This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochcmical assay to estimate the secretion of mucin by suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures. Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labellcd material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect. A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-depcndently stimulated bulk mucin release. Syncrgistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues. In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.

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Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

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