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Background: Apremilast, a selective inhibitor of the enzyme phosphodiesterase 4, is efficacious for psoriasis. However, detailed in vivo effects of apremilast on psoriasis remain to be elucidated., Objective: To examine the in vivo effects of apremilast on psoriasis., Methods: Psoriasiform dermatitis was induced by applying imiquimod (IMQ) on the murine shaved back skin for six days. Mice were treated with apremilast or vehicle intraperitoneally daily., Results: Apremilast alleviated IMQ-induced psoriasiform dermatitis clinically and pathologically on days 3-6 by reducing infiltration of antigen-presenting cells and interleukin (IL)-17A-positive cells and increasing infiltration of Foxp3-postive cells into the skin on day 6, although a significant increase in IL-10 mRNA level was not observed on day 2. In addition, mRNA expression of IL-17A, IL-17F, and IL-22 was lower in the skin of IMQ-applied mice treated with apremilast than in those without apremilast on day 2, and apremilast inhibited infiltration of IL-17A-producing γδ T cells into the dermis on day 6. Furthermore, apremilast induced regulatory T cells and regulatory B cells in the spleen but not in the draining lymph nodes., Conclusion: Apremilast downregulated IL-17 production and induced splenic regulatory B cells and regulatory T cells in an IMQ-induced psoriasiform dermatitis mouse model., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: A novel polyomavirus, the Merkel cell polyomavirus (MCPyV) has been implicated in the pathogenesis of Merkel cell carcinoma (MCC); however, the prevalence of MCPyV in Japan has not been extensively investigated., Objective: To clarify the prevalence of MCPyV in Japanese patients with MCC., Methods: MCPyV DNA was examined by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples from 26 patients with MCC diagnosed in four medical centers in Japan. Immunohistochemistry was simultaneously performed using a monoclonal antibody against the viral large T (LT) antigen. FFPE samples from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) were also analyzed as controls., Results: Twenty-three out of 26 cases (88.5%) were positive for MCPyV DNA by PCR. The amplified products harbored 4 patterns of mutations. Phylogenetic analysis demonstrated that one of our strains was closely related to the other Japanese strains previously reported. The LT antigen was expressed in various degrees in 20 of 26 cases (76.9%) by immunohistochemistry. Histological type had little relation to CM2B4 positivity, whereas 3 of 5 trabecular-type tumors showed no staining. The immunoreactivity for CM2B4 did not correlate with the relative viral DNA load. In BCC and SCC, the LT antigen was immunohistochemically positive, but MCPyV DNA was not detected by PCR. The cells around some MCC and non-MCC tumors were stained with CM2B4 with a distribution similar to CD20- and CD45RO- (especially CD8-) positive lymphocytes., Conclusion: MCPyV was highly positive in Japanese patients with MCC. It is of note that the positive rate differs depending upon the detection method., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Clouston syndrome is a rare autosomal dominant condition characterized by hypotrichosis, nail dystrophy, and occasionally palmoplantar keratoderma. The disease is caused by mutations in GJB6 gene, which encodes a gap junction protein connexin 30 (Cx30)., Objective: To disclose the molecular basis of Clouston syndrome in a Lebanese-German family, and also to determine precise expression of Cx30 in normal skin of humans and mice, as well as transcriptional regulation for the GJB6 expression., Methods: We searched for mutations in the GJB6 gene using DNA of the family members with Clouston syndrome. We performed immunostaining to localize the Cx30 expression in normal human skin and mouse embryos. In addition, we did a series of in vitro studies to investigate if the GJB6 could be a direct transcriptional target gene of p63., Results: We identified a recurrent heterozygous mutation c.31G>C (p.Gly11Arg) in the GJB6 gene in the Lebanese-German family with Clouston syndrome. Immunostaining in normal human skin sections demonstrated predominant expression of Cx30 in hair follicles, nails, and palmoplantar epidermis, which partially overlapped with p63 expression. We also showed co-expression of Cx30 and p63 in developing mouse hair follicles and nail units. In cultured cells, the GJB6 expression was significantly upregulated by ΔNp63α isoform. Further in vitro analyses suggested that ΔNp63α was potentially involved in the GJB6 expression via binding to the sequences in intron 1 of the GJB6 gene., Conclusion: Our data further underscore the crucial roles of Cx30 in morphogenesis and development of skin and its appendages., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Survival analysis of a large series of patients with mycosis fungoides (MF) and Sézary syndrome (SS) has not been performed in Japan. Revision to the staging system for MF and SS was recently published., Objective: To determine the long-term prognosis of Japanese patients with MF and SS, to identify factors predictive of survival, and to evaluate the prognostic significance of the revised staging system., Methods: We performed a retrospective cohort study of 100 Japanese patients with MF and SS managed at the dermatology division of Niigata University Hospital between April 1, 1982 and March 31, 2006. We estimated survival according to the patient's clinical characteristics including the stages, and assessed their prognostic significance., Results: Survival rates of Japanese patients with MF and SS were similar to those shown in previous studies conducted in the United States and Europe. Prognosis of patients with skin tumor (stage IIB) and extracutaneous involvement (stage IV) was significantly worse than that of those with early-stage disease (stages IA-IIA), but erythrodermic MF patients without blood involvement (stage IIIA) showed excellent survival. Independent prognostic factors in multivariate analyses were higher age and the presence of skin tumor and extracutaneous disease., Conclusion: Patient age and stage are the most important clinical prognostic factors in Japanese patients with MF and SS. The revised staging system is useful for predicting survival of the patients, but at least a subpopulation of stage IIIA patients may have a favorable prognosis., (Copyright 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Dermatophytes are closely related keratinophilic fungal pathogens and are the causative agents of a superficial cutaneous infection called dermatophytosis (ringworm). A lack of gene manipulation techniques has prevented detailed analyses of the mechanisms of host invasion by dermatophytes. We have introduced the tetracycline-regulatable (TR) gene expression system into dermatophytes to facilitate functional analyses of genes essential for growth and virulence. As the TR gene expression system consists of two plasmid vector components, two dominant selectable markers are required for genetic transformation. In dermatophytes, only the hygromycin B phosphotransferase gene (hph) is available as a selectable marker., Objective: We investigated the possibility of G418 resistance as a secondary selectable marker for genetic transformation in dermatophytes., Methods: A series of plasmid vectors carrying the neomycin phosphotransferase gene (nptII) were introduced into the protoplasts of Trichophyton mentagrophytes, one of the most clinically important dermatophyte species, by polyethylene glycol (PEG)-mediated transformation. Transformants were selected on selective medium containing G418 at 300-500 microg/ml., Results: Molecular biological analyses indicated that colonies appearing on the selective medium harbored nptII in their chromosomes. Colonies produced from protoplasts transformed with the enhanced green fluorescent protein (eGFP) gene-T. mentagrophytes cyclophilin cDNA (TmcypB) fusion vector also exhibited GFP fluorescence throughout their mycelia, but accumulation of the GFP-TmCYPB fusion protein in specific intracellular compartments was not observed., Conclusions: This study has provided a new selectable marker for genetic transformation in dermatophytes.

Background: Tenascin-C is a large, hexameric extracellular matrix glycoprotein that is expressed during embryogenesis, carcinogenesis and wound healing. In normal adult human skin the expression level of tenascin-C is low, but levels are elevated in skin tumors and rise significantly in the dermal compartment during wound healing. Although the expression of tenascin-C could be upregulated by inflammatory cytokines, the role of tenascin-C in atopic dermatitis (AD) is still unclear., Objective: To identify genes that plays a role in AD., Methods: We screened for differentially expressed genes in lesional and non-lesional skin of AD patients using DNA microarray. Then we monitored with quantitative PCR the expression of the novel disease related genes in human keratinocytes or pinnae from NC/Nga mice., Results: We found that tenascin-C gene expression was expressed at higher levels in lesional skin compared to non-lesional skin of the patients, whereas it was not upregulated in the skin of psoriatic patients or healthy controls. In human cultured keratinocytes, tenascin-C was markedly upregulated by IL-4 and IL-13, and moderately upregulated by IFN-gamma. Tenascin-C expression was also upregulated in the AD-like skin lesions induced in NC/Nga mice ears by intradermal injection of mite antigen, and this upregulation was inhibited by prednisolone., Conclusion: These results suggest that upregulation of the tenascin-C expression is specific to AD lesions, and that tenascin-C may therefore play a critical role in regulating the underlining inflammatory processes, which are involved in the pathology of AD.

Background: Previous studies indicate that FXIIIa+ proliferative cells are the cells constituting DFs, however, in spite of the high incidence of DFs, there is a little information in the literature regarding ultrastructural characteristics of the FXIIIa+ dendritic cells on DFs., Objectives: In this study, we examined the fine structures and potential heterogeneity among the subgroup of factor XIIIa (FXIIIa) positive dendritic cells consisted of eleven cases of dermatofibroma (DF)., Methods: Immunohistochemical, electron microscopical, and immunoelectron microscopical techniques were utilized., Results: We demonstrated (i) the immunohistochemical labeling of FXIIIa and CD68 in the DFs. The reactivity was stronger in histiocytic lesions than in fibroblastic lesions. On the other hand, the labeling of HHF35 was mainly in fibroblastic lesions. (ii) The fibroblastic and histiocytic cells on DFs displayed the same basic fine structures; moderate or abundant rough endoplasmic reticulum (RER), lipid droplets and/or bundles of myofilaments in varying proportions. Macular adherence connections between neighboring cells were common. (iii) They also showed the similar features to dermal dendritic cells (DDC), which have been well characterized with long cytoplasmic processes, abundant RER, fibronexus-like plaques and pinocytotic vesicles. (iv) FXIIIa expressions were found within the cytoplasm of both fibroblastic and histiocytic cells in association with the nucleus by immunoelectron microscopy. The labeling was stronger in the histiocytic cells and the cells expressing elongated cytoplasmic processes than in the fibroblastic cells., Conclusion: The FXIIIa+ dendritic cells might be an essential cell of DF, and might have potential to develop HHF35+ fibroblastic or CD68+ histiocytic cells, under appropriate stimuli. The FXIIIa+ dendritic cells might be originated from DDC.

Background: Mouse epidermis contains a population of gammadelta T cells, termed dendritic epidermal T cells (DETCs), which uniformly express the invariant Vgamma3 T cell receptor. Certain DETC lines were reported to respond to Gram-negative bacteria in the presence of immobilized anti-CD3 monoclonal antibody or to lipopolysaccharide (LPS) in the presence of B cell lines., Objective: To determine whether DETCs express the primary signaling receptor for LPS, Toll-like receptor (TLR) 4-MD2., Methods: We analyzed expression of TLR4-MD2 in three independent DETC lines as well as in freshly isolated DETCs., Results: All DETC lines expressed TLR4 and MD2 transcripts and TLR4-MD2 protein complex intracellularly, but none expressed TLR4-MD2 on the cell surface. By immunoblotting, only the immature form of TLR4 protein was detected in the DETC lines. The DETC lines did not respond to LPS even in the presence of immobilized anti-CD3 monoclonal antibody. Freshly isolated DETCs and their fetal thymic precursors also lacked cell surface expression of TLR4-MD2, but a small subpopulation of dermal Vgamma3 T cells isolated from croton oil-painted skin expressed TLR4-MD2 on the cell surface. Similarly, Vgamma3 T cells emigrated from organ-cultured epidermis expressed cell surface TLR4-MD2., Conclusions: These results demonstrate that DETCs do not constitutively express cell surface TLR4-MD2, but TLR4-MD2 expression may be up-regulated when DETCs emigrate from epidermis during cutaneous inflammation.

Background: Atopic dermatitis (AD) and psoriasis are common inflammatory skin diseases. Although many reports implicate Th2 cytokines in the pathophysiology of AD and Th1 cytokines in psoriasis, the precise etiology of these diseases remains elusive., Objective: We investigated novel AD- or psoriasis-related genes to further understand the pathogenesis of these diseases., Methods: We performed a comprehensive analysis of mRNA expression in skin biopsies from AD or psoriasis patients using DNA microarrays. Quantitative PCR was then used to monitor the expression of novel disease-related genes in human keratinocytes or pinnae from NC/Nga mice., Results: Levels of mRNA for IDO (indoleamine 2,3-dioxygenase) and kynureninase, enzymes constituting the tryptophan degradation pathway, were found to be upregulated in the skin lesions as compared to the uninvolved skin of patients with AD or psoriasis. Expression of these two genes was induced in human epidermal keratinocytes stimulated with IFN-gamma in vitro. Moreover, in NC/Nga mice, the expression of kynureninase mRNA in the ear skin was induced following development of AD-like skin lesions., Conclusion: The tryptophan degradation pathway may play an important role in the pathophysiology of AD and psoriasis.

Background: Sh3yl1, which contains one Src homology (SH) 3 domain, has been previously identified from mouse skin and considered to play an important role in hair follicle formation by interacting with other proteins., Objective: We performed this study to identify proteins capable of associating with Sh3yl1., Methods: We screened a mouse skin cDNA library using the SH3 domain of Sh3yl1 as bait in the yeast two-hybrid system., Results: We identified a 420-amino acid-long protein containing a proline-rich stretch and five carboxyl-terminal SH3 domains, which we have termed Sh3d19. We confirmed the interactions between Sh3yl1 and Sh3d19 by in vitro binding assays. Northern blot analysis showed that Sh3d19 transcripts in mouse skin were expressed in accordance with the hair cycle. Furthermore, RNA in situ hybridization studies demonstrated that its transcripts were detected predominantly in the medulla cells at the level corresponding to the keratogenous zone of the hair follicles during the mid and late anagen phases., Conclusion: Sh3d19 is a novel adaptor protein that may be involved in the development of medulla cells during the anagen phase.

Epidermal keratinocytes secrete cytokines, chemokines, and anti-microbial peptides in response to various microbial pathogens and their components including lipopolysaccharide (LPS). To identify the receptor(s) involved in the anti-microbial responses of epidermal keratinocytes, we analyzed expression of CD14, Toll-like receptor 2 (TLR2), and TLR4 on cultured normal human epidermal keratinocytes (NHEK). Although CD14 and TLR2 mRNA were expressed in cultured NHEK, only TLR2 was detected on the cell surface. Cultured NHEK did not express TLR4 mRNA or protein. Commercial LPS preparations could stimulate epidermal keratinocytes to produce beta-defensin-2 and IL-8, and the LPS response was inhibited with mAb specific for TLR2, but not for CD14 or TLR4. Repurified LPS and lipid A did not stimulate epidermal keratinocytes, whereas peptidoglycan (PGN) from Gram-positive bacteria and yeast cell wall particle induced beta-defensin-2 and IL-8 production. Thus, cultured NHEK express functional TLR2, but not CD14 or TLR4, and the "LPS" response of epidermal keratinocytes shown in the previous studies might be mediated by TLR2-dependent recognition of non-LPS bacterial components contaminating in commercial LPS preparations. In the normal human skin, however, epidermal keratinocytes expressed both TLR2 and TLR4. Because TLR4 was induced in epidermal keratinocytes by in vitro stimulation with PGN from Gram-positive bacteria, constitutive expression of TLR4 on epidermal keratinocytes in vivo might also be induced by continuous recognition of the resident skin flora containing Gram-positive bacteria through TLR2.