Katsuyuki Takeda

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.

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.

Culture of human outer root sheath cells from plucked hair follicles in serum-free conditions

We succeeded in culturing human outer root sheath cells (ORSC) in serum-free conditions without a biological feeder layer. The combination of collagen type IV substrate and modified MCDB 153 medium supplemented with bovine pituitary gland extract (K-GM medium) could support the growth of ORSC. These cells can be serially cultivated for at least 4 passages and stored in liquid nitrogen with good recovery. Thus, a large series of experiments using ORSC may be run simultaneously.

Eosinophilic Polymyositis Induced by Tranilast

A 38‐year‐old man with asthma developed eosinophilic polymyositis following the administration of Tranilast, an antiasthmatic agent. Low grade fever, erythematous rashes on the entire body, dysphagia, blood eosinophilia, elevations of serum creatine phosphokinase and myoglobin levels, and inverted T waves in the electrocardiogram were noted. A muscle biopsy showed focal degeneration of muscle fibers with an infiltrate of eosinophils and lymphocytes. A rechallenge with Tranilast resulted in erythema formation, blood eosinophilia, and elevations of some serum muscle enzymes and myoglobin levels. Tranilast was considered to be the causative agent. This is the first reported case of Tranilast‐induced eosinophilic polymyositis.

The effect of 1,25-dihydroxyvitamin D3 on the growth and differentiation of cultured human outer root sheath cells from normal subjects and patients with vitamin D-dependent rickets type II with alopecia.

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth and differentiation of cultured human hair outer root sheath cells (ORSC) from normal subjects and patients with vitamin D-dependent rickets type II (DDR-II) with alopecia. 1,25(OH)2D3 dose-dependently suppressed the plating efficiency, clonal growth, and DNA synthesis of normal ORSC. It enhanced the cornified envelope formation and caused morphological changes in the cells. All results indicated the existence of specific receptors for 1,25(OH)2D3 in the ORSC, and suggest that 1,25(OH)2D3 is a potent inhibitor of proliferation of ORSC as well as a stimulator of terminal differentiation. However, the cells from DDR-II patients with alopecia did not respond to 1,25(OH)2D3, suggesting a lack of the specific receptors in the cells. The differences in the cellular response to the hormone between the normal ORSC and those from the patients were apparent and easily distinguishable, therefore this experiment may be a rapid and simple diagnostic test for DDR-II patients with alopecia. Large number of hairs were difficult to obtain from patients with alopecia, and we developed a new culture method to accomplish these studies from a few plucked hair follicles. Our system may be useful in the culture of ORSC from limited number of follicles, and could be utilized to analyse the cellular characteristics of ORSC in patients with hair diseases.

Blood Vessels and Immunoreactive Substance P‐Containing Nerve Fibers in Rat Skin Treated Topically with Clobetasol Propionate, a Corticosteroid

After applying topically a cream (0.1 ml) containing corticosteroid (clobetasol propionate), on rat back skin, we examined the morphological alterations of blood vessels, substance P‐containing nerve fibers, and cutaneous mast cells. After 3, 6, 10, 15, 30, and 60 min and 4 h, the skin treated was cut out with a sharp knife after killing the animals. The skin pieces were processed into conventional histological sections cut vertically and examined by staining immunohistochemically with anti‐substance P serum, by staining with toluidine blue for mast cell granules, and by estimating morphometrically the average areas of vascular cavity and the number of substance P fibers in the dermis. In the dermis and subcutaneous tissue of untreated skin, we found many immunoreactive SP‐containing nerve fibers and mast cells in close association. Three to ten min after the treatment, the average area of the vascular cavities steadily increased, and SP‐positive fibers became less frequent in the dermis. In concomitant with those events, cutaneous mast cells discharged their granules. Thereafter, the average area of vascular cavities gradually decreased to a minimum at 4 h after the treatment. In contrast, both SP‐containing fibers and mast cells reestablished their initial states after the same duration.

A New Method for Culturing Human Hair Follicle Cells on Floating Mixed Collagen Membranes

A new method for culturing human hair follicle cells without a biological feeder layer has been developed.

Development of the Hair in the Rat: In vivo and in Transplanted Tissue

Differentiation of the hair was examined in the head skin of rat embryos aged from days 12.5 to 18.5 of gestation.

Transcutaneous PO2 and PCO2 Measurements in Various Skin Lesions

Low tcPO2 levels were observed in various skin lesions, indicating that most pathological changes in the skin induce such a reduction. In contrast, the tcPCO2 changed less, with the exception of marked elevations in bullous or prenecrotic lesions. This procedure may have practical applications in clinical dermatology, particulary in quantitating the response to therapy and in predicting a necrotic change.