Eiko Ichikawa

Association between polymorphisms in the SPINK5 gene and atopic dermatitis in the Japanese

Atopy, which is characterized by increased levels of immunoglobulin E (IgE) against common environmental allergens, is considered the strongest predisposing factor for asthma and atopic dermatitis (AD). Mutations in the gene encoding serine protease inhibitor Kazal-type 5 (SPINK5) are responsible for Netherton syndrome, a rare skin disorder characterized by greatly elevated IgE levels with atopic manifestations. A recent study of Caucasian AD families showed that maternally derived alleles of the SPINK5 gene are associated with development of AD and asthma, suggesting the parent-of-origin effect for the development of atopic diseases in the SPINK5 gene. We studied the possible association of the SPINK5 gene for the development of atopic diseases by determining the genotypes of five polymorphisms in a Japanese population. Ttransmission disequilibrium tests revealed an association of SPINK5 polymorphisms with AD but not with asthma. Our data indicate that the SPINK5 gene is associated with AD across ethnicities.

Mutation and association analysis of the interferon regulatory factor 2 gene (IRF2) with atopic dermatitis

AbstractInterferon regulatory factor 2 (IRF-2) is a member of a family of transcriptional factors involved in the modulation of cellular responses to interferons (IFNs) and viral infection as well as in the regulation of cell growth and transformation. Irf2 knockout mice show T helper 1 (Th1) cell development defect and spontaneous development of an inflammatory skin disease. To determine if there are any mutations in IRF2 associated with development of atopic dermatitis (AD), we screened for mutations in the 5′ flanking and coding regions of IRF2 in AD patients and control subjects by single-strand conformational polymorphism (SSCP) analysis. We found three mutations in the promoter region ([−829C>T, −830C>T], −684C>T, and −467G>A), one silent mutation in exon 9 (921G>A), and a 10-bp deletion in the 3′ untranslated region (1739[ATCCC]8>6). Among them, the −467G allele and the haplotype of the −467G, 921A, and 1739(ATCCC)8 alleles were transmitted preferentially to AD-affected children (P = 0.02 and P = 0.007, respectively). Our data suggest that IRF-2 plays some role in the development of AD in the Japanese population.

Expression of angiogenic factors in neurofibromas

Abstract:  We studied the expression of angiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor, platelet‐derived growth factor and hepatocyte growth factor) in cutaneous neurofibroma samples from patients with neurofibromatosis‐1. Immunohistochemical staining and the reverse transcribed polymerase chain reaction (RT‐PCR) method demonstrated that vascular endothelial and basic fibroblast growths factor are highly expressed in neurofibroma cells at both the protein and mRNA level. These data suggest that vascular endothelial and basic fibroblast growth factors may contribute to both the angiogenesis and hypervascularity of neurofibromas.

Eccrine syringofibroadenoma in a patient with a burn scar ulcer

A 72‐year‐old woman with a burn scar on the calves of both legs developed an ulcer on her right heel, surrounded by multiple verrucous nodules and plaques. She had experienced similar verrucous lesions on both legs in the burn scar areas. Although the clinical diagnosis was Marjolins ulcer, histologically the ulcer region showed thick fibrous tissue without any atypical epithelial cells. The verrucous lesions were consistent with the diagnosis of eccrine syringofibroadenoma (ESFA). Moreover, an ESFA‐like growth pattern was seen in the elevated margin of the ulcer. Our findings suggest that these lesions developed as a result of reactive eccrine duct hyperplasia followed by skin tissue remodelling.

Immunohistochemical localization of keratins and involucrin in solar keratosis and Bowen's disease

The present study was conducted to determine the patterns of immunohistochemical characterization of keratin (K) and involucrin in solar keratosis and Bowens disease in order to clarify the abnormal differentiation or maturation of the tumor cells in these precancerous epithelial dermatoses. Seventeen human anti-cytokeratin antibodies and an anti-involucrin antibody were used to examine 15 cases of solar keratosis and 18 cases of Bowens disease. Formalin-fixed and paraffin-embedded sections were stained with these antibodies by the avidin-biotin-peroxidase technique. In solar keratosis, keratin and involucrin distribution was similar to that in normal epidermis, whereas in Bowens disease the keratin distribution varied among individual cases. The dyskeratotic cells in Bowens disease showed a reduction or loss of staining with these antibodies, and they were occasionally positive for keratin 19. These observations suggest that there is a difference in keratin and involucrin expression between solar keratosis and Bowens disease and that the atypical cells of Bowens disease exhibit a diversity of differentiation.

Hereditary complement (C9) deficiency associated with dermatomyositis

A 28‐year‐old Japanese woman with hereditary complement (C9) deficiency and dermatomyositis is reported. She had a 3‐year history of facial erythema and a 1‐month history of progressive muscle weakness. Clinical and laboratory findings were suggestive of dermatomyositis; muscle biopsy confirmed an inflammatory myopathy. An unexpected finding, however, was the low titre of serum haemolytic complement (CH50). Treatment with prednisolone resulted in marked clinical improvement but did not affect the CH50 titre. Further investigation revealed a selective and total absence of the ninth complement component (C9), with direct DNA sequence analysis revealing a non‐sense mutation at Arg95 of the C9 gene. This case demonstrates that the muscle lesions of dermatomyositis can occur in the presence of a complement defect that would prevent the formation of the C5b‐9 membrane attack complex.

NF1 gene silencing induces upregulation of vascular endothelial growth factor expression in both Schwann and non-Schwann cells.

Neurofibromatosis type I (NF1) is associated with typical hypervascular tumors, including neurofibroma, glioma, malignant peripheral nerve sheath tumors (MPNST) and glomus tumors. Previously, we and other groups reported that neurofibromas showed high‐level expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor involved in neovascularization. However, the molecular mechanism underlying the upregulation of VEGF in neurofibromas remains unclear. In this study, we examined the effects of Nf1 gene silencing on VEGF expression in Schwann cell and non‐Schwann cell line and the upstream mTOR‐HIF‐1α – VEGF pathway in Schwann cell line. The results indicated that Nf1 gene silencing by lentiviral‐mediated RNA interference resulted in elevated expression of VEGF, HIF‐1α and phosphorylated mTOR at the protein level. The results obtained from Nf1 gene silencing in murine Schwann cell line analogously suggest that NF1 gene haploinsufficiency in human tumor Schwann cells may directly elicit upregulation of VEGF expression without the tumor microenvironment by activation of the mTOR‐HIF‐1α – VEGF pathway. We also showed that interleukin‐6 is upregulated in Nf1 gene knock‐down Schwann cells at the protein level.

Cutaneous malignant fibrous histiocytoma of the face.

An 88‐year‐old Japanese female visited our department with an asymptomatic nodule on the right cheek. Other than treatment for hypertension for 15 years, the patient had been healthy. She had noticed the nodule one year earlier, and it had slowly enlarged to 25 × 25 × 5 mm, with redness, an irregular‐surface with ulcer formation, and was bony‐hard with no tenderness ( Fig. 1 ). There were no regional lymphadenopathies. These clinical features suggested a soft tissue tumor or basal cell carcinoma. Histopathological examinations revealed that the tumor was visible from the upper dermis beneath the flattered and partly eroded epidermis, and showed atypical and pleomorphic tumor cells with large, irregularly shaped nuclei, and variable amounts of cytoplasm ( Fig. 2 ). Some had bizarre nuclei, and occasional mitotic figures. These histopathological findings were suggestive of malignant fibrous histiocytoma. Magnetic resonance imaging showed that the tumor extended from the upper dermis to the subcutaneous tissue, was attached to the right maxillary bone, and involved the right orbit inferior margin. Apparent invasion to the bone was not seen. The tumor was resected and included skin, subcutaneous tissue, lower orbital adipose tissue, and fascia. A skin graft was taken from the thigh to cover the tumor resection. The resected tumor specimen measured 28 × 28 × 15 mm. Although the histopathological examination results were similar to the biopsy results, the tumor cells were arranged in irregular, intertwining bands, known as a storiform pattern in some areas, and focal necrosis was also seen. The tumor cells were markedly atypical with bizarre nuclei. Subcutaneous fat tissue was widely involved, and the tumor extended into the muscle tissue. In addition, the margin was positive for tumor cells in the deeper area of the tumor. Two weeks after the first operation, the patient had developed a small, firm nodule in the center of the skin graft that rapidly enlarged to form a 10‐mm tumor, which was likely the regrowth of the remaining tumor cells. A wide excision was considered, but because of her age and the extensiveness of the excision, containing maxillary bone, alternative treatment was pursued. Radiation therapy was performed five times per week, with a total dose of 60 gray. During the radiation therapy, the tumor enlargement stopped, and a skin ulcer on the grafted skin was made. The tumor started to decrease in size 1 month after the radiation therapy, and it continued to reduce in size until no tumor mass was seen by magnetic resonance imaging. No recurrence and no metastasis have occurred during the 3 years since cessation of the radiation therapy ( Fig. 3 ).

A Case of Cutaneous Malignant Fibrous Histiocytoma

A 54‐year‐old Japanese man with cutaneous malignant fibrous histiocytoma on the back is reported. He not only had a past history of thyroid cancer 1 year prior to the onset of the skin tumor, but also had simultaneous bladder cancer. Despite the early, wide resection, the prognosis was rapid and progressive. Histologically, the primary lesion of the skin tumor was difficult to differentiate from dermatofibrosarcoma protuberans; however, the recurrent and the metastatic lesions changed in appearance.

Effects of Activin A on the Growth of Neurofibroma-Derived Cells from a Patient with Neurofibromatosis Type 1

Activin is a dimeric protein that was isolated from ovarian fluid as a stimulator of follicle-stimulating hormone [1]. Activin is a member of the transforming growth factor ß superfamily and known to be involved in the control of cellular proliferation, differentation and metabolism [1]. Three different isoforms of activin have been identified: activin A (ßAßA), activin B (ßBßB) and activin AB (ßAßB) [2]. Two types of activin receptors are known: type I receptors and type II receptors [3]. Because activin A stimulates fibroblast proliferation [4], it is reasonable to speculate that it may also make some contributions to neurofibroma enlargements in neurofibromatosis type I (NF-1) patients. Follistatin, which binds activin and inhibits activin action [1], may also be used as a therapeutic material, if activin A stimulates neurofibroma-derived cell proliferation much more than control fibroblasts. To investigate whether activin A or its antagonist, follistatin, may be effective for the treatment of neurofibromas in NF-1 patients, we examined the effects of activin A on the growth of neurofibromaderived cultured cells. Cutaneous neurofibromas were taken surgically from a 36-year-old male patient with NF-1 and the neurofibroma tissues were cultured in Dulbeco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (passage 0, P0). Control fibroblasts were also cultured from the surrounding normal dermis of the same patient. Those cells (P2 or P3) were subjected to the following [3H]thymidine incorporation assays. Briefly, neurofibroma-derived cells or control fibroblasts were seeded (1.5 ! 105 cells/well) onto 24-well plates. After a 48-hour culture in serum-free DMEM, the medium from each well was replaced with log dilutions of activin A (10–13 – 10–9 M of activin A, kindly provided by Dr. Yuzuru Etoh, Ajinomoto Co., Yokohama, Japan) in serum-free DMEM. After a further 24-hour culture, those cells were labeled with 1 ÌCi/ml of [3H]thymidine for 2 h. The incorporated radioactivity was measured, using a liquid scintillation system. The results are shown in figure 1. A maximum effect of activin A on neurofibroma-derived cell proliferation was observed at the concentration of 10–12 M, with a 2.5 times higher incorporation than that of cells without activin A. Similarly, the maximum effect of activin A on control fibroblast proliferation was observed at the concentration of 10–11 M, with a 4.5 times higher incorporation than that of cells without activin A. This is very consistent with the previous report that the maximum effect of activin A on human lung fibroblast proliferation was observed at the concentration of 10–11 M [4]. Immunoblot analysis of activin receptor Fig. 1. Effects of activin A on the growth of neurofibroma-derived cells. Neurofibroma-derived cells (