Medicine (Northwest Academic Centre) - Theses
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ItemThe role of placental heparan sulphate proteoglycans in the pathogenesis of pre-eclampsia( 2015)Introduction: Uncomplicated pregnancies represent a hypercoaguable state. However, placental thrombosis is rare in these pregnancies which suggests that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia not only demonstrate an exaggerated increase in procoagulant activity which may contribute to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies, but there is evidence of substantial cell growth, differentiation and angiogenic functional defects. Proteoglycans are abundantly expressed within the placenta compared to other human tissues. Particularly of interest are heparan sulphate proteoglycans which have important anticoagulant, anti-inflammatory and angiogenic properties. Hypothesis: Altered abundance, structure or function of placental heparan sulphate proteoglycans contributes to the development of pre-eclampsia by: increasing placental thrombin generation, interfering with cellular growth, differentiation, disrupting normal angiogenesis and altering growth factor interactions. Aims : 1) To determine the mRNA expression, protein abundance and cellular localisation of placental heparan sulphate proteoglycans from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. 2) To investigate the functional consequence of reduced HSPGs in placental cells 3) To determine the differences in the abundance and structure of heparan sulphate proteoglycans and heparan sulphate GAGs from placentae obtained from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. Methods: 1) The mRNA, protein abundance and cellular localisation of placental heparan sulphate proteoglycans was determined using real-time PCR, western immunoblotting and immunofluorescence, respectively. 2) To investigate the functions of reduced heparan sulphate proteoglycans, a cell culture model using short interference RNA was used. Cellular growth will be assessed using the xCELLigence system, thrombin generation using the calibrated automated thrombogram system and angiogenesis will be determined using a matrigel based assay. Cellular differentiation and apoptosis will be determined using real-time PCR. Growth factor signalling will be determined using a real-time PCR growth factor array. 3) To isolate proteoglycans from placenta, anion exchange chromatography will be utilised. Enzymatic digestion will be used to isolate the glycosaminoglycans, and enzyme-linked immunosorbant assays will be undertaken to determine the abundance of PGs and GAGs. Results: The mRNA expression of heparan sulphate proteoglycans, Syndecan 1, Syndecan 2, Glypican 1 and Glypican 3 are significantly reduced in the placentae of women whose pregnancies are complicated by pre-eclampsia. A cell culture model was utilised to determine the functions of heparan sulphate proteoglycans in the placenta. Successful downregulation of heparan sulphate proteoglycans was achieved in the cell lines using short interference RNA treatment, and a number of functions were significantly altered as a result. The downregulation of Syndecan 1, Glypican 1 and Glypican 3 resulted in significant alterations in the downstream growth factor targets. Reduced Syndecan 2 expression resulted in a significant reduction in the thrombin generation potential of endothelial cells. Investigation into the abundance and structure of glycosaminoglycans within the placenta, demonstrated heparan sulphate glycosaminoglycans to be significantly reduced in pregnancies complicated with pre-eclampsia compared to gestation matched controls. Conclusion: The reduction in heparan sulphate proteoglycans expression observed in pregnancies complicated with pre-eclampsia may be responsible for the altered growth factor interaction commonly observed in preeclamptic pregnancies. This study has provided us with a greater understanding of the biological role of heparan sulphate proteoglycans within the human placenta and its potential implications in the pathogenesis of pre-eclampsia.
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ItemVitamin D and HIV: exploring determinants of 25-hydroxyvitamin D and altered vitamin D metabolism in HIV-infected adults( 2015)Background: Vitamin D deficiency is a widespread global problem of particular importance for people with HIV. Vitamin D is an important hormone for endocrine and extra-endocrine functions, including modulating the immune system. Vitamin D deficiency is associated with reduced bone mineral density and infectious diseases, both common in people with HIV. Maintaining optimal vitamin D levels may help to reduce the risk of developing these diseases. The overall research questions for this thesis are: Firstly, which factors influence vitamin D metabolites, and secondly, what effect does vitamin D status have on bone and immunological outcomes, in people living with HIV infection? Method: To answer these questions, five studies were performed: 1. Cross-sectional comparison of 25(OH)D levels in HIV-infected and uninfected individuals in southern Australia. 2. Cross-sectional analysis of the determinants of 25(OH)D in people with HIV in Queensland and Melbourne, Australia. 3. Cross-sectional analysis of parathyroid hormone levels in antiretroviral-treated individuals. 4. Cross-sectional analysis of the effect of 25(OH)D on bone mineral density in antiretroviral-treated individuals. 5. Longitudinal analysis of CD4 cell count trajectory in antiretroviral-untreated individuals. Results: 1. People with HIV were more likely to be vitamin D deficient when compared with people without HIV in southern Australia. 2. Determinants of 25(OH)D levels: a. UV index and location are important determinants of 25(OH)D levels in people with HIV in Australia. b. Antiretroviral therapy impacts 25(OH)D levels: i. Efavirenz is associated with lower 25(OH)D levels, particularly in those with dyslipidaemia. ii. Protease inhibitors are associated with higher 25(OH)D levels. 3. The effect of tenofovir on parathyroid hormone levels depends on sex and ethnicity; parathyroid hormone levels were higher in non-white males using tenofovir. 4. Influence of ART and 25(OH)D on 1,25(OH)2D levels and bone mineral density: a. People using tenofovir have higher 1,25(OH)2D levels and people using protease inhibitors have lower 1,25(OH)2D levels. b. There is an interaction between tenofovir and 25(OH)D status on 1,25(OH)2D levels and bone mineral density. Vitamin D deficiency increases the odds of low bone mineral density only in those not using tenofovir. 5. Vitamin D deficiency may reduce the time to CD4 decline to <350 cells/μL in people living with HIV and untreated with antiretroviral therapy. Conclusions: Traditional risk factors for vitamin D deficiency and bone mineral density appear to be more important than HIV-related factors in people with HIV. In spite of this, antiretroviral therapy clearly alters vitamin D and bone metabolism. Understanding the mechanisms behind these alterations may assist in modifying the negative effects of vitamin D deficiency.
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ItemSun exposure and type 2 diabetes mellitus: Can sun exposure lower type 2 diabetes risk?( 2015)Background: Lower serum 25-hydroxyvitamin D (25OHD) levels have been consistently associated with increased type 2 diabetes mellitus (T2DM) prevalence and incidence in systematic reviews and meta-analyses of observational studies, however this association has not consistently been replicated in vitamin D supplementation trials. This disparity may be due to a number of different factors: lack of power in the trials due to small sample size, insufficient duration of dosing, baseline vitamin D or glycaemic status differing between studies, low supplementation compliance or insufficient vitamin D dose. Alternatively, lower 25OHD levels may be a product, rather than a cause of ill-health, or they may share pathology earlier in life or in disease progression so that supplementing with vitamin D in adulthood has no effect on disease outcome. This body of work presents another explanation: given that sun exposure is the most influential contributor to serum 25OHD levels, observational studies may be reporting an effect of sun exposure, rather than vitamin D, on T2DM. Therefore vitamin D supplementation trials may be failing to capture any additional benefits of sun exposure through non-vitamin D pathways. The aim of this body of work was to investigate the possible association between sun exposure and T2DM endpoints reported in scientific literature as well as in an original analysis. A major objective of the original analysis was to determine whether or not any association found between sun exposure and T2DM incidence was through non-vitamin D pathways. Methods: Following a literature review, a systematic review of observational studies reporting on associations between sun exposure variables and T2DM-related endpoints was conducted. The potential of an association between sun exposure- measured using ambient ultraviolet radiation (UVR), and five-year cumulative incidence of T2DM was explored using a prospective, national diabetes cohort (AusDiab). A causal mediation analysis was undertaken to explore whether or not there were effects of ambient UVR on cumulative T2DM incidence, via non-vitamin D pathways. Results: The systematic review revealed that high-level evidence for an association between sun exposure and T2DM-related outcomes was lacking. There was moderate-level evidence for greater sun exposure reducing T2DM incidence. The opposite was found in the original analysis using the AusDiab cohort: ambient UVR was associated with increased T2DM incidence (OR=1.17, 95% CI: 1.01-1.36, p=0.04). This association was independent of an effect of age, sex, body mass index, physical activity, ethnicity, smoking status and serum 25OHD levels, but was likely to be confounded by area-level determinants of health due to the nature of the exposure variable. The major limitations of this work were that the sun exposure measures were suboptimal. Time-of-year measures were the most common sun exposure variables contained in the systematic review, and ambient UVR at the site of participant recruitment was the proxy for sun exposure in the original analysis. Conclusion: There is likely to be a complex relationship between sun exposure and T2DM. The direction of the association between sun exposure and T2DM incidence, as well as delineation of the mechanistic pathways through which this association may exist, are yet to be confirmed. Future studies are encouraged to use person-level sun exposure measurements, and findings from such studies may influence sun protection policy.
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ItemThe treatment of prosthetic joint infection with debridement, prosthesis retention and biofilm-active antibiotics( 2014)Background: Prosthetic joint infection (PJI) is a serious infection that is difficult to cure and is associated with significant morbidity. The pathogenesis of PJI involves bacteria growing in biofilm adherent to the prosthesis surface, making them resistant to eradication with standard antibiotics. Recent evidence demonstrates successful treatment of early PJI with surgical debridement and retention of the prosthesis (DAR) and the biofilm-active antibiotic combination of rifampicin and a fluoroquinolone for staphylococcal infections. However, there are few studies investigating appropriate antibiotics to use in combination with rifampicin for PJI caused by staphylococci resistant to fluoroquinolones or which antibiotics to use for organisms other than staphylococci. Little is known about functional outcomes, quality of life (QOL) or complications after treatment of PJI. The aim of this thesis is to provide further evidence to help guide management in these areas. Methods: This thesis synthesizes three of my recent studies published in peer-reviewed journals and one study presented at a national scientific meeting. In the first study, outcomes were analysed for consecutive patients with staphylococcal PJI treated with DAR and a combination of rifampicin and fusidic acid. The second examined consecutive patients with a Gram-negative bacillus PJI treated with DAR and ciprofloxacin-based regimens. In the third study, consecutive patients treated for hip PJI with DAR and biofilm-active antibiotics were matched to controls that had hip arthroplasty with no infection, and their function, QOL and complications compared. In the fourth study, a large prospective hip and knee arthroplasty cohort was analysed to determine if PJI treated with DAR and biofilm-active antibiotics was predictive of adverse QOL outcomes. Results: Of 20 patients with staphylococcal PJI, treatment failure occurred in two patients. The cumulative risk of treatment failure after 1 year was 11.76% (95% CI 3.08–39.40%). Ten of 11 patients with infections involving methicillin-resistant Staphylococcus aureus had successful outcomes. Of 17 patients with Gram-negative bacillus PJI, treatment failure occurred in two patients. In only one patient was a relapsed Gram-negative infection responsible for the failure and this patient had not been treated with ciprofloxacin. The 2-year survival rate free of treatment failure was 94% (95% CI, 63–99%). In 19 hip PJI cases there was significant improvement 12-months post-arthroplasty in function according to Harris Hip Score and QOL according to the 12-item Short Form Health Survey Physical Component Summary. There was no significant difference in the improvement between 76 controls and the 19 cases. Medical complications occurred more frequently in cases than controls but the rate of surgical complications was the same. Of 2134 patients in a hip and knee arthroplasty cohort there were 37 patients with PJI treated with DAR and biofilm-active antibiotics. On multivariate logistic analysis, PJI treated this way was not predictive of adverse QOL outcomes according to SF-12 scores, however pre-arthroplasty SF-12, female gender, knee arthroplasty and a comorbidity index were. Conclusions: DAR in combination with rifampicin and fusidic acid is effective and should be considered for patients with early staphylococcal PJI, including those with infections involving fluoroquinolone-resistant organisms. DAR in combination with ciprofloxacin is effective for patients with early Gram-negative bacillus prosthetic joint infection. Treatment of PJI with DAR and biofilm-active antibiotics was well tolerated and results in good improvements in function and QOL.
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ItemCross‐priming of TCD8+ specific for cell‐associated antigens( 2014)TCD8+ of the adaptive immune system play critical roles in the host defence against viruses and other pathogens through elimination of infected host cells. After infection, TCD8+ proliferate and adopt effector functions following recognition of specific antigenic‐peptides in the groove of a MHC class I molecule expressed by antigen presenting cells (APC). Professional APCs such as dendritic cells (DCs) are specialized for the priming and activation of naïve TCD8+. DCs are the key cell type responsible for T cell priming. They can either be directly infected and present viral antigens (direct‐priming) or phagocytose infected cell debris and process and present phagocytosed antigens to the specific TCD8+ (cross‐priming). Little is known about the actual contribution of direct versus cross‐priming during an immune response against viral infections. Understanding how DCs regulate TCD8+ responses is central for our ability to favourably manipulate the immune system as well as develop effective targeting strategies for optimal anti‐viral and antitumoral vaccination. To determine the contributions of direct versus cross‐priming to the clearance of an in vivo viral infection, we generated a Cre‐indicator transgenic mouse model utilising cutaneous HSV‐Cre infection to conditionally trigger model antigen expression in infected cells. This was expected to enable us to discriminate between virally infected and hence direct‐presenting DCs from uninfected and cross‐presenting ones in vivo for the first time. Unexpectedly, all generated transgenic mouse lines showed various levels of tolerance towards our model antigen due to undesired protein leakiness at steady state. However, TCD8+ in these neo‐transgene expresser transgenic mice possessed antigen ignorant properties and were non‐specifically activated following acute viral infection and the introduction of other innate immune cell ligands. Therefore, although these generated transgenic mice could not be used for their initial study purpose, their varying expression levels of model antigen make them an excellent model for studying peripheral tolerance induction and its maintenance. Cross‐priming is especially important for anti‐tumour immunity as tumour cells, although carrying tumour associated antigens, do not activate naïve TCD8+ efficiently due to an absence of co‐stimulatory molecules. Remarkably, our group has recently shown that influenza A virus (IAV) infection of allogeneic cells led to tumour protection due to enhanced cross‐priming of TCD8+ specific to cellular antigen. We have previously demonstrated that this enhancement was partially mediated through TLR7 sensing and entirely dependent on MyD88 and IFN signalling pathways, yet independent of the IL‐1β‐inflammasome. To further increase our understanding of cross‐priming enhancement mechanisms found in our system, we have additionally investigated the involvement of other immunological mechanisms in this thesis. Here, we show that IAV enhanced crosspriming is independent from the IL‐18‐inflammasome signalling pathway but that TCD4+ helper play a surprisingly important role for optimal enhancement. Also, through investigations using Batf3‐/‐ mice, we not only confirm the specificity of CD8α+ and CD103+ DC subsets for cross‐presenting, but also demonstrated that there are two types of cross‐priming outcomes: a baseline cross‐priming that is innate signalling independent and an innate immune signal enhanced crosspriming pathway. Interestingly, both types of cross‐priming events were abolished in Batf3‐/‐ mice. This knowledge will be useful to aid future efforts to develop more robust cancer vaccines. Finally, efficient antigen processing and presentation of the TCD8+ epitope is a central factor that determines the extent of specific T‐cell responses. Our group has identified the most immunodominant T‐cell response in the Balb/c mice after IAV infection. Interestingly, this novel epitope is encoded by the intronic region of the non‐structural protein mRNA. To dissect the mechanism of such epitope translation, as being either through spliced mRNA translation or generation using an alternative open reading frame (AltORF), we disabled splicing mechanisms and discovered that generation of the novel epitope occurred through AltORF translation. We will focus on the identification of the exact mechanism(s) underlying the efficient generation of this novel epitope as such mechanisms may provide a great opportunity for developing more efficient IAV T‐cell based vaccine strategies.
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ItemLong term effects of dietary calcium intake on fractures, mortality, cardiovascular diseases and abdominal aortic calcification: a prospective cohort study( 2014)High calcium intake can prevent age-related bone loss; however, its efficacy in fracture prevention is not clear. Some recent studies have associated calcium supplementation with adverse cardiovascular events (CVE). Scientific evidence on the long term effects of dietary calcium intake (DCI) and fracture or CVD risk lacks consistency.
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ItemTo validate a preoperative risk stratification model developed at Barwon Health for mortality and major morbidity in major colorectal surgery( 2013)Colorectal surgery is associated with significant mortality, with reported rates of 1% to 2% in elective surgery and 5% to 10% in emergency surgery. The majority of the colorectal workload is attributed to colorectal cancer and according to the National Health and Medical Research Council guidelines, being the second leading cause of cancer related death in Australia. Each year at least 12,600 new cases are diagnosed with an estimated annual increase of 0.3%. Therefore in the past decade, there has been much initiative to create a prognostic model that could predict patient’s mortality and morbidity before colorectal surgery. Currently there are three known models; Association Française de Chirurgie index score, Barwon Health 2009 model and the American College of Surgeons National Surgery Quality Improvement Program colorectal risk calculator. These prognostic models were created for the purpose of informed consent, to discuss a patient’s risk for surgery and to identify high risk patients for early medical, high dependency unit or intensive care unit admission. Although all three models have reported reliable results in predicting patient mortality, these models have never been validated with another independent dataset in Australia. The purpose of validating the prognostic models was to ensure they were reliable at predicting patient’s mortality in other institutions. However only the Association Française de Chirurgie score and the Barwon Health 2009 model were validated and neither predicted mortality accurately. The American College of Surgeons National Surgery Quality Improvement Program colorectal risk calculator was not validated as it was password protected and available only to the participating hospitals. As there were no reliable models for colorectal surgery, the Colorectal preOperative Surgical Score was created. The Colorectal preOperative Surgical Score is a 4-variable pre-operative model that predicts a patient’s mortality risk following emergency or elective colorectal surgery. This model was successfully validated at two independent institutions. We hope to use this model to guide pre-operative counselling with individual patients and as a simple tool for risk stratification of outcomes as reported in colorectal surgical audit.
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ItemOsteo-cise: Strong Bones for Life: a multi-component, community-based exercise, falls prevention and osteoporosis education program, an 18-month RCT( 2012)Multi-component exercise programs incorporating high-intensity progressive resistance training (PRT), moderate-to-high impact weight-bearing activities and high-challenge balance training are recommended for optimising the bone and falls-related risk factors for fracture. However, few studies have evaluated their combined effectiveness in a ‘real world’ community setting. Furthermore recent evidence suggests that high velocity (power) training may be more effective for improving physical function and bone health than traditional slow-speed PRT, and thus should be the key focus of fracture prevention programs. Within this thesis, three studies were conducted. The first cross-sectional study investigated the relationship between lifetime loading activity and trabecular bone microarchitecture and condylar size at the proximal tibia assessed by 3-Tesla Magnetic Resonance Imaging. Increasing age was significantly associated with a deterioration in trabecular microarchitecture, but no association between loading activity and any bone parameter was observed at this site. Studies 2 and 3 are described below. Study 2: Osteo-cise: Strong Bones for Life: A 12-month multi-faceted community-based exercise, osteoporosis education and behavioural change program on bone mineral density and risk factors for falls Aims: To investigate the effects of a multi-component exercise and osteoporosis education program on bone density (aBMD), body composition, functional power and performance, and falls in older adults at risk of falls and fracture. Methods: 162 men and women aged 66.9 ± 6.0 (mean ± standard deviation) years were randomised to the: 1) Osteo-cise program (n=81) or 2) control group (n=81). Exercise comprised high velocity PRT, weight-bearing (60-180 impacts/session) and balance training 3 days per week for 12 months. Dual energy X-ray absorptiometry (DXA) was used to assess total body, arm and leg lean and fat mass and aBMD at the hip and spine at baseline and 12 months. Leg and back 1-RM muscle strength and functional power (Timed Stair Climb) and performance (Four Square Step test [FSST], Functional Reach, Timed Up and Go, 30 Second Sit to Stand [30-STS]) were assessed at baseline, 6 and 12 months. Results: 93% of participants completed the study and groups were matched at baseline. Exercise attendance averaged 59%. Exercise resulted in a significant 1-1.1% net gain in femoral neck and lumbar spine aBMD vs. controls. There were significant exercise-induced net gains in back (13% [95% CI 6, 20]) and leg (10% [3, 16]) muscle strength, stair climbing power (5% [95% CI 1, 9]), 30-STS performance (16% [9, 22]) and dynamic balance (FSST) (-6% [-10, -1]), but no differences between groups in weight, total body or regional (arm and leg) lean mass or fat mass or falls. Conclusion: This multi-component intervention program was effective for improving aBMD, functional muscle power and performance in older adults at risk of fracture. Study 3: Osteo-cise: Strong Bones for Life. An 18-month multi-faceted community-based exercise, osteoporosis education and behavioural change program on musculoskeletal health and risk factors for falls: Translating research into practise Aims and Methods: As per Study 2. The final 6 months of the program was a ‘translation’ phase, whereby the program continued to be carried out by the health and fitness centres with minimal input from research staff to assess the feasibility of the program in the ‘real-world’. Results: Exercise compliance average 45% during the last 6 months and 55% over 18 months. From 12 to 18 months, the Osteo-cise group maintained aBMD at the lumbar spine and femoral neck aBMD, while the control group significantly lost aBMD at the femoral neck (1.3% over 18 months). The Osteo-cise group also maintained their net gain in muscle strength, arm lean mass, functional muscle power and performance while no significant differences in falls rates were observed. Conclusion: Exercise-induced gains in aBMD, functional muscle power and performance were maintained at 18 months providing support for the viability of this program in the ‘real world’ community setting.
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ItemBarriers and enablers in the treatment and prevention of osteoporosis: exploring knowledge, beliefs, attitudes and cultural models among consumers and medical practitioners( 2011)This study explored knowledge, beliefs, attitudes and cultural models of osteoporosis, in the context of health communication. The purpose of the study was to generate new knowledge that may contribute towards development of health communications about osteoporosis. The study was conducted within the Geelong Osteoporosis Study, in south-eastern Australia, and involved consultation with fracture patients, community members, general medical practitioners and orthopaedic surgeons. The study’s qualitative methodology was predicated upon the medical literature regarding osteoporosis and fragility fracture, and contemporary social and theoretical discourses on the body, health, illness, disease and risk. Materials included brief interviews, in-depth interviews, focus groups and field notes, and the data from each participant type were subjected to two methods of analysis from: thematic analysis, domain analysis, framework analysis, analytic comparison and case study analysis. There was widespread uncertainty among women and men in the study as to the nature and definition of osteoporosis as well as its management and prevention. The doctor–patient relationship was suggested as an important source of health information and education for men, while the women cited a wide range of sources, including libraries, the internet and the media, among their preferences for seeking information about osteoporosis. Gender was a critical factor in the framing of communications regarding osteoporosis, as was paradigmatic perspective. The GPs were ambivalent in their beliefs and attitudes about osteoporosis and its investigation and management; they did not consistently investigate for osteoporosis, though when they did so they recognised that effective investigative technologies and treatment options were available. Their ambivalence derived from structural factors, such as financial barriers (real or perceived) to investigation and adverse media reports about osteoporosis medications. The GPs were confident in the efficacy of the medications they were prescribing to prevent fracture. This confidence was not shared among the orthopædic surgeons, who were more confident about patient adherence to prescribed osteoporosis medications. The study identified the cultural model ‘Osteoporosis has low salience’: consumers saw osteoporosis as something that caused women to be ‘bent over’ in old age, while fracture patients thought of it as a risk of future fracture and the attendant pain, loss of mobility etc. The medical professionals viewed osteoporosis as risk for fracture: to orthopædic surgeons it presented an endpoint at which they had a specific role to play (the repair of fracture), whereas to GPs it represented yet another chronic disease of which they needed to be vigilant in their limited encounters with patients. The study was significant, being the first qualitative investigation of osteoporosis and fragility fracture from multiple perspectives in Geelong. Its results are significant in that they: contribute new knowledge to a field currently lacking in empirical data, namely regarding osteoporosis in men; point to deficits in consumer knowledge about osteoporosis as a health condition; highlight medical practitioners’ concerns about the investigation and management of osteoporosis; and provide valuable inputs into practical considerations in the development of health messages about osteoporosis and related fracture, for both consumers and health professionals.