IL-4/ IL-13 directed microglial activation and differentiation in response to LPS-induced neuroinflammation
- Authors: Ackerdien, Shiraz
- Date: 2024-04
- Subjects: Inflammation , Inflammation -- Treatment , Anti-inflammatory agents
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63606 , vital:73554
- Description: Microglia activation is a common hallmark of neuroinflammation that occurs during pathogen invasion or lipopolysaccharide (LPS)-induced inflammation. A neuroinflammatory response is elicited by the release of proinflammatory cytokines which stimulates microglia in an autocrine manner to be polarized into classically activated, pro-inflammatory M1 cells. Prolonged exposure to the inflammatory response can have disastrous effects on the central nervous system (CNS). However, microglia can alternatively be polarized into the activated M2 anti-inflammatory phenotype, but the exact molecular mechanism mediating this phenotypic switch remains poorly understood. Studies have shown that interleukin (IL)-4 can induce the M2 phenotype and activate the signal transducer and activator of the transcription 6 (STAT6) signalling pathway that in turn provokes a beneficial Th2 immune response. Since IL-4 and IL-13 share a common IL-4 receptor alpha (IL-4Rα) chain, it is possible that alternative microglia differentiation and its anti-inflammatory action also involve IL-13. This study aimed to investigate how IL-13 and STAT6 signalling orchestrates the microglial response and differentiation associated with LPS-induced inflammation. Furthermore, the molecular mechanisms that relieve LPS-induced neuroinflammation and neural protection through IL-13-enhanced BDNF signalling were also investigated. C8-B4 microglial cells were induced with LPS to exhibit an M1 pro-inflammatory phenotype or stimulated with IL-4 and/or IL-13 to exhibit an M2 anti-inflammatory microglial phenotype. The cell viability following LPS, IL-4, and/ or IL-13 exposure was determined. The LPS-induced neuroinflammatory response and the anti-inflammatory response induced by IL-4 and IL-13 which promotes STAT-6 signalling were determined by measuring TNFα, IL-1β, and BDNF protein concentrations using ELISA assays. The polarising effects of LPS and IL-4/IL-13 cytokines were also examined via changes in the expression of Iba-1, CD206, CD86, and STAT-6 determined by immunofluorescence analysis. These changes were further investigated by quantifying the mRNA transcripts of TNFα, IL-1 β, Arg-1, CD206, IL-4R, and STAT-6 and BDNF using qRT-PCR. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-04
- Authors: Ackerdien, Shiraz
- Date: 2024-04
- Subjects: Inflammation , Inflammation -- Treatment , Anti-inflammatory agents
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63606 , vital:73554
- Description: Microglia activation is a common hallmark of neuroinflammation that occurs during pathogen invasion or lipopolysaccharide (LPS)-induced inflammation. A neuroinflammatory response is elicited by the release of proinflammatory cytokines which stimulates microglia in an autocrine manner to be polarized into classically activated, pro-inflammatory M1 cells. Prolonged exposure to the inflammatory response can have disastrous effects on the central nervous system (CNS). However, microglia can alternatively be polarized into the activated M2 anti-inflammatory phenotype, but the exact molecular mechanism mediating this phenotypic switch remains poorly understood. Studies have shown that interleukin (IL)-4 can induce the M2 phenotype and activate the signal transducer and activator of the transcription 6 (STAT6) signalling pathway that in turn provokes a beneficial Th2 immune response. Since IL-4 and IL-13 share a common IL-4 receptor alpha (IL-4Rα) chain, it is possible that alternative microglia differentiation and its anti-inflammatory action also involve IL-13. This study aimed to investigate how IL-13 and STAT6 signalling orchestrates the microglial response and differentiation associated with LPS-induced inflammation. Furthermore, the molecular mechanisms that relieve LPS-induced neuroinflammation and neural protection through IL-13-enhanced BDNF signalling were also investigated. C8-B4 microglial cells were induced with LPS to exhibit an M1 pro-inflammatory phenotype or stimulated with IL-4 and/or IL-13 to exhibit an M2 anti-inflammatory microglial phenotype. The cell viability following LPS, IL-4, and/ or IL-13 exposure was determined. The LPS-induced neuroinflammatory response and the anti-inflammatory response induced by IL-4 and IL-13 which promotes STAT-6 signalling were determined by measuring TNFα, IL-1β, and BDNF protein concentrations using ELISA assays. The polarising effects of LPS and IL-4/IL-13 cytokines were also examined via changes in the expression of Iba-1, CD206, CD86, and STAT-6 determined by immunofluorescence analysis. These changes were further investigated by quantifying the mRNA transcripts of TNFα, IL-1 β, Arg-1, CD206, IL-4R, and STAT-6 and BDNF using qRT-PCR. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-04
Isolation, purification and partial characterisation of cancer procoagulant from placental amnion-chorion membranes and its role in angiogenesis inflammation and metastasis
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
- Authors: Krause, Jason
- Date: 2014
- Subjects: Coagulation , Amnion , Chorion , Metastasis , Inflammation , Neovascularization
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10350 , http://hdl.handle.net/10948/d1020897
- Description: Cancer procoagulant (EC 3.4.22.26) is an enzyme that is derived from tumour and foetal tissue, but not normal tissue. It is a direct activator of factor X and has been isolated from amnion-chorion membranes as well as from extracts and cells from human melanoma. The presence of cancer procoagulant has been associated with the malignant phenotype, as well as having a particularly high activity in metastatic cells. Cancer procoagulant activity is elevated in the serum of early stage breast cancer patients and decreased to normal in the advanced stages of the disease. In this study, cancer procoagulant was successfully isolated from amnion-chorion membranes and purified to homogeneity. The molecular weight of cancer procoagulant was determined using SDS-PAGE and was found to be 68 kDa. Cancer procoagulant was delipidated and it was shown that its activity was increased by the presence of lipids in a dose-dependent manner. Recovery of cancer procoagulant after delipidation is poor, consequently, a larger mass of sample is required to obtain sufficient amounts of delipidated material for N-terminal amino acid analysis. The optimum pH of cancer procoagulant was determined to be pH 8 and its optimal temperature was found to be 50°C. Novel synthetic substrates were designed to assay for cancer procoagulant activity. Currently, 2 potential candidates have been identified, namely, PQVR-AMC and AVSQSKP-AMC. Cancer procoagulant-induced expression of cytokines is differently modulated in the less aggressive MCF-7 cell line as compared to the metastatic and more aggressive MDA-MB-231 cell line. There are marked similarities in the inflammatory response produced by cancer procoagulant in hTERT-HDLEC and MDA-MB-231 cells, which are both associated with migratory capacity. Furthermore, cancer procoagulant-induced PDGF-β expression in hTERT-HDLEC and MDA-MB-231 cells could point to involvement of cancer procoagulant in wound healing and metastatic spread, respectively. Cancer procoagulant induced the motility of MDA-MB-231, MCF-7 and hTERT- cells in vitro in a time- and dose-dependent manner. Together, these results suggest that cancer procoagulant plays a role in the migration of breast cancer cells as well as the migration of endothelial cells.
- Full Text:
- Date Issued: 2014
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