Medicine (RMH) - Theses
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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.
Hereditary Haemorrhagic Telangiectasia: Genotype, phenotype and clinical practice
Hereditary Haemorrhagic Telangiectasia (HHT) is caused by pathogenic mutations in one of several genes, most commonly ENG, ACVRL1 or SMAD4. Characterised by dysregulated angiogenesis in multiple organs, it typically presents as bleeding from nasal telangiectasia and gastrointestinal arteriovenous malformations (AVMs). Lung, brain and liver AVMs are also common. As the genes causing HHT are integral to many fundamental cellular pathways additional to angiogenesis, it is likely that occult phenotypic features exist, but are incompletely explored. The studies described in this thesis aimed to document, explore and extend the phenotype of HHT in the Royal Melbourne Hospital HHT patient cohort, utilising basic disease pathophysiology to hypothesise the presence of additional occult phenotypic features, and to explore novel options for therapy. As Vascular Endothelial Growth Factor (VEGF) is known to be mechanistically involved in the abnormal angiogenesis of HHT, monoclonal antibody against VEGF has been used to treat epistaxis. Adverse reactions to systemic use have stimulated interest in topical intranasal administration, although results of previous studies have been mixed. We hypothesised that efficacy may be masked by nasal crusting, preventing the drug from reaching its target receptors. In a pilot open-label study (Chapter 2), topical Bevacizumab, when administered after standardised washout, reduced epistaxis severity. Given access to best care, HHT patients have normal longevity, but Quality of Life (QoL) may be overestimated. Using standardised tools, QoL assessment (Chapter 3) indicates significant disease impact, particularly on pain and discomfort. The propensity to develop AVMs in internal organs in HHT reportedly varies between the two commonest causative genes, ENG and ACVRL1. Lung AVMs are reported more commonly in ENG mutations, and central nervous system AVMs in ACVRL1 mutations. Disturbed angiogenesis may extend to aortic abnormalities. Analysis of aortic dimensions in HHT patients (Chapter 4), across the three most common HHT genes, revealed a limited association between SMAD4 mutation and proximal aortic enlargement. All HHT mutations result in perturbed TGF-Beta physiology, and TGF-Beta is integral to multiple immune system functions. While major immune disturbance is unlikely, decreased natural killer (NK) cell function could compromise normal immune surveillance capacity without being overtly obvious. NK cell function in HHT is explored in Chapter 5. Chapter 6 explores potential renal involvement in HHT, with no phenotype found. As the physiological link between BMP9 and ALK1 could predict disturbed calcification in HHT, circulating calciprotein particles were assayed. Beyond the sequencing of individual genes, Chapter 7 describes utilisation of massively parallel sequencing to attempt to define genotype in patients with classic phenotype but negative routine four-gene testing. Undertaking clinical research in rare diseases requires an understanding of patients’ perspective. Time constraints limit researcher-patient dialogue in the clinic, especially when multiple organ systems demand attention. The “family day” of Chapter 8 was designed to facilitate trilateral communication between patients, researchers and clinicians, and to evaluate its effectiveness.
Early intervention models of diabetes care to address adverse glycaemia in hospital
Diabetes affects one quarter of individuals in hospital and contributes to worse clinical and economic outcomes. Acute hyperglycaemia causes immune dysfunction, proinflammatory and prothrombotic changes, and endothelial dysfunction, leading to increased risk of hospital-acquired infections, and cardiovascular and renal complications. Acute hypoglycaemia also causes proinflammatory and prothrombotic changes, endothelial dysfunction, and neuroglycopaenia-related complications. ‘Adverse glycaemia’ describes both extremes of hypoglycaemia and hyperglycaemia (defined as glucose <4 or >15 mmol/L), that are associated with adverse pathophysiology and adverse outcomes in hospital. Adverse glycaemia remains common in hospital patients due to various barriers including clinical inertia. This thesis aimed to develop and investigate a strategy of proactive care and early diabetes intervention to address adverse glycaemia in hospital. A glucose alert system, comprising a novel clinical escalation tool coupled with alert-capable networked glucose meters, was developed to decrease clinical inertia (Chapter 3). In a 14-week, pre- and post- implementation study, the glucose alert system increased nursing and medical staff actions in response to adverse glycaemia, and this translated to a reduction in the incidence of hyperglycaemia. Networked glucose meter technology was then implemented on eight noncritical medical and surgical care wards at the Royal Melbourne Hospital, enabling detailed baseline assessment of inpatient glycaemia (Chapter 4). In this first detailed glucometric analysis of an Australian hospital, our cohort was found to have a higher incidence of hyperglycaemia but a lower incidence of hypoglycaemia compared to benchmarks in the United States hospitals. A novel glucometric measure of ‘adverse glycaemic days’, defined as patient-days with glucose <4 or >15 mmol/L, was proposed as a useful metric for benchmarking, and as a tangible concept for educating health professionals about safe glycaemic control in hospital. A comprehensive early intervention model of diabetes care was developed and investigated in the Randomised study of a Proactive Inpatient Diabetes Service (RAPIDS) (Chapter 5). The early intervention model included remote glycaemic surveillance and proactive management of all diabetes patients, by an inpatient diabetes team within 24 hours of admission. RAPIDS, a 24-week cluster randomised trial with a baseline period, involving 1002 consecutive patients, is amongst the largest randomised trials of inpatient diabetes care to date. Early intervention decreased the incidence of adverse glycaemic days by 28%, and decreased severe hyperglycaemia (patient-days with mean glucose >15 mmol/L) by 55%. This intervention was associated with an 80% relative risk reduction (and 4% absolute risk reduction) of developing hospital-acquired infection. Lastly, a prediction tool to enable early identification of diabetes inpatients at high risk for persistent adverse glycaemia was developed (Chapter 6). A prediction tool based on four clinical factors available at admission (glucose at admission, glucose-lowering treatment regimen, glycosylated haemoglobin and glucocorticoid medication), accurately identified high-risk patients, and may assist delivery of targeted management. The studies describe models of clinical care which may be implemented as stand-alone or as a bundle of interventions. The findings support the strategy of proactive care to improve inpatient glucose. Proactive and early intervention models of care which improve glycaemia may improve the care of individuals with diabetes in hospital.
Advance care planning in rehabilitation settings
Advance care planning (ACP) is the process whereby patients are able to communicate with family and healthcare providers regarding future health care choices in the event that they do not have the capacity to do so. In Australia, people are living longer with an increasing aging population and higher rates of chronic illnesses. Significant costs are involved in managing people with chronic illnesses including hospitalisation, rehabilitation and supportive care. ACP is known as an effective tool used in future care planning, and has many known benefits, including improving patient autonomy, enhanced shared decision-making processes with clinicians, better satisfaction with end-of-life (EOL) care, reduced rehospitalisation rates, and improved quality of life for patients and families. Despite known benefits of ACP, its evidence in rehabilitation settings and in specific populations such as cancer patients remains limited in the literature. There have been previous educational efforts with ACP in outpatient rehabilitation settings such as pulmonary and cardiac rehabilitation. However, beyond this, existing literature remains scarce on overall evidence of ACP in rehabilitation settings, in those nearing the EOL, and in those at risk of cognitive impairment. The main aim of this thesis is to investigate the evidence of ACP interventions in patients with cancer, including those with brain tumours (BT) and in rehabilitation settings. Four studies were developed in this thesis to address current gaps in evidence-based practice in ACP in these populations. A standardised framework was utilised to develop protocols for these studies. Firstly, a systematic review was developed to look into existing research regarding the efficacy of ACP interventions in patients with BT who are often managed in rehabilitation settings, at risk of cognitive impairment and nearing EOL. Two studies were then designed to investigate the experience of ACP in patients with BT. Of these two studies, one represented a pilot study to explore the experience of ACP in patients with BT in a tertiary hospital in Australia, and another was an extension of this study to further explore the experience of ACP in a larger cohort and included carer burden. Lastly, an ACP program was implemented in an inpatient rehabilitation setting in a tertiary hospital setting to address staff and systemic barriers to implementation and to improve ACP uptake. Gaps in evidence in relation to barriers and facilitators to ACP implementation in rehabilitation, and types of ACP interventions that can be implemented were identified and recommendations made to further develop and enhance ACP programs, and guide future research. Study 1 presented a systematic review of ACP in patients with primary malignant brain tumours (pmBT). There was only “low to moderate” evidence for ACP studies in this cohort. Only a single randomised controlled trial (RCT) evaluated a video decision support tool in facilitating ACP in patients with pmBT which showed a beneficial effect in promoting comfort care and confidence in decision-making. Positive effects of ACP included lower hospital readmission rates and intensive care unit utilization. None of the studies assessed mortality outcomes associated with ACP. The study highlighted that although there were some beneficial effects of ACP in pmBT population, the literature remains limited in this area with lack of intervention studies, and further studies with appropriate study design, outcome measures and defined interventions are required. Studies 2 and 3 investigated the awareness and experience of patients with BT in discussing ACP. Study 2, an initial pilot study, investigated the experience of patients with BT in discussing ACP, identified main symptoms experienced, physical and functional status, perceived quality of life (QoL) and level of coping. Qualitative analysis indicated good QoL and the use of problem focused coping strategies, however findings demonstrated limited awareness, understanding and documentation of ACP and variable views on appropriate timing of ACP discussions. Study 3 is an extension of this initial pilot study and further investigated a larger cohort of patients with BT, with the addition of assessing carer burden. The majority of patients had high grade gliomas, and there was a moderate level of carer burden. The ongoing limited ACP discussions between patients and healthcare professionals demonstrate the need for increased awareness of ACP in clinical practice, and also encourages the neuropalliative-rehabilitation model of care approach which integrates care amongst treating teams including neurology, neurosurgery, oncology, rehabilitation and palliative care to be able to provide timely ACP information to patients, This study also highlighted the need for multifaceted system-wide interventions in implementing ACP. Study 4 was designed to develop, implement and evaluate the effectiveness of an ACP program in an inpatient rehabilitation setting. The implementation of this program showed that a structured ACP program is feasible and effective in improving the prevalence of ACP discussions between rehabilitation patients with chronic illnesses and/or multiple comorbidities and rehabilitation staff, however short-term impacts on Medical Enduring Power of Attorney (MEPOA) nomination and Advance Directive (AD) completion rates remained inconclusive. This was the first study using process evaluation in assessing an ACP program and its feasibility. In conclusion, limited evidence still exist with ACP in patients with BT and in inpatient rehabilitation settings. The implementation of a structured ACP program is effective in increasing ACP discussions between rehabilitation patients and staff, and emphasises a multifaceted approach that is required for its feasibility. It is also easily replicated in other rehabilitation settings. Future larger and longer term follow up studies are still required to assess impact on other outcomes including QoL, quality of care and economic costs.
Non-pharmacological management of chronic pain in multiple sclerosis and rehabilitation outcomes
Multiple sclerosis (MS) is a chronic, inflammatory and demyelinating autoimmune condition which affects the central nervous system. Multiple sclerosis can present with a wide variety of symptoms and can affect a patient’s mobility, sensory and cognitive functions. Pain in multiple sclerosis is common and can present as either acute or chronic pain. Chronic pain is pain which lasts greater than 3-6 months and has significant implications on a patient’s health, disability and quality of life. Causes for chronic pain in MS can range from nociceptive somatic pain to neuropathic pain and specific chronic pain syndromes which include trigeminal neuralgia and peripheral neuropathy. Currently, there is a lack of literature on chronic pain in multiple sclerosis. Given the prevalence of pain, its significant impact on patients and the need for greater evidence for non-pharmacological interventions, further information is required to help with improving identification, assessment and management of chronic pain in multiple sclerosis. In this thesis, four studies address current gaps in the literature and help progress evidence-based practice. Study 1 is a systematic review of the effectiveness of non-pharmacological management of chronic pain in multiple sclerosis. Despite there being very low quality of evidence, it remains unknown what type of non-pharmacological interventions reduce the intensity of chronic pain and improve quality of life and function in multiple sclerosis. This study acknowledges the gaps in the current research, the inherent difficulties associated with the review of non-pharmacological interventions, methodological limitations and appropriate study designs. Study 2 is a 10-year longitudinal study on chronic pain in multiple sclerosis in an Australian cohort. This study showed that over time there was a greater representation of bilateral bodily pain, greater subjective worse pain and deterioration in the quality of life and increase in carer burden. This study suggests that over-time chronic pain is persistent and has a significant impact on people with multiple sclerosis. Study 3 is a retrospective study on the effectiveness of interdisciplinary management of chronic pain in multiple sclerosis and central nervous system disorders. This study accessed clinical data from an Australian and New Zealand-based electronic pain clinic database. From this database; patient outcomes were compared between those with multiple sclerosis and central nervous system disorders and those without these comorbidities. This study showed that those with multiple sclerosis and central nervous system disorders could also benefit from interdisciplinary management and have improvements pain severity scores, catastrophisation, depression, anxiety and stress scores and improvement in pain self-efficacy. Study 4 is a prospective single-blinded randomised controlled trial using transcranial direct stimulation for chronic neuropathic pain in multiple sclerosis. This study showed that those who had received transcranial direct stimulation compared to sham treatment had an improvement in pain visual analogue scale but no change in affective outcome measures, quality of life scores and depression, anxiety and stress scores. In addition to this; the most common adverse effect was mild tingling which was only during stimulation. This study suggests that transcranial direct stimulation is a safe and feasible non-pharmacological intervention for chronic neuropathic pain in multiple sclerosis. This work was completed to test the hypothesis that non-pharmacological interventions can improve patient outcomes. The study conclusions can help with evidence-based recommendations, future-research directions and clinical implementation of non-pharmacological interventions.