Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Exploring the impact of ovariectomy on hair growth: can ovariectomized mouse serve as a model for investigating female pattern hair loss in humans?

Female pattern hair loss (FPHL), a type of hair disease common in pre- and postmenopausal women, is characterized by thinning of hair to O-type, mainly at the crown. Although a mouse model of this disease has recently been established, its details are still unknown, and thus, warrants further analysis. In this study, 3 week-old and 7- to 8 week-old C57BL/6 female mice were divided into two groups: one group underwent ovariectomy (OVX), while the other underwent sham surgery. In the 3 week-old mice, the dorsal skin was collected at seven weeks of age, while in the 7- to 8 week-old mice, it was collected at 12 and 24 weeks of age. In the former group, both the pore size of the hair follicles (HFs) and diameter of the hair shaft of telogen HFs decreased upon OVX; while in the latter group, these factors increased significantly. Notably, the thickness of the dermis and subcutis increased significantly in the OVX group. It needs to be further elucidated whether OVX mouse could serve as an ideal mouse model for FPHL, but our results upon evaluation of skin thickness indicate that it could be used to establish a novel treatment for non-hair-related diseases, such as post-menopause-related skin condition.

Stress and Nasal Allergy: Corticotropin-Releasing Hormone Stimulates Mast Cell Degranulation and Proliferation in Human Nasal Mucosa.

Psychological stress exacerbates mast cell (MC)-dependent inflammation, including nasal allergy, but the underlying mechanisms are not thoroughly understood. Because the key stress-mediating neurohormone, corticotropin-releasing hormone (CRH), induces human skin MC degranulation, we hypothesized that CRH may be a key player in stress-aggravated nasal allergy. In the current study, we probed this hypothesis in human nasal mucosa MCs (hM-MCs) in situ using nasal polyp organ culture and tested whether CRH is required for murine M-MC activation by perceived stress in vivo. CRH stimulation significantly increased the number of hM-MCs, stimulated both their degranulation and proliferation ex vivo, and increased stem cell factor (SCF) expression in human nasal mucosa epithelium. CRH also sensitized hM-MCs to further CRH stimulation and promoted a pro-inflammatory hM-MC phenotype. The CRH-induced increase in hM-MCs was mitigated by co-administration of CRH receptor type 1 (CRH-R1)-specific antagonist antalarmin, CRH-R1 small interfering RNA (siRNA), or SCF-neutralizing antibody. In vivo, restraint stress significantly increased the number and degranulation of murine M-MCs compared with sham-stressed mice. This effect was mitigated by intranasal antalarmin. Our data suggest that CRH is a major activator of hM-MC in nasal mucosa, in part via promoting SCF production, and that CRH-R1 antagonists such as antalarmin are promising candidate therapeutics for nasal mucosa neuroinflammation induced by perceived stress.

Human epithelial stem cell survival within their niche requires "tonic" cannabinoid receptor 1-signalling-Lessons from the hair follicle.

The endocannabinoid system (ECS) regulates multiple aspects of human epithelial physiology, including inhibition/stimulation of keratinocyte proliferation/apoptosis, respectively. Yet, how the ECS impacts on human adult epithelial stem cell (eSC) functions remains unknown. Scalp hair follicles (HFs) offer a clinically relevant, prototypic model system for studying this directly within the native human stem cell niche. Here, we show in organ-cultured human HFs that, unexpectedly, selective activation of cannabinoid receptor-1 (CB1)-mediated signalling via the MAPK (MEK/Erk 1/2) and Akt pathways significantly increases the number and proliferation of cytokeratin CK15+ or CK19+ human HF bulge eSCs in situ, and enhances CK15 promoter activity in situ. In striking contrast, CB1-stimulation promotes apoptosis in the differentiated progeny of these eSCs (CK6+ HF keratinocytes). Instead, intrafollicular CB1 gene knockdown or CB1 antagonist treatment significantly reduces human HF eSCs numbers and stimulates their apoptosis, while CB1 knockout mice exhibit a reduced bulge eSCs pool in vivo. This identifies "tonic" CB1 signalling as a required survival stimulus for adult human HF eSCs within their niche. This novel concept must be taken into account whenever the human ECS is targeted therapeutically.

A Case of Nevoid Acanthosis Nigricans Successfully Treated with Topical Ketoconazole Plus Urea.

Dear Editor, Nevoid acanthosis nigricans (AN) is a rare form of benign AN that can be mostly found as a solitary lesion distributed along Blaschko's lines (1). It is not associated with any known syndrome, endocrinopathy, drugs, or internal malignancy. Treatments include retinoid, calcipotriol, and laser treatments (2). Herein we report a case of nevoid AN successfully treated with topical ketoconazole plus urea. A 15-year-old woman presented with a 3-year history of asymptomatic plaques on her abdomen that were increasing in size. She had no medical history and no family history and was not obese. Physical examination revealed dark-brownish pigmented plaques on the midline and right side of her abdomen (Figure 1, a). Potassium hydroxide test was negative. Thyroid function test, antinuclear antibody test, and liver and renal function tests were within normal limits. Histological examination of skin biopsy showed hyperkeratosis and papillomatosis with minimal acanthosis and a mild perivascular lymphocytic infiltration in the superficial dermis (Figure 1, b). Some melanophages were observed in the superficial dermis. Based on the clinical features and these histological findings, a diagnosis of nevoid AN was established. Additionally, there were numerous hyphae and spores in the stratum corneum that were confirmed by Grocott staining (Figure 1, c) and periodic acid-Schiff staining (Figure 1, d). Fungal infection was suggested, and the result of a potassium hydroxide test was considered to be pseudo-negative. Topical ketoconazole cream was initially administrated for one month, and the rough surface was markedly improved (Figure 1, e). Subsequently, topical 20 % urea cream was used and the area of skin lesion decreased in size after 6 months (Figure 1, f). We discontinued ketoconazole cream after 2 months. To the best of our knowledge, this is the first case of nevoid AN successfully treated with topical ketoconazole plus urea. Some cases of AN appear to have an associated endocrinopathy (1). However, genetic factors may also play a role in the pathogenesis of AN. It has been reported that mosaic mutation in fibroblast growth factor 3 (FGFR3) is associated with nevoid AN (3). All known mutations in FGFR3 are gain-of-function mutations, and the activity of the FGFR3 signal correlates with the severity of AN. Involvement of fungal infection has not been reported in the pathogenesis of nevoid AN. We did not identify the fungal species in our patient, but Malassezia infection was suggested. In general, potassium hydroxide test can reveal only yeast forms of Malassezia, and pseudo-negative results may often occur. The abundant hyphae and spores in the stratum corneum are a characteristic pathological feature of Malassezia infection, and the obvious effects of ketoconazole may support the Malassezia infection. Since Malassezia is known to promote cytokine production in human keratinocytes (4), an autocrine FGFR3 signal might accelerate the proliferation of keratinocytes such as myeloma cells (5). Urea is the most widely used moisturizer and keratolytic agent, and has been utilized for the treatment of various hyperkeratotic cutaneous diseases. We successfully treated nevoid AN with the combination of topical ketoconazole and urea. This combination therapy may have fewer side-effects than previous reported treatments and could be considered as an optional treatment. Acknowledgment: The patients in this manuscript have given written informed consent to publication of their case details.

Exploration of biomarkers to predict clinical improvement of atopic dermatitis in patients treated with dupilumab: A study protocol.

BACKGROUND: Atopic dermatitis (AD) is a common eczematous skin disorder that profoundly reduces the quality of life due to intractable pruritus. Excellent therapeutic success of the anti-interleukin 4 receptor-α antibody dupilumab in clinical trials and a real-world clinical context indicates the crucial roles of interleukin (IL)-4 and IL-13 in the pathogenesis of AD. Along with the clinical improvement in skin scores and pruritus, dupilumab significantly and progressively reduces and normalizes the upregulated expression of T helper type 2 signatures such as Chemokine (C-C motif) ligand (CCL)17, CCL18, CCL22, and CCL26 in the lesional skin of AD. However, no blood/serum biomarkers are known to predict good or poor outcome in patients with AD treated with dupilumab. METHODS: Patients are at least 18 years of age and have moderate-to-severe AD with Eczema Area and Severity Index (EASI) ≥16, Investigator's Global Assessment ≥3, and body surface area ≥10%. We are going to enroll more than 130 subjects from 18 medical facilities. Clinical objective findings will be evaluated by EASI. Subjective symptoms will be assessed by Patient-Oriented Eczema Measure, Numerical Rating Scale for Pruritus (Pruritus-NRS), Skin Comfort-NRS, and Treatment Satisfaction-NRS. We will measure 18 blood/serum biomarkers including % eosinophils in blood cell count, lactate dehydrogenase, total IgE, soluble interleukin 2 receptor, CCL17, CCL18, CCL22, CCL26, CCL27, IL-13, IL-22, IL-24, IL-25, IL-31, IL-33, thymic stromal lymphopoietin, periostin, and squamous cell carcinoma antigen-2. The clinical evaluation and biomarker sampling will be performed at 0, 2, 4, 8, and 16 weeks of dupilumab treatment. We will also perform proteomic analysis (of roughly 300 proteins) of the patients' sera obtained at 0 and 2 weeks of treatment. The primary endpoint is the association between "baseline levels of 18 biomarkers" and "% change from baseline of EASI at 16 weeks of dupilumab treatment." DISCUSSION: This is the first clinical trial to explore the biomarkers, including potential proteomic markers, most strongly associated with improvement in EASI in patients with moderate-to-severe AD treated with dupilumab for 16 weeks (B-PAD study). A limitation is that we will only enroll Japanese patients.

The Mast Cell-SCF-CB1 Interaction Is a Key Player in Seborrheic Keratosis.

Mast cell (MC) is an important player in the development of skin diseases, including atopic dermatitis, psoriasis, and urticaria. It is reported that MC infiltration and activation are observed around various types of tumors and speculated that MCs play key roles in their pathogenesis. As MCs in human seborrheic keratosis (SK) have not been well investigated, here we focused on the MCs in SK. The number of c-Kit and tryptase-positive MCs was significantly increased around the SK compared with the marginal lesion. Degranulated MCs were also increased around the tumors. Furthermore, MC growth factor, stem cell factor (SCF), expression within the SK was significantly upregulated compared with the marginal lesion. Interestingly, one of the cognitive regulators of SCF expression, cannabinoid receptor type 1 (CB1) immunoreactivity was downregulated within the SK. Our results suggest that MCs play important roles in the pathogenesis of SK and that SCF can be also deeply involved in the development of SKs. Our current results highlight the CB1-SCF-MC interaction as a novel mechanism of SK development and this also will be utilized for developing a novel treatment.

Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Survey of Japanese dermatological vasculitis specialists on cases of cutaneous arteritis (cutaneous polyarteritis nodosa).

We developed a questionnaire to examine the findings of cutaneous arteritis among dermatological specialists experienced in vasculitis as certified by the Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. We sent a questionnaire to 12 dermatological facilities identified through the revised Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. Retrospective data obtained from 84 patients at the 12 dermatological facilities between 2012 January 2016 December were evaluated. The 84 patients were categorized into two groups, a systemic steroid treatment group (group 1, n = 52) and a no systemic steroid treatment group (group 2, n = 32). C-reactive protein in group 1 patients was significantly higher than that in group 2 patients. Frequency of fever, arthritis, myalgia- and peripheral neuropathy in group 1 was significantly higher than that in group 2. We propose that these symptoms could serve as early markers for the transfer from cutaneous arteritis to systemic polyarteritis nodosa. We further suggest that patients who are subsequently associated with cerebral hemorrhage and infarction, who are originally diagnosed as having cutaneous arteritis, could progress to systemic polyarteritis nodosa. The study demonstrated that it is important for dermatologists to detect these findings early in order to establish an accurate diagnosis and a timely treatment.

A Case of Dapsone-induced Mild Methemoglobinemia with Dyspnea and Cyanosis.

Dear Editor, Dapsone is a dual-function drug with antimicrobial and antiprotozoal effects and anti-inflammatory features (1). In dermatology, it is a first choice for conditions such as leprosy, IgA pemphigus, dermatitis herpetiformis, and linear IgA bullous dermatosis, or an adjunctive treatment for, e.g. bullous pemphigoid (BP) and pemphigus vulgaris (1). However, dapsone is associated with some adverse effects, including methemoglobinemia (1). Methemoglobin (MetHb) concentrations of less than 15% usually cause no symptoms in patients with normal hemoglobin concentrations (2). Herein, we report the case of a patient with BP who developed dyspnea because of dapsone-induced methemoglobinemia that was as mild as 4.7%. A 93-year-old man was diagnosed with BP based on skin manifestations (Figure 1, a and b), histopathological findings (Figure 1, c and d), and anti-BP180 NC16A antibody titer determined by chemiluminescence enzyme immunoassay (279 U/mL) 3 years earlier. His comorbidities included diabetes mellitus, chronic heart failure, right pleural effusion, and brain infarction. The patient had been successfully treated with oral prednisolone, so the steroid was tapered to 4 mg/day. The blisters recurred, however, and new ones kept developing even though the prednisolone was increased to 25 mg/day. Dapsone (75 mg/day) was begun as adjunctive treatment, and new blister formation ceased. At one week from dapsone initiation, the patient developed dyspnea, and his oxygen saturation as measured by pulse oximetry decreased to 88% on room air. At presentation, his blood pressure was 118/78 mmHg, the heart rate was 95 beats/minute, and axillary temperature was 36.3 °C. Neurological examination and consciousness findings remained unchanged compared with findings before dyspnea onset. Chest examination showed normal breath and heart sounds, but lip and peripheral cyanosis was present. Blood tests revealed a white blood cell count of 12,920/µl; red blood cells, 370×104/µl; hemoglobin, 11.7 g/dl; and CMV antigenemia (or C7-HRP), negative. Chest CT and echocardiography indicated no remarkable change compared with imaging from one year earlier. Arterial blood gas analysis showed a pH of 7.454, PaO2 63.1 mmHg, PaCO2 35.4 mmHg, HCO3- 24.3 mmol/L, SaO2 92.4%, and MetHb of 4.7%. These findings indicated a saturation gap (difference between SpO2 and SaO2) induced by MetHb. Upon cessation of dapsone, MetHb levels and SpO2 returned to normal and the dyspnea resolved, implicating dapsone in the methemoglobinemia (Figure 1, e). Differential diagnoses were pulmonary disease, heart disease, neuromuscular disease, sepsis, and drug intoxication. These possibilities were ruled out by the physical examination, drug history, vital signs, blood tests, and chest CT and echocardiography. In normal individuals, MetHb levels are less than 1% (2). Healthy patients with normal hemoglobin concentrations develop cyanosis at MetHb level of 15-20%, dyspnea at 20-50%, and coma at 50-70%, and die at more than 70% (2). However, patients with hematologic disease, acidosis, or cardiopulmonary diseases, for example, present with symptoms even with MetHb levels less than 15% (2,3). We inferred that our patient presented with dyspnea even under mild methemoglobinemia because he had anemia, chronic heart failure, and right pleural effusion. The occurrence of dapsone-induced methemoglobinemia with obvious symptoms is rare (1,4). Clinicians should be aware that methemoglobinemia symptoms are influenced not only by MetHb concentrations but also by comorbidities.

Homeostatic Function of Dermokine in the Skin Barrier and Inflammation.

Dermokine is a chiefly skin-specific secreted glycoprotein localized in the upper epidermis, and its family consists of three splice variants in mice and five in humans. To investigate the pathophysiological impact of dermokine, we generated mice deficient for two (ßγ) or all dermokine isoforms (αßγ). Both variants, especially dermokine αßγ-deficient mice exhibited scale and wrinkle formation resembling ichthyosis accompanied by transepidermal water imbalance at the neonatal stage. Several dermokine αßγ-deficient mice died by postnatal day 21 when reared under low humidity. Moreover, the cornified envelope was vulnerable, and skin barrier lipid ceramides were reduced in the epidermis of dermokine αßγ-deficient mice. cDNA microarray and quantitative reverse transcriptase-PCR assays of the epidermis revealed the upregulation of small proline-rich protein and late cornified envelope family members, as well as antimicrobial peptides in the dermokine αßγ-deficient mice. These barrier gene signatures were similar to that seen in psoriasis, whereas recent studies demonstrated that congenital ichthyosis has gene profiles resembling psoriasis. In line with these findings, adult dermokine αßγ-deficient mice exhibited aggravated phenotypes in psoriasis-like dermatitis models but not in allergic dermatitis models. Dermokine may play a regulatory role in inflammatory dyskeratotic diseases, such as congenital ichthyosis and psoriasis, in the crosstalk between barrier dysfunction and inflammation.

Acne keloidalis in an Asian female patient.

Although AK is uncommon in Asians, this diagnosis must be suspected if specific clinical picture is seen. Early treatment is advised to avoid scarring alopecia but to avoid firmness of these plaques. Although we need to further investigate, IL-6 could be a new target for the development of novel treatments.

Re-investigating the Basement Membrane Zone of Psoriatic Epidermal Lesions: Is Laminin-511 a New Player in Psoriasis Pathogenesis?

Psoriasis is a complex chronic inflammatory skin disease characterized by epidermal thickening on the basis of increased keratinocyte proliferation and insufficient apoptosis. Laminins are important components of the basement membrane (BM) and impact on epidermal keratinocyte growth/apoptosis. Although several laminins are involved in the pathogenesis of psoriasis, it is still controversial about the expression patterns of laminin isoforms and which laminins are important in the development of psoriasis. Because laminin-511 and -332 are key BM components in human skin, and laminin-511 stimulates human hair follicle growth, we asked whether the BM zone in psoriasis shows any laminin-related abnormalities. This showed that the BM expression of laminin-511 and -332 was significantly increased within the skin lesion of psoriasis. Immunofluorescence microscopy revealed that laminin-511, -332, and collagen type IV proteins were also significantly increased in psoriasis-like skin lesions of Imiquimod-treated mice. Transmission electron microscopy showed a few gaps of lamina densa, and its thickness was significantly increased. Finally, laminin-511 treatment significantly stimulated the proliferation and inhibited apoptosis of HaCaT cells, while laminin-α5 chain gene knockdown decreased proliferation and induced apoptosis. These phenomenological observations raise the question of whether laminin-511-controlled keratinocyte growth/death may be a previously overlooked player in the pathogenesis of psoriatic epidermal lesions.

Novel splice site mutation in the LIPH gene in a patient with autosomal recessive woolly hair/hypotrichosis: Case report and published work review.

Autosomal recessive woolly hair is a relatively rare hereditary hair disorder characterized by sparse, short, curly hair. This condition is known to be caused by mutations in the LIPH gene, LPAR6 gene or KRT25 gene. In the Japanese population, most patients with autosomal recessive woolly hair carry one of two founder mutations in the LIPH gene, c.736T>A (p.Cys246Ser) or c.742C>A (p.His248Asn). However, occasionally, individuals with this condition carry compound heterozygous mutations, typically one founder mutation and another mutation. In this study, we describe a patient with a compound heterozygous mutation in the LIPH gene at c.736T>A and c.1095-3C>G. The latter mutation created a novel splice site. This was the fourth splice site mutation to be described in the LIPH gene. Furthermore, we performed an in vitro transcription assay in cultured cells, and demonstrated that the c.1095-3C>G mutation led to a frame-shift, which created a premature termination codon at the protein level (p.Glu366Ilefs*7). Finally, we summarized the mutations previously reported for the LIPH gene. Our findings provide further clues as to the molecular basis of autosomal recessive woolly hair.

Sorafenib stimulates human skin type mast cell degranulation and maturation.

BACKGROUND: Sorafenib is a multi-kinase inhibitor for treating advanced hepatocellular and renal cell carcinomas by targeting various types of receptors and signaling molecules, including vascular endothelial growth factor receptors, platelet-derived growth factor receptor, and Raf-1. Sorafenib may cause diverse cutaneous adverse reactions, including hand-foot reaction, facial and scalp eruptions, alopecia and pruritus. However, the mechanism of these adverse effects has not been well-investigated. OBJECTIVE: Mast cells (MCs) are reported to be associated with various types of skin diseases. To investigate the mechanism of sorafenib-induced cutaneous adverse effects, we focused on MCs in situ. METHODS: We evaluated skin samples of organ cultured normal human skin treated with sorafenib using c-Kit, tryptase, and stem cell factor (SCF), Ki-67, and TUNEL immunohistochemistry as well as quantitative real-time polymerase chain reaction to evaluate MC number, degranulation, proliferation, and apoptosis in situ. RESULTS: Sorafenib significantly increased the number and degranulation of skin-type MCs compared with the vehicle-treated control group in situ. However, sorafenib did not affect MC proliferation and apoptosis, suggesting that it stimulated MC maturation from resident precursors. Furthermore, sorafenib increased SCF expression in situ. The increase in MC number by sorafenib was abrogated by co-administration of SCF neutralizing antibody or the phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, but not the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, PD98059. This suggests that SCF is involved in sorafenib-induced MC maturation. In addition, the compensatory upregulation of PI3K-signaling from inhibition of MAPK signaling by sorafenib might stimulate MC maturation in situ. We also evaluated MCs within the skin samples from patients with drug eruptions by sorafenib administration. The total and degranuated MCs number as well as SCF expression was significantly increased compared to healthy individuals. CONCLUSION: Our results contribute to a better understanding of the mechanism by which sorafenib induces adverse cutaneous reactions via activation of skin-type MC degranulation and maturation. This activation appears to be related to PI3K signaling and SCF production, which could be a new targets for treating sorafenib-induced adverse reactions.