A Mouse Model of Trichophyton Inflammation Based on Trichophytin-induced Contact Hypersensitivity.

The prevalence of Trichophyton-induced superficial skin mycosis is very high among human patients. Dermatophytes generally infect the epidermis, especially the stratum corneum, forming scales, hyperkeratosis, and vesicles. The important roles played by the immune system in Trichophyton infection are detection of fungal invasion and elimination of fungi.These immune mechanisms are presumed to involve not only innate immunity but also acquired immunity. Therefore, there is a substantial need for studies on treatment methods based on new basic knowledge, and the elucidation of immunological mechanisms of Trichophyton-induced inflammatory reactions is especially important.However, since Trichophyton cannot colonize on the mouse skin, we tried to develop a model for Trichophyton inflammation induced by trichophytin extracted from Trichophyton mentagrophytes using a method based on contact hypersensitivity.Trichophytin is a crude extract that mainly contains fungal cell wall constituents including ß-glucan and zymosan. In this model, TLR2, TLR4, and dectin-1 were highly expressed, and production of IL-17A and IL23 was observed. This indicates that we succeeded in inducing fungal-specific inflammation in the mice.In this review, we introduce a mouse Trichophyton inflammation model developed to investigate the immunological mechanisms of Trichophyton-induced inflammatory reactions. In addition, we report results of evaluation of anti-inflammatory and anti-itching effects of anti-fungal agents using the inflammation model.

A Case of Cutaneous Mycosis Caused by Scedosporium dehoogii on an Immunocompromised Patient.

This report describes a 77-year-old man with cutaneous mycosis caused by Scedosporium dehoogii while taking oral betamethasone and tacrolimus for the treatment of rheumatoid arthritis. At examination in our clinic, the patient had multiple cystic lesions and nodules with slight tenderness, varying in size up to 4 cm, on his left knee and shin. He had not noticed any traumatic injury at the site of the lesions. Fungal cultures of samples taken from the abscesses, scales, and crusts of the lesions yielded white, later grayish brown, fluffy surfaced colonies. Partial sequencing of the ß-tubulin gene confirmed the species of the isolate. The patient was initially treated with oral voriconazole and local hyperthermia, but experienced hepatic injury 2 weeks later. His treatment was changed to itraconazole (ITC) and local hyperthermia, followed by a combination of ITC and terbinafine. The patient recovered completely during the 12-month course of treatment.

Polyclonality of Trichophyton rubrum Isolates in aDermatophytosis Patient with Multiple Lesions.

We cultured 15 isolates of Trichophyton rubrum and one isolate of Trichophyton mentagrophytes from an 82-year-old male tinea patient with multiple lesions. To determine whether feet lesions were the source of dermatophytes of other tinea lesions, we extracted total cellular DNA from the T. rubrum isolates（13 from feet, two from right waist and buttock）. PCR targeting the non-transcribed spacer（NTS）region of ribosomal RNA gene was performed. Molecular polymorphisms were detected by length variation of amplicons.Four molecular types were found among the 15 isolates. The predominant type, which we previously named Type III, comprised seven isolates cultured from both feet and from left waist and buttock. This was followed by Type VI, five isolates; Type V, two isolates; and Type IV, one isolate. Apart from type III, which was cultured from both feet, isolates were cultured from one foot only. The patient was successfully treated for all types with a six-month course of oral terbinafine and topical luliconazole. The molecular typing supported the notion that tinea pedis was the source of tinea corporis in the patient.

Glycyrrhetinic acid inhibits contact hypersensitivity induced by trichophytin via dectin-1.

Trichophyton infection is highly prevalent and tends to be recurrent. Therefore, it is important to develop new therapeutic agents. Previously, we established a mouse model of Trichophyton-induced contact hypersensitivity (CHS) and demonstrated that dectin-1 was involved in inflammation induced by trichophytin, the Trichophyton antigen. Here, we used that model to investigate glycyrrhetinic acid (GA) from plants of the genus Glycyrrhiza as a potential anti-inflammatory agent against superficial mycoses. GA suppressed swelling and the expression of inflammatory cytokines, including macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA. Anti-MIP-2 antibody suppressed trichophytin-induced inflammation, and antidectin-1 antibody suppressed zymosan-induced MIP-2 production in keratinocyte cells. These results suggest that MIP-2 is produced by dectin-1 activation and is involved in inflammation associated with CHS to trichophytin. GA also suppressed zymosan-induced MIP-2 and interleukin (IL)-8, production in mouse and human macrophages and keratinocytes. Furthermore, GA suppressed the phosphorylation of spleen tyrosine kinase (Syk) and inhibitor of nuclear factor-kappa B (IκBα) and the degradation of IκBα in zymosan-simulated RAW264.7 cells. The results of this study suggest that GA suppresses inflammation induced by trichophytin, partly by the downregulation of Syk phosphorylation.

Case of phaeohyphomycosis producing sporotrichoid lesions.

A 90-year-old Japanese woman, taking prednisolone (5-10 mg/day) for polyarthritis, presented to our hospital with multiple subcutaneous lesions on her left arm in 2009. Her history included excision of a phaeomycotic cyst on the left middle finger in 2007. There were three subcutaneous nodules approximately 15 mm in diameter around her left wrist and a large soft cystic lesion measuring 80 mm × 60 mm on her left elbow. A granuloma with neutrophilic infiltration was detected in the deep dermis of a biopsy specimen. Chains composed of round brown cells and short pseudomycelia were found in the granuloma. Fungal cultures from the samples confirmed Exophiala sp. to be the causative agent. Treatment with terbinafine and local hyperthermia seemed effective as all the lesions tended to subside. However, the patient died due to pneumonia approximately 1 month after commencement of therapy.

Molecular epidemiology of a major subgroup of Arthroderma benhamiae isolated in Japan by restriction fragment length polymorphism analysis of the non-transcribed spacer region of ribosomal RNA gene.

Arthroderma benhamiae vectored by small animals, such as household pets, causes tinea lesions on human skin. The number of tinea cases caused by this species is increasing in Japan. We attempted to develop a simple molecular method for strain discrimination, which is expected to be useful in molecular epidemiology. Out of the 61 strains of A. benhamiae registered at our institute, 46 A. benhamiae strains showed very high degrees of sequence similarity on cluster analysis of the internal transcribed spacer regions of ribosomal RNA (rRNA) genes. These 46 strains, including 22 strains isolated from Japan, were further used for strain typing by analyzing the non-transcribed spacer (NTS) region of the rRNA gene. Polymerase chain reaction was performed using a primer pair designed for amplification of a part of the NTS region, and the amplicons were successfully discriminated by restriction fragment length polymorphism (RFLP) analysis performed using MvaI. RFLP analysis showed 11 NTS types (NTS1-NTS11) among the 46 strains. Out of the 22 Japanese strains, 10 were of the NTS8 type; 6, of the NTS1 type; 3, of the NTS2 type; and 3, of the NTS5 type. Molecular typing showed consistency of NTS types among the strains isolated from different lesions on the same patient, among the strains derived from the same family, and among the strains from pets and their owners. We observed that 3 out of the 4 NTS types among the Japanese strains were detected outside Japan as well.

Endothelin receptor antagonist bosentan improves the dermal sclerosis in a patient with systemic sclerosis.

We report a case of systemic sclerosis with interstitial pneumonia and severe pulmonary hypertension treated with bosentan, an endothelin receptor antagonist. Within a short period of administration of the bosentan the skin sclerosis improved.

Analysis of Trichophyton antigen-induced contact hypersensitivity in mouse.

BACKGROUND: Trichophyton-induced superficial skin mycosis is a common infectious human disease, but the immunological mechanism against Trichophyton infection is unclear with regard to many points. Since Trichophyton cannot colonize mice, guinea pigs were used in previous experiments on Trichophyton infection. However, it is difficult to perform immunological and genetic analyses in guinea pigs. OBJECTIVE: The objective of this study was to establish a mouse Trichophytin-associated inflammation model of superficial skin mycosis in which immunological and genetic analyses can be performed. METHODS: We established a mouse Trichophyton-induced contact hypersensitivity model by applying Trichophytin, the Trichophyton antigen, extracted from Trichophyton mentagrophytes, to mice. Using a Th1-dominant strain, C57BL/6, and a Th2-dominant strain, BALB/c, we investigated the expression of inflammatory cytokines and receptors of the innate immune system for fungi, TLR4, TLR2, and dectin-1, and their influences on responses of the acquired immune system. RESULTS: In C57BL/6 mice, expressions of IFN-γ and IL-17 A in regional lymph nodes and IL-1ß, IFN-γ, IL-6, and IL-23 in the inflammatory auricular skin were enhanced by Trichophytin challenge, suggesting that not only Th1 cells but also Th17 cells were induced. In BALB/c mice, expressions of IL-4 in regional lymph nodes, and TSLP and IL-4 in the auricular skin were enhanced by Trichophytin challenge. Interestingly, dectin-1-neutralizing antibody inhibited the promotion of IFN-γ production in C57BL/6 mice, and dectin-1-expressing immune cells had crucial actions in Trichophyton-induced IFN-γ production. CONCLUSION: These results suggest that inflammatory mediators differently regulate Trichophytin-induced contact hypersensitivity on the basis of the status of host immunity.

Molecular epidemiology of Trichophyton tonsurans strains isolated in Japan between 2006 and 2010 and their susceptibility to oral antimycotics.

Trichophyton tonsurans has been isolated among judo practitioners, wrestlers, and sumo wrestlers during an epidemic of tinea corporis and tinea capitis in Japan. A previous study using restriction fragment length polymorphism (RFLP) analysis of the non-transcribed spacer (NTS) region of the ribosomal RNA gene revealed that different sources for the causative fungus in epidemics among judo practitioners and among wrestlers. Many different fungal strains have since been isolated from practitioners of these sports. The present study evaluated fungal characteristics of strains newly isolated between July 2006 and December 2010 using this molecular method. PCR-RFLP analysis using MvaI and AvaI was performed on 263 strains, composed of 186 isolates from judo practitioners, 32 from wrestlers, 30 from sumo wrestlers, 5 from other sports, 7 from family members or friends of the sports practitioner patients, and 3 from sporadic (non-epidemic) cases. Four molecular types, NTS I, II, III, and VII were detected. Of these, NTS I was the most predominant, occurring in 243 of 263 strains (92.4%). All of the 30 strains isolated from sumo wrestlers were classified as NTS I, suggesting that the epidemic among sumo wrestlers originated from an earlier epidemic among judo practitioners. Thirteen strains were classified as NTS II; all were related to wrestling and were isolated mainly from Chubu and Kansai areas in the central part of Honshu island. NTS III was detected in 6 strains, and one strain classified as NTS VII was isolated from a sporadic case of tinea capitis in a Peruvian immigrant. The minimum inhibitory concentrations (MICs) of terbinafine, itraconazole, fluconazole, and griseofulvin on 10 strains of NTS I and NTS II and 4 strains of NTS III were examined; there were no differences in MIC between these molecular types.

Letter: Two cases of metastases to the scalp bone mimicking epidermoid cyst.

Metastatic skin tumors present with a variety of clinical manifestations. We herein present two cases of metastases to the scalp, invading the skull, which showed epidermoid cyst-like appearances.

Intravital imaging of IL-1beta production in skin.

IL-1 is a prototypic inflammatory cytokine that has pathogenic roles in various skin disorders. Although Langerhans cells (LCs) have been reported to express IL-1beta mRNA upon application of contact sensitizers, it remains unclear whether other cell types produce IL-1beta in skin. Thus, we sought to directly identify IL-1beta-producing cells in living animals by construction of transgenic mice expressing DsRed fluorescence protein gene under the control of IL-1beta promoter. Little DsRed fluorescence signal was detected in skin under steady-state conditions. Striking increases in DsRed signal were observed after topical application of a contact sensitizer, oxazolone, which also induced markedly elevated IL-1beta mRNA and protein expression. DsRed signal was expressed primarily by CD45(+)/CD11b(+) myeloid leukocytes in both epidermal and dermal compartments and was detected only in small fractions of epidermal LCs. Interestingly, DsRed(+) cells emerged preferentially as clusters around hair follicles. Intravital confocal imaging experiments revealed highly motile potentials of DsRed(+) cells-they constantly crawled around hair follicles via amoeba-like movements with a mean velocity of 1.0+/-0.4 microm min(-1) (epidermis) or 2.7+/-1.4 microm min(-1) (dermis). The newly developed in vivo imaging system represents a useful tool for studying spatial regulation of IL-1beta production in skin.

Dual therapeutic efficacy of vinblastine as a unique chemotherapeutic agent capable of inducing dendritic cell maturation.

Our recent unbiased functional screen of 54 chemotherapeutic drugs unveiled striking heterogeneity in their effects on dendritic cells (DC). Most notably, vinblastine (VBL) was found to induce phenotypic and functional maturation of DCs in vitro. Here, we sought to determine whether VBL exhibits "dual" therapeutic efficacy in living animals by directly killing tumor cells and by boosting host immunity via DC maturation. Local injection of VBL in a low dose into the skin of C57BL/6 mice induced in situ maturation of epidermal Langerhans cells. When coinjected with a model antigen, ovalbumin (OVA), VBL enhanced OVA-specific cellular and humoral immune responses. When injected directly into the OVA cDNA-transduced E.G7 tumors, VBL augmented clonal expansion of OVA-reactive CD8 T cells and CTL activities. In B16 melanoma model, intratumor VBL injection induced apoptosis of melanoma cells, phenotypic maturation of tumor-infiltrating DCs, and significant CTL activities. Although complete clearance was never achieved, growth kinetic of B16 melanoma was markedly reduced in C57BL/6 mice by intratumor VBL injection. Importantly, the same treatment was far less efficacious in immunocompromised severe combined immunodeficient mice, indicating the requirement of intact host immunity. Our results introduce a new concept that VBL may be used to design "immunostimulatory" chemotherapy regimens.