In vitro and in vivo antiinflammatory activities of C10 substituted anthralin derivatives.
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Animals↗
Biomedical subjects
B Shroot
Publications and source records attributed to B Shroot.
Identification and subcellular distribution of cornified envelope precursor proteins in the transformed human keratinocyte line SV-K14.
SV-40 transformed human foreskin keratinocytes (line SV-K14) develop under conditions of serum starvation the competence to form cornified envelopes that are characteristic of terminally differentiating epidermal cells. In this cell line, the final assembly of the envelope does not occur spontaneously but must be induced using a calcium ionophore. Five potential precursor proteins with molecular weights of 140K, 90K, 61K, 53K, and 36K, respectively, could be detected in the extracts of envelope competent and noncompetent cells. The 61 kD and the 36 kD precursors were specifically decorated in immunoblots when using an antiserum directed against the purified cornified envelope of SV-K14 cells. The 140 kD protein was identified as involucrin by means of a commercial anti-involucrin antibody. Part of the 61 kD protein was found to be inserted into the plasma membrane after the cells gained envelope competence. The set of precursor proteins used by SV-K14 cells differed markedly from those described in the literature for epidermal cells in vivo and for normal human keratinocytes in vitro. Furthermore, cyanogen bromide cleavage of purified envelopes from transformed and normal keratinocytes revealed a completely different peptide pattern. This indicates that the exact molecular composition of the cornified envelope may not be strictly determined and may vary according to the availability of potential substrate proteins at the very moment when the cross-linking enzyme, the plasma membrane associated transglutaminase, becomes functional.
Cell Line↗
Induction of ornithine decarboxylase activity in hairless rat epidermis as a pharmacological model: validation of the animal model.
We use a mutant hairless Sprague Dawley rat to evaluate the capacity of retinoids to inhibit the epidermal ornithine decarboxylase activity induced by sellotape stripping. In order to minimize the variability introduced by the animals in our model we decided to validate the hairless rats used. A number of animal parameters were examined using a single lot of 50 males and 50 females aged from 4 to 11 weeks and acclimatized to laboratory conditions. The body weight growth curves were established. Nude animals present two periods of hair growth, the first at 6-7 weeks and the second at about 10-11 weeks. Hair development is more pronounced in males. No histological change was observed in the stratum corneum but an increase in epidermal thickness was noted in males aged 9 weeks. Removal of the stratum corneum by sellotape stripping was more effective and reproducible in the females, as determined histologically. Sellotape-stripping induction of ornithine decarboxylase in the epidermis was higher in rats aged 5-6 weeks and reached a plateau in animals aged 6-12 weeks. Individual variations obtained were lower in females (about 5%-10% in females and 10%-20% in males). The present research suggests that female rats aged about 8 weeks provide maximum reproducibility of response and ease of use.
Animals↗
Psoriatic hair follicle cells. III. Characterization of aberrant morphology in differentiating cultures.
Psoriatic and control human hair follicle keratinocytes were cultured on bovine eye lens capsules in Epicult dishes for a period of 5-6 weeks and examined using light microscopy. The following morphological differences between cultures were observed: 1. The lower cell layers contained predominantly flattened cells in psoriatic cultures instead of roundish in control cultures. 2. The differentiation pattern was irregular in psoriatic cultures instead of regular in control cultures. 3. The differentiated zone of psoriatic cultures was more compact and thicker in comparison to normal cultures. These differences might allow discrimination between normal and psoriatic cultures.
Cells, Cultured↗
Glucocorticoids: binding affinity and lipophilicity.
The relative binding affinity of 35 steroids for the glucocorticoid receptor was determined in experiments in which the competition of various unlabeled steroids with either [6,7-3H]dexamethasone or [1,2-3H]hydrocortisone for the cytosolic glucocorticoid receptor of cultured human keratinocytes was measured. The data obtained were correlated with steroid lipophilicity, measured as the partition coefficient of the steroid between 1-octanol and pH 7.4 aqueous buffer. The introduction of various substituents on the steroid molecule induced changes in the binding affinity and was associated in some cases with concomitant changes in steroid lipophilicity. The substitution by a 17 alpha-OH or 21-OH group leads in all cases to a decrease in steroid lipophilicity and to an increase in affinity. In contrast, 17 alpha-OAc and especially 21-OAc substitution on hydrocortisone and betamethasone causes a decrease in the steroid affinity for the receptor and an increase in steroid lipophilicity. The elongation of the ester chain from acetate to valerate in both position C-17 and C-21 leads to the increase in both the binding affinity for the receptor and the lipophilicity of steroids. However, all 21-esters showed lower binding affinity than the parent alcohol. The binding affinity of the highly lipophilic 17 alpha, 21-diester was found to be lower than that of the 17 alpha-ester but higher than that of the 21-ester or of the parent alcohol. Only in the series of 17 alpha- and 21-esters is there a correlation between the binding affinity of steroids for the glucocorticoid receptor and their lipophilicity.
Cells, Cultured↗
Receptor-linked adenylate cyclase in the membranes of cultured human epidermal keratinocytes.
The beta-adrenergic receptors, previously shown to be present on the membranes of cultured human epidermal keratinocytes, were found to be functionally coupled to membrane-bound adenylate cyclase. Using membrane preparations, the enzyme could be activated by guanosine triphosphate (GTP), the stable GTP analog GPP(HN)p, and NaF, all of which are known to activate the adenylate cyclase without interacting with membrane receptors. Binding of catecholamine agonists (epinephrine, norepinephrine, and isoproterenol) to the beta-adrenergic receptors is followed by an increase in the activity of adenylate cyclase. This activation could be reversed (or prevented) by beta-adrenergic antagonists, but was unaffect by the presence of alpha-adrenergic ligands (either agonists or antagonists). The activation by catecholamines appears to be directly related to receptor occupancy, since the activation constant (Ka) of adenylate cyclase for the three catecholamines was found to be very similar to the equilibrium dissociation constant (Kd) determined from competition binding experiments. The activation of adenylate cyclase under these conditions appears to be restricted to the catecholamine agonists only. The non-catecholamine beta-adrenergic agonists (salbutamol, terbutaline) did not show any measurable activation of adenylate cyclase, even though these agonists were shown previously to bind to the beta-adrenergic receptors on keratinocyte membranes with the expected affinities.
Adenylyl Cyclases↗
Psoriatic hair follicle cells. IV. Calmodulin levels in freshly isolated and cultured human scalp hair follicle cells.
Calmodulin levels were measured by radioimmuno-assay in freshly isolated and cultured psoriatic human scalp hair follicle cells. The mean value +/- SEM for calmodulin was 1.97 +/- 0.15 ng calmodulin micrograms-1 protein for 16 control subjects whereas calmodulin levels were significantly increased in psoriatic hair follicles, 2.93 +/- 0.26 ng calmodulin micrograms-1 protein (uninvolved skin) for 18 patients and 3.09 +/- 0.21 ng calmodulin micrograms-1 protein for involved skin derived hair follicles for 17 of these patients. In vitro, 3-week-old cultures of psoriatic keratinocytes contained less DNA and more calmodulin per DNA than their normal counterparts. When 6 week-old cultures of psoriatic and control hair follicle keratinocytes were compared, this difference disappeared. These results are related to the state of differentiation of these cultures.
Calmodulin↗
A rapid and simple test system for the evaluation of the inhibitory activity of retinoids on induced ornithine decarboxylase activity in the hairless rat epidermis.
In mouse skin, antiproliferative agents including retinoids inhibit induction of ornithine decarboxylase activity by a variety of hyperproliferative stimuli. In the hairless rat skin, ornithine decarboxylase activity was induced by ten successive strippings with cellotape and by topical application of 12-O-tetradecanoyl-phorbol-13-acetate. Topical application of all trans-retinoic acid (25 nmol/cm2) immediately after the tape stripping of the skin significantly inhibited the induction of ornithine decarboxylase activity at all time points measured. The inhibition by all trans-retinoic acid of ornithine decarboxylase induced by cellotape stripping was dose dependent as was found to be the case for arotinoid, retinol, Ro-10-1670, motretinid, 13-cis-retinoic acid, etretinate, and vitamin A. Oral administration of all trans-retinoic acid also inhibited the ornithine decarboxylase activity induced by cellotape stripping. We propose the assay of ornithine decarboxylase activity in the hairless rat epidermis after tape stripping for a rapid evaluation of new retinoids.
Animals↗
Pharmacology of topical retinoids.
The goal of research into retinoic acid is to maintain or improve the pharmacologic activities of topically applied retinoic acid while decreasing both local and systemic toxicity and improving chemical stability. Data are presented from a wide range of in vitro and in vivo test systems for retinoic acid, its congeners such as etretinate, and recently synthesized derivatives. The in vitro tests involved cell culture and biochemical assays: rat testes cytosol (receptor binding), mouse embryo teratocarcinoma cells (morphologic evaluation of differentiation), transformed human keratinocytes (cross-linked envelope formation), and polymorphonuclear leukocytes (lipoxygenase enzyme). The in vivo tests were carried out in the hairless rat (ornithine decarboxylase inhibition), the rabbit (irritation), and the guinea pig (ultraviolet erythema). Results from these experiments were used to determine biologic indices for the test substances. Subsequently, these test systems were used to evaluate a series of new compounds designed to control the chemical nature of a key double bond in the retinoic acid structure. Results are presented to show that different actions on the proliferative, differentiating, and inflammatory processes can be obtained by progressive aromatization of the retinoic acid structure.
Administration, Topical↗
Hyper-induction of skin ornithine decarboxylase activity in psoriatic patients.
Ornithine decarboxylase activity was measured in skin from 14 normal and 24 psoriatic subjects (with stable plaque type psoriasis vulgaris) using punch biopsies carried out immediately (0 h) and 6 h after stimulation by cellotape stripping. At 0 h, no significant difference was found between normal and involved or uninvolved psoriatic skin, but a significant increase was measured 6 h after tape stripping. This increase was significantly greater in uninvolved and involved psoriatic skin compared with normal skin. Preliminary results indicated that these changes were localized in the epidermis.
Adolescent↗
Reduced levels of histidine and urocanic acid in suction blister fluids from patients with psoriasis.
Metabolites of histidine were determined by high performance liquid chromatography in suction blister fluids from lesions and normal appearing skin of patients with psoriasis and from healthy subjects. There was a significant decrease in the levels of histidine and urocanic acid in the samples obtained from patients with psoriasis as compared to healthy subjects. Virtually only the E-isomer of urocanic acid was detected.
Adult↗
Free radicals in skin exposed to dithranol and its derivatives.
The generation of persistent free radical species derived from antipsoriatic drugs in their target tissue under use conditions is reported. Dithranol (anthralin, 1,8-dihydroxy-9-anthrone) and chemical analogues were applied topically to the ear of the pig and the time course of the production of the resulting free radicals was followed by electron spin resonance spectroscopy in biopsy samples. The spectral characteristics observed correspond to data previously reported in vitro for the anthralin-10-yl radical. The role of these radical species in the mode of action of the drug is discussed.
Animals↗
Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes.
When confluent cultures of the transformed human keratinocyte line SV-K14 are shifted to serum-free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross-linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane-bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.
Acyltransferases↗
Growth and differentiation of human keratinocytes cultured on eye lens capsules.
The efficiency of the outgrowth of human epidermal and hair-follicle-sheath keratinocytes was studied using three different growth substrates: plastic, type-I collagen and bovine eye lens capsules (the Epicult system). It was shown that the eye lens capsule is the best substrate, since a higher percentage of cultures showed outgrowth, and the outgrowth of epidermal keratinocytes was much more rapid. This effect is related to the faster migration (not proliferation) of cells grown on lens capsules as compared to the two other substrates. The view that lens capsules can replace the basement membrane present in vivo was supported by the finding that two basement-membrane components, i.e., laminin and fibronectin, are present on lens capsules. It was shown that, in cultures grown on lens capsules, bullous-pemphigoid antigen is restricted to the basal layer, indicating that the differentiation of these cells is comparable to that of keratinocytes grown on irradiated, non-viable pig dermis.
Animals↗
Differentiation of keratinocytes in vitro: a new culture vessel mimicking the in vivo situation.
A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO2 in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO2, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiation in vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.
Animals↗
Lipoxygenase products of arachidonic acid in human inflamed skin.
Monohydroxy acids (HETEs) and leukotriene B4 (LTB4) metabolites of arachidonic acid were measured in skin of healthy volunteers after ultraviolet B irradiation, and in the uninvolved skin of psoriatics after topical dithranol application. Exudate was collected from suction bullae on control and inflamed abdominal skin, and analysed for 12-HETE and PGE2 by GC-MS and LTB4 by bioassay. 12-HETE and PGE2 were raised at 24 h but not at 72 h after u.v.B irradiation: control and 24 h values were 13.7 and 41.5 ng ml-1 (P less than 0.05, n = 6) for 12-HETE respectively, and 4.5 and 30.2 ng ml-1 (P less than 0.01, n = 6) for PGE2. Dithranol application raised PGE2 levels from 23.1 ng ml-1 in control exudate to 62 ng ml-1 (P less than 0.01, n = 6) at 24 h before declining to base levels at 72 h. However, 12-HETE was raised at 72 h (200 ng ml-1, P less than 0.01, n = 5) but not at 24 h (104 ng ml-1) compared to control levels (50 ng ml-1, n = 5). The levels of the LTB4 were low (less than 100 pg ml-1), and no significant increases were observed. Arachidonic acid in inflamed skin can be metabolised by the cyclo-oxygenase and lipoxygenase pathway. It is probable that the lipoxygenase product 12-HETE is involved in these inflammatory reactions.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗