Medicine (RMH) - Theses
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ItemThe impact of prenatal and postnatal factors on infant stunting and growth in East New Britain, Papua New Guinea( 2023-12)Background Stunting, defined as low-height-for-age, affects 165 million children with a global prevalence of 22% for children under 5. Stunting during the first 1000 days of life is associated with childhood mortality and cognitive and physical impairments that are often permanent and irreversible. The causes of stunting and poor growth are poorly understood in most settings. Papua New Guinea has some of the world’s poorest maternal and child health indicators. The national stunting rate is one of the highest globally affecting nearly half of all children under 5. East New Britain (ENB) is a province in the islands region where poor growth is an understudied problem. There are no recent stunting estimates for ENB and no studies on the causes of stunting. This thesis aims to address knowledge gaps on the prevalence and predictors of stunting in ENB, PNG. Methods This PhD study was part of the Healthy Mothers, Healthy Babies (HMHB) research program. This program, established in 2015, included a longitudinal cohort study of mothers and infants, recruited during pregnancy, and followed up to 12 months post-partum. Recruitment of mothers occurred at antenatal clinics at 5 health facilities. Women >16 years of age living in the facilities’ catchment areas, attending the clinic for the first time in the current pregnancy, regardless of gestational age, were eligible to participate. At each site, women were randomly selected. Data from this longitudinal study was used for all chapters of this thesis. Data on infant stunting was analysed cross-sectionally to identify risk factors for stunting at birth, 6 and 12 months. A simultaneous latent growth curve model was developed to describe infant growth and identify factors associated with growth velocity for weight and length. A mediation analysis was conducted to identify mediators on the pathway between maternal education and infant stunting. To further investigate causes of stunting, an exploratory analysis aimed to identify associations of maternal metabolomic profiles, or specific maternal metabolic with low birth weight and stunting at birth using maternal serum samples collected during pregnancy. Results High rates of stunting were observed among infants increasing from 15% at birth to 29% at 12 months. Among both mothers and infants there was a high prevalence of known risk factors for stunting, including maternal nutritional deficiencies and infections. Preterm birth was the strongest predictor of stunting at birth and birth weight was the strongest predictor of infant stunting at 6 months. Maternal anthropometric characteristics, height and MUAC, and maternal education were associated with stunting at 6 or 12 months. Differences in growth velocity between boys and girls were identified and some similar risk factors for growth velocity, as for stunting, were identified, including maternal height and MUAC. Despite the breath of data collected, no post-natal risk factors, including infant illness, breastfeeding and complementary feeding, were associated with infant stunting or growth. Different maternal metabolites and metabolomic pathways were associated with either low birth weight or stunting suggesting that different metabolic profiles or pathways contribute to different aspects of fetal growth. Individual metabolites, including cysteine, glycine and ornithine, were associated with infant birth weight or stunting at birth with substantial effect sizes. Conclusions This study showed a high prevalence of infant stunting and the prevalence of indicators of poor health were high among mothers and infants. Improving the health of mothers in ENB must be a priority if the high rates of infant stunting are to decline. Novel epidemiological and analysis methods identified risk factors for growth velocity distinct from stunting and elucidated casual pathways. These methods can be used to identify targets for interventions and the evaluate the effect of interventions on infant growth and stunting.
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ItemContribution of CCL17 as an inflammatory mediator in rheumatoid arthritis( 2023-10)Rheumatoid arthritis (RA) is an inflammatory, progressive, and destructive autoimmune disease. During RA, intra-articular as well as extra-articular manifestations result in morbidity and extreme cases can lead to mortality. Currently available RA therapies, such as glucocorticoids, methotrexate and TNF-inhibitors are costly and accompanied by significant adverse side effects, as well as limited effectiveness in some patients, highlighting the need for new therapies. Pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF), are among the important causative mediators in the development and progression of RA. Preclinical studies identified that CCL17 is a downstream mediator of GM-CSF. GM-CSF-induced CCL17 expression in monocytes and macrophages is via the epigenetic demethylase, Jumonji domain-containing 3 protein (JMJD3), and the transcription factor, interferon regulatory factor 4 (IRF4). While GM-CSF is one of the most essential cytokines for inducing CCL17 expression, TNF has also been implicated in CCL17 expression and pain development in preclinical models of arthritis in conjunction with GM-CSF. Furthermore, GM-CSF and TNF are produced by numerous cell types in response to various stimuli. Data suggested the possibility that TNF can engage in a cytokine loop, thus potentially linking TNF biology to the GM-CSF-CCL17 pathway. However, there is little evidence about the key cells involved in this linkage. CCL17 is a potential target for treating RA, as it accumulates in the serum and synovial fluid of patients. It is speculated that CCL17 regulates T cell migration to joints via the CCR4 receptor. This receptor is common for both CCL17 and CCL22 cytokines. In contrast to CCL17, CCL22 is decreased in the RA. At the commencement of this PhD project, no specific CCL17-suppressing drugs were available in the clinics. In addition, the cells that might be responsible for CCL17-driven pathology were unknown. In this PhD thesis, FDA-approved drugs were screened in the presence of GM-CSF to identify drugs with the potential to specifically suppress CCL17 expression, but not that of CCL22. Among the 1,500 screened drugs, 5 drugs suppressed GM-CSF-induced CCL17 expression and maintained/increased GM-CSF-upregulated CCL22 in both human monocytes and mouse macrophages, with no toxicity to the cells. The identified drugs were tested in the Zymosan-induced arthritis model (ZIA) to explore their potential to ameliorate arthritic pain and disease. Among them, four drugs significantly inhibited CCL17-dependent pain with a trend towards decreasing the disease in the ZIA model. Analysis of the mechanistic pathways of these drugs in the presence of GM-CSF demonstrated that inhibition of GM-CSF-induced CCL17 expression was due to suppression of both STAT5 activity and IRF4 protein expression. Dendritic cells are thought to be a key cell type that expresses CCL17 in RA patients. There is an imbalance in Tregs/Th17 cells in RA synovial samples which suggests the possibility of a chemotactic function of the increased CCL17 on Th17 cells. However, the potential relationship(s) between CCL17 and immune cells within the RA synovium has not been explored. In this PhD, it was found that macrophage marker (CD68) was the most prevalent marker that co-expressed with CCL17 and its receptor CCR4 in synovial membranes. Significantly, elevated expression of CCL17 was observed in RA synovium compared to that from healthy controls. The thesis showed that the effect of TNF on the expression of CCL17 induced by GM-CSF is different between human and mouse macrophages. Interestingly, TNF alone had no positive effect on CCL17 expression in human monocytes/macrophages as well as in mouse macrophages. In contrast to CCL17 expression, CCL22 was significantly upregulated by TNF in monocytes and macrophages. In conclusion, this thesis indicated the promising effects of repurposed FDA-approved drugs to ameliorate arthritic pain which could be due to inhibition of the GM-CSF/CCL17 pathway. These drugs might be able to be used in future clinical trials. Furthermore, it was shown that the type of immune cells that express CCL17 in response to other cytokines, such as TNF, would be different between humans and mice. This finding once more emphasises the complex role of TNF in inflammation. Furthermore, it was demonstrated that, in addition to T cells, macrophages are among the cells that are important in the pathogenesis of RA, supported by the co-expression of both CCL17 and CCR4 in synovium tissue samples from RA patients.
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ItemContribution of CCL17 as an inflammatory mediator in rheumatoid arthritis( 2023-10)Rheumatoid arthritis (RA) is an inflammatory, progressive, and destructive autoimmune disease. During RA, intra-articular as well as extra-articular manifestations result in morbidity and extreme cases can lead to mortality. Currently available RA therapies, such as glucocorticoids, methotrexate and TNF-inhibitors are costly and accompanied by significant adverse side effects, as well as limited effectiveness in some patients, highlighting the need for new therapies. Pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF), are among the important causative mediators in the development and progression of RA. Preclinical studies identified that CCL17 is a downstream mediator of GM-CSF. GM-CSF-induced CCL17 expression in monocytes and macrophages is via the epigenetic demethylase, Jumonji domain-containing 3 protein (JMJD3), and the transcription factor, interferon regulatory factor 4 (IRF4). While GM-CSF is one of the most essential cytokines for inducing CCL17 expression, TNF has also been implicated in CCL17 expression and pain development in preclinical models of arthritis in conjunction with GM-CSF. Furthermore, GM-CSF and TNF are produced by numerous cell types in response to various stimuli. Data suggested the possibility that TNF can engage in a cytokine loop, thus potentially linking TNF biology to the GM-CSF-CCL17 pathway. However, there is little evidence about the key cells involved in this linkage. CCL17 is a potential target for treating RA, as it accumulates in the serum and synovial fluid of patients. It is speculated that CCL17 regulates T cell migration to joints via the CCR4 receptor. This receptor is common for both CCL17 and CCL22 cytokines. In contrast to CCL17, CCL22 is decreased in the RA. At the commencement of this PhD project, no specific CCL17-suppressing drugs were available in the clinics. In addition, the cells that might be responsible for CCL17-driven pathology were unknown. In this PhD thesis, FDA-approved drugs were screened in the presence of GM-CSF to identify drugs with the potential to specifically suppress CCL17 expression, but not that of CCL22. Among the 1,500 screened drugs, 5 drugs suppressed GM-CSF-induced CCL17 expression and maintained/increased GM-CSF-upregulated CCL22 in both human monocytes and mouse macrophages, with no toxicity to the cells. The identified drugs were tested in the Zymosan-induced arthritis model (ZIA) to explore their potential to ameliorate arthritic pain and disease. Among them, four drugs significantly inhibited CCL17-dependent pain with a trend towards decreasing the disease in the ZIA model. Analysis of the mechanistic pathways of these drugs in the presence of GM-CSF demonstrated that inhibition of GM-CSF-induced CCL17 expression was due to suppression of both STAT5 activity and IRF4 protein expression. Dendritic cells are thought to be a key cell type that expresses CCL17 in RA patients. There is an imbalance in Tregs/Th17 cells in RA synovial samples which suggests the possibility of a chemotactic function of the increased CCL17 on Th17 cells. However, the potential relationship(s) between CCL17 and immune cells within the RA synovium has not been explored. In this PhD, it was found that macrophage marker (CD68) was the most prevalent marker that co-expressed with CCL17 and its receptor CCR4 in synovial membranes. Significantly, elevated expression of CCL17 was observed in RA synovium compared to that from healthy controls. The thesis showed that the effect of TNF on the expression of CCL17 induced by GM-CSF is different between human and mouse macrophages. Interestingly, TNF alone had no positive effect on CCL17 expression in human monocytes/macrophages as well as in mouse macrophages. In contrast to CCL17 expression, CCL22 was significantly upregulated by TNF in monocytes and macrophages. In conclusion, this thesis indicated the promising effects of repurposed FDA-approved drugs to ameliorate arthritic pain which could be due to inhibition of the GM-CSF/CCL17 pathway. These drugs might be able to be used in future clinical trials. Furthermore, it was shown that the type of immune cells that express CCL17 in response to other cytokines, such as TNF, would be different between humans and mice. This finding once more emphasises the complex role of TNF in inflammation. Furthermore, it was demonstrated that, in addition to T cells, macrophages are among the cells that are important in the pathogenesis of RA, supported by the co-expression of both CCL17 and CCR4 in synovium tissue samples from RA patients.
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ItemElectrophysiologic remodelling and arrhythmias in endurance athletes: prevalence and mechanisms( 2023-03)Introduction Endurance sport participation has many health benefits including improved cardiovascular fitness and longer life expectancy. It is a paradox that endurance sport participation is also associated with cardiac arrhythmias. The true prevalence and incidence of arrhythmias in endurance athletes is difficult to define due to variable definitions of endurance sport participation and identifying athletes with arrhythmias. There are several proposed mechanisms by which endurance sport may predispose athletes to arrhythmias. Aims The aim of this thesis is to characterise what electrophysiologic changes are normal in endurance athletes, define the risk of arrhythmias attributable to endurance sport participation and investigate the mechanistic causes for an increased burden of arrhythmias. Results To characterise normal electrophysiologic changes in endurance athletes we recruited 223 current and former athletes as well as fifty-two nonathletic control participants. We found that bradycardia and notably ventricular pauses are very common in athletes and rare in non-athletes. We found no significant association of ectopic burden with endurance sport. To investigate the mechanisms by which endurance sport leads to bradycardia in athletes we performed an experimental study in thirty volunteers split equally between nonathletes, endurance athletes and endurance athletes with extreme bradycardia. By administering autonomic blockade, we confirmed that bradycardia in athletes is not due to autonomic influence. Furthermore, this is the first study which has shown high vagal tone does not explain extreme resting bradycardia in athletes. To better define the risk of atrial fibrillation attributable to endurance sport, we performed a case control study which strictly defined past endurance sport participation. We recruited 121 former elite rowers who competed at a national, world championship or Olympic level and compared them to an age, gender and ethnicity matched cohort from the UK Biobank at a ratio of 100:1. Despite less traditional risk factors we found the prevalence of atrial fibrillation 6.7 times higher in the athlete cohort and incidence was 2.8 times higher over two years of follow up. Using a polygenic risk score we were able to show that genetic risk plays a role in which athletes develop atrial fibrillation in a similar manner to non-athletes. In our last study we attempted to identify why some athletes develop atrial fibrillation. In our previous study we demonstrated that genetic risk plays a role in which athletes develop atrial fibrillation but does not appear to explain the excess burden of atrial fibrillation in endurance athletes. For this study we performed a case control study with fifty-three current and former endurance athletes with atrial fibrillation age and gender matched to fifty-three athletes without atrial fibrillation. We found that endurance sports athletes with atrial fibrillation have similar risk factor profiles, body composition, training history, physical fitness and electrophysiologic remodelling to endurance athletes without atrial fibrillation. Conclusion Endurance sport training leads to profound electrophysiologic remodelling which is not explained by autonomic tone. The risk of atrial fibrillation in markedly increased in endurance athletes but the mechanisms which lead to the excess burden of atrial fibrillation in endurance athletes remain elusive.
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ItemPharmacological Treatment of delirium in hospitalised older adults; changing the natural history( 2023-08)Delirium is a neuropsychiatric syndrome characterised by the acute onset of fluctuating cognitive disturbances including awareness and attention deficits, hallucinations, delusions, and circadian rhythm changes. Delirium is a common condition in hospitalised older adults that portends a poor prognosis and provokes many malign complications. Under-diagnosis is a problem in Delirium despite its prevalence. Delirium lacks any therapy that improves the natural history of the disorder: while antipsychotics are frequently used for symptoms, clear evidence of benefit is lacking and the superiority of any single agent over another is unknown. Once established then, Delirium’s course cannot be modified. Circadian rhythm disturbances are common in Delirium. Melatonin is a pineal gland hormone that contributes to circadian regulation, and secretion is perturbed in Delirium. Melatonin is available as an oral medication, approved for use in sleep disorders. Melatonin has been used to prevent Delirium onset but has not been evaluated for treatment of established Delirium. This thesis examined the prevalence and prevalence of under-diagnosis of Delirium, finding it large. The thesis examined melatonin for treatment of Delirium severity in hospitalised older patients. An effect on a comprehensive rating scale of Delirium symptoms was not found.
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ItemNo Preview AvailableInvestigating the relationship between glycaemia and infections in the hospital setting.( 2023-09)Diabetes mellitus affects up to one third of individuals admitted to hospital. Diabetes and its consequence of hyperglycaemia impede the normal functioning of the immune system and impair vascular function. They are thus associated with increased rates of adverse clinical outcomes in the inpatient setting including healthcare-associated infection. Intervention in the hospital setting to reduce hyperglycaemia may thus prevent the development of infections and other adverse consequences. This thesis describes a broad program of research that sought to reduce hyperglycaemia occurring in hospital with the goal of reducing healthcare-associated infection. In order to carry out this randomised controlled trial it was necessary to perform multiple research studies utilising various methods, to inform the design and enable the conduct of the trial. While there are multiple studies considering the impact of diabetes and hyperglycaemia on outcomes in certain disease states, heterogeneity of definitions and populations precludes ready appreciation of associations and their magnitude. A systematic review and meta-analysis of the literature was thus performed regarding the impact of diabetes and hyperglycaemia on outcomes in hospitalised patients with community-acquired pneumonia, finding that while hyperglycaemia was most strongly associated with adverse outcomes during the inpatient stay, diabetes had the greater association with post-discharge adverse outcomes (chapter 4). To enable the measurement and remote review of glucose across the hospital, a networked blood glucose monitoring system was implemented on all inpatient wards of the hospital. During implementation nurses were surveyed on their perceptions regarding in-hospital glucose monitoring, yielding unexpected results of a perception of greater importance amongst less experienced and non-educator nurses (chapter 5). Following program implementation the accurate use of glucose meters was tracked and found to deteriorate following training and with COVID-19 pandemic preparations (chapter 6). Additionally, glucometric benchmarking was performed and compared to earlier benchmarking, showing the positive effect of quality improvement projects in the intervening period (chapter 7). COVID-19 had significant impacts upon health services and people with diabetes. Performing a multi-centre study across Melbourne in 2020 revealed that unlike international experiences, neither diabetes nor hyperglycaemia was independently associated with increased adverse outcomes (chapter 8). A prospective cohort study was performed of all patients who received glucose testing at our institution (chapter 9). This study showed that hyperglycaemia was independently associated with healthcare-associated infection, acute kidney injury, and stroke occurring in the inpatient setting. Furthermore, the use of propensity weighting suggested a causative role for hyperglycaemia in the relationships. Finally, a randomised controlled trial of early intervention with an electronic specialist-led model of diabetes care was performed (chapter 10). This trial showed that such early intervention can both reduce glucose and healthcare-associated infection in patients with and without diabetes admitted to hospital. The studies in this research program describe a research journey of capacity building and problem defining towards designing and trialling a model of inpatient care that enabled glycaemic reduction with the prevention of healthcare-associated infection. Implementation of a similar model of care in other settings may improve the care of people with diabetes and hyperglycaemia admitted to hospital.
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ItemAdvancing Translational Care in Genetic Dilated Cardiomyopathy: Genotype-Phenotype Correlations, Natural History and Risk Prediction( 2023-03)Dilated Cardiomyopathy (DCM) is a common heart disorder characterised by reduced left ventricular (LV) systolic function that is not due to coronary artery disease. DCM can be familial, acquired, or idiopathic, though increasingly, all DCM is thought to have some genetic basis. Genetic testing for DCM has expanded in recent years due to improved technologies, reduced cost and increasing awareness. However, translation of clinical results remains challenging, particularly in relation to interpreting test results, enacting gene-based management approaches, and devising best monitoring and surveillance strategies for at-risk individuals. Further research is required to (a) define genotype-phenotype correlations, (b) understand the expected natural history of disease, (c) explore the role of environmental factors in disease manifestations, and (d) evaluate methods of risk prediction in genotype positive, unaffected individuals. In this thesis, these research areas were addressed in a series of studies divided into two research streams. Stream 1 focuses on genotype phenotype correlations with a focus on arrhythmic DCM and SCN5A DCM. First, in Chapter 2 a systematic approach was used to assess the strength of evidence for the putative cardiomyopathy genes and establish phenotypic correlates for each. The data from this work was published in a larger review Fatkin D, .. Peters S., Kovacic J.C. Contemporary and Future Approaches to Precision Medicine in Inherited Cardiomyopathies, 2021, J Am Coll Card. DCM genes that produce an arrhythmic phenotype have specific clinical implications and are emerging as important in personalised therapy of cardiomyopathies. In Chapter 3 the evidence for arrhythmic DCM genes was reviewed in a paper titled Peters S, .. Fatkin D. Arrhythmic Genotypes in Familial Dilated Cardiomyopathy: Implications for Genetic Testing and Clinical Management, 2019, Heart & Lung Circulation. One of the arrhythmic DCM genes of note was SCN5A, which appears to be the only arrhythmic DCM gene with a genotype directed therapy. In Chapter 4 a long-term followup of a family with the recurring SCN5A variant p.R222Q was performed, titled Peters S, .. Fatkin D. Longterm Efficacy and Safety of Sodium Channel Antagonists in p.R222Q SCN5A related Arrhythmic Dilated Cardiomyopathy, 2021, JACC Clin Electrophysiol. In Chapter 5 a systematic analysis of SCN5A variants with an association to DCM was performed, identifying unifying patterns of genotype and phenotype. This work was published in Peters S, .. Fatkin D. Arrhythmic Phenotypes are a Defining Feature of Dilated Cardiomyopathy-Associated SCN5A Variants: A Systematic Review, 2022, Circulation Genomic and Precision Medicine. Stream 2 of this thesis focuses on environmental factors, natural history and subclinical disease in familial DCM, utilizing a deeply characterised cohort of individuals from the Royal Melbourne Hospital (RMH) with confirmed genetic DCM. In Chapter 6 (manuscript in preparation), the role of environmental factors in the natural history of familial DCM is assessed. In Chapter 7, a focus specifically on pregnancy as an important environmental risk factor was performed, and this work was published in a research letter titled Peters S, .. Zentner D. Pregnancy Outcomes in Females with Dilated Cardiomyopathy-Associated Rare Genetic Variants, 2022, Circulation Genomic and Precision Medicine. In Chapter 8 (manuscript in preparation), specialised cardiac imaging techniques were used to evaluate for subclinical disease among genoptype positive, phenotype negative individuals. Genotype positive, phenotype negative individuals had lower average global longitudinal strain, and higher average LV volumes compared to genotype negative controls. Further, a significant interaction between genotype, environmental factors and GLS was found, indicating, again, that genotype positive individuals may be particularly vulnerable to environmental stressors. The studies in this PhD have gone some way to advancing translational care in genetic dilated cardiomyopathy in relation to genotype-phenotype correlations and risk prediction. This research paves the way for prospective longer term evaluations of genetic DCM cohorts to establish personalised treatment pathways.
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ItemThe role of neutrophils in immunity to malaria( 2023-07)The global malaria burden continues to remain high with most deaths occurring in young children. Understanding the protective immune mechanisms against malaria is imperative for the development of a highly efficacious vaccine since RTS,S and R21 which are recommended malaria vaccines, have modest efficacy and are limited to young children. Recent reports indicate that neutrophils may potentially play a key role in protective immunity against malaria via antibody-mediated interactions with Fc-gamma receptors (FcgR) expressed on their surface. Additionally, complement proteins may promote parasite clearance via interaction with antibodies. This thesis presents new findings on antibody interactions with complement (C1q) and FcgRs on neutrophils in immunity against severe malaria. It further presents new information on neutrophil phenotypes in response to naturally acquired malaria as well as phenotypes and transcription profiles in controlled human malaria infections (CHMI), and in vitro models. First, cytophilic IgG1 and IgG3 antibody subclasses, and antibodies that can fix C1q, or engage FcgRs to induce phagocytic clearance of merozoites were investigated. Additionally, opsonic phagocytosis of merozoites by neutrophils was quantified. These investigations were carried out on samples from young Papua New Guinean (PNG) children (n = 383) presenting with severe malaria versus uncomplicated malaria. In children presenting with severe malaria, cytophilic antibodies, antibodies that can fix C1q, or engage FcgRs against whole merozoites and neutrophil phagocytosis of merozoites opsonized by serum antibodies were lower than children with uncomplicated malaria. There was a strong association between high antibody responses to merozoites that fix complement or bind FcgRs, and reduced odds of severe disease. Second, cryopreserved neutrophils from children and adults in PNG, were phenotyped by examining receptors relevant to functions and malaria immunity. Neutrophils from children infected with P. vivax malaria (n = 37) had increased CD66b expression compared to uninfected children. Increased statistical power by combining uninfected children (n = 29) and uninfected adults (n = 35) into one group and comparing them with P. vivax and P. falciparum (n = 25) infected children further highlighted increased CD64 expression on neutrophils from malaria infected children compared to uninfected group. Neutrophils from uninfected children had distinct phenotypic differences from uninfected adults. There were also some differences identified in neutrophil phenotypes in males compared to females infected with P. falciparum. In addition, neutrophil phenotypes from uninfected adults and uninfected children varied substantially from neutrophils from malaria-naive adult Australian residents (n = 11), revealing increased CD64, CD54, and CD66b, and decreased CD62L, CD16, and CD32a expression, although CD32a expression in malaria-exposed children did not differ from malaria-naive adults. Neutrophil phenotype changes relevant to effector function and immunity to malaria model were assessed at multiple time points in a controlled human experimental P. falciparum infection trial. The study included 8 healthy Australian adult participants who were treated at day 8 of malaria infection. Neutrophil phenotypic changes occurred as early as day 4, prior to substantial blood-stage parasitemia. There were significant and substantial changes in neutrophil phenotypes following treatment to clear parasitemia and some changes persisted weeks post-treatment. To further understand neutrophil responses to malaria, phenotype changes in response to direct interaction with merozoites in an in vitro model were examined. There were no phenotype changes after neutrophils were incubated with unopsonized merozoites or with merozoites opsonized with serum from malaria-naive donors. In contrast, distinct changes in phenotypes were observed after incubation of neutrophils with merozoites opsonized by naturally-acquired antibodies from children. These changes included upregulated CD66b and downregulated CD64, CD32a, CD16, and CD62L expression. Finally, transcriptional analysis of neutrophil gene sets relevant to functions and clearance of malaria parasites were explored among samples from a controlled P. falciparum experimental infection trial in healthy Australian adults (n = 12) and using in vitro models. CHMI data revealed upregulation of gene sets including phagocytosis pathways and cytokine mediated signalling pathways. In vitro transcriptional analysis of neutrophils incubated with merozoites opsonized by purified IgG from malaria-exposed children showed upregulated gene sets including respiratory burst and cytokine activity compared to neutrophils incubated with merozoites opsonized with IgG from malaria-naive individuals. However, gene set enrichment analysis did not reveal any significant change in phagocytosis gene sets. The data generated in this thesis advances our knowledge on antibody mechanisms involved in immunity to malaria and provides new knowledge on the phenotypes and functions of neutrophils in response to malaria. Given that emerging data are increasingly indicating an important role for neutrophils in naturally-acquired and vaccine-induced immunity, this knowledge will be valuable for informing the development of more efficacious malaria vaccines.
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ItemDissecting the antibody responses in pregnancy malaria( 2023-09)Abstract Malaria remains a major public health problem in tropical and subtropical areas of the world. It is a leading cause of morbidity and mortality in these areas, especially in young children and pregnant women. Pregnant women, despite lifetime malaria exposure, have increased susceptibility to Plasmodium falciparum malaria. This is due to the ability of parasites to sequester in the placenta by expressing VAR2CSA, a P. falciparum erythrocyte membrane protein 1 (PfEMP1) that is upregulated during pregnancy. With repeated exposure, pregnant women develop antibodies to VAR2CSA that are associated with reduced adhesion of infected erythrocytes (IEs) to the placenta, which may improve pregnancy outcomes. However, the development and function of these antibodies may depend on a combination of factors, including transmission intensity, cross species immunity, and maternal genetics. This PhD thesis aims to dissect diverse aspects of antibody responses to malaria in pregnancy and factors that may affect acquisition and functions of these antibodies. Aim 1 in this thesis examined antibody responses to P. falciparum and P. vivax antigens in pregnant women residing in low malaria transmission areas in the Brazilian Amazon region (chapter 4 and 5). The total and opsonic IgG antibody levels to pregnancy specific whole parasite surface antigens on CS2-IEs (CS2VSA), individual recombinant VAR2CSA domains and to other malaria antigens were assessed in the plasma of 408 Brazilian pregnant women using various assay formats including flow cytometry, enzyme linked immune assay (ELISA), and multiplex assays. Antibodies to CS2VSA were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other, but not with antibody to CS2VSA or history of infection. We did not find a significant association between antibodies to CS2VSA with pregnancy outcomes. However, antibodies to individual VAR2CSA DBLs and certain P. falciparum merozoite antigens were significantly associated with lower odds of maternal anaemia (OR range 0.38-0.54, p<0.038). Aim 2 explored whether exposure to P. vivax antigens could lead to the production of cross-reactive antibodies to VAR2CSA. The acquisition of antibodies specific to VAR2CSA is generally assumed to occur through exposure to P. falciparum during pregnancy. More recent evidence has suggested that individuals exposed to P. vivax can also acquire antibodies to VAR2CSA. However, this evidence is based on antibodies to recombinant proteins and may be an artifact of the protein expression system. Therefore, in chapter 6 of this thesis, we investigated a hypothesis that P. vivax exposure does not result in antibodies that bind to VAR2CSA on the surface of the IEs and the acquisition of antibodies to VAR2CSA is restricted to pregnancy. The study cohort included P. vivax only infected primigravid women, P. falciparum infected multigravid women, uninfected pregnant women, men, and children from Brazil. When antibodies were measured to surface antigens on P. falciparum IEs, the P. vivax infected women showed significantly lower levels of antibodies to whole parasite CS2VSA in comparison to P. falciparum-infected women (p<0.0001). Additionally, the levels of CS2VSA-specific IgG in primigravid women infected with P. vivax was not significantly different compared to antibody levels in plasma samples from uninfected women. However, it was observed that pregnant women regardless of infection status and gravidity exhibited comparable levels of IgG response to recombinant VAR2CSA DBL3 expressed in P. pastoris and VAR2CSA DBL5 expressed in Chinese hamster ovary (CHO) cells. Intriguingly, men and children also exhibited antibodies against the recombinant VAR2CSA DBL3 domain at levels similar to those observed in pregnant women. In summary, antibodies to CS2VSA did not appear to result from exposure to P. vivax. Nonetheless, antibodies to individual recombinant VAR2CSA domains are not restricted to pregnancy or exposure to P. falciparum. Aim 3 investigated the effect of allotypic variations on the transplacental transfer and functions of malaria-specific IgG3 antibodies. Antibody allotypes are polymorphisms in the constant regions of immunoglobulin heavy and light chains. IgG3 allotypic variations can have a significant impact on associated Fc-mediated effector functions and transplacental transfer of malaria specific antibodies. We identified a novel IgG3 allotype in a cohort of pregnant women from PNG which has a histidine-to-glutamine replacement at residue 433 (IgG3-Q433). The maternal IgG3 gene was sequenced in a total 558 women to identify the IgG3-Q433 polymorphism. A total of 298 pairs of maternal and cord serum samples were analysed to investigate the effect of this allotypic variation on transplacental transfer of IgG3 antibodies. Mother to cord transfer ratios were calculated. We used four VAR2CSA DBL3-specific monoclonal antibodies with different heavy chain constant regions (IGHG1*01, IGHG3*01, IGHG3*30 and IGHG3*30-H435R) to assess the effect of IgG3-Q433 polymorphism on the FC-mediated effector functions, including complement fixation and antibody dependent cellular phagocytosis. In brief, the novel IgG3 variant displayed enhanced ability to induce phagocytosis by neutrophils and THP-1 cells, but it did not show an increased ability to activate the complement system. Additionally, there was no significant difference in antibody transfer from mother to baby between the novel variant and common IgG3 and IgG1 allotypes (Chapter 7). In summary, this thesis analysed diverse aspects of antibody responses to malaria in pregnancy and investigated factors that may affect acquisition and Fc- mediated effector functions of these antibodies. It explored whether these antibodies confer protection against malaria associated pregnancy outcomes or just serve as markers of prior exposure. Furthermore, this thesis investigated the effects of IgG3 allotypic variations on the transplacental transfer of antibodies and the associated Fc-mediated functions. Continued research in this area will add to our understanding of pregnancy associated malaria and potentially reveal novel therapy and vaccine strategies to combat this disease.
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ItemMedication use of anti-cholesterol drugs and cognitive decline in ageing( 2022-08)Projections estimate 131.5 million will be living with dementia by the year 2050. Alzheimer’s disease is the leading cause of dementia, accounting for about two-thirds of all cases and is a global public health priority. Alzheimer’s disease risk escalates with age and lacks efficacious drugs. Statins are prescribed to lower cholesterol levels and the risk of adverse cardiovascular events. Some epidemiological studies have linked statin use to improved cognitive functioning. Nonetheless, literature in this field is conflicting. Thus, investigations of long-term statin use from midlife have been suggested in recent systematic reviews. To the best of our knowledge, only one study has examined a female-specific cohort, and only few studies have investigated this relationship over a period of at least ten years, and prior to this thesis, no study has examined this relationship in women over a 24-year period. Chapters 1 and 2 examined the current literature regarding statin-cognition relationship. Previous systematic review suggests a mixed and inconclusive relationship between statins and cognitive outcome: however, this thesis provided the first evidence of this relationship extending to the prodromal phase of neuropathological change. A comprehensive systematic review and meta-analysis into statin and cognition found that the significant difference reported in the current statin studies was mainly driven by male statin users. This review highlighted the importance of having long-term statin studies and the urgent need to focus on female statin users. Chapter 4 presented a cross-sectional analysis of the relationship between statin and cognition in participants of the Women’s Healthy Ageing Project. Independent of underlying vascular risk, current statin users, initiation of statin use by women (1-4 years of use) was associated with the greatest deterioration in global cognitive function. This effect is not simply reflective of the lipid levels in the women. Chapters 5 and 6 investigated the longitudinal effect of statin consumption on the cognition of Australian ageing women over time. Participants from the longitudinal Women’s Healthy Ageing Project completed assessments from 1992 to 2016. Statin use by women was associated with greatest decline in episodic memory, global cognitive function and visuospatial ability. Non-statin users were also mostly seen to have a better cognition than statin users across this 24-year period. Type of statins do appear important and the effect of statins on cognition could take up to many years before it is noticeable, highlighting the importance of needing an extensive duration (more than 20 years) of longitudinal studies. This thesis presented the first systematic review and meta-analysis in highlighting the paucity of statin studies with long duration and lack of inclusion of female participants, who are at a much greater risk than their male counterparts. This thesis was the first to examine this relationship in a female only cohort in more than 20 years of follow-up. The results of this thesis, in concert with previous literature, suggest statin has a substantial role in jeopardizing our cognition and the detrimental effects of statins could take many years to be apparent. However; it is clear that the complex connection between statin and brain health requires further research to elucidate the underlying mechanism governing this relationship.