Medicine (St Vincent's) - Theses
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Itemβ-cell function, insulin sensitivity and non-esterified fatty acid (NEFA) dynamics after intra-portal allogeneic islet transplantation( 2010)BACKGROUND: Intra-portal allogeneic islet-transplantation is an effective means of biologic insulin-therapy in T1DM with debilitating hypoglycemia. While whole-pancreas-transplantation is known to restore normal biphasic, pulsatile insulin secretion and the incretin-response; little is known of islet-function and the metabolic milieu post-islet-transplantation. Few studies have compared recipients with T2DM subjects. AIMS: We aimed to evaluate Si pre- and post-transplantation as well as biphasic secretion, the incretin-response, glucose-sensitivity and NEFA suppression during an OGTT; comparing these results with controls and type 2 diabetics. METHODS: 3 islet-recipients in their 1st year of insulin-independence, 10 non-diabetic controls and 4 T2DM patients (requiring OHAs) each underwent 3 different glucose challenges on 3 separate days over 3-4 months, 4-6 weeks apart. The 3 procedures were a 4hr 75g-OGTT, an isoglycemic IVGTT and an FSIGT. Plasma glucose, insulin, C-peptide and NEFA were assayed. The islet-recipients underwent a non-insulin-modified FSIGT (enabling estimation of 2nd phase), the results of which were compared with historical controls similarly assessed. The rest underwent an insulin-modified-FSIGT. 2-step hyperinsulinemic-euglycemic-clamps were performed pre-transplantation and post-insulin-independence. RESULTS: Although recipients maintain an HbA1c ≤ 6% post-transplant without exogenous insulin, mean 2hr-glucose post-OGTT was 13 mmol/L. Clamp Si was unimpaired post-transplantation despite use of tacrolimus/mycophenolate. Post-transplantation, FSIGT second-phase secretion was restored to a greater extent than first (87% vs 46% normal). AIRg post-transplant was diminished compared with controls but ~8.5 times better than that in T2DM. During the OGTT while both early (0-30min) and late (30-240min) phase secretion were significantly and similarly reduced in recipients and T2DM compared with controls, more insulin was secreted per mmol glucose during late phase than early phase in recipients but not in type 2 diabetics. Islet-recipients had an oral DI ~26% of normal (4-fold higher thanT2DM). The incretin-response was reduced post-transplant and in T2DM (recipients 49%, T2DM 47%, controls 80%). Glucose-sensitivity was 26.3% of normal in recipients. NEFA suppression was unexpectedly normal and better than that in T2DM. Insulin secretion in recipients was worse during the OGTT (21% of normal) than the FSIGT (77% of normal). CONCLUSIONS: Our findings indicate that islet-recipients have a hybrid form of diabetes with features in common with T1DM (unimpaired Si) and T2DM (metabolic stability without exogenous insulin), and features unique to the post-transplant state (normal NEFA dynamics).Post-transplant diabetes is characterized by quantitative and qualitative β-cell secretory defects classically associated with the T2DM phenotype–reduced first-phase, incretin-response and glucose-sensitivity with preserved second-phase. Islet-recipients however have greater functional β-cell secretory-capacity and better insulin-sensitivity than OHA-requiring T2DM subjects. The presence of these secretory defects in recipients infused with healthy donor beta-cells indicate these are acquired functional β-cell defects common to all forms of dysglycemia resulting from a final common pathway of β-cell dysfunction/apoptosis. Despite these severe defects, insulin-independence is preserved - indicating that an oral DI 1/4th normal, in these highly insulin-sensitive subjects is sufficient for normoglycemia under free-living conditions. The impaired insulin response during the oral challenge compared with the intravenous challenge may be secondary to defects in incretin mechanisms and/or secretion of anti-incretin factors during the OGTT. Normal NEFA levels early post-transplant imply reduced risk of glucolipotoxicity and its deleterious effects on Si and graft-function. Our work highlights the important contribution of insulin-sensitivity and second-phase secretion towards maintaining normoglycemia post-transplant and hence the need to monitor these parameters. Abbreviations: type 1 diabetes (T1DM), type 2 diabetes (T2DM), oral hypoglycaemic agents (OHAs), oral glucose tolerance test (OGTT), isoglycemic intravenous glucose tolerance test (IVGTT), frequently-sampled intravenous glucose tolerance test (FSIGT), non-esterified fatty acids (NEFA), HbA1c (glycated hemoglobin), disposition index (DI), AIRg (acute insulin response to glucose), insulin-sensitivity (Si)
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ItemThe evaluation of endoscopic technologies in the investigation and management of colonic neoplasia and obscure gastrointestinal bleeding( 2010)The aim of this work is to evaluate the use of endoscopic technology in colorectal neoplasia and obscure gastrointestinal bleeding. The focus is on the specific evaluation of cold forceps polypectomy, balloon enteroscopy, narrow band imaging and capsule endoscopy. The accuracy of cold forceps polypectomy for diminutive polyps (≤5mm) is assessed in a prospective study using a novel protocol. Patients referred for colonoscopy with one or more diminutive polyps, underwent cold forceps polypectomy followed by endoscopic mucosal resection of the polypectomy site with a 2-3mm resection margin. The two specimens were collected in separate pathology jars and assessed by a pathologist blinded to the study protocol. Comparison between the two specimens allowed for unequivocal assessment of resection efficacy. Fifty-two patients were enrolled and fifty-four polyps were resected. The complete resection rate with cold forceps polypectomy was 39%. Histology was predictive of resection efficacy, with the resection rate for adenomas at 62% compared to 24% for hyperplastic polyps, P=0.008. Polyp size and the number of bites of the forceps were not predictive of resection. A pilot study is then presented, assessing the feasibility of a protocol comparing narrow band imaging and chromoendoscopy, for dysplasia surveillance in patients with chronic colitis. The secondary aim of the study was to assess the accuracy of the Kudo classification (using narrow band imaging) in predicting the neoplastic potential of detected lesions. Twenty-two patients were enrolled in the study. The colon was divided into segments, using the flexures. Each segment was independently examined using narrow band imaging followed by examination with chromoendoscopy. Seven dysplastic lesions were detected in four patients. Both narrow band imaging and chromoendoscopy detected six of the seven dysplastic lesions. Narrow band imaging failed to diagnose one dysplastic lesion detected by chromoendoscopy. Both narrow band imaging and chromoendoscopy detected far more benign lesions than neoplastic. The sensitivity and specificity of the Kudo classification for neoplasia, (using narrow band imaging) was 60% and 79% respectively with a negative predictive value of 95%. Attention is then turned to capsule endoscopy, in a prospective assessment of the accuracy of faecal occult blood tests (FOBT) as a screening test pre-capsule endoscopy in obscure gastrointestinal bleeding. Patients referred for capsule endoscopy underwent two FOBTs within a fortnight preceding capsule endoscopy. The results of the FOBTs were correlated with clinically significant findings on capsule endoscopy. The primary aim of the study was to calculate the predictive value of FOBT in this setting. Fifty-six patients were enrolled. Fifty four percent had occult obscure gastrointestinal bleeding and the remainder overt obscure gastrointestinal bleeding. The overall yield of capsule endoscopy for clinically significant findings was 41%. The combination of immunochemical and guaiac FOBT had only modest sensitivity (63%), specificity (59%), negative predictive value (68%) and positive predictive value (54%). The last project presented in this thesis, is a randomised controlled study comparing single balloon enteroscopy (SBE) and double balloon enteroscopy (DBE) for the investigation and treatment of small bowel conditions. Patients referred for balloon enteroscopy were randomized in 1:1 allocation to single balloon enteroscopy or double balloon enteroscopy. Ninety-nine patients were enrolled and in total, 110 procedures were undertaken, 46 with SBE and 64 with DBE. The primary endpoint was diagnostic yield. Secondary endpoints were therapeutic yield, depth of insertion and procedural characteristics. The diagnostic yield of single and double balloon enteroscopy for small bowel findings was similar at 46% and 41% respectively, P=0.60. Therapeutic interventions were undertaken in 28% of those with SBE and 23% of those with DBE, P=0.57. The depth of insertion was longer for antegrade double balloon enteroscopy, however this was not statistically significant (200cm for SBE and 250cm for DBE, P=0.13). Insertion depth for retrograde procedures was 70cm median for SBE and 85cm for DBE, P=0.86. Despite shorter endoscope setup times with single balloon enteroscopy (3min vs. 11min, P<0.005), total procedure times were similar at median 90 minutes for SBE and 85 minutes for DBE, P=0.84. Cost identification analysis confirmed that the initial set up costs for SBE were considerably lower than those for DBE for hospitals with compatible Olympus equipment, however the overall procedure costs over time were similar. Each of these studies has addressed unique questions involving the use of endoscopic technology in the areas of colorectal neoplasia as well as obscure bleeding, which is a common presentation of small bowel neoplasia.
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ItemEpidemiology, natural history and impact of inflammatory bowel disease in Australia( 2010)The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract. International studies have demonstrated a dramatic rise in the incidence of these conditions over the past several decades. There are no corresponding studies from Australia to determine the local incidence. CD has been shown to have a complicated disease course, with high requirements for surgery and progression to disability. Psychological morbidity is also common, with a negative impact on health related quality of life. International studies have also demonstrated high direct and indirect health economic costs associated with CD. Australian data in each of these areas is either limited or totally absent. The aim of this work was to address the deficiencies in each of these key areas. The first Australian population-based study of IBD incidence was conducted prospectively over a one-year period in the region of Barwon. This utilised an extensive capture-recapture methodology, with near complete case identification and rigorous verification of diagnosed cases. The IBD incidence rates observed are among the highest reported in the world literature. The natural history of CD was then assessed in a retrospective, inception cohort study over a five-year period from the time of diagnosis. There was the progressive development of a more complicated disease phenotype, with high requirements for surgery. The results from surgery were not durable, with 60 percent needing escalation of therapy within 5 years. 41 percent of the cohort met the definition of disabling disease, with the presence of perianal fistulae at diagnosis highlighted as a key risk factor. This was followed by two cross-sectional, questionnaire-based, cohort studies to assess psychological health and health related quality of life (HRQoL) in CD. The first study was conducted in patients from the Inflammatory Bowel Disease Clinic at St. Vincent’s Hospital, Melbourne. The second was in a cohort of patients from the same institution who had required the formation of an ileostomy for CD. Both studies revealed high rates of depression, anxiety and poor HRQoL. These negative factors were contributed to by increased disease activity, but the strongest predictive factor was found to be the use of a negative, maladaptive coping style. Some patients with a stoma have adapted well, but others found it to be a negative experience, with ongoing concerns regarding sexuality and body image. The health economic costs of CD were then established using prospective cost diaries. Both direct and indirect costs were high, in keeping with the complicated natural history of CD. These studies highlight that IBD is common in Australia, and that CD has a complicated natural history, with negative impacts on psychological health and HRQoL, and with high economic costs. There is a need for increased public awareness as well as ongoing research and funding to improve clinical care.
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ItemRheumatoid arthritis, cardiovascular disease and inflammation.... and the effects of rheumatoid arthritis drug therapies on this musketeer trio( 2010)Rheumatoid arthritis (RA) is associated with a significantly higher rate of cardiovascular (CV) disease and mortality (1). The higher CV risk appears to be independent of traditional CV risk factors (2). Atherosclerosis is now recognised as an inflammatory disease, and the relationship between RA and CV disease may partly be explained by chronic systemic inflammation (3). Inflammation plays a role at all stages of the atherosclerotic process from the earliest stages of endothelial dysfunction to plaque development and eventually to plaque complications such as rupture and thrombosis which lead to clinical events such as heart attack and stroke (4). Medications commonly taken by RA patients may exacerbate or improve CV risk. Methotrexate improves RA disease activity, inflammation and CV mortality in RA (5). Non-steroidal anti-inflammatory medications (NSAIDs), cyclo-oxygenase inhibitors (COX-2 inhibitors) and TNFα-inhibitors are commonly used in RA with varying degrees of inflammation suppression. Their effect on CV risk in RA might therefore assist in our understanding of the complex relationship which we hypothesise exists between RA, CV disease and inflammation. The aim of this thesis was to investigate vascular dysfunction in RA and the relationship to markers of inflammation, and to assess whether intervention with NSAIDs, COX-2 inhibitors and TNFα-inhibitors has an effect on vascular function in RA. In a cross sectional study of 106 RA and control subjects pulse wave analysis (PWA) was performed. A subgroup of RA patients and controls had known coronary artery disease (CAD). Vascular measures were correlated with markers of inflammation (6).In a double blind placebo controlled trial, 37 RA patients were randomised to a two week course of COX-2 inhibitor, rofecoxib, NSAID, indomethacin or placebo. Flow mediated dilatation (FMD) and PWA were assessed before and after the two week treatment course (7). In a further intervention study 26 RA patients were randomised to TNFα-inhibitor Infliximab or placebo infusions. Vascular assessments included pulse wave velocity (PWV), PWA, carotid intima media-thickness (CIMT) and Carotid artery plaque (CAP). Follow up was to 56 weeks (8). Results from the cross sectional study confirmed that vascular function was impaired in RA and in the subgroup of patients with CAD when measured with PWA. Vascular function correlated with acute phase proteins when analysing the study group as a whole. In terms of short term intervention, COX inhibition with either rofecoxib or indomethacin did not significantly improve vascular function at two weeks as measured by FMD and PWA. Markers of inflammation also did not improve with these treatments. Post-hoc analysis of the infliximab intervention study using multivariate ANOVA modelling showed significant reduction in PWV over 56 weeks. RA disease activity and markers of inflammation also improved with this treatment. There was no change in PWA, CIMT or carotid plaques. In conclusion, patients with RA have impaired vascular function. This was independent of traditional CV risk factors but correlated with markers of inflammation. A short term intervention with a COX-2 inhibitor or NSAID did not improve vascular function at two weeks when measured with PWA in RA patients. Intervention with TNFα inhibitor improved PWV in RA patients at 56 weeks. These findings support the concept that chronic inflammation is associated with vascular dysfunction and this may relate at least in part to the increased CV risk in RA. Therapies effective in suppressing inflammation not only improve RA disease activity but also vascular function.
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ItemThe clinical role of novel biological agents and the in vivo immunological effects of immunomodulatory drugs (IMiDs) in the management of multiple myeloma( 2010)Over the last decade, the introduction of novel therapeutic agents including thalidomide, lenalidomide and bortezomib has revolutionised the treatment and outlook of multiple myeloma (MM), an incurable plasma cell malignancy with significant morbidity. Our experience with these novel agents has yet to improve however, with regards to the optimal scheduling and combination therapy that would maximise response duration and survival. Furthermore, the exact anti-myeloma mechanisms of these agents are poorly understood, and many of the proposed mechanisms in vitro have yet to be corroborated in vivo. The work presented in this thesis aims to improve our current understanding of the in vivo immune modulation by lenalidomide in patients with MM, and to fine tune current treatment regimens based on our matured experience with these novel therapeutic agents, so as to minimise toxicity and maximise response and survival outcome. The first chapter (1A) reviews the current treatment paradigm of MM, and the current accepted role of thalidomide, lenalidomide, bortezomib and histone-deacetylase inhibitors. The current understanding of MM pathophysiology and the mechanism of actions of novel agents is reviewed in chapter 1B, providing a background for the correlative immunological studies in chapter 3B. The work from chapters 1A and 1B have culminated in 2 published review manuscript in Drugs and Aging 2007 and Leukemia 2009, respectively (see preface). The second chapter explores the long-term outcome to thalidomide-based treatment to show that depth of response to thalidomide correlates to survival outcome. This chapter highlights the importance complete remissions in the quest for durable responses in the era of novel therapeutic agents. The work from this chapter has been published as a manuscript in Leukemia & Lymphoma 2009. The following chapters (3,5,6) are a series of investigator-driven phase II trials exploring optimal scheduling of lenalidomide or bortezomib with dexamethasone in the treatment of relapsed or refractory MM, as well as the safety and preliminary efficacy of a novel bortezomib-romidepsin combination therapy. To enable the ongoing optimisation of treatment schedules or combinations of novel-agent, an understanding of their individual in vivo effects is important. Correlative immunological studies were therefore performed, as outlined in chapter 3B, to investigate the in vivo immunological changes in patients treated with immunomodulators, and how immune stimulation can be optimised to further improve clinical outcome. Indeed it was found that the concurrent use of dexamethasone abrogates lenalidomide-induced NK cell stimulation, and may subvert the full immune stimulatory capacity of lenalidomide. These findings have been submitted as a manuscript to Blood at the time of this thesis submission, and have been presented at several international scientific meetings. The insight that was gained from this correlative immunological study also resulted in a review manuscript on drug-mediated and cellular immunotherapy in MM, was published in Immunotherapy 2010. Overall, the work from this thesis aims to contribute to the shaping of optimal treatment strategies for MM, as well as an improved understanding of the in vivo effects of lenalidomide. We remain in an era of exploration of the role of novel therapeutic agents in the treatment of MM, and how to best manipulate sequential or combination therapy with the ultimate aim of cure, if not durable disease control.
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ItemThe potential role of inflammatory cells in neoadipogenesis( 2010)Reconstruction of soft tissue defects presents a major challenge in clinical practice. Current treatment strategies for large and small volume replacement of such defects are less than satisfactory. Generation of endogenous stable vascularized adipose tissue engineered constructs can have an enormous clinical impetus. In order to study adipose tissue engineering in vivo, a mouse tissue engineering model that consists of a ‘sealed’ silicone chamber supplied with a dedicated flow-through epigastric pedicle and incorporating Matrigel matrix supplemented with fibroblast growth factor-2 was developed at the O’Brien Institute. Previous research using this model revealed that sealed chambers containing just matrix resulted in minimal adipogenesis. However, when a viable free fat graft was inserted into the chamber together with the matrix, resulted in significant vascularized adipose tissue in the chamber that was subsequently found to be host-derived. The free graft was mostly necrotic and was infiltrated by inflammatory cells. When the graft was replaced by the sterile inflammagen zymosan, adipogenesis was induced in an inverse dose-dependent manner. This data supports several studies that link adipogenesis and inflammation in obesity. Mast cells and macrophages are important inflammatory cells that participate in inflammatory responses and may be related to endogenous adipogenesis. They also participate in angiogenesis which is closely related to adipogenesis. The potential roles of mast cells and macrophages in angiogenesis and adipogenesis were investigated using this mouse tissue engineering model. This was performed using two mast cell-deficient mouse strains and knocking out macrophages in another strain using clodronate liposomes. Mast cells were not found to be important in angiogenesis and adipose tissue development in this model, though there was a significant strain difference in fat development. On the other hand, macrophages play a critical role in both angiogenesis and adipose tissue development in this model. Normal white adipose tissue is innervated by sympathetic nerve fibres that have important physiological functions. There is a neuroimmune link between sympathetic nerve fibres, nerve growth factor and inflammatory cells such as mast cells and macrophages. Sympathetic nerve regeneration in the mouse tissue engineering model was described in this thesis. Sympathetic nerves did not correlate with the mast cell numbers and inversely correlated with macrophage density at six weeks. However, sympathetic nerve regeneration positively correlated with the percentage of new adipose tissue developed. Further in vitro studies revealed that both undifferentiated and differentiated stromal vascular fraction induced neurite outgrowth from SCG suggesting that cells, possibly preadipocytes, differentiated adipocytes, vascular endothelial cells and inflammatory cells may support sympathetic nerve development in the chamber. This thesis identifies the importance of macrophages and the insignificance of mast cells in vascularized adipose tissue development. It also describes sympathetic nerve regeneration in association with vascularized adipose development.