Medicine - Doctoral Theses

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Now showing 1 - 5 of 92
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    Characterisation of the effect of electroporation and electrochemotherapy on cancer cells and immune cells in the tumour microenvironment
    (University College Cork, 2022-10) Bendix, Maura; Brint, Elizabeth K.; Houston, Aileen M.; Amu, Sylvie; Forde, Patrick; Health Research Board; Breakthrough Cancer Research
    Lung cancer is the leading cause of cancer-related death worldwide, with the lung cancer incidence rate expected to rise further. Despite recently developed novel therapy options, 5-year survival rates for lung cancer patients remains below 20% generally and below 5% for late-stage diagnosis, thus additional therapy options are still needed. Electrochemotherapy (ECT), the application of an electric pulse to deliver chemotherapy drugs into cells, could be a new treatment option for lung cancer patients. ECT is a locally very effective treatment, with local tumour reduction of up to 85%, while the systemic effects are more varied. For clinical application ECT treatment modalities have been standardized since 2006, after the ESOPE study, which optimized ECT parameters to 8 pulses at 1000V/cm with 100µs pulse length at 1Hz frequency and either bleomycin or cisplatin as the drug of choice. To evaluate whether ECT could be a potential treatment option for lung cancer patients’ ECT parameters, the needed electric field strength and the needed drug and drug concentration, were optimized for in vitro lung cancer research. In our study, we initially developed a standard operating protocol (SOP) to determine the optimal electric field strength for a given cancer cell line in vitro, while keeping the other ESOPE parameters constant. The developed SOP combined short-, medium-, and long-term assays to fully visualize the impact treatment, at a given field strength, has on the tested cancer cell line. This evaluation showed that human lung cancer cell lines (A549, H460 and SK-MES 1) and the human pancreatic Pan02 cell line have an optimal electric field strength of 800V/cm, the melanoma A375 (human) and B16F10 (murine) cell lines as well as the murine pancreatic Mia-PACA2 cell line have an optimal electric field strength of 700V/cm, while the murine Lewis Lung carcinoma (LLC) cell line has an optimal electric field strength of 1300V/cm. In addition, our study findings demonstrate that cisplatin at 11µM would be the drug of choice when using ECT for lung cancer treatments. In recent years while the importance of the immune system in lung cancer development and treatment results has become increasingly clear, little is known about how ECT treatments impact immune cells. Therefore, the impact of ECT on murine T cells, dendritic cells (DCs) and macrophages was evaluated in vitro. Our data indicates that while T cells are able to tolerate electric field strengths of up to 1400V/cm, DCs and macrophages are significantly negatively impacted by electric field strengths exceeding 800V/cm. Further investigation on the impact of ECT on dendritic cells demonstrated that DCs die via necrosis following ECT treatment, while ECT at electric field strength exceeding 1000V/cm leads to DC maturation and activation of the surviving cells. In addition, DCs remain partially functional following ECT treatment in a stimuli and treatment dependent manner with distinctively different sets of genes upregulated 4-hours post treatment at 800V/cm compared to treatment at 1000V/cm and 1300V/cm. Taken together, our data indicates that it is worthwhile to further investigate ECT as a potential therapy option for lung cancer patients, while more attention needs to be paid to the impact ECT has on immune cells in order to maximize treatment results.
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    A population-based epidemiologic study of adult neuromuscular disease in the Republic of Ireland
    (University College Cork, 2014) Lefter, Stela; Ryan, Aisling; Hardiman, Orla
    This research project generates for the first time prevalence rates (PR) for all adult neuromuscular diseases (NMD) as well as the incidence rates (IR) for Guillain-Barre syndrome (GBS) in the Republic of Ireland (ROI). Multiple case ascertainment sources were used to achieve as complete as possible case ascertainment to accurately estimate the country-wide prevalence. Acquired demyelinating polyneuropathies represented the biggest cohort (26.0%) followed by muscular dystrophies (22.5%) and myasthenia gravis (19.7%). For GBS, in the 20 year period (1992 to 2012), comparable with other international studies, incidence rates of 0.3-1.3 per 100,000 person-years were attained. Higher PR figures for chronic inflammatory demyelinating polyneuropathy and sporadic inclusion body myositis were found (5.87 per 100,000 and 11.7 per 100,000 in those older than 50 year, respectively) compared with previous studies. The PR for myasthenia gravis in ROI is comparable with countries such as Australia, Italy and Norway but is lower than in Northern Ireland. The PR for Charcot-Marie-Tooth disease and limb girdle muscular dystrophies were similar with United Kingdom PR; however, a definite diagnosis was attained in only one third of Irish patients. Given the fact that paediatric cases were excluded from our study, lower PR figures were obtained for dystophinopathies. Interestingly, the PR for myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy (FSHD) in the ROI was lower than in United Kingdom but not considerably. A high PR for periodic paralysis (PP) was found in our study, nearly ten times higher for Hyper PP and as twice as high for Hypo PP when compared with figures from England; however, the ROI figures were derived from large families suggesting a founder effect. Of all the inherited NMD, 46% of cases had a definite histopathology and/or genetic diagnosis. The combined PR for inherited and acquired NMD was high, 62.6 per 100,000, when compared with other chronic neurological diseases in ROI. During the course of the study, a definite genetic diagnosis was achieved in families with rare previously unreported NMD in ROI, such as Laing distal myopathy, Andersen Tawil syndrome and FSHD2. This research defines the burden of NMD in the ROI, raises awareness of these conditions, will facilitate earlier intervention where appropriate and will provide a strong argument for service provision for these patients. This research project will serve as a useful originator for future research studies, especially with regard to obtaining genetic confirmation of currently undefined cases.
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    Characterisation of the role of the IL-36 family of cytokines in the pathogenesis of colon cancer
    (University College Cork, 2022-06-26) Baker, Kevin J.; Brint, Elizabeth K.; Houston, Aileen M.; Irish Research Council
    The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines. These cytokines have now been identified to play a role in the pathogenesis of many inflammatory diseases and are increasingly being implicated in tumourigenesis. Given the pluripotent nature of other IL-1 family members and the relationship between inflammation and tumorigenesis, here we have investigated the effects of IL-36 signalling in colorectal cancer. In this study we demonstrate that expression of IL-36 family member mRNA and protein is significantly increased in colorectal cancer tissue compared to adjacent colonic non-tumour tissue. Colon cancer cell lines express IL-36 family genes differentially, and these genes are inducible with Toll-like Receptor ligands and pro-inflammatory cytokines. Following stimulation with IL-36 agonists, colon cancer cell lines increase expression of pro-inflammatory genes, especially genes involved in myeloid cell chemotaxis. Colon cancer cells lines are more responsive to IL-36β and IL-36γ in comparison to IL-36α. In vitro assays showed stimulation of colon cancer cell lines with IL-36 agonists augmented several pro-tumorigenic phenotypes such as cellular migration, invasion and proliferation in both 2D and 3D models. In pre-clinical models of colon cancer, intraperitoneal injection of the IL-36 Receptor antagonist (IL-36Ra) significantly reduced tumour burden using the subcutaneous CT26 tumour model in syngeneic Balb/mice. This was associated with a decrease in Ki-67 expression by tumour cells in the IL-36Ra-treated group relative to untreated control tumours, suggesting the inhibition of the pro-proliferative signalling of IL-36 agonists resulted in the decreased tumour size. Moreover, colon cancer cells lacking the IL-36R also showed reduced tumour growth and reduced Ki-67 expression in vivo. IL-36 agonist administration also resulted in a tumour reduction in mice, although this was not as effective as IL-36Ra administration and did not alter Ki67 expression levels in tumour tissue but rather acted through immune infiltration of tumours. Taken together, this data suggests that targeting IL-36R signalling may be a useful targeted therapy for colorectal cancer patients with IL-36R+ cancer cells. In order to further understand the effects of IL-36 cytokine signalling in the context of immune cells, co-cultures of macrophages and colon cancer cells were completed in vitro. The THP-1 model of macrophages showed minimal changes in response to IL-36 agonist stimulation. M1 macrophage cells significantly reduced spheroid formation of HT29 cells, with addition of IL-36 agonists facilitating recovery of spheroid size back to untreated size, indicating colon cancer cells are more responsive to IL-36 stimulation than macrophages when in co-culture in this model. Preliminary work using HL-60 cells as models of neutrophils showed IL-36 can augment cancer-cell induction of neutrophil NETosis, potentially contributing to immune evasion and metastasis. Transcriptomic analysis of publicly available patient cohorts revealed increased expression of IL-36 family members in malignant intestinal tissue in comparison to paired healthy tissue. Moreover, IL-36R expression is associated with poorer patient survival rates in colon cancer. Our DEG analysis of tumours expressing high levels of IL-36R mRNA revealed a possible role for the IL-17/IL-22/IL-23 signalling axis in colon cancer involving IL-36 signalling. Together, this study demonstrates that IL-36 signalling in colon cancer may contribute to disease progression and that inhibition of this signalling, in subgroups of patients stratified according to cancer cell expression of the IL-36R, may benefit survival rates, as shown in our in vivo pre-clinical models of colon cancer.
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    Phenotyping for a personalised approach to CVD prevention
    (University College Cork, 2022-03-01) McEvoy, John William; Eustace, Joe; Fanning, Liam J.
    Cardiovascular disease (CVD) prevention requires interventions at the level of the population (public health) and at the level of individuals (medical care). Therapeutic options to reduce the risk of CVD among at risk individuals are increasingly available, but access to and implementation of these evidence-based therapies is suboptimal and costs to health systems are often a deterrent to payers. Therefore, advanced phenotyping of individuals, for example using biomarkers of subclinical CVD, in order to better personalize their preventive care is an attractive approach. In the corpus of work presented for this PhD by prior publication, a series of eleven published papers is included that examines the role of biomarkers of subclinical CVD (specifically high-sensitivity Troponin measured in blood and coronary artery calcium imaging) in the management of persons with hypertension and abnormalities in diastolic blood pressure.
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    Effect of the α4β7 integrin blocking antibody vedolizumab on the clinical and host immune response in patients with inflammatory bowel disease
    (University College Cork, 2022-02-05) Judge, Ciaran; Nally, Ken; Melgar Villeda, Silvia; Buckley, Martin
    Background: Inflammatory bowel disease (IBD) is a chronic, relapsing and remitting condition affecting the gastrointestinal tract. Development of IBD is thought to arise from a complex interplay of genetic susceptibility, environmental contributors, and disordered immune responses to host microbial components. Improvements in our understanding of these interactions have identified novel therapeutic targets and therapeutic interventions which will address unmet clinical need and the therapeutic gap in IBD. However, currently, response and remission rates remain static, reflecting the need and potential for future developments. Vedolizumab (VDZ) is a humanised monoclonal anti-integrin targeting the α4β7 molecule. It is designed to have gut-specific anti-inflammatory effects by blocking the interaction between α4β7+ lymphocytes and mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) on gut endothelial cells. It is thought that this may reduce lymphocyte trafficking specifically into inflamed gut tissue in IBD. Despite advances in our understanding of its clinical efficacy and its proposed mechanism of action the exact mechanism of action of this drug remains uncertain. Aim: The aim of this MD thesis is to characterise the effect of treatment with VDZ on patients with IBD at the clinical and immune system level over three studies, to determine if there are clinical or immune features that may predict or enhance response to therapy, or that may create new opportunities for treating this challenging group of conditions. Methods: Study One was a cohort study evaluating the clinical outcomes of patients with IBD treated with VDZ from 9 Irish hospitals. Study Two was designed as a prospective cohort study to assess the impact of VDZ monotherapy on the gut mucosal immune response. Mucosal biopsies were obtained from patients about to start VDZ therapy for active IBD and isolated cells from tissue were analyzed by flow cytometry to assess both mucosal α4β7+ macrophages (CD45, CD11c, CD14, CD163, CD64, CD66b, HLA DR, SIRPa, CD103, α4β7) and α4β7+ lymphocytes (CD3, CD4, CD8, CCR7, CD45 RA, αEβ7, α4β7). Study Three took a similar approach to investigate potential differences in the gut mucosal immune response of patients already established on a combination therapy (CBT) of VDZ with another anti-TNF biological therapy (e.g. VDZ + infliximab, or VDZ + adalimumab) for treatment-refractory IBD, versus those established on VDZ-monotherapy, or patients who were VDZ-naïve. Results: Analysis of 129 patients in Study One supported the clinical efficacy and safety of VDZ in IBD, with 6-month corticosteroid-free clinical remission rates of 31% and 48% in patients with ulcerative colitis (UC) and Crohn’s disease (CD), respectively; and adverse event rates of 1% and 11%, respectively. Study Two, although limited by effects of Covid-19 pandemic in Ireland, revealed that there were more α4β7+ monocytes and lymphocytes in inflamed vs non-inflamed GI mucosa, and more α4β7+ lymphocytes in mucosal samples from patients with CD vs UC. Small numbers of CD64+α4β7+ monocytes were also identified in the inflamed tissue. Study Three revealed a significant ongoing trend towards a higher percentage of α4β7+ T cells in those treated with VDZ-CBT in comparison to both other groups, in addition to an overall increase in the number of αEβ7+ cells in both CD4+ and CD8+ compartments in those treated with VDZ-CBT. Conclusion: Study One demonstrated the clinical utility of VDZ in the treatment of IBD in an Irish cohort of patients, providing valuable real-world data in line with international studies. Study Two supports reports that α4β7+ lymphocytes are more prevalent in inflamed tissue in IBD, and suggests that α4β7+ monocytes may play a role in mucosal inflammation. Study Three demonstrates ongoing presence of α4β7+ cells despite combination biologic therapy, perhaps due to a more refractory inflammatory phenotype, or the ‘re-wiring’ of inflammatory pathways. This project provides valuable information on the clinical utility of VDZ in the treatment of IBD, in addition to novel insight into the cellular and immunological effects of this important medication.