Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation

The Role of Properdin in Cellular and Humoral Responses – in vitro and in vivo analyses

Complement is part of the innate immune defence. It can be activated via three pathways, the classical pathway, the lectin pathway, and the alternative pathway. It comprises soluble factors and receptors. Properdin (Factor P) is a soluble component of the alternative complement pathway which acts as an important positive regulator of complement activation that stabilises the alternative pathway convertases (C3bBb) and C3bBb5b in the feedback loop of the alternative pathway, protecting them from rapid inactivation. The thesis is interested to see the role of properdin in cellular and humoral immune responses by in vitro and in vivo analyses. Firstly, properdin was examined for its global activity by characterisation of promoter activity of the human gene for properdin which involved using bioinformatics and molecular biology. The promoter activity was measured by dual-luciferase reporter system. The findings appeared to have activity in the 670bp properdin plasmid construct in U937 non-LPS transfection but the transfection upon LPS stimulation was not successful. Next, by using a properdin-deficient mouse line as a tool, it is interested to investigate the role of properdin in immunity by using properdin-deficient mice as model in pneumococcal vaccination studies, in vitro characterisation of dendritic cells and mycobacterium infectious studies in bone marrow-derived dendritic cells culture. In vaccination studies demonstrated that vaccination proved efficacious as both properdin-deficient and WT had increased in total IgM level and specific IgM level after the vaccination as measured by commercial ELISA for total IgM and specific ELISA for PPS2 IgM. In the absence of properdin, specific anti-polysaccharide antibodies of the IgM type are made. Vaccinated properdin-deficient mice do not differ from wild type in their immunoglobulin response to the pneumococcal polysaccharide vaccine. Meanwhile properdin-deficiency had a benefit in survival, independent of vaccination. For the in vitro characterisation of dendritic cells, dendritic cells are derived from bone marrow and spleen culture, and flow cytometry measured dendritic cells phenotype surface markers. Both bone marrow and spleen dendritic cells derived from properdin deficient mice are impaired to be activated and mature as dendritic cells compared to wild type mice. The study presently concludes that the presence of properdin is essential to allow dendritic cells to develop their activated phenotype and properdin is a relevant player in dendritic cell mediated immune response. Further investigation of function of generated BM-derived DC of WT and properdin-deficient towards Mycobacterium tuberculosis and Mycobacterium bovis BCG. In overall findings of mycobacterial viability, secretion of TNF-α and intracellular containment of mycobacterium in BMDC, it is concluded that properdin has no role of in the immune response of BM-derived DC towards Mycobacterium tuberculosis and Mycobacterium bovis BCG. In conclusion, having properdin is essential to help the complement system as part of defence mechanism against infection. Additionally, properdin could play a ‘double-edged’ role

The role of properdin in cellular and humoral responses : in vitro and in vivo analyses

Due to third party copyright restrictions the published articles have been removed from the appendix of the electronic version of this thesis. The unabridged version can be consulted, on request, at the University of Leicester’s David Wilson Library.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Tumor suppressive effects of GAS5 in cancer cells

In recent years, long non-coding RNAs (lncRNAs) have been shown to play important regulatory roles in cellular processes. Growth arrests specific transcript 5 (GAS5) is a lncRNA that is highly expressed during the cell cycle arrest phase but is downregulated in actively growing cells. Growth arrests specific transcript 5 was discovered to be downregulated in several cancers, primarily solid tumors, and it is known as a tumor suppressor gene that regulates cell proliferation, invasion, migration, and apoptosis via multiple molecular mechanisms. Furthermore, GAS5 polymorphism was found to affect GAS5 expression and functionality in a cell-specific manner. This review article focuses on GAS5’s tumor-suppressive effects in regulating oncogenic signaling pathways, cell cycle, apoptosis, tumor-associated genes, and treatment-resistant cells. We also discussed genetic polymorphisms of GAS5 and their association with cancer susceptibility

Tumor Suppressive Effects of GAS5 in Cancer Cells

In recent years, long non-coding RNAs (lncRNAs) have been shown to play important regulatory roles in cellular processes. Growth arrests specific transcript 5 (GAS5) is a lncRNA that is highly expressed during the cell cycle arrest phase but is downregulated in actively growing cells. Growth arrests specific transcript 5 was discovered to be downregulated in several cancers, primarily solid tumors, and it is known as a tumor suppressor gene that regulates cell proliferation, invasion, migration, and apoptosis via multiple molecular mechanisms. Furthermore, GAS5 polymorphism was found to affect GAS5 expression and functionality in a cell-specific manner. This review article focuses on GAS5’s tumor-suppressive effects in regulating oncogenic signaling pathways, cell cycle, apoptosis, tumor-associated genes, and treatment-resistant cells. We also discussed genetic polymorphisms of GAS5 and their association with cancer susceptibility