Aging of epidermis. I. Morphological changes and their quantitative analysis

Morphological changes in the epidermis caused by aging and sun exposure were studied by light microscopy and micrometric techniques. The following findings were obtained. 1. Corneocytes of the sun exposed skin of young healthy subjects were smaller than those of aged persons. 2. The epidermal rete ridges of the sun-exposed skin of old subjects disappeared and flattened. 3. The thinning and flattening of the epidermis of old subjects were more prominent in sun exposed skin than in sun protected skin. These data suggest that sun exposure accelerates some morphological changes of epidermis considered due to aging.     

Three-dimensional reconstruction and stereometric analysis of Langerhans cells in mouse epidermis.

In the presented studies stereometric analysis and spatial reconstruction was performed on two Langerhans cell (LC) types. One was free of LC-I and the other contained LC-II Birbeck granules in the perinuclear space. The presented stereometric analysis demonstrated significant differences between the so-distinguished two cell types. Differences were observed not only in the number and distribution of Birbeck's granules but also in the areas of smooth and rough endoplasmic reticulum, in the area of vesicles surrounding Golgi apparatus, in the volume of cisterns of the apparatus, and in the ratio of cell nucleus area to its volume. Differences noted between the two cell types were of quantitative character. They might result from different stages of differentiation of the cells from their precursors in the epidermis or from distinct functional stages of the cells.     

Presence of two typical DNA-binding nonhistone proteins in psoriatic scales contrary to normal human dermis,epidermis and horny layer

Summary The composition of DNA-binding proteins (DBP) was shown to be tissue-specific and to vary at different stages of gene expression. As the accelerated epidermopoesis in psoriasis indicates changed gene activities, DBP of psoriatic scales were compared with those of normal human epidermis, dermis and horny layer.Each skin fraction is characterized by its own DBP pattern, indicating different cell species. 1. The DBP of normal human epidermis shows only a small accordance with the DBP of human dermis and implies their difference in origin, function and cell types. 2. Psoriatic scale DBP and epidermal DPB contain more corresponding proteins which can be deduced from the scale's origin from epidermis. However, the composition of all proteins differs to a great extent. This either occured during parakeratotic keratinization or reflects differences of normal to psoriatic epidermis. Imposing for psoriatic scale DBP are two protein bands with molecular weights of 84,000 and 90,000 daltons. Evidently both are not present in the DBP of other skin layers. 3. The horny layer contains a very small amount of DBP which might represent DNases to a major part. The small DBP content in horny layer confirms the previous supposition of psoriatic scales, to be mostly derived from the preserved nuclei of the parakeratotic scale layer.

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Abbreviations DBP DNA-binding proteins - NHP nonhistone proteins Supported by grant of the Deutsche Forschungsgemeinschaft     

Localization of calbindin-D28k in normal and incised mouse skin: immunohistochemical and immunoblot analysis

The subcellular localization of the protein calbindin-D28k in normal and incised mouse skin was investigated by immunohistochemical staining and immunoblot analysis. In normal skin, the presence of calbindin-D28k was demonstrated in both the nucleus and the cytoplasm of epidermal keratinocytes. Higher levels of calbindin-D28k were detected in the nucleus than in the cytoplasm. Following incision, the levels of calbindin-D28k were significantly decreased in epidermal keratinocytes, particularly in the nucleus, compared with those in normal skin. The immunohistochemical staining and immunoblot analysis showed nuclear calbindin-D28k to be decreased or absent during a 10-day observation period following skin incision. Based on these findings, it is suggested that calbindin-D28k may be distributed in both the nucleus and cytoplasm of epidermal keratinocytes in mice, and this protein may be involved in active cell proliferation of the epidermis induced by skin incision. Received: 7 March 1997     

Determination and correlation of in vitro viability for hairless mouse and human neonatal whole skin and stratum corneum/epidermis

Background and design: Viable tissue is essential to assess the rate and extent of biotransformation during percutaneous absorption in vitro. We assessed the viability of hairless mouse whole skin (WS) and stratum corneum/epidermis (SCE) and human neonatal SCE following separation from the dermis by EDTA phosphate-buffered saline (EDTA-PBS) incubation or by heat treatment by measuring the conversion of dextrose to lactate. Lactate concentrations in receptor fluid samples were determined using a Sigma diagnostic lactate determination kit. A standard curve was prepared and samples assayed spectrophotometrically at 340 nm using a lambda 2β spectrophotometer. Standard curves were prepared for each experiment and correlation coefficient values ( r ) were calculated. Results: Our results showed that heirless mouse SCE was associated with glucose conversion to lactic acid at an increased rate if incubated in EDTA-PBS for 4 h and used immediately. Lactate production was greater with the dermis present (EDTA-PBS WS). The rate of glucose to lactate conversion in hairless mouse SCE was 20–25% of that found in WS. Compared with Dulbecco’s modified PBS (DMPBS)-treated WS controls, the rate of lactate production in EDTA-PBS-treated WS was nearly a 50% less. Heat treatment in water at 60° C to separate SCE from hairless mouse WS appeared to eliminate viability. Viability of hairless mouse SCE, as measured by glucose conversion to lactate, was comparable to human neonatal SCE. Conclusions: These results suggest that the dermis is a significant contributor to glucose metabolism and that incubation in EDTA-PBS is a contributing factor to the overall decrease in metabolic capacity of the tissue. As a result of these findings, hairless mouse SCE appears to be useful as a model for human neonatal SCE in percutaneous absorption studies. Received: 1 September 1995     

Differential expression of heat shock protein 70 (HSP70) and heat shock cognate protein 70 (HSC70) in human epidermis

Autoimmunity and microbial agents have been suggested as playing a pathogenetic role in psoriasis. Since immune responses to microbial infections are often directed towards heat shock proteins (HSP), we investigated the expression of three HSP families in normal and inflamed human skin. Specimens from ten patients with psoriasis and three patients with positive patch tests for nickel and from five healthy volunteers were analysed by means of immunohistochemistry. The patterns observed were qualitatively similar in these conditions showing only minor quantitative differences. Psoriatic epidermis exhibited the highest level of expression. HSP27, HSP70 and heat shock cognate protein 70 (HSC70) were readily detectable. HSP27 was homogeneously distributed throghout the epidermis, whereas HSP70 was restricted to the basal layer and HSC70 primarily to the suprabasal layers. Other HSPs were detected to a lesser degree and showed a more irregular pattern. Thus, the qualitative expression pattern of HSPs seems to be constant between different skin conditions, but the expression of constitutive and inducible HSP70 depends on the differentiation state of keratinocytes.Abbreviations BiP immunoglobulin heavy chain binding protein - GrP glucose regulated protein - HSC70 heat shock cognate protein 70 - HSP heat shock protein - HSP70 heat shock protein 70     

Comparison of DNase,DNA-polymerase and RNA-polymerase activities present in the DNA-bindung proteins of normal human dermis,epidermis, horny layer and psoriatic scales

Summary DNA-bindung proteins (DBP) of normal human dermis, epidermis, horny layer and psoriatic scales represent a tissue-specific group of mostly nuclear nonhistone proteins. To analyse their function, the different DBP fractions were examined concerning the presence of DNase, DNA-polymerase and RNA-polymerase activities.DBP of normal epidermis and horny layer contain four different DNases. One DNase of both DBP fractions is active only at pH 5.0. Three DNases of epidermal DBP are active at a pH-range from 5.0–8.5, while the corresponding DNases of horny layer-DBP are most active at pH 7.4. Probably these DNases have changed their pH-optimum during keratinisation. DBP of psoriatic scales include no activity of these three DNases and the pH 5.0-DNases seem to have reduced DNA-affinity. Human dermis DBP contain quite another set of four DNases which hardly can be correlated to the DNases of epidermal DBP.DNA-polymerase activities are present in each fraction and derive from different DNA-polymerases. Two DNA-polymerases with pI-values of 4.5 and 9.3 may correspond to - and -DNA-polymerase of eukaryotes, respectively. Further activity of proteins which are focussed at pH 6.5–7.2 and 8.2 could be detected. The proteins represent either tissue-specific DNA-polymerases or further thymidine monophosphate incorporating enzymes. Contrary, RNA-polymerase activity could not be enriched from correlating extracts by DNA-cellulose chromatography.

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Abbreviations (d)ATP (deoxy)adenosine 5-triphosphate - (d)CTP (deoxy)cytine 5-triphosphate - DBP DNA-binding proteins - DNase deoxyribonucleate 5-oligonucleotidohydrolase (EC 3.1.4.5) = deoxyribonuclease - EDTA ethylendiaminetetraacetic acid sodium salt - (d)GTP (deoxy)guanosine 5-triphosphate - RNase ribonucleate 5-oligonucleotidohydrolase (EC 3.1.4.22) = ribonuclease - TCA trichloroacetic acid - TTP thymidine 5-triphosphate - TMP thymidine 5-monophosphate - UTP uridine 5 triphosphate - UMP uridine 5 monophosphate Supported by grant of the Deutsche ForschungsgemeinschaftSupported by grant of the Deutsche Forschungsgemeinschaft     

A review and mathematical analysis of circadian rhythms in cell proliferation in mouse, rat, and human epidermis.

The data from published studies of circadian rhythms in epidermal cell proliferation in mice, rats, and humans were reanalyzed. They were calculated as the percent difference from the mean at six timepoints over 24 h. Composite circadian rhythm curves were plotted from the combined data for each species for S-phase and M-phase. Each group of studies showed a general consensus on timing, and the composite curve showed a regular sinusoidal pattern. The rhythms in mice and rats were the same, whereas those in humans were in the opposite phase and had reduced amplitudes. In the rodents, S-phase peaked at about 3:30 A.M. and M-phase peaked at about 8:30 A.M. In humans S-phase peaked at about 3:30 P.M. and M-phase peaked at about 11:30 P.M. If the timing of the circadian rhythms in cell proliferation can be firmly established, it may be possible to schedule drug treatments to take advantage of the differences in cell proliferation rates at different times of day.     

Heat-separation of normal human skin for epidermal and dermal prostaglandin analysis

Summary Heat-separation was introduced as a simple, reliable method of obtaining pure epidermis and dermis for prostaglandin (PG) analysis. Heating of normal human skin at 60°C for 1 min resulted in a distinct separation of the epidermis from the dermis. After heat-separation the mean concentration ±SEM of PGE₁ activity in normal epidermal tissue was 2906±281 pg/mg dry weight. The PGE₁ level in the corresponding dermal samples was 30±4 pg/mg dry weight and the mean leakage of PGE₁ from the tissue into the buffer used during heating was 426±54 pg/ml.This work was supported by grant 512-8125 from the Danish State Research Foundation     

Flow cytometric sorting and keratin analysis of human epidermal basal cells

A prerequisite condition for the elucidation of the expression of keratin subunits in human basal cells is obtaining highly purified basal cell fractions. A method has therefore been developed for the isolation of the basal cell sub-population from normal human epidermis at very high purity. Epidermal cell suspensions obtained by trypsinization at 4°C were first enriched in basal cells by selective attachment to collagen. The cells were then stained in suspension with KLI monoclonal antikeratin antibody which had been shown to be reactive with suprabasal cells. The suprabasal cells contaminating the suspensions were then eliminated by selective flow cytometry sorting and basal keratinocyte populations of 99.5% purity were obtained. Analysis of the keratin composition of these cells showed that the large majority of basal cells contained two keratins, one from each keratin subfamily: type I acidic, 50 Kd [keratin no 14 as defined by Moll et al. (1)] and type II basic, 58 Kd [no 5]. Nevertheless, a small subpopulation of basal cells (2%) was shown to express, in addition to the 50/58 Kd subunits, the 56.5/65–67 Kd keratins (nos 10, 1–2) which are regarded as markers for cells committed to terminal differentiation.     

Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflammation. I. IL-1 gene family

Acute disruption of the cutaneous permeability barrier with either solvents or tape-stripping stimulates a homeostatic metabolic response in the subjacent nucleated layers of the epidermis that results in a rapid restoration of normal permeability barrier function. When the aged epidermal permeability barrier is stressed, it reveals a diminished capacity for recovery, in comparison to young epidermis, analogous to other organs in the aged when stressed. Although the signals that regulate this homeostatic response by the epidermis have not yet been resolved, acute permeability barrier disruption stimulates release of prestored IL-1alpha, and increased production of potentially regulatory cytokines, including IL-1alpha and TNFalpha in the epidermis. In these studies, we addressed the hypothesis that cytokine dysregulation explains the permeability barrier abnormality in aged epidermis, assessing the regulation of IL-1 and TNF signaling in aged vs young mice. To determine whether the IL-1 family of cytokines plays a key role in the permeability barrier abnormality of the aged, permeability barrier recovery rates were compared in transgenic mice lacking the functional IL-1 type 1 receptor vs wild-type mice at various ages. Knockout of the IL-1 type 1 receptor exacerbates the defect in permeability barrier homeostasis that is seen in age-matched, wild-type counterparts. Furthermore, the sluggish permeability barrier recovery in aged epidermis is associated with, and at least in part attributable to, altered expression of the IL-1 family of cytokines and receptors both under basal conditions and after acute barrier perturbations. Whereas modulations in cytokine expression with epidermal permeability barrier perturbation are qualitatively similar in aged epidermis, they greatly differ quantitatively. In contrast, examination of TNFalpha mRNA and protein basally, and following barrier perturbation revealed no alterations in aged epidermis. Together, these results show that selective alterations in the IL-1 family of cytokines occur with aging and that defects in IL-1 signaling may contribute to the epidermal permeability barrier abnormality of aged skin.     

Image analysis for quantification of nucleolar organizer regions in basal cell carcinoma and seborrheic keratosis

Background/aims: Nucleolar organizer regions (NORs) have recently attracted much attention because of claims that their frequency within nuclei is significantly higher in malignant cells than in normal, reactive, or benign neoplastic cells. The purpose of this paper is to analyze a method allowing selection of the best morphometric criterion for quantifying AgNORs (Silver stained Nucleolar organizer Regions) under conventional observation conditions, by light microscopy.
Methods: The various parameters including NORs counting in cutaneous tumors using image analysis system were studied.
Results: There were significant differences in mean numbers of AgNORs per nucleus, mean ratio of AgNORs area per nucleus area, mean ratio of greatest AgNORs area per nucleus area, mean nucleus area per a AgNOR, and CV (Coefficient of Variation) of AgNORs area between basal cell carcinoma and seborrheic keratosis.
Conclusion: Study of AgNORs using the image analysis system is a useful tool for diagnosis of cutaneous tumors.     

Clinical and histopathological characteristics of basal cell carcinoma in Korean patients.

Seventy-eight Korean patients with basal cell carcinoma (BCC) between 1984 and 1998 were retrospectively examined at Ewha Womans University Tongdaemun Hospital, Seoul, Korea. We analyzed the annual incidence, age and sex distribution, site of the lesions, clinical appearance, including the proportion of clinically pigmented tumors, modalities of treatment, incidence of recurrence and metastasis of the tumors, the histopathological patterns, and whether solar elastosis, microscopic pigmentation, or adamantinoid feature were associated. The male-to-female ratio was 0.902, and the average age of the patients at first examination was 58.2 years. Eighty percent of the tumors occurred on the head and neck, most commonly on the nose (26.9%), followed by the cheek, eyelid, and upper lip. Ulcerated nodules were the most common clinical presentation. Clinically, 55% of the tumors were pigmented. Six tumors recurred; none metastasized. Surgical excision was the most common modality of treatment. The most frequent histopathological pattern was the solid type (60.3%), followed by the superficial (11.5%) and fibrosing (9.0%) types. The occurrence of the superficial type was significantly associated with truncal lesions (p < 0.001). Solar elastosis was present in 62.1% of the tumors on the head and neck, compared with 8.3% in those of the trunk and limbs (p < 0.001), indicating the significance of sun exposure in the pathogenesis of BCC on exposed areas. Microscopic pigmentation was seen in 69.2% of the tumors. The focal adamantinoid feature was found in 14.1%, which is much higher than the previously reported incidence.