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- ItemCharacterisation 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 ResearchLung 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.
- ItemCharacterisation 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 CouncilThe 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.
- ItemReal-time bioaerosol analysis in the healthcare environment(University College Cork, 2020-01) Fennelly, Mehael; Sodeau, John R.; Prentice, Michael B.; O’Connor, David; Healthcare Infection SocietyAirborne infection has been difficult to study in hospitals. Conventional sampling methods for airborne organisms are limited in sample time intervals (minutes to hours) and conventional culture requirements, restricting organism detection and only allowing retrospective analysis (days). This limits their usefulness in analysing air quality and risks of airborne transmission of infection. They provide limited data for standard setting and assessing the effect of interventions designed to increase air quality and decrease airborne infection risks. Direct continuous bioaerosol sampling is an established technology used to characterise ambient external air. Portable instruments such as the Wideband Integrated Bioaerosol Sensor (WIBS) combine laser particle size and shape detection with signals of particle viability (fluorescence from amino acids and NAD(P)H) characteristic of bioaerosols. This aim of this thesis was to investigate the utility of continuous monitoring approaches including WIBS and other instruments to characterise indoor air bioaerosols in hospital environments and evaluate the results of interventions designed to increase air quality. The WIBS-4A was used to characterise airborne biological particles in a 4-bedded hospital respiratory ward bay over a 4-week period before and during a plasma air treatment intervention designed to increase air quality. Twice-daily conventional impaction and settle plates and surface swabs were carried out in parallel with continuous WIBS bioaerosol monitoring. No statistical difference between conventional culture counts was detected during the plasma air treatment period compared with the control. Cumulative continuous monitoring plotted diurnally revealed raw numbers of airborne fluorescent particles were lowest at night, with four striking recurrent fluorescent particle peaks during the daytime when the number of particles increased by over 200-fold compared to the nocturnal minimum. These peaks corresponded to observed nebuliser use on the ward. WIBS analysis of the two nebulised therapy drugs used on the ward defined a characteristic fluorescence signature for nebuliser aerosols. This allowed design of a threshold filter to remove interferent nebulised drugs from fluorescent particle counts which did not eliminate bacteria when applied to experimentally aerosolised bacteria. Both raw and filtered WIBS data (excluding nebulised drug particles) showed a statistically significant ~28% reduction in fluorescent particles, (P<0.05), during the operation of the plasma disinfection unit. The clinical significance of this requires further study. The effect of footfall counts on bioaerosol concentrations was also monitored by deploying an infra-red footfall counter in tandem with the WIBS instrument. Both devices were successful in identifying that the highest footfall count coincided with the highest bioaerosol concentrations observed on the ward, which also coincided with the main morning staff shift change and handover. The cumulative filtered count data was used to devise a statistical threshold which could be the basis of a standard for the environment tested. The WIBS-4A was used in conjunction with the nebulised drug signatures to show that a portable extractor tent (Demistifier 2000, Peace Medical) was 100% efficacious in preventing spread of nebulised bronchodilator drug aerosols. This confirmed that use of Extractor tents prevents spread of drug particles from nebulised therapy. Air DNA samples were taken on six separate days over three months on the respiratory ward, and a preliminary analysis suggested that in most cases the largest single group at Phylum level were Firmicutes (Clostridiaceae/Clostridiales Families). Because these bacteria are potentially of gastrointestinal origin, it was hypothesized the source could be a communal lavatory that was present within the ward bay. A week-long WIBS campaign was therefore undertaken in a communal office toilet to investigate aerosol production from different toilet activities. Increased fluorescent particles were found in lavatory air on flushing after defaecation compared to other activities. Previous studies reporting the effect of toilet lids have found that they prevent the spread of visible droplets on flushing, however the effect on smaller particles was less clear cut. This study found that placing the lid down before flushing the toilet reduced the number of airborne fluorescent particles produced by flushing following defaecation, however it significantly increased particle size, shape, particle fluorescent intensity and residency time of defaecation-related particles in the air. A hypothesis is presented to account for this, involving acoustic reverberation magnification of flush-related turbulence by the lid, and implications of this for toilet design are discussed. This thesis highlights the ability of continuous bioaerosol detection by instruments such as WIBS to provide biologically meaningful characterisation of the healthcare environment. This characterisation facilitates airborne particle source attribution, allows provisional standard setting, and provides a powerful mode of assessment of the results of interventions designed to increase air quality.
- ItemEarly milk diet of infants and the effect on their body composition and growth and development in the first two years of life(University College Cork, 2018) Smith, Hazel Ann; Murray, Deirdre M.; Leahy-Warren, Patricia; National Children's Research Centre (NCRC)Background: Nutrition in the first few months of life has important effects on long-term growth. The aim of this PhD was to investigate the effect of an infant’s milk diet (both formula and breastmilk intake) in the first two months of life on body composition at two months of age, growth in the first two years of life and neurodevelopment at two years; and to examine whether breast- and formula-fed infants differ at birth, confounding the true effect of breastfeeding. Methods: Secondary data analysis of the feeding patterns, growth and development of children in the Cork BASELINE Birth Cohort Study. Descriptive and multivariate (multi-linear and logistic regression) analysis was employed. Results: Admission to the neonatal intensive care unit had the greatest negative impact on exclusively breastfeeding at two months (adjusted odds ratio = 0.20 (95% CI 0.05, 0.83)). Nearly twice as many exclusively formula-fed infants experienced early rapid growth (ERG) at two months compared to exclusively breastfed infants, n=87 (30%) vs n=56 (16.9%), respectively. Infants that experienced ERG saw an increase in their weight-for-height (wfh) z-score at 24 months compared to infants that did not experience ERG, β=0.39 (95% CI 0.19, 0.54). Breastfed infants had a higher mean(SD) birthweight to formula-fed infants, 3.56(0.42)kg versus 3.46(0.44)kg, respectively. However, breastfed infants had a lower mean(SD) percentage fat mass at birth compared to formula-fed infants, 10.01(3.71)% versus 12.05(4.06)%. Conclusion statement: By two months of age few Irish infants are exclusively breastfed. Formula supplementation and admission to the neonatal intensive care unit in the maternity hospital shortened breastfeeding duration. Formula feeding increased the odds of ERG and experiencing ERG at two months increased a child’s wfh z-score at 24 months. Breastfed infants were different in growth and body composition at birth in our cohort.
- ItemInvestigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation(University College Cork, 2015) Lyons, Caitríona M.; Brint, Elizabeth K.; Houston, Aileen M.; Science Foundation IrelandBackground/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.