Medicine (RMH) - Theses
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Surgical Antimicrobial Prophylaxis Prescribing in Australia
Abstract Background: Antimicrobial stewardship (AMS) programs are well established initiatives for the rationalisation and conservation of antimicrobial use. Surgical antimicrobial prophylaxis (SAP) has been identified as the most common indication for antimicrobial use in Australian hospitals, with low rates of appropriateness in both public and private hospitals. There is a paucity of literature targeting surgical AMS programs that are applicable to both sectors. Aims: The aim of this PhD is to explore challenges in SAP prescribing and strategies for the optimisation of SAP prescribing in Australia. Developing an understanding of current SAP prescribing practices and factors that influence SAP prescribing decisions, informs a framework for surgical AMS. The framework aims to address identified issues and support implementation and sustainability of surgical AMS across all Australian hospitals. Methods: A mixed-methods design integrating quantitative and qualitative research (within a pragmatic paradigm) constituted the overarching methodological approach in this thesis. Five projects were completed. In project 1, the implementability of the national SAP guidelines was assessed. In projects 2, 3 and 4, multiple logistic regression analyses were adopted across the Surgical National Antimicrobial Prescribing Survey dataset to investigate any potential association between patient, hospital and surgical factors’ and SAP appropriateness. Finally, in project 5, a qualitative case study explored the phenomenon of SAP decision-making across the professional groups involved in SAP prescribing and administration, i.e., surgeons, anaesthetists, theatre nurses and pharmacists. Barriers and enablers of prescribing decisions were identified through use of validated theoretical frameworks. Based on the integration and triangulation of all results, a framework for surgical AMS in Australia is proposed. Results: There are limited surgical AMS interventions identified in the current literature, and a framework to support surgical AMS (and outcome measures such as surgical site infection surveillance) in Australia does not currently exist. SAP prescribing practices continue to demonstrate high rates of inappropriateness across a range of surgical specialties and surgical procedures. Identified targets for improvement include; procedural dose timing, post-procedural prescription duration, and surgical procedures with noted high rates of inappropriateness, broad-spectrum antimicrobial use, and SAP administration when not required. The qualitative research identified six themes in clinicians’ perceptions about decision-making for antimicrobial use across surgical settings and highlighted the need for resources to support adequate documentation and communication about SAP prescribing decisions, engagement with senior clinicians who have significant influence upon their juniors’ prescribing practices, and the need for meaningful data addressing common fears and misperceptions regarding SAP durations to generate prescribing behaviour change. Conclusion: The surgical AMS framework provides a theoretical and evidence-based approach to improving patient quality of care and outcomes at a local level whilst also addressing the pressing issue of AMR at a national and global level. The implementation of surgical AMS is critical for the optimisation of antimicrobial use across public and private surgical settings in Australia. The framework for surgical AMS focuses on three key strategies; engagement and collaboration with stakeholders, advocating for a multi-disciplinary collaboration; development, implementation and evaluation of meaningful data and outcome measures; and the adoption of a multi-faceted quality improvement program.
Frailty: risk stratification, measurement and outcomes in surgical and critically ill patients
Surgical and intensive care populations are ageing worldwide. Frailty, a state of increased vulnerability to stress, is increasingly common in older surgical and critically ill patients. In these cohorts, frailty predisposes to increased complications, mortality, and longer lengths of stay. Implementation of frailty screening can aid case finding, to select patients for more in-depth frailty measurement. This can assist identification of at-risk surgical and critically ill patients, and help identify which areas of health are more affected by frailty in these populations. Using routinely collected hospital data may allow construction of frailty indices, which can accurately and reproducibly measure frailty in granular detail, and help clarify the interplay between age, medical comorbidities, and frailty in individual patients. Aims of this PhD were to: 1. Review the role of frailty indices in the measurement of frailty in critically ill and surgical populations. 2. Determine which areas of health are adversely affected by frailty in surgical and intensive care patients. 3. Investigate the correlation between the screening Clinical Frailty Scale and multi-dimensional frailty measurement tools. 4. Explore whether frailty can be measured retrospectively from the clinical record. 5. Examine the prevalence and impact of frailty in intensive care unit populations in Australia and New Zealand. 6. To develop and validate a frailty index from medical admission data to measure frailty in surgical and intensive care unit patients. Phases of research and results The phases of research to accomplish these aims were: Phase 1: A systematic literature review was undertaken, assessing the role of frailty indices in the measurement of frailty in intensive care and surgery. Studies were identified through systematic review of MEDLINE, EMBASE, CINAHL databases, including studies which utilised a frailty index containing at least 30 health deficits. Outcomes assessed included mortality, complications, length of stay and discharge location. Study and frailty index quality were assessed, with findings narratively described. Phase 1 Results (Chapter 2): Thirteen studies utilising frailty indices were identified, nine prospective and four retrospective. Frailty index quality was high in 11 of the 13 studies. Frailty indices identified patients at risk of increased death, surgical complications, increased length of stay, and discharge to residential care. The term “frailty index” was found to be misapplied to a number of measurement tools not fulfilling criteria to be true frailty index scales. Phase 2: Two concurrent prospective cohort studies were conducted in a tertiary metropolitan hospital, in an intensive care unit and peri-operative department. Adult patients aged >/= 50 years (ICU) or >/= 65 years (surgery) admitted between February and June 2017 were eligible for inclusion. Frailty (Clinical Frailty Scale, Edmonton Frail Scale, Frailty Index) were measured at baseline. Outcomes included mortality, discharge to residential care, ICU and peri-operative complications, six-month mortality and residential location. Phase 2 Results (Chapters 4, 5, 8) : Three-hundred and thirty-six patients were enrolled in a four-month period, 160 ICU patients, 218 surgical patients, with 42 patients in both cohorts. Frailty prevalence (measured via the Edmonton Frail scale) was 36% in ICU patients, and 24% in surgical patients. Mortality in frail patients was higher in both ICU and surgical cohorts (ICU: 24% vs. 9%, p = 0.010; surgery = 10% vs. 2%, p = 0.019). Patients with frailty were less likely to be discharged home and more likely to be discharged to in-patient rehabilitation, and to be residing in assisted living facilities at six month follow up. Phase 3: Based on the results from the cohorts enrolled in Phase 2 above, the Clinical Frailty Scale was compared to the multi-dimensional Edmonton Frail Scale, with agreement measured via Kappa co-efficient, and correlation via Spearman’s correlation coefficient. The affected health domains of patients with frailty were compared with those of patients without frailty. Phase 3 Results (Chapter 4): Clinical Frailty Scale and Edmonton Frail Scales were highly correlated in ICU patients (Spearman correlation coefficient = 0.85; 95% CI, 0.81 to 0.88), with high agreement (kappa coefficient = 0.78; 95% CI, 0.68 to 0.88), and in surgical patients (Spearman correlation coefficient, 0.81; 95% CI, 0.77 to 0.86; kappa coefficient, 0.76; 95% CI, 0.70 to 0.81). Frail patients had worse health status across the full spectrum of frailty domains, in particular functional dependence, malnutrition, and prior hospital admissions. Phase 4: A secondary analysis of an existing dataset was conducted to examine the feasibility and inter-rater reliability of retrospectively determining a Clinical Frailty Scale from the medical record of critically ill patients. One-hundred and forty-four ICU patients had CFS scores independently assigned by four blinded investigators, with inter-rater agreement between CFS scores examined via quadratic weighted Cohen’s kappa coefficients. Phase 4 Results (Chapter 6): Of 144 enrolled patients, 137 (95%) were able to have a CFS score assigned retrospectively from the medical record. Cohen’s kappa coefficient for inter-rater reliability between frailty assessors was 0.67, confirming substantial agreement. Frailty measurement was thus deemed feasible from the ICU clinical record. Phase 5: A retrospective population-based cohort study was conducted, analysing data from the Australian and New Zealand Intensive Care Society Adult Patient Database. All patients aged >/= 80 years on admission to ICU between 1 January 2017 and 31 December 2018 were included in the study. The database was interrogated for the Clinical Frailty Scale on admission, with a mixed effects logistic regression fitted to the primary outcome of in-hospital mortality. Secondary outcomes were length of stay (hospital and ICU), re-admission to ICU, and discharge destination (including new chronic care or nursing home admission). Phase 5 Results (Chapter 7): 15,613 patients aged >/= 80 years were included from 131 ICUs; 6,203 patients (40%) were frail. Patients with frailty had higher illness severity, and were more likely to be admitted emergently to ICU with sepsis or respiratory failure. Mortality was higher in patients with frailty (17.6% vs. 8.2%, p < 0.001; adjusted mortality OR [95% CI] = 1.87 [1.65 – 2.11], p < 0.001). Patients with frailty had longer lengths of stay in-ICU and in-hospital, and were more likely to be newly discharged to nursing home/chronic care (4.9% vs. 2.8%, p < 0.001). Phase 6: Based on the results from the cohorts enrolled in Phase 2 above, I developed a frailty index from routine data collected on hospital admission, and tested in both surgical and ICU cohorts. The diagnostic performance of the frailty index against existing frailty tools for both screening (the Clinical Frailty Scale) and measurement (the Edmonton Frail Scale) was assessed. The discriminative ability of the frailty index for mortality was compared to existing risk predictions tools, including the Acute Physiology and Chronic Health Evaluation (APACHE) III score (ICU) and the P-POSSUM score (surgical patients). Phase 6 Results (Chapter 8): A 36-item frailty index was constructed, able to be completed for all patients. Correlation between Edmonton and Clinical Frailty scales was strong for both ICU and surgical patients. The frailty index had good discriminative ability for prediction of mortality, comparable with the performance of the APACHE-III illness severity score in ICU (AUC-ROC [95% CI] = 0.75 [0.64 – 0.85] vs. 0.80 [0.72 – 0.88]) and the P-POSSUM score in surgery (AUC-ROC = 0.76 [0.61 – 0.91] vs. 0.81 [0.71 – 0.92]). Conclusions Frailty is common in critically ill and surgical patients, affecting the full spectrum of health domains, and predisposing to poor outcomes. The Clinical Frailty Scale accurately screens for frailty in these cohorts, is able to be measured retrospectively from the clinical record, and can be used to determine frailty in critically ill patients at a population registry level. Frailty indices derived from routine hospital data are able to measure frailty in the peri-operative and ICU setting; electronic medical records show promise in automating such measurement.
Immunologic manipulation to prevent or treat relapse of haematological malignancies after allogeneic stem cell transplantation
Allogeneic haematopoietic stem cell transplantation (alloSCT) is a potent form of immunotherapy and an important treatment modality for patients with haematological malignancies. While alloSCT is a curative therapy for many patients, relapse remains a significant cause of treatment failure in a considerable proportion of patients. There is therefore an unmet need to improve the outcomes of patients after alloSCT by identifying patients at high risk of relapse and to develop effective strategies to prevent relapse in these patients. In addition, the outcome of patients with relapsed haematological malignancies after alloSCT remains poor and there is significant scope to develop novel strategies to treat relapse after alloSCT. In this thesis I investigate immunological biomarkers of relapse and explore novel strategies to prevent and treat relapse of haematological malignancies after alloSCT through two investigator initiated clinical trials. I describe that donor/recipient T-cell chimerism is significantly associated with relapse after both myeloablative and non-myeloablative alloSCT in a continuous fashion, however its utility is limited by poor sensitivity. In addition, there remains a subgroup of patients with low-risk disease who can remain in long-term remission despite mixed T-cell chimerism. In another piece of work, I explore the dynamics of T-cell receptor (TCR) repertoire reconstitution post-alloSCT and demonstrate that contrary to previous reports, a restricted TCR repertoire is not associated with early relapse of acute myeloid leukaemia (AML) after alloSCT. As an interesting aside, TCR diversity is significantly and adversely impacted by early cytomegalovirus (CMV) viremia and this effect persists late post-transplant. Therapeutic strategies to prevent relapse of haematological malignancies after alloSCT are required and in this context I explore the feasibility of low-dose lenalidomide to prevent relapse of AML and myelodysplastic syndromes (MDS) post-alloSCT. Preliminary results of this investigator-initiated phase 1 dose escalation study, which remains open to recruitment, have demonstrated that to date lenalidomide 5mg twice per week commencing at day 40 post-alloSCT is safe and tolerable. Finally, I describe the safety and efficacy of nivolumab, an inhibitor of the programmed death 1 (PD-1) receptor, for the treatment of relapsed haematological malignancies after alloSCT. In this investigator-initiated clinical trial, nivolumab induced highly potent alloimmune responses with complete remissions observed in highly refractory haematological malignancies albeit with a considerable risk of GVHD. Importantly, immunophenotypic and gene expression markers of T-cell activation may be able to identify patients who are primed to develop GVHD following nivolumab treatment and therefore guide the use of this powerful treatment strategy to those patients least likely to develop significant toxicities.
A Recipe for Disaster: Impact of extreme weather on nutrition and metabolic health, with a case study of Aboriginal and Torres Strait Islander communities in Northern Australia
BACKGROUND: To date, the effects of extreme weather events (EWEs) on specific nutrients within the population’s diet have not been quantified. With climate change projected to increase the severity of extreme weather across the globe, it is necessary to understand how the global nutrient supply has historically responded, and which subpopulations are most at risk. One particular subpopulation of importance is Aboriginal and Torres Strait Islander people, who are already more susceptible to nutritional insult and metabolic syndrome than their non- Indigenous counterparts. While extreme weather is not the primary driving force in poor health outcomes, I hypothesise that it further exacerbates cardiometabolic health burdens globally, and especially among vulnerable populations. METHODS: In my first study, I conducted superposed epoch analysis to calculate the percentage change in nutrient supply during the year of an EWE relative to its five-year window. I composited the results globally and by United Nations designated low-income subgroups. I also reported changes in terms of Recommended Dietary Allowance (RDA) for children aged 1-3 years. In my second study, I constructed a holistic model with data from 104 Aboriginal and Tor- res Strait Islander communities in the Northern Territory from 2010-2015. I used varimax- rotated Principal Component Analysis to extract and condense information from 218 variables for physical environment, census, and climate data. I then conducted MM-estimator regression to model the extreme heat impacts on cardiometabolic-related rates for emergency room, inpatient admission, primary healthcare, and mortality. RESULTS: In the first study, most micronutrient supplies exhibited modest negative percentage change during the year of an EWE, including folate, magnesium, niacin, phosphorus, potassium, thiamin, vitamin B6, vitamin C, and zinc. Effects were magnified among Landlocked Developing Countries, which exhibited significant nutrient supply changes ranging from 1.6 – 8.0% of average supply. The observed nutrient supply deficits were found to be a large percentage (up to 41.5%) of what a healthy child’s average sufficient dietary intake level should be. The most compelling finding of the second study is that holding all else fixed, a unit in- crease in climate PC1 score is associated with a 77.8% change in emergency room rates, 6.7 percentage point increase in inpatient rates, and 9.3 percentage point increase in primary healthcare rates. Thus, a larger cardiometabolic health burden is strongly correlated with communities that experience relatively lower apparent temperature and humidity but greater maximum heatwave severity, e.g. Alice Springs area. CONCLUSION: The EWE effects on nutrient supply are modest in isolation; however, in the context of nutrient needs for healthy child development, the effects observed are substantial. Children are a particularly vulnerable subpopulation of interest, given how certain nutritional deficiencies during gestation and the first five years of life can have irrecoverable consequences for health, growth, and development. In the second study, I found that relative heat – or sudden rises in temperature above that of the recent past – poses the most significant threat to cardiometabolic health among Indigenous communities in northern Australia. Relative heat, rather than absolute heat, should therefore be the focus of public health preparation and response. Both studies echo previous literature in stressing the urgency with which the international community must tackle the issue of nutritional and metabolic health among vulnerable populations, especially because this health landscape is poised to worsen with climate change.
Novel Personalised Determinants of Atrial Substrate in Atrial Fibrillation
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia encountered in clinical practice in developed countries, with a rising incidence reaching epidemic proportions. Beyond adverse impacts on quality of life, AF is associated with significant morbidity, heart failure, stroke and a markedly higher risk of mortality. Current understanding incorporates contributions from focal triggers and remodelled atrial substrate, however the precise interactions between these mechanisms remain incompletely understood. The paradigm of AF management over the last decade has evolved to a more lifestyle directed and holistic approach rather than basic pharmacological rate versus rhythm control measures. However, the AF disease process is multifactorial and the optimal treatment of particularly persistent forms of AF continues to be elusive. This thesis aims to evaluate novel mechanisms and influences on electroanatomic atrial substrate contributing to AF which may form the basis for emerging strategies in personalised AF therapy. Initially, we assess the genetic predisposition to AF by effects on atrial substrate and post AF ablation outcomes. We explore the impact of novel pacing strategies and sex-differences on atrial substrate in patients with AF. Finally, we define the influence of AF on sinus node and crista terminalis characteristics. Chapter 1 delineates the role of genetics in AF, a rapidly progressive area in cardiovascular medicine. We then explore the evolving understanding of pathogenesis of the AF mechanism with an emphasis on the impact and importance of atrial substrate, sex-differences and sinus node remodelling. Chapter 2 investigates the impact of genetic susceptibility in patients with AF to electrical and structural remodelling and outcomes. Chapter 2 is a prospective cohort study of 102 patients undergoing AF ablation who undergo genetic sequencing for a 4q25 single nucleotide variant (SNV) and high-density electroanatomical mapping of their left atria. The genetic aspects were completed under the supervision of Prof Diane Fatkin’s Inherited Heart Diseases Laboratory at the Victor Chang Cardiac Research Institute. We document long-term outcomes 2 years post ablation utilising high intensity monitoring including insertable loop recorders and regular Holter monitors. We compare carriers and non-carriers to determine whether there are differences in electrophysiological and conduction properties between groups. We conclude that the 4q25 variants is associated with adverse atrial remodelling characterised by greater conduction heterogeneity and presence of complex fractionated signals with poorer long-term outcomes. Chapter 3 and 4 examine the impact of pacing strategies on electroanatomic atrial substrate in patients with AF. Chapter 3 describes rate-dependent conduction differences in maps created at different cycle-lengths in 56 patients with a history of AF. It observes globally greater atrial substrate at a faster cycle-length across multiple electrophysiologic parameters including voltage, conduction velocity and complex signals. Chapter 4 then evaluates the impact of direction-dependent conduction in 17 patients with AF when pacing from the pulmonary vein. It concludes a highly regional increase in atrial substrate posteriorly. Together, these data suggest the dynamic nature of atrial substrate maps with marked variation according to changes in pacing rate and direction. Chapter 5 focusses on the comparison of electrophysiologic properties underpinning sex-based differences in AF as despite having a lower incidence of AF compared with men, women carry higher risks of stroke and adverse AF-related outcomes. We perform a cross-sectional electroanatomic mapping study of 93 patients with AF and 45 control patients with SVT. Interestingly, in both patients with and without AF, women have a greater degree of atrial substrate when compared with men. Coupled with these substrate differences, we demonstrate that women had higher single and multi-procedure arrhythmia-recurrence following AF ablation. Chapter 6 presents an ultra high-density mapping study investigating the characteristics of the sinus node and anatomically-determined regions of right atrial substrate in relation to AF. We conduct a mapping study on 25 patients with AF and 25 age-matched controls undergoing SVT ablation. Key findings included more significant sinus node remodelling in patients with AF and persistent forms of AF in particular characterised by progressive caudal shifts in sinus node activation, loss of multicentricity, lower sinus node voltage and greater ‘latent’ substrate at the crista terminalis. Chapter 7 concludes the thesis by summarising the pertinent translational findings and implications for the clinical outlook of each study. Moreover, the future directions of novel mechanisms of AF may help pave the way for personalised AF strategies to better treat AF.
Exploring the immunology of small molecule inhibitors in chronic lymphocytic leukaemia
Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the western world. CLL is associated with profound immunodeficiency which leaves patients with insufficient anti-tumour immunity and is often compounded by immunosuppressive treatments. Recent advances in the development of targeted small molecule inhibitors, including Bruton’s tyrosine kinase (BTK) and B-cell lymphoma 2 (Bcl-2) inhibitors, has provided new, highly effective and less immunosuppressive treatment options. However, targeted therapies have not yet provided a cure for CLL, and there is still a need to improve the depth and durability of patient responses. Immunotherapies harness the patient’s own immune system to target cancer cells and are an attractive modality for combination with targeted therapies. However, by both on- or off-target mechanisms targeted therapies also been shown to have the potential to disrupt normal immune populations. This study aimed to understand the effects of targeted therapies on the immune system, thereby providing insight into the potential opportunities and pitfalls for the combination of immunotherapy with targeted therapies in CLL. This thesis explored the immunological impacts of the first-generation BTK inhibitor ibrutinib, as well as two more selective second-generation inhibitors zanubrutinib and acalabrutinib. The Bcl-2 inhibitor venetoclax was also investigated both as monotherapy and in combination with BTK inhibition. In Chapter 3, gene expression analysis was used to faithfully replicate a comprehensive immune profile comparable to more time consuming and costly flow cytometry analysis. Chapter 4 demonstrated that BTK and Bcl2 inhibitors had distinct effects on the immune profile of patients after long-term treatment with targeted therapies. Chapter 5 utilised in vitro functional assays to demonstrate how effector functions of T cells and natural killer (NK) cells, including cytotoxicity, cytokine production, and proliferation, are impaired by BTK inhibitors whereas NK cells, but not T cells were sensitive to venetoclax treatment. Subsequently, Chapter 6 explored the effect of the immunomodulatory drugs lenalidomide and a PD-1 inhibitor BGB-A317 in combination with ibrutinib, showing that T cell activation is modulated by both ibrutinib and the immunotherapies. These finding presented in this thesis demonstrate that targeted therapies have clear and distinct immunological effects. Ibrutinib broadly impair the function of multiple immune populations and in particular cellular cytotoxicity, suggesting that more selective BTK inhibitors are better candidates for combination with immunotherapies. Venetoclax affected the survival of specific T cell and NK cell populations, suggesting that long-term venetoclax treatment may result in alterations in the immune profile that may be exploited by combination therapy. These findings highlight the importance of understanding the immunological impacts of targeted therapies when incorporating immunotherapies into treatment combinations.
Refining an in vivo prion disease model to facilitate characterization of the toxic molecular species and mechanisms of acute neurotoxicity
Prion diseases are rare, fatal, transmissible neurodegenerative diseases associated with misfolding and aggregation of the host encoded cellular prion protein (PrPC). They naturally occur in a wide range of mammalian species, including human beings. The most common human prion disease phenotype is Creutzfeldt Jakob disease (CJD), which can arise sporadically, be genetic or be acquired, with sporadic CJD (sCJD) the most common, occurring at a rate of around 1-2 per million per year. The other forms of human prion disease such as Gerstmann Staussler Scheinker syndrome (GSS) and fatal familial insomnia (FFI) are much less common. In Australia, acquired human prion disease accounts for only <1% of human prion disease. Acquired human prion diseases include Kuru, iatrogenic CJD (iCJD) and the variant form of CJD that was transmitted to humans via consumption of affected cattle. Kuru has occurred exclusively in the Fore linguistic group of Papua New Guinea Eastern Highlands and the neighboring peoples with whom they are intermarried. This disease is now most likely eradicated stemming from the effective outlawing of cannibalistic mourning rituals amongst natives. The main pathogenic event in prion disease is the misfolding of PrPC into the altered disease-associated isoform (PrPSc), which accumulates in the CNS typically as extracellular deposits, also constituting the transmissible agent, resulting in synaptic dysfunction and neuronal death through unresolved pathogenic mechanisms. PrPC is encoded by prion protein (PRNP) gene. Human PrPC is translated with 253 amino acids wherein the first 22 N-terminal signal peptide amino acids are removed from PrPC after its transport to endoplasmic reticulum, while the last 23 C-terminal amino acids are cleaved off during the addition of a glycosyl-phosphatidylinositol (GPI) anchor, which serves to attach the protein to the outer surface of cell membrane. The post-translational conformational change of PrPC to PrPSc is associated with simultaneous loss of normal alpha helical content and considerable gain of beta strand secondary structure largely explaining the altered biochemical properties of PrPSc such as detergent insolubility, relative protease resistance and high tendency to aggregate. The misfolding event can be predisposed to by mutations in the PRNP gene associated with genetic prion disease, the acquired presence of PrPSc through horizontal transmission events or occur through a stochastic misfolding event (sCJD). PrPC to PrPSc conversion is an incompletely understood autocatalytic, self-propagation process that requires very close association of the two isoforms such that PrPSc serves as a template for conversion. Over the last few decades, the field of prion research has advanced, with various new techniques including in vivo/in vitro models to better characterise prion disease and better understand pathogenesis. Although not all scientific findings neatly aligned to the protein only hypothesis over the years, the essential validation of this hypothesis over the last 10-15 years has established PrPSc as the principal, if not exclusive, constituent of the transmissible agent (prion) and the principal causative agent of prion disease pathogenesis. Despite meritorious progress in our understanding of prion biology many fundamental questions remain unresolved such as the primary function of PrPC in the brain, the molecular pathway sub-serving PrPC conversion to PrPSc and detailed understanding of the biophysical characteristics of the species underpinning disease transmission and their relationship with those species associated with neurotoxicity. For the development of targeted, effective treatments for prion and indeed other neurodegenerative diseases, the importance of a comprehensive understanding of the pathophysiological mechanisms cannot be over-stated. Serving this purpose, in vivo and in vitro models have been, and remain, critical to helping our understanding in relation to many aspects of prion biology and pathobiology. In the neurodegeneration research field, prion animal models are considered arguably the most authentic in vivo models that exist and have provided valuable and multifarious insights into pathogenesis; however, they are expensive, and their use generally requires lengthy periods with often incomplete delineation of evidence of direct PrPSc neurotoxicity contributing to pathogenesis. To try to overcome some of these limitations but maximise the recognised likely translational value of in vivo prion models, this thesis describes the use and further refinement of an in vivo prion acute neurotoxicity model involving bilateral stereotaxic hippocampal injection of mice with prions from two strains (M1000 and MU02) followed by a battery of cognitive behavioral testing developed in my laboratory that occurs within an eclipse period during which there is no net de novo prion propagation and subsequent brain region specific biochemical interrogation of pre-and post-synaptic markers. The acute in vivo prion neurotoxic model was developed to be utilised to investigate cognitive behavioural changes, as well as correlating molecular pathogenesis within 16 days following acute exposure to prions derived from terminal mouse brains. In previous studies of a PhD student (Senesi 2015), it was demonstrated that stereotaxic injection of 2 micro litre volumes 10% brain homogenates containing M1000 prions into the hippocampal CA1 region corresponded with modest behavioural changes. In my PhD studies, I utilised the brains of these mice to selectively probe for alteration of candidate pre- and post-synaptic markers in the hippocampus and/or cerebral cortex. Such alterations, especially NMDAR2B, pCREB, synaptophysin, and PSD95 reduction in the hippocampus and/or cerebral cortex of M1000 prion injected mice provide insights into possible acute neurotoxicity with the possible disruption of hippocampal dependent learning and cerebral cortical function. In addition, as part of my PhD studies, I also aimed to try to further refine this acute in vivo prion neurotoxicity model to enhance its capacity to induce acute neurotoxicity. To achieve this, I stereotaxically injected a larger 4 micro litre volume of 10% brain homogenate from mice dying of M1000 prion disease into each hippocampal CA1 region, followed by assessment using the same battery of behavioural tests used when 2 micro litre volumes were injected. Unexpectedly, my results demonstrated that Open Field and Barnes Maze testing did not display any evidence of acute M1000 neurotoxicity; however, there was some suggestive evidence of hippocampal dependent visual and spatial memory impairment in Y-Maze testing of M1000 injected mice. In contrast, conditioned fear memory assessments showed evidence of significant (albeit transient) impairment of generating extinction memory in M1000 injected mice at 24 hour following induction of associative learning. In other studies of my PhD research, I evaluated whether acute behavioural and biochemical changes observed when employing the M1000 prion strain would be observed when using another prion strain. To achieve this, I used the MU02 prion strain in the in vivo acute prion neurotoxicity model utilising selected behavioural studies, as well as comprehensive biochemical assessment of molecular pathogenesis over the same four time points examined when using M1000 prions. Although I did not observe any alteration in conditioned fear memory extinction, suggesting variation in the sensitivity of prion strains to behavioural testing when using the in vivo acute prion neurotoxicity model, I did observe selective alteration of pre- and post-synaptic markers in the hippocampus and/or cerebral cortex. My biochemical interrogation showed early stage astrocyte activation in MU02 injected mouse hippocampus and cerebral cortex, which has never been reported in acute in vivo prion models and also suggests that astrocytes may be involved in acute MU02 prion pathogenesis. NMDR subunits also showed significant alteration in MU02 injected mice hippocampus and cerebral cortex supporting that ionotropic glutamate receptors mainly NMDR2A and NMDAR2B are sensitive to acute MU02 prion exposure. The final principal aim of my PhD studies was to assess the relevance of any acute biochemical alterations observed through the in vivo acute prion neurotoxicity model to M1000 pathogenesis occurring late in natural prion disease evolution. To achieve this, I stereotaxically injected into each hippocampal CA1 region 2 micro litre of 10% brain homogenate from mice dying of M1000 prion disease and followed the mice until they reached terminal prion disease requiring euthanising. I then undertook the same detailed biochemical assessment of pre- and post-synaptic markers in the hippocampus and cerebral cortex of these mice. The results demonstrated that most of the pre- and post-synaptic markers were significantly altered in the hippocampus of M1000 injected mice supporting that the hippocampus is very susceptible to chronic M1000 prion pathogenesis. Importantly, the biochemical alterations observed at the terminal stage of disease were essentially identical to the acute molecular pathogenic changes observed in the hippocampus. My findings at the terminal stage of M1000 prion disease offer molecular pathogenic insights into the underpinnings of the behavioural impairments including visual spatial memory deficits observed at the terminal stage in this model. In conclusion, my PhD studies employing an in vivo acute prion neurotoxicity model have: characterized the acute biochemical aberrations in the hippocampus and cerebral cortex focusing on synaptic constituents when employing two different mouse adapted (M1000 and MU02) human prion strains; examined potential correlations of the biochemical changes with observed acute behavioural deficits; and assessed the relevance of the acute biochemical changes to those observed at the terminal stage of prion disease. Future studies would ideally supplement my biochemical observations by employing other techniques such as morphological analyses of the hippocampus, amygdala and/or cerebral cortex to validate and extend my tentative acute pathogenic insights and also the performance of electrophysiological studies of the amygdala and/or hippocampus, especially after conditioned fear memory testing, to try to characterise any synaptic dysfunction occurring as a consequence of the biochemical changes and how this may relate to any acute behavioural alterations. Further refinement of the in vivo acute prion model appears necessary to enhance the acute neurotoxicity so that it can more robustly induce acute behavioural impairments, including independently of prion strain. These refinements could potentially involve enrichment of toxic PrPSc species from brain homogenates for stereotaxic injection by utilizing fractionation and/or immunoprecipitation techniques but also the use of techniques that allow longer-term introduction of toxic PrPSc species such as through osmotic infusion pumps. Moreover, parallel assessments of potentially more sensitive behavioural tests, such as touch screen paradigms would appear worthy of consideration.
Novel mechanisms of Atrial Fibrillation
This thesis has 3 distinct strands comprising of 5 separate studies, all of which evaluate novel mechanisms of atrial fibrillation (AF) in humans. The 3 strands include the impacts of sleep apnoea (OSA) (Chapters 2-4), epicardial adipose tissue (EAT) (Chapter 5) and the crista terminalis (CT) (Chapter 6) on AF mechanism. The first chapter summarizes multiple perspectives that examine the epidemiologic, mechanistic and therapeutic associations between AF and a variety of novel factors that include obstructive sleep apnoea (OSA), epicardial adipose tissue (EAT) and the unique contribution of the crista terminalis, and provide a framework for the subsequent 5 studies. While OSA has been observed to associate with AF, the composition of sleep and its relevance to AF remains poorly described. Chapter 2 evaluates the impact of the various parameters of sleep on the AF phenotype. Specifically, the impact of hypoxia on the atrial substrate is examined. These data suggest that hypoxic burden is an important factor for determining the progression of AF and provides new insights for the pathophysiologic relationship between these 2 common disorders. Chapters 3 and 4 examine the impacts of OSA and OSA management on the atrial substrate for AF. By utilizing high density mapping, chapter 3 describes the dose dependent relationship of OSA severity on the electroanatomic substrate for AF and its relationship with the AF phenotype. It observes that the strongest association between OSA and AF are observed in the paroxysmal AF cohort with severe OSA. Chapter 4 then evaluates the impacts of OSA management on reversal of atrial remodeling in OSA by utilizing a randomized controlled trial design. OSA management reverses atrial remodeling in AF. Together, these data suggest a tacit relationship between OSA and atrial remodeling, with potential for clinical reversal. Chapter 5 focuses on the impacts of EAT content on the atrial substrate in humans assessed using radiological, electrophysiologic, histologic and molecular techniques. The histologic and molecular aspects of this project were completed with the assistance of the Cardiac Phenomics Laboratory of Professor Lea Delbridge at the University of Melbourne. We observed that local EAT depots associate with local electrophysiologic, histologic and molecular changes, implying a mechanistic relationship between the 2 factors. Chapter 6 presents an epicardial high density mapping study evaluating the role of the crista terminalis (CT) for maintaining persistent AF. It assessed the electrophysiologic substrate in patients without AF and then describes the dynamics of atrial activation during persistent AF in a separate patient cohort. In addition to identifying local electrophysiologic anisotropy, this project observed increased prevalence of circuitous activation at the CT relative to other atrial regions that often associates with lines of transient block. These observations raise the proposition that the CT forms an important substrate for maintaining persistent AF. Chapter 7 concludes by summarizing the key findings of the studies and their clinical implications. Further, it paves the way for future work that might progress our understanding of AF, especially in light of new and novel mechanisms.
Manipulation of Natural Killer cells to improve stem cell transplant outcomes
Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an effective and potentially curative treatment for many haematological malignancies due to its graft-versus-leukemia (GVL) effect. In order to achieve sustained engraftment after donor cell infusion, conditioning regimens before transplantation are required to deplete recipient immune cells. Myeloablative total body irradiation before transplantation is commonly used as an immunosuppressive method to allow recipients achieve donor cell engraftment. However, myeloablative conditioning (MAC) is organ toxic and tissue damage resulting from high dose of irradiation triggers pro-inflammatory cytokine release and inflammation inducing donor T cells activation, expansion then attack of host organs, which is defined as graft-versus-host disease (GVHD), the major complication and contributing factor to treatment-related mortality (TRM) after transplantation. Therefore, to improve prognosis and apply alloHSCT to patients who are not eligible for alloHSCT based on conventional intensive conditioning regimens, reduced intensity conditioning (RIC) was developed in recent years. Researchers have found patients with RIC treatment exhibited reduced TRM, but higher relapse rate compared to the MAC group, and results from experiments on mouse models revealed recipients that received RIC rejected the donor graft. In summary, although RIC has the potential to mitigate GVHD post-transplantation due to reduced toxicity to organs, it is insufficient to ensure sustained engraftment and GVL effect on its own. Previous studies have proven that the GVL effect is intertwined with GVHD, therefore it is a clinical aim to achieve a balance between the two, ensuring engraftment and tumor cell eradication, meanwhile mitigating complications and prolonging survival. RIC is less toxic to organs and has the potential to reduce GVHD but cause graft failure due to insufficient inhibition of the recipient immune system. Our previous study has found that Natural Killer (NK) cells were the primary residual cell population after reduced TBI, and more radio-resistant than CD8+ T cells in recipient mice. Mouse alloHSCT recipients with complete inhibition of NK cell cytotoxic function via perforin knock out exhibited rapid donor cell engraftment but early onset of acute GVHD. Moreover, NK cell adoptive therapy is becoming increasingly utilized as an anti-tumor immunotherapy, in addition to the supplementation of donor T cells in a HSCT scenario. Recent studies demonstrated infusion of donor NK cells is beneficial to promote donor cell engraftment and augment GVL effect while reduce GVHD by inhibiting alloreactive donor T cells and killing recipient APCs. Collectively, although RIC is less toxic to organs and has the potential to reduce GVHD, it is insufficient to deplete recipient immune cells resulting in graft failure after alloHSCT. Manipulation of recipient and donor NK cells combined with RIC using murine models allows us to explore the optimal method to maintain donor cell engraftment with preserved GVL effect and minimal GVHD, which may be further applied to clinical trials. We hypothesized that improved alloHSCT outcomes could be achieved using reduced intensity conditioning (RIC) in combination with pharmacological inhibition of recipient NK cells and introduction of donor NK cells to 1) promote engraftment; 2) reduce GVHD; and 3) exert GVL effect.
Dissecting the cellular mechanisms of NMDA receptor antagonist-induced disruptions to working memory and gamma oscillations
Schizophrenia is a chronic, heterogeneous psychiatric disorder characterised by the experience of a suite of positive, negative and cognitive symptoms. Neural oscillations in the gamma frequency range (30-80 Hz) are associated with some of the same cognitive process disrupted in schizophrenia. Gamma oscillations arise from a circuit between inhibitory parvalbumin-positive (PV+) interneurons and excitatory pyramidal cells. PV+ interneurons and gamma oscillations are both altered in schizophrenia, inviting the hypothesis that PV+ interneuron dysfunction drives gamma deficits and that this underlies cognitive impairment in schizophrenia. Schizophrenia-like behavioural impairments and gamma disruptions can both be modelled in rodents by administering NMDA receptor (NMDAr) antagonists, such as MK-801 and ketamine. NMDAr hypofunction rodent models have been widely used to explore the functional relevance of PV+ interneuron dysfunction and gamma oscillatory disruptions to cognitive impairment. The first aim of this thesis was to further examine whether gamma oscillatory disruptions are mechanistically linked to cognitive impairment. Second, this thesis examined whether NMDAr antagonists act at the cell types that generate gamma activity to disrupt gamma and cognition. I first assessed whether gamma and working memory impairments induced by MK-801 could both be recovered with an agonist of the metabotropic glutamate receptor type 2/3 receptor (mGluR2/3), LY379268. MK-801 impaired performance on the trial-unique nonmatching-to-location (TUNL) test of working memory in the rodent operant touchscreen system and decreased auditory evoked gamma power. It also increased ongoing gamma power and regional gamma coherence. Pre-treatment with LY379268 recovered the MK801-induced increase in ongoing gamma power and regional gamma coherence but failed to improve the reduction in evoked gamma power and working memory. This suggested that aberrant ongoing gamma and regional gamma coherence may not be mechanistically linked to working memory impairment. Next, I examined the contribution of the NMDAr on PV+ interneurons and pyramidal cells to working memory impairment induced by MK-801. Excision of the obligatory subunit of the NMDAr, GluN1, was driven in either PV+ interneurons or forebrain pyramidal cells, expressing calcium/calmodulin-dependent protein kinase II alpha (CaMKIIa). PV GluN1 KO and CaMKIIa GluN1 KO mice performed at the same level as their wildtype (WT) littermates on the TUNL test of working memory. Interestingly, PV GluN1 KO mice were sensitised to the MK-801-induced decrease in accuracy and increase in perseveration on the task. In contrast, the response to MK-801 was no different in the CaMKIIa GluN1 KO mice compared to WTs. This suggests that NMDAr hypofunction at PV+ interneurons or pyramidal cells is not sufficient to impair working memory. Further, while the NMDAr on neither cell type exclusively mediates the effects of MK-801 on working memory, NMDAr hypofunction on PV+ interneurons may sensitise circuits for NMDAr hypofunction at other cell types to impair working memory. Lastly, I examined whether ablation of PV+ interneurons in the medial prefrontal cortex (mPFC) could disrupt gamma activity and if this manipulation would affect the MK-801-induced deficits in gamma. To achieve this, diphtheria toxin (DT) was infused into the mPFC of mice expressing the diphtheria toxin receptor (DTR) exclusively in PV+ interneurons, expected to drive loss of that cell only in the infused region. Infusion of DT did not alter ongoing gamma, evoked gamma or regional gamma coherence. It did, however, blunt the MK-801-induced increase in ongoing gamma power, while the decrease in evoked power and increase in regional coherence were unaffected. This suggests that PV+ interneurons may play a redundant role in maintaining gamma activity under normal conditions, but this cell type contributes to the increase in ongoing gamma power induced by NMDAr hypofunction. To summarise, my thesis shows that the effects of NMDAr antagonists on ongoing gamma, evoked gamma and regional gamma coherence are likely mediated by different mechanisms. Further, the different gamma disruptions may be functionally relevant to specific behavioural impairments arising from NMDAr hypofunction. More specifically, NMDAr hypofunction at PV+ interneurons can be linked to increased ongoing gamma power but may not contribute to the decrease in evoked gamma power. Further, elevated ongoing gamma power might not be mechanistically linked to working memory impairment. Lastly, NMDAr hypofunction at PV+ interneurons may not be sufficient to impair working memory, but it could exacerbate the effects of NMDAr hypofunction at other cell types on this process. In all, a complex relationship appears to exist between NMDAr hypofunction, PV+ interneuron dysfunction, gamma deficits and cognitive impairment. This guides future studies in identifying biomarkers and potential treatment targets for the cognitive symptoms of schizophrenia.
Investigating the immune landscape in gastric cancer
This thesis investigates the relationship between the immunological microenvironment and clinical outcomes of patients diagnosed with gastric cancer (GC). I conducted a comprehensive study integrating immune cellular and molecular analyses of tumour tissues as well as paired peripheral bloods to investigate the role of T cells on clinical outcomes. Gene expression data from gastric tumours (n=100) and non-tumour gastric tissue (n=50) from a prospectively collected cohort (MAUGIC) was analysed using multiple bioinformatics tools to reveal that immune-related pathways and genes were enriched in gastric tumours. This enrichment emphasise T cells play a fundamental role in tumour biology and warrant detailed examination. The immune landscape of gastric cancer was based on a whole-slide multiplex immunohistochemistry (mIHC) platform which allowed both density and distance analysis of immunological components of the microenvironment. A novel algorithm measuring spatial distance relationships between cells, termed Intercellular Spatial Analysis Tool (ISAT) was developed. ISAT calculated the parameter, median intercellular nearest (MIN) distance, to reveal spatial characteristics relevant to dynamics of the tumour microenvironment. It was revealed that the EBV positive and microsatellite unstable (MSI) molecular subtypes showed a robust immune response. The difference in immune characteristics was not strong between intestinal and diffuse subtypes. The association of gastric cancer patients’ outcomes and the immune context in the tumours was further explored using mIHC in FFPE sections (n=56) and the transcriptome profiling data from paired tumours (n=40). It was discovered increased CD4+FOXP3+ T cell density in tumour correlated with prolonged survival. ISAT algorithm revealed CD4+FOXP3+ T cells clustered with CD8+ T cells rather than tumour cells. High density of CD4+FOXP3+ T cells and CD8+ T cells (High-High) predicted prolonged patient survival, and this was validated in an independent cohort (n=84). Gene expression profiling from paired tumour samples showed an interferon-gamma gene signature that was up-regulated in these High-High tumours and this gene signature was validated in two public gastric cancer datasets (n=876 total). Importantly, it was further revealed the High-High group also had prognostic benefit in genomically stable (GS) and chromosomal instability (CIN) molecular groups. No data to date showed these two groups were associated with activated host immunity. However, CIN and GS tumours represent the majority of gastric cancer patients (70% combined). The potential immune dysfunction mechanism/s in the High-High and Low-Low GS/CIN tumours on the systemic level was further explored by T-cell receptor sequencing and mass cytometry techniques using tumour tissue and matched peripheral blood. It was revealed that the High-High GS/CIN tumours were coupled with an increased interferon-gamma response, antigen presentation, dendritic cell differentiation and PDL1 up-regulation in the local tumours, as well as enrichment of Tbet+ CD4+ T cells and central memory CD4+ T cells circulating in the peripheral blood. In contrast, the Low-Low GS/CIN exhibited a high frequency of PDL1+ dendritic cells in the peripheral blood and low immune infiltrates in the tumour. In conclusion, using a combination of gene expression analysis, multiplex immunohistochemistry, T-cell receptor sequencing and mass cytometry, I have found a novel immunological clustering between CD8+ T cells and CD4+FOXP3+ T cells that identify patients with good prognosis and may serve as a novel biomarker to broaden targeting of immune checkpoint therapy beyond EBV/MSI GC, to include a significant number of GC patients with GS/CIN subtype. The findings from this study will significantly facilitate disease diagnosis and prognosis and aid in establishing precise immunotherapy treatment to individual gastric cancer patients, concurrently improving their overall survival outcome.
Head control and infant plagiocephaly: towards prevention
The thesis investigated infant head control development for a new approach to prevention of deformational plagiocephaly associated with infants sleeping in supine (Back-to-Sleep) and reduced rates of Sudden Infant Death Syndrome (SIDS). Standard prevention advice is counter positioning and prone play or tummy time but has limited evidence. Reports of delayed development associated with plagiocephaly led to examination of infant head control as there had been no previous studies for this within a dynamical systems framework. The objective was to explore infant plagiocephaly aetiology and prevention strategies in relation to head control development in infants and develop more effective prevention advice. Mixed methods were used, including reviews, a survey of plagiocephaly awareness in community health professionals and an audit of specialist plagiocephaly clinics. Observational studies investigated the pull to sit test in three cohorts of infants: 1) infants with plagiocephaly; 2) 4-month-old infants; and 3) infants aged 1-4 months. A fact sheet for infants aged 2-8 weeks was developed and evaluated by an expert focus group. Reviews confirmed that the aetiology unclear, but the environment was a factor and tummy time promotes only prone related motor skills. The survey of community Maternal Child Health (MCH) nurses (n=183) and paediatric physiotherapists (n=16) confirmed a gap in plagiocephaly prevention advice. MCH nurses’ primary strategy was referral, thus delaying intervention at a critical age. A 3-year audit of specialist plagiocephaly clinics (n=4000 reviews, n=1990 individuals) revealed delays in consultation. Infants with plagiocephaly, aged 7 SD2.5 months (n=24) showed that they had poor antigravity head control and adopted an extended posture in supine for the pull to sit. A cross-sectional study of normal infants aged 4-months (n=53) showed variation in pull to sit. Over half had poor chin tuck (51%), early head lag (66%) and extended posture (57%). (3) An investigation of head control development over time of infants in the community aged 1-4 months (n=32) including some with plagiocephaly (n=5), all achieved antigravity head flexion control with chin tuck and flexed posture for pull to sit by 3-4 months. The variations observed in the ability of infants to flex their head and neck against gravity or engage associated arms, trunk and hip flexion posture for pull to sit enabled a new operational definition of head control. The 1 to 4-month normal group achieved head control with postural adjustments, chin tuck and flexion, due to the early experience and modelling by a physiotherapist. A new Baby’s Head Shape Fact Sheet: face time plus tummy time equals head control, approved by an expert focus group at the Royal Children’s Hospital (RCH), is to counter infants spending awake time in supine and not developing competent antigravity head control. It is designed for use by Maternal Child Health (MCH) Nurses and physiotherapists in for infants 2 to 8 weeks, the aim being to encourage parents to interact with their baby, encourage early volitional head rotation and development of antigravity neck strength for postural transitions. The implementation of the Baby’s Head Shape Fact Sheet forms a starting point for research to reduce rates of plagiocephaly.