Toshio Tazawa

ANTI‐HAIR KERATIN MONOCLONAL ANTIBODY (HKN‐2)

BALB/c mice were immunized with human hair fibrous proteins. Then their spleen cells were fused with Sp2/0‐Agl4 mouse myeloma cells. Antibody‐producing hybridomas were selected by ELISA method and cloned by limiting dilution. A monoclonal antibody was chosen and designated as HKN‐2. Immunohistochemically, on frozen sections of normal human skin, HKN‐2 was found to recognize the suprabasal cells of epidermis and hair follicle, the cells in the keratogenous zone of hair shaft, the sebaceous cells and the ductal and myoepithelial cells of sweat glands. The basal cells of the epidermis and lower hair follicle, hair matrix cells and secretory cells of sweat glands did not react with HKN‐2. An immunoelectron microscopic method showed that the positive reaction to HKN‐2 was located on the tonofilaments in the cytoplasm. It was concluded that there might be a common antigenic determinant between hair and other skin epithelial tissues. A complexity in keratin manifestation in each epithelial tissue of the skin was suspected.

Immunohistochemical study on keratin expression in certain cutaneous epithelial neoplasms: basal cell carcinoma, pilomatricoma, and seborrheic keratosis

We investigated immunohistochemically 20 basal cell carcinomas (BCC), five pilomatricomas, and nine seborrheic keratoses using anti-BCC keratin monoclonal antibody (BKN-1) and anti-hair keratin monoclonal antibodies (HKN-2, HKN-4∼-7). The neoplastic cells in all the cases of BCC were always uniformly stained by BKN-1, HKN-2, and HKN-4, indicating that the BCC cells display a constant antigenicity of keratin, which may be different from that of the normal epidermis. Although no fluorescence by HKN-6 or HKN-7 was seen in any cases of BCC, HKN-5 partially but strongly stained the neoplastic nests in most cases of BCC; BCC may have differentiation toward the lower part of hair follicular epithelium. In pilomatricoma, all the anti-keratin monoclonal antibodies showed a similar staining pattern; the differentiating neoplastic cells undergoing transition from basaloid to eosinophilic were positively stained by each antibody in all the cases. This finding of pilomatricoma corresponds to that of the differentiating cells in the inner hair layers, especially in the hair cortex. In seborrheic keratoses, no fluorescence was recognized with HKN-5∼-7, which stain the lower follicular cells in the normal human skin. The staining patterns of seborrheic keratosis by BKN-1, HKN-2, and HKN-4 were similar to those of the normal interfollicular epidermis. These anti-keratin monoclonal antibodies seem to be useful for the investigation of the direction of differentiation of skin adnexal neoplasms.

Investigation of germinative cells in generating and renewed anagen hair apparatus in mice using anti-bromodeoxyuridine monoclonal antibody

To elucidate the cell kinetics in generating and renewed anagen hair apparatus in mice, S-phase cells in dorsal skin of new-born and 14 to 25-day-old mice were labeled with bromodeoxyuridine (BrdU) and stained immunohistochemically using anti-BrdU monoclonal antibody. In primary hair germ, lower and outer cells were positively stained. In hair peg, the positive cells were divided into upper and lower portions. In bulbous hair peg, the lower positive cells gathered at the suprapapillary area, whereas the upper positive cells were located in the outer part of the hair apparatus. The latter positive cells were further divided in the middle and upper parts of the epithelial cord. The upper cells were going to form a sebaceous gland. From the suprapapillary group of germinative cells, hair cortex through the innermost cell layer of the outer root sheath were considered to be produced. The positive cells of the outer middle group may be important for elongation of hair apparatus and produce outer root sheath cells. At the end of anagen phase, first the suprapapillary cells became negative and then the outer root sheath cells became negative. BrdU-positive cells reappeared at the bottom of telogen hair apparatus and formed a secondary hair germ. Then, a similar cell kinetics was repeated in the renewed anagen hair apparatus. The germinative cells were separated into a few groups during the generation and cyclic changes of hair apparatus. Each cell group formed the individual part of the hair apparatus in a coordinate fashion, resulting in dynamic changes of the hair apparatus.

Monoclonal antibody analysis of keratin expression in carcinomas of sweat glands

Characteristics of keratins of live carcinomas of sweat gland origin were immunohistochemically investigated with several antikeratin monoclonal antibodies with differing specificities. Specimens were obtained from two cases of mucinous carcinoma of the skin, two cases of classic type of eccrine adenocarcinoma, and a case of eccrine porocarcinoma. The tumor cells of mucinous carcinoma expressed only simple epithelial keratins. In a case of eccrine adenocarcinoma, simple epithelial keratin 19 was diffusely expressed. The expression of the other simple epithelial keratins was confined to the luminal cells, whereas the remaining tumor cells further expressed stratified epithelial keratins. Eccrine porocarcinoma and a second case of eccrine adenocarcinoma did not express simple epithelial keratins, although stratified epithelial keratins were diffusely expressed. These data suggest that carcinomas of sweat glands express various combinations of simple and stratified epithelial keratins. Development of additional data along these lines may help to further define their classification.

Differential analysis of the human anagen hair apparatus using lectin binding histochemistry

SummaryCell differentiation in the human anagen hair apparatus was examined by lectin-binding histochemistry using seven different lectins: Con A, WGA, RCA-I, PNA, SBA, DBA and UEA-I. Con A and WGA positively stained almost all the cells in the hair apparatus. RCA-I and PNA positively stained the outer cells of the outer root sheath (ORS), but they did not stain the innermost cells (IMCs) of the ORS in the suprabulbar region. However, in the isthmus region, the IMCs showed positive staining with RCA-I, and more intense staining with PNA than that of the outer ORS cells. The ORS cells, including the IMCs, were negative with SBA and DBA below the suprabulbar region, whereas the IMCs became more strongly positive with these two lectins than the other ORS cells in the isthmus region. UEA-I strongly stained the IMCs, but not the outer ORS cells in the hair bulb. The latter cells became positive for UEA-I above the suprabulbar region. These findings indicate that the surface glycoconjugate distribution of the IMCs differs from that of the outer ORS cells. It is concluded that the IMCs of the ORS may undergo an independent cell differentiation process.

Intraepidermal pilar epithelioma: a new dermatopathologic interpretation of a skin tumor.

An intraepidermally developed epithelial cell tumor, forming multiple nests, was examined to identify its cytologic characteristics. Histochemically, the tumor cells contained neither glycogen nor lipid substance. By N-(7-dimethylamino-3-methyl-4-coumarinyl)maleimide staining, the cytoplasm of the tumor cells in the periphery of each nest was rich in SH groups but not in SS linkages, whereas centrally located homogeneous tumor cells contained SS diffusely but no SH. The tumor cells showed no activity of phosphorylase and a weak activity of succinic dehydrogenase. Immunohistochemically, antihair keratin monoclonal antibodies specific for hair cells decorated the tumor cells, but carcinoembryonic antigen staining showed no positivity. Ultrastructurally, the tumor cells underwent a keratinization forming a fingerprint pattern of keratin filaments; however, membrane-coating granules and marginal bands were not formed. These intraepidermal tumor cells may have cytologic natures similar to those of hair cortical cells. The term intraepidermal pilar epithelioma is proposed as a diagnosis for this tumor.

Anti-keratin Monoclonal Antibody against Basal Cell Epithelioma Keratin: BKN-1

A BALB/c mouse was immunized with fibrous proteins (FP) extracted from basal cell epithelioma (BCE) and anti‐keratin monoclonal antibodies were produced by a hybridoma technique with a mouse myeloma cell line. One monoclonal antibody was designated as BKN‐1.

In vitro Keratin Expression of Hair Cells

Human hair follicles were isolated from the scalp by dispase and collagenase treatment and dispersed into a cell suspension by trypsin. These cells proliferated well and could be subcultured 7 to 8 times. The medium used was MCDB 153 HAA medium further supplemented with some amino acids, hydrocortisone, insulin, EGF, and bovine brain extract. The concentration of Ca++ was adjusted to 0.1 mM. Immunohistochemically, these cells were proved to possess keratins specific to hair forming cells.