first 2,600 infants included in the study, which is due to complete recruitment in April 2011. Among infants participating in the HealthNuts study, there was a high prevalence of egg allergy (9.3%, 95% CI 8.2-10.5). There was no evidence that maternal dietary factors were associated with infant egg allergy. Of the infant dietary factors examined, only timing of egg introduction was associated with egg allergy risk. Introduction of cooked egg into the infant diet at 4-6 months of age was associated with a 3-fold decreased risk of egg allergy compared with introduction after 12 months of age. Other infant dietary factors previously suggested to be associated with general allergy risk, namely duration of breastfeeding and age at introduction of first solid foods, were not associated with egg allergy. Factors previously associated with increased microbial exposure, including siblings, childcare and pet ownership, were associated with a reduced risk of egg allergy. There was evidence of a dose-response effect for siblings and egg allergy, with an increasing number of siblings associated with a further reduction in egg allergy: one sibling, OR0.72 (95% CI 0.53-0.97), two siblings OR 0.57 (95% CI 0.35-0.92) and three or more siblings OR 0.47 (95% CI 0.20-1.09). Attending childcare before 7 months of age was associated with a reduced risk of egg allergy compared with never having attended childcare (OR 2.05, 95% CI 0.97-4.35), while there was no evidence of a protective effect of childcare attendance between 7-12 months of age. Having either a cat or dog at home at one year of age was associated with a reduced risk of egg allergy among infants with a history of eczema (OR 0.49, 95% CI 0.31-0.78) but not among those without a history of eczema (OR 1.01, 95% CI 0.62-1.62). My results show that egg allergy is common in infants and suggest several possible risk factors related to infant diet and potentially microbial exposures which may help to explain recent rise in food allergy prevalence. The protective effect of introduction of egg at 4-6 months of age has implications for infant feeding guidelines internationally and, if confirmed, suggests that changes in infant feeding practices could have the potential to reduce the prevalence of egg allergy.