Yukio Kitano

Expression of basement membrane components in skin equivalents--influence of dermal fibroblasts.

We have made a skin equivalent constructed of fibroblasts embedded in a type I collagen, with an overlying stratified keratinocyte epithelium to examine formation of the basement membrane. We assessed the influence of the existence and species of fibroblasts in the collagen gel. Cultured human keratinocytes were well attached to the dermal equivalent. Plating efficiency was not clearly different among several types of gel. On the control and mouse fibroblast gel, sheet formation was delayed and epithelial stratification on the human fibroblast gel was more remarkable than on the control gel. On the human fibroblast gel, we observed the expression of basement membrane components (bulbous phemphigoid antigen, laminin, type IV collagen and fibronectin) between the sheet of cultured keratinocytes and the human fibroblast gel earlier than those on the control gel and mouse fibroblast gel. Type VIII collagen was not observed in any of the models at 4 weeks.

Immediate contact hypersensitivity induced by repeated hapten challenge in mice

An immediate reaction was investigated during repeated challenge testing for contact hypersensitivity to dinitrofluorobenzene ( DNFB) in BALB/c mice. The mice were sensitized to DNFB on back skin and repeatedly challenged with the same hapten on the left ear at 1 week intervals. The ear after the 5th challenge showed biphasic responses which consisted of an immediate and a delayed‐type reaction. The reactions were hapten specific. Mast cell‐deficient WBB6F1 W/WV mice did not show any immediate reaction, while congenic normal mice showed both immediate and delayed‐type reactions. Histologically, numerous dermal mast cells were found in the left ear of repeatedly challenged BALB/c and WBB6F1 normal mice, while there were few mast cells in the ear of WBB6F1 W/WV mice. Anti‐ DNP IgE antibodies were detected in BALB/c, WBB6F1 normal and W/WV mice after repeated challenge with DNFB. Intradermal injection of anti‐IgE antibodies in the repeatedly DNFB‐challenged ear elicited an immediate reaction. These results suggest that immediate contact hypersensitivity develops through the production of anti‐ DNP IgE antibodies and an increase in dermal mast cells after repeated challenge with DNFB.

Expression of Cyclin D1 and p53 Protein in Various Malignant Skin Tumors

BACKGROUND Overexpression of cyclin D1 and p53 protein has been reported in many types of malignant tumors. OBJECTIVES We investigated whether cyclin D1 was detected immunohistochemically in various types of malignant tumors of the skin, comparable with p53 protein. METHODS Immunohistochemical staining of cyclin D1 and p53 protein was applied to squamous cell carcinoma, malignant melanoma and malignant fibrous histiocytoma and various kinds of benign skin tumors. RESULTS Cyclin D1 was positive only in malignant tumors at the same incidence as p53 protein. CONCLUSION Cyclin D1 immunohistochemical staining may be a malignant marker for various skin tumors.

Coexpression of p21Waf1/Cip1 and p53 in sun‐exposed normal epidermis, but not in neoplastic epidermis

Summary Wild‐type p53 accumulation induced by DNA damaging agents such as ultraviolet (UV) radiation. γ‐irradiation and drugs, may arrest the cell cycle until DNA damage is repaired. p21Waf1/Cip1 is a cyclin‐dependent kinase (CDK) inhibitor induced by wild‐type p53. CDK is activated by cyclin and progresses the cell cycle. On the other hand. CDK inhibitors inhibit CDK activity to arrest the cell cycle. Thus, p21Waf1/Cip1 is thought to mediate the signal of p53 induced by DNA damaging agents to arrest the cell cycle. p21Waf1/Cip1 is induced by wild‐type, but not mutant p53. To investigate p21Waf1/Cip1 regulation by p53 in epidermis in vivo, immunohistochemical staining of p21Waf1/Cip1 and p53 were conducted in chronically sun‐exposed normal epidermis and in neoplastic epidermis. p21Waf1/Cip1 expression was found to be coincident with the p53‐positive regions or not coincident with the p53‐positive regions in chronically sun‐exposed normal epidermis, whereas there was only low or undetectable p21Waf1/Cip1 expression in any regions including the p53‐positive regions of solar keratosis and squamous cell carcinoma of the skin. This suggests that wild‐type p53 and p21Waf1/Cip1 may play a part in chronically sun‐exposed normal epidermis response to UV exposure, whereas p21Waf1/Cip1 cannot be induced by mutated p53 in solar keratosis and squamous cell carcinoma of the skin.

Electron microscopic study of cultured cells from the murine hair tissues: cell growth and differentiation

SummaryThe cultured hair cells from 4-day-old C3H mice were studied by electron microscopy. The hair roots isolated from the skin by collagenase digestion were dispersed into a cell suspension by treatment with a mixture of trypsin and ethylenediaminetetraacetate. The cells were cultured in MCDB-153 (a medium containing seven growth factors) for 1, 3, 6 or 13 days. The number of cultured cells on day 3 was twice that on day 1, and stayed at the same level until day 13. By electron microscopy, some of the cells cultured for 1 day were seen to be undifferentiated and others already showed differentiation into various hair structures. Such differentiated cells disappeared on day 3 and most of the cells cultured for 3 days were undifferentiated. Cells cultured for 6 days were differentiated showing inner root sheath cell, hair cortical cell and medulla cell structures. The characteristics of these cultured cells corresponded well to those of in vivo cells of the hair tissues from the back skin of 7-day-old C3H mice. On day 13 degeneration occurred in the cultured cells. In none of these cultures were mesenchymal cells, such as fibroblasts, found. The present electron microscopic study reveals that immature cells obtained from mouse hair tissues proliferate in vitro and differentiate into several subpopulations corresponding to those of in vivo cell layers of hair tissues. The present culture technique may be useful for studies of hair cell growth and differentiation.

Effects of several growth factors on cultured neurofibroma cells

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by abnormalities affecting multiple tissues derived from the neural crest. The peripheral neurofibromas are numerous and sometimes reach several hundred in number. In this study, the possible involvement of several growth factors in neurofibroma growth was investigated in vitro. When explants of neurofibroma tissue were cultured, macrophage-like cells with pseudopodia migrated out first, and later took on a slender fusiform shape. These cells contained S-100 protein and were identified as Schwann cells. They did not proliferate under standard culture conditions. Nerve growth factor (NGF) was helpful in maintaining the differentiated phenotype of Schwann cells, but did not stimulate their proliferation. Immunohistochemical staining for type IV collagen revealed that some large flattened polygonal cells had a mesh of type IV collagen on the surface. These cells were perineurial cells. The proliferation of cells derived from neurofibroma was stimulated by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-alpha). In comparison with skin fibroblasts, the cells derived from neurofibroma responded to these growth factors at considerably lower concentrations. Stimulation by EGF at physiological concentrations indicated the possible involvement of EGF in the development of neurofibromas.

Elevation of serum-soluble E-selectin in atopic dermatitis

Atopic dermatitis (AD) is characterized by alterations in cellular and humoral immunity. The objective of this study is to determine whether soluble E-selectin (sE-selectin) plays a role in AD. We examined the serum sE-selectin levels in patients with atopic dermatitis (n = 23), patients with urticaria (n = 9), and normal healthy individuals (n = 15). The severity of the disease in the AD patients was graded using an established clinical scoring system. We found that sE-selectin levels were significantly higher in atopic dermatitis than in urticaria (P < 0.001) or normal controls (P < 0.0001). In addition, there was a significant correlation between serum sE-selectin and the clinical score (R = 0.73, P < 0.0001). Clinical improvement was associated with a decrease in both the clinical score (P < 0.01) and serum sE-selectin (P < 0.01). E-selectin was recognized on the vascular endothelial cells of the erythematous lesions of AD patients. These results indicate that sE-selectin may play a role in AD.

Immunohistochemical detection of cyclin D and cyclin A in human hyperproliferative epidermis.

Cyclin was first identified in sea urchin eggs as a protein that is synthesized prior to repeated cell division alter fertilization, and it disappears rapidly on completion of cell division [3]. Cyclin, when combined with cdc2, activates cdc2 kinase which then phosphorylates various proteins to regulate the cell cycle [10]. Such a mechanism is common to all eukaryotic cells [ 11 ]. Subtypes exist for both cyclin and cdc2 in mammalian cells and the advance of the cell cycle is controlled by the combination of these subtypes [13]. Five cyclin subtypes (A to E) have so far been identified. These cell cycle proteins are thought to be abnormal in carcinoma cells. It has been suggested that cyclin A and cyclin D may be oncogenic proteins. In one case of hepatocellular carcinoma, it was reported that the hepatitis B virus gene is inserted into the intron of the cyclin A gene [14]. The cyclin D gene was first reported as a gene (PRAD1) that is activated by chromosome inversion in cells of parathyroid tumours [9]. At about the same time, it was reported as a gene (CYL) that is induced in the G1 phase by stimulation of the growth factor specific to macrophages [8] and also as a human gene (cyclin D gene) that can rescue G1 phase-deficient yeast cells [7, 16]. Furthermore, it has been reported that the cyclin D gene is overexpressed by chromosome translocation or gene amplification in B cell lymphoma [15], squamous cell carcinoma of the head and neck [6], mammary carcinoma [6] and oesophageal cancer [5]. These findings indicate that cyclin D may be an oncogenic cyclin. In the present study, we examined the immunohistochemical localization of cyclin D and cyclin A in normal human epidermis and various epidermal hyperproliferative diseases including psoriasis vulgaris, seborrhoeic

Organization and disorganization of actin filaments in human epidermal keratinocytes: Heat-shock treatment and recovery process

SummaryWe investigated alterations of actin organization due to heat shock and recovery from the collapse in human epidermal keratinocytes. Exposure of keratinocytes to elevated temperature caused the rapid disintegration of actin filaments. With a heat-shock treatment at 45° C for 10 min, actin filaments disappeared and granular actin was distributed diffusely in the cytoplasm. After return to 37° C, recovery of actin organization was observed. Completely disintegrated granular actin assembled to form actin dots, which tended to group. The grouping actin dots often took a circular, semicircular or arched form. Filamentous actin then extended out from the actin dots. Fine short bundles of actin filaments had a rippled appearance or were polygonal in structure, with actin filaments converged at many points. On the seventh day after heat-shock treatment, actin organization had almost returned to the pre-heat-shock condition, with diffusely distributed actin filaments. In previous studies, we observed such aberrant structures in human malignant keratinocytes and human epidermal keratinocytes treated with 12-O-tetradecanoylphorbol-13-acetate. The observations presented here indicate that those structures are not specific to malignancy or to the process of malignant transformation, but represent less mature and aberrant organizations of actin.

Elevation of serum major basic protein in patients with atopic dermatitis

Serum levels of major basic protein (MBP) were measured in patients with atopic dermatitis (AD). The severity of the disease in the AD patients was examined using our clinical scoring system. AD patients showed significantly elevated serum levels of MBP compared with normal controls. There were significant correlations between serum MBP and clinical score. These data indicate that serum MBP could be a possible marker for the disease activity.