Tatsuya Hisajima

Protective effects of farnesol against oral candidiasis in mice

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 μmol/mouse showed a protective effect against oral candidiasis in a dose‐dependent manner, at least as estimated by symptom scores of tongues. At 9 μmol/mouse it decreased bodyweight loss. Histological studies of 2.25 μmol/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.

Suppression of anti‐Candida activity of macrophages by a quorum‐sensing molecule, farnesol, through induction of oxidative stress

Farnesol is well known as a quorum‐sensing molecule of Candida albicans. To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro. Murine macrophages, when cultured in the presence of 56–112 μM of farnesol for 1–2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti‐oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol‐treated macrophages was shown by fluorescence of DCFH‐DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti‐Candida activity, perhaps through induction of ROS.

Morphological relationships between peptidergic nerve fibers and immunoglobulin A-producing lymphocytes in the mouse intestine.

Immunoglobulin A (IgA) lymphocytes are present close to the nerve fibers in the lamina propria of the small intestine, and the administration of lipopolysaccharides (LPSs) increases the number of these cells and IgA secretion to the lumen. In the present study, we demonstrated that the nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) were close to the IgA lymphocytes in the mouse ileum lamina propria. Three hours after intraperitoneal administration of LPSs, IgA lymphocytes close to VIP nerve fibers, those close to basement membrane, and those close to both VIP nerve fibers and basement membrane were increased in number. Further, all IgA lymphocytes seen in the ileum lamina propria expressed the receptors for VIP, VIPR1, and VIPR2. Electron microscopy revealed that varicosities were in close apposition to the lymphocyte plasma membrane. The present study suggests that VIP/NPY/CGRP neurons in the submucosal plexus have a close anatomical relationship to IgA lymphocytes, playing a role in the secretion of IgA and intestinal fluid in response to stimulation by lipopolysaccharides, pathogens, or toxins.

Invasion process of Candida albicans to tongue surface in early stages of experimental murine oral candidiasis

We analyzed the morphologic and microbiologic aspects of the process of adhesion and invasion in the early stages of Candida albicans oral infection in a murine system. ICR mice were anesthetized by intramuscular injection with chlorpromazine chloride and then orally inoculated by swabbing with the C. albicans yeast cells. Their tongues were resected 1-3h after inoculation, washed sequentially with a physiological saline and 0.25% trypsin-solution and then homogenized. The number of viable C. albicans cells on the tongue surface was counted and fround to increase from 1-3h after inoculation. Most of the Candida cells attached to the tongue surface were present in clusters, mainly located in the gaps between lingual papillae and were covered with a mucoidal substance. By 3h after inoculation, these clusters frequently formed mycelia and could not be easily detached from the tongue surface by trypsin treatment. Observation of SEM and histological sections stained by Fungiflora Y revealed that the Candida hyphae at 3h stretched out of the cluster and entered the tongues through the surface. These results indicate that Candida hyphae begin to invade the tongue surface within 3h after inoculation and suggest that the mucus-like substance covering these cells may have an important early role in the interaction between the Candida cells and the tongue mucosal epithelium.

Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes

The frequency of targeted gene disruption via homologous recombination is low in the clinically important dermatophyte, Trichophyton mentagrophytes. The Ku genes, Ku70 and Ku80, encode key components of the nonhomologous end-joining pathway involved in DNA double-strand break repair. Their deletion increases the homologous recombination frequency, facilitating targeted gene disruption. To improve the homologous recombination frequency in T. mentagrophytes, the Ku80 ortholog was inactivated. The nucleotide sequence of the Ku80 locus containing a 2788-bp ORF encoding a predicted product of 728 amino acids was identified, and designated as TmKu80. The predicted TmKu80 product showed a high degree of amino acid sequence similarity to known fungal Ku80 proteins. Ku80 disruption mutant strains of T. mentagrophytes were constructed by Agrobacterium tumefaciens-mediated genetic transformation. The average homologous recombination frequency was 73.3 +/- 25.2% for the areA/nit-2-like nitrogen regulatory gene (tnr) in Ku80(-) mutants, about 33-fold higher than that in wild-type controls. A high frequency (c. 67%) was also obtained for the Tri m4 gene encoding a putative serine protease. Ku80(-) mutant strains will be useful for large-scale reverse genetics studies of dermatophytes, including T. mentagrophytes, providing valuable information on the basic mechanisms of host invasion.

Pathological analysis of the Candida albicans-infected tongue tissues of a murine oral candidiasis model in the early infection stage.

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.

A murine model of esophageal candidiasis with local characteristic symptoms.

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× 107 viable cells of Candida albicans by intra‐esophageal injection with a round‐head needle on syringe. From day 3 to day 6 post inoculation, 105‐106 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.