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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.

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.

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.

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.

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.

研究グループは、ストレスホルモンであるコルチコトロピン放出ホルモン(CRH)が、ヒト鼻粘膜内のアレルギー発症に関わっている肥満細胞の増殖と脱顆粒を誘導することを明らかにしました。今回、本研究グループはCRHが粘膜型肥満細胞を活性化してアレルギー性疾患の病態に関与する可能性を考え、ヒト鼻ポリープ組織培養系を用いた研究を行いました。本研究ではまず、鼻ポリープ組織培養系にCRHを添加したところ、肥満細胞数と脱顆粒が増加したことを発見しました。また、同細胞の増殖因子であるStem cell factor(SCF)の粘膜上皮内での発現の上昇も確認しました。そして、このCRHによる肥満細胞への影響は、CRHR1遺伝子ノックダウン、CRHR1阻害薬またはSCF中和抗体添加によって阻害されました。さらに拘束ストレスモデルマウスを用いた実験も行い、ストレス群のマウス鼻腔粘膜内で肥満細胞数と脱顆粒の増加も確認し、これはCRHR1阻害薬の鼻腔投与で阻害されたことも発見しました。この成果により、CRHR１を介したCRH−肥満細胞のシグナル制御は、ストレス関連の難治性アレルギー疾患の新規治療薬の開発につながることが期待されます。, 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.

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.

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.

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.

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.

Systemic sclerosis (SSc), or scleroderma, is a multisystem autoimmune disorder characterized by vasculopathy and fibrosis in the skin and internal organs, most frequently in the esophagus and lungs. Hitherto, studies on SSc pathogenesis centered on immune cells, vascular cells, and fibroblasts. Although dysregulated keratinocytes in SSc have been recently reported, the contribution of epithelial cells to pathogenesis remains unexplored. In this study, we demonstrated the induction of SSc-like molecular phenotype in keratinocytes by gene silencing of transcription factor Friend leukemia virus integration 1 (Fli1), the deficiency of which is implicated in SSc pathogenesis. Keratin 14–expressing epithelial cell–specific Fli1 knockout mice spontaneously developed dermal and esophageal fibrosis with epithelial activation. Furthermore, they developed remarkable autoimmunity with interstitial lung disease derived from thymic defects with down-regulation of autoimmune regulator (Aire). Importantly, Fli1 directly regulated Aire expression in epithelial cells. Collectively, epithelial Fli1 deficiency might be involved in the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified roles of dysregulated epithelial cells in SSc pathogenesis.

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 (βγ) or all dermokine isoforms (αβγ). Both variants, especially dermokine αβγ-deficient mice exhibited scale and wrinkle formation resembling ichthyosis accompanied by transepidermal water imbalance at the neonatal stage. Several dermokine αβγ-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 αβγ-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 αβγ-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 αβγ-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.

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.

Several studies have suggested that pre-dialysis care is associated with clinical outcomes. However, little has been reported on the influence of pre-dialysis care on the psychological adjustment to dialysis. The purpose of this study was to evaluate the impact of pre-dialysis care on psychological adjustment to dialysis and clinical characteristics. In this cross-sectional study, we enrolled 52 patients who started hemodialysis at our hospital. They were divided into two groups according to the time of referral to our hospital: the early referral group (over 1 year prior to first dialysis: 19 patients, mean age 69.3 ± 11.1) and the late referral group (within 1 year prior to first dialysis: 33 patients, mean age 72.3 ± 8.9). We measured the clinical characteristics and evaluated the psychological adjustment to dialysis by Shontz's stage theory. Compared with the late referral group, the early referral group had a significantly better clinical characteristics concerning blood pressure (140.2 ± 23.7 vs. 156.9 ± 23.3 mmHg, P = 0.0150), hemoglobin (10.3 ± 1.5 vs. 9.4 ± 1.0 g/dL, P = 0.0078), and phosphorus (4.5 ± 1.5 vs. 5.5 ± 1.3 mg/dL, P = 0.0166). In addition, psychological adjustment to dialysis evaluated by Shontz's stage theory was significantly better in the early referral group (P = 0.017). Our results indicate that nephrology pre-dialysis care affects not only blood pressure, anemia, and phosphorus control but also the psychological adjustment to dialysis.

P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.

Hair is one of the smallest organs, but has many important functions to mammals. Hair morphogenesis occurs through the reciprocal exchange of epithelial and mesenchymal signals. There are some reports about the expression of laminin-511 and -332 during hair morphogenesis, but are no reports of the chronological expression and function of laminin-511 and its counter regulator laminin-332 during hair morphogenesis. Our results of immunoblotting revealed that laminin-332 proteins were detected at stage 0 and downregulated during stage 1 to stage 2, and then recovered at stage 3. However, laminin α5 expression was constant throughout stages 0-3. According to the results of semi-quantitative RT-PCR, the mRNA expression of all laminin-332 subunits increased gradually from stage 0 to stage 2, while the mRNA expression of all laminin-511 subunits remained constant from stage 0 to stage 3. Our results suggest that the proper expression of laminin-332 and laminin-511 may regulate appropriate hair morphogenesis.

In this study, we generated a new set of monoclonal antibodies (mAbs) to bovine and human type VII collagen (COL7) by immunizing mice with bovine cornea-derived basement membrane zone (BMZ) fraction. The four mAbs, tentatively named as COL7-like mAbs, showed speckled subepidermal staining in addition to linear BMZ staining of normal human skin and bovine cornea, a characteristic immunofluorescence feature of COL7, but showed no reactivity with COL7 by in vitro biochemical analyses. Taking advantage of the phenomenon that COL7-like mAbs did not react with mouse BMZ, we compared immunofluorescence reactivity between wild-type and COL7-rescued humanized mice and found that COL7-like mAbs reacted with BMZ of COL7-rescued humanized mice. In ELISAs, COL7-like mAbs reacted with intact triple-helical mammalian recombinant protein (RP) of COL7 but not with bacterial RP. Furthermore, COL7-like mAbs did not react with COL7 within either cultured DJM-1 cells or basal cells of skin of a bullous dermolysis of the newborn patient. These results confirmed that COL7-like mAbs reacted with human and bovine COL7. The epitopes for COL7-like mAbs were considered to be present only on mature COL7 after secretion from keratinocytes and deposition to BMZ and to be easily destroyed during immunoblotting procedure. Additional studies indicated association of the speckled subepidermal staining with both type IV collagen and elastin. These unique anti-COL7 mAbs should be useful in studies of both normal and diseased conditions, particularly dystrophic epidermolysis bullosa, which produces only immature COL7.

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.

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway., Author Summary Ectodermal dysplasias refer to a large group of inherited disorders characterized by developmental defects in tissues of ectodermal origin. The study of these conditions has been instrumental in the discovery of biological pathways involved in the regulation of epithelial tissue morphogenesis. In this report, through the delineation of the molecular basis of a novel form of autosomal recessive ectodermal dysplasia, we identified a new key player in ectodermal development. We detected a number of mutations in TSPEAR co-segregating with abnormal hair and tooth development in three families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR was found to be strongly expressed in murine hair and tooth. Using a reporter assay, we showed that it regulates Notch activity. Accordingly, NOTCH1 expression was altered in patient skin, and NOTCH1, as well as many of its known targets, was down-regulated in TSPEAR deficient keratinocytes. Moreover, Tspear silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch pathway. As such, these new data are likely to lead to further investigations aimed at characterizing the role of Notch signaling pathway in other forms of ectodermal dysplasias as well as acquired hair and tooth pathologies.

Background Little is known about the effect of neuropeptides on basophils, which are important effector cells in immune and allergic responses. Objective This study aimed at revealing the role of α-melanocyte–stimulating hormone (α-MSH) on basophil function. Methods Expression of melanocortin receptors and proopiomelanocortin (POMC) was analyzed by means of RT-PCR, Western immunoblotting, fluorescence-activated cell sorting, and double-immunofluorescence analysis. Signal transduction studies included cyclic AMP and Ca 2+ mobilization assays. Basophil activity was assessed based on CD63 surface expression and cytokine release. Results MC-1R expression was detectable in basophils isolated from human peripheral blood, as well as in basophils within nasal tissue. In isolated basophils from human blood, truncated POMC transcripts were present, but there was no POMC protein. Treatment of basophils with α-MSH increased intracellular Ca 2+ but not cyclic AMP levels. α-MSH at physiologic doses potently suppressed basophil activation induced by N-formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate, or grass pollen allergen in whole blood of healthy or allergic subjects, respectively. The effect of α-MSH on basophil activation was MC-1R mediated (as shown by blockade with a peptide analogue of agouti-signaling protein) and imitated by adrenocorticotropic hormone but not elicited by the tripeptides KPV and KdPT, both of which lack the central pharmacophore of α-MSH. Moreover, α-MSH at physiologic doses significantly suppressed secretion of 3 proallergic cytokines, IL-4, IL-6, and IL-13, in basophils stimulated with anti-IgE, N-formyl-methionyl-leucyl-phenylalanine, or phorbol 12-myristate 13-acetate. Conclusion Our findings highlight a novel functional activity of α-MSH, which acts as a natural antiallergic basophil-response modifier. These findings might point to novel therapeutic strategies in treating allergic diseases.

Epidermal nevi are congenital cutaneous hamartomas caused by embryonic somatic mutations. Ultrastructural features of adult epidermal nevi have rarely been investigated. Herein, we report a case involving a Japanese adult who had epidermal nevi with right congenital blindness and a right accessory nipple. The histopathologic and ultrastructural studies showed divergent abnormal epidermal structures in both melanocytes and keratinocytes. Our case indicates the need to further investigate histopathologic, ultrastructural, and genetic associations in adult epidermal nevi.

Hair follicles (HFs) maintain a peripheral, functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis, whose most proximal element is corticotropin-releasing hormone (CRH). The mast cell (MC)-rich connective-tissue sheath (CTS) of mouse vibrissa HFs harbors MC precursors. Differentiation of these MC precursors into mature MCs can be induced by stem cell factor (SCF). We have investigated whether the MC progenitors of normal human scalp HF CTS respond to stimulation with CRH. Microdissected anagen HFs and full-thickness scalp skin were treated with CRH (10(-7) M). CRH treatment induced the degranulation of CTS MCs, in addition to increasing the number of CTS MCs in full-thickness skin and HF organ cultures in situ. In the latter, cells with characteristic MC features emigrated from the CTS. CRH-receptor protein expression in the CTS was colocalized with Kit expression on some CTS MCs in situ. CRH treatment upregulated SCF mRNA and protein expression within the HF epithelium. In skin organ culture, CRH-induced degranulation of CTS MCs was abolished by anti-SCF antibody. We demonstrate that human skin is an extramedullary reservoir for MC precursors, and we have identified a regulatory loop between CRH and SCF signaling. This highlights a previously unpublished finding about neuroendocrine control of human MC biology.

Excessive UVR ranks among the most harmful environmental influences on human skin. However, the direct impact of UVR on human skin appendages remains to be systematically investigated. Organ-cultured human anagen hair follicles in vitro were irradiated, and reduction of hair shaft elongation, premature catagen entry, and reduced hair matrix keratinocyte proliferation were observed upon irradiation with UVB (20/50 mJ cm(-2)). At 20 mJ cm(-2), apoptotic cell death prevailed (casp-3/p53 activation), whereas at 50 mJ cm(-2), necrotic cell death was predominant (lactate dehydrogenase increase). Mitochondrial common deletion and oxidatively damaged genomic DNA (8-OH-dG) was mainly observed at 20 mJ cm(-2). Follicular melanogenesis and ACTH immunoreactivity drastically declined, but alpha-melanocyte-stimulating hormone remained unchanged, whereas transforming growth factor-beta(2) expression shifted from the outer toward the inner root sheath. Both the number of Giemsa+ mast cells and the degree of mast-cell degranulation increased in the connective tissue sheath (CTS), and CD117 immunoreactivity of CTS cells and matrix keratinocytes was upregulated. Thus, UVR differentially modifies hair growth and cycle, promotes cell death, and induces complex regulatory events in human hair follicles in vitro. The leads from this human organ model, which is a living and human tissue interaction system under physiologically relevant in situ conditions, may encourage its use for general investigation of UV-induced effects as well as for testing possible agents for their UV-protective potency.

Treatment with cyclophosphamide and steroids for idiopathic membranous nephropathy (IMN) is effective in Caucasian patients, but the cumulative cyclophosphamide dosage exceeds 10 g and includes steroid pulse therapy. Adverse effects and difficulties with repeating treatment are major limitations. We studied the long-term outcomes of low-dose cyclophosphamide and prednisolone therapy in Japanese patients, who were thought to have relatively benign IMN compared with Caucasian patients.This is a prospective cohort study of 103 consecutive Japanese patients with IMN and nephrotic syndrome. Patients were treated with cyclophosphamide (50 mg/day for the first 3 months and 25 mg/day for the next 3 months) and prednisolone (30 mg/day for the first week and the dosage was gradually tapered to withdraw by 2 years). Additional therapies were allowed for initial treatment failure or relapse.With a mean observation period of 8.5 years, 90 patients (87.4%) achieved proteinuria of1 g/day and 78 (75.7%) achieved complete remission. A total of 27 patients did not respond to initial treatment and 30 patients had relapses after remission. Of these patients, 39 received additional therapies. At the last observation, 12 patients had developed renal insufficiency (S-Cr1.5 mg/dL) but only 2 patients had reached renal death. Multivariate analysis revealed that the duration without remission was the strongest risk factor for renal prognosis. There were 14 deaths, and 8 patients developed cancers during the observation period.Treating nephrotic IMN in Japanese patients with low-dose cyclophosphamide and prednisolone is beneficial for long-term renal prognosis with relatively few adverse effects.

In this report, we describe a patient who contracted fatal cryptococcosis after the induction of hemodialysis. A 76-year-old man was hospitalized to initiate hemodialysis. On admission, clinical findings showed no signs of any infections, and hemodialysis was inducted favorably. On the 6th hospital day he suddenly had a dyspnea and died from acute respiratory failure having a dyspnea for only 6 h. By microscopic examination at autopsy, we detected microemboli in the pulmonary capillary arteries caused by Cryptococcus and that the embolic source was a multiple-abscessed spleen. To the best of our knowledge, this is the first reported case of pulmonary capillary microembolism caused by cryptococcemia.

For many years, extracellular matrix (ECM) was considered to function as a tissue support and filler. However, we now know that ECM proteins control many cellular events through their interaction with cell-surface receptors and cytoplasmic signaling pathways. For example, they regulate cell proliferation, cell division, cell adhesion, cell migration, and apoptosis. We focus in this review on a laminin isoform, laminin-332 (formerly termed laminin-5), a major component of the basement membrane (BM) of skin and other epithelial tissues. It is composed of 3 subunits (alpha3beta3 and gamma3 and interacts with at least two integrin receptors expressed by epithelial cells (alpha3beta1 and alpha6beta4 integrin. Mutations in either laminin-332 or integrin alpha6beta4 result in junctional epidermolysis bullosa, a blistering skin disease, while targeting of laminin-332 by autoantibodies in cicatricial pemphigoid leads to dysadhesion of epithelial cells from their underlying connective tissue. Abnormal expression of laminin-332 and its integrin receptors is also a hallmark of certain tumor types and is believed to promote invasion of colon, breast and skin cancer cells. Moreover, there is emerging evidence that laminin-332 and its protease degradation products are not only found at the leading front of several tumors but also likely induce and/or promote tumor cell migration. Thus, in this review, we focus specifically on the role of laminin-332 and its integrin receptors in adhesion, proliferation, and migration/invasion of cancer cells. Finally, we discuss strategies for the development of laminin-332-based antagonists for the treatment of malignant tumors.

The extracellular matrix (ECM) was long thought to be merely a structural tissue support and/or a filter. However, recent studies have suggested that ECM proteins regulate many intracellular and extracellular events, including cell growth, cell adhesion, cell division, cell movement, and apoptosis. They do so through activation of several families of cell surface receptor, including the integrins and syndecans. The focus of this review is on two laminin isoforms expressed in the skin. Laminins are an important molecular component of the basement membranes in a variety of tissue types. They have a cruciform shape, and are composed of three chains-alpha, beta, and gamma. Keratinocytes of the skin secrete numerous laminin isoforms, including laminin-511 and laminin-332. The latter are known to affect the behaviour of keratinocytes through binding to membrane-penetrating receptors (outside-in signal transduction). Conversely, the expression, secretion and assembly of laminin-rich matrices is regulated by cell surface receptors through inside-out signal transduction. We will review how integrins regulate laminin matrix assembly and the signals elicited by laminins that support either migration or stable adhesion of keratinocytes. We will also discuss recent data indicating that laminins plays key regulatory roles in the development of skin appendages and contribute to the pathogenesis of skin cancer.