Medicine (St Vincent's) - Theses
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ItemImpaired awareness of hypoglycaemia in type 1 diabetes: Challenges to achieving metabolic control and advances in therapeutic options to replace beta cell function( 2021)Impaired awareness of hypoglycaemia (IAH) affects an estimated 20% of people living with type 1 diabetes and increases the risk of severe hypoglycaemia six-fold. The increased susceptibility to hypoglycaemia results from defective physiological defences in response to a fall in blood glucose levels, leading to IAH and a self-perpetuating cycle of recurrent hypoglycaemia. People with IAH and recurrent severe hypoglycaemia have high morbidity and mortality, though this remains to be fully defined. The management of individuals with IAH is complex. A staged clinical approach has been proposed which includes educational, technological and transplantation interventions, aiming to avoid hypoglycaemia. Novel diabetes therapies and technologies, in particular closed-loop insulin delivery systems, also known as the ‘artificial pancreas’, may be a viable alternative therapeutic option to manage these high-risk individuals. This thesis focuses on IAH in adults with type 1 diabetes, aiming to better define mortality risk in people living with IAH and recurrent severe hypoglycaemia, and to better understand strategies to address challenges to achieving optimal glycaemic control. The original research conducted in this thesis also investigates the efficacy of hybrid closed-loop technology and a novel insulin formulation to optimise overall glucose control, and specifically, for individuals with IAH, to minimise hypoglycaemia and potentially ameliorate hypoglycaemia awareness. The first study of this thesis explored mortality rates and cause of death in adults with IAH and recurrent severe hypoglycaemia who were considered for islet transplantation. This study found that hypoglycaemia-related mortality was high in those who did not undergo islet transplantation, which is considered gold standard for this vulnerable group. These findings highlight the importance of seeking alternative technology-based therapeutic options for those who are deemed not suitable for transplantation or for those awaiting transplantation. The findings from this study also justify the importance of the subsequent studies of this thesis. The following two studies of this thesis investigated whether novel technology, specifically advanced hybrid closed-loop algorithms and faster-acting insulin formulations, which are two important components critical to the success of a closed-loop system, can improve glucose control further in a well-controlled general adult population with type 1 diabetes. Overall high glucose time-in-range, high time spent in closed-loop, positive user acceptability with no major safety concerns demonstrated promising progression in the evolution of these technologies, and warrants broader evaluation in a group with IAH. The fourth randomised crossover study investigated glucose control and counterregulatory responses using a hybrid closed-loop system in adults with IAH when undertaking moderate- and high-intensity exercise. This is a particularly challenging area for people with IAH due to the risk of exercise-associated hypoglycaemia. Closed-loop use during exercise was safe and effective with minimal hypoglycaemia, despite an overall attenuated counterregulatory response to exercise, though the cortisol response to high-intensity exercise was preserved. The final study brought together elements of the preceding studies to comprehensively evaluate adults with IAH and recurrent severe hypoglycaemia, comparable to those who meet criteria for islet transplantation. Findings demonstrated that a hybrid closed-loop system improved overall glucose control without an increase in hypoglycaemia, reduced glucose variability, reduced quantitative composite hypoglycaemia scores, and partially improved glucose counterregulatory responses without restoration of hypoglycaemia awareness compared with standard diabetes therapy. This thesis adds a substantial body of knowledge towards the current understanding of IAH and its associated burden, and the strengths and limitations of hybrid closed-loop insulin delivery for the management of adults with IAH. This work contributes added knowledge towards better delineating and improving decision algorithms to allocate closed-loop systems or transplantation to the most appropriate recipients. Until a biological cure is achieved, ongoing advances in both automated insulin delivery systems and beta cell replacement with transplantation will continue to improve biological and psychosocial outcomes for this vulnerable group of people living with type 1 diabetes.
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ItemSubstituting beta cell function in type 1 diabetes( 2017)Matching exogenous insulin dosing to the varying metabolic requirements of people with type 1 diabetes is crucial for optimising health and minimising the burden of diabetes self-care. Advances in insulin formulations, insulin delivery systems and glucose monitoring technology have resulted in improvements in glucose control and in increased automation of therapy. However, subcutaneous insulin administration is significantly limited by its non-physiological delivery. Continuous subcutaneous delivery of rapid-acting insulin analogues via pump is well established in clinical care. Despite this, pharmacokinetic and pharmacodynamic responses to small insulin pump basal rate changes—typical of those implemented in clinical practice—have not previously been established. There is additional complexity associated with exercise due to changes in insulin sensitivity, absorption and action. While blood glucose meters represent a proven technology for point glucose measurement, their use is painful, requires user initiation and does not provide predictive information. These shortcomings are in part addressed by continuous glucose monitoring technology; however, the performance of the present generation of glucose sensors has substantial limitations. Hence, maintenance of glucose homeostasis in type 1 diabetes remains a therapeutic challenge. This research investigated the utility of effectively employing insulin pump and glucose sensor technology to optimise metabolic control and improve diabetes outcomes for adults with type 1 diabetes. This thesis shows that after small insulin pump basal rate changes, there are substantial delays until changes in circulating insulin levels occur. Moreover, for small rate changes of equal magnitude, it takes longer to achieve change in circulating insulin after a rate reduction than after an increase. Adjustment of basal insulin delivery to minimise hypoglycaemia with exercise was investigated. Findings demonstrated that very large reductions in basal insulin delivery are required to achieve a timely decrease in circulating insulin for aerobic exercise; when pre-exercise glucose levels are low-normal, supplemental carbohydrate ingestion may also be necessary to avoid hypoglycaemia. In a cross-sectional study, insulin pump users were observed to have more favourable vascular health profiles than those treated with insulin injections; these differences are possibly explained by multiple factors independent of the insulin delivery modality. To improve glucose sensor performance, a novel sensor combining two distinct sensing methodologies was developed and investigated. Feasibility of the novel sensor was confirmed, and its accuracy compared favourably with glucose sensors available at the time the research was undertaken. This thesis expands the current understanding of insulin delivery via pump and glucose sensing technology for people with type 1 diabetes. Until type 1 diabetes prevention and cure are achieved, the optimisation of insulin dose adjustment in parallel with the further development of glucose sensing technology is still required to mimic healthy pancreatic beta cell function.
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ItemInduction of antigen-specific tolerance and development of autoreactive T cells in an experimental model of autoimmune diabetes( 2016)Immune responses to proinsulin initiate anti-islet autoimmunity in non-obese diabetic (NOD) mice and possibly in humans. This results in autoimmune destruction of insulin secreting beta cells leading to type 1 diabetes (T1D). Therapies that bolster immune tolerance to islet antigens are highly desirable, however such approaches have failed to prevent clinical T1D. The major aim of this thesis was to determine a stage of life when antigen-specific tolerance is most effective in preventing anti-islet immune responses. Chapter 2 describes generation and validation of transgenic NOD mice engineered to express islet antigens proinsulin (TIP mice) and IGRP (TII mice) in the antigen presenting cells (APCs) in a tetracycline dependent manner. MHC class II IEα promoter in combination with tet-OFF transactivator induced robust, doxycycline dependent and APC specific expression of proinsulin and IGRP in TIP and TII mice respectively. TIP mice expressing proinsulin did not develop insulitis and were protected from cyclophosphamide-induced diabetes, suggesting that proinsulin expression in TIP mice was sufficient to induce functional antigen-specific tolerance. In chapter 3, we examined the impact of antigen-specific tolerance on the development of autoreactive T cells and spontaneous diabetes by expressing islet antigens proinsulin and IGRP in the APCs during defined periods of life in TIP and TII mice. Our results indicate that tolerance to proinsulin in early life until the weaning period is sufficient to prevent diabetes development in TIP mice. The protection from diabetes was not due to dominant tolerance, but mainly due to a significant reduction in the insulin reactive T cells. Although insulin reactive T cells were not completely absent, the residual autoreactive T cells lacked pathogenic potential. By tracking IGRP reactive T cells in TII mice we demonstrate that IGRP T cells also emerge during early life. These data suggest that early life is a vulnerable period for escape of islet reactive T cells, and that boosting immune tolerance to islet antigens during this time imparts durable protection from islet autoimmunity. Immune tolerance to proinsulin-2 imparts robust protection from autoimmune diabetes in the NOD mice. Whether dampening immune responses to proinsulin-1 would influence diabetes development in NOD mice remains to be investigated. Chapter 4 describes the generation of transgenic NOD mice that express proinsulin-1 in the APCs (TIP-1 mice) in a tetracycline dependent manner. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in number in TIP-1 mice indicating immune tolerance. Although immune response to downstream antigen IGRP was reduced in TIP-1 mice, tolerance to proinsulin-1 was unable to prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, despite being highly conserved to proinsulin-2, tolerance to proinsulin-1 only partially prevents islet-autoimmunity in NOD mice, which suggests an ongoing residual immune response to proinsulin-2 epitopes in TIP-1 mice.
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ItemDissecting the interferon response required for triggering autoimmune diabetes( 2016)Type 1 diabetes (T1D) is characterized by the autoimmune destruction of β cells in the islets of Langerhans by immune cells. The interferon (IFN) family of cytokines has been implicated in diabetes pathogenesis, possibly via the activation of innate immune pathways. However, how IFNs contribute to the pathogenesis of autoimmune diabetes remains unclear. The overall aim of this thesis is to dissect the role of IFNs in the triggering of autoimmune diabetes using the non-obese diabetic (NOD) mouse model. Chapter 2 describes the contribution of type I IFN to the development of autoimmune diabetes in NOD mice. The islets of NOD mice displayed a unique mRNA expression pattern of IFN-induced genes that peaked at 3-4 weeks of age. Genetic ablation of type I IFN receptor in NOD mice (NOD.Ifnar1-/-) significantly reduced the expression of these genes. However, lack of IFNAR1 did not affect the development of diabetes. The overlapping function of type III IFN could be a reason for the lack of effect of IFNAR1-deficiency. Another reason could be that the natural level of type I IFN produced in wild-type NOD mice is insufficient to have an effect on diabetes development. In Chapter 3, the possible role of type III IFN in autoimmune diabetes was examined. β cells isolated from humans and mice expressed the type III IFN receptor and β cells were able to respond to type III IFN stimulation. Type III IFN was detected in the islets in NOD mice. These results indicate that type III IFN may contribute to diabetes development in NOD mice. Future experiments using NOD mice deficient in the type III IFN receptor will determine whether type III IFN contributes to the development of autoimmune diabetes. Engagement of pattern recognition receptors (PRRs) with exogenous and endogenous danger signals can trigger type I IFN production. Endogenous danger signals, such as aberrant cytosolic DNA, are normally eliminated to prevent unnecessary induction of PRRs. Nucleases, such as the 3’ exonuclease TREX1, are important in degrading aberrant DNA, and have been implicated in protection from autoimmunity. TREX1 is normally found in the SET complex, and one protease that can activate this complex is granzyme A. In Chapter 4, I show that granzyme A deficiency resulted in increased diabetes in NOD mice. Single-stranded DNA accumulated in the cytoplasm of dendritic cells and NK cells in the islets and spleens, and this was observed more frequently in NOD.Gzma-/- mice. Consistent with poor clearance of DNA and increased PRR activation, expression of IFN-induced genes was higher in the islets of NOD.Gzma-/- mice than NOD mice at 4 weeks of age. When NOD.Gzma-/- mice were crossed with NOD.Ifnar1-/- mice, diabetes returned to the rate observed in NOD mice. Overall, the data indicate that the natural level of type I IFN produced in wild-type NOD mice is not sufficient to cause diabetes, but excessive type I IFN can increase diabetes development in NOD.Gzma-/- mice. The results provide mechanistic insight into the triggering of autoimmune diabetes, suggesting that aberrant accumulation of cytoplasmic DNA and type I IFN production are important. In this study, these were caused by loss of granzyme A, but it is also possible that virus infection could similarly activate type I IFN and possibly also type III IFN to trigger T1D.
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ItemThe development and regulation of islet-specific T cells in an experimental model of autoimmune diabetes( 2014)Type 1 diabetes (T1D) is an autoimmune disease. T cells specific for β-cell antigens such as proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) are important in mediating the disease. The aims of this thesis were to study the development and regulation of T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Chapter 2 describes the development of IGRP-specific CD8+ T cells in autoimmune diabetes. IGRP-specific T cells in the mouse were tracked using a sensitive MHC-tetramer based magnetic enrichment. There was an increase in the number of IGRP-specific T cells in the peripheral blood and lymphoid tissue as mice age, and the increase correlated with insulitis progression. These cells had an effector-memory phenotype, which was only acquired in the inflammatory environment of the islets, and not the draining lymph nodes. Islet-specific T cells could also migrate from islets into the periphery. In the development of autoimmune diabetes, important changes to IGRP-specific T cells during the pathogenesis of diabetes occur not in the draining lymph nodes but in the islets, where they expand and differentiate into effector-memory T cells, and emigrate to the periphery, where they can report progression of islet pathology. Tumour Necrosis Factor (TNF) is an inflammatory cytokine that has been implicated in the pathogenesis of autoimmune diabetes. In chapter 3, we investigate the effects of TNF-TNFR1 signalling deficiency on the development of autoimmune diabetes, by using a NOD mouse deficient in TNF receptor 1 (TNFR1). TNFR1-/- islets grafted onto kidney capsule of diabetic mice were destroyed, showing that TNFR1 deficiency on β-cell did not confer protection against immune destruction. The specific effects of TNFR1 deficiency on the immune system of NOD mice were also examined. Adoptively transferred β-cell specific T cells proliferated normally in the pancreatic lymph nodes, but failed to migrate into the pancreas of TNFR1-/- recipient mice. Notably, analysis of immune cell subsets by flow cytometry showed an increased percentage of CD4+CD25+Foxp3+ T regulatory cells in TNFR1 deficient mice. Depletion of CD4+CD25+ regulatory T cells using GK1.5 CD4 depleting mAb restored diabetes in NOD8.3/TNFR1-/- mice. These results suggest that blockade of TNF signalling suppresses diabetes by increasing regulatory functions of the immune system. T cell responses to insulin (INS) are crucial in development of T1D. Chapter 4 of the thesis examines insulin-specific T cells in NOD mice, and in NOD mice tolerant to proinsulin II (NODPI), which do not develop diabetes or insulitis. There was no significant difference in the absolute number of insulin-specific CD8+ T-cells in NOD and NODPI mice. INS-specific CD8+ T-cells in NOD mice expanded significantly more in response to stimulation by peptide compared to NODPI. In vivo cytotoxic activity in NODPI was reduced compared to NOD. The absolute number of INS-specific CD4+ T-cells in NOD and NODPI mice was similar. The proportion of regulatory INS-specific CD4+ T cells that were Foxp3+ was also similar. INS-specific CD4+ T cells in the NOD and NODPI were tested on whether they could help CD8+ T-cells mediate diabetes. NODRAG1-/-/8.3 developed diabetes rapidly after NOD CD4+ T-cells were transferred (median=32days). 7/8 of recipients did not develop diabetes when NODPI CD4+ T-cells were transferred. In a NOD mouse tolerant to proinsulin, insulin-specific CD4+ and CD8+ T-cells were detected, suggesting that the main mechanism of tolerance is not deletion. It is more likely that these cells could have impaired function.
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ItemCharacterisation of susceptibility to Listeria monocytogenes infection in the non-obese diabetic (NOD) mouse( 2012)The immune system has evolved the ability to prevent infection by a wide range of pathogens while maintaining tolerance to self-tissues. Due to the strong selective pressure imposed by microbial pathogens, susceptibility to infection can be modulated by a large number of genetic loci. It is postulated that, at least in some cases, allelic variants for particular loci confer increased resistance to pathogens while simultaneously increasing the risk of developing autoimmune diseases. A number of recently discovered disease loci appear to act as “genetic pivot points” between pathogen defence and autoimmune pathogenesis. It is anticipated that characterising these loci will provide novel insights regarding the interplay between immune and autoimmune responses, as well as reveal potential therapeutic targets for treating both infectious and autoimmune diseases. Murine models provide a complementary approach to human studies for investigating genetic and cellular mechanisms that underlie susceptibility to infectious and autoimmune diseases. The non-obese diabetic (NOD) mouse strain is one of the best-characterised models of type 1 diabetes (T1D), an autoimmune disease caused by the destruction of insulin-producing pancreatic β cells. Similar to humans, predisposition to T1D is attributed to multiple genetic loci in NOD mice. Intriguingly, non-diabetogenic mouse strains can also harbour diabetogenic alleles for some T1D susceptibility loci. Using congenic mice, our laboratory confirmed that non-diabetogenic C57BL/6 (B6) mice harbour a diabetogenic allele for a T1D susceptibility locus on chromosome (Chr) 13, termed Idd14. Coincidentally, the Idd14 locus overlapped with Listr2, a proposed susceptibility locus for Listeria monocytogenes, which is an extensively studied intracellular bacterial pathogen. Notably, B6 mice are resistant to L. monocytogenes infection, whereas NOD mice are reported as susceptible. We therefore hypothesised that allelic variation for this interval, which increases T1D risk, would confer resistance against L. monocytogenes in NOD mice. The goal of this study was to investigate the biological and genetic effects of allelic variation for Idd14/Listr2 in NOD mice for L. monocytogenes infection and T1D. Towards this goal, the first aim was to investigate the immunological basis of susceptibility to L. monocytogenes in NOD mice. In comparison to infected B6 mice, infected NOD mice exhibited reduced antigen-specific CD8+ T-cell responses, which was associated with apparent deficiencies in dendritic cells. Infected NOD mice also exhibited exacerbated neutrophilia, a potential compensatory mechanism in susceptible hosts. The second aim was to determine the role of neutrophils during L. monocytogenes infection. Specific depletion of neutrophils impaired bacterial clearance in susceptible NOD mice, but not in resistant B6 mice, demonstrating that neutrophils are essential for controlling L. monocytogenes infection in susceptible hosts. In addition, NOD macrophages exhibited impaired antimicrobial function. These results indicate that neutrophils may compensate for deficient T-cell and macrophage responses to ensure host survival during L. monocytogenes infection. The third aim was to confirm the Listr2 locus using congenic NOD mouse strains, which harbour different B6-derived Chr 13 interval, and determine if the refined intervals for Listr2 and Idd14 continue to co-localise. Concurrently, it was found that the Idd14 locus could be dissected into two sub-loci, Idd14.1 and Idd14.2. Listr2 was confirmed and co-localised with Idd14.1 to an overlapping interval (~ 18 Mb) on Chr 13. This co-localisation raises the intriguing possibility that allele variation for the same gene(s) within this interval affects both infection and autoimmune disease susceptibility.