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Identifier 000410070
Title Διερεύνηση της παράδοξης δράσης των εχινοκανδινών εναντίων των υφομυκήτων του γένους Aspergillus
Alternative Title Dissecting the echinocandin paradoxical activity against the fungus aspergillus
Author Ιωάννου, Πέτρος
Thesis advisor Σαμώνης, Γεώργιος
Abstract INTRODUCTION: Aspergilli are saprophytic hyphomycetes that pose a lethal threat to the immunocompromised patients, such as patients with hematological or solid malignancies or organ recipients receiving immunosupression. Echinocandins are a new category of potent antifungal agents against Aspergilli in vivo; however they have mediocre in vitro activity against these fungi, such as in in vitro susceptibility studies. This dissertation is trying to dissect the mechanisms that cause this lag in the in vitro and the in vivo activity of these antifungal agents against Aspergilli. MATERIALS AND METHODS: Several Aspergilli strains were used, along with some Candida strains as controls, and susceptibility tests against echinocandins and voriconazole were done to 6 different Aspergillus strains in standard susceptibility conditions for hyphomycetes but also in cell culture medium in order to simulate the in vivo conditions in the tissues of patients infected with Aspergillus. The same tests were repeated with one Aspergillus fumigatus strain with caspofungin in several media (the media were different as to buffer content (Hepes or MOPS) and concentration, pH, CO2 and glucose concentration), and then in media with and without bovine serum (Fetal Calf Serum – FCS), with and without Bovine Serum Albumin (BSA) or BSA conjugated to FITC (Fluorescein isothiocyanate) – a fluorescent molecule. In every experiment the Minimum Effective (concentration of echinocandin that leads to production of short branched stubby hyphae) or Inhibitory Concentration (MEC or MIC respectively) was noted, and the metabolic activity of 18 the fungi was based on its ability to reduce XTT ((2,3)-bis (2-Methoxy 4-Nitro 5- Sulphenyl) (2H) Tetrazolium Carboxanilide) to a chromogenic product or after addition of CFDA (6-carboxyfluorescein diacetate) and estimation of the fluorescence by the living biomass. Similar experiments were performed with anidulafungin and micafungin in a smaller extent, in order to check if the phenotype takes place with these echinocandins as well. The data were analyzed statistically and the different conditions were compared based on the MECs, the concentration needed to reduce the metabolic activity by 50% (Effective Concentration 50 – EC50), and the slope of the curves generated with a non-linear regression (Hill’s equation). Confocal microscopy experiments were performed with and without FITC-BSA in Aspergillus fumigatus a) in different stages of its germination, b) with and without previous exposure to caspofungin c) with and without previous exposure to high concentrations of BSA. Additional confocal microscopy experiments were performed in Aspergillus fumigatus with and without BSA, with caspofungin covalently bound to a fluorescent molecule as well as scanning electron microscopy experiments with the same fungus with and without BSA, with and without caspofungin. Finally, immunofluorescence confocal microscopy experiments were performed with Aspergillus fumigatus hyphae grown with different concentrations of caspofungin, with and without FCS, stained with a primary murine anti-b-glucan antibody and a secondary fluorescent anti-murine antibody. RESULTS: The susceptibility tests of 6 Aspergillus strains to antifungals showed that caspofungin demonstrates an increased activity in the cell culture medium that 19 resembles the conditions found in the organisms of patients infected with Aspergillus; this is not the case with voriconazole. This increased activity is documented by the smaller MECs and EC50s in those conditions. I a series of experiments performed in order to find the factor that mediates this lag in the caspofungin activity in the two different media, FCS and more specifically BSA was found to mediate this effect. This was confirmed with scanning electron microscopy experiments that extensively described the damage at the hyphae and the hyphal tips. Next, confocal microscopy experiments were performed, and showed that BSA is bound on Aspergilli, mostly at the germinating forms, and that caspofungin binds more at the surface of the fungi in the presence of BSA, while the increased caspofungin mediated b-glucan exposure is even higher when BSA is present in the medium. Surprisingly, the abovementioned phenotype was not confirmed with anidulafungin or micafungin. DISCUSSION: The experiments described in this dissertation show that caspofungin activity is increased in a cell culture medium that resembles more the physiologic conditions in the tissues of patients infected with Aspergilli. Surprisingly, this is not the case with anidulafungin or micafungin. This increased caspofungin activity, as shown in the experiments in this dissertation, is associated with the increased BSA binding on the fungal cell wall of Aspergillus, especially in the germinating forms of the fungus. In the presence of caspofungin, BSA binding on the fungal cell wall is even more increased and in the presence of BSA, caspofungin binding on the fungal cell wall is increased, implying the possibility of BSA acting as a carrier molecule for caspofungin, leading in a positive feedback loop, since the increased binding and 20 activity of caspofungin would lead to even higher binding of BSA on the fungal cell wall. Furthermore, since one of the current explanations of the lag in the in vivo and the in vitro activity of echinocandins in the literature is the caspofungin induced increased b-glucan exposure on the fungal cell wall surface, we performed confocal microscopy experiments to Aspergillus fumigatus exposed to increasing caspofungin concentrations and found that when the fungus had been exposed to BSA, b-glucan exposure was higher. These two mechanisms could explain the lag in the in vivo and the in vitro activity of echinocandins.
Language Greek
Subject Ασπεργίλλος
Issue date 2017-07-26
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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