Nurse's Certificate

Presented to "Miss Frances O'Reilly"

Novel functions for FAS in the regulation of germinal centre and antibody responses

Inactivating mutations in the death receptor FAS or its ligand (FASL) cause Autoimmune Lymphoproliferative Syndrome (ALPS) and lupus-like autoimmune diseases. FAS is thought to safeguard against autoantibody production by delivering death signals to self-reactive B cells that arise in the germinal centre (GC). Nevertheless, studies performed over the past two decades to elucidate a role for FAS in the regulation of GC B cell selection, differentiation and self-tolerance have yielded conflicting results. In this thesis a high resolution in vivo mouse model, specifically the adoptive transfer and challenge of anti-hen egg lysozyme (SWHEL) B cells lacking expression ofFAS, was used to definitively address the role of FAS in regulating GC B cell responses.The results indicated that FAS was in fact not required for the deletion of self-reactive B cells that arise in the GC reaction. Instead, it was discovered that FAS functions by preventing the development of a previously unrecognized lymphocyte population designated "rogue" GC B cells (GCr). GCr B cells somatically mutate and survive despite losing antigen reactivity and differentiate into large populations of plasma cells with unconventional specificities, including autoantibody-secreting clones. Significantly, lgE+ plasma cells were particularly increased, leading us to identify a major cohort of ALPS patients with hyper-lgE. Data presented in this thesis indicate that GCr B cells are a major driver of autoantibody production and provide a mechanistic explanation for the linked production of lgE and autoantibodies in autoimmune disease

DNA replication timing: impact on the cancer genome and epigenome

Alterations of the epigenome are increasingly being recognised as ubiquitous features ofcancer development. While considerable research has focused on mapping changes tothe cancer epigenome, few studies have integrated the contribution of DNA replication.As cancer undergoes unrestrained proliferation, DNA replication and the concomitantpropagation of epigenetic marks represents a unique environment to alter the genomeand epigenome. DNA replication itself follows a highly organised ‘replication timing’program whereby genomic domains are replicated in a specific temporal order during Sphase,from early to late. To address the impact of DNA replication timing on cancer, Iinvestigated the reciprocal relationships between replication timing and the cancergenome and epigenome, with a particular focus on DNA methylation.First, I found that DNA replication timing is a key feature that defines regions of thegenome prone to genetic and epigenetic aberrations. Using a well-developed prostatecancer cell model system and integrative genomic analyses, I demonstrated that earlyreplicating loci are predisposed to translocations, and late-replicating loci to DNAhypomethylation, alterations in heterochromatin, increased single nucleotide variantsand chromosomal rearrangements. These results have led me to propose a 3D model ofepigenome organisation relative to replication timing to explain the nature of the cancergenomic landscape.Next, to examine causation between alterations in the epigenome and alterations inreplication timing, I investigated replication timing in a DNA methyltransferase(DNMT1/3B) knockout model. I showed that the degree of replication timing changedepended on the degree of DNA methylation loss, and overall, loss of DNA methylationreduced the precision of replication timing. Finally, I developed a novel techniquecalled RepliBis-Seq to investigate the role of DNA maintenance methylation acrossreplication timing. I revealed that reduced maintenance methylation fidelity is biasedtowards late replication timing in normal cells and may be a mechanism contributing tothe global DNA hypomethylation observed in cancer.Altogether, my study shows that alterations in the epigenome can modulate DNAreplication timing, and conversely, DNA replication timing modulates the genome andepigenome. Ultimately, this research shows that DNA replication timing is an importantfeature to be considered in the genomic and epigenomic deregulation of cancer

Assessment of Left Ventricular Contractility and Loading Conditions Under Continuous-flow Left Ventricular Assist Device Support

Continuous-flow left ventricular assist devices (cf-LVADs) now form a cornerstone of the treatment of advanced heart failure. With their increasing use, improving our understanding of the mechanical, histopathological and physiological interactions between these devices and the patients in whom they are implanted is paramount.Initially, through non-invasive assessment of the impact of dynamic manoeuvres on the pump flow waveform and left ventricular dimensions, I demonstrate that changes in afterload pressure, posture and intrathoracic pressure have significant and highly variable effects on pump flow.The relationship between the intrathoracic pressure changes, loading conditions and pump flow is then assessed invasively, with low preload, low arterial resistance and increased ventricular-arterial coupling predictive of pulsatility loss and suction events during modified Valsalva manoeuvre.Using a pulsatile mock loop circulation, I assess the contribution of the outflow conduit to pump afterload. Haemodynamically significant gradients can be generated across an unobstructed HVAD outflow graft, and their magnitude predicted using an empirically derived model incorporating conduit diameter, mean pump flow, systolic dQdt and conduit length.I then demonstrate in vivo that some degree of tissue ingrowth into the cf-LVAD outflow graft due to acute inflammatory, chronic inflammatory, fibrotic or neointimal reaction is a near-universal phenomenon and is associated with a small but measurable decrease in pump flow and flow pulsatility over time.In order to enable an integrated assessment of left ventricular contractility, energetics and loading conditions, I describe a method to derive pressure-volume loops using non-invasive inputs that are readily available in the clinic setting. This method is validated invasively by assessing its ability to detect predictable pharmacodynamic effects of intravenous Milrinone.Finally, I utilise this pressure-volume derivation in order to assess the haemodynamic effects of exercise, revealing increased left ventricular contraction that is likely driven by increased preload, and profoundly diminished unloading effect of increased pump speed during exercise.Overall, this thesis sheds light on the complex interplay between ventricular contractility, loading conditions and cf-LVAD performance under ‘real-world’ conditions, with significant implications for both clinical practice and future research in this area

Vitamin D in critically ill patients

The pleiotropic functions of vitamin D, the clinical impact of deficiency and effect of supplementation have been a research focus in many specialties of ambulatory medicine. However, its prevalence in critical illness was not known. The parathyroid-vitamin D- calcium axis is the only endocrine axis that had not been previously described in critically ill patients. To study the effects of correction of deficiency, the dose, route and method of vitamin D supplementation needed investigation. Critically ill patients are heterogeneous and response to supplementation may not be consistent in all cohorts. This information is key to inform the design of a randomised controlled trial of supplementation. This thesis was a program of research that included a prospective observational study to describe serial changes in the parathyroid-vitamin D-calcium axis and the association with clinical outcomes in a sample of critically ill patients. This was followed by a randomised trial comparing two doses of intramuscular vitamin D to study the effectiveness and safety of supplementation. Subsequently, a sample of patients requiring life-sustaining extracorporeal support (ECMO) was studied to study pharmacokinetic profiles of vitamin D in patients with extreme disease severity. The observational study found marked changes in the parathyroid-vitamin D-calcium axis, which was associated with adverse outcomes. The supplementation study showed that a single intramuscular injection of cholecalciferol corrected vitamin D deficiency safely in critically ill patients. Vitamin D repletion was accompanied by a reduction in pro-inflammatory responses. The study on ECMO patients found them to be almost universally deficient in vitamin D. Supplementation by the single intramuscular dose corrected deficiency in only half these patients.Vitamin D deficiency is common in critical illness and is associated with adverse outcomes. Supplementation can be achieved effectively and safely in most patients. Groups of critically ill patients, such as those on ECMO are more susceptible to vitamin D deficiency and supplementation in this group is not easily achieved. This novel work and increasingly emerging evidence in the international literature provide a basis to inform the design of a high-quality comparative effectiveness randomised controlled trial of vitamin D supplementation in critically ill patients

An investigation of in vivo antigen-specific B cell responses

To optimize the initial wave of antibody production against T-dependent antigens, primary B cell clones with the highest antigen affinity are selected to generate the largest extrafollicular plasmablast (PB) responses. The mechanism behind this remains undefined, primarily due to the difficulty of analysing low-frequency antigen-specific B cells during the earliest phases of the immune response when key differentiation decisions are made. In this thesis a high resolution in vivo mouse model, namely the adoptive transfer and challenge of anti-hen egg lysozyme (SWHEL) B cells, was used to characterize in detail the first six days of a T-dependent B cell response and to identify the steps at which initial antigen affinity has a major impact. Antigen-specific B cells proliferated within splenic follicles from days 1.0-3.0 before undergoing a dynamic phase of multi-lineage differentiation (days 3.0-4.0) that generated switched and unswitched populations of germinal centre (GC) B cells, early memory B cells, and extrafollicular PBs. Differences in antigen affinity of >50-fold did not markedly affect the early stages of the response, including the differentiation and extrafollicular migration of PBs. However, high affinity PBs underwent significantly greater expansion within the splenic bridging channels and red pulp, due to both increased proliferation and decreased apoptosis. This thesis also describes an adaptation to the adoptive transfer strategy utilizing the SWHEL adoptive transfer model that enables the investigation of tolerance mechanisms that exist in the periphery. Within the GC, the random nature of somatic hypermutation gives rise to the possibility that B cells may generate self-reactive specificities. A description is provided of the design and production of a novel in vivo mouse model (HEL4X transgenic) engineered to study autoreactivity acquired during the GC reaction. Preliminary experiments are also described which demonstrated that upon encounter of a ubiquitously-expressed cross-reactive autoantigen, high- but not low-affinity autoreactive B cell clones generated in the GC undergo rapid deletion. This model provides an ideal system for defining the mechanisms that mediate inactivation of autoreactive B cells in the GC and how these cells may escape and differentiate into plasma cells that may potentially produce pathogenic autoantibody responses

Antiretroviral therapy and treatment cascade outcomes in a cohort of adults living with HIV in Australia

Successful HIV treatment requires daily, lifelong antiretroviral therapy (ART). Unfortunately, almost 20% of patients in Australia have uncontrolled HIV and so risk AIDS, death and transmitting HIV. The impact of socioeconomic variables on ART failure is poorly understood in a high-income, universal healthcare environment. In addition, many patients take concomitant medications that might affect ART adherence. To better understand ART failure, an Australia-wide cohort was developed and enrolled 522 adults on suppressive ART. At baseline, 15% missed ART at a rate previously associated with virological failure over time. Suboptimal adherence was associated with being Australian-born, not in a relationship, reaching the Medicare safety net, living in subsidised housing, receiving home-care services, linkage to HIV community services, and asking to start ART. 75% of the cohort took ?1 concomitant medication, and 23% had polypharmacy of concomitant medications. In total, 54% had polypharmacy or an ART drug interaction. Polypharmacy was associated with clinical trial participation, kidney disease, major comorbidity, hospital/general-practice based HIV care, and benzodiazepine use. For the first time in patients on ART, polypharmacy of concomitant medication was significantly associated with numerous adverse effects.To further understand the socioeconomic/psychosocial impact on ART failure, a case-control study compared 39 participants failing ART to 120 controls drawn from the main cohort. Ten socioeconomic/psychosocial indicators associated with ART failure, of which three (quality of life, foregoing necessities for financial reasons, and cost barriers to accessing HIV care) were independently associated.After 12 months, nearly 20% of the cohort failed ART, mainly for toxicities requiring an ART switch. ART failure was associated with; interrupting concomitant medications or non-diagnosis of a sexually transmitted infection in the year prior to baseline, psychiatric illness at baseline, or a new comorbidity or kidney impairment after baseline. Virological failure, but not ART failure overall, was associated with suboptimal baseline adherence. This was the first study to comprehensively demonstrate that socioeconomic variables are associated with suboptimal ART adherence in this setting. These variables are easily assessed in the clinic. Identifying patients at risk of ART failure will allow focused deployment of supportive resources to improve the Australian HIV treatment cascade

The role of macrophages in the regulation of β-cell function

Loss of β-cell function determines the development of T2D, as β-cells are the only site of insulin production. Macrophages are associated with β-cells, and are the major contributors of cytokines and islet inflammation in type 2 diabetes. Cytokines acutely stimulate, and chronically inhibit, insulin secretion. Islet inflammation is associated with β-cell failure, however the role of macrophages and cytokines in affecting β-cell function at stages preceeding T2D is not well characterised. Thus, the main aim of this thesis was to characterise the role of macrophages in regulating β-cell function in these stages.Islet inflammation was found to be absent in mice fed a high-fat diet, at the level of innate immune cell populations, and expression of inflammatory markers. An unbiased screen of the gene expression profile of islet-associated macrophages confirmed no major inflammatory alteration, and an upregulation of genes associated with the extracellular matrix, and trophic factors. This gene expression profile was compared to macrophage populations throughout the body, and was found to be unique.Depletion of macrophages in mice fed a HFD led to the unexpected induction of an acute inflammatory state. This improved β-cell function acutely, and appears to impair β-cell function over the longer term. This illustrated the role of inflammation in regulating β-cell function, and stressed the need for caution when using models to deplete cells in vivo, especially in the study of metabolism. The role of PKCε in macrophage biology was investigated in relation to the regulation of β-cell function in the HFD-fed mouse. While no specific effect of deletion of PKCε was found in macrophages, deletion of PKCε improved β-cell function in mice fed a HFD. Thus, an alteration in macrophage function may affect β-cell function in this state. In summary, this research has shown that: (1) islet-associated macrophages have a unique gene expression profile and may facilitate β-cell hyperplasia; (2) islet inflammation is not present before the onset of β-cell failure; (3) inflammation acutely stimulates and chronically inhibits insulin secretion in vivo; (4) PKCε is not central to macrophage activation; (5) a subtle alteration in macrophage function can affect β-cell function