Mizuki Goto

Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection

Defensins are small cationic proteins that harbor broad-spectrum microbicidal activity against bacteria, fungi and viruses. This study examines the effects on pathogens of the epidermis engineered to express human beta-defensin 3 (HBD3) to combat bacterial infections. First, we examined the localization of HBD3 in the epidermis and observed HBD3 in the intercellular spaces and lamellar bodies of the upper epidermal layers. This result showed HBD3 expressed and assembled in the outer layers of the epidermis was suspected to counter the invading microorganisms. Next, we established a keratinocyte cell line that stably expressed HBD3 and found that the culture medium showed antibacterial activity. Furthermore, we prepared an epidermal sheet of these cells with the HBD3 gene and grafted this onto a dermal wound on a nude rat. The HBD3 engineered epidermis demonstrated significant antimicrobial activity. Skin ulcers without epidermis are constantly exposed to invading microorganisms. Biopsy samples of re-epithelizing epidermis from patients with skin ulcers were collected, and HBD3 mRNA level measured in the epidermis. The epidermal samples from the ulcer skin expressed 2.5 times higher levels of HBD3 transcript than those in the control skin. These results, taken together, indicate that the therapeutic introduction of the HBD3 gene into somatic cells may provide a new gene therapy strategy for intractable infectious diseases.

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane‐associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1‐positive cells occurs in a caerulein‐induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in “ducts in foci”, a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.

Apocrine hidrocystoma : a case report and analysis of 167 Japanese cases

Correspondence Apocrine hidrocystoma: a case report and analysis of 167 Japanese cases An 81-year-old woman presented with a deep-purple cystic tumor, measuring 15 mm × 15 mm, on her right cheek (Fig. 1). Puncture of the tumor resulted in the extravasation of a clear but straw-colored fluid. The histopathologic examination showed cystic spaces in the mid-dermis, with some papillary projections. The epithelial lining of the cyst was composed of columnar cells showing a “decapitation” secretion indicative of apocrine secretion (Fig. 2). Periodic acid–Schiff-positive, diastase-resistant granules were found in the secretory cells. An outer layer of spindle-shaped cells was partially S-100 protein positive. Although total excision is the most common treatment, this would have been difficult in our patient, who had severe senile dementia. We therefore excised, by scissors, only the roof of the tumor. A faint scar remained on her cheek at her 2-month follow-up visit. Apocrine hidrocystoma (cystadenoma) commonly occurs as a solitary cystic lesion on the head and neck. In 1968, Mehregan described 17 cases of a benign nevoid tumor as apocrine cystadenoma, and noted that the tumor was considered as an adenomatous cystic proliferation of apocrine glands and not a simple retention cyst. The first Japanese case was reported in 1968. So far, several review articles on Japanese cases of apocrine hidrocystoma have been published. Hosaka and Shishiba summarized 141 cases that appeared in the Japanese literature between 1968 and 1998. In the 5 years from 1999 to 2003, 25 new cases were reported in Japan. We counted a total of 167 Japanese cases of apocrine hidrocystoma, including the present case. Of these, 86 occurred in men and 80 in women (one was not described). The age distribution was as follows: < 10 years, four cases; 10–19 years, three cases; 20–29 years, 12 cases; 30–39 years, 28 cases; 40–49 years, 31 cases; 50–59 years, 35 cases; 60– 69 years, 30 cases; 70–79 years, 19 cases; > 80 years, four cases (one was not described). The tumor seems to occur in any age group. In 123 cases (73.7%), the tumor was found on the face or scalp (Table 1). The periorbital location seems to be the favored site. In only 15 cases (9.0%) was the tumor found in apocrine gland-bearing regions, such as the axilla and groin. Multiple apocrine hidrocystomas seem to be uncommon, as reported previously. Only two cases presented

Elimination of Epiplakin by Gene Targeting Results in Acceleration of Keratinocyte Migration in Mice

ABSTRACT Epiplakin (EPPK) was originally identified as a human epidermal autoantigen. To identify the function of epiplakin, we generated epiplakin “knockout” mice. These mice developed normally, with apparently normal epidermis and hair. Electron microscopy after immunostaining revealed the presence of EPPK adjacent to keratin filaments in wild-type mice, suggesting that epiplakin might associate with keratin. The appearance and localization of keratin bundles in intact epidermal keratinocytes of EPPK−/− mice were similar to those in wild-type mice. Wounds on the backs of EPPK−/− mice closed more rapidly than those on the backs of wild-type and heterozygous mice. The outgrowth of keratinocytes from skin explants from knockout mice was enhanced compared to outgrowth from explants from wild-type mice, even in the presence of mitomycin C, suggesting that the difference in keratinocyte outgrowth might be due to a difference in the speed of migration of keratinocytes. At wound edges in wild-type mice, EPPK was expressed in proliferating keratinocytes in conjunction with keratin 6. In EPPK−/− mice, no similar proliferating keratinocytes were observed, but migrating keratinocytes weakly expressed keratin 6. EPPK was coexpressed with keratin 6 in some keratinocytes in explant cultures from wild mice. We propose that EPPK might be linked functionally with keratin 6.

Epiplakin accelerates the lateral organization of keratin filaments during wound healing

BACKGROUND Epiplakin (EPPK) belongs to the plakin family of cytolinker proteins and, resembling other members of the plakin family such as BPAG1 (an autoantigen of bullous pemphigoid) and plectin, EPPK has plakin repeat domains (PRDs) that bind to intermediate filaments. Elimination of EPPK by gene targeting in mice resulted in the acceleration of keratinocyte migration during wound healing. EPPK is expressed in proliferating keratinocytes at wound edges and, in view of its putative function in binding to keratin, we postulated that the keratin network in EPPK-null (EPPK(-/-)) mice might be disrupted during wound healing. OBJECTIVE To examine this hypothesis and to determine the precise localization of EPPK in relation to keratin filaments, we compared the non-wounded and wounded epidermis of wild-type and EPPK(-/-) mice. METHODS Non-wounded epidermis and wounded epidermis from wild-type and EPPK(-/-) mice were examined by immunofluorescence staining and electron microscopy before and after double immunostaining. RESULTS EPPK was colocalized with keratin 17 (K17) more extensively than with other keratins examined in wounded epidermis. The expression of K5, K10, K6, and K17 was the same in EPPK(-/-) mice after wounding as in normal mice, but diameters of keratin filaments were reduced in EPPK(-/-) keratinocytes. Electron microscopy after immunostaining revealed that EPPK colocalized with K5, K10 and K6 after wounding in wild-type mice. CONCLUSION Our data indicate that EPPK accelerates keratin bundling in proliferating keratinocytes during wound healing and suggest that EPPK might contribute to reinforcement of keratin networks under mechanical stress.

Increased Fragility, Impaired Differentiation, and Acceleration of Migration of Corneal Epithelium of Epiplakin-Null Mice

PURPOSE To investigate the effects of gene ablation of epiplakin on the homeostasis of corneal epithelium in mice. METHODS Light and transmission electron microscopic histology, immunohistochemistry, and real-time RT-PCR were carried out to evaluate the effects of the loss of epiplakin on structure and gene expression of cell-cell adhesion-related components in mice. Integrity against mechanical intervention and wound-healing response of corneal epithelium were also tested. RESULTS Epiplakin protein was detected in the cells of the basal layer of corneal epithelium. Morphologically basal-like cells were observed in the suprabasal layer of adult epiplakin-null corneal epithelium, suggesting an impaired intraepithelial cell differentiation. Such abnormality was not detected in mice before the age of postnatal day 14. Epiplakin-deficient epithelium exhibits fragility against mechanical intervention as compared with wild-type epithelium. Although cell proliferation is suppressed, migration-dependent wound healing is promoted in epiplakin-null epithelium. E-cadherin expression was suppressed by the loss of epiplakin in the epithelium. CONCLUSIONS Lacking epiplakin affects cell differentiation of the corneal epithelium, as well as its proliferation activity and its structural integrity. The mechanism of acceleration of cell migration in the epiplakin-null corneal epithelium is to be further investigated, although suppression of expression of E-cadherin might be included.

Analysis of 256 cases of basal cell carcinoma after either one‐step or two‐step surgery in a Japanese institution

Basal cell carcinoma (BCC) is a common skin cancer that arises from the cells of the basal layer of the epithelium or from the external root sheath of the hair follicle. In the present report, 256 cases treated surgically between 1999 and 2008 in our department were retrospectively analyzed. The most frequent BCC locations included the face (77.8%), especially the nose (26.9%) and eyelids (21.5%). Incomplete excisions occurred in 21 cases. Two patients experienced local recurrence; one of these patients exhibited a bone metastasis while the other had a metastasis of the parotid gland without the local recurrence. The rate of local BCC recurrence was 0.78%, which is lower than that described in previous reports. We categorized BCC into four histological types: superficial, solid, adenoid and infiltrative. The solid type was the most frequent histological type (62.1%). For preventive recurrence, we treated BCC patients with two‐step surgery when the tumor was large or histologically invasive. At the first step, we excised the tumor with a sufficient safety margin, and at the second step, we performed reconstruction after the histological confirmation that no remnant malignant cells were in the tumor margins. In the present report, no local recurrence occurred in patients following the two‐step surgery. Therefore, two‐step surgery is recommended for tumors at locations and with histological types related to frequent recurrence.

Inability to detect sentinel lymph node metastasis due to an obstruction of the lymphatics by metastatic Merkel cell carcinoma

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Atypical pemphigus with exclusively anti-desmocollin 3-specific IgG antibodies

ejd.2012.1762 Auteur(s) : Yutaka Hatano1, Takashi Hashimoto2, Shunpei Fukuda2, Kazushi Ishikawa1, Mizuki Goto1, Yoshitaka Kai1, Naoko Takeo1, Osamu Okamoto1, Sakuhei Fujiwara1 fujiwara@oita-u.ac.jp 1 Department of Dermatology, Oita University, Idaigaoka1-1 Hasama, Yufu 879-5593, Japan 2 Department of Dermatology, Kurume University School of Medicine, Kurume University, Kurume 830-0011, Japan Antibodies against desmocollin 3 (Dsc3), together with other autoantibodies have been detected in some cases [...]

Epidermotropic CD8+ cytotoxic T-cell lymphoma exhibiting a transition from the indolent to the aggressive phase, accompanied by emergence of CD7+ cells and formation of neutrophilic pustules

A 47‐year‐old‐man presented with rashes on his trunk and limbs, and a diagnosis of parapsoriasis was made. Ten years later, the rashes had progressed gradually to form plaques and tumours. Gene rearrangement studies revealed monoclonality of the T‐cell receptor β‐chain (TCR‐Jβ)1 gene, and results of flow cytometry and immunohistochemical examination confirmed a diagnosis of epidermotropic CD8+ cytotoxic T‐cell lymphoma. The clinical course of the disease remained indolent for some time, but about 2 years later, neutrophilic pustules formed on the surface of the skin lesions, and tumours developed in the patient’s testes. Using flow cytometry, emergence of CD7+ cells was found. The patient died the following year of respiratory failure due to brain herniation. On postmortem examination, CD8+ tumour cells were found in the brain. This case demonstrates an unusually protracted indolent phase in a patient with cutaneous aggressive epidermotropic CD8+ cytotoxic T‐cell lymphoma; its transition into the aggressive phase was accompanied by emergence of CD7+ cells and formation of neutrophilic pustules.