Localization of the Darier disease gene to a 2-cM portion of 12q23-24.1.

Positional cloning with microsatellite markers allowed further localization of the Darier disease gene to a 2-cM interval of chromosome 12, 12q23-24.1, between the polymorphic loci D12S234 and D12S129. A region this size is suitable for construction of a contig to identify the Darier disease gene. Use of a polymorphic intronic marker for nitric oxide synthetase 1 gene, which maps to the same chromosomal area as the Darier gene, allowed exclusion of that gene as the Darier disease gene.

Keratinocytes from junctional epidermolysis bullosa do adhere and migrate on the basement membrane protein nicein through alpha 3 beta 1 integrin.

BACKGROUND: Junctional epidermolysis bullosa (JEB) encompasses several genodermatoses characterized by skin blistering, and possibly disturbed wound healing. Although the molecular defects underlying JEB are not known, we have demonstrated previously that nicein, an adhesive laminin-related basement membrane component, is immunologically altered in the very severe JEB of Herlitz type (H-JEB), and was expressed to a lesser extent in skin from patients with inversa JEB (I-JEB). In this study, we assessed adhesion and migration of H-JEB and I-JEB keratinocytes on exogenous nicein and laminin to get insights on the biologic function defective in JEB skin. EXPERIMENTAL DESIGN: Adhesion of cultured epidermal keratinocytes from H-JEB and I-JEB patients was assayed by quantitation of cell attachment 1 hour after seeding into microtiter wells coated with nicein or laminin. Cell migration and modulation by function-blocking antibodies to integrins was quantified by computer-assisted image analysis of the tracks left by the cells in a phagokinetic assay using gold particles coated with nicein or laminin. RESULTS: In spite of the fact that H-JEB keratinocytes do not produce normal immunoreactive nicein, they were able to adhere on exogenous nicein similarly to normal and I-JEB keratinocytes which produce nicein. Adhesion of both JEB and normal keratinocytes to laminin was weak compared with nicein. At low and high concentrations of nicein, a reduced migration response occurred with H-JEB keratinocytes whereas I-JEB cells behaved like their normal counterparts. Integrin alpha 3 beta 1 was dominantly involved in adhesion and migration of all these cells. Laminin did not support the migration of either JEB or normal keratinocytes. CONCLUSIONS: H-JEB and I-JEB keratinocytes which produce no or less nicein than normal keratinocytes are able to adhere and migrate on exogenous nicein. Integrin alpha 3 beta 1 which is specifically involved in migration and adhesion of keratinocytes on nicein does not appear altered in JEB. These data indicate that defective nicein rather than modifications of the nicein-recognizing receptor play a central role in the pathogenesis of H-JEB.

Localization of the gene for Darier disease to a 5-cM interval on chromosome 12q.

Darier disease is an autosomal dominant abnormality of epidermal differentiation characterized clinically by the presence of hyperkeratotic papules on the skin and histologically by the loss of cell cohesion and by disorderly keratinization. Two groups recently found evidence that the gene whose mutations underlie this disease is located at chromosome 12q23-q24.1, a site on chromosome 12 that clearly is distal to the type II keratin gene cluster. We report here evidence for sublocalization to a 5-cM region of that site in an additional ten families of European and Middle Eastern ancestry with a combined lod score in excess of 20.

Immunohistopathological study of autoimmune pemphigus. Lack of strictly specific histological and indirect immunofluorescence criteria for paraneoplastic pemphigus.

Paraneoplastic pemphigus (PNP) is a form of pemphigus considered to bear characteristic immunohistopathological features (vacuolar-interface dermatitis, keratinocyte dyskeratosis and reactivity of patients' sera with transitional epithelia). The present study was undertaken in order to investigate whether these criteria are specific enough so as to allow the diagnosis of PNP in the absence of clinical data. A retrospective study of 66 biopsies of pemphigus revealed that one third of them comprised histological signs of PNP; one of the corresponding sera reacted with mouse bladder epithelium. However, no evidence of a neoplastic disease was present in any of the patients, and Western blotting further excluded the diagnosis of PNP. These results suggest that some of the features considered characteristic of PNP are not strictly specific for this variety of pemphigus; hence this diagnosis cannot be reliably established by histology or immunofluorescence but requires biochemical studies.

Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the alpha 2 integrin subunit.

The migration of human keratinocytes across the would bed is an early and critical event in the reepithelialization of cutaneous wounds. Epidermal growth factor (EGF) has been shown to accelerate the healing of fresh, split-thickness cutaneous wounds when applied topically. The mechanism(s) by which this accelerated healing occurs remains unknown. Using an assay that directly evaluates human keratinocyte locomotion without confounding the possibility of cell proliferation, we examined the influence of EGF on human keratinocyte motility. Both recombinant epidermal growth factor (rEGF) and transforming growth factor-alpha (TGF-alpha) promoted human keratinocyte locomotion when the cells were apposed to connective tissue matrices of collagen or fibronectin, important components of the wound bed. Other growth factors studied did not enhance keratinocyte migration. Blocking the EGF/TGF-alpha receptor on the cell surface of keratinocytes with specific antibody inhibited the stimulation of keratinocyte locomotion by rEGF and TGF-alpha. Flow cytometry analysis of keratinocytes migrating on type I collagen in the presence of rEGF or TGF-alpha revealed increased expression of the alpha 2 integrin subunit on the keratinocyte surface. The alpha 2 beta 1 integrin mediates keratinocyte migration on collagens type I and IV, and inhibition of migration via antibody blockade of the alpha 2 beta 1 integrin can be partially overcome by increasing the concentration of rEGF present in the medium. Our study demonstrates that the growth-independent stimulation of keratinocyte locomotion via regulation of integrin expression may be one mechanism by which EGF accelerates the reepithelialization of human cutaneous wounds.

Re-epithelialization. Human keratinocyte locomotion.

Keratinocyte migration and proliferation both play a role in covering skin wounds by the process of re-epithelialization. Connective tissue components are a powerful influence on keratinocyte locomotion. The mechanisms of keratinocyte locomotion and cellular division are independent. Both connective tissue components and soluble factors may serve to enhance keratinocyte migration. In addition to the field of growth factors, we would like to suggest that there should be the recognition of an entire new class of agents called migration factors.

Cutaneous mesenchymal tumour with haemangiopericytoma-like features.

A case of cutaneous mesenchymal haemangiopericytoma-like tumour affecting a 17-year-old male is presented. Clinical features were non-specific. The tumour was studied by light, electron microscopy and immunohistochemistry. The results of these studies suggested the diagnosis of a mesenchymal, poorly-differentiated tumour showing features of haemangiopericytoma. The clinical course after surgery was uneventful after a 9-month follow-up period.

[Interleukin-2 immunotherapy and cutaneous manifestations]. / Immunothérapie par interleukine 2 et manifestations cutanées.

The cutaneous manifestations which follow the administration of recombinant interleukin-2 (rIL-2) are well known, but their nature and severity have not yet been fully studied. The authors report the dermatological changes observed in 12 patients treated with rIL-2 for renal cancer. The predominant lesion was generalized erythema followed by desquamation.

Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices.

Human keratinocytes synthesize interstitial collagenase, a 72-kDa gelatinase, and a recently described 92-kDa gelatinase/type IV collagenase. We examined the synthesis of this novel enzyme by basal keratinocytes apposed to plastic, basement membrane collagen (type IV), and interstitial dermal collagen (type I). Samples of conditioned medium were electrophoresed on a 10% polyacrylamide, gelatin-ladened zymogram. Protein bands with gelatin-cleaving properties were identified by clarification of the gel and quantified by densitometry. A 92-kDa band had marked gelatinolytic activity and increased in culture over 72 h. The identification of this 92-kDa band as type IV collagenase was demonstrated by Western immunoblotting using monospecific antibody to the 92-kDa type IV collagenase. Keratinocytes apposed to type I collagen exhibited a threefold increase in the synthesis of the 92-kDa enzyme compared to cultures apposed to type IV collagen and a 1.5-times increase compared to plastic. The specificity of this enhancement was shown by constant levels of other proteins (e.g., the 72-kDa gelatinase). This study demonstrates that cell-matrix interactions modulate the synthesis of a recently described, keratinocyte-derived, 92-kDa gelatinase and that specific collagen types (I versus IV) have opposite effects upon the synthesis of this enzyme.

Keratinocyte migration is partially supported by the cell-binding domain of fibronectin and is RGDS-dependent.

Fibronectin (FN) plays a key role in cell attachment, embryonic development, and wound healing. In this respect, it is known that FN promotes keratinocyte migration. The aim of this study was to examine specific FN domains (120-kD cell-binding fragment, 45-kD collagen fragment, and 40-kD heparin fragment) and a biologically active peptide within the molecule (RGDS) for their ability to influence human keratinocyte (HK) locomotion. HKs were plated on gold salts coated with different substrates (type IV collagen, FN with or without the RGDS peptide, and the three FN fragments). After 20 h, locomotion tracks were quantified by computer-assisted image analysis that determines the area of each microscopic field occupied by migration tracks, a so-called migration index (MI). MIs on type IV collagen and FN were 39.14 +/- 2.8% and 30 +/- 0.4%, respectively. The maximal MIs on the collagen-binding domain and heparin-binding domain of FN were similar to our negative controls (plastic and albumin): 3 +/- 1%. In contrast, the maximal MI on the cell-binding fragment of FN was 18.45 +/- 2.1%. The effect of the cell-binding domain on keratinocyte motility was found to be dose dependent. Moreover, we could specifically inhibit the FN-driven locomotion using the RGDS sequence contained in the cell-binding fragment. We did not observe a synergistic effect (i.e., a higher MI) when we added the three fragments in a same dish. These results suggest i) that the cell-binding fragment of FN partially supports HK locomotion, ii) that other untested FN domain(s) should act in synergy with the cell-binding fragment to promote keratinocyte locomotion, or alternatively iii) that the FN function of promoting cell migration resides within the FN cell-binding domain, but the proper presentation of this domain to the cell requires an intact, native FN molecule, and iv) that the RGDS sequence is essential for HK movement.

[Fibronectins]. / Les fibronectines.

Fibronectins are complex glycoprotein macromolecules whose molecular weight is 440 kilodaltons. These proteins, found throughout the body, are soluble in biological fluids and insoluble in connective tissue. They contribute to tissue morphogenesis through multiple interactions with cells and extracellular matrix components. Fibronectins play a key role in cell attachment and remodeling during embryonic development, in cell migration and anchorage during wound healing, and in immune responses. This review discusses the structure of fibronectins, their integrin cell receptors, their biologic properties and their implications for diseases.

Human keratinocyte locomotion: the effect of selected cytokines.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are two powerful mitogens for human keratinocytes that also have been shown to promote the healing of in vivo wounds. Transforming growth factor-beta (TGF-beta) markedly inhibits human keratinocyte proliferation and growth and yet has been shown to promote wound healing. Using a migration assay that evaluates pure cell locomotion independently from cell proliferation, we examined the influence of EGF, bFGF, and TGF-B on human keratinocyte locomotion. Although these agents had profound influences upon the growth potential of keratinocytes in parallel thymidine incorporation assays, they had no significant effect upon keratinocyte locomotion when cells were apposed to either tissue culture plastic or a collagen substratum. In contrast, we found that bovine pituitary extract (BPE), a poorly defined mitogen that is commonly used in keratinocyte cultures, could stimulate keratinocyte locomotion when the cells were apposed to a collagen substrate. These studies demonstrate that i) keratinocyte locomotion and proliferation operate by completely independent mechanisms, ii) the positive effects upon wound healing by EGF, bFGF, and TGF-beta are not due to a direct promotion of keratinocyte locomotion, and iii) that one or more components of BPE are capable of directly promoting keratinocyte locomotion on collagen.

Autoimmunity to type VII collagen.

The epidermolysis bullosa acquisita (EBA) antigen is type VII collagen that is found within the anchoring fibrils of the basement membrane zone beneath stratified epithelia. Autoimmunity to the EBA antigen/type VII collagen has been associated with three diseases: EBA, bullous systemic lupus erythematosus (SLE) and a subset of linear IgA bullous diseases. Although some systemic diseases in which autoimmunity is thought to play a role (eg, inflammatory bowel disease, thyroiditis) have been reported in association with EBA, so far there have not been systemic diseases associated with autoimmunity to type VII collagen other than SLE. In the case of EBA and bullous SLE, it appears that many patients may have a genetic predisposition toward autoimmunity because they share a human leukocyte antigen (HLA) major histocompatibility (MHC) class II cell surface protein, HLA-DR2. The full clinical spectrum of EBA and perhaps other diseases in which there is an association with autoimmunity to type VII collagen is currently being defined.

Salt-split human skin substrate for the immunofluorescent screening of serum from patients with cicatricial pemphigoid and a new method of immunoprecipitation with IgA antibodies.

In patients with cicatricial pemphigoid, immunoglobulins (usually IgG) and complement are deposited within the dermoepidermal junction and are detected by direct immunofluorescent staining of perilesional mucous membrane and/or skin with fluorescein-labeled antibodies to human immunoglobulins. Although rare, some patients also have circulating low-titer, anti-basement membrane zone autoantibodies. In this study, we report 11 patients with the clinical, histologic, and immunologic criteria for cicatricial pemphigoid who had circulating anti-BMZ autoantibodies as demonstrated by positive indirect staining of a normal human skin that had been fractured through the dermoepidermal junction by prolonged incubation in a cold, 1 mol/L sodium chloride solution. On this salt-split human skin substrate, 9 of the 11 patients (82%) had autoantibodies that bound to the epidermal roof, one serum stained only the dermal floor, and one serum stained both sides of the separation. The predominant class of immunoglobulin in the patients' sera that bound to the substrate was IgA; IgA was the single immunoglobulin in 55% and was associated with IgG in 18%. IgG was the only immunoglobulin detected in 27% of the cases. No specific protein was detected by either Western immunoblot or a new IgA immunoprecipitation procedure.

GP37 is different from filaggrin.

The identity of two differentiation markers of human epidermis, filaggrin and a Concanavalin A (Con-A) reactive glycoprotein of 37 kD, has been studied. Human epidermis was extracted in Nonidet P-40 buffer, and the soluble proteins were separated by two-dimensional electrophoresis. Con-A reactive glycoproteins were identified by incubating gels with iodinated lectin followed by autoradiography. Identical, parallel gels were electrophoretically transferred to nitrocellulose paper and filaggrin-related molecules labeled by the specific monoclonal antibody AKH1. We found that the 37-kD Con-A reactive component was resolved by two-dimensional gel electrophoresis into several glycoproteins and that the lectin Con-A does not bind to filaggrin. Under these conditions, the anti-GP37 serum failed to identify any component. However, when applied to human keratinocyte culture extract, AKH1 and the anti-GP37 serum reacted in a similar way. These data show 1) that the 37-kD band is not homogeneous but contains distinct markers of differentiation (filaggrin and Con-A reactive glycoproteins) and 2) that the GP37 antibody's specificity is for the filaggrin precursor.