Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions.

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o-expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to beta- and gamma-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o-cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.

Loss of epithelial integrity resulting from E-cadherin dysfunction predisposes airway epithelial cells to adenoviral infection.

Epithelial intercellular adhesion is fundamental to the formation of the airway epithelial protective barrier. In this respect, cadherins are important because these adhesion molecules regulate formation and maintenance of epithelial intercellular junctions. To study the importance of airway epithelial integrity in determining susceptibility to virus infection, we used a replication-incompetent adenovirus, RAd35, and an E-cadherin specific function-blocking antibody, SHE78-7, to disrupt intercellular contacts in human bronchial epithelial cell line 16HBE14o- and primary bronchial epithelial cells. After exposure of 16HBE14o- cell cultures to SHE78-7, disruption of the transepithelial permeability barrier was indicated by a loss of transepithelial electrical resistance and an associated increase of mannitol, inulin, and dextran paracellular flux. Subsequent exposure of SHE78-7-treated cell cultures to RAd35 showed a remarkable increase in adenoviral infection as assessed by beta-galactosidase reporter gene expression. In cultures exposed to SHE78-7, disruption of E-cadherin function resulted in infection equivalent to that in control cultures using 16-fold lower viral titers. These studies show that manipulation of E-cadherin function provides a specific means of altering epithelial integrity that in turn determines resistance of airway epithelia to adenoviral infection.

A microplate assay measuring chloride ion channel activity.

A microplate chloride ion channel assay, using N-(6-methoxyquinolyl) acetoethyl ester (MQAE) fluorescence changes has been developed. Forskolin stimulation of T84 cells caused cAMP-dependent, increased Cl- loss. Forskolin responses after 6 min gave an EC50 of 0.27 +/- 0.05 microM, illustrating the reproducibility of the assay. Forskolin exposure of CFPAC-1 cells, containing delta F508 CFTR, and CFPAC-1 PLJ4.7 cells, transfected with WT-CFTR stimulated Cl- secretion only from the latter, showing that the assay can be used to measure CFTR function. Stimulation of CFPAC-1 cells with ionomycin increased Cl- efflux, demonstrating functional Ca(2+)-mediated Cl- secretion in these cells. Ionomycin also induced a dose-responsive Cl- efflux from T84 cells that, unlike the forskolin response, was Ca2+ dependent. Removal of Na+ ions severely inhibited basal and stimulated Cl- efflux from T84 cells. However, furosemide did not affect forskolin-stimulated JCl, although the magnitude of the Cl- loss was reduced. The Stern-Volmer constant for MQAE fluorescence in T84 cells was calculated as 28.3 +/- 0.9 M-1 and the [Cl-]i in untreated T84 cells was determined as 52.4 +/- 0.6 mM. Stimulation of T84 cells with ionomycin and forskolin before inducing Cl- efflux allowed calculation of initial efflux rates without interference by second messenger generation and ion channel activation kinetics.

A new tracheal tube for difficult intubation.

We have compared a new Portex tracheal tube with the Oxford tube in performing simulated grade 3 difficult intubations. The Portex tube was modified so that the bevel faced backwards, as in the Oxford tube. A gum elastic introducer was used with both tubes. The time taken and number of attempts needed were recorded, with changes in arterial pressure, heart rate and incidence of sore throat. Both tubes were successful in avoiding the problem of obstruction at the cords, which occurs when a standard Magill tube is used with an introducer. Thus the new tube has the merits of the Oxford tube without the disadvantages of rubber. It is suitable for both easy and difficult intubations with advantages in safety, cost and convenience. An unexpected but important finding was a clear learning effect, despite both investigators being familiar with the technique at the outset. Over the course of the study, intubation time decreased progressively (P < 0.001). This provides new evidence of the need for trainees to practise the art of intubation when the cords are not visible. Our estimate of the learning "half-life" was 15 intubations; we conclude that 30 simulated grade 3 intubations would be a reasonable objective for trainees before handling high-risk cases.

Simple assays of retinoid activity as potential screens for compounds that may be useful in treatment of psoriasis.

Human epidermal cell cultures were used to study the effects of retinoids on keratinocyte differentiation. Keratin profiles were studied by quantitative gel electrophoresis of culture extracts, whereas the extent of envelope formation was assessed in an enzyme-linked immunosorbent assay (ELISA) using an antibody that specifically recognizes keratinocyte envelopes. Exposure of cultures to a variety of different retinoids produced both dose-dependent decreases in keratin 16 with consequent increases in the keratin 14: keratin 16 ratio, and a decrease in envelope formation. The order of activity in both assays was similar: arotinoid ethyl ester (Ro 13-6298) greater than or equal to arotinoid acid (Ro 13-7410) much greater than all trans retinoic acid (Ro 1-5488) greater than acitretin (Ro 10-1670) greater than or equal to etretinate (Ro 10-9359), the only difference being that acitretin was slightly more active than etretinate in the keratin assay whereas these retinoids were equi-active in the envelope assay. Analysis of the lesional keratins of psoriasis patients showed that etretinate caused a reduction in keratin 16 and an increase in the keratin 14:keratin 16 ratio, although the magnitude of these changes and their correlation with clinical improvement was variable. As the in vitro assays reported here are simple and quick, they allow rapid screening of compounds for retinoid-like activity.

Epidermal keratin levels during oral 1-alpha-hydroxyvitamin D3 treatment for psoriasis.

Recently the treatment of psoriasis with vitamin D3, its metabolites or analogues, has been reported to be clinically effective and free from side effects. We have quantified the changes in the levels of epidermal lesional keratins in 15 psoriatic patients receiving oral 1 alpha(OH)D3 treatment. Lesions were sampled before treatment commenced and at monthly intervals for 4-6 months. Clinical resolution occurred in 7 patients within this time; 3 other patients showed incomplete lesion resolution and the remaining 5 patients showed a complete lack of response within the treatment period. Lesion resolution, as judged by clinical criteria, was accompanied by significant changes in the levels of three of the keratin polypeptides and smaller changes in others. Keratin 2 increased to levels greater than those in normal epidermis, while keratins 16 and 18 decreased to normal levels. Changes in the levels of keratins 1 and 5 were small and those of keratins 7, 10 and 14 minimal. These changes were compared with values found during lesion resolution with other therapies used in psoriasis, i.e. topical dithranol, PUVA, oral etretinate and hydroxyurea and were highly reminiscent of those observed during PUVA therapy but contrasted with those during etretinate treatment. The decrease in level of keratin 16, a hyperproliferation marker, suggests that 1 alpha(OH)D3 inhibits keratinocyte proliferation, but at the same time the overproduction of keratin 2, a major keratin of the granular cells, indicates that there is an increase in the number of cells in the later stages of differentiation.

In vitro growth rates of epidermal cells derived from the skin of psoriatic patients and non-psoriatic controls.

The growth of epidermal cells derived from clinically normal skin of psoriatic patients and controls has been studied in culture. Growth rates were measured in secondary cultures established with 5 x 10(4) cells/35 mm plate without feeder layers by determining the plating efficiency, and cell yield and surface area of growth at the end of a 14-day culture period. There was no significant correlation of plating efficiency, cell yield/colony or surface area/colony with the sex or age of the donor in either the normal or psoriatic groups. The morphological development of normal and psoriatic epidermal cell cultures was similar. Comparisons of plating efficiency, cell yield/colony and surface area/colony for the normal and psoriatic groups revealed no significant difference.

Sister chromatid exchange frequency in human epidermal cells in culture treated with 8-methoxypsoralen and long-wave UV radiation.

The effects of 8-methoxypsoralen with long-wave ultraviolet radiation on the sister chromatid exchange frequency in human epidermal cells in culture was investigated. With a constant amount of radiation the number of exchanges increased in an approximately linear manner with increasing concentrations of 8-methoxypsoralen up to 0.3 micrograms/ml. Above this concentration there were fewer dividing cells and an apparent departure from linearity in the dose-response curve. These results show that 8-methoxypsoralen concentrations equivalent to those found in the serum of patients undergoing photochemotherapy, in conjunction with UVA radiation, cause striking increases in sister chromatid exchange frequency in human epidermal cells in vitro.

An investigation of the influence of the extracellular environment on the regeneration of the normal and dystrophic mouse muscle in vitro.

The regenerative response of normal and dystrophic muscle cultured with fetal spinal cord has been investigated. Nearly all the normal muscle explants regenerated, but the dystrophic muscle gave a much more varied response as only 30% regenerated, and 38% showed total explant degradation with loss of all recognisable muscle elements. Forty-five % of the dystrophic explants, produced 'pseudostraps' in which there was an apparent block of myoblast fusion. Cultures in which cellular contact between spinal cord tissue and the normal or dystrophic muscle was prevented showed less regeneration than when it was allowed. However, the incidence of regeneration in these cultures was much higher than when the muscle was cultured in isolation. Direct cellular contact between the cord and the muscle is, therefore, not necessary to initiate regeneration which is presumably mediated in these cultures by changes in the chemical environment. This is substantiated by the almost complete absence of regeneration in cultures of muscle alone, where the fibres were shown to be devoid of nuclei within 5 days. The experiments have shown that under our conditions, a myogenic defect is apparent in the dystrophic muscle. This does not exclude a possible neuronal involvement in the aetiology of the disease, although we have no evidence to support it.