The Toll-like receptor (TLR) family has important roles in microbial recognition and dendritic cell activation. TLRs 7 and 9 can recognize nucleic acids and trigger signalling cascades that activate plasmacytoid dendritic cells to produce interferon-alpha (IFN-alpha) (refs 7, 8). TLR7/9-mediated dendritic cell activation is critical for antiviral immunity but also contributes to the pathogenesis of systemic lupus erythematosus, a disease in which serum IFN-alpha levels are elevated owing to plasmacytoid dendritic cell activation. TLR7/9-induced IFN-alpha induction depends on a molecular complex that contains a TLR adaptor, MyD88, and IFN regulatory factor 7 (IRF-7) (refs 10-14), but the underlying molecular mechanisms are as yet unknown. Here we show that IkappaB kinase-alpha (IKK-alpha) is critically involved in TLR7/9-induced IFN-alpha production. TLR7/9-induced IFN-alpha production was severely impaired in IKK-alpha-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction was decreased but still occurred. Kinase-deficient IKK-alpha inhibited the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7. Furthermore, IKK-alpha associated with and phosphorylated IRF-7. Our results identify a role for IKK-alpha in TLR7/9 signalling, and highlight IKK-alpha as a potential target for manipulating TLR-induced IFN-alpha production.

The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD) and colonizes the skin surface of patients with AD. With the goal of reducing the number of Malassezia cells, we investigated the antifungal activities of a therapeutic agent for AD, tacrolimus, and the azole agents itraconazole and ketoconazole against Malassezia species in vitro. We examined 125 strains of the 11 currently accepted Malassezia species by using the agar dilution method. All strains of the 11 Malassezia species were very susceptible to both azole agents, with MICs ranging from 0.016 to 0.25 g/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 g/ml. Two of the major cutaneous floras, Malassezia globosa and Malassezia restricta, have several genotypes in the intergenic spacer region of the rRNA gene; the azole agents had slightly higher MICs for specific genotype strains of both microorganisms. A combination of azole agents and tacrolimus had a synergistic effect against Malassezia isolates, based on a fractional inhibitory index of 0.245 to 0.378. Our results provide the basis for testing these agents in future clinical trials to reduce the number of Malassezia cells colonizing the skin surface in patients with AD.Although lipophilic yeasts, Malassezia spp., colonize the skin surface of healthy individuals, they may also cause seborrheic dermatitis (SD), pityriasis (tinea) versicolor, and Malassezia folliculitis and may exacerbate atopic dermatitis (AD) (1). AD is a common chronic inflammatory skin disease. The standard treatment of AD is topical corticosteroids and topical immunomodulating agents, although some patients do not respond to these treatments. Cutaneous microorganisms are considered an exacerbating factor. Although large numbers of lipophilic Malassezia species organisms colonize the skin surfaces of both AD patients and healthy subjects, anti-Malassezia-specific immunoglobulin E antibody is detected only in AD patient sera (14,16,32). This is probably owing to the disrupted barrier function of the skin surface and the effects of scratching on sensitization to the organisms (30). The application of topical antifungal agents to AD patients decreases Malassezia colonization and the severity of eczematous lesions (2), suggesting that Malassezia species play a role in atopic dermatitis. In addition, several candidate Malassezia antigens have been implicated in the pathogenesis of AD (15,19,20,23,34).In 1996, the taxonomy of the genus Malassezia was revised by Guého et al. (8). The authors described seven species (Malassezia furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. sympodialis, and M. pachydermatis). Subsequently, Japanese researchers found another four new species: Malassezia dermatis (25), M. yamatoensis (28), M. japonica (27), and M. nana (11) were isolated from an AD patient, SD patients, a healthy individual, and an animal, respectively, between 2002 and 2004. At present, 11 species have been accepted in this genus. By use of the r...

With the exception of Malassezia pachydermatis, Malassezia species require a lipid for growth, and are part of the human cutaneous microflora. In dermatology, Malassezia species are important fungi clinically, as they are associated with pityriasis versicolor, seborrheic dermatitis (SD), Malassezia folliculitis, and atopic dermatitis (AD) (2-4). Although M. furfur was previously thought to be the causative agent or trigger factor in all of these skin diseases, Guého et al. (6) reclassified this microorganism into several species in 1996. Based on the new taxonomy of the genus Malassezia, several studies have examined the relationship between the newly defined species and skin diseases (1,7,8,12). Our research group has also analyzed the cutaneous microflora of AD patients and healthy subjects using a molecular-based non-culture method (17,19). During these studies, we isolated two new Malassezia species from Japanese AD patients and healthy subjects: M. dermatis (18) and M. japonica (20). Subsequently, we analyzed the cutaneous microflora of SD patients and found an additional new Malassezia species. In this paper, we propose a new species, M. yamatoensis, and describe the distribution of this microorganism in the skin of SD and AD patients, and healthy subjects. Materials and MethodsStrains. Two Malassezia strains (M 9985 and M 9986) were isolated from a lesion on the wing of the nose of a 30-year-old male SD patient. OpSite transparent dressings (Smith and Nephew Medical, Ltd., Hull, U.K.) were applied to the skin of the SD patient and then transferred onto modified Leeming and Notman agar (10 g glucose, 10 g peptone, 8 g bile salts (OXOID, Hampshire, U.K.), 2 g yeast extract, 0.5 g glycerol monostearate, 15 g agar, 10 ml glycerol, 5 ml Tween 60, 20 ml olive oil, and 50 mg chloramphenicol (Sankyo, Tokyo) per liter) as recommended by the Centraalbureau voor Schimmelcultures, and incubated at 32 C.rDNA sequencing and phylogenetic analysis. In the rRNA gene, D1/D2 26S rDNA, the internal transcribed spacer (ITS) region including 5.8S rDNA, and inter- Abstract: Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non-culture-based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.

The lipophilic yeast Malassezia globosa is one of the major constituents of the mycoflora of the skin of patients with atopic dermatitis (AD). We compared the genotypes of M. globosa colonizing the skin surface of 32 AD patients and 20 healthy individuals for polymorphism of the intergenic spacer (IGS) 1 region of the rRNA gene. Sequence analysis demonstrated that M. globosa was divided into four major groups, which corresponded to the sources of the samples, on the phylogenetic tree. Of the four groups, two were from AD patients and one was from healthy subjects. The remaining group included samples from both AD patients and healthy subjects. In addition, the IGS 1 region of M. globosa contained short sequence repeats: (CT) n , and (GT) n . The number of sequence repeats also differed between the IGS 1 of M. globosa from AD patients and that from healthy subjects. These findings suggest that a specific genotype of M. globosa may play a significant role in AD, although M. globosa commonly colonizes both AD patients and healthy subjects.Malassezia species are lipophilic yeasts that are part of the normal human cutaneous commensal flora; they are isolated from sebaceous gland-rich areas of the skin, particularly on the chest, back, and head. They are also associated with several cutaneous diseases, including atopic dermatitis (AD), folliculitis, pityriasis versicolor, and seborrheic dermatitis (1, 7). In a taxonomic revision in 1996, the genus Malassezia was classified into seven different species: M. furfur, M. globosa, M. restricta, M. obtusa, M. pachydermatis, M. slooffiae, and M. sympodialis (9). Recently, we described an eighth species, M. dermatis, which was isolated from Japanese patients with AD (27). Since the taxonomic revision of the genus Malassezia, several studies have examined the distribution of the newly defined species of Malassezia on healthy human skin and lesions of skin diseases (2, 10, 21). However, culture media or sampling techniques often affect analyses of the Malassezia microflora. In a previous study, we used a nonculture method as an alternative to fungal culture to analyze the distribution of cutaneous Malassezia species (25). M. globosa and M. restricta were detected in approximately 90% of AD patients, and M. furfur and M. sympodialis were detected in approximately 40% of the subjects. In healthy subjects, M. globosa, M. restricta, and M. sympodialis were detected in approximately 40 to 60% of the subjects; M. furfur was found in only 4% of the subjects; and no other Malassezia species were detected. Therefore, these four species are common inhabitants of the skin of both AD patients and healthy individuals. In addition, while anti-Malassezia immunoglobulin E (IgE) antibody was detected in more than 90% of AD patients, no antibody was found in healthy subjects. Based on these results, M. globosa and M. restricta are thought to play a significant mycological role in AD. M. globosa is also part of the major microflora on the skin of healthy individuals. We used the intergenic space...

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-Bdeficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this un-