SACM - United Kingdom
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9667
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Item Restricted In-Vitro Effects of Recombinant IL-37 protein on Bladder cancer cell line Growth and Proliferation(University of East London, 2024-08) Alamri, Ashwaq; Gobena, EdessaIntroduction: Interleukin-37 (IL-37) is a cytokine of the IL-1 family, recognized for its anti-inflammatory and immunomodulatory functions. Despite growing interest in IL-37’s role in different types of cancers, its impact on bladder cancer growth and proliferation has not been thoroughly investigated. This study aims to explore the effects of recombinant IL-37 protein on the viability, proliferation, and apoptosis of T24 bladder cancer cell lines, addressing a significant gap in current cancer research. Methods: In this in vitro study, T24 bladder cancer cells were treated with different concentrations of recombinant IL-37 protein (1 μg/ml, 0.5 μg/ml, and 0.25 μg/ml) with negative control (0 μg/ml). Cell viability and apoptosis were assessed using flow cytometry, and the levels of pro- inflammatory cytokines IL-6 and IL-8 in the cell culture supernatants were quantified using enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test to determine the significance of the results. Results: The results showed that IL-37 induces apoptosis in a dose-dependent manner, with the highest concentration (1 μg/ml) significantly reducing the percentage of viable cells from 90% to 71% and increasing the apoptotic cell population from 7% to 27%. Additionally, IL-37 treatment modulated the secretion of IL-6 and IL-8, with a significant increase in IL-6 production at 0.25 μg/ml (P ≤ 0.05) compared to the control. However, IL-37 did not significantly inhibit IL-8 production across the different treatment groups. (P ≤ 0.05). Conclusion: This study demonstrates that IL-37 not only promotes apoptosis in bladder cancer cells but also alters the tumor microenvironment by modulating cytokine secretion. These findings suggest that IL-37 may hold therapeutic potential as a novel approach to bladder cancer treatment. Further research is necessary to validate these results in vivo and to elucidate the precise molecular mechanisms through which IL-37 exerts its anti-tumor effects.57 0Item 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 Role of C-reactive protein in airway smooth muscle dysfunction relevant to obstructive lung disease.(University of Leicester, 2024-07-31) Alanazi, Amani; Saunders, RuthC-reactive protein (CRP), is an inflammation marker, often elevated in airways conditions such as asthma and COPD. This research investigated the impact of CRP on airway smooth muscle (ASM) cells, which are crucial in the airway remodelling and hyperresponsiveness which is observed in these conditions. By using primary human ASM cells, this study has shown that purified CRP reduced cell number, increased cell size and intracellular complexity, and maintained cell viability. However, these findings were not replicated with recombinant CRP, which lacks endotoxin, suggesting that endotoxin contamination in the purified CRP may have played a role. Lipopolysaccharide (LPS) treatment, an endotoxin component, resulted in increased intracellular complexity but did not completely replicate the other effects of purified CRP on ASM cells. Moreover, a CRP-neutralizing antibody did not reverse the changes induced by purified CRP, indicating the potential involvement of contaminants. The differences between purified and recombinant CRP highlight the challenges in isolating the true effects of CRP from those of other inflammatory agents. Future research will give priority to endotoxin removal or neutralization, using higher concentrations of both CRP and the neutralizing antibody. Additionally, the study will focus on exploring potential synergistic effects between CRP and LPS on ASM cells. Further investigations are needed to fully understand the role of CRP in the ASM dysfunction and the underlying mechanisms, including apoptosis, hypertrophy, and mediator secretion. In conclusion, this study suggests that CRP has the potential to contribute to ASM dysfunction but underscores the importance of strict experimental controls to distinguish its effects from potential contaminants like endotoxin. The research emphasizes the need for further exploration of the complex interplay between CRP, endotoxin, and ASM cells to elucidate their individual or combined contribution to ASM dysfunction in lung diseases.84 0Item Restricted The impact of macrophage migration inhibitory factor inhibition on acute myeloid leukaemia(University of Westminster, 2024-05-02) Aljuhani, Talah Akram; Saia, MarcoAcute myeloid leukaemia (AML) is one of the most aggressive and challenging malignancies that is characterised by rapid blast cell proliferation in the bone marrow and peripheral blood. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is overexpressed in various types of solid tumours. Despite considerable evidence regarding the function of MIF in different tumours and cancers, limited research has been conducted on its role in AML. This study aimed to explore MIF’s role in AML, specifically in relation to cell proliferation, cell cycle, and apoptosis. Additionally, the effects of a 50 µM ISO-1 MIF inhibitor on restricting AML cell growth by inhibiting MIF were assessed. Herein, THP-1 cells demonstrated high surface CD44 and CXCR4 receptor expression and low surface CD74 and CXCR2 receptor expression, suggesting the contribution of CD44 and CXCR4 receptors in MIF activation, thereby AML development. Additionally, the cell proliferation rate was decreased, the apoptotic cell count was increased, the percentage of treated cells in the pre-G0-G1 phase was increased, and the percentages of cells in the G0-G1, S, and G2-M phases were decreased. This suggests an impact of the ISO-1 inhibitor on decreasing AML proliferation, inducing apoptosis, and regulating the cell cycle. These results can have a positive impact on understanding MIF’s role in AML pathogenesis and improving AML treatment efficiency using potential therapeutic targets that can have a considerable impact on the survival rate and health outcomes of patients.16 0Item Restricted Pathological and Immunological Changes in Host Cells in Response to Leishmania mexicana Infection(Nottingham Trent University, UK, 2019-04) Alhajri, Salah Mahrous; Selman, AliAbstract Introduction: Leishmaniasis is a group of parasitic diseases caused by obligate intracellular protozoa of the genus Leishmania, with more than 20 pathogenic species. Leishmania infects approximately 12 million people annually in 98 countries. The deaths associated with this disease ranges between 20,000 - 30,000 per year (WHO, 2018). Therefore, the need for treatments or vaccines get more urgent. Macrophages are the ultimate host cell for the Leishmania parasite where it survives and multiplies. Though, a lot is known on how the Leishmania parasite survives and multiplies inside macrophages, there are still aspects related to pathophysiological and immunological responses to their infection that need further investigation to aid in the development of new vaccines or drugs for this disease. In this study, a virulent and avirulent L.mexicana model was developed to examine their interaction with bone marrow derived macrophages (BMDM) from susceptible (Balb/c) and resistant (C57) mice in vitro. Methods: Virulent L.mexicana parasite MNYC/BZ/62/M379 (P1) was maintained by subcutaneous inoculation of Balb/c mice. Avirulent L.mexicana passage twenty (P20) was produced by sub culturing of L.mexicana passage one (P1) twenty times in vitro. The effects of 20 continuous passages on virulence-associated gene (LPG1, LPG2, A2, CHAT1, CPB2, CPB2.8, CPC, GP63, LACK, and MAPK9) were investigated using qPCR. The expression of LPG and PS was also investigated using flow cytometry and immunofluorescence analysis. Growth characteristics and morphology of avirulent (P20) and virulent (P1) L.mexicana parasites grown in two media (Schneider's Drosophila and RPMI1640) in vitro were investigated under different culture conditions (temperature, and oxygen) using light immunofluorescence microscopy, EM and AFM. Differentiation into amastigotes under several conditions was investigated by estimation of the number of amastigotes. The infectivity of the parasites at each passage was also assessed by hemocytometry and Alamar blue assay. Survival of parasites inside macrophages was assessed visually by labelling the parasites with CFSE stain and the ability to form PV in BMDM from C57 and Balb/c mice. qPCR was used assess the expression pro-inflammatory cytokine expression (TNF- α, IL-6, IL-1β and TGF-β) and ELISA was used for estimation of TNF-α in the culture supernatants. Annexin V stain and flow cytometry analysis was used to assess apoptosis of infected cells. qPCR was used to assess the expression of genes associated with apoptosis (Bax, BCL 2, Caspase 1, Caspase 8, Caspase 9 and PD 1). The effect of supernatants derived from cultures of infected BMDM on the P1 and P20 promastigotes growth and virulence genes regulation was also investigated by qPCR. v Results: Twenty passages of L.mexicana in vitro caused significant changes in parasite morphology, ability to differentiate into amastigotes and downregulation of all tested virulence associated genes. Expression of LPG decreased, and PS increased on the surface of L.mexicana promastigotes. P20 infected both Balb/c and C57 BMDM but failed to survive inside BMDM both mice strains. P1 survived and inhibited apoptosis accompanied by significant downregulation of Caspase 8 by qPCR. Both P1 and P20 induced the release of TNF-α as confirmed by qPCR and ELISA. P1 promastigotes incubated in conditioned media derived from Balb/c BMDM infected with P1, enhanced their growth accompanied by upregulation of LPG1, CHT1, CPB2 and CPB2.8. While, incubation in conditioned media derived from C57 BMDM infected with P1 inhibited their growth and caused downregulation of LPG1, LPG2 CPB2, CPB2.8, CHT1 and A2. Conclusion: Culturing of L.mexicana in vitro for 20 passages has produced significant changes in their ability to differentiate from promastigotes into amastigotes, the ability to survive in macrophages and regulation of apoptosis associated genes. Supernatants produced by BMDM infected with P1 enhanced the growth rate of P1 promastigotes, when derived from Balb/c and inhibited their growth when derived from C57 cells. Understanding differences between P1 and P20 and their interaction with mammalian host may help in identifying the virulence factors of P1 L.mexicana which may aid in development of vaccines or drugs against this disease.12 0