Untersuchungen zur Regulation sekretorischer Aspartatproteinasen in einem oralen Candidose-Modell und in vivo. [Investigations on the regulation of secreted aspartyl proteases in a model of oral candidiasis in vivo]

By means of RT-PCR and specific primers the expression of SAP1-6 and SAP8 was investigated with respect to the time course in an in vitro candidosis model based on reconstituted human mucosal epithelium. Corresponding morphological alterations of the epithelium were documented by light microscopy. The detection of Sap was performed immunoelectron microscopically using a monoclonal antibody. In the oral candidosis model SAP1 and SAP3 transcripts were detected 42 h after inoculation corresponding to first histopathological changes. Additional SAP6 expression was observed six hours later concomitantly with germ-tube formation. Later on SAP2 and SAP8 transcripts were found after 60 h. On protein level it was possible to demonstrate Sap antigens within Candida and markedly deteriorated epithelial cells. Initial experiments with proteinase mutants and proteinase inhibitors showed reduction of histological damage. In a clinical specimen obtained from a twenty nine-year-old female patient suffering from acute oral candidosis SAP1, 3 and 6 could be demonstrated corresponding to the findings in vitro after 48 h. Investigating a clinical specimen obtained from a lesion of chronic oral candidosis in an HIV-infected patient also showed SAP2 expression. On the basis of our results a relationship between the expression on of particular SAP genes and the turn up of lesions looks as probable as a relevant contribution to the in vivo infection

Influence of Human Serum on Antifungal Pharmacodynamics with Candida albicans

Antifungal susceptibilities (NCCLS, approved standard M27-A, 1997) were determined for the reference strain ATCC 90028 and 21 clinical isolates of Candida albicans with varying levels of fluconazole susceptibility using RPMI 1640 (RPMI) and 80% fresh human serum–20% RPMI (serum). Sixty-four percent (14 of 22) of the isolates tested demonstrated significant decreases (≥4-fold) in fluconazole MICs in the presence of serum, and the remaining eight isolates exhibited no change. Itraconazole and ketoconazole, two highly protein-bound antifungal agents, had MICs in serum that were increased or unchanged for 46% (10 of 22) and 41% (9 of 22) of the isolates, respectively. All 10 isolates tested against an investigational antifungal agent, LY303366, demonstrated significant increases in the MIC required in serum, while differences in amphotericin B MICs in the two media were not observed. Four of 10 isolates tested demonstrated fourfold higher flucytosine MICs in serum than in RPMI. Postantifungal effects (PAFEs) and 24-h kill curves were determined by standard methods for selected isolates. At the MIC, fluconazole, itraconazole, ketoconazole, flucytosine, and LY303366 kill curves and PAFEs in RPMI were similar to those in serum. Isolates of fluconazole-resistant C. albicans required lower MICs in serum than in RPMI, without relative increases in fungal killing or PAFEs. Isolates tested against amphotericin B demonstrated significantly reduced killing and shorter PAFEs in serum than in RPMI without observable changes in MIC. In conclusion, antifungal pharmacodynamics in RPMI did not consistently predict antifungal activity in serum for azoles and amphotericin B. Generally speaking, antifungal agents with high protein binding exhibited some form of reduced activity (MIC, killing, or PAFE) in the presence of serum compared to those with low protein binding