Blister fluid from epidermolysis bullosa letalis induces dermal-epidermal separation in vitro

Normal human skin was cultured with blister fluid (BF) from 2 patients with epidermolysis bullosa letalis (EB letalis). After 12-24 h incubation, initial separation was observed at the dermal-epidermal junction. Complete separation was seen after 48-72 h incubation. Epidermal and dermal structures themselves were well preserved up to 72 h. Electron microscopy revealed that the separation took place through the lamina lucida. These findings corresponded to in vivo histologic pictures of the skin lesions. The activity in BF was lost by lowering (pH 4) or elevating (pH 9) pH of the media or by prolonged autoclaving (5h). Boiling and dialization did not affect the activity of BF. Certain protease inhibitors, such as alpha 2-macroglobulin, aprotinin (trasylol), soybean trypsin inhibitor, phenylmethylsulfonylfluoride, or 50% fetal bovine serum, inhibited or depressed the separation, while triamcinolone acetonide, n-ethylmaleimide, or EDTA did not. Patient's serum (case 2) and normal human sera partially inhibited cleft formation. It was proposed that in EB letalis some factors in BF, probably protease(s), play an important role in the induction and/or spread of blister.