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Background Atopic dermatitis (AD) is a chronic inflammatory skin disease that often cannot be completely controlled by modern medicine. Since multiple factors are intricately involved in the pathogenesis of AD, wide-ranging research is required for further advancement of AD treatment. Epidermal keratinocytes are the forefront to the external environment and play a pivotal role in the initiation of immune reaction against exogenous invasion. Objective Thymic stromal lymphopoietin (TSLP) is a keratinocyte-derived cytokine that induces differentiation and activation of type 2 helper T cells and innate lymphoid cells, cardinal effectors in pathophysiology of AD. We previously reported that ΔNp63, a p53-related molecule, regulates the expression of TSLP receptors and suggested the entity of a potential TSLP autocrine loop in the AD epidermis. In this study, we further explored the significance of p53 family transcription factors in TSLP production from human keratinocytes. Method Expression profile of p73, a p53-related molecule, was evaluated in human AD tissue by immunohistochemistry. In addition, the function of p73 in producing TSLP was investigated with in vitro cultured keratinocytes via molecular biological analysis. Results ΔNp73 was abundantly expressed in the AD epidermis and increased the release of TSLP via NF-κB activation. Furthermore, the Toll-like receptor 3 signal enhanced ΔNp73 expression and thereby induced TSLP expression. Conclusion Our results indicate that ΔNp73 is an additional participant in the mechanism of TSLP production. Amending the aberrant state of keratinocytes, represented by overexpression of ΔNp73, can be a novel therapeutic target of AD.

Cytokeratin 19 fragment 21-1 (CYFRA 21-1) has been used as a tumor marker for several malignancies. However, to date, no studies have assessed whether CYFRA 21-1 could be a useful marker for extramammary Paget's disease (EMPD). The present study aimed to evaluate the significance of CYFRA 21-1 as a serum tumor marker for EMPD progression. Concentrations of serum CYFRA 21-1 and carcinoembryonic antigen (CEA) in 13 cases of EMPD were measured prior to undergoing treatment at Sapporo Medical University Hospital from January 2014 to May 2016. Four of the 13 patients had lymph node metastases at diagnosis, but none had distant metastases. Immunohistochemistry indicated that all 13 primary tumors and four metastatic tumors in lymph nodes were positive for cytokeratin 19. Although none of the 13 patients showed high serum CEA levels, six patients (46.2%) had elevated serum CYFRA 21-1. Furthermore, CYFRA 21-1 was reduced in association with post-treatment tumor reduction in all six patients. Among these six patients, four developed recurrence and metastasis during the follow-up period. CYFRA 21-1 was re-elevated in all four of these patients; however, serum CEA was elevated only in the patient with distant metastasis. These results suggest that CYFRA 21-1 is more sensitive compared with CEA, and can be useful as a tumor marker for evaluating tumor progression and treatment efficacy in patients with EMPD.

Aim To clarify the relationship between programmed cell death ligand 1 (PD-L1) expression in cutaneous squamous cell carcinoma (cSCC) and clinicopathological variables. Methods We examined PD-L1 expression in tumor cells (TCs) and tumor infiltrating immune cells (ICs) in 46 cases of cSCC by immunohistochemistry. In each case, we employed two methods-intensity and proportion scores-to evaluate PD-L1 expression in TCs. For the evaluation of PD-L1 expression in ICs, only the proportion score was used. Association between PD-L1 expression and clinicopathological variables was analyzed using Fisher's exact test. Results High intensity scores in TCs were observed in 18 of the 46 cases (39.1%) and low intensity scores were observed in 28 cases (60.9%). Applying the proportions, using cut-off values of ≥1% and 50%, positive scores in TCs were observed in 36 (78.3%) and 20 cases (43.5%), respectively. PD-L1-positive ICs were observed in 29 (63%) and seven cases (15.2%), using cut-off values of ≥1% and 10%, respectively. The high intensity scores in TCs correlated with lymph node metastasis (P = 0.008) and female gender (P = 0.017), although positive proportions in TCs or ICs were not significantly related to lymph node metastasis. A multivariate analysis showed that high intensity of PD-L1 expression in TCs was an independent risk factor for lymph node metastasis. Conclusions The results suggested that high intensity of PD-L1 expression in TCs is associated with lymph node metastasis in cSCC.

Background Rhododendrol (RD), a skin-whitening agent, is believed to be associated with cases of cosmetics-related leukoderma that have been reported in Japan. Recently, we have shown that RD is catalyzed by tyrosinase to produce putative toxic metabolites RD-catechol and RD-cyclic catechol. Objective To examine the cytotoxicity and production of reactive oxygen species (ROS) in melanocytic cells by RD and its metabolic products. Methods The growth inhibitory effect of RD or its metabolite on the normal human epidermal melanocyte (NHEM) and B16F1 cells was assessed by cell counting or WST assay. ROS production was detected by flow cytometry and confocal microscopy after cells were treated with 2′,7′-dichlorofluorescein and RD or its metabolite. Results Growth of NHEM derived from African American (NHEMb) and B16F1 cells was suppressed by 300 μM or more RD. Growth inhibitory activity of RD (IC50 of B16F1: 671 μM) was weaker than hydroquinone (IC50 of B16F1: 28.3 μM) or resveratrol (IC50 of B16F1: 27.1 μM). Flow cytometric analysis detected ROS production in the NHEMb and B16F1 cells exposed to RD. However, neither RD nor H 2 O 2 increased the subG1 fraction of these melanocytic cells. RD-catechol and RD-cyclic catechol inhibited growth of NHEMb and B16F1 cells much more strongly than did RD. RD-catechol, as well as RD, produced ROS detected by both flow cytometry and immunostaining, while RD-cyclic catechol produced a hardly detectable amount of ROS in B16F1 cells. Conclusions These results suggest that RD exerts the cytotoxicity in melanocytic cells through its oxidative metabolites and that ROS plays a role in RD-mediated cytotoxicity.

Our purpose is to clarify the physiological role of leptin in hair cycle as leptin reportedly causes activation of Stat3, which is indispensable for hair cycling. While hair follicles in dorsal skin of 5-week-old C57/BL6 mice had progressed to late anagen phase, those in dorsal skin of 5-week-old leptin receptor deficient db/db mice remained in the first telogen and later entered the anagen at postnatal day 40, indicating that deficiency in leptin receptor signalling delayed the second hair cycle progression. Next, we shaved dorsal hairs on wild-type mice at postnatal 7 weeks and injected skin with mouse leptin or a mock. After 20 days, although mock injection showed no effect, hair growth occurred around leptin injection area. Human leptin fragment (aa22-56) had similar effects. Although the hair cycle of ob/ob mice was similar to that of wild-type mice, injection of mouse leptin on ob/ob mice at postnatal 7 weeks induced anagen transition. Immunohistochemically, leptin is expressed in hair follicles from catagen to early anagen in wild-type mice, suggesting that leptin is an anagen inducer in vivo. Phosphorylation of Erk, Jak2 and Stat3 in human keratinocytes was stimulated by leptin and leptin fragment. In addition, RT-PCR and ELISA showed that the production of leptin by human dermal papilla cells increased under hypoxic condition, suggesting that hypoxia in catagen/telogen phase promotes leptin production, preparing for entry into the next anagen. In conclusion, leptin, a well-known adipokine, acts as an anagen inducer and represents a new player in hair biology.

A 51-year-old man had been followed up for approximately 10 years at our clinic for persistent itchy eczematous lesions on his limbs and trunk. At the first visit, physical examination revealed itchy lichenificated and eczematous eruptions and many pruriginous papules over his entire body. Laboratory data showed remarkably high levels of immunoglobulin E (20,000 IU/mL). He was diagnosed as having severe atopic dermatitis (AD). Topical treatment with a potent corticosteroid (class I-II) did not significantly decrease the eruptions and occasional administration of a systemic corticosteroid elicited only a subtle response. The pruriginous lesions did not respond at all to topical or systemic corticosteroids. We used semiquantitative reverse transcription polymerase chain reaction to examine the expression of glucocorticoid receptor (GR)alpha and GRbeta mRNA in peripheral blood mononuclear cells (PBMC) of this patient and compared those with six other patients with AD and three normal controls. As a result, the expression of GRalpha was remarkably decreased compared to normal controls and the other AD patients. Therefore, his poor response to corticosteroid was at least partially caused by this GRalpha decrease in the PBMC.

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.

Background We report a case of an urticarial reaction after drinking alcohol beverages. The patient was a 47-year-old man suffering urticarial and anaphylactoid reaction to alcohol for two years. These reactions were observed at every alcohol beverages intake. Case Summary We performed a prick test with diluted ethanol, alcohol beverages and their metabolites (acetaldehyde, acetic acid). Only acetic acid showed a positive result. Oral challenge test with diluted-ethanol caused pruritus and swelling of his lips. An oral challenge test with 8% diluted Shochu (Japanese distilled alcohol from rice or wheat) caused wheals on his upper back. Discussion Only acetic acid, a metabolite of alcohol, induced a positive prick test in the patient with alcohol-induced urticaria. This result was not observed in normal volunteers. An oral challenge test with diluted-alcohol or Shochu showed a positive wheal reaction in a dose dependent-manner which suggests that urticaria seen in this patient might be induced by alcohol-intolerance. However possible allergic reaction to acetaldehyde could not be excluded.

In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and in vitro studies were also performed to examine the functional relevance of ΔNp63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63(lo/-)). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63(lo/-) keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.

Background: The type of earwax-wet or dry-is determined by a single nucleotide polymorphism (SNP)-538G>A (Gly180Arg)-in the ABCC gene (ABCC11), which encodes an ATP-binding cassette domain. Since axillary osmidrosis is closely related to earwax type, some dermatoses may be associated with earwax type. Objectives: We analyzed the association between the prevalence of a series of dermatoses and earwax phenotype. Further, we investigated ABCC11 localization in the skin. Methods: A total of 660 patients who visited Nopporo Dermatology Clinic in Hokkaido were enrolled in this study. The patients were interviewed to determine their earwax type-wet or dry-and a medical practitioner diagnosed dermatoses. The association between the prevalence of dermatoses and earwax type was analyzed statistically. ABCC11 localization in the skin was immunohistochemically evaluated using anti-ABCC11 antibody. Results: Wet earwax type was more prevalent in patients with acne than in those without acne and the odds ratio was 5.36. On the other hand, dry earwax was more prevalent in patients with atopic dermatitis than in those without it, and the odds ratio was 1.86, which was non-significant. In the immunohistochemical staining, ABCC11 was found to be strongly expressed in the sebaceous glands of wet earwax type skin specimen. Conclusion: Wet earwax phenotype is a high-risk factor for acne. Therefore, hyper-secretion of lipids from sebaceous glands elicited by ABCC11 may play an important role in the pathogenesis of acne.

To analyze immunoregulation of autoreactive T cells specific for epidermal skin antigens, we crossed transgenic mice expressing ovalbumin selectively in keratinocytes under the keratin 5 promoter (K5-mOVA) with mice expressing a K(b)-restricted OVA-specific T cell receptor transgene (OT-I). In athymic double-transgenic mice, OT-I cells developed extrathymically and, at 8-12 weeks of age, initiated severe epidermal damage mimicking toxic epidermal necrolysis (TEN). In contrast, euthymic double-transgenic mice showed thymic deletion of OT-I cells, had few of these cells in the periphery, and never developed skin changes mimicking TEN. Adoptive transfer of OT-I cells isolated from euthymic double-transgenic mice induced TEN in athymic K5-mOVA single-transgenic mice. This spontaneous disease in athymic double-transgenic mice was prevented by transferring lymph node cells from euthymic mice, but was not prevented when CD4(+) or CD25(+) cells were depleted from this population. Although purified CD4(+)CD25(+) cells scarcely prevented the skin disease induced by adoptive transfer of OT-I cells, they efficiently prevented the disease when co-transferred with CD11c(+) dendritic cells. These results suggested that thymus-derived regulatory T cells cooperate with CD11c(+) dendritic cells to prevent life-threatening skin damage such as TEN.

The epidermis, which covers the surface of all mammals, serves as a front line of defense against the invasion of pathogenic microbes and acts as a crucial site for innate immune responses. Various antimicrobial molecules are expressed not only on the surfaces of monocytes but also on epithelial cells. β-Defensins, a family of antimicrobial peptides, are produced by several types of epithelial cells, including keratinocytes. However, the induction pathways for β-defensins in keratinocytes are not fully understood. We hypothesized that bacterial components would trigger the expression of β-defensins in keratinocytes through a toll-like receptor (TLR)–MyD88 signaling pathway that plays important roles in innate immunity. Production of TNF-alpha and IL-1 alpha following stimulation with lipopolysaccharide or bacterial lipopeptides was completely abolished in TLR2&TLR4-doubly deficient keratinocytes and in MyD88-deficient keratinocytes. Expression of murine β-defensin was upregulated by bacterial lipopeptides in wild-type keratinocytes, while it was attenuated in TLR2-deficient keratinocytes. To evaluate the in vivo role of TLRs in keratinocytes, we inoculated Staphylococcus aureus into the tail skin from TLR2-deficient mice that had been grafted on the dorsal skin of syngeneic mice. The grafted skin from TLR2-deficient mice resulted in erosion. These studies strongly suggest that the TLR2–MyD88-dependent pathway in keratinocytes is essential for antimicrobial activity in vivo.

Transgenic mice were generated to establish an animal model for T-cell-mediated autoimmune skin disease. A membrane-bound form of OVA (mOVA) was specifically expressed under the control of the keratin 5 (K5) promoter in the epidermal and hair follicular keratinocytes of mice. Syngeneic, wild-type mice rejected the skin grafts of K5-mOVA mice with the generation of OVA-specific CTL. To study the CTL response against K5-mOVA skin, we used OT-I transgenic mice, which produce K(b)-restricted, OVA-specific CD8+ T cells. Accelerated rejection of K5-mOVA skin was demonstrated when transplanted onto OT-I mice. Furthermore, OT-I cells, when adoptively transferred into K5-mOVA mice, underwent activation and vigorous proliferation in the skin-draining lymph nodes. A bone-marrow-reconstitution assay demonstrated that K(b) presentation by bone-marrow-derived cells, but not epithelial cells, was required for this response, indicating that cross-priming was the basis for immunity in this model. Finally, transferred OT-I cells, activated by cross-priming, targeted the skin of K5-mOVA mice, resulting in development of skin lesions that were reminiscent of toxic epidermal necrolysis. We conclude that our system provides a useful model for autoimmune skin diseases and will aid understanding of the pathomechanism of drug eruption, viral exanthema, and graft-versus-host disease.

ejd.2011.1377 Auteur(s) : Reiko Tobita1 reiko.kikkawa@sunny.ocn.ne.jp, Yasuyuki Sumikawa1, Kaoru Imaoka1, Susumu Murata1, Itaru Dekio1, Riruke Maruyama2, Eishin Morita1 1 Department of Dermatology, Shimane University School of Medicine, 89-1 Enya-cho Izumo, Shimane 693-8501, Japan 2 Department of Organ Pathology, Shimane University School of Medicine, 89-1 Enya-cho Izumo, Shimane 693-8501, Japan Lichen scrofulosorum (LS) is a rare tuberculid that usually develops in children and young adults. It [...]

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.