Saudi Cultural Missions Theses & Dissertations
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Item Restricted The role of IL-17 in CD95L-driven nonapoptotic pathology during respiratory disease(University of Liverpool, 2024-07) ALAidarous, Sondus; Stewart, JamesCD95L can induce a pathological nonapoptotic pathway and promote Th17 migration when cleaved from the cell surface by proteases. The targeted inhibition of this nonapoptotic pathway by blocking the calcium-inducing domain to impede IL-17 migration could be of therapeutic benefit in inflammation, as seen in a murine lupus model. We aimed to investigate the CD95L nonapoptotic pathway and factors involved in the cleavage of CD95L in the context of SARS-CoV-2 infection and pulmonary fibrosis. Three approaches were used: in vitro studies, in vivo murine models and human clinical sample-based studies. In vitro, the Jurkat cell line was stimulated with PHA, LPS, recombinant MMP9 and mouse sera to optimise conditions for the expression and cleavage of CD95L. MMP9 was investigated as a putative cleavage factor of CD95L and was found at higher levels in naïve lung supernatant than in serum. In a SARS-CoV-2 murine model, the effect of inhibitory peptide treatment on mice intranasally infected with 104 PFU SARS-CoV-2 was assessed. IL-17 serum levels in the peptide-treated group were significantly lower than in the control group, yet this difference did not translate to disease progression, measured by viral load, weight loss, histopathology and survival. IL-17 and sCD95L levels were tested in SARS-CoV-2 patient samples from a biobank cohort and healthy control samples. There was no difference between the groups, and sCD95L was undetectable in the SARS-CoV-2 patient group. Our hypothesis on pulmonary fibrosis was tested through a model of bleomycin-induced fibrosis in BALB/c mice across 36 days. Animals were challenged with 4x105 of murine gammaherpesvirus (MHV) intranasally to aid fibrosis development on day 0. Bleomycin was administered twice at 40 mg/kg on day 8 and day 17. Inhibitory peptide treatment, control peptide treatment or mock treatment was given the day after bleomycin administration. Post-mortem, cardiac bleeds and lung and liver tissue were collected. IL-17, MMP9 and sCD95L were measured by ELISA. Right lungs were processed using Trizol for qPCR, and left lungs, along with livers, were prepared for histopathology. No difference was found between groups in terms of IL-17, MMP9 or sCD95L serum levels, gene expression of il-17 or ccr6, fibrosis scores based on Masson’s trichrome staining, and weight loss or survival rate. However, a significant correlation was seen between weight loss and the fibrosis score. In human pulmonary fibrosis serum samples, IL-17 levels were barely detectable and did not differ from those in healthy controls. Interestingly, sCD95L levels were significantly higher and MMP9 levels were significantly lower than in healthy controls. In summary, blocking the calcium-inducing domain could reduce IL-17 levels but did not affect disease progression in a SARS-CoV-2 model. Moreover, these findings could not be translated to other respiratory diseases, like pulmonary fibrosis.38 0Item Restricted Host parasite interactions in Trichuris infections(The University of Manchester, 2024-06-27) Othman, Abdulrazzag Abdulaziz A; Else, Kathryn J; Hepworth, MatthewInfection with T. trichiura affects around 500 million people worldwide. Currently, no effective vaccines against worm infection are available to prevent infection of humans. The murine species of Trichuris, Trichuris muris, serves as an excellent model of human trichuriasis, and is used widely to study the host immune response against Trichuris. It is well documented that resistance to T. muris infection in mice requires a T helper 2 (Th2) type of immune response with T helper 1 (Th1) cells dominating in susceptible individuals. Understanding how Th2 immune responses are generated, is an unmet need which will facilitate vaccine development. It was previously thought that B cells are not essential for worm expulsion. However, our lab has recently shown that B cells play an important role in the generation of protective immunity. Thus B cells not only important in antibody production but also play a regulatory role in the generation of protective Th2 responses to Trichuris muris. This PhD thesis explores the cellular sources of interleukin-10 (IL-10) during T. muris infection, focusing on subsets of T cells, macrophages, and B cells using the IL-10 reporter (Vert-X) mice. Follicular B cells and marginal zone B cells were found to be the main source of IL-10 in the mesenteric lymph nodes at day 21 post-infection. This pattern was shifted at day 35 post-infection, when B1a, B1b, and marginal zone B cells became the main IL-10 producers. These data suggest the importance of B cell-derived IL-10 in controlling immunopathology and facilitating long term immune regulation. In large intestine, follicular B cells were identified as the primary IL-10 producers in the lamina propria leukocytes at day 21 post-infection. Building on these findings, the thesis goes on to investigate the role of B cell-derived IL-10 in immune responses and pathology in a low dose Trichuris muris infection using a transgenic CD19creIL10flfl mouse model that lacks IL-10 production in B cells. Notably, the absence of B cell-derived IL-10 made mice less able to support a chronic infection, without altering gut pathology. Interestingly, the absence of B cell-derived IL-10 was associated with a reduction in IL-6 mRNA and in some cases reduced IFN-γ levels in the gut, alongside an increased in FoxP3+ Treg cells. The study also extended to exploring the importance of B cell-derived IL-10 in the context of a high dose T. muris infection. No significant differences were identified between mice where B cells could and could not make IL-10 in terms of gut pathology and the kinetics of worm expulsion. However interestingly, in the absence of B cell derived IL-10 mice had high numbers of M2 macrophages in the gut tissue which correlated with non-significantly reduced levels of IFN-γ mRNA. This research has informed our understanding of the contribution of B cell-derived IL-10 to the host immunity to T. muris. It highlights the necessity for further investigation to understand the mechanisms by which the B cell-specific IL-10 deficient mouse presents with elevated intestinal numbers of Foxp3+ Treg cells and elevated M2 macrophages in the context of low and high dose T. muris infection respectively17 0Item Restricted CHARACTERIZATION OF THE ROLE OF T REGULATORY CELLS IN THE IMMUNE RESPONSE TO BORRELIA BURGDORFERI INFECTION(Saudi Digital Library, 2022-12-20) Almatrook, Alaa; Nardelli, DeanLyme disease, caused by the spirochete Borrelia burgdorferi, is the most common tickborne infectious disease in the United States. The most common complication of Lyme disease is the development of Lyme arthritis. A dysregulated T cell immune response might contribute to Lyme arthritis development and persistence post-treatment in some patients. In addition, the host immune response may be incompetent in clearing the infection in some patients. Here, the hypothesis that T regulatory cells prevent Lyme arthritis development by containing T cell responses yet promote B. burgdorferi persistence by repressing the anti-pathogen immune response was tested. We show, using a Lyme arthritis-resistant mouse model, that T regulatory cells prevent Lyme arthritis development at various stages of Borrelia burgdorferi infection. Specifically, we demonstrate that T regulatory cell depletion before or after infection leads to tibiotarsal joint swelling and histopathology. We also provide evidence that T regulatory cells prevent Lyme arthritis by regulating pathogenic T helper 17 immune responses. Further, we demonstrate that T regulatory cells are involved in the humoral response to clear B. burgdorferi. We show that T regulatory cell depletion after infection increased B. burgdorferi-specific IgM antibodies and enhanced B. burgdorferi tissue clearance. However, T regulatory cell depletion after infection did not affect the generation of B. burgdorferi-specific IgG antibodies or the germinal centers. Our findings support and extend existing knowledge by demonstrating that T regulatory cells assist in restraining the T cell immune response to B. burgdorferi to prevent Lyme arthritis and that T regulatory cells may hinder the protective humoral immune response to B. burgdorferi and promote their survival.12 0Item Restricted TIM3 is a context-dependent coregulator of cytotoxic T cell function(Saudi Digital Library, 2023-12-05) Alamir, Hanin; Wuelfing, ChristophCytotoxic T lymphocytes (CTLs) are essential effectors in the antiviral and antitumour immune response and attractive targets in cancer immunotherapy. Although CTLs can directly recognise and kill tumour cells, CTLs become suppressed in the tumour microenvironment. This project investigated the inhibitory receptor T cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is expressed on T cells after chronic antigen exposure and marks the most exhausted tumour infiltrating CTLs in multiple solid tumours. However, it is unclear whether TIM3 directly regulates CTL function. In addition, despite its predominantly inhibitory role in vivo, TIM3 can promote cellular activation in T and non-T cells, and the roles of putative ligands in TIM3 function are disputed. Therefore, we aimed to determine the effect of TIM3 on direct CTL antitumour function and how the TIM3 ligands Galectin9 (GAL9) and CEACAM1 regulate its function. We employed three-dimensional (3D) tumour spheroids that effectively induce CTL suppression similar to the in vivo tumour microenvironment in comparison to conventional two-dimensional (2D) tumour cell culture. In the 3D spheroid model, TIM3 significantly inhibited CTL cytotoxicity and cytoskeletal polarisation as a key mechanism of effective cytolysis in murine and human CTLs. In contrast, in the 2D tumour model, TIM3 stimulated CTL cytotoxicity, cytoskeletal polarisation, and secretion of the immune-stimulatory cytokine interferon γ (IFNγ). Expression of GAL9 and CEACAM1 in trans on tumour cells further suppressed the CTL killing ability in the 3D spheroid model and enhanced costimulatory function in 2D. CEACAM1 in cis neutralised TIM3 functions in both 3D and 2D. We suggest that TIM3 functions as a context-dependent coregulatory receptor, as supported by the engagement of its ligands GAL9 and CEACAM1. In a largely stimulatory signalling context of a CTL, TIM3 functions as a costimulator, and in a more inhibitory context, TIM3 functions as a coinhibitor.8 0Item Restricted Three Dimensional Printed Immunomodulatory Scaffolds with Controlled Drug Release for Bone Regeneration(Saudi Digital Library, 2023-10-24) Majrashi, Majed; Yang, Jing; Ghaemmaghami, AmirLarge bone defects pose significant challenges in orthopaedic surgery, necessitating the exploration of innovative repair technologies beyond traditional treatments like autografts, allografts, and synthetic substitutes, each fraught with specific challenges. Tissue engineering and regenerative medicine have emerged as promising fields, employing bioactive materials, growth factors, and cellular components to emulate natural bone properties and functions. Notably, additive manufacturing techniques contribute to these advancements by customising 3D-printed scaffolds enhancing patient-specific treatments. Recent studies underscore the significant influence of immune responses in bone regeneration, an area still in its infancy. Particularly, the modulation of immune reactions through specialised biomaterials and the strategic delivery of anti-inflammatory agents like dexamethasone present a novel approach to support bone healing processes, avoiding the systemic side effects of traditional drug administration. In this thesis, novel inks were developed to sustain the release of dexamethasone from a 3D-printed scaffold to modulate the immune response and osteogenesis. Excipients with surfactant properties, including the poloxamers F127, F68, L31, sorbitan monooleate Span80, and sucrose acetate isobutyrate (SAIB), were added to PCL to test their ability to sustain drug release. All these inks were fabricated into scaffolds by using direct ink writing 3D printing technique. The fabricated scaffolds were then characterised by SEM, DSC, FTIR, and ToF-SIMS. Macrophages and mesenchymal stem cells (MSCs) were cocultured to investigate the effects of the controlled release of dexamethasone on the modulation of macrophage polarisation and osteogenic differentiation of MSCs. Notably, blending PCL with 40% wt/wt (SAIB) has improved dexamethasone-cyclodextrin dispersal and facilitated a sustained 35-day release dominated by first-ordered and Higuchi models. In this modified environment, investigations into macrophage-mesenchymal stem cell (MSC) interactions revealed that controlled dexamethasone release significantly influenced macrophage behaviour and MSC osteogenic differentiation. M1 macrophages boosted early alkaline phosphatase production (ALP) at (7 days), while later stages (21 days) saw dexamethasone's predominance. Bone morphogenic protein-2 (BMP-2) was significantly increased at day 21; meanwhile, interleukin-6 (IL-6) decreased at the same time. Moreover, the released dexamethasone switched the phenotype of macrophages from M1 to M2 at day 21, evidenced by the increased level of mannose receptor and decreased expression of calprotectin receptor. These results offer new insight into macrophage-MSC cross-talk and demonstrate the potential of drug-release scaffolds to modulate inflammation and enhance bone regeneration.21 0Item Restricted The role of ASC inflammasom in Pancreatic Cancer(Saudi Digital Library, 2022-09-17) Kashgari, Bassam; Jenkines, BrendanPancreatic ductal adenocarcinoma (PDAC) is an aggressive and highly lethal malignancy. The vast majority of PDAC patients are diagnosed at an advanced and incurable stage of the disease. Even with the best current treatments, the prognosis for advanced PDAC is less than one year. While molecular targeted treatment is widely used in many malignancies with impressive results, no such “precision medicine” approach is currently used for PDAC. The dysregulation of the immune system plays a role in the progression of the disease; in fact, chronic pancreatic inflammation is a risk factor for PDAC. Inflammasomes are key regulators of innate immunity in chronic inflammatory disorders and autoimmune diseases, but their role in PDAC remains ill-defined. The genetically engineered KPC mouse model mimics the molecular and cellular immunological and oncogenic processes that drive human PDAC. The genetic abolition of the Pycard gene, encoding ASC, dramatically reduced tumor growth in different age groups of KPC mice. This phenotypic reduction was associated with reduced levels of inflammasome activity and maturation for the downstream target of inflammasomes Interleukin (IL)-18 (but increased IL-1B). Subsequently, the elevated levels of IL-18, inflammation, proliferation, and apoptosis were reversed upon the genetic disruption of ASC expression in the KPC mouse model in different development stages of PDAC. Furthermore, our clinical data supports that ASC inflammasomes are over-activated and associated with poor patient survival and responsiveness to chemotherapy in PDAC. In conclusion, the findings in this work show that elevated ASC levels are a key feature of PDAC and supports the role of ASC as a tumor promoter in PDAC (e.g. tumor incidence and PDAC characteristics) through mediating IL-18 rather than IL-1B. Understanding the currently ill-defined role of ASC inflammasomes in PDAC may provide novel biomarkers and druggable candidates for targeted therapy for PDAC patients.a14 0