Cytokine signalling functions of human soluble IgE receptors in peripheral blood mononuclear cells from normal and hyper-allergic individuals and in B-lymphoblastoid and monocytic cell lines
- Authors: Askew, Sandra Lyn
- Date: 2006
- Subjects: Ligands , Cell receptors , Cellular signal transduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10305 , http://hdl.handle.net/10948/455 , Ligands , Cell receptors , Cellular signal transduction
- Description: CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.
- Full Text:
- Date Issued: 2006
- Authors: Askew, Sandra Lyn
- Date: 2006
- Subjects: Ligands , Cell receptors , Cellular signal transduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10305 , http://hdl.handle.net/10948/455 , Ligands , Cell receptors , Cellular signal transduction
- Description: CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.
- Full Text:
- Date Issued: 2006
Metabolic effects brought about by tricyclic antidepressants and the contribution of a medicinal plant in alleviating high fat diet induced insulin resistance in male wistar rats
- Authors: Chadwick, Wayne
- Date: 2006
- Subjects: Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10329 , http://hdl.handle.net/10948/461 , Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Description: Type II diabetes is becoming a growing problem in developed countries worldwide. The median age for diagnosis was around sixty, but recent surveys have shown that the entire age distribution curve shifting left. The incidence of type II diabetes is thought to be parallel with the growing rate of obesity associated with an unhealthy western diet. Type II diabetes is an expensive disease to manage, it is for this reason that cheaper medication needs to be investigated in the form of traditional plants, such as Sutherlandia frutescens. Prescription medication, such as tricyclic antidepressants, may also increase body weight thereby playing a role in obesity. The cause of weight gain in such cases may go unrecognized or lead to cessation of the medication with or without the practitioner’s knowledge or approval. It is therefore necessary to investigate the causative agents responsible for the excessive weight gain. Drinking water containing extracts of S. frutescens or metformin was administered to two groups of eleven insulin resistant male Wistar rats. The insulin resistant control group received water without any medication. Rats were sacrificed after 8 weeks allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. The effect of the medication and the diet on muscle post receptor insulin signaling proteins was determined through Western blots. Liver proteomics was also performed using 2-D electrophoresis. In a separate experiment 26 male Wistar rats were exposed to strepotozotocin toxin, 7 of these rats received intravenous insulin treatment, 7 rats received S. frutescens extract and 7 rats received a combination of both medications, the remaining 5 received no treatment and served as the control. Rats were sacrificed after 6 days allowing for fasting blood glucose, insulin and tissue glycogen content determination. Two groups of 14 male Wistar rats received amitriptyline or trimipramine (common tricyclic antidepressants) in their drinking water, the control group (30 rats) received water without any medication. The rats’ weight and food consumption was monitored throughout the trial and their oxygen consumption was also determined. Rats were sacrificed after 6 weeks or 14 weeks of medicinal compliance allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. S. frutescens treatment normalized circulating serum insulin levels and significantly increased the rate of glucose clearance. Certain post receptor insulin signaling proteins were also significantly increased relative to the insulin resistant control group. 2-D electrophoresis identified the normalization of protein levels associated with the urea cycle. S. frutescens was also able to, independently; maintain normoglycaemic levels in the strepotozotocin treated group. The tricyclic antidepressants significantly increased blood glucose levels while significantly reducing tissue glycogen levels for both sacrifice periods. Serum insulin remained unchanged while a significant increase in insulin degradation and insulin degrading enzyme levels were found for both antidepressants. S. frutescens shows promise as a low cost antidiabetic medication for future use. Although the antidepressants did not promote weight gain, the increase in blood glucose levels may be cause for concern in patients with a pre-disposition toward developing diabetes.
- Full Text:
- Date Issued: 2006
- Authors: Chadwick, Wayne
- Date: 2006
- Subjects: Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10329 , http://hdl.handle.net/10948/461 , Rats -- Metabolism , Diabetes -- Research , Medicinal plants -- South Africa
- Description: Type II diabetes is becoming a growing problem in developed countries worldwide. The median age for diagnosis was around sixty, but recent surveys have shown that the entire age distribution curve shifting left. The incidence of type II diabetes is thought to be parallel with the growing rate of obesity associated with an unhealthy western diet. Type II diabetes is an expensive disease to manage, it is for this reason that cheaper medication needs to be investigated in the form of traditional plants, such as Sutherlandia frutescens. Prescription medication, such as tricyclic antidepressants, may also increase body weight thereby playing a role in obesity. The cause of weight gain in such cases may go unrecognized or lead to cessation of the medication with or without the practitioner’s knowledge or approval. It is therefore necessary to investigate the causative agents responsible for the excessive weight gain. Drinking water containing extracts of S. frutescens or metformin was administered to two groups of eleven insulin resistant male Wistar rats. The insulin resistant control group received water without any medication. Rats were sacrificed after 8 weeks allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. The effect of the medication and the diet on muscle post receptor insulin signaling proteins was determined through Western blots. Liver proteomics was also performed using 2-D electrophoresis. In a separate experiment 26 male Wistar rats were exposed to strepotozotocin toxin, 7 of these rats received intravenous insulin treatment, 7 rats received S. frutescens extract and 7 rats received a combination of both medications, the remaining 5 received no treatment and served as the control. Rats were sacrificed after 6 days allowing for fasting blood glucose, insulin and tissue glycogen content determination. Two groups of 14 male Wistar rats received amitriptyline or trimipramine (common tricyclic antidepressants) in their drinking water, the control group (30 rats) received water without any medication. The rats’ weight and food consumption was monitored throughout the trial and their oxygen consumption was also determined. Rats were sacrificed after 6 weeks or 14 weeks of medicinal compliance allowing for fasting blood glucose, insulin and tissue glycogen content determination. Glucose uptake was also determined using [3H] deoxyglucose. S. frutescens treatment normalized circulating serum insulin levels and significantly increased the rate of glucose clearance. Certain post receptor insulin signaling proteins were also significantly increased relative to the insulin resistant control group. 2-D electrophoresis identified the normalization of protein levels associated with the urea cycle. S. frutescens was also able to, independently; maintain normoglycaemic levels in the strepotozotocin treated group. The tricyclic antidepressants significantly increased blood glucose levels while significantly reducing tissue glycogen levels for both sacrifice periods. Serum insulin remained unchanged while a significant increase in insulin degradation and insulin degrading enzyme levels were found for both antidepressants. S. frutescens shows promise as a low cost antidiabetic medication for future use. Although the antidepressants did not promote weight gain, the increase in blood glucose levels may be cause for concern in patients with a pre-disposition toward developing diabetes.
- Full Text:
- Date Issued: 2006
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