Epidermolysis Bullosa Dystrophica-Recessive: A Possible Role Of Anchoring Fibrils In The Pathogenesis

The purpose of this study was to define the ultrastructural defects and pathogenesis of epidermolysis bullosa dystrophica-recessive (EBD-R). The only consistent ultrastructural alteration found in EBD-R was an absence of anchoring fibrils. In many specimens of nonblistered, nontraumatized EBD-R skin, absence of anchoring fibrils was the only ultrastructural abnormality observed. The possibility that lack of anchoring fibrils was a secondary change resulting from previous blistering and scarring was eliminated by our observation that anchoring fibrils were consistently absent in the never previously blistered skin of two newborns with EBD-R. In experimentally traumatized skin, the epidermis and dermis separated in the region of the epidermal-dermal junction normally occupied by anchoring fibrils. Basal lamina and dermal microfibril bundles appeared to be normal. Using recombinant grafts, we demonstrated that anchoring fibrils were not formed by EBD-R dermis when combined with EBD-R epidermis or normal epidermis. Anchoring fibrils were formed when normal dermis was combined with normal and EBDR epidermis. These studies indicate that the defect in EBD-R resides in the dermis and that the defect may he associated with impaired formation of anchoring fibrils

Monoclonal Antibodies to Anchoring Fibrils for the Diagnosis of Epidermolysis Bullosa

Murine monoclonal antibodies to human anchoring fibrils reacted with human and monkey cervix, tongue, esophagus, and vagina. Rat, mouse, and guinea pig tissues were negative. In 11 patients with dystrophic recessive epidermolysis bullosa there was no reaction by immunofluorescence and immunoelectron microscopy. Other forms of epidermolysis bullosa had normal reactivity

Epidermal-dermal Interactions in Adult Skin

Epidermal-dermal interactions are important determinants of embryonic development in skin. This review examines the role of such epidermal-dermal interactions in the conservation of epithelial specificity in adult skin. The basic epidermal keratinization program as defined as a proliferative basal cell population, orderly stratified cytodifferentiation of the cells and production of stratum corneum can be expressed by adult epidermis without the continued presence of a specific dermis. This is evidenced by the ability of epidermis to differentiate fully in association with nondermal connective tissues. Several other epithelia including cornea and esophagus express their specific differentiative characteristics without the continued presence of specific connective tissue. On the other hand, certain regional epithelial specificities in adult skin may be determined by the dermis. These regional epidermal specificities involve alterations in epidermal thickness, size of epidermal compartments including the stratum corneum, and the folding pattern at the epidermal-dermal interface. Possible mechanisms by which dermis could control these regional characteristics are discussed. Epidermal-dermal recombination techniques have been used to define the site of gene action in a variety of inherited skin abnormalities in animals. Similar studies are reported utilizing adult human skin in long-term cultivation on nude athymic mice. The abnormal gene in lamellar ichthyosis exerts its effect directly on the epidermis. Generalized exfoliative psoriasis was also studied by recombination techniques. Both epidermis and dermis were required for the maintenance of psoriatic morphology, suggesting a complex polygenic mechanism or one involving both genetic and environmental factors

Double Immunofluorescence Microscopy: A Method for Localizing Immune Deposits in Skin Diseases Associated with Linear Basement Membrane Zone Immunofluorescence

Direct immunofluorescence microscopy has shown that a linear pattern of immunoglobulin and/or complement deposition at the cutaneous basement membrane zone is a characteristic feature in a number of acquired bullous diseases and is occasionally observed in systemic lupus erythematosus. Immunoelectron microscopy has shown the linear pattern of immunofluorescence may be produced by immune deposits located either above the basal lamina (in the lamina lucida) or below the basal lamina (in the upper dermis). Distinguishing between these sites of immune reactant deposition may be of value in differential diagnosis. In this study we report a double immunofluorescent method by which skin biopsies with linear IgG immunofluorescence due to deposits above the basal lamina (bullous pemphigoid) could be distinguished from biopsies with deposits beneath the basal lamina (bullous systemic lupus erythematosus and epidermolysis bullosa acquisita). When skin sections were treated sequentially with rhodamine-labeled anti-human IgG followed by fluorescein-labeled antilamina lucida (pemphigoid) antibody and examined by fluorescence microscopy, the following results were obtained. In biopsies with IgG deposits in the lamina lucida, a single green fluorescent band was observed. In tissues with subbasal lamina deposits, either parallel and contiguous bands of green and yellow-orange fluorescence or a single band of yellow-orange fluorescence was observed. The method is simpler, quicker, and less expensive than immunoelectron microscopy and should be a useful technique for evaluating skin diseases with linear immunofluorescence at the basement membrane zone

Adherens Junctions: Demonstration in Human Epidermis

Adherens junctions are intercellular and cell-matrix junctions that, like desmosomes and hemidesmosomes, mediate adhesion of cells to each other or to matrix structures. These junctions have been detected recently in cultured human keratinocytes, indicating that they may be of importance in epidermis. To investigate the localization of adherens junctions in normal epidermis, we examined human epidermis, human oral mucosa, and monkey esophagus for the presence of vinculin, a major protein of the intracellular plaques of adherens junctions that is thought to be present in all adherens junctions. 'Western blot analysis demonstrated vinculin in extracts of epidermis. Immunohistochemistry of vinculin in these tissues displayed two distinct locations for adherens junctions: i) at the dermal-epidermal junction, and ii) in the region of cell-cell contacts in all layers of the epidermis. The location of vinculin in the region of the epidermal-dermal junction is reminiscent of the distribution of vinculin-containing focal contacts in cultured keratinocytes, and the intercellular staining of vinculin in epidermis is consistent with the presence of vinculin in adherens junctions in cultured keratinocytes at sites of cell-cell contact. These results demonstrate that adherens junctions are present in human epidermis, oral mucosa, and monkey esophagus. Vinculin-containing junctions in epidermis may be important in the pathogenesis, of skin diseases involving alterations in intercellular integrity

Characterization of “Neo-Dermis” Formation Beneath Cultured Human Epidermal Autografts Transplanted on Muscle Fascia

Cultured human keratinocyte autografts were transplanted to burn wounds that had been completely excised down to muscle fascia such that all cutaneous elements were removed from the wounds. Healing autografts were biopsied from days 6-153 in five patients, and the "neo-dermis" beneath the autografts was examined by immunofluorescent staining using antibody probes to connective tissue molecules, by histochemical staining for elastin fibers, and by electron microscopy. We found that the neo-dermis contained most of the major connective tissue elements early in the post-transplantation period. However, regardless of the time examined, there was a paucity of elastin fibers and poor organization of linkin (microthread-like fibers) in the neo-dermis beneath autografts. The perturbations of these connective tissue components in the neo-dermis may play a role in the poor recoil and elastic properties of burn wounds treated with autografts

Epidermolysis Bullosa Acquisita Antigen, a Major Cutaneous Basement Membrane Component, Is Synthesized by Human Dermal Fibroblasts and Other Cutaneous Tissues

The epidermolysis bullosa acquisita (EBA) antigen is identified as 2 chains: a 290,000-dalton protein and a less prominent 145,000-dalton protein. The 290,000-dalton chain is synthesized by human keratinocytes in culture. In this study, we show that the 290,000-dalton chain is synthesized by human skin fibroblasts and cutaneous human tumors. In contrast, HT1080 cells, a human sarcoma cell line known to produce matrix molecules (such as laminin and type IV collagen), does not synthesize the EBA antigen. Further, the EBA antigen is absent from serum and blood components, placenta, amnion, lung, and the EHS tumor, a murine sarcoma that produces large amounts of laminin, type IV collagen, nidogen, entactin, and basement membrane proteoglycan but is present in cutaneous tumors of adnexal and epithelial origin. These data suggest that while the EBA antigen is synthesized by both human skin keratinocytes and fibroblasts and is therefore not specific for a primordial germ layer, it does appear to be specific for tissue containing a stratified squamous epithelium

A Mouse Monoclonal Antibody Against a Newly Discovered Basement Membrane Component, the Epidermolysis Bullosa Acquisita Antigen

A mouse monoclonal antibody, H3a, directed against the newly described epidermolysis bullosa acquisita (EBA) antigen was obtained using hybridoma techniques. The distribution of the monoclonal antibody is identical to that of the polyclonal serum antibody of patients with EBA. By immunofluorescence, a linear band is seen at the dermal-epidermal junction and, by immunoelectron microscopy, immune reaction products are present in the lamina densa and sublamina densa regions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot analysis shows that the monoclonal antibody recognizes 290 and 145 kilodalton proteins present in the immunizing junctional extract, identical with the newly discovered EBA antigen. This monoclonal antibody should be useful in the further isolation and characterization of the EBA antigen

Neonatal Foreskin Substrate Has Limitations for the Immunofluorescent Screening of Monoclonal Antibodies

Two monoclonal antibodies to type IV collagen showed a marked decrease in the labeling of the dermal-epidermal junction of neonatal foreskin while the basement membrane around dermal blood vessels was brightly stained. In contrast, these antibodies labeled the junction and dermal blood vessels with approximately equal intensity when adult skin of nonforeskin site was used as substrate. Other antibodies to matrix molecules (bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and laminin) showed excellent staining of both the dermal-epidermal junction and dermal blood vessels in both neonatal foreskin and adult skin. Further, the ultrastructural appearance of the substrates appeared identical. The implication is that neonatal foreskin is not a good substrate to use for the routine screening of monoclonal antibodies to matrix components by indirect immunofluorescence since a "false negative" evaluation may occur