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OBJECTIVE: The early pathological process of Candida infection and immunological responses in tongues of the mice with experimental oral candidiasis was analysed. METHODS: CD-1 mice, pretreated by prednisolone were orally inoculated with Candida albicans. Symptoms were monitored by measuring the area of white tongue coating and number of viable Candida cells in oral cavity. The histopathological analysis was carried by PAS-stain and immunofluorescent staining. IL-4, IL-12p70, IFN-γ, TNF-α in recovered from the homogenates of the tongues were measured by ELISA. RESULTS: The fungus invaded the tongue surface of the mice and white patches developed within 24h after inoculation. Histopathological examination indicated the presence of local acute inflammation in superficial tissues of tongues covered by mycelium of C. albicans. Pathological exacerbation was observed from 24 to 48 h after the inoculation and from then the symptoms of oral candidiasis appeared to move into the recovery phase. Inflammatory cells mainly consisting of neutrophils was accumulated and located under the lesions covered by Candida-hyphae. An increase in IL-12p70 and IFN-γ in tongue homogenates was observed at 48 h after inoculation. CONCLUSIONS: The worst condition in the pathological process in experimental oral candidiasis was found 48 h after C. albicans inoculation. When the surface of the Candida-inoculated tongues was covered with Candida-hyphae, a dense accumulation of neutrophils was observed under the lesions and homogenates of the tongues contained increased levels of IL-12p70 and IFN-γ. These suggested that local pathological condition of Candida-infected tongues may be affected by neutrophils accumulation and increased levels of some cytokines.

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The therapeutic efficacy of tea tree oil (TTO), Melaleuca alternifolia, and its main component, terpinen-4-ol, were evaluated in a murine oral candidiasis model. Prednisolone -pretreated mice were orally infected with a fluconazole-susceptible (TIMM 2640) or a resistant (TIMM 3163) strain of Candida albicans to induce oral candidiasis. TTO or terpinen-4-ol was administrated with a cotton swab 3 h and 24 h after candida infection. These treatments clearly showed a decrease in the symptom score of tongues and in the viable candida cell number in the oral cavity at 2 d after azole-susceptible C. albicans infection, although the degree of the efficacy was less than that of fluconazole. Even against oral candidiasis caused by azole-resistant C. albicans, TTO and terpinen-4-ol were similarly effective, while fluconazole appeared ineffective. These results suggest that TTO and terpinen-4-ol may have the potential of therapeutic ability for mucosal candidiasis which may also be applicable to C. albicans oral candidiasis induced by the azole-resistant strain.

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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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We assessed the potential of oligonol, a low molecular polyphenol formulation prepared from lychee fruits, for treatment of oral candidiasis using a murine model. Oligonol at concentration of more than 313 microg/ml inhibited the mycelial growth of Candida albicans in vitro. When 50 microl of oligonol (20 mg/ml ) was administered three times into the oral cavity of orally Candida -infected mice, the number of viable Candida cells in the oral cavity was reduced significantly and the score of lesions on the tongue recovered on day 2. These findings suggest that oligonol could have potential as a food component supporting anti- Candida treatment.

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The Japanese Society for Medical Mycology (JSMM) method used for testing the antifungal susceptibility of yeast, the MIC end point for azole antifungal agents, is currently set at IC(80). It was recently shown, however that there is an inconsistency in the MIC value between the JSMM method and the CLSI M27-A2 (CLSI) method, in which the end- point was to read as IC(50). To resolve this discrepancy and reassess the JSMM method, the MIC for three azoles, fluconazole, itraconazole and voriconazole were compared to 5 strains of each of the following Candida species: C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei, for a total of 25 comparisons, using the JSMM method, a modified JSMM method, and the CLSI method. The results showed that when the MIC end- point criterion of the JSMM method was changed from IC(80) to IC(50) (the modified JSMM method) , the MIC value was consistent and compatible with the CLSI method. Finally, it should be emphasized that the JSMM method, using a spectrophotometer for MIC measurement, was superior in both stability and reproducibility, as compared to the CLSI method in which growth was assessed by visual observation.

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We examined the effects of spices and herbs on Candida albicans growth using in vitro assay and therapeutic activity of some selected herbal preparations against murine oral candidiasis. All tested samples: lemongrass (Cymbopogon citratus), lemon balm (Melissa officinalis), thyme (Thymus vulgaris), rosemary (Rosmarinus officinalis), roselle (Hibiscus sabdariffa), green tea (Camellia sinensis), and cassia (Cinnamomum cassia) inhibited Candida mycelial growth in vitro. The results of this assay showed that the anti-Candida activity of lemongrass, green tea, and cassia is stronger than that of the other tested herbs. Oral administration of lemongrass or green tea did not result in significant improvement in the murine oral candidiasis, while the administration of cassia improved the symptoms and reduced the number of viable Candida cells in the oral cavity. The results of in vitro Candida growth assay including GC/MS analysis suggested that cinnamaldehyde in the cassia preparation was the principal component responsible for the inhibitory activity of Candida mycelial growth. These findings suggest that oral intake of a cassia preparation is a clinical candidate for a prophylactic or therapeutic tool against oral Candida infection.

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In order to evaluate an effective administration method of essential oils for vaginal candidiasis, efficacy of vaginal application of essential oils against murine experimental candidiasis was investigated. The effect on vaginal inflammation and Candida growth form was also studied. Vaginal candidiasis was established by intravaginal infection of C. albicans to estradiol-treated mice. These mice intravaginally received essential oils such as geranium and tea tree singly or in combination with vaginal washing. Vaginal administration of clotrimazole significantly decreased the number of viable C. albicans cells in the vaginal cavity by itself. In contrast, these essential oils did not lower the cell number. When application of geranium oil or geraniol was combined with vaginal washing, the cell number was decreased significantly. The myeloperoxidase activity assay exhibited the possibility that essential oils worked not only to reduce the viable cell number of C. albicans, but also to improve vaginal inflammation. The smear of vaginal washing suspension suggested that more yeast-form cells appeared in vaginal smears of these oil-treated mice than in control mice. In vitro study showed that a very low concentration (25 microg/ml) of geranium oil and geraniol inhibited mycelial growth, but not yeast growth. Based on these findings, it is estimated that vaginal application of geranium oil or its main component, geraniol, suppressed Candida cell growth in the vagina and its local inflammation when combined with vaginal washing.

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Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.

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We examined the alleviation of cyclophosphamide-induced immunodepression by the antlered form of Ganoderma lucidum (G. lucidum AF) and also evaluated the anti-tumor and anti-metastatic effects of G. lucidum AF in cyclophosphamide-treated mice. G. lucidum AF alleviated cyclophosphamide-induced decrease in body weight, natural killer (NK) activity, interferon (IFN)-gamma production, and cytotoxic T lymphocyte (CTL) activity, and inhibited the abnormal increase and decrease in interleukine (IL)-4 level due to cyclophosphamide administration. Post-treatment with cyclophosphamide and G. lucidum AF significantly inhibited tumor growth in MM 46-bearing mice. When Lewis lung carcinoma cells were injected into mice after a cyclophosphamide administration, metastasis of these cells to the lung was increased, but G. lucidum AF suppressed it. The anti-tumor and anti-metastatic effects of the combination of G. lucidum AF and cyclophosphamide might influence the modulatory effects of G. lucidum AF on both cellular and humoral immunity. These findings suggest that G. lucidum AF would be beneficial in alleviating the reduction of immune response by chemotherapeutic anti-cancer drugs.

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In order to estimate predisposing activity of oral application of beclomethasone dipropionate (BDP)-containing mucoadhesive films for oral candidiasis, the effects of BDP on growth of Candida albicans were examined in vivo and in vitro. Murine neutrophils inhibited the mycelial growth of C. albicans in vitro, but this anti-Candida activity was clearly suppressed by the presence of 10(-6) M of BDP. In vitro, a BDP-release test showed that the amount of BDP released from BDP-containing films into the fluid phase increased in a time- and concentration-dependent manner and reached about 10-15% of the total amount of BDP in the film within 30 min. When the BDP-containing film was attached to the tongues of mice orally infected with C. albicans, oral infection by C. albicans deteriorated, but not as severely as in mice systemically immunosuppressed with prednisolone. Based on these findings, we also discuss the problems associated with the clinical application of BDP-film as an anti-inflammatory tool.

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A simple method to establish a murine esophageal candidiasis model that displayed characteristic symptoms of the condition was developed using the sedative agent, chlorpromazine. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. One day later, the mice received chlorpromazine to keep them in a sedated state for about 3 hr. Under the sedated condition, they were infected with 4 x 10(7) viable cells of Candida albicans by intra-esophageal injection with a round-head needle on syringe. From day 3 to day 6 post inoculation, 10(5)-10(6) colony forming units of C. albicans were recovered from the esophageal tube of each mouse and whitish, curd-like patches were observed on most of the inner surface of the tube. Histological examination showed that C. albicans in esophageal lesions grew mainly in mycelial form. In this experimental model, intragastric administration of an itraconazole oral solution (20 mg/kg/day) was clearly effective. This model would provide a useful tool to investigate the pathogenesis of C. albicans esophageal infection and the efficacy of various antifungal agents microbiologically and symptomatically.

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We established a novel murine model of pharyngeal candidiasis maintaining stable yeast population and local symptoms characteristic of pharyngeal thrush. The persistent Candida-infection was prolonged by inhalation of beclomethasone dipropionate corticosteroid. The severity of infection lesions was evaluated by determining viable cell number of Candia albicans and scores representing symptomatic curd-like white patch on pharyngeal tissue. The utility of this model was shown by the disappearance of lesions and fungal cells after treatment with fluconazole (FLCZ). The model would be useful for evaluating new chemotherapeutic or immunotherapeutic approaches against pharyngeal candidiasis, as well as in pathological studies.

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OBJECTIVE: The therapeutic activities of cyclodextrin-associated itraconazole oral solution (itraconazole OS) by two administration routes in experimental oral and oesophageal candidiasis in mice were examined and compared. METHODS: Using experimental oral and oesophageal candidiasis models in ICR mice, we investigated the efficacy of oral and intragastric administration of itraconazole OS and checked the concentration of itraconazole and its metabolite hydroxyitraconazole (OH-itraconazole) in tongues or oesophagus tissue after administration of itraconazole OS. RESULTS: Oral administration of itraconazole OS at doses of 0.8, 4.0 or 20 mg/kg/day clearly decreased the number of viable Candida albicans cells in the oral cavity of mice with oral candidiasis in a dose-dependent manner at 3 days after infection. Intragastric administration of itraconazole OS at doses of 4 and 20 mg/kg/day once a day were also effective but to a lesser degree than that of oral administration. In the oesophageal candidiasis model, oral administration of itraconazole OS displayed superior therapeutic efficacy to the intragastric route. In coincidence with the greater efficacy, itraconazole was detected in lesional tissues after oral administration of itraconazole OS. CONCLUSIONS: Oral administration of itraconazole OS displayed therapeutic efficacy against murine oral and oesophageal candidiasis superior to that achieved by intragastric administration. This can be explained by there being higher concentrations of itraconazole in tongues or oesophagus tissues after administration of the suspension by the oral route.

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We investigated the anti-tumor effects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF significantly countered the depression of splenic CD8+ cells and protected the decrease in interferon-gamma (IFN-gamma) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer.

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To obtain experimental evidence on the therapeutic efficacy of essential oils in aromatherapy for inflammatory diseases, we examined the effects of geranium oil on carrageenan-induced and collagen II-induced inflammation in mice, to assess acute and chronic anti-inflammatory activities of the oil. Single intraperitoneal injection of 5 mu L of geranium oil clearly suppressed the carrageenan-induced footpaw edema and increase in tissue myeloperoxidase activity, and repeated administration of the oil suppressed collagen-induced arthritis. These results revealed that geranium oil suppressed both acute and chronic inflammatory responses in mice.

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The efficacy of itraconazole (ITZ) solubilized in hydroxypropyl-beta-cyclodextrin (ITZ-IV) was examined in a murine model of invasive pulmonary aspergillosis (IPA). Immunosuppressed mice were infected by the intratracheal inoculation of Aspergillus fumigatus conidia (2 x 10(6) conidia/mouse). Their body weight rapidly decreased and they died within 6 days after infection. Intravenous administration of various doses of ITZ-IV was started 24 h after infection and was continued once a day for 4 days. ITZ-IV at daily doses of 10, 20, or 40 mg/kg was as effective as the intraperitoneal administration of amphotericin B (AMPH) at a dosage of 1 mg/kg daily in improving survival. ITZ-IV (20 mg/kg per day), as well as AMPH (1 mg/kg per day) significantly lowered the fungal burden in the pulmonary tissues. Histological improvement was seen within 2 days after the beginning of administration of ITZ-IV (20 mg/kg per day). In mice intravenously given a single dose of ITZ-IV (20 mg/kg), the blood level and pulmonary tissue level of ITZ plus its active metabolites, mainly hydroxyitraconazole (OH-ITZ), decreased gradually after the injection, but after 4 h their concentration was still between 1.4 microg/ml (ITZ) and 1.9 microg/ml (OH-ITZ), concentrations that were approximately 10 to 20 times greater than the minimum inhibitory concentration (MIC) of ITZ for challenging the strain of A. fumigatus (0.16 microg/ml). These results support the clinical usefulness of ITZ-IV for the treatment of IPA in immunocompromised patients.

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The immunological functions of Ganoderma lucidum antlered form (AF) (Rokkaku-Reishi in Japanese), a variant type of Ganoderma lucidum, were investigated in C57BL/6 mice treated with cyclophosphamide (CY). Ganoderma lucidum AF alleviated CY-induced decrease in body weight and abnormal increase in blood neutrophil level, when the mice were fed a diet containing 2.5% Ganoderma lucidum AF starting one week before CY treatment (150 mg/kg, ip). The recovery of CD8+ and NK1.1+ cells in the spleen was accelerated in Ganoderma lucidum AF group compared to the control group. Ganoderma lucidum AF also both alleviated CY-induced splenic lymphopenia and suppressed the abnormal increase in splenocytes 7 days after CY treatment. These results suggest that ingestion of Ganoderma lucidum AF is beneficial for improvement of quality of life reduced by anti-cancer chemotherapeutic drugs such as CY.

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BACKGROUND: Previous studies suggested that essential oils suppressed the adherence response of human neutrophils in vitro and that intraperitoneal application of geranium oil suppressed the neutrophil accumulation into peritoneal cavity in vivo. Usually, essential oils are applied through skin in aromatherapy in inflammatory symptoms. The purpose of this study is to assess the effects of cutaneous application of essential oils on the accumulation of neutrophils in inflammatory sites in skin of mice. METHODS: Inflammation with accumulation of inflammatory cells was induced by injection of curdlan, a (1-->3)-beta-D-glucan in skin or peritoneal cavity of mice. Essential oils were applied cutaneously to the mice immediately and 3 hr after intradermal injection of curdlan. The skin with inflammatory lesion was cut off 6 hr after injection of curdlan, and the homogenates were used for myeloperoxidase (MPO: a marker enzyme of neutrophil granule) assay. RESULTS: The MPO activity of the skin lesion induced by curdlan was suppressed dose-dependently by cutaneous application of geranium oil. Other oils such as lavender, eucalyptus and tea tree oils also suppressed the activity, but their activities seemed weaker than geranium. Juniper oil didn't suppress the activity CONCLUSION: Cutaneous application of essential oils, especially geranium oil, can suppress the inflammatory symptoms with neutrophil accumulation and edema.

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We examined the effect of a clove (Syzygium aromaticum) administered by two different routes on Candida albicans growth, using a murine oral candidiasis model. When the clove preparation was administered into the oral cavity of Candida-infected mice, their oral symptoms were improved and the number of viable Candida cells in the cavity was reduced. In contrast, when the clove preparation was administered intragastrically, oral symptoms were not improved, but viable cell numbers of Candida in the stomach and feces were decreased. These findings demonstrate that oral intake of an herbal food, clove, may suppress the overgrowth of C. albicans in the alimentary tract including the oral cavity.

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