Effects of methotrexate in vitro on epidermal cell proliferation

Epidermal cell migratory activity and epidermal cell proliferation are clearly dissociable in explant culture. Epidermal cell migration requires a non-dialysable, 65,000 mol.wt factor which is destroyed at 100 C but is stable at 80 C for at least 30 min. In the presence of dialysed serum or heated serum (80° C for 30 min) or DNA synthesis inhibitors (methotrexate or hydroxyurea), cells will migrate from the explant but will not proliferate. At least two factors are required for normal proliferation under these restricted conditions—an adequate supply of DNA precursers, i.e. nucleosides, and a heat labile (80° C) non-dialysable serum component. Methotrexate in concentrations of 1.0 μg/ml or greater added to cultures in normal fetal calf serum significantly inhibited mitoses; however, when added to serum dialysed to remove thymidine, mitotic inhibition occurred at a concentration of 0.1 μg/ml of methotrexate and when added to dialysed serum and kept in dialysed serum, inhibition occurred with 0.01 μg/ml of methotrexate. Methotrexate did not inhibit outgrowth. Hydroxyurea also inhibited mitoses but did not effect outgrowth.     

Abnormal epidermal cell proliferation on the elbow in psoriatic and normal skin

Summary The influence of various zinc concentrations on the osmotic resistance of erythrocytes was studied in six normal adults. Sodium chloride solutions ranging from 0.1% to 0.7% were prepared, each containing 0 g% zinc, 50 g% zinc, 100 g% zinc, 500 g%, and 1000 g% zinc. Heparinstabilized venous blood was added to the various solutions at 25°C, and the degree of haemolysis (erythrolysis) after 1 h was determined by measurement of the absorbance at 540 nm. Neither the absence, nor the presence of relatively high zinc concentrations were found to influence the degree of haemolysis. The results indicate that haemolytic anaemia which was observed in zinc-deficient patients is not due to a zinc-dependent change in the osmotic resistance of erythrocytes.     

The epidermal cell proliferation in lichen planus.

The proliferative activity of epidermal structures was determined in 6 patients suffering from lichen planus (i.p.). By using the in vitro 3H-thymidine labelling technique, semithin Epon sections of lesional and perilesional uninvolved skin were compared autoradiographically with normal controls. The following results were obtained. The labelling index of epidermal cells was significantly increased within the i.p. papules. The mean values ranged from 41% in acanthotic i.p. to 23% in atrophic i.p. whereas in adjacent unaffected skin (15% and 11% respectively) as well as in normal controls (6%) a lower 3H-index was observed. As regards damage to basal cells within i.p. lesions, a factor--the relation between the number of basal cells in involved and uninvolved skin in reference to the epidermal length--was calculated in order to correct the autoradiographic results in involved skin. The differences between i.p. papules, adjacent skin and controls remained statistically significant even after correction. The intradermal eccrine ducts revealed a significantly increased labelling index in i.p. lesions as compared with perilesional and normal skin. In this respect no difference could be observed between the acanthotic and the atrophic variant of i.p. These findings indicate that replacement of damaged basal cells in i.p. is achieved by an increase in actively dividing keratinocytes in both epidermis and skin appendages within the lesion.     

Autoradiographic in vitro studies on diurnal variation in human epidermal cell proliferation

Summary Five men with healthy skin ranging in age from 24 to 37 years were investigated for the presence of diurnal variation in epidermal cell proliferation by the in vitro³H-thymidine labeling technique. The following parameters were studied: The basal cell LI; the numbers of labeled basal and supra-basal cell nuclei as well as the total number of labeled nuclei situated above a basal membrane length of 100 µm.All autoradiographic parameters showed marked diurnal variations, the amplitudes of which differed individually. The basal³H labeling index always showed the highest diurnal variation. In all patients diurnal variations of the basal and suprabasal cell proliferation occurred simultaneously and in equal direction. However, each test person showed maxima and minima of epidermal DNA synthesis at different times. No synchronism could be found when comparing the individual variations of cell proliferation. Several possible reasons for the asynchronous in vitro behaviour of epidermal cells will be discussed.

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Zusammenfassung An 5 hautgesunden Männern im Alter von 24-37 Jahren wurde durch in vitro-³H-Thymidinmarkierung geprüft, ob tageszeitliche Schwankungen der epidermalen Proliferation bestehen. Untersucht wurden folgende Parameter: der³H-Index der Basalzellkerne, die Anzahl markierter Basalzellkerne/100 µ Basalmembranlänge, die Anzahl markierter Suprabasalzellkerne/100 µ Basalmembranlänge und die Gesamtanzahl markierter Zellkerne/100 µ Basalmembranlänge.Sämtliche autoradiographische Parameter zeigten deutliche diurnale Schwankungen, deren Amplitude individuell variierte. Die ausgeprägteste tageszeitliche Variabilität wies in allen Fällen der basale³H-Index auf. Die im einzelnen beobachteten diurnalen Schwankungen der basalen und suprabasalen Proliferation traten gleichzeitig auf und verliefen gleichsinnig. Jedoch zeigte jede Versuchsperson zeitlich unterschiedliche Maxima und Minima der epidermalen DNS-Synthese, so daß keine diurnale Synchronizität der individuellen Proliferationsschwankungen nachgewiesen werden konnte. Einige mögliche Gründe für das desynchrone Verhalten der Epidermiszellen in vitro werden diskutiert.

     

Inhibitors of epidermal cell DNA synthesis in surviving pig skin in vitro

Keratome slices of domestic pig skin were used to study the DNA synthesis phase of epidermal cell DNA synthesis. Cyclic AMP and agents which elevate intracellular concentrations of cyclic AMP have no direct effect on the "S" phase of DNA synthesis. Theophylline, isobutylmethylxanthine, and adenosine inhibit DNA synthesis immediately by a mechanism which is reversible and is not dependent on cyclic AMP. This inhibition is not associated with an increase in intracellular thymidine phosphates. Hydroxyurea, however, inhibits DNA synthesis immediately and does produce an elevated pool of thymidine phosphates.     

Cytophotometric studies of epidermal proliferation in psoriatic and normal skin.

Cytophotometric measurements of single-cell DNA content were used to study human epidermal cell proliferation in vivo. It was found that Feulgen-DNA profiles can be used to assess proliferative activity in involved, uninvolved, and nonpsoriatic skin. Profiles of involved psoriatic skin were bimodal as is characteristic of actively proliferating populations. This was due to the presence of cells with twice (2C--presynthetic) or four times (4C--post-synthetic) the amount of DNA of the gametes, separated by the intermediate values of cells undergoing scheduled DNA synthesis. Profiles of uninvolved psoriatic as well as nonpsoriatic epidermis were unimodal with the majority of cells containing a 2C amount of DNA incating relatively low levels of proliferative activity. The observed variations in proliferative activity of these samples are discussed in terms of two alternative models. Since radioisotopes are not required, this technique presents a useful approach to studying human epidermal proliferation in vivo.     

On circadian rhythms in human epidermal cell proliferation.

The ascertainment of consistent diurnal variations in human epidermal cell proliferation may have important implications for the treatment of many skin diseases. For the evaluation of diurnal rhythms in the growth of human epidermis, skin biopsies were taken every 4th hour for 48 h from each of two persons under synchronized living conditions. The epidermal cell proliferation was assessed by the fraction of cells in S and in G2-M phase as determined by measurements of the DNA content in the individual cells in single-cell suspensions. The existence of diurnally consistent body functions in the test persons was verified by monitoring the excretion of cortisol by urine. The fraction of cells in G2-M phase indicated circadian rhythmicity for the first 32 h of the test period. No regular variation according to time of day could be established in the fraction of cells in S-phase.     

In vitro uptake of L-dopa and catecholamines into the epidermal Langerhans cell.

The Langerhans cells are capable of taking up L-dopa and the catecholamines dopamine and noradrenaline when exposed to these substances in vitro. Within the cell L-dopa is found in the cytoplasm as well as in the nucleus, whereas the catecholamines are confined to cytoplasmic granules. The L-dopa uptake is most probably carrier-mediated and the hypothesis is brought forward that L-dopa enters the cell by exchange diffusion. At present little is known about the nature of the amine uptake mechanism.     

Trypsin induces epidermal proliferation and inflammation in murine skin

Human keratinocytes are known to express the protease-activated receptors, PAR-1 and PAR-2. Activation of PAR-1 results in increased proliferation, whereas PAR-2 activation results in decreased keratinocyte proliferation. Trypsin activates PAR-1 and in higher concentrations, PAR-2. The aim of this study was to evaluate the overall effect of trypsin on keratinocyte proliferation in a mouse in vivo and in vitro model. Daily topical application of 0.3-300 pmol trypsin/cm2 on hairless mouse skin induced dose-dependent epidermal hyperproliferation as determined by an increase in 5-bromo-2'-deoxyuridine incorporation of up to eight-fold in basal keratinocytes and an up to three-fold increase in keratinocyte layers. This was accompanied by an increased transepidermal water loss. These effects of trypsin were abolished by the addition of the trypsin inhibitor n-p-tosyl-l-lysine-chloromethyl ketone. Histological analysis revealed acanthosis, hypergranulosis, and spongiosis in the epidermis as well as vasodilatation and an inflammatory infiltrate in the upper dermis. In the murine keratinocyte cell line PAM-212 activation of PAR-1 with specific activating peptides resulted in a calcium influx and an increase of proliferation, whereas activation of PAR-2 caused a diminished proliferation. Incubation with trypsin, PAR-1-, and PAR-2-activating peptides induced cytokine-induced neutrophil chemoattractant (KC) mRNA expression as a marker for inflammation in PAM-212 in a dose-dependent manner. In conclusion, our results suggest that trypsin induces in vivo epidermal proliferation and inflammation. Proliferation seems not to be signaled by PAR activation, but PAR-2-induced KC chemokine expression may contribute in part to trypsin-induced inflammation.     

Active epidermal movement in human skin in vitro

Epidermal cells migrate from the edges of adult human skin in culture during the first 48 h. In order to determine whether mitotic acitivity was responsible for this cell movement the autoradiographic labelling index for cells in DNA synthesis was determined for skin at different stages of culture. The labelling index of skin maintained for 48 h but labelled with tritiated thymidine in the first 4 h, did not differ significantly from skin both maintained and labelled for 4 h only. Skin maintained for 48 h but labelled in the last 4 h showed a much lower labelling index. No normal mitotic figures were seen in the epidermis of skin maintained for 48 h but exposed to colcemid for the last 4 h of culture. It has been concluded that at least in short-term organ culture of adult human skin there is a phase of mitotic arrest and that epidermal migration during the first 48 h is an active process independent of mitotic activity.     

In vitro examination of cell proliferation in dermatofibroma and in the overlying epidermis

Summary In nine typical cases of dermatofibroma autoradiographic methods were used to examine the proliferative activity in the tumor and in the overlying epidermis. The number of DNA-synthesizing cells in the dermal nodular lesions was extremely low (0–0.1%). In seven of nine skin lesions acanthosis was to be seen in the overlying epidermis. The quantity of DNA-synthesizing cells in the epidermis overlying the dermatofibroma was not related to the thickness of the epidermis and remained independent, whether the predominant element in the tumor is fibrillar or cellular. In this study we were able to demonstrate the existence of a direct relation between the degree of epidermal proliferation and the distance between the tumor and the epidermis. The duration of DNA-synthesis was constant.Herrn Prof. Dr. G. W. Korting zum 60. Geburtstag     

Alginate oligosaccharides modulate cell morphology, cell proliferation and collagen expression in human skin fibroblasts in vitro

Abstract Effects of alginate oligosaccharides on cell proliferation and expression of collagen in cultured skin fibroblasts were studied. The oligosaccharides were found to suppress fibroblast proliferation to half the level in control cultures at a dose of 10 mg/ml during a period of 5 days. The inhibition was accompanied by a change in cell shape. The inhibition of cell proliferation was reversible, since depletion of these oligosaccharides led to a recovery of cell motility. Treatment of confluent cells with 10 mg/ml oligosaccharides for 5 days resulted in a reduction in collagen synthesis to one half of that in control cultures and inhibition of steady state levels of α₁(I), α₂(I), α₁(III) and α₁(VI) collagen mRNAs. These results suggest that alginate oligosaccharides are potential modulators of dermal fibroblasts and may provide a useful tool for the treatment of disorders related to abnormal collagen metabolism. Received: 16 October 1998 / Received after revision: 8 February 1999 / Accepted: 12 February 1999     

Is epidermal cell proliferation in psoriatic skin grafts on nude mice driven by T-cell derived cytokines?

Plasminogen activity and DNA synthesis by epidermal cells have been reported to be doubled in psoriatic skin grafts compared with grafts of normal skin 6 weeks after transplantation to nude mice. In our study human lymphocytes disappeared from such grafts within 48 h whilst some DR-positive human dendritic cells were retained in the grafts for up to 4 weeks. However, the grafts were infiltrated by Thy 1.2+ mouse lymphocytes within 6 days and this infiltration persisted at a moderate level throughout the observation period. It consisted of perivascular aggregates, scattered dermal and papillary T cells, and some mouse T cells were also found in the epidermal compartment. Grafts of psoriatic and non-psoriatic control skin were infiltrated to a similar extent, suggesting a low-grade rejection response against the human xenografts. These findings raise the possibility that psoriatic keratinocytes are responding abnormally to inflammatory cytokines released by mouse lymphocytes reacting against the skin grafts.     

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

Background

With the availability of large-scale genome-wide association study (GWAS) data, choosing an optimal set of SNPs for disease susceptibility prediction is a challenging task. This study aimed to use single nucleotide polymorphisms (SNPs) to predict psoriasis from searching GWAS data.

Methods

Totally we had 2,798 samples and 451,724 SNPs. Process for searching a set of SNPs to predict susceptibility for psoriasis consisted of two steps. The first one was to search top 1,000 SNPs with high accuracy for prediction of psoriasis from GWAS dataset. The second one was to search for an optimal SNP subset for predicting psoriasis. The sequential information bottleneck (sIB) method was compared with classical linear discriminant analysis(LDA) for classification performance.

Results

The best test harmonic mean of sensitivity and specificity for predicting psoriasis by sIB was 0.674(95% CI: 0.650-0.698), while only 0.520(95% CI: 0.472-0.524) was reported for predicting disease by LDA. Our results indicate that the new classifier sIB performs better than LDA in the study.

Conclusions

The fact that a small set of SNPs can predict disease status with average accuracy of 68% makes it possible to use SNP data for psoriasis prediction.