杉浦 一充

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

Background: IL36RN encodes the IL-36 receptor antagonist (IL-36Ra), and loss-of-function mutations in IL36RN define a recessively inherited autoinflammatory disease named "deficiency of IL-36Ra" (DITRA). DITRA causes systemic autoinflammatory diseases, including generalized pustular psoriasis (GPP), an occasionally life-threatening disease that is characterized by widespread sterile pustules on the skin, fever and other systemic symptoms. GPP can present at any age, and provocative factors include various infections, medicines and pregnancy. Objective: We aimed to elucidate the role of toll-like receptor 4 (TLR4) signaling in DITRA and to innovate an efficient treatment for DITRA. Methods: We generated Il36rn(-/-) mice and treated them with TLR4 agonist to establish DITRA model mice. Furthermore, we administrated TLR4 antagonist TAK-242 to the model mice to inhibit the DITRA symptoms. Result: II36rn-1 mice treated by TLR4 agonist showed autoinflammatory symptoms in skin, articulation and liver. Thus, we established model mice for DITRA or GPP that show cutaneous, articular, and hepatic autoinflammatory symptoms typical of DITRA or GPP: sterile pustules on the skin, liver abscesses and enthesitis of the hind paws. Additionally, these symptoms were canceled by TAK-242 administration. We demonstrated the inhibitory effects of the TLR4 antagonist TAK-242 on the autoinflammatory symptoms exhibited by the DITRA models. Conclusion: We suggested that blockage of TLR4 signaling is a promising treatment for DITRA and GPP. (C) 2017 Elsevier Ltd. All rights reserved.

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

© 2016 Japanese Dermatological Association Pili torti is an extremely rare hair phenotype characterized by short length of hairs with hair shafts being easily broken. However, the mechanism of fragility in pili torti is unclear. In this study, we examined the underlying morphological features responsible for pili torti formation using transmission electron microscopy (TEM). We used pili torti samples from a patient with Björnstad syndrome and normal hairs from a healthy subject as a comparison. The macroscopic morphological features of the samples agreed with the results of a previous study showing that pili torti is twisted, flattened, thin and with partial trichorrhexis. Young's modulus of the samples was lower than that of normal hairs. Because the cross-sectional area of the pili torti samples was also smaller than that of normal hairs, it was clarified that the tensile strength of pili torti is 2.1-times lower than that of normal hair. Assessment of morphological features by TEM showed that the cuticle layers of the samples had wavy shapes with different thicknesses. Additionally, the cortex in the samples showed loose keratin intermediate filaments (IF). Our results suggested that these abnormalities in pili torti had already occurred below the infundibulum. Thus, the weakness of pili torti in tensile strength is thought to result from loose IF because of dysformation of disulfide bonds.

© 2017 British Association of Dermatologists Patients with deficiency of interleukin-36 receptor antagonist (DITRA), due to mutation of IL36RN, exhibit psoriatic phenotypes, typically generalized pustular psoriasis (GPP). We report a paediatric patient with DITRA, whose cutaneous lesions varied from psoriasis vulgaris in infancy to annular pustular psoriasis with acute exacerbation to GPP at 13 years of age. Conventional systemic treatments for GPP, which include oral retinoids, ciclosporin and methotrexate, are controversial in paediatric cases, because of their adverse effects and uncertain long-term consequences. Granulocyte monocyte apheresis, a process associated with few adverse events, promptly controlled the GPP of our paediatric patient, and has potential as a suitable alternative treatment for paediatric patients with DITRA.

IMPORTANCE We found CARD14 mutations (2 de novo novel mutations and another previously reported mutation) in 3 of 3 patients with pityriasis rubra pilaris (PRP) type V, but not in patients with PRP of other types. Our findings, combined with the published literature, suggest that type V PRP, both familial and sporadic, can be caused by CARD14 mutations. Detailed clinical observation revealed that all 3 patients displayed unique patchy macular brown hyperpigmentation. OBJECTIVE To further determine how often patients with PRP have pathogenic mutations in CARD14 and to elucidate which clinical subtype of PRP is caused by CARD14 mutations. DESIGN, SETTING, AND PARTICIPANTS We sequenced the entire coding regions of CARD14 in genomic DNA from patients with 5 clinical subtypes of PRP. The detailed clinical features were analyzed in all the patients. The pathogenicity of each mutation was evaluated by several computational predictions. PRP was classified into 6 subgroups, types I to VI, based on clinical criteria. We categorized all the patients with PRP into the clinical subtypes using the classic PRP classification; 22 cases of PRP with varying subtypes were studied. MAIN OUTCOMES AND MEASURES The prevalence of CARD14 mutations in each subtype of PRP was evaluated. Clinical features and characteristics of patients with PRP with CARD14 mutations were analyzed. RESULTS Overall 22 patients with PRP were included in our study (12 men, 10 women; mean [SD] age, 26 [18] years). Among 3 patients with PRP type V, all were found to have CARD14 mutations: 2 de novo novel mutations (p. Cys127Ser and p. Gln136Leu), and another previously reported mutation (p. Gly117Ser). All were close to the reported pathogenic domains. In silico analysis of all 3 mutations suggested that they are functionally relevant to pathogenesis. All 3 patients displayed unique patchy macular brown hyperpigmentation additionally to other typical features of PRP. Patients with PRP type I and type IV, 1 patient each, had the rare variants in CARD14. CONCLUSIONS AND RELEVANCE Pityriasis rubra pilaris type V is a distinct variant of PRP that is caused by CARD14 mutations. In addition, a rare variant of CARD14 might also be implicated in the pathophysiology of other forms of PRP.

Darier's disease (DD, keratosis follicularis: OMIM#124200) is an autosomal dominant skin disorder characterized by multiple dark brown keratotic plaques and warty papules covered by thick crusts. Most cases of DD are caused by mutations in ATP2A2, which is expressed in both the skin and the brain. ATP2A2 encodes the cardiac muscle SERCA2a protein and the ubiquitously expressed SERCA2b. SERCA2 plays an important role as a calcium pump. It is thought that a mutation in ATP2A2 causes dyskeratosis and abnormality of cell-cell adhesion. Here, we report five DD patients from five independent families who presented or were referred to the Nagoya University Hospital in the past five years. We detected five mutations in ATP2A2, including a previously unreported mutation. We observed no apparent genotype/phenotype correlation between types and sites of the ATP2A2 mutations and DD phenotypes in the present series of DD patients. Genetic diagnosis from ATP2A2 mutation search is useful for the definite diagnosis of DD, although it is difficult to predict the severity and prognosis of skin symptoms from the results of the ATP2A2 mutation analysis in DD patients.

© 2015, Springer Science+Business Media New York. Dermatomyositis (DM) is a common idiopathic inflammatory myopathy. The pathogenesis is considered to be microangiopathy affecting skin and muscle. The cutaneous manifestations of DM are the most important aspect of this disease, and their correct evaluation is important for early diagnosis. The skin signs are various: Some are pathognomonic or highly characteristic, and others are compatible with DM. Recently, DM has been categorized into several disease subsets based on the various autoantibodies present in patients. Sometimes, characteristic cutaneous manifestations are strongly associated with the presence of specific autoantibodies. For example, anti-Mi-2 antibody is associated with the classic features of DM, including heliotrope rash, Gottron’s papules, the V-neck sign, the shawl sign, cuticular overgrowth, and photosensitivity. Frequent cutaneous features in anti-transcriptional intermediary factor 1 gamma (TIF1γ)-positive patients are diffuse photoerythema, including “dusky red face,” while skin ulcerations, palmar papules (inverse Gottron), diffuse hair loss, panniculitis, and oral pain and/or ulcers are sometimes associated with anti-melanoma differentiation-associated gene 5 product (MDA5) antibody. Here, we review important cutaneous manifestations seen in patients with DM, and we examine the relationship between the skin changes and myositis-associated autoantibodies. Correct evaluation of cutaneous manifestations and myositis-associated autoantibodies should help the clinician in the early diagnosis of DM, for a quick recognition of cutaneous signs that may be the symptom of onset before muscle inflammation.

© 2016 The Authors. Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders characterized by joint and skin laxity and tissue fragility (1). Dermatan 4-O-sulfotransferase-1 (D4ST1) deficiency, a recently delineated form of EDS caused by bi-allelic loss-offunction mutations in the carbohydrate sulfotransferase 14 gene (CHST14), is clinically characterized by multiple congenital malformations (craniofacial abnormalities, multiple congenital contractures, congenital heart/eye/ gastrointestinal defects) and progressive fragility-related manifestations (skin hyperextensibility and fragility, large subcutaneous haematomas, recurrent dislocations, progressive skeletal deformities) (2). Biochemical and pathological investigations on patients’ skin specimens suggest multisystem fragility caused by impaired assembly of collagen fibrils resulting from dermatan sulphate (DS) depletion in the decorin glycosaminoglycan (GAG) side chain (2). The disorder is currently called “EDS musculocontractural type 1” (MIM#601776) or “D4ST1-deficient EDS” (2). We report here a 45-yearold Japanese woman with the disorder.

Background: Harlequin ichthyosis (HI), one of the most severe genetic skin disorders, is autosomal recessively inherited. Mutations in ABCA12, which encodes ATP-binding cassette transporter A12 (ABCA12), are known to be the cause of HI. It is very difficult to make precise genetic diagnosis when an exon deletion mutation overlaps the site of another causative point mutation. This combination of mutations may lead us to conclude incorrectly that the patient has the point mutation homozygously, a phenomenon called "apparent homozygosity". Objective: To demonstrate that the present HI patient has apparent homozygosity in ABCA12 mutations. Methods: We performed direct sequencing of gDNA in the entire coding region, including exon-intron boundaries, of ABCA12 in the HI patient and her parents. To further elucidate the mutations in the patient, parental mutation segregation study was done and SNP analysis was conducted on the region flanking ABCA12 in the patients and her parents. Quantitative PCR of gDNA in exon 11 of ABCA12 was also performed. Direct sequencing of cDNA from exon 9 to exon 13 and of gDNA between intron 9 and intron 11 of ABCA12 was done in the HI patient and her parents. AResults: Direct sequencing of gDNA in the entire coding region, including exon-intron boundaries, of ABCA12 seemed to indicate that the patient had the novel homozygous nonsense mutation c.1216A>T (p.Lys406X) in exon 11. However, mutation segregation analysis, SNP analysis, qRTPCR of gDNA in exon 11 of ABCA12 and direct sequencing of cDNA from exon 9 to exon 12 of ABCA12 and of gDNA between intron 9 and intron 11 of ABCA12 in the HI patient and her parents demonstrated that the present patient was compound heterozygous for two ABCA12 mutations: c.1216A>T (p.Lys406X) in exon 11 and g.111346_113217del1872 (p.Leu355_Lys428del, Gln354fs7*) which was overlapping exon deletion mutations involving exons 10 and 11. Conclusion: When direct sequencing indicates that a patient from a non-consanguineous family has an apparently homozygous non-founder point mutation, the homozygosity may be "apparent homozygosity", and we should keep in mind the possibility of overlapping exon deletion mutation. (C) 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Lupus erythematosus profundus (LEP), which is a variant of chronic cutaneous lupus erythematosus (CLE), is seen in approximately 2 +/- 3% of CLE patients, and only 10% to 20% of LEP patients present with systemic LE (SLE). LEP shows subcutaneous nodules with or without discoid LE (DLE). Linear LEP, a very rare variant of LEP, was first reported in 1991 in Japanese and in 1998 in English. Since LEP sometimes leaves skin depressions or scars as a result of atrophy of adipose tissue, early and adequate treatments are necessary. Here, we introduce an LEP case in which magnetic resonance imaging (MRI) was quite effective in evaluating a lesion that had been considered to be linear DLE.

Research on the molecular genetics and pathomechanisms of autosomal recessive congenital ichthyosis (ARCI) has advanced considerably and several causative genes and molecules underlying the disease have been identified. Three major ARCI phenotypes are harlequin ichthyosis (HI), lamellar ichthyosis (LI), and congenital ichthyosiform erythroderma (CIE): Skin barrier defects are involved in the pathogenesis of ARCI. In this review, the causative genes of ARCI and its phenotypes as well as recent advances in the field are summarized. The known causative molecules underlying ARCI include ABCA12, TGM1, ALOXE3, ALOX12B, NIPAL4, CYP4F22, PNPLA1, CERS3, and LIPN. It is important to examine genetic associations and to elucidate the pathomechanisms of ARCI to establish effective therapies and beneficial genetic counseling. Next-generation sequencing is a promising method that enables the detection of causative disease mutations, even in cases of unexpected concomitant genetic diseases. For genetic diagnosis, obtaining mRNA from hair follicle epithelial cells, which are analogous to keratinocytes in the interfollicular epidermis, is convenient and minimally invasive in patients with ARCI. We confirmed that our mRNA analysis method using hair follicle samples can be applied not only to keratinization disorders, but also to other genetic diseases in the dermatology field. Studies that suggest potential next-generation therapies using ARCI model mice are also reviewed. (C) 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.

Generalized pustular psoriasis (GPP) is characterized by sudden fever and extensive erythema with pustules and occurs in patients with or without preceding psoriasis vulgaris. We report an 83-year-old man showing irregularly shaped erythema with pustules on the trunk and extremities. He initially had no fever and came to our clinic a few days after the onset of the skin lesions because of high fever and general malaise. We found an extension and new development of erythema and pustules on the whole body. The patient also manifested night delirium. Histological examination revealed neutrophil infiltration into the upper epidermis, which formed a spongiform pustule of Kogoj. Pustular fluid cultures were negative for bacteria. We diagnosed GPP without preceding psoriasis vulgaris. Mutation analysis revealed no significant mutations in IL36RN and CARD14. Previous reports indicated that onset of GPP at the age of 83 years is definitely rare. In older individuals, general disease characteristics include an atypical clinical course, an especially slow appearance and cure, and mental disorder. Our case also revealed such characteristics. Thus, it is necessary to be aware of the clinical course and mental problems in elderly patients with GPP.

Background: Anticentromere antibodies (ACAs) have been observed in patients with autoimmune hepatitis (AIH) and hepatitis C virus (HCV)-related chronic liver disease (CLD-C) as well as those with primary biliary cirrhosis (PBC). However, little is known about the differences in immune responses to the centromere proteins among these liver diseases. Objective: By synthesizing recombinant proteins consisting of the N- and C-termini of major centromere proteins, we investigated the humoral responses against them in each disease. Results: Eight of the 754 (1%) patients with CLD-C, 14 of the 57 (25%) patients with PBC and six of the 38 (16%) patients with AIH were seropositive for ACAs. There were no significant differences in ACA titers determined by an indirect immunofluorescent method among the groups of patients with CLD-C, PBC and AIH. However, the analysis of immunoreactivities against each recombinant protein revealed that the titers of IgG-subclass autoantibodies against the C-terminus of centromere protein (CENP)-B were significantly higher in the CLD-C patients than in the AIH patients. Likewise, the titers of IgM-subclass autoantibodies against the N-terminus of CENP-A were significantly higher in the PBC group than in the CLD-C group. The ACA-positive patients who developed liver cirrhosis had significantly higher titers of the IgA-subclass autoantibodies against the C-terminus of CENP-C than those who did not. Conclusion: These findings suggest that immunoreactivities against the fragments of centromere proteins show distinct patterns among CLD-C, PBC and AIH and that the determination of immunoreactivities against the centromere proteins may be useful for the prediction of disease progression. (C) 2014 Elsevier Masson SAS. All rights reserved.

IMPORTANCE Acute generalized exanthematous pustulosis (AGEP) is a rare and severe type of drug eruption. Dihydrocodeine phosphate is a semisynthetic opioid analgesic. Recently, recessive mutations in IL36RN have been identified in generalized pustular psoriasis (GPP). To date, 4 cases of AGEP and IL36RN mutation without previous history of psoriasis vulgaris (PV) have been reported. OBSERVATIONS A woman in her 60s with PV presented with diffuse erythema, nonfollicular pustules, and fever. She had been treated with dextromethorphan hydrobromide hydrate, amoxicillin hydrate, clarithromycin, dihydrocodeine phosphate, tipepidine hibenzate, and tulobuterol tape for a cough and common cold. Based on histopathologic results and a positive result in a drug provocation test with dihydrocodeine phosphate, she was diagnosed with AGEP. A heterozygous IL36RN mutation c.28C>T (p.ArglOX) was also confirmed by mutation analysis. CONCLUSIONS AND RELEVANCE This is the first report of dihydrocodeine phosphate-induced AGEP. In this case, helper T cells, type 17, might have been activated because of morphine and underlying PV, followed by increased production of interleukin (IL) 36. However, because of the IL36RN mutation, IL-36 signaling was uncontrolled, which might have resulted in the occurrence of AGEP. An IL36RN mutation might underlie several different pustular skin eruptions, including AGEP and GPP, and further accumulation of patient data is required.