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Title: The effect of biomaterials on the regulation of chemokine receptor expression
Author: Fujiyoshi, Keiko
ISNI:       0000 0001 3485 2699
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2005
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Prior to the analysis using biomaterials, the basic mechanism of the regulation of CXCRI, CXCR2, CCRI and CCR2~ expression on neutrophils, monocytes/macrophages, B lymphocytes and T lymphocytes in whole blood culture wasinitially studied. Firstly, the analysis of chemokine receptor expression on each leukocyte with fMLP and PMAstimulations showed the dominant expression of CXCR1 and CXCR2 on_ neutrophils and CCRI and CCR2 on monocytes/macrophages whilst the receptor expression wassignificantly lower on B and T lymphocytes. Subsequently, the effects of IL-8 and MCP-2 and 3 on the regulation of respective CXCR and CCR expression were analysed using FACS, and demonstrated the difference in the rate of the down regulation of CXCR and CCR. However, unlike CXCR expression, the pattern of CCR expression was notclearly observed, thus the level of CCR1I and CCR2 mRNA expression in monocytes/macrophages using quantitative RT-PCR was analysed which subsequently showed the decreased level of CCR1 and CCR2 mRNAafter 3 hours. By understanding the basic pattern of CKCR1, CXKCR2, CCR1 and CCR2 expression, the effect of nickel, cobalt, and copper ions on the regulation of chemokine receptor expression was subsequently analysed. The FACSanalysis of CXCR1 and CXCR2 expression on neutrophils demonstrated the significant differences in the effect of each metal ion. For example, 10ppm of copper ions induced immediate down regulation of CXCRI1 whilst the up regulation of CXCR2 was recorded with 10ppm of cobalt ions after 30 minutes. Nickel ions however, showed no effects on the expression of CXCR1 and CXCR2. The analysis of CCR1 and CCR2 expression with quantitative RT-PCR and FACS demonstrated no significant effects of those metal ions on the level of CCR1 and CCR2 mRNA,but FACSanalysis showed MFI of CCR1 with 10ppm of cobalt ions after 30 minutes wasrelatively higher than other stimulants. Simultaneously, the qualitative visualisation was carried out using confocal laser scanning microscopy which showed the pattern of the CCRI expression and also proved the regulation of receptor expression was not due to the cytotoxic effects of metal ions. Asthe metal ions can be considered to chemically affect the regulation of chemokine receptor expression, the physical effect of particulates was also studied. The analysis was conducted with endodontic sealant Grossman, NiCr and CoCr alloy particulates by including parameters such as opsonisation and volumetric concentrations of particulates. Firstly, the expression of CXCR1 and CXCR2 were examined with Grossman andthe results demonstrated that the opsonised Grossman decreased the MFI of CXCR1 expression, but the same weight of NiCr alloy particulates showed no significant effect on the chemokine receptor expressions. Subsequently, the effect of different volumetric concentrations of NiCr and CoCr alloy particulates was observed and demonstratedthatcell:particulates ratio of 1:1 of non opsonised NiCr alloy particulates induced down regulation of CXCR1, and 1:0.25 of opsonised NiCr induced up regulation of CXCR2. CoCr alloy particulates, however, showed no effects on CXCR expression. It has been long knownthat the chemotactic response can be impaired by phagocytic activities due to the down regulation of chemokine receptor expression. Despite of the statement above and other studies, the current analysis in a whole blood culture showed no significant changes in the expression of chemokine receptor. Thus, CXCR1 expression was subsequently analysed with purified neutrophils, and mixed culture of purified neutrophils and monocytes/macrophages. The results demonstrated that CXCR1 expression on purified cells significantly decreased after 30 minutes in comparison with the expression in whole blood culture. Consequently, the cause of the constant expression of CXCR1 in a whole blood culture was demonstrated by determining the concentration of G-CSF using ELISA and showedthe significant differences of the concentration of G-CSF between those cultures, suggesting that the constant expression of CXCR1 in a whole blood culture can be partly due to G-CSFreleased from the activated monocytes/macrophages.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral