The effects of lactoferrin (LF), an antimicrobial protein secreted in body fluids, and its peptides in combination with azole antifungal agents were investigated by the micro-broth-dilution method in a study of Candida albicans. In the case of LF, its pepsin hydrolysate (LFhyd) or the LF-derived antimicrobial peptide LactoferricinR B (LF-B), the concentrations required to inhibit the growth of Candida decreased in the presence of relatively low concentrations of clotrimazole (CTZ). The minimum inhibitory concentration (MIC) of all azole antifungal agents tested was reduced by 1/4-1/16 in the presence of a sub-MIC level of each of these LF-related substances. Polyene and fluoropyrimidine antifungal agents did not show such a combined effect with these LF-related substances. The anti-Candida activity of LF or LF-B in combination with CTZ was shown to be synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against C. albicans.
A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HC1 (0.04 N) to extract Candida cell-bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of 3H-glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.
Anti‐Candida activity of murine neutrophils and its regulation by immunomodulators were studied in vitro. Murine neutrophils which were prepared from peritoneal‐exudated cells inhibited the growth of Candida albicans at an effector: target (E/T) ratio of 30/1 or above. This anti‐Candida activity of neutrophils was augmented by lipopolysaccharide from Escherichia coli, murine tumor necrosis factor (TNF), murine interferon‐γ (IFN‐γ) and murine granulocyte macrophage colony‐stimulating factor (GM‐CSF) but not by granulocyte colony‐stimulating factor (G‐CSF) added to the incubation medium. Greater extent of augmentation was obtained when TNF plus GM‐CSF or INF‐γ plus GM‐CSF were used in combination. These results indicate that anti‐Candida activity of murine neutrophils is regulated similarly to that of the human neutrophils reported previously. Therefore murine peritoneal neutrophils can be used as a favorable substitute for human neutrophils in studies on protective machinery against C. albicans infection.
Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.
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