赤松 浩彦

Background: In recent years, it has been reported that stem cells exist in the mesenchymal tissues of the bone marrow and adipose. These stem cells are thought to express specific cell surface markers such as CD44, CD54, CD105, CD90, and CD271 and have been confirmed to be pluripotent. Furthermore, although it has been reported that stem cells are also present in the dermis, their cell surface markers and characteristics are not fully understood. Objective: To confirm the presence of stem cells in the dermis and their ability, employing the mesenchymal stem cell markers which have previously been reported as an indication. Methods: We analyzed the percentages of CD44 (+), CD54 (+), CD90 (+), CD105 (+), and CD271 (+) cells in the dermis of neonatal mice (HR-1 mouse) by performing immunostaining and FACS. Secondly, we isolated each type of marker-positive and -negative cells from dermal tissues and evaluated their proliferation potential and their ability to differentiate into adipocytes, osteoblasts, and chondrocytes. Results: According to the immunostaining and FACS results, we confirmed that stem cells that express CD44, CD54, CD90, CD105, and CD271 are present in the dermal tissues of neonatal mice. In addition, when we measured the proliferation and differentiation potentials of each type of marker-positive cells, it was revealed that cells expressing CD54 or CD271 have a high proliferation potential and are able to differentiate into adipocytes, osteoblasts, and chondrocytes. Conclusions: These results indicated that dermal tissues contain stem cells that express CD44, CD54, CD90, CD105, and CD271 which are stem cell markers. More precisely, it was suggested that both CD54 (+) and CD271 (+) stem cells have high proliferation and differentiation potentials. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Obtaining good adherence to acne therapy is a challenge for all dermatologists. We studied 428 acne patients in Japan to determine the likelihood of good adherence and factors associated with medication-taking. This study utilized a simple validated questionnaire to assess risk of poor adherence; information about patient and treatment characteristics was also collected. There was an overall rate of poor adherence in 76% of subjects. Adherence to topical medication was poor in 52% of those treated with a topical agent only (n = 123). Among those taking combination therapies (n = 275), adherence to the topical portion of therapy was poor in 49% of subjects. The likelihood of poor adherence to oral medication was higher, both when administered alone (n = 30, 93% poor adherence) and when given as part of a combination regimen (n = 275, 86%). Factors with an impact on adherence included satisfaction with treatment (p = 0.023) and the experience of side effects (p = 0.027). Patients who felt they had a good understanding of acne and its treatment were more likely to have good adherence. These data suggest that there is significant room for improvement in acne adherence in Japan, as in other areas of the world, and that improved education may enhance adherence. Copyright (C) 2011 S. Karger AG, Basel

Malassezia is a component of normal cutaneous resident microbiota. The aim of this study was to quantitatively clarify the differences in cutaneous Malassezia microbiota in healthy subjects by sex, body part and season. Samples were collected from the forehead, cheek, upper chest and upper back of 20 healthy men and 20 healthy women (average age 32 years) in summer and winter by the swab method. Malassezia DNA was analyzed using a real-time PCR system. As a result, in sex, body parts and season, men, the upper trunk and summer showed the highest total numbers of cutaneous Malassezia species on average. There were also differences depending on the analytical method. The predominant species were M. restricta on the face of men, M. globosa and M. dermatis on the upper trunk of men, and M. globosa and M. sympodialis on the upper trunk of women. This study clarified that the cutaneous Malassezia microbiota of healthy subjects differed by sex, body part and season.

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics. To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4(+) cells and Fzd7(+) cells were different from those of Kit. cells (precursor of melanocytes: melanoblasts). Fzd4(+). and Fzd7(+) cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit(+) cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4(+) and Fzd7(+) cells following Kit cells during differentiation. These results suggested that Fzd4(+) and Fzd7(+) cells were more immature than melanoblasts, therefore raising the possibility that Fzd4(+) and Fzd7(+) cells are MSCs. (c) 2010 Elsevier Inc. All rights reserved.

Background: The existence of multipotent stem cells in subcutaneous adipose tissue has been reported. We previously confirmed that p75 neurotrophin receptor (p75NTR; CD271)-positive cells in subcutaneous adipose tissue possessed multipotency, although changes of the characteristics in p75NTR-positive adipose-derived stem cells (ASCs) with aging remain unclear. Objective: To investigate the effect of aging on p75NTR-positive ASCs. Methods: The number of p75NTR-positive ASCs in subcutaneous adipose tissue of ICR mice aged 3-24 weeks was analyzed by immunostaining and flow cytometry. Subsequently, the cells were isolated and their ability to attach to the cell culture dish, proliferation rate (doubling time) and the expression of senescence-associated beta-galactosidase (SA-beta gal), a cellular senescence marker, were assessed. Age-related changes in the differentiation potential of p75NTR-positive cells in adipogenic, osteogenic, chondrogenic and myogenic lineage were also investigated. Results: The number of ASCs per unit of tissue weight in adipose tissue and the attachment rate of isolated cells decreased with aging. No difference in the cell proliferation rate and the percentage of SA-beta gal-positive cells was detected. Although the efficacy of differentiation into adipogenic and osteogenic lineages slightly decreased with aging, the differentiation potential into chondrogenic and myogenic lineages was not changed. Conclusion: The number of ASCs per unit of tissue weight decreased in aged mice. However, the cells possessed proliferation and differentiation potentials almost equal to those of young mice even though the differentiation potentials showed a tendency of decrease. These results raise the possibility that stem cell functions, self-renewal and multipotency, are maintained regardless of aging. (C) 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH(3)MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AA(center dot)) increased with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AA(center dot) decreased with the application of fullerene. The co-application of AA and fullerene, which suppressed AA(center dot) in vitro, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48 h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AA(center dot) peak (AA(center dot)/H(center dot) > 4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by U-V-B irradiation, and the development of an AA(center dot) inhibitor such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Background: Malassezia is a particularly important factor in the occurrence of atopic dermatitis (AD). Aim: The aim of this study was to quantitatively clarify the Malassezia species isolated from AD patients by gender, body part and analytical method in detail. Methods: The subjects were 20 AD males and 47 AD females. Samples were collected from lesion and nonlesion areas on the face and upper trunk of AD patients. Malassezia DNA was analyzed using a real-time PCR system. Results: The cutaneous Malassezia microbiota in AD patients differed by gender, body part and analytical method. Conclusions: The present results indicate the possibility that the influence of Malassezia antigens is different according to gender and body part. Copyright (C) 2010 S. Karger AG, Basel

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro. However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro. All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro. This experiment clarified that P. acnes influences the differentiation of NHEK in vitro. As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.

Malassezia folliculitis [MF] is caused by the invasion of hair follicles by large numbers of Malassezia cells, but it remains unclear which Malassezia species are involved in the disease. To clarify this situation, Malassezia species isolated from lesions of MF patients were analyzed by both culture and non-culture methods. In addition, Malassezia species recovered from the non-lesion areas of the skin of MF patients and skin samples of healthy subjects were included in this study. The test population consisted of 32 MF patients and 40 healthy individuals. The lesions were obtained using a comedone extractor, while swabs were employed to obtain skin samples from non-lesion areas of the patients and healthy subjects. Malassezia DNA was analyzed using a real-time PCR technique. The detection limit of the culture method was 5 CFU/cm(2) as opposes 50 cells/cm(2) with non-culture procedures. The predominant species recovered from MF lesions were M. globosa and M. sympodialis by culture method analysis, and M. restricta, M. globosa, and M. sympodialis with non-culture methods. These results were in agreement with those found with samples from non-lesion skin areas of MF patients and healthy subjects. This study clarified that MF is caused by Malassezia species that are part of the cutaneous microflora and not by exogenous species.

【目的】ストレスによるアトピー性皮膚炎(以下AD)増悪の機序を明らかにするために，正常マウスとADマウスに慢性ストレスを与え，検討した。<BR>【方法】HR-1マウスとADモデルであるHR-ADfマウスに対し拘束ストレスを2週間与え，皮膚肥満細胞脱顆粒率，皮膚組織中サブスタンスP(SP)を測定した。<BR>【結果】肥満細胞脱顆粒率は両者でストレス負荷後に有意な差で増加した。SPはHR-1ではストレス前後で差はなかったが，HR-ADfではストレス負荷により有意な差で上昇していた。<BR>【結語】慢性ストレスはHR-1マウスと比較し，HR-ADfマウスに対してより多くの皮膚の肥満細胞脱顆粒を引き起こし，皮膚組織中SPを上昇させた。