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Human Beta-defensins: Differential Activity against Candidal Species and Regulation by Candida albicans

¹ Department of Biological Sciences and Department of Periodontics, ³ Department of Community Dentistry, Case Western Reserve University School of Dental Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4905, USA; and
² Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH, USA;

View larger version (12K): [in this window] [in a new window]   Figure 1. Real-time PCR analysis of hBD-2 and -3 mRNA induction in NHOECs following challenge with C. albicans SC5314 and respective mutants. C. albicans SC5314 (parent), HLC54 (suppressed hyphal mutant), and HLC84 (reconstituted hyphal expression) were incubated with NHOEC monolayers at a multiplicity of infection of 0.1:1 (Ca:HOEC), 24 hrs, followed by real-time PCR analysis. White bar, hBD2; black bar, hBD3. N = 4. *Significantly different from negative control and ** from SC5314 (p < 0.05; t test).

View larger version (30K): [in this window] [in a new window]   Figure 2. Differential activity of human beta-defensins against candidal species. (A–F) Human beta defensin killing of candidal species. Recombinant hBD-1, -2, and -3 were incubated at increasing concentrations (0–10 µM) with strains of C. albicans (OPC76, OPC84; OPC99; SC5314), C. krusei (A91, MRL-214), C. parapsilosis (A58, ATCC 22019), and C. glabrata (90030, 2255), respectively, 37°C, 3 hrs, followed by serial dilutions, plating on Sabouraud Dextrose agar plates, and counting of colonies 48 hrs later. (A) rhBD-1 killing of C. albicans strains; (B) rhBD-1 killing of Candida krusei, C. parapsilosis, and C. glabrata strains; (C) rhBD-2 killing of C. albicans strains; (D) rhBD-2 killing of Candida krusei, C. parapsilosis, and C. glabrata strains; (E) rhBD-3 killing of C. albicans strains; and (F) rhBD-3 killing of Candida krusei, C. parapsilosis, and C. glabrata strains. Results are calculated as a percentage of colony-forming units when compared with untreated controls. Data are presented as mean ± SD of 3 (n = 3) independent experiments. (G,H) β-defensin inhibition of Candida glabrata adherence to human oral epithelial cells. C. glabrata strain 90030 (G) or C. glabrata strain 2255 (H) was incubated with either rhBD-1, -2, or -3 (2.5 or 5 µM), at 37°C, for 3 hrs, followed by assay for adherence to confluent monolayers of OKF6/Tert cells. Results are calculated as a percentage of adhering fungi when compared with untreated controls. Data are presented as mean ± SD of 3 (n = 3) independent experiments. Asterisks show a significant difference from control values at *P < 0.05 and **P < 0.005 by Student’s unpaired t test.

View larger version (47K): [in this window] [in a new window]   Figure 3. Confocal analysis of C. albicans OPC 84 and C. glabrata 90030 after incubation with rhBD-2. C. albicans OPC 84 or C. glabrata 90030 yeast cells were respectively incubated with rhBD-2, at 37°C, for 48 hrs. Cells were then incubated with a staining solution containing Alexa Fluor 488 conjugated Concanavalin A (ConA) (50 µg/mL) (Molecular Probes, Eugene, OR, USA) and FUN® 1 (20 µM) (Molecular Probes) in PBS, 37°C, 30 min. Stained cells were observed with the use of a dual scanning confocal microscopy system (LSM 510, Zeiss, Oberkochen, Germany). (A) Normal C. albicans cells showing uniform membrane thickness (green boundaries) and normal metabolic activity (red inside yeast cells); (B,C) C. albicans after incubation with rhBD-2, showing a lack of FUN 1 staining within the cells, indicating severe metabolic deficiencies, and evidence of thinning of the cell walls (B, arrows); (C) shows leakage of cytoplasmic material (arrow); (D) C. glabrata 90030 showing uniform membrane thickness (green boundaries) and normal metabolic activity (red inside yeast cells); (E) C. glabrata after incubation with rhBD-2 does not show appreciable differences in either envelope thickness, metabolic activity, or cytoplasmic debris when compared with untreated cells (D).

Journal of Dental Research, Vol. 84, No. 5, 445-450 (2005)
DOI: 10.1177/154405910508400509


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