Characterization of the beta-adrenergic receptors of cultured human epidermal keratinocytes.

The presence of beta-adrenergic receptors has been demonstrated in membrane preparations from passaged human epidermal keratinocytes. The receptors were characterized in terms of density and binding properties. Using the titrated beta-adrenergic antagonists dihydroalprenolol and propranolol, the equilibrium dissociation constant (Kd) was found to be about 1.4 nM for the two antagonists with a receptor density of approximately 280 fmol/mg membrane protein. Stereospecificity of the binding sites was shown by the much lowered affinity to D-isoproterenol as compared to that of L-isoproterenol. By the use of subtype specific antagonists, the receptors were classified as beta 2 adrenoceptors. This finding is supported by the relative order of affinities of the agonists isoproterenol greater than epinephrine greater than norepinephrine. The Kd value for dihydroalprenolol was approximately the same when determined from equilibrium binding studies or from association and dissociation kinetics, suggesting that the ligand binding is a single step bi-molecular reaction.

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.

Anthralin: chemical instability and glucose-6-phosphate dehydrogenase inhibition.

The chemical stability of the antipsoriatic drug, anthralin (1,8-dihydroxy-9-anthrone), in solution has been studied using high-performance liquid chromatographic analysis. The time course for decomposition in solution has been correlated with that of the inhibition of glucose-6-phosphate dehydrogenase, one of the most widely documented biochemical properties associated with anthralin. Solutions of anthralin in aqueous buffer (37 degrees, pH 7.5, under light protection) completely within 4 hr giving the 10,10'-dimer (40%), no detectable 1,8-dihydroxy-9,10-anthraquinone, and a greatly increased potency of inhibition of glucose-6-phosphate dehydrogenase. This increased inhibitory potency could not be explained by formation of the dimer which, like anthralin and its quinone, were shown to be only weak inhibitors of the enzyme. In acetone solution exposed to light and air, anthralin decomposed completely within 4 days, in part via the dimer as intermediate. The final solution had the characteristic color of anthralin-brown, contained the quinone (20%), and like decomposed aqueous solutions of anthralin, completely inhibited glucose-6-phosphate dehydrogenase. The results show that neither anthralin, nor either of its two identified decomposition products, is the potent toxic species against glucose-6-phosphate dehydrogenase.

Equine herpesvirus type 2: prevalence and seroepidemiology in foals.

Whole blood and serum were collected from foals to determine the prevalence of Equine herpesvirus type 2 (EHV 2) infection in foals, age at which infection can first be identified and serological responses to infection. Equine herpesvirus type 2 was isolated from peripheral blood mononuclear cells (PBMC) from 68 of 69 foals, 1-8-months-old, sampled once. Virus isolation was performed twice at intervals of 2-7 months on PBMCs from 33 foals and EHV2 was isolated on both occasions in all but one foal (negative, then positive). Regression analysis of log2-transformed reciprocal serum EHV2 virus neutralising (VN) titres revealed that in foals age 1-7 months, EHV2 VN antibody titre was positively correlated with age (r = 0.94). Paired serum samples were obtained from 58 foals, with the first samples collected age 1-6 months and the second samples collected 2-4 months later. There were significant (P < 0.05) increases in mean VN titres to EHV2 in foals sampled initially at age 1-4. Eight foals had blood sampled prior to sucking and at age 7, 20, 30 and 45 days. Each foal was negative for EHV2 in PBMC and each foal had a negative serum EHV2 VN titre immediately after birth. Each foal was positive for EHV2 in PBMC by age 45 days, with the earliest isolation at 25 days. Tracheal aspirate fluid and peripheral blood were collected from 20 foals without clinical signs of respiratory disease and from 30 foals with clinical signs of lower respiratory disease. In 20 foals without clinical signs of respiratory disease, EHV2 was isolated from tracheal aspirates (1/20 foals) and PBMC (20/20 foals) and in 30 foals with such clinical signs, from trachea aspirates (20/30 foals: P < 0.01) and from PBMC (30/30 foals). In one 6-month-old foal, EHV1, but not EHV2, was isolated from the tracheal aspirate, 3 months after EHV2 had been isolated from a tracheal aspirate. These results demonstrate a greater prevalence of EHV2 in lower respiratory secretions in foals with clinically apparent lower respiratory disease, but a cause and effect relationship between the virus and lower respiratory disease remains to be elucidated. It is noteworthy, however, that of virus isolations performed on 50 tracheal aspirates, a virus (EHV1) other than EHV2 was isolated only once.

Signs of sympathetic denervation associated with a thoracic melanoma in a horse.

Sympathetic denervation in a 20-year-old, gray, Thoroughbred-Percheron gelding was manifested by cutaneous hyperthermia and sweating over the right side of the body, demarcated by a line from the withers to the elbow and extending cranially. There was cutaneous hyperthermia over the right side of the head, but other signs of Horner's syndrome (sweating, ptosis, miosis, enophthalmos) were not present. The pattern of cutaneous hyperthermia and sweating was consistent with sympathetic denervation localized to the cervicothoracic ganglion, and thoracic radiographs revealed increased density in the craniodorsal thorax. Cytologic evaluation of a sample of pleural effusion revealed mesothelial cells containing melanin and cells suggestive of melanocytes or melanoblasts. Treatment with oral cimetidine and intrapleural cisplatin was not successful. A necropsy was not performed, but the clinical findings supported a diagnosis of thoracic melanoma involving the cervicothoracic ganglion.

Epidermal growth factor-like activity in mares' milk.

Epidermal growth factor (EGF)-like activity was measured in mares' colostrum and milk by radioreceptor assay. Milk samples were collected from 22 mares 1 or more times during early lactation. Samples of colostrum were taken after parturition and before the foal first suckled (presuckle), within 6 hours after the foal first suckled (postsuckle), and on days 1, 2, 4, and 8 of lactation. In the 5 mares from which milk samples were obtained at each sampling time, presuckle colostral mean EGF-like activity (17.8 ng/ml) was greatest (P less than 0.05). The mean values for EGF-like activity at all other sampling times were not significantly different from each other (postsuckle colostrum, 9.7 ng/ml; day 1, 9.6 ng/ml; day 2, 8.5 ng/ml; day 4, 8.0 ng/ml; day 8, 7.8 ng/ml).

The in vivo fate of topically applied dithranol in the skin of the hairless rat. A comparison of continuous and short contact application.

The fate in vivo of topically applied 1,8-dihydroxy-9-anthrone (dithranol, anthralin) was investigated in the skin of the hairless rat, using a specially designed drug delivery system (film). The film was applied on intact skin as well as on skin with an impaired barrier function (stripped skin). The distribution of the drug was examined either after continuous application or at selected times after short contact periods. The exposed skin was extracted with diisopropylether and free dithranol, its dimer and quinone assayed by quantitative HPLC analysis. The incorporation of trace amounts of 3H-dithranol and 14C-dithranol in the vehicle made it possible to quantify the fraction of penetrated drug which was insoluble in ether. With continuous application to intact skin (up to 24 h), extractable dithranol rapidly reached a plateau level (15 min) and was concentrated in the stratum corneum. Substantial dimer formation occurred in both normal and stripped skin. Ether insoluble material rapidly predominated over soluble material, especially when the stratum corneum was absent. However, on short contact application (0.5-1 h), ether soluble material (dithranol in the stratum corneum) was quantitatively predominant in the intact skin. Removal of the vehicle after a short contact time resulted in the disappearance of dithranol from the skin (normal and stripped). In intact skin, the drug was converted into ether insoluble material. In the stripped skin, this insoluble fraction remained constant over the duration of the experiment (24 h).

Repeated application of dinitrochlorobenzene to the ears of sensitized guinea pigs: a preliminary characterization of a potential new animal model for contact eczema in humans.

We have evaluated a subchronic model of contact hypersensitivity in the guinea pig to mimic human chronic/recurrent eczema. Repeated challenges of the ears of previously sensitized guinea pigs with 0.1% dinitrochlorobenzene (once a week for 4 weeks) induced a typical oedema response, which increased during the first 48 h after each challenge. Crusts were detectable (48 h after challenge) and histological observations (72 h after challenge) revealed hyperplasia, papillomatosis, hyperkeratosis and some mononuclear cell infiltrates in the dermis. In agreement with clinical observations in humans, topical treatment of challenged animals with corticosteroid (1% hydrocortisone) reduced the oedema, hyperplasia, papillomatosis, and leucocyte infiltrates, while application of 5% bufexamac (a non-steroidal drug) was associated with a slight enhancement of the inflammatory response. Thus, this model presents clinical and histological similarities with human eczema. Its pharmacological relevance is also suggested, although further investigations are required to better define its selectivity.

A novel in vitro model for the study of human keratinocyte/leucocyte interactions under autologous conditions.

Keratinocyte/leucocyte interactions have become an area of intense investigations in the last decade. However, few convenient in vitro models are available at present. We have therefore designed a novel in vitro system for autologous human keratinocyte/leucocyte co-culture. Non-invasive epidermal cell sampling was achieved by using outer root sheath cells from hair follicles. After one passage, pure keratinocyte cultures (no Langerhans cells or melanocytes) were obtained. Co-culture experiments were performed on a Transwell system: keratinocytes were grown on the porous cupula, and then laid on to wells containing leucocytes. Alternatively, leucocytes can be added to the cupula when contact interactions between the two cell types are to be investigated. Using this system, we demonstrated that Phaesolus vulgaris phytohaemagglutinin-activated T lymphocytes (with 10% monocytes) in the lower compartment induced intercellular adhesion molecule 1 (ICAM-1) and HLA-DR expression, and inhibited methyl-3H-thymidine incorporation in normal human autologous keratinocytes cultured on the cupula. These changes were mediated by soluble factors (no cell contacts between keratinocytes and leucocytes), and required lymphocyte activation. This is the first direct in vitro evidence for leucocyte-induced ICAM-1 and HLA-DR expression on keratinocytes. This system is a potential tool for the study of keratinocyte/leucocyte interactions.(ABSTRACT TRUNCATED AT 250 WORDS)