EFFECT OF UVA IRRADIATION ON B16 MELANOMA CELLS IN MONOLAYER CULTURE

B16 murine melanoma cells in monolayer culture were exposed to a single dose of long wave ultraviolet light (UVA). The number of viable melanoma cells was decreased at 10 hrs post irradiation in parallel to the increase in UVA dose (10⁴~10⁷ ergs/cm²). After ten hours, the cultured cells entered the logarithmic growth phase. The doubling time of the cells exposed to the low dose of UVA was similar to that of nonirradiated cells, but the exposure to higher doses of UVA (10⁵~10⁶ ergs/cm²) elongated the doubling time of these cells. Still higher doses of UVA exposure (10⁷ ergs/cm²) caused no further elongation of doubling time. The tyrosinase activity of these melanoma cells was also measured using an oxygen electrode. The tyrosinase activity of each irradiated cell at ten hours was significantly increased, but no significant increase of tyrosinase activity per mg protein was observed, since protein content per cell increased at the same time. The increase in tyrosinase activity was still observed after ten hours in cells exposed to higher amounts of UVA. These results suggest that UVA repressed cell division and induced protein synthesis to prepare for melanogenesis.     

EFFECTS OF CYTOCHALASIN B ON EPIDERMAL CELLS IN VITRO

Summary.— Effects of cytochalasin B have been studied on organ cultures and dispersed cell cultures of adult guinea-pig ear skin, using electron microscopy and time-lapse cinephotomicrography. Cytochalasin B failed to inhibit epiboly at 1 ug/ml, caused slight inhibition at 2 ug/ml and total inhibition at 5 ug/ml. These concentrations did not appear to affect ruffling of membranes or internal cell movements. Some effect was demonstrated at 20 ug/ml. These concentrations, however, caused a general toxic effect on the cells. Ultrastructurally, there was possibly some reduction in the microfilament system, but this was far from dramatic. Abnormalities caused were increased numbers of Lysosomes and dilation of the endoplasmic reticulum.     

ORGAN CULTURE OF ADULT HUMAN SKIN; EFFECT OF CULTURE TEMPERATURE

In the organ culture of adult human skin, long-term preservation of cultured epidermis was obtained when the culture temperature was shifted from 37°C to 32°C. The epidermal cell kinetics were compared for both culture temperatures using the findings of mitotic rate, mitotic distribution and autoradiographic labelling index with tritiated-thymidine (³H-TdR). As a result, it appeared that a lowered culture temperature controlled the cell cycle in vitro to maintain long-term, active epidermal proliferation.     

A COMPARISON OF IN VIVO AND IN VITRO METHODS OF IDENTIFYING HUMAN EPIDERMAL CELLS IN DNA SYNTHESIS

Summary.— For identifying epidermal cells in DNA synthesis there are 2 rather different methods in use: subcutaneous injection of ³H thymidine in vivo , and short term incubation of skin slices with ³H thymidine in vitro.
A direct comparison of these methods on the skin of the same individuals at the same times shows little difference in the results obtained. If anything, the in vivo method is slightly more sensitive.     

EFFECT OF RETINOL ON MURINE EPIDERMAL DENDRITIC CELLS

Numerous immunoregulatory activities have been attributed to retinoids. The present study examined the effect of a single application of retinol (RO) on immunocompetent cells in murine epidermis. The inner epidermis of the left ear of C57/BL6J mice was painted with RO (50 ug/ml) in ethanol, while the right (control) ear received ethanol alone. Mice were sacrified at 0,12,24,36 or 48 hours, and epithelial sheets of the treated areas prepared. Two cell populations were detected using enzyme histochemistry and indirect immunofluorescence: Langerhans cells (ATPase⁺, Ia⁺), and cells expressing Thy 1.2 antigen (ATPase-, la-). In all cases ATPase⁺ cells were more numerous that Ia⁺ cells, however, neither RO nor alcohol treatment modulated la⁺or ATPase⁺ expression on Langerhans cells. In contrast, R O treatment increased the number of Thy 1.2⁺ cells detected at 12 and 24 hr post-treatment when compared to baseline and alcohol controls. These results indicate that retinol is capable of modulating antigen expression on murine dendritic cells in vivo.     

GUINEA PIG ENDOTHELIAL CELLS IN CULTURE

In pursuit of a model for the studies of endothelial cells in a readily available experimental animal, culture methods and the in vitro characteristics of endothelial cells isolated from guinea pig aorta are described. Endothelial cells were harvested by trypsinization with perfusion techniques and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Their passage cultivation was possible for more than six months and they have maintained the in vitro morphological characteristics of endothelial cells. The cell doubling time of the passaged endothelial cells was 115–131 hours. The growth of guinea pig aorta endothelial cells was satisfactory in Eagle's minimal essential medium containing guinea pig serum and fetal calf serum, but not horse and calf serum. Of the various concentrations of fetal calf serum in Eagle's minimal essential medium, 3% fetal calf serum did not activate cell growth and 10% fetal calf serum induced the best growth; in 20% concentration of fetal calf serum the cell growth rate decreased as compared with the rate in 10% fetal calf serum. DNA synthesis (³H-thymidine uptake) of the cells in 20% fetal calf serum was less than that in 10% fetal calf serum. These results have no relation to cell generation nor to the cell density. With respect to the type of media supplemented with 10% fetal calf serum, Dulbecco's modified Eagle's medium, RPMI ?1640 and Eagle's minimal essential medium were satisfactory for the cultivation of guinea pig aorta endothelial cells, but Medium-199 was unsuitable. By adding endothelial cell growth supplement (75, 150 mcg) or fibroblast growth factor (100, 200 ng) to Eagle's minimal essential medium containing 3% fetal calf serum, the cell growth was stimulated approximately three times. Fibronectin-coated wells did not activate cell growth.     

A COMPARATIVE STUDY OF THE FUNCTIONS OF EPIDERMAL LANGERHANS CELLS IN HAIRLESS MICE AND NUDE MICE

An experimental examination of contact allergic hypersensitivity in hairless mice and nude mice was carried out using 2,4-dinitro-1-chlorobenzene. In normal nude mice without T-cells, we were unable to detect apposition of mononuclear cells to Langerhans cells. In hairless mice, however, we could detect apposition of mononuclear cells to Langerhans cells after both sensitization and challenge procedures. Accordingly, hypotheses are proposed that mononuclear cells are T-cells, and that Langerhans cells play the role of target cells which do not traverse the epidermis but remain within it. When we studied untreated hairless mice, a few immature Langerhans cells and indeterminate dendritic cells could be seen. Three hours after the challenge with 2,4-dinitro-1-chlorobenzene, however, a great number of mature Langerhans cells were recognizable. The investigation of nude mice yielded reciprocal data.     

EPIDERMAL LANGERHANS CELLS IN VARIOUS SKIN DISEASES (2)

We compared the density of epidermal Langerhans cells (LC) in specimens taken from involved areas and peripheral normally pigmented skin in vitiligo, idiopathic guttate hypomelanosis (IGH) and photoleukomelanoderma, principally by means of staining for ATPase. Ia antigen staining was used as supplementary identification method for LC. A decrease in the density of LC in peripheral normally pigmented skin was observed in 4 of 8 specimens from normoimmunological vitiligo, and in 4 of 5 specimens from dysimmunological vitiligo. A remarkable decrease in the density of LC in involved areas was observed in IGH. The differences in LC density between vitiligo and IGH were highly suggestive of possible differences in the pathogenesis of leukoderma. We also discussed some possible roles of LC in the pathogenesis of vitiligo.     

THE ULTRASTRUCTURE OF LICHEN AMYLOIDOSUS WITH SPECIAL REFERENCE TO THE EPIDERMAL CHANGES

Summary.— The ultrastructure of lichen amyloidosus has been studied with special reference to the epidermal changes. The amyloid deposits often came into close proximity to the lower epidermis and obscured the basal lamina. Occasionally, breaks in the basal lamina occurred so that amyloid fibrils came into direct contact with the cytoplasm of basal cells. Small deposits of amyloid were sometimes seen in the inter-cellular spaces between basal cells. Focal areas of liquefaction degeneration were observed within a number of basal cells. A few keratinocytes in the upper epidermis contained deposits of amyloid-like fibrils. It is suggested that these amyloid-like fibrils probably represent degenerate tonofilaments rather than true amyloid.     

DNA SYNTHESIS IN CULTURED EPIDERMAL CELLS FROM PSORIATIC AND NORMAL SKIN

While it is generally accepted that psoriatic epidermis in vivo shows an increase in the number of DNA-synthesizing cells and mitoses, the published data from in vitro studies disagree as to whether or not psoriatic epidermal cells in vitro are more proliferative than normal epidermal cells. In this study, DNA synthesis was measured autoradiographically using cultured epidermal cells from both the involved and uninvolved skin of 8 psoriatic patients as well as from 8 normal individuals. Our results showed no statistically significant differences in DNA synthesis among the three groups.     

THE ROLE OF EPIDERMAL LYSOSOMES IN MELANIN PHYSIOLOGY

SUMMARY.— Melanosomes are formed within melanocytes and transferred via dendrites to keratinocytes. In caucasoids, most melanosomes are aggregated in membrane-bound organelles (melanosome complexes). These aggregates have phagocytic capacity, stain positively with aryl sulphatase and acid phosphatase, contain melanosomes apparently undergoing degradation, and are lysosomes. In addition to melanosome complexes, individual melanosomes, both in melanocytes and keratinocytes, also exhibit lysosomal properties of phagocytosis and hydrolytic enzyme activity. In contrast, negro melanosomes are mostly singly dispersed and are rarely aggregated in complexes. Melanosome dispersal contributes to the darker colour and superior sunlight protection of negro skin. Degradation by lysosomal enzymes explains the limitation of melanin principally to the basal layer.
In skin with severe solar degeneration, particularly in xeroderma pigmentosum, pigment production is often apparently adequate since hypertrophic melanocytes and dendrites are conspicuous. However, pigment transfer from melanocytes to keratinocytes does not occur normally. Some keratinocytes contain little melanin while others show an increase. Melanosomes within keratinocytes of actinically damaged skin tend to be concentrated within larger than normal complexes. Skin colour and sunlight protection appear to depend not only upon the rate of melanin formation but also upon distribution and rate of degradation.     

地塞米松和硫唑嘌呤对小鼠Thy-1~+树突状表皮细胞的影响

Thy-1阳性树突状表皮细胞(Thy-1~+dEC)是一种存在于正常小鼠表皮内的树突状细胞。应用抗Thy-1抗原的单克隆抗体和间接免疫荧光技术,我们证实:(1)外用地塞米松或硫唑嘌吟均可使小鼠Thy-1~+dEC密度明显降低(P<0.01);(2)系统应用不同剂量的地塞米松可使小鼠Thy-1~+dEC密度明显降低,(P<0.01),其降低程度与剂量呈直线负相关。     

THE GROWTH OF ADULT HUMAN SKIN IN VITRO

Summary.— In this report we investigate the role of serum in organ culture of adult human skin. The migratory overgrowth of dermis by epidermal cells is shown to depend on the presence of high molecular weight (mol. wt.) components of serum. Regeneration of the epidermis after 19 days' incubation occurs in the presence of 10% whole serum in the growth medium. DNA synthesis is found to increase in long-term incubations in medium including serum components but not in the presence of Minimal Essential Medium (MEM) only.     

EFFECT OF TOPICAL TRETINOIN ON EPIDERMAL LANGERHANS'CELLS IN PHOTODAMAGED SKIN

Background. Topical tretinoin has been successfully applied to treat photoaging; however, a decrease in the number of Langerhans’cells (LC) has been reported after its topical application in Macaque skin. A study was performed to evaluate the possible effect of topical tretinoin on the number of LC in human beings. Methods. Eight patients were studied. Topical tretinoin was applied in progressively increasing concentrations: 0.025% for 1 month, 0.05% for one month and 0.1% for 4 months. A skin biopsy from the malar area was taken before this therapy and 6 months later. To study LC, 4 p frozen sections were stained with the anti-CD1 antibody. Results. The number of CD1+ cells did not change when they were counted per unit of epidermal length, but they decreased when they were counted per unit of epidermal surface. Conclusions. These results indicate that topical tretinoin might damage epidermal Langerhans’cells, when it is applied for long periods of time; future studies are necessary to clarify this point.