赤松 浩彦

A placebo-controlled, randomized, double-blind, parallel-group, comparative, multicenter study was conducted to investigate the efficacy and safety of benzoyl peroxide (BPO) gel, administrated once daily for 12 weeks to Japanese patients with acne vulgaris. Efficacy was evaluated by counting all inflammatory and non-inflammatory lesions. Safety was evaluated based on adverse events, local skin tolerability scores and laboratory test values. All 609 subjects were randomly assigned to receive the study products (2.5% and 5% BPO and placebo), and 607 subjects were included in the full analysis set, 544 in the per protocol set and 609 in the safety analyses. The median rates of reduction from baseline to the last evaluation of the inflammatory lesion counts, the primary end-point, in the 2.5% and 5% BPO groups were 72.7% and 75.0%, respectively, and were significantly higher than that in the placebo group (41.7%). No deaths or other serious adverse events were observed. The incidences of adverse events in the 2.5% and 5% BPO groups were 56.4% and 58.8%, respectively; a higher incidence than in the placebo group, but there was no obvious difference between the 2.5% and 5% BPO groups. All adverse events were mild or moderate in severity. Most adverse events did not lead to study product discontinuation. The results suggested that both 2.5% and 5% BPO are useful for the treatment of acne vulgaris.

Background: Adipose-derived stem cells (ASCs) are a robust, multipotent cell source. They are easily obtained and hold promise in many regenerative applications. It is generally considered that the function of somatic stem cells declines with age. Although several studies have examined the effects of donor age on proliferation potential and pluripotency of ASCs, the results of these studies were not consistent. Objective: This study tested whether the donor age affects the yield of ASCs from adipose tissue, as well as the proliferation and differentiation potentials of ASCs. Methods: This study used ASCs obtained from adipose tissues of 260 donors (ages 5-97 years). ASCs were examined for individual differences in proliferation, and adipogenic, osteogenic and chondrogenic differentiation potentials in vitro. Characteristics of ASCs from each donor were evaluated by the principal component analysis (PCA) using their potential parameters. Results: Analyses on ASCs demonstrated that adipogenic potentials declined with age, but proliferation, osteogenic and chondrogenic potentials were not correlated with age. Interestingly, in all ASC potentials, including adipogenesis, individual differences were observed. Principal component analysis (PCA) revealed that individual differences became evident in the elderly, and those variations were more prominent in females than in males. Conclusions: This study demonstrated age-related changes in the potentials of ASCs and revealed that the individual differences of ASCs become significant in people over 60 years of age (for females over 60, and for males over 80). We believe that it is important to carefully observe ASC potentials in order to achieve effective regenerative medicine treatments using ASCs. (C) 2017, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271(+) cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time course of wound closure, CD271(+) cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271(+) cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271(+) cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271(-) cells, suggesting that CD271(+) cells play important roles by producing growth factors. This study also investigated dermal CD271(+) cells in patients with chronic skin ulcers. Dermal CD271(+) cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271(+) cells are closely associated with wound healing.

To clarify the relationship between major cutaneous microorganisms (Propionibacterium, Staphylococcus and Malassezia spp.) and acne vulgaris (acne), we examined the microbiota quantitatively in the follicular contents of inflammatory acne and on the facial skin of patients with acne. Fifteen Japanese untreated acne outpatients were studied. The follicular contents from inflammatory acne lesions of the face were collected using a comedo extractor. The skin surface samples were obtained by the swab method from 10 cm(2) of facial skin. The microbiota was analyzed using polymerase chain reaction. The microbiota in follicular contents was similar to that on the skin surface, namely, there were large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. Moreover, the number of Malassezia spp. on the skin surface was correlated with that of inflammatory acne and that in follicular contents. This study clarified that there are large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. in follicular contents. These results suggest the possibility that not only Propionibacterium acnes but also other cutaneous resident microorganisms are related to acne. Particularly, we considered that Malassezia spp. is closely related.

Background: Solar lentigines (SLs) are characterized by hyperpigmented macules, commonly seen on sun-exposed areas of the skin. Although it has been reported that an increase in the number of melanocytes and epidermal melanin content was observed in the lesions, the following questions remain to be answered: (1) Is acceleration of melanogenesis in the epidermis caused by an increased number of melanocytes or the high melanogenic potential of each melanocyte? (2) Why does the number of melanocytes increase? Objective: To elucidate the pathogenic mechanism of SLs by investigating the number, melanogenic potential and proliferation status of the melanocyte lineage in healthy skin and SL lesions. Methods: Immunostaining for melanocyte lineage markers (tyrosinase, MART-1, MITF, and Frizzled-4) and a proliferation marker, Ki67, was performed on skin sections, and the obtained images were analyzed by image analysis software. Results: The expression level of tyrosinase to MART-1 of each melanocyte was significantly higher in SL lesions than healthy skin. The numbers of melanocytes in the epidermis, melanoblasts in the hair follicular infundibulum and melanocyte stem cells in the bulge region were increased in SL; however, no significant difference was observed in the Ki67-positive rate of these cells. Conclusion: The melanogenic potential of each melanocyte was elevated in SL lesions. It was suggested that the increased number of melanocytes in the SL epidermis might be attributed to the abnormal increase of melanocyte stem cells in the bulge. (C) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Systemic sclerosis [scleroderma (SSc)]-associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc-induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose-derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor 1 (TGF-1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte-conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis-like conditions. We concluded that in the skin affected by bleomycin-induced fibrosis, increased TGF-1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.

Hydroquinone (HQ) is a chemical compound that inhibits the functions of melanocytes and has long been known for its skin-whitening effect. According to previous studies, the Tyrosinase (Tyr) activity inhibitory effect and melanocyte-specific cell toxicity are known depigmenting mechanisms; however, details of the underlying mechanisms are unknown. Arbutin (Arb) is also known for its Tyr activity inhibitory effect and is commonly used as a skin-whitening agent. However, the detailed depigmenting mechanism of Arb is also not yet fully understood. Few studies have attempted to elucidate the effects of HQ and Arb on undifferentiated melanocytes. In this study, we examined the effects of HQ and Arb throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell (ESC) culture system to induce melanocytes. The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated, and the late stage of differentiation, in which melanogenesis became active. On the other hand, Arb had no effect on the differentiation of melanocytes, and only suppressed melanogenesis by specifically suppressing elevations in Tyr expression in the late stage of differentiation.

Morphea is a type of localized scleroderma. It is a skin disease involving the development of fibrosis in the dermis and subcutaneous fat tissue beneath without a visceral lesion, and the cause is still unclear. An involvement of epithelial-mesenchymal transition (EMT) has been reported as a cause of tissue fibrosis, but this was mostly observed in pulmonary and hepatic fibrosis, and the involvement of EMT in a skin disease, morphea, has not been studied. Thus, we analyzed the involvement of EMT in skin fibrosis in morphea patients using pathological techniques. Skin lesions of six morphea patients were analyzed (five female and one male patient). As a control, non-light-exposed skin lesions of 11 healthy females were analyzed. Concretely, tissue samples were prepared from these subjects and subjected to immunostaining of transforming growth factor (TGF)-beta 1, alpha-smooth muscle actin (alpha-SMA) and fibronectin, which have been reported to be associated with fibrosis, and Snail1 and E-cadherin, which are considered to be involved in EMT, and expressions of these were analyzed. In morphea patients, dermal expression of TGF-beta 1, alpha-SMA and fibronectin, which are involved in fibrosis, was enhanced, and, at the same time, enhanced expression of Snail1 and reduced expression of E-cadherin, which are involved in EMT, were observed in the dermal eccrine glands. These findings suggested the progression of EMT in the dermal eccrine glands in morphea.

To take advantage of the therapeutic potential of embryonic stem cells (ESCs), it is necessary to regulate their differentiation in response to defined factors. In this study, in order to explore novel molecules that regulate the differentiation of ESCs, we investigated whether collagen hydrolysate, collagen-characteristic amino acids, glycine (Gly), L-proline and trans-4-hydroxy-L-proline (L-Hyp); or dipeptides, proline-hydroxyproline and hydroxyproline-glycine regulate the differentiation of mouse embryoid bodies (EBs). We identified that treatment with collagen hydrolysate or Gly repressed the expression of the mesendodermal markers, Brachyury and Foxa2 in EBs and maintained the undifferentiated state of mESCs in a feeder-free monolayer culture. In contrast, L-Hyp promoted the expression of Brachyury, Mixl1, Gsc and Foxa2 in EBs. And the treatment with L-Hyp promoted cardiac differentiation within EBs, which was proven by the spontaneous contraction of cardiomyocytes and the expression of the cardiac markers, alpha-MHC, MLC-2v and Nkx2.5. Results suggest that L-Hyp is a promising new inducer for reproducible and efficient differentiation of mesendoderm lineages. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

Backgroud: : More effective treatment strategies are needed for the chronic skin ulcers. Recently, it has been reported that clinical application of stem cells improve wound healing. Objective: We aimed to determine the dynamic time-course movement of epidermal stem cell markers especially p75 neurotrophin receptor (p75NTR) and Integrin beta-1 in wound healing process. Furthermore, we also investigated the presence of these markers in human. Methods: Epidermal Integrin beta-1(+) and p75NTR(+) cells were counted in wound healing process in mice. Both cells were also counted in human skin specimen obtained from chronic skin ulcers and healthy controls. Growth factor gene expression levels by purified mouse epidermal p75NTR. cells were also analyzed using real-time RT-PCR. Results: Integrin beta-1(+) and p75NTR(+) cells were proliferated from 3 days after wounding. Reepithelization was completed 7 days after wounding, and the numbers of cells were returned to the baseline levels by 14 days after wounding. Integrin beta-1(+) cells were proliferated in the basal layer, and p75NTR(+) cells were proliferated in the upper layer of epidermis. In human skin, Integrin beta-1(+) and p75NTR(+) cells were 81% +/- 12% and 36% +/- 15% of the basal cells, respectively. In patients with chronic skin ulcers, the percentage of Integrin beta-1(+) cells in the epidermis was identical to healthy controls. Surprisingly, p75NTR(+) cells were significantly decreased in chronic skin ulcer patients (1.2% +/- 2.6%; p < 0.0005) compared to healthy controls. Purified mouse epidermal p75NTR(+) cells expressed higher transforming growth factor-beta2 and vascular endothelial growth factor-alpha transcripts and lower epidermal growth factor transcripts than p75NTR(-) cells. Conclusion: These results suggest that Integrin beta-1(+) and p75NTR(+) cells play an important role in wound healing process, and that p75NTR may be a key molecule and a candidate for new therapeutic target besides preexisting molecules for chronic skin ulcer patients. (C) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future. © 2013 Inoue et al.

We investigated the optimum application for evaluating skin irritation response by using samples of irritants commonly used as additives in cosmetics and other common household products. We studied 47 volunteers (16 men and 31 women). We selected three types of surfactant, one moisturizer, one anti-infective agent and one oil solution. Using Finn chambers on Scanpor tape, we performed the patch test. A total of 0.015 mL of each sample was applied to the Finn chamber. For liquids, circular filter paper was soaked in 0.015 mL of the sample. Samples were placed on the upper back of participants, and closed for 4, 24 or 48 h. A patch application time of 24 h is sufficient to detect primary skin irritation from irritants in cosmetics and other common household products. In addition, we found that skin irritation reactions were strongest at 24 h after patch removal and that the reaction tended to be weaker at 48 h after patch removal. Patch testing to evaluate irritants should be performed by means of a 24-h patch test with a follow-up reading at 24 h after patch removal. An application time of 24 h places less of a burden on patients than a 48-h patch test. © 2013 Japanese Dermatological Association.

A Kampo prescriptions, hochuekkito (HET) has been utilized for treating functional conditions such as general fatigue, compromised state and gastrointestinal motility disorder. Recently, HET has attracted the attention of dermatologists because of its clinically positive effects in atopic dermatitis (AD) treatment. To explain this positive effect of HET, we examined its protective ability against oxidative skin stress using a murine model. The dorsal region of 8-week-old male HR-1 hairless mice, which were raised on a HET (0%, 2% and 10%) mixed diet, was irradiated once with 70 mJ/cm2 of ultraviolet (UV)-B light. After 4 days, transepidermal water loss (TEWL) and stratum corneum water content (SCWC), were determined as a measure of degree of skin dysfunction. To estimate the amount of active oxygen generated, the stratum corneum catalase activity (SCCA) and stratum corneum carbonylated protein (SCCP) content in the tape-stripped stratum corneum samples were measured. We also measured the H2O2 scavenging ability of HET, and analyzed the changes in the expression levels of several inflammation and oxidative stress-related genes in the skin of HET-fed mice. In control mice, exposure to UV-B led to significant increases in TEWL and SCCP and significant decreases in SCWC and SCCA. These UV-B-induced changes were reduced in mice administrated HET, and the reduction was HET dose-dependent. Our results suggested that HET offered a protective effect against UV-B-induced skin damage. We also found that HET had relatively low ability to scavenge H 2O2, and expression level of cyclooxygenase-2 mRNA decreased in HET-fed mouse. © 2013 Japanese Dermatological Association.

UV radiation is a well-known inducer of epidermal pigmentation that is utilized in therapy for vitiligo, one of the skin depigmentation disorders. Although it has been reported that melanocyte stem cells (McSCs) play essential roles in hair pigmentation, the relationship between McSCs and epidermal pigmentation remains unclear. Repetitive UVB irradiation on the dorsal skin of F1 mice of HR-1 × HR/De caused apparent epidermal pigmentation, and it was characterized by increase in the number of melanocytes. Interestingly, differentiation of McSCs into melanoblasts in hair follicles was followed by induction of epidermal melanocyte differentiation. Administration of a neutralizing antibody for Kit receptor that depletes resident melanoblasts could not suppress increased number of melanocytes. UVB irradiation also induced robust expression of Wnt7a as well as Kitl in epidermis, and β-catenin translocation into nucleus in McSCs. Intradermal injection of IWR-1 (inhibitor of Wnt response 1), a chemical inhibitor of β-catenin activation, and small interfering RNA (siRNA) against Wnt7a suppressed increase in the number of epidermal melanocytes. Taken altogether, it was demonstrated that Wnt7a triggered McSCs differentiation through β-catenin activation, and Kitl might induce following migration of melanoblasts to epidermis. These findings will help in developing therapeutic technologies for vitiligo and other pigmentary disorders. © 2013 The Society for Investigative Dermatology.

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 X 10(5) cells) and the Malassezia species (1 X 10(6) cells) were co-cultured, and IL-1 alpha, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and beta-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not beta-1,3-glucan expression.

Malassezia globosa is a major pathogen of Malassezia folliculitis (MF) and the predominant species on human skin. The aim of this study was to clarify the differences between M. globosa and other cutaneous Malassezia species, M. restricta, M. dermatis, M. sympodialis and M. furfur. The optimum growth temperature, effects of compounds of sweat and free fatty acids on growth, and lipase activities of five cutaneous Malassezia species were determined. The growth of M. globosa was promoted strongly by the compounds of sweat and high temperature unlike that of other cutaneous Malassezia species. This result clarified that M. globosa tended to grow actively in summer conditions more than other cutaneous Malassezia species. Furthermore, M. globosa showed high lipase activity. We consider these characteristics of M. globosa to relate to the pathogenesis of MF.

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.