Medicine (St Vincent's) - Theses
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Understanding the effects of Radiotherapy and Surgical injury on Local immunity and Lymphatic Architecture and Function
This thesis aims to provide insight into the pathophysiological mechanisms behind two important clinical problems encountered in patients surviving oncological treatments: 1) Infections in previously radiated sites and 2) Lymphoedema that follows surgical injury to lymphatics or radiation of nodal basins (RTx). Both infection and lymphoedema have significant impact on patient morbidity and quality of life (QOL). Lymphoedema, in particular, has a significant “psychological and social health cost”, and is associated with significant costs in providing long term conservative management to prevent symptom progression and maintenance of QOL outcomes for patients. Therefore, socioeconomic implications of these diseases certainly warrants further investigation of the disease pathophysiology and potential avenues for prevention and treatment. This thesis provides a review of knowledge regarding normal structure, function and development of the lymphatic system, lymphangiogenesis and lymphatic remodelling and how these processes relate to lymphoedema. In addition, the processes of antigen capture and transfer to lymph nodes for presentation to lymphocytes will also be addressed as a background to understanding infections in radiated sites. Subsequently, methods to investigate lymphatic morphology utilising a murine Prox1-eGFP model and confocal imaging were detailed, as was an automated segmentation and analysis method to allow high throughput analysis of lymphatic vessel morphology following sugical or RTx injury in detail. Functional techniques utilised to assess antigen presentation and fluid transport to draining lymph nodes (dLNS)have also been described in detail. We have demonstrated in this thesis that RTx significantly reduces CCL21 expression by LECs and has subsequent effects on antigen presentation from RTx tissue. These results have implications for the increased rates of infection noted after RTx. Moreover, RTx significantly impairs early wound lymphangiogenesis in vivo. While surgical injury alone induces remodelling of both lymphatic capillaries and deep collecting lymphatic vessels (CLVs) distal to sites of surgical, combined injury with RTx and Surgery markedly exacerbates these remodelling responses. By using longer time points, we have also explored possible mechanisms behind chronic lymphoedema, and have demonstrated that RTx continues to impair wound lymphatic regeneration in combination with severe surgical injury. Furthermore, the distal limb appears to demonstrate evidence of progressive remodelling at late time points following lymphatic injury. As with earlier time points, combined injury with RTx and Surgery, appears to promote exacerbated secondary lymphangiogenesis in the distal limb at later time points. Potential molecular mechanisms behind these differences between the groups have then been subsequently explored through RNA sequencing and multiple PCR analyses of total tissue mRNA. The broader implications of the findings of this thesis to clinical lymphoedema and RTx are then addressed, as well as potential future directions for research to build on the findings of this thesis.
Associations between Hepatitis B virus mutations and infection kinetics, host responses and outcomes
The hepatitis B virus (HBV) has infected more than 2 billion people worldwide and is associated with a significant global burden of disease due to its main associated complications of cirrhosis and hepatocellular carcinoma (HCC). The risk of developing cirrhosis or HCC is highly variable, with some people who have quiescent disease throughout their lifetime, whilst others develop aggressive disease associated with high mortality and morbidity rates. HBV is prone to mutations during viral replication and, as a result, it acquires multiple mutations due to pressures either from exogenous vaccines or therapeutic agents or from the host’s immune system. This propensity to mutate has resulted in the formation of genotypes and viral variants. Genotypes of HBV have a sequence variation of 8% or more and they have a specific geographical distribution. The most common viral variants of HBV are the basal core promoter (BCP) and precore (PC) mutations. BCP diminishes the production of the e antigen (HBeAg) whilst PC abolishes it completely. The role of the HBeAg is not completely understood. It is not required for infectivity or any major steps of the viral life cycle. It is thought to be an immune-modulator although the mechanisms of immune modulation are yet to be elucidated. There is a robust body of epidemiological evidence that links viral variants and genotypes to distinct clinical phenotypes. However, the actual host-virus mechanistic interactions are unknown. The HBV, in itself, is not generally cytopathic. It is the immune response to the virus that is thought to be responsible for the inflammatory response and subsequent scarring and liver fibrosis. Currently, there is no definitive cure for chronic hepatitis B (CHB) infection. Antiviral therapy suppresses viral replication but does not eliminate the virus. Due to its close interaction with the immune response, an understanding of the host-virus immuno-pathogenesis is needed in order to explore novel immune-based therapies. The first aim of this thesis was to understand the characteristics of CHB in Australia. Using a population sample from a large tertiary Victorian hospital, a detailed study of the virological characteristics including genotypes, and BCP and PC mutations was performed. We focused on the association between virological factors including genotypes and mutations and clinical phenotypes. Genotypes A, B, C and D were the most prevalent, with a clear preservation of the relationship between genotype and country of origin of the patient. Genotypes B and C were the most common, a reflection of the large migrant population from South East Asia. HBeAg-negative CHB was common and was strongly associated with the presence of the PC variant. A large proportion of patients with HBeAg-negative CHB had an active hepatitis and they were in the immune escape phase. BCP and PC mutations were highly prevalent and were independently associated with the presence of advanced fibrosis, regardless of age, sex or genotype. These findings suggest that there may be a role for sequencing for these mutations in clinical practice and use them as biomarkers. Based on this clinical observation, we also hypothesized that HBeAg was modulating the immune response to enable chronicity in early stages of disease but then becomes a liability in later phases. This formed the basis of studies of the immune response with a specific focus on HBeAg. The second aim of this thesis was to explore some of the key immune pathways that modulate HBV infection. The hypothesis was that HBeAg modifies innate host immunity to establish chronic infection in the early phases of disease. Much of the literature in HBV has focused on the adaptive immune response but we were interested in the early phases of the natural history, where HBeAg is almost universally present and its absence is associated with reduced infectivity. The role of HBeAg in the early stages of HBV infection was studied using an immunocompetent mouse model transfected with HBeAg- negative strains of HBV. Pertinent findings included a sudden, early drop in viraemia in HBeAg- negative mutants compared to wild type (WT) HBV. A corresponding transaminitis strongly suggested that in absence of HBeAg, there was a more robust innate immune and inflammatory response to the virus. Complementation studies confirmed that the effect was due to HBeAg and no other viral components. Using gene-modified animals and selective antibody depletion, a series of studies were performed to examine individual arms of the innate immune response. We showed that the Type 1 interferon (IFN-I) response was much stronger in absence of HBeAg. Correspondingly, transcriptomic analysis revealed the downregulation of multiple IFN-I pathways and proteome/ phosphor-proteome studies showed changes in the expression of interferon regulating genes associated with HBeAg. Collectively our data showed that HBeAg allows the virus to evade the innate immune response by antagonising IFN-I in the initial stages of infection. The emergence of HBeAg- negative mutants in the later stages of the natural history of CHB suggests that HBeAg presents a liability to the virus. In our mouse model, following a significant drop in viraemia in the initial phases of infection, there was a recrudescence in viral load in the later stages of disease in HBeAg deficient mutants. We hypothesized that in the later, adaptive immune response, HBeAg is an immune target and there is a strong selective pressure that favours HBeAg- deficient viral variants. A series of experiments using gene-targeted animals was performed to examine the roles of various mediators of the adaptive immune system. These studies showed that the absence of HBeAg was associated with reduced function of CD4+ T cells and a reduction in TNF and IFN- gamma activity. HBeAg deficiency was also associated with an escape from the antibody response. In conclusion, HBeAg is an important antigen in the modulation of the immune response in HBV. Sequencing for HBeAg negative variants may have a role in clinical practice and further longitudinal, prospective studies are warranted. Further dissection of the proteomic and transcriptomic signatures associated with HBeAg negative mutants and their correlation with clinical samples would be the next steps to elucidate the intricate role of HBeAg in modulating disease behaviour and outcomes.
Studies investigating the clinical impact and immunological host response to viral eradication in hepatitis C infection
Australia now has improved access to HCV treatment for individuals with all stages of liver disease. There is limited data however, documenting any long-term clinical benefit of viral eradication in those with early stage liver disease. We report on the long-term outcomes of a well-characterised cohort of CHC subjects with predominantly early stage liver disease, whom paired liver fibrosis assessments were performed more than 10 years apart. We show in a real-world setting, that both early and curative HCV treatment halts fibrosis progression. Our data supports the early treatment of all people with CHC regardless of liver fibrosis stage, to prevent long-term liver sequelae. In subjects with acute / subacute HCV infection, we identify predictors of innate immunological response in peripheral blood mononuclear cells, to differentiate spontaneous clearers to those who progress to chronic infection. Our data strongly implicates TLR4 signaling in the persistence of HCV infection. Those who developed chronic infection had higher TLR4 expression on peripheral monocytes and NK cells and increased IFN-gamma response to TLR4 stimulation. We also observed increased TLR7 responsiveness in this group. We confirm the previously noted associations of IFNL4 genotype and plasma IP-10. Our data presents TLR4 as a potential biomarker for predicting clearance. This would clinically translate to individuals presenting with acute / subacute HCV infection, being able to defer drug therapy and its associated morbidity and cost. In subjects with chronic HCV infection, we demonstrate a clear effect of direct antiviral agent (DAA) mediated viral suppression therapy on patterns of TLR signaling in subjects with chronic HCV-1. We show that peripheral monocytic TLR2, TLR4 and TLR7 signaling is down-regulated early on in treatment, with a strong trend to higher baseline TLR signaling being associated with viral clearance with the DAAs. We are the first study to demonstrate a relationship between TLR signaling activity and IFN-free therapy for HCV. We hypothesise that the HCV virus directly stimulates the TLR pathways to induce an antiviral effect, with higher TLR signaling evident among those who respond to DAA therapy. This was accompanied by a reduction in PBMC ISG expression, NK activation markers and plasma levels of inflammatory cytokines / chemokines, with restoration of the innate immune response. Despite excellent treatment options, the mechanisms responsible for viral eradication in HCV have remained poorly defined. Previous data suggest a link between innate immunity and HCV pathogenesis, as well as spontaneous viral clearance. The data suggested that antiviral therapy alone was not sufficient to clear virus and supported a key role for innate immunity contributing to viral clearance. Our results strongly implicate TLR signaling / expression in HCV viral clearance. We believe TLR agonists must be considered as a potential HCV vaccine candidate and further research in this area is required.
Activation of islet inflammation by cytokine signalling in pancreatic beta cells: understanding the role of protein tyrosine phosphatases
Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. Furthermore, PTPN1 inhibition reduced IFN-g induced MHC-I expression on the surface of NODPI beta-cells and reduced the ability of autoreactive NOD8.3 CD8+ T-cells to destroy isolated NOD/Lt islets. These studies showed that PTPN1 is a positive regulator of cytotoxic signalling in NIT-1 cells and NODPI islets and promotes immune cell mediated death, suggesting it may be a potential therapeutic target for type 1 diabetes. Chapters 5&6 study the role of PTPN6 in cytokine signalling regulation in NIT-1 cells. PTPN6 inhibition was found to enhance TNF-a induced NIT-1 cell death independent of IFN-g and IL-1b in vitro. TNF-a induced JNK signalling was enhanced with PTPN6 inhibition which resulted in reduced anti-apoptotic BCL-2 protein expression and enhanced caspase-3 cleavage. Pan-caspase inhibition prevented TNF-a induced cell death suggesting that cells were dying through apoptosis. TNF-a induced cell death was also prevented with RIPK1 inhibition which prevented enhanced caspase-8 cleavage in PTPN6 deficient cells. Collectively these studies showed that PTPN6 negatively regulates TNF-a induced intrinsic and extrinsic apoptosis of beta-cells in vitro. Overall, the data indicate that PTPs play a nonredundant role in the regulation of cytotoxic signalling in NIT-1 cells and NODPI islets. This finding is consistent with results in other disease pathologies. The results provide a mechanistic insight into how PTPN1 and PTPN6 have opposite roles in regulating cytokine signalling, highlighting how PTPN1 antagonism and PTPN6 agonism may prove beneficial in reducing beta-cell death in vitro. Whether these results are directly translatable into in vivo models of autoimmune diabetes remains undetermined. The use of PTPN1 inhibitors or PTPN6 agonists currently under development would allow direct translation of these results.
The contribution of the pulmonary circulation and cardiac function to exercise intolerance in people with diabetes
Diabetes mellitus (DM), and in particular type 2 DM (T2DM), is approaching epidemic proportions globally. Relative to people without diabetes, exercise performance is reduced by approximately 20% in T2DM subjects universally, whereas impairment in type 1 DM (T1DM) is less consistently reported. Risk of cardiovascular disease (CVD) is significantly elevated in both T1DM and T2DM, and thus emphasis is placed on cardiovascular constraints to exercise capacity, including subclinical cardiac dysfunction and the entity of diabetic cardiomyopathy (DMC). A clear bidirectional association between heart failure and T2DM exists, with numerous pathophysiological mechanisms identified in animal models. Longitudinal data suggests a low prevalence of heart failure in T1DM, on the other hand, which insinuates the pathological mechanisms thought to underpin DMC may be exclusive of chronic hyperglycemia which unifies the diagnosis of T1DM and T2DM. The pulmonary microvasculature is targeted in diabetes, meanwhile, and may be an under-appreciated contributor to exercise limitation. It is thought that the lungs are protected from deleterious effects of subclinical microvascular disease by virtue of their substantial size, although the ability to assess pulmonary microvascular function during exercise has been limited. This thesis assesses exercise performance (VO2peak) in people with T1DM and T2DM, evaluates the contribution of cardiac function and the pulmonary circulation using comprehensive traditional and novel echocardiographic analyses, and investigates the effect of phosphodiesterase type 5 (PDE5) inhibitor Sildenafil on exercise capacity. VO2peak is demonstrated to be impaired in only the T2DM cohort of the study group, and independently associated with sedentary lifestyle and reduced left ventricular end diastolic volume. Comprehensive resting and exercise echocardiographic measurements of biventricular systolic function and LV diastolic function, in addition to speckle-tracking global longitudinal strain and LV twist mechanics, are normal. The novel echocardiographic assessment of pulmonary microvascular function by measuring the pulmonary transit of agitated contrast (PTAC) identifies less PTAC (low-PTAC) in DM subjects, and associations with reduced RV function, higher pulmonary artery pressures, and lower exercise capacity. Finally, Sildenafil improves a number of central hemodynamic parameters but does not improve VO2peak. Results of this thesis support the argument that in the presence of impaired exercise performance and longstanding DM, subclinical cardiac dysfunction and DMC should not be universally anticipated. Diagnosis of pulmonary microvascular dysfunction using PTAC is feasible and readily accessible, and pulmonary microvascular disease is associated with reduced exercise capacity. Nonetheless, quantification of pulmonary microvascular disease remains imperfect, and the pulmonary vasodilator Sildenafil is ineffective in improving exercise performance presumably due to the mismatch between the pathophysiology thought to underpin diabetic lung disease and the more proximal smooth muscle vascular target of Sildenafil.
Molecular Investigation of the Fanconi Anemia DNA repair pathway
Chemotherapeutic drugs often kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs) that inhibit DNA replication or transcription. Cells from patients with the genetic disorder Fanconi Anaemia (FA) are extremely sensitive to such DNA crosslinking agents. A critical step in the repair of ICLs is the biochemical modification of FANCI:FANCD2 protein by monoubiquitination. This monoubiquitination reaction is defective in 95% of FA patients, however the exact function of this process remains unclear. This thesis aims to better investigate how FANCI:FANCD2 monoubiquitination is regulated. To determine how monoubiquitinated proteins regulate the FA DNA repair pathway, several targets of monoubiquitination including FANCI:FANCD2, PCNA trimer and nucleosomes ubiquitinated by BRCA1:BARD1 were purified using Avi-ubiquitin. This thesis presents a minimal approach to purify challenging DNA repair proteins, which could potentially be applied to elucidate signalling mechanisms of other ubiquitination-mediated DNA repair pathways. Biochemical reconstitution of the FA pathway using recombinant human proteins revealed that FANCI:FANCD2 monoubiquitination is dependent on DNA and low ionic strength buffers. In addition, electrophoretic mobility shift assays revealed that monoubiquitination locks FANCI:FANCD2 on double-stranded DNA (dsDNA). Affinity pulldown of monoubiquitinated FANCI:FANCD2 confirmed that the complex remained bound to dsDNA, reflecting the monoubiquitination’s role in stabilising the FANCI:FANCD2 complex. The increased affinity of monoubiquitinated proteins on dsDNA suggests a conformational change on FANCI:FANCD2 after monoubiquitination. Using a newly developed Avi-tag-ubiquitin construct, the first successful purification of dually-monoubiquitinated FANCI:FANCD2 complex bound to DNA was reported. Strikingly, single-particle electron microscopy analysis revealed that monoubiquitinated FANCI:FANCD2 forms oligomers on dsDNA. This unexpected discovery hints that the function of monoubiquitination is to lock FANCI:FANCD2 oligomers on chromatin to initiate DNA repair. FANCI phosphorylation was previously reported to play a key role in stimulating FANCI:FANCD2 monoubiquitination. Mass spectrometry and western blot using phospho-specific antibodies revealed that ATR-mediated FANCI phosphorylation at specific serine-glutamate (SQ) clusters regulate both ubiquitination and deubiquitination of FANCI:FANCD2 complex. These results indicate a mechanistic view where FANCI phosphorylation is required for ubiquitination-mediated DNA repair which gives insights into the biology of FA pathway and potential chemosensitiser development. Collectively, the results presented in this thesis elucidate the role of monoubiquitination in locking FANCI:FANCD2 on DNA to form oligomers to initiate DNA repair. This work uncovers a new role of monoubiquitinated FANCI:FANCD2 during interstrand crosslink repair and provides new potential chemotherapeutic targets.
Novel aspects of commonly encountered pituitary adenomas in clinical practice
Clinically relevant pituitary adenomas are common in the general population occurring in 1 per 865-1470 people. Prolactinomas and non-functioning pituitary adenomas account for more than 80% of pituitary adenoma types. My thesis is composed of several studies, undertaken at a single centre specialising in the multidisciplinary care of pituitary diseases, investigating clinical issues pertinent to the management of patients with the above adenomas. The findings of these studies contribute to the understanding of outcomes in the local practice, as well as being an Australian contribution to the international experience of managing these adenomas. Firstly, I examine the issue of valvular heart disease in prolactinoma patients treated with cabergoline therapy. By studying a local cohort and by a systematic review I found that the prevalence of cabergoline-associated valvular heart disease (CAV) in prolactinoma patients to be extremely low, with just three confirmed cases amongst 1800 patients described in the literature. I have shown evidence that a simple annual cardiovascular examination is a suitable screening tool for this rare condition and made recommendations on when to consider a diagnostic echocardiogram. Secondly, I examine two clinical issues affecting patients with surgically treated non-functioning pituitary macroadenomas (NFPMAs): the issues of regrowth and recurrence, and of hormonal outcomes. In the largest Australian cohort of cases with NFPMAs to be described, it was found that residual disease is a common finding post-surgery. In cases with residual disease, regrowth occurred in 40% of these cases compared to only 12.5% in cases without residual disease, at a median of 3.6 years of follow-up. Not surprisingly, larger baseline adenoma size was a predictor of regrowth and recurrence. In multivariate analysis, the presence of residual disease and younger age (under 41 years) at presentation were independent risk factors for regrowth and recurrences. Based on this risk factor of younger age, I have recommended that these cases have lifelong radiological follow-up. Results of hormonal outcomes in cases with NFPMAs treated with surgery showed the novel finding of gender differences in hormonal outcomes, with males having a higher prevalence of multiple (more than two) hormone deficiencies (MHD) at presentation than females, and after surgery. In particular, pre-menopausal females had very few hormone deficiencies post-operatively and demonstrated a propensity to recover function. These gender subgroup differences are the first to be noted in the literature and have important clinical implications particularly for pre-menopausal females where fertility preservation is an issue. I also found that pre-menopausal females had smaller adenomas than males suggesting that they may present earlier in the natural history of the disease. In multivariate analysis, larger adenoma size remained a significant factor associated with post-operative MHD. The final part of this thesis is an exploratory study of intrasellar pressure (ISP) and how it relates to adenoma size and hormonal outcomes in cases with NFPMAs undergoing surgery. I did not find that ISP was correlated to adenoma size, but results did suggest that raised ISP may play a role in more frequent hormone deficiencies at presentation. In this study adenoma size was again noted to be showing a trend towards influencing hormonal outcomes. The findings from this study will require a larger cohort in order to better quantify how ISP and adenoma size interact to contribute to the pathophysiology of hormone deficiencies. In these three studies of NFPMAs adenoma size has emerged as an important factor influencing surgical and hormonal outcomes; smaller adenomas are more likely to be fully resected and less likely to result in hormone deficiencies. This thesis lays the foundations for advocating for the consideration of early surgical intervention of NFPMAs (when adenomas are smaller and earlier in the natural history of the disease), in order to optimise surgical and hormone outcomes. The above should now be an important clinical consideration in the multidisciplinary care of these patients.
Echocardiographic evaluation of the evolution of cardiac dysfunction in the community and risk of heart failure, using the SCReening Evaluation of the Evolution of New Heart Failure (SCREEN-HF) cohort
Heart failure (HF) is a major cause of morbidity and mortality in the community. Outcomes for HF with reduced ejection fraction (HFrEF) remain poor despite available treatments, and no medical therapy has been shown to improve survival in HF with preserved ejection fraction (HFpEF). Consequently, HF prevention is urgently required to curb the growth of HF in our communities. HF prevention requires that we improve our ability to identify individuals at high-risk of HF who may benefit from preventative measures. Although cardiovascular disease prevention has traditionally focused on risk assessment using cardiovascular risk factors, HF risk is further increased by the additional presence of cardiac structural and functional abnormalities. Cardiac dysfunction detected by echocardiography may therefore aid in improving risk stratification in HF. The SCReening Evaluation of the Evolution of New Heart Failure (SCREEN-HF) study is a community-based evaluation of individuals aged 60 years or over with cardiovascular risk factors followed longitudinally for the development of HF. Baseline transthoracic echocardiographic examinations were performed to document the asymptomatic cardiac dysfunction which preceded the development of incident HFpEF and HFrEF, and follow-up echocardiography was performed approximately four years later to document age-related cardiac changes. Using data from the SCREEN-HF cohort, this thesis investigated the clinical and echocardiographic predictors of incident HFpEF and HFrEF, in order to gain insights into HF development, and the role of echocardiography in the risk assessment of HFpEF and HFrEF. Contrasting clinical predictors of HFpEF and HFrEF provided insight into their underlying mechanisms. Risk factors associated with atherosclerosis, including smoking and abdominal adiposity, predicted only HFrEF, while pro-inflammatory conditions such as diabetes, obesity and renal dysfunction, and additionally high white cell count predicted only HFpEF. These pro-inflammatory risk factors were present approximately four years prior to clinical HFpEF, suggesting that focused risk factor management is an important strategy in HFpEF prevention. Baseline echocardiographic data from over 3000 subjects was used to investigate the role of echocardiography in HFpEF and HFrEF risk assessment. Diastolic dysfunction is an integral feature of HFpEF and diastolic dysfunction determined using current guideline algorithms was shown to predict incident HFpEF. In addition, individual echocardiographic parameters reflecting diastolic dysfunction, long axis systolic dysfunction and increased left ventricular mass predicted both HFpEF and HFrEF, while left ventricular enlargement and low left ventricular ejection fraction predicted only HFrEF. Using combined echocardiographic abnormalities, a low- and high-risk subgroup for HFpEF and HFrEF were identified, and echocardiography also assisted in differentiating between HFpEF and HFrEF risk. Older individuals are more likely affected by HFpEF than HFrEF. Using serial echocardiographic examinations from over 2300 subjects, age-related changes in cardiac structure and function were evaluated to provide insight into the tendency for older individuals to develop HFpEF. The pattern of age-related cardiac remodeling, particularly in women, was found to parallel the cardiac changes seen in HFpEF, providing one explanation for the propensity of older women to develop HFpEF. This thesis provides insights into HF development and provides evidence for the utility of echocardiography in HF risk stratification. These results serve as a platform on which echocardiography-guided management of HF risk can be further evaluated.
Predicting response to anti-TNF therapy in Crohn's disease: clinical and metabolic studies
Crohn’s disease is a chronic, disabling inflammatory condition that affects the gastrointestinal tract that is associated with significant morbidity. Up to 80 percent of patients require surgery at some point in their lives. A third of patients require surgery within five year of diagnosis even in the era of biologic therapy. Ant- Tumour Necrosis Factor alpha (Anti-TNF) therapies form the backbone of drug therapy in patients with moderate to severe Crohn’s disease and remain the most effective biologic drugs available. There are currently three classes of biological drugs available on the Pharmaceutical Benefits Scheme (PBS) for Australians with many more under development. Up to thirty percent of patients fail to have an adequate response to anti-TNF therapy and loss of response occurs at a rate of ten percent per year. There are at present no highly sensitive biomarkers of response to anti-TNF therapies. Without biomarkers to assist with selecting the most effective biologic for a particular patient, there is a risk of exposing patients to ineffective drug therapies with unnecessary side-effects and costs. This thesis comprises a range of clinical and scientific studies that seek to identify predictors of loss of response to anti-TNF therapies in Crohn’s disease. Two types of loss of response have been identified: Primary nonresponse (PNR), is defined as a lack of response to the initial drug therapy as determined at 12 weeks post induction, and Secondary Loss of Response (SLOR) is defined as the loss of response after the patient has had an initial response to the drug. Based on the clinical observation that patients with altered body composition and those with strictures have a variable to response to anti-TNF therapy, we sought to identify whether there were specific clinical, endoscopic, histologic and biochemical parameters that correlated with response to anti-TNF therapy amongst patients from whom longitudinal body composition and endoscopic data had been collected. A prospective cohort study of patients who had been newly started on anti-TNF therapies was also undertaken. In this cohort samples of urine, blood and faeces were taken prior to anti-TNF therapy delivery (baseline) and then at 3 monthly intervals thereafter. A combination of bioinformatic analyses and novel techniques evaluating the metabolome were applied to the cohort, in order to find clinical factors and biomarkers that might correlate with therapeutic response to anti-TNF therapy. This series of studies presented in this thesis on clinical and metabolic predictors of outcomes in patients receiving anti-TNF therapy for Crohn’s disease reveals new insights into the pathophysiology of Crohn’s disease and has identified biomarkers for the prediction of therapeutic response – thereby helping to come a step closer towards the ultimate goal of precision medicine.
Topographic rod function in intermediate age-related macular degeneration
Background Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the elderly. In the early stages of disease, dark adaptation problems are more significant for patients than visual acuity impairment. For decades, studies of rod function related to dark adaptation issues suggested that rod function could be useful as a functional marker for differentiating AMD severity and monitoring disease progression. However, there remains many unanswered questions about how best to investigate rod function in the early stages of AMD. Earlier studies were not able to phenotype AMD cases to the extent we can today as they relied only on colour fundus photographs to determine AMD subgroups. We now have the opportunity to look at different AMD phenotypes using multimodal imaging techniques. These advances have added clarity to the phenotyping of AMD, as high-risk features such as reticular pseudodrusen (RPD), hyperreflective foci (HRF), and nascent geographic atrophy (nGA) can now be identified. At the same time as the advances in detecting anatomical changes were made, instruments that could measure rod function in a more thorough way were improving. Previously, instruments measured rod function at only a single retinal location, or could only detect large losses in sensitivity due to a lack of dynamic range. Recent advances have seen perimeters that are able to measure rod function at multiple locations in the one setting and also have a larger dynamic range to detect subtle rod dysfunction. I have used one of these new tools in my studies. Aims Two-color dark-adapted chromatic perimeter (DACP) is a novel device, designed and manufactured in Melbourne, Australia. It was first used in 2015 when I commenced my PhD. This perimeter measures rod function at multiple locations with sufficiently large range of stimulus intensities to detect subtle changes seen in early AMD. My thesis aimed to investigate the ability of the DACP to reliably detect and record rod sensitivities. I then utilized this new perimeter to investigate the ability of rod function to act as a robust functional biomarker that could be used to determine AMD disease severity and to monitor disease progression, and to compare rod function in AMD cases to normal control participants. I was also able to separate my AMD cohort into those with or without RPD, a high-risk AMD phenotype, to investigate the impact of this phenotype on rod function. Both static and dynamic rod functional changes were recorded at baseline visits and then again at 6- and 12-months. The results of this study will contribute to our understanding of functional loss in AMD and help clinicians and researchers when designing research protocols aiming to evaluate rod functional impairment before vision loss. Methods This study was conducted between 2015 and 2018 at the Macular Research Unit, Centre for Eye Research Australia. During that time, three main studies were completed. An initial assessment of test-retest reliability of the DACP was followed by a cross-sectional study comparing the severity of rod dysfunction based on point wise sensitivity (PWS) and point wise sensitivity difference (PWSD) of two color sensitivities. AMD cases were divided into an AMD group with traditional drusen only and an RPD group, and rod function was evaluated at multiple ring eccentricities within 24º of the central macula. A further 12-months evaluation of static (PWSD) and dynamic rod functional changes (rod intercept time (RIT) and rod recovery rate (RRR)) within the central 12º was conducted in participants with intermediate AMD with and without RPD as well as normal control eyes. Results The DACP did not have ceiling or floor issues during the intrasession and intersession testing, with a coefficient of repeatability of ± 5 dB. Intermediate AMD (iAMD) with the presence of RPD was found to be associated with poorer rod function compared with iAMD without RPD and normal control eyes, but only within the central macular 8º. Rod dysfunction was more significant if the eyes were prebleached before undertaking the sensitivity measurements. In a longitudinal study, dynamic rod functional changes, ie. RRR, was the only parameter that changed over 12 months, and only in the iAMD without RPD group. Change was found only at the peripheral 12º ring eccentricities. Conclusion The DACP offers a great opportunity to evaluate topographic rod function when eyesight is still normal. The findings from this study support that concept of rod function being impaired in the early stages of AMD. The presence of RPD in cases of iAMD were associated with further reductions in rod function. Measuring dynamic rod function offers a more sensitive functional marker in determining severity of AMD within the central 8 degrees of the macula, but changes over time were only detected at more peripheral locations. Longer follow-up will be beneficial to determine how rod function deteriorates over time and correlates with progression to vision loss.
An Australian population-based study of the incidence and outcomes of hepatocellular carcinoma: the Hepatomas of Melbourne Epidemiological Research (HoMER) study
Liver cancer, the world’s second highest cause of cancer death, is reportedly increasing in incidence in developed countries including Australia. Hepatocellular carcinoma (HCC), the predominant type of liver cancer, has a complex epidemiology involving the interplay of many dynamic risk factors including chronic viral hepatitis B and C, alcohol-related liver disease and non-alcoholic fatty liver disease. Recent trends in these risk factors, changes in Melbourne’s population demographics, together with advances in HCC diagnostics are thought to be increasing HCC incidence, although this may be currently underreported by the cancer registry. Accurate local epidemiology is required to inform healthcare policy for the appropriate allocation of resources for treatment, prevention and research that will improve clinical and economic outcomes. The major study of this thesis, to my knowledge, is the first in the world to determine the population incidence of HCC using clinical case capture independent of the cancer registry. In doing so, it is also the first study to validate the completeness and accuracy of a cancer registry in reporting HCC, which is critical given that the HCC epidemiology literature has been mostly dependent on local registry data. Over 12 months (July 2012 to June 2013), there were 272 new cases of HCC identified from multiple primary sources including multidisciplinary meeting reviews, hospital inpatient, outpatient and emergency attendance at any of Melbourne’s seven tertiary hospital networks, pathology, radiology and pharmacy databases. After cross- referencing with the Victorian Cancer Registry cases for the same period, the HCC incidence determined by this study was found to be twice as high as that reported by the registry. The recruited population-based prospective cohort of HCC patients then provided the opportunity to examine clinical outcomes across the breadth of presentation, demographics and institutional expertise. This allowed for the determination of factors associated with improved survival, in particular, the influence of HCC surveillance participation. The commonest risk factors for HCC were chronic hepatitis C (41%) and alcohol-related liver disease (39%) followed by chronic hepatitis B (22%). While participation rates were low (40%), surveillance was associated with earlier tumour stage at diagnosis, being offered curative therapies, and improved survival probability. In the era of advanced diagnostic imaging and therapeutics for HCC, only some of which is publicly funded, the economic cost of the HCC disease burden is an important consideration in cost-effectiveness analyses. This HCC incidence cohort provides the basis for the first direct costings study of HCC management in Australia, with associated clinical factors that determine high cost. The highest costs occurred at both of ends of the disease spectrum, with early stage disease curable by liver transplantation or surgical resection, as well as in the sickest patients with advanced disease and only palliative options available. Thus, investment of healthcare expenditure towards disease prevention and early detection would likely be most cost-effective with improved survival and reduced morbidity achieved. This world-first research, using a novel methodology for clinical case capture, determined the population-based incidence of HCC in Melbourne, confirmed the underreporting of incidence by the cancer registry and contributed to the correction of registry classification practices. For cancers with rapidly changing epidemiology such as HCC, accurate and contemporary local data is important in disease prevention and management and in guiding healthcare policy and research.
Quality of life outcomes following surgery for extremity sarcoma in Victoria, Australia: an observational prospective cohort study
This study was part of the Victorian Sarcoma Service (VSS) – a statewide Australian service comprising multidisciplinary teams dedicated to managing sarcoma. We followed a cohort of 76 people from the time of diagnosis of a new, surgically resectable extremity sarcoma (ES) to one year after surgery. Baseline characteristics that were collected included medical, sociodemographic and employment-related information, as well as information about received and perceived adequacy of social support. Physical function limitations, quality of life (QoL) as well as levels of depression, anxiety and stress were assessed at baseline, six months after surgery and one year after surgery. About a third of our cohort reported symptoms suggestive of psychological distress at each of the time points assessed. A history of previous psychiatric morbidity was associated with symptoms of depression, anxiety and stress throughout the cancer journey, perhaps suggesting increased vulnerability to distress. Other associations with distress included poor self-reported QoL, financial stress, increased levels of pain and higher levels of physical function limitations. A quarter of our participants reported poor QoL. Factors associated with poor QoL included psychological distress and physical function impairment. We found that majority of our cohort was engaged in paid employment prior to the diagnosis of ES. After a year from surgery, less than a third had returned to their usual work whilst almost a quarter did not return to work at all. Those that had difficulty returning to work reported difficulty keeping up with work early on, even before surgery. The most common reason for not returning to one’s usual level of work was due to the complications or side effects of ES and / or treatment – including pain, stiffness, weakness and fatigue. We identified that a significant proportion of people did not return to work due to mental health related reasons. Furthermore, there was a strong association between difficulty with performing work-related tasks and psychological distress, financial stress, physical function and poor QoL. Overall, we have identified that employment related difficulties are common and become apparent early on. We propose that early screening for employment-related difficulties and appropriate counselling with or without job re-training may have some benefit in improving outcomes for people with ES.