The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4␣-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14␣-demethylase inhibition, leading to secondary degenerative changes. Ergosterol is an important structural component of fungal cell membranes, maintaining membrane fluidity and a permeability barrier, and is essential for fungal cell viability (1-3). Several classes of antifungal drugs target ergosterol biosynthesis. Among these, triazole antifungals (e.g., itraconazole) and imidazole antifungals (e.g., clotrimazole and miconazole) inhibit sterol 14␣-demethylase (14-DM) in the ergosterol biosynthesis pathway (4). The consequent ergosterol depletion affects cell membrane integrity and function and is believed to inhibit fungal cell growth and affect morphology.Efinaconazole, a novel triazole antifungal drug currently under development as a topical treatment for onychomycosis, has demonstrated efficacy in patients with toenail onychomycosis in two phase 3 clinical trials (5). Onychomycosis and other superficial mycoses are caused mainly by dermatophytes (e.g., Trichophyton rubrum and Trichophyton mentagrophytes) and yeast (e.g., Candida albicans). Efinaconazole possesses similar or higher antifungal activity against T. rubrum and T. mentagrophytes (MIC range, 0.00098 to 0.031 g/ml) and a broader spectrum of activity than those of currently marketed antifungals used in onychomycosis (6). We investigated the effects of efinaconazole on fungal ergosterol biosynthesis and dermatophyte hyphal morphology.In the present study, T. mentagrophytes strain SM-110 and C. albicans strain ATCC 10231 were used. The MICs of efinaconazole (Kaken Pharmaceutical), itraconazole, and clotrimazole (SigmaAldrich) were determined by the broth microdilution method using morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640, as described in CLSI documents M38-A2 (7) and M27-A3 (8), using visual endpoint readings of 80% growth inhibition at 4 days and 50% growth inhibition at 48 h, respectively. Efinaconazole was 4-fold more active than itraconazole against T. mentagrophytes SM-110 (MICs of 0.0039 and 0.016 g/ml, respectively). Similarly, efinaconazole was 8-fold more active than clotrimazole against C. albicans ATCC 10231 (MICs of 0.00098 and 0.0078 g/ml, respectively). The two strains showed typical susceptibilities to the antifungals tested, consistent with previous findings for these species (6, 9).Ergosterol biosynthesis assays were conducted by modifying the methods of Vanden Bossche et al. (10) and Ryder et al. (11). T. mentagrophytes (2 ϫ 10 8 microconidia/ml...

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In vivo effects of basic fibroblast growth factor (bFGF) on bone formation was examined in rats. Daily systemic injections of 100 micrograms/kg bFGF for 7 days caused a marked stimulation of endosteal bone formation in both cortical and secondary cancellous bone areas. Histological examinations revealed that the sequence of responses to the injections of bFGF consisted of three phases: an early increase in the number of preosteoblastic cells over the osteoblastic cell layer (days 1-3), recruitment of osteoblasts from preosteoblastic cells (days 3-5), and an increase in new bone formation (days 5-7). These histological changes in the endosteum correlated closely with histomorphometrical parameters of bone formation, and the endosteal mineral apposition rate was almost unaffected during the initial 4 days but was markedly enhanced after this period. Immunohistochemical examinations using antitransforming growth factor (TGF)-beta 1 antibody demonstrated that immunostaining of preosteoblastic cells for TGF-beta already increased 1 day after bFGF treatment. Distribution of TGF-beta in osteoblasts and bone matrices began to increase on day 3, and all the osteoblasts and new bone matrices were intensively immuno-stained on day 7. These results demonstrate that systemic injections of bFGF in rats stimulate endosteal bone formation, and that the stimulation of bone formation is preceded by an initial increase in preosteoblastic cells with later recruitment of osteoblasts from these cells. Because the distribution of TGF-beta in the endosteal cells is increased by bFGF, the effect of bFGF may at least in part be mediated by TGF-beta. However, the precise mechanism of action of bFGF on bone formation remains to be clarified.

Predominant benign plasmacytoid myoepithelial cells in pleomorphic adenoma and malignant plasmacytoid myoepithelioma cells were investigated morphologically. The cells of both tumors were plasmacytoid in appearance and sheet-like. Immunohistochemically, they were positive for keratin, vimentin, and S-100 protein, and negative for alpha-smooth muscle actin. In the malignant cells, large nuclei with irregular nuclear membranes and distinct nucleoi and occasional intranuclear inclusions and nuclear grooves were seen. Ultrastructural findings showed that the benign cells were richer in intermediate filaments and had fewer mitochondria. The intranuclear inclusions and nuclear grooves of the malignant cells were caused by invagination of the irregular nuclear membranes. Taken in their entirety, the above light microscopical nuclear findings may be useful as an adjunct for distinguishing malignant from benign plasmacytoid neoplastic myoepithelial cells of the salivary gland.

Spontaneous mammary adenocarcinoma was observed in a 12-week-old female SD rat. A movable mass in the right cervical region was found at 11 weeks of age, and the rat was sacrificed the following week. The mass was located in the vicinity of the right salivary gland and measured 38 mm × 26 mm × 16 mm in gross size. It was a firm whitish mass, with a cut surface that was also whitish in appearance. Histopathologically, neoplastic cells formed glandular structures that contained secreted eosinophilic material. Ultrastructurally, similar secreted material and lipid droplets were in the cytoplasm of the neoplastic cells. Immunohistochemically, the neoplastic cells were positive for cytokeratin 8, cytokeratin 18 and estrogen receptor α. Based on these findings, the tumor was diagnosed as a mammary gland adenocarcinoma, and we therefore conclude that this tumor type can occur spontaneously in female SD rats as young as 12 weeks of age.