The effect of human soluble FceRII on the RPMI 8866 B-Lymphoblastoid and the U937 Monocyte cell lines
- Authors: Daniels, Brodie Belinda
- Date: 2003
- Subjects: Developmental inmmunology , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11084 , http://hdl.handle.net/10948/322 , Developmental inmmunology , Cellular control mechanisms
- Description: Due to the diverse functions of Fc eRII, such as its roles in cellular adhesion, growth and differentiation of B and T lymphocytes, rescue of B cells from apoptosis and release of cytotoxic mediators, it is clear why it is believed to be a central molecule in allergic response. Because of its important role in the regulation of IgE production, FceRII may be the primary cause of certain allergic conditions. This study attempted to express and purify a recombinant human soluble FceRII to test its effect on a B-lymphoblastoid (RPMI 8866) and a monocytic (U937) cell line. The protein was expressed in Escherichia coli inclusion bodies, before being refolded and purified in a single gel chromatography step. This pure protein was then tested for biological activity by testing its IgE binding func tion. Once proven functional, it was used to test its effect on the cell lines at three concentrations for its apoptotic rescue properties and its cytokine effects. The recombinant protein did not seem to have any significant effect on the apoptotic rescue of either cell line. While the recombinant sFceRII appeared to have a slight effect on the stimulation of IL-1ß and TNFa in the RPMI 8866 cells, there was no apparent effect on the production of NF?B. In U937 cells, the protein did not seem to have any effect on the stimulation of IL-1ß, TNFa or NF?B. However, the cytokine effects of the recombinant protein were tested on isolated PBMCs from a healthy individual and a hyper-IgE syndrome patient. The recombinant protein was able to stimulate the production of cytokines in both individuals’ PBMCs, proving that it has the same effect as the natural protein. The upregulation of these cytokines indicates that the recombinant protein is able to stimulate the immune system. Therefore, this recombinant soluble FceRII protein could possibly be used for immune therapy.
- Full Text:
- Date Issued: 2003
- Authors: Daniels, Brodie Belinda
- Date: 2003
- Subjects: Developmental inmmunology , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11084 , http://hdl.handle.net/10948/322 , Developmental inmmunology , Cellular control mechanisms
- Description: Due to the diverse functions of Fc eRII, such as its roles in cellular adhesion, growth and differentiation of B and T lymphocytes, rescue of B cells from apoptosis and release of cytotoxic mediators, it is clear why it is believed to be a central molecule in allergic response. Because of its important role in the regulation of IgE production, FceRII may be the primary cause of certain allergic conditions. This study attempted to express and purify a recombinant human soluble FceRII to test its effect on a B-lymphoblastoid (RPMI 8866) and a monocytic (U937) cell line. The protein was expressed in Escherichia coli inclusion bodies, before being refolded and purified in a single gel chromatography step. This pure protein was then tested for biological activity by testing its IgE binding func tion. Once proven functional, it was used to test its effect on the cell lines at three concentrations for its apoptotic rescue properties and its cytokine effects. The recombinant protein did not seem to have any significant effect on the apoptotic rescue of either cell line. While the recombinant sFceRII appeared to have a slight effect on the stimulation of IL-1ß and TNFa in the RPMI 8866 cells, there was no apparent effect on the production of NF?B. In U937 cells, the protein did not seem to have any effect on the stimulation of IL-1ß, TNFa or NF?B. However, the cytokine effects of the recombinant protein were tested on isolated PBMCs from a healthy individual and a hyper-IgE syndrome patient. The recombinant protein was able to stimulate the production of cytokines in both individuals’ PBMCs, proving that it has the same effect as the natural protein. The upregulation of these cytokines indicates that the recombinant protein is able to stimulate the immune system. Therefore, this recombinant soluble FceRII protein could possibly be used for immune therapy.
- Full Text:
- Date Issued: 2003
Progestin receptor heterogeneity in a breast cancer cell line
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
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