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Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue
B P Lockhart1, T P Smith, C J Bailey
1Department of Biochemistry, Trinity College, Dublin, Eire.
Abstract:
An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a 'direct' fluorescence procedure using FTC-toxin, but not by an 'indirect' procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the 'direct' and 'indirect' procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the 'indirect' procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a 'direct' procedure using FTC-antiprofilaggrin but only weakly by an 'indirect' double antibody procedure. Insensitivity to 'indirect' procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.
Insights
Direct fluorescence effectively detects epidermolytic toxin targets in skin keratohyalin granules. Indirect methods are insensitive, suggesting a specific property of the keratohyalin granule site.
Area of Science:
- Biochemistry
- Cell Biology
- Dermatology
Background:
- Epidermolytic toxins are key virulence factors in certain skin infections.
- Keratohyalin granules are crucial structures in epidermal differentiation and barrier function.
- Understanding toxin-host interactions at the cellular level is vital for developing targeted therapies.
Purpose of the Study:
- To compare the efficacy of direct and indirect immunofluorescence methods for detecting epidermolytic toxin targets in epidermal cells.
- To investigate the accessibility of toxin targets within keratohyalin granules.
- To elucidate the properties of the keratohyalin granule site that influence detection methods.
Main Methods:
- Direct and indirect immunofluorescence assays using fluorescein isothiocyanate (FTC)-labeled reagents.
- Dispase digestion to isolate epidermal cell layers (basal, spinous, granular).
- Trypsin treatment to assess cell permeability and target accessibility.
Main Results:
- Direct fluorescence detected epidermolytic toxin targets in keratohyalin granules, while indirect methods showed poor sensitivity.
- Profilaggrin, a keratohyalin granule component, was also more readily detected by direct fluorescence.
- Toxin targets were accessible in permeable (trypsin-isolated) granular cells but not in impermeable (dispase-isolated) cells.
Conclusions:
- Direct immunofluorescence is superior for detecting epidermolytic toxin targets in keratohyalin granules.
- The keratohyalin granule site exhibits inherent insensitivity to indirect detection procedures.
- Cell permeability significantly impacts the accessibility of toxin targets within the epidermis.