Yoshio Urano

The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells

The effect of cultured normal human dermal papilla cells (DPCs) and conditioned medium prepared with cultured DPCs on chemotactic migration of human hair outer root sheath cells (ORSCs) was examined quantitatively. ORSCs showed significantly increased migration toward both cultured DPCs and the conditioned medium suggesting that DPCs produce and secrete a paracrine factor(s), which attracts hair follicle epithelial cells. Some soluble factors, which are reportedly produced by DPCs, such as insulin-like growth factor-I (IGF-I), hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1), were also examined. ORSCs showed dramatically increased migration toward IGF-I and HGF at concentrations of 1-10 ng/ml. On the other hand, neither VEGF nor TGF-beta1 showed any effect on the chemotaxis of ORSCs. It is interesting that all factors involving mitogenic activity did not always have chemotactic activity for ORSCs. This is the first report to establish that IGF-I and HGF have not only a growth stimulatory but also a chemotactic effect on ORSCs. In addition, the method presented here may help to simplify chemotaxis assays of any type of epithelial keratinocytes with poor mobility.

Culture of cells derived from the human sebaceous gland under serum-free conditions without a biological feeder layer or specific matrices

We succeeded in serially culturing cells derived from human sebaceous gland (sebocytes) under serum-free conditions. Sebaceous glands were isolated from dispase-treated facial skin specimens and cultured using two different methods, explant culture and dispersed cell culture, in KGM. In both types of culture the sebocytes proliferated rapidly without a biological feeder layer or specific matrices. It was possible to cultivate the cells serially for at least three passages (explant culture) and six passages (dispersed cell culture), and to store the cells in liquid nitrogen with good recovery. Analytical thin-layer chromatography revealed that the cells synthesized a large amount of sebum-specific lipids, squalene and wax esters in vitro. Both testosterone and 5α-dihydrotestosterone significantly stimulated the proliferation of the sebocytes. The methods presented here may permit a large series of studies on the function of sebaceous glands or cells derived from sebaceous glands.

Sweet’s syndrome associated with chronic myelogenous leukemia: Demonstration of leukemic cells within a skin lesion☆☆☆

We report a case of acute febrile neutrophilic dermatosis, Sweets syndrome, associated with chronic myelogenous leukemia (CML) in which we found rearrangement of the bcr gene in DNA obtained from a skin lesion as well as in blood DNA by Southern blot analysis. This indicated the presence of CML cells within the skin lesion. To our knowledge, this is the first report in which the presence of CML cells is shown within skin lesions of Sweets syndrome. In our patient, leukocyte alkaline phosphatase activities returned to normal levels when he was suffering from Sweets syndrome and decreased again to below normal levels after it subsided. Whether the normalization of leukocyte alkaline phosphatase activity is common among CML patients with Sweets syndrome remains to be determined.

Immunohistological analysis of P53 expression in human skin tumors

The p53 expression in various skin tumors was immunohistologically evaluated using two mouse monoclonal anti-p53 antibodies, PAb421 and PAb1801. The p53 expression was not detected in the normal epidermal cells. Nuclear staining suggested that the p53 expression was observed in 10 of 26 squamous cell carcinomas (SCCs) from 24 patients, in one undifferentiated carcinoma, one proliferating trichilemmal cyst, one malignant proliferating trichilemmal tumor and in one metastatic carcinoma of breast cancer. None off four cases of Bowens disease (SCC in situ) showed nuclear staining. In the SCCs, five of 20 primary lesions, three of four recurrent lesions and both of two metastatic lesions had positive nuclei. There was one case of SCC in which a primary lesion was negative but a recurrent lesion was positive. Thus, p53 expression was more frequently observed in SCCs at more clinically advanced stages. This may suggest that p53 has some relevance to progression of SCC. Nuclear staining was not detected in any of the following cases: two cases of seborrheic keratosis, one eccrine poroma, one keratoacanthoma, 11 basal cell epitheliomas, two mammary Pagets disease, three genital Pagets disease, one sebaceous carcinoma, four malignant melanomas, six lymphomas, two leukemia cutis and two angiosarcomas.

Co‐culture of Human Hair Follicles and Dermal Papillae in a Collagen Matrix

Human hair follicles, either alone or in combination with dermal papillae, were cultured in a collagen matrix. When plucked hair follicles were cultured alone, spike‐like structures composed of outer root sheath cells started growing around the follicle and then radiated into the gel. When isolated dermal papillae were embedded close to the follicles, spikes started growing earlier and grew more rapidly than without the papillae. In cultures of excised follicles from which the dermal papilla had been removed, epithelial cells (possibly hair bulb cells) started growing out from the bulbous portion and then also formed spikes. In the presence of a papilla, the spikes elongated toward the papilla, finally reaching and surrounding it. These findings suggest that dermal papilla cells produce a factor(s) that enhances growth of follicular epithelial cells and also attracts those cells.

Lower eyelid reconstruction with a cheek flap supported by fascia lata.

The use of a cheek rotation flap is a well-known method for reconstruction of a large defect of the lower eyelid. In this technique, a separate lining tissue supporting the cheek flap is required for full-thickness reconstruction. Previously, a chondromucosal graft or conchal cartilage has been used to support this flap. Recently, we have used a homologous or autologous fascia lata as support for the cheek flap instead of rigid tissues like cartilages. A fascia lata strip is fixed with tolerable tension to the medial canthal tendon and lateral orbital rim. The inner surface of the fascia and the cheek flap is lined with a buccal mucosa graft to decrease irritation of the conjunctiva and cornea. We present here seven patients in whom this procedure was used for lower eyelid reconstruction following resection of a malignant skin tumor. Based on follow-ups of 7 to 22 months, the functional and aesthetic results have been good in all cases. This procedure may be applicable for total or subtotal reconstruction of the lower eyelid.

Immunohistochemical demonstration of peptidylarginine deiminase in human sweat glands.

Human skin is known to contain protein-bound citrulline. This is the product of enzymatic deimination of arginine residues catalyzed by peptidylarginine deiminase. We probed frozen sections of human skin with a rabbit antiserum raised to rat skeletal muscle peptidylarginine deiminase using the avidin-biotin-peroxidase complex technique. This led us to interesting findings. No staining was observed in epidermis, inner root sheaths of hair follicles, sebaceous glands, and hair erector muscle. However, we noticed specific staining of the cytoplasm of secretory and myoepithelial cells of both eccrine and apocrine sweat glands. The procedure also stained neoplastic cells present in specimens dissected from extramammary Pagets disease. The data mean that peptidylarginine deiminase may be used as a new marker in the classification of skin neoplasms showing sweat gland differentiation. Possible localization of multiple types of peptidylarginine de-iminases in human skin is discussed.

Microcystic adnexal carcinoma. Case report with an immunohistochemical study.

We report the case of 71-year-old Japanese woman with microcystic adnexal carcinoma (MAC) of the nose. Our histological and immunohistochemical observations suggest that MAC has both pilar and eccrine sweat gland differentiation.

Lack of somatic mutation in the PTEN gene in squamous cell carcinomas of human skin

The tumor suppressor gene PTEN is deleted and/or mutated in a variety of tumors and the susceptibility gene for Cowden disease. Loss of heterozygosity of chromosome 10q23, where PTEN resides, in squamous cell carcinomas (SCCs) of human skin and the association of SCC with Cowden disease were reported previously. In the present study, we screened for mutations of PTEN in SCCs by polymerase chain reaction single strand conformation polymorphism analysis to examine whether PTEN is involved in the carcinogenesis of SCC. None of 21 SCCs showed somatic mutations in the coding regions of PTEM. Instead the same allelic variation was detected in two cases without any clinical features of Cowden disease. Our results indicate that inactivation of PTEN does not play an important role in the carcinogenesis of SCC.

A novel PTEN mutation in a Japanese patient with Cowden disease

Cowden disease (CD) is an autosomal dominant syndrome characterized by multiple hamartomatous lesions and an increased risk for malignancies. Recent evidence has indicated that the PTEN gene, encoding a protein tyrosine phosphatase, is the CD susceptibility gene. However, another line of evidence has suggested that CD might be genetically heterogeneous. Clinical features of CD are variable, and there are interfamilial differences in the expression of skin lesions . Therefore, information on PTEN mutations in CD patients should be accumulated to clarify the genotype–phenotype correlation. In the present study, we found heterozygous germline mutations of PTEN in all of three Japanese patients with CD examined, indicating no genetic heterogeneity among our patients. The mutations included two non‐sense mutations of R335X and R130X, and a mis‐sense mutation of C136R. To the best of our knowledge, the C136R mutation has not previously been reported in CD patients. This novel mutation was located outside the core motif of the phosphatase domain of PTEN protein, where most of the missense mutations previously reported in CD patients were clustered. Mucocutaneous manifestations were far fewer in the patient with this mutation than in the patients with nonsense mutations. Whether the phenotypic difference in mucocutaneous features was due to the different mutations remains unclear.