Immunofluorescence mapping of antigenic determinants within the dermal-epidermal junction in the mechanobullous diseases

The classification of mechanobullous diseases often depends on the electron microscopic distinction of intradermal (dermolytic), junctional and intraepidermal sites of cleavage. Electron microscopy is tedious and time consuming. In this report we describe a different approach to the determination of the cleavage plane by using a method which recognizes subtle differences in the localization of antigenic structures relative to the cleavage plane. Cryostat sections of lesional and extralesional skin of 3 patients with dermolytic epidermolysis bullosa, 3 with epidermolytic epidermolysis bullosa and 8 with junctional epidermolysis bullosa were examined by immunofluorescence, with specific antisera against type IV collagen (localized within the basal lamina); against laminin (noncollagenous protein, localized in the lamina lucida); and with bullous pemphigoid antibodies (directed against the bullous pemphigoid antigen localized in the lamina lucida). All specimens were also examined by electron microscopy. In dermolytic epidermolysis bullosa (where cleft formation occurs intradermally) type IV collagen, laminin and the bullous pemphigoid antigen were consistently found in the roof of the blister, whereas in junctional epidermolysis bullosa (where the cleft occurs in the lamina lucida) type IV collagen and laminin were found on the floor of the blister whereas bullous pemphigoid antigen was present mainly on the roof, but focally also on the floor, of the blister. In epidermolytic epidermolysis bullosa (where the cleft is intraepidermal) all antigens were localized below the cleavage plane. In all cases electron microscopy confirmed the level of cleft formation predicted from the immunofluorescence mapping of the antigenic sites. The described method equals electron microscopy in accuracy but it is more rapid and simpler to perform.     

Clinical variability in dystrophic epidermolysis bullosa and findings with scanning electron microscopy

In dystrophic epidermolysis bullosa, the genetic defect of anchoring fibrils leads to cleavage beneath the basement membrane and its consequent loss. A 46 year-old female patient presented blisters with a pretibial distribution associated with nail dystrophy. Her two children had hyponychia and anonychia, which affected all toe nails and the thumb, forefinger and middle finger. DNA sequencing identified in exon 75 of COL7A1 gene a pathologic mutation: c.6235G>A (p.Gly2079Arg). Immunomapping of a blister demonstrated collagen IV (basal membrane) in the blister roof and collagen VII in its floor, confirming dystrophic epidermolysis bullosa. Scanning electron microscopy of an inverted blister showed net-forming collagen attached to the blister roof . The variability found in this family has already been reported and confirms, on a clinical basis, the nail subtype as a dystrophic variant.     

弹力纤维染色在营养不良型大疱性表皮松解症中的诊断价值

 目的:通过免疫组织化学染色回顾性诊断营养不良型大疱性表皮松解症（DEB）患者，并了解弹力纤维在DEB和大疱性类天疱疮(BP)患者皮损中的分布情况，进一步探讨弹力纤维染色在DEB诊断中的临床意义。方法： 收集17例DEB和3例BP患者皮损标本和临床资料,采用免疫组织化学染色方法检测角蛋白14、层粘蛋白5和胶原蛋白Ⅳ、Ⅶ在DEB患者中的表达，弹力纤维染色检测弹力纤维在DEB患者和BP患者皮损中的分布情况。结果：17例DEB患者皮损中，角蛋白14和层粘蛋白5在真、表皮裂隙的表皮侧表达，胶原蛋白Ⅳ在表皮侧表达或表达缺失，而胶原蛋白Ⅶ表达缺失。弹力纤维在17例DEB患者真皮浅中层均缺失，而在BP患者真皮乳头处减少。结论：DEB可根据角蛋白14、层粘蛋白5、胶原蛋白Ⅳ和胶原蛋白Ⅶ在皮损中的免疫组化检测结果诊断。弹力纤维在DEB和BP患者皮肤中分布不同，弹力纤维染色可作为DEB和BP的鉴别诊断依据之一。     

Intra-epidermal retention of type VII collagen in a patient with recessive dystrophic epidermolysis bullosa

An infant born with severe blisters on the limbs, face, trunk, and oral mucosa was diagnosed by light and electron microscopy to have recessive dystrophic epidermolysis bullosa. Transmission electron microscopy showed that the basal lamina remained with the epidermis and that the floor of the blister was exposed collagen of the papillary dermis. No banded anchoring fibrils were observed along either the roof or the floor of the blister; however, small filamentous structures, possibly immature anchoring fibrils, extended down from the lamina densa along the blister roof. Some basal and suprabasal keratinocytes contained large vesicles filled with filamentous matrix of variable electron density. Immunofluorescent staining of skin for type VII collagen showed sparse and irregular staining of type VII collagen along the blister roof, and intense intracellular labeling for type VII collagen in clusters of epidermal cells in basal and suprabasal layers. Type VII collagen appeared to be synthesized by keratinocytes but not secreted.     

Animal model for dermolytic mechanobullous disease: sheep with recessive dystrophic epidermolysis bullosa lack collagen VII

A severe congenital mechanobullous disease with dermolytic blistering and recessive inheritance is described in sheep. The affected animals of wild and inbred flocks of the breed Weisses Alpenschaf (WAS) have blisters of skin, oral mucosa, tongue, and esophagus at birth or within the first week of life. Exungulation occurs early, and severe erosions in the mouth lead to difficulty in feeding. Electron microscopic examination revealed sub-lamina densa splitting in natural or fresh friction blisters and absence of identifiable anchoring fibrils in clinically uninvolved skin. Antigen mapping localized laminin and collagen IV to the blister roof. Indirect immunofluorescence staining with antibodies to collagen VII, the major structural component of the anchoring fibrils, demonstrated a complete absence of reaction in clinically uninvolved tissues of the affected sheep, whereas in normal sheep a strong linear fluorescence was seen at the epithelial-mesenchymal basement membrane zone. Dermal extracts of normal sheep contained intact collagen VII, but epidermal and dermal extracts from the affected sheep lacked this collagen or its fragments in immunoblotting experiments. Based on genetic, clinical, ultrastructural, and immunochemical findings, the sheep disorder corresponds to the severe mutilating subtype of recessive dystrophic epidermolysis bullosa in humans and can be used as an animal model to investigate the human disorder.     

Three new cases of transient bullous dermolysis of the newborn

We present 3 new patients with transient bullous dermolysis of the newborn (TBDN), which is a form of dystrophic epidermolysis bullosa. TBDN may be diagnosed by electron microscopy showing a sublamina densa cleavage; immunofluorescence antigenic mapping demonstrating bullous pemphigoid antigen, laminin- 1, and type IV collagen along the epidermal roof of subepidermal clefts; and indirect immunofluorescence with monoclonal antibodies revealing intraepidermal type VII collagen. Although intraepidermal type VII collagen has been reported in other forms of dystrophic epidermolysis bullosa, we believe that the presence of type VII collagen in a striking intraepidermal granular array is a finding unique to TBDN. Our cases demonstrate the importance of immunodermatologic studies in the diagnosis of bullous disorders that are seen at birth because accurate diagnosis carries prognostic implications. This variant of epidermolysis bullosa, in contrast to other forms of dystrophic epidermolysis bullosa, is a benign, self-limited disease.     

Comparative scanning electron microscopy of bullous diseases

The purpose of this study is to compare scanning electron microscopy findings of the blister roof in three distinct bullous diseases: one intraepidermal acantholytic (pemphigus foliaceus); one due to hemidesmosomal dysfunction (bullous pemphigoid); and one secondary to anchoring fibril dysfunction - type VII collagen (dystrophic epidermolysis bullosa). In pemphigus foliaceus, acantholytic phenomena were readily demonstrated. In bullous pemphigoid, the epidermis had a solid aspect. In dystrophic epidermolysis bullosa a net was seen in the blister roof.     

Acquired epidermolysis bullosa with the clinical feature of Brunsting-Perry cicatricial bullous pemphigoid

A 56-year-old woman with the typical clinical feature of cicatricial bullous pemphigoid of the Brunsting-Perry type was studied. Histologic examination of a lesion skin biopsy specimen demonstrated a subepidermal blister. Direct immunofluorescence microscopy revealed linear deposits of IgG, IgM, and C3 located on both the roof and the floor of the blister. Immunofluorescence antigen mapping using cryostat sections of a spontaneous blister and antisera against defined basement membrane components localized the bullous pemphigoid antigen and type IV collagen in the roof of the blister. This dermal type of blister formation was confirmed by electron microscopy, which showed the cleavage level below the lamina densa. In direct immunoelectron microscopy, granular deposits of C3 and IgG were found attached to and just beneath the lamina densa in a pattern identical to the distribution of anchoring fibrils. These findings are diagnostic of acquired epidermolysis bullosa, a blistering disease that has much more clinical heterogeneity than previously suggested.     

Basement membrane and fibroblast aberration in blisters at the donor, graft, and spontaneously healed sites in patients with burns.

BACKGROUND AND DESIGN--Blisters that developed on spontaneously healed wounds and grafts in 13 patients with burns were analyzed by light, fluorescence, and electron microscopy. RESULTS--Blisters developed on the dermal side of the dermoepidermal junction and occurred more frequently in donor site and healed mesh graft than in split-thickness sheet graft. The four major components of the basement membrane zone (bullous pemphigoid antigen, laminin, type IV collagen, and epidermolysis bullosa acquisita antigen) were reduced in quantity and irregularly deposited at blister sites. Immediately adjacent to the blisters, epidermolysis bullosa acquisita antigen appeared normal in quantity, while laminin, type IV collagen, and bullous pemphigoid antigen levels appeared slightly reduced. Mononuclear infiltrates and autoantibodies were not detected by light microscopy or direct-indirect immunofluorescence, respectively. Ultrastructurally, adjacent dermal fibroblasts demonstrated swollen rough endoplasmic reticulum and vacuolization. CONCLUSIONS--We speculate that blister development in patients with burns is related to defective reorganization of the basement membrane zone in association with dermal fibroblast aberration during wound healing.     

Location of basement membrane Type IV collagen beneath subepidermal bullous diseases

Using standard immunohistochemical methods, routinely processed sections, and a polyclonal antibody to Type IV collagen, we have determined the location of Type IV collagen, a substance located in the lamina densa of basement membrane, in a spectrum of acquired subepidermal bullous diseases. Type IV collagen was attached to the blister roof in five cases of well-established epidermolysis bullosa acquisita and to the blister base in 25 cases of bullous pemphigoid, four cases of dermatitis herpetiformis and 12 cases of porphyria cutanea tarda. Immunohistochemical localization of Type IV collagen in epidermal-dermal basement membrane is a simple, rapid and reliable technique which can be utilized to exclude and possibly to confirm the diagnosis of epidermolysis bullosa acquisita in routinely fixed paraffin-embedded tissues.     

Epidermolysis bullosa: hereditary skin fragility diseases as paradigms in cell biology

Recent research into the molecular basis of epidermolysis bullosa has provided a unique insight into a variety of mechanisms in normal cell biology, such as cell-matrix interactions, and has uncovered an excellent model for studies on keratin intermediate filaments. The simplex forms of epidermolysis bullosa are caused by mutations in the genes for the basal epidermal keratins, K5 and K14. Most mutations affect highly conserved parts of the molecules, illustrating their importance in normal keratin filament assembly and integrity. Mutations in corresponding regions of the differentiation-associated keratins, K1 and K10 can also occur in epidermolytic ichthyosis. Both recessive and dominant forms of dystrophic epidermolysis bullosa result from mutations in an anchoring fibril collagen gene, COL7A1. Junctional epidermolysis bullosa is caused by mutations in the genes encoding different chains of the novel laminin isoform, nicein/ kalinin, also known as laminin 5, which is associated with the anchoring filament-hemidesmosome complex of the basement membrane zone. These recent findings strengthen the evidence for the role of nicein/kalinin and type VII collagen in adherence and stabilization of the dermo-epidermal junction.     

Scanning electron microscopy of a blister roof in dystrophic epidermolysis bullosa

In dystrophic epidermolysis bullosa the genetic defect of anchoring fibrils leads to cleavage beneath the basement membrane, with its consequent loss. We performed scanning electron microscopy of an inverted blister roof of a case of dystrophic epidermolysis bullosa, confirmed by immunomapping and gene sequencing. With a magnification of 2000 times a net attached to the blister roof could be easily identified. This net was composed of intertwined flat fibers. With higher magnifications, different fiber sizes could be observed, some thin fibers measuring around 80 nm and thicker ones measuring between 200 and 300 nm.     

Persistent subepidermal blistering in split-thickness skin graft sites. Ultrastructural and antigenic features simulating dystrophic or immunofluorescence-negative acquired epidermolysis bullosa

We describe a child who began developing subepidermal blisters in the recipient sites of split-thickness skin grafts; this process has continued for almost a year and continues to spare nongrafted skin. Routine histologic and immunofluorescence mapping studies demonstrated this disorder to be a relatively noninflammatory one characterized by sub-lamina densa blister formation. Results of direct immunofluorescence were negative. By electron microscopy, anchoring fibrils were sparse in number and in some areas appeared malformed; otherwise, the basement membrane zone was morphologically unremarkable. Bullous pemphigoid antigen, laminin, type IV collagen, epidermolysis bullosa acquisita antigen, and LDA-1 were all normally expressed along the dermoepidermal junction. In contrast, KF-1 antigen was absent. These findings suggest a disease process confined to skin graft recipient sites with features identical to those previously described with recessive dystrophic or immunofluorescence-negative acquired epidermolysis bullosa.     

Epidermolysis bullosa junctionalis progressiva in three siblings

Three siblings of Swiss origin with epidermolysis bullosa junctionalis progressiva are described. The following clinical features were present from school age; dystrophy of the nails, non-scarring blistering of the skin, mild skin atrophy, hypodontia and dental caries. Light microscopy showed subepidermal blistering. Direct immunofluorescence was negative. On indirect immunofluorescence staining of a fresh spontaneous blister, bullous pemphigoid antigen and laminin were localized to the blister roof, and collagen IV and collagen VII to the blister base, indicating junctional splitting. Electron microscopy revealed a normal dermo-epidermal junction zone, including normal hemidesmosomes. There were no deposits of electron-dense amorphous material.     

Immunohistochemical localization of epidermal basement membrane laminin and type IV collagen in bullous lesions of dermatitis herpetiformis

Antibodies against the human basement membrane proteins, laminin and the 7-S domain of type IV collagen, were used to study the epidermal basement membrane in lesional skin from four patients with dermatitis herpetiformis. The staining pattern of both antigens was mostly fragmented and sometimes absent on papillary microabscesses, but when present it was attached to the epidermal basal cells. On papillary microblisters and larger blisters the staining of both antigens showed discontinuities and was located in the floor of the blister, except for two cases where tiny fragments of laminin staining were also seen in the roof of larger blisters. These results suggest that blister formation in dermatitis herpetiformis takes place between the epidermal basal cells and the basement membrane.     

Lack of type VII collagen in unaffected skin of patients with severe recessive dystrophic epidermolysis bullosa

Type VII collagen, the major structural component of the anchoring fibrils, was assayed in normal unaffected skin of patients with different forms of hereditary epidermolysis bullosa. Immunofluorescence staining with affinity-purified polyclonal antibodies to type VII collagen revealed a complete absence of staining in the skin of patients with severe dystrophic recessive epidermolysis bullosa. In all other forms, localized recessive dystrophic, dominant dystrophic, junctional and simplex forms there was an intense continuous linear staining of type VII collagen at the dermoepidermal junction. Also, obligate heterozygote carriers of the gene for severe dystrophic recessive form showed a normal pattern of staining. As internal controls and to define the clinical diagnosis, staining with antibodies to type IV collagen, laminin and bullous pemphigoid antigen was also performed. All these antibodies showed a normal staining pattern indicating an intact general morphology of the dermoepidermal junction zone. These results suggest that there is a defect of type VII collagen in patients with severe recessive dystrophic epidermolysis bullosa. The data also suggest that the group of recessive dystrophic epidermolysis bullosa may be heterogeneous not only clinically, but also at the molecular level.     

Expression of integrins in junctional and dystrophic epidermolysis bullosa.

Recently, monoclonal antibodies (MoAb) have been raised against a family of adhesive membrane receptors (R) for extracellular matrix molecules known as integrins. In order to ascertain whether these adhesive proteins are normally expressed in inherited epidermolysis bullosa (EB) dermal epidermal junction, we studied the reactivity of MoAb recognizing receptors for VLA-1 (R for unknown ligand), VLA-2 (R for collagen), VLA-3 (R for collagen, laminin, fibronectin), VLA-4 (R for unknown ligand), VLA-5 (R for fibronectin), VLA-6 (R for laminin), VNR alpha, and VNR beta (R for vitronectin) on cryostat skin sections from EB patients and normal controls and on cytospins of normal epidermal cell suspensions with indirect immunohistochemical methods. Two cases of junctional EB (EBj) (lethal and non-lethal), three cases of dominant dystrophic EB (EBdd), two cases of recessive dystrophic EB (EBdr), and two normal controls skin sections and cell suspensions entered the study. No significant modification of the distribution of these adhesive receptors was observed in junctional and dystrophic EB skin. Both in normal and EB specimens MoAb against VLA-2, VLA-3, and VNR alpha determinants showed reactivity with the total cytoplasmic membrane of basal keratinocytes and basement membrane zone. Interestingly, anti-VLA-6 MoAb was characterized by an intense linear staining of the dermal-epidermal junction with the same localization on the roof of the blisters in EBj, EBdd, and EBdr as bullous pemphigoid (BP) serum. On the basis of these results we suggest that anti-VLA-6 MoAb could be used instead of BP serum for immunohistochemical detection of the cleavage of blisters in EB.     

Evidence for a structural abnormality of collagen VII in a patient with dystrophic epidermolysis bullosa inversa.

Recent studies indicate that in skin of patients with dystrophic epidermolysis bullosa (EB) inversa, anchoring fibrils have an abnormal ultrastructure, but the major protein of these fibrils, collagen VII, is expressed and detectable with antibodies at the dermo-epidermal junction. For molecular characterization of this rare EB phenotype, skin biopsies from a patient with dystrophic EB inversa were investigated with indirect immunofluorescence, immunoelectron microscopy, and immunoblotting. Ultrastructural analysis of clinically uninvolved skin showed sublamina densa splitting. In unblistered areas, focal groups of anchoring fibrils that appeared loosely polymerized and without a distinct crossbanding pattern were observed. Indirect immunofluorescence staining with antibodies to collagen VII exhibited a linear fluorescence at the dermo-epidermal junction and at the roof of a spontaneous blister. Immunoelectron microscopy demonstrated staining of the poorly assembled anchoring fibrils, but no significant reaction in areas where no fibrillar structures could be discerned. In contrast to normal control skin, immunoblotting showed immunoreactive collagen VII in both epidermal and dermal extracts. Moreover, the dermis-associated collagen VII appeared as a distinct doubleband that contained the tissue form of collagen VII (250-300 kD) and an additional band with a slightly smaller molecular weight. In epidermal extracts one band, of the size of the tissue form, was detected. The studies on the present patient suggest that a structural abnormality of collagen VII that prevents its aggregation to stable dimers or polymerization to distinct anchoring fibrils may contribute to the etiopathogenesis of dystrophic EB inversa.     

Epidermolysis bullosa acquisita: diagnosis by optic immunofluorescent demonstration of junctional antigens and vitamin E treatment

In a patient with epidermolysis bullosa acquisita the characteristic dermolytic cleavage was demonstrated by electron microscopy and by mapping of antigenic determinants (type IV collagen, laminin, bullous pemphigoid antigen) of the dermal-epidermal junction. The latter method represents a rapid and reliable way to determine the cleavage plane in diseases which display subepidermal blister formation at the light-microscopic level. The classification of epidermolysis bullosa acquisita is still under dispute. Due to its highly characteristic clinical, ultrastructural, and immunologic features and pending further experimental data, epidermolysis bullosa acquisita should be regarded as a separate disease entity; its lumping together with cicatricial pemphigoid, as proposed by some authors appears speculative. Therapy of epidermolysis bullosa acquisita is generally regarded as difficult; following a 3-year course of high dose vitamin E therapy our patient underwent complete clearing; the possibility of a spontaneous remission, on the other hand, cannot be unequivocally ruled out.