Masuyoshi Saito

A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects.

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.

Sequence Diversity of the Intergenic Spacer Region of the rRNA Gene of Malassezia globosa Colonizing the Skin of Patients with Atopic Dermatitis and Healthy Individuals

ABSTRACT 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.

Antifungal Activities of Tacrolimus and Azole Agents against the Eleven Currently Accepted Malassezia Species

ABSTRACT 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 μg/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 μg/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.

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin

BACKGROUND Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function. OBJECTIVE To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems. METHODS We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99. RESULTS The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis. CONCLUSION Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.

Pirfenidone suppresses keloid fibroblast-embedded collagen gel contraction

Keloid is a clinically intractable disease that causes disfigurement, itching, and pain due to abnormal proliferation of fibroblasts and production of collagen. Pirfenidone is a novel anti-fibrotic agent that inhibits the progression of fibrosis occurring in the keloid lesions of the lung and kidney. In order to examine whether pirfenidone has a therapeutic effect on keloid lesions, we prepared an in vitro wound contraction model with keloid fibroblasts. The gel contractility of a mixture of keloid fibroblasts and an acid-soluble collagen solution was examined with/without transforming growth factor (TGF)-β1 in the presence or absence of pirfenidone. Real time RT-PCR was performed to detect mRNA expression of TGFB1, CTGF, aSMA, and Col1A1 quantitatively in keloid fibroblasts incubated with/without TGF-β1 in the presence or absence of pirfenidone. The contractility of keloid fibroblast-embedded collagen gel was increased after the addition of TGF-β1. Pirfenidone suppressed gel contraction with TGF-β1 dose dependently. TGF-β1 stimulated mRNA expression of TGFB1, CTGF, aSMA, and Col1A1 in keloid fibroblasts, while pirfenidone significantly inhibited mRNA expression of CTGF and aSMA in the identical cells. These findings suggest that pirfenidone suppresses the contraction of keloid-derived fibroblasts by inhibiting the down-stream pathway of TGF-β1, thus demonstrating its therapeutic utility for the treatment of keloid lesions.

The basidiomycetous yeasts Cryptococcus diffluens and C. liquefaciens colonize the skin of patients with atopic dermatitis.

Our previous research showed that lipophilic yeasts, Malassezia species, colonize the skin of patients with atopic dermatitis (AD) at a high frequency. In this study, we found that two basidiomycetous yeasts, Cryptococcus diffluens and C. liquefaciens, colonize the skin significantly more frequently in AD patients than in healthy subjects. Transparent dressings were applied to the skin of 36 AD patients and 30 healthy subjects and then transferred onto Sabouraud dextrose agar. Colonies recovered from the medium were identified by DNA sequence analysis of internal transcribed spacer regions and the D1/D2 26S rRNA gene. C. diffluens and C. liquefaciens were isolated from 42% (15/36) and 33% (12/36) of AD patients and from 20% (6/30) and 20% (6/30) of healthy subjects, respectively. In addition, fungal DNA was extracted directly from the dressings and amplified in a specific nested PCR assay. C. diffluens and C. liquefaciens DNA were detected in dressings from 97% (35/36) and 86% (31/36) of the AD patients and 47% (14/30) and 37% (11/30) of the healthy subjects, respectively. These findings show that Malassezia spp. are not the only yeasts that colonize the skin of AD patients; Cryptococcus spp. also are present in a high proportion of patients. The role of these microorganisms in AD is as yet unknown, but the current findings, in combination with previous results, indicate that C. diffluens, C. liquefaciens, M. globosa, and M. restricta together colonize the skin surface of AD patients at a high frequency.

Food-dependent exercise-induced anaphylaxis with a high level of plasma noradrenaline.

Ingesting certain foods sometimes triggers anaphylaxis when followed by exercise (food‐dependent exercise‐induced anaphylaxis, FDEIA). Specific food‐induced mucocutaneous urticaria may also progress to anaphylaxis (oral allergy syndrome, OAS). A positive skin test and/or radioallergosorbent test (RAST) to the foods suggest involvement of immunoglobulin (Ig)E‐anaphylaxis in both disorders. The triggering foods and initial target organs are usually different in each case. In the present study, a 32‐year‐old male reported dyspnea accompanied by wheals, and symptoms of low blood pressure while walking after eating Chinese noodles and donuts. He also reported uncomfortable sensations in his mouth and throat after ingesting melon. Exercise challenge tests were administered. Serum histamine, plasma adrenaline, noradrenaline and dopamine were measured pre‐ and post‐test. No symptoms were induced by exercise or by the ingestion of any single food item before exercise. However, numerous wheals appeared when exercise followed the combined ingestion of foods. Likewise, the sequence of eating pancakes and then exercising resulted in numerous wheals and anaphylaxis. Olopatadine hydrochloride and ketotifen fumarate completely inhibited this anaphylaxis. The skin prick tests resulted in fruit‐induced erythema and wheals. The results of these tests with wheat, butter and sugar were negative, and no symptoms were induced by the exercise test after ingestion of watermelon, melon or apple. The anaphylactoid symptoms were accompanied by a significant increase of plasma noradrenaline. In this case, not only wheat, but sugar and butter may induce the onset of FDEIA. There was no significant correlation between the intensity of the symptoms and the serum histamine levels in the present case. Noradrenaline may be involved in the onset of FDEIA, since noradrenaline may selectively inhibit T‐helper (Th)1 functions while favoring Th2 responses. The tests showed no cross‐reactivity between the causative foods of OAS and FDEIA, indicating that the mechanisms of onset are different between them.

Novel mutation in the ATP2A2 gene in a Japanese Darier's disease patient with extremely hyperkeratotic lesions.

the affected cells. In pedigree 1 and sporadic case 1, we detected the same missense mutation within the GJB4 gene (c.292C>T, R98C), which has also been reported by Common. We speculate it may be a hotspot of mutations. We did not identify any mutation of the GJB3 and GJB4 gene in sporadic patient 2, which may shed light on genetic heterogeneity of EKV. In summary, we identified one novel mutation of the GJB3 gene and one reported mutation of the GJB4 gene in two pedigrees and one sporadic case. Our findings provided an addition to the EKV mutation database and will contribute further to the understanding of EKV genotype ⁄ phenotype correlations and to the pathogenesis of this disease.

Two cases of malignant melanoma of the toe developed skin ulcers following local injection of natural beta‐interferon

islands was higher than those for Th cells and CTL from preto post-treatment. These phenomena might account for the ineffective local and systemic control of tumor progression. The increased infiltration of regulatory T cells in the outer layer of the tumor mass may inhibit CTL to infiltrate into the inner layer of the tumor mass and/or avoid antitumor function of CTL. Increased infiltration of NK cells, NKT cells, Th cells and CTL into tumor islands requires that these cells pass through the fibrous or dense collagenous capsule to the inside of the tumor. To achieve this, adequate stimulation of TLR9 in tumorinfiltrating pDC and B cells with CpG-ODN followed by induction of antitumor immune cells is required. The combination therapy in our case elicited only a limited immune response and further treatment options are required for improving the clinical effect through strong induction of antitumor immune cells and alteration of the tumor microenvironment. Akiko OZAWA,* Tomoko NOMIYAMA, Noriaki NAKAI,* Gunther HARTMANN, Hideya TAKENAKA, Saburo KISHIMOTO, Norito KATOH Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Osaka General Hospital of West Japan Railway Company, Osaka, Japan, and Institute of Clinical Chemistry and Pharmacology, University of Bonn, Bonn, Germany *These authors contributed equally to this work.

Herpetiform pemphigus with anti-Dsg 1 and full-length BP180 autoantibodies

ejd.2012.1643 Auteur(s) : Yuta Kurashige1 kurasige@tokyo-med.ac.jp, Yoshihiko Mitsuhashi1, Masuyoshi Saito1, Shunpei Fukuda2, Takashi Hashimoto2, Ryoji Tsuboi1 1 Department of Dermatology, Tokyo Medical University, 6-7-1 Nishishinjuku Shinjuku-ku, Tokyo 160-0023, Japan 2 Department of Dermatology, Kurume University, Kurume, Japan Herpetiform pemphigus (HP) has been considered an unusual clinical variant of pemphigus. HP shows vesicles arranged on the periphery of annular erythemas in a manner similar [...]