Epidermolysis bullosa: variability of expression of cicatricial pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita antigens in clinically uninvolved skin

Indirect immunofluorescence was performed on skin from 13 patients with epidermolysis bullosa (EB) (simplex, 6; junctional, 2; dystrophic, 5) to compare the expression of 3 basement membrane components, bullous pemphigoid (BP) antigen, cicatricial pemphigoid (CP) antigen, and epidermolysis bullosa acquisita (EBA) antigen, in clinically uninvolved tissue. In addition, expression of laminin, type IV collagen, and KF-1 antigen was also evaluated. Whereas laminin, type IV collagen, and KF-1 antigen were each detectable in EB skin in a manner identical to that previously reported, marked variability was noted in the expression of BP, CP, and EBA antigens. However, no correlation was noted comparing lack of expression of any one of these latter antigens with either of the remaining two. Of these 3 antigens, BP antigen was the least often detectable, particularly in skin from patients with EB simplex. The lack of detectable BP antigen in EB simplex skin appeared to correlate with more extensive disease involvement and/or younger patient age. These findings may, therefore, limit the usefulness of BP serum in immunofluorescence mapping. Additionally, the disparity in expression of basement membrane antigens defined by BP and CP sera suggests that BP antigen and CP antigen are distinct entities.     

Epidermolysis bullosa acquisita and associated symptomatic esophageal webs

Epidermolysis bullosa acquisita (EBA) is a well-characterized, subepidermal blistering disorder associated with autoimmunity to type VII collagen, which is the collagen localized to anchoring fibrils within the dermoepidermal junction of skin. Although the full clinical spectrum of EBA is still being defined, it is known that the clinical features of EBA may be reminiscent of hereditary dystrophic epidermolysis bullosa, a scarring blistering disease of children that is commonly associated with esophageal stenosis. We describe a patient with EBA who had both an acral-predominant mechanobullous disease akin to dystrophic epidermolysis bullosa and an inflammatory, widespread bullous eruption reminiscent of bullous pemphigoid in association with esophageal webs and dysphagia. Although esophageal involvement is common in dystrophic epidermolysis bullosa, a review of the literature shows that this is the first bonafide case of EBA with symptomatic esophageal disease.     

Identification of a de novo glycine substitution in the type VII collagen gene in a proband with mild dystrophic epidermolysis bullosa

The dystrophic forms of epidermolysis bullosa result from different types and combinations of mutations in the type VII collagen gene (COL7A1). We describe a novel glycine substitution arising as a de novo mutation in a proband with a clinically mild form of dystrophic epidermolysis bullosa and no family history of any blistering disease. This report underscores the predominance of glycine substitutions in the dominantly inherited forms of dystrophic form epidermolysis bullosa, and heightens our awareness of unusual modes of inheritance. This information is critical for accurate genetic counseling and determination of recurrence risk in families with dystrophic EB.     

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.     

Behavior of epidermolysis bullosa fibroblasts in a hydrated collagen lattice

To determine if an altered ability to contract a hydrated collagen lattice is characteristic of fibroblasts from patients with recessive dystrophic epidermolysis bullosa (RDEB), we examined contraction by fibroblasts from normal subjects and patients with RDEB, dominant dystrophic epidermolysis bullosa (DDEB), and dominant epidermolysis bullosa simplex (DEBS). An extremely broad range of contractility (normal, poor, and hypercontraction) was observed in all types of epidermolysis bullosa (EB). When contraction in control fibroblasts was defined as the mean +/- 2 SD, (all control values were within this range) and the data were analyzed by the chi-square test, only 32% of EB cells fell within this range, with 47% poorly contractile and 21% hypercontractile. These data, derived from 34 patients, indicate that no single genetic defect resulting in altered contractility in the 3 distinct types of EB is likely. Neither cell viability, collagenase expression, nor PGE2 synthesis as correlated with gel contraction in any group. Indomethacin had no effect on contraction in RDEB. It is possible that the genetic defects in EB cause blister formation in vivo and may lead in some way to an abnormal interaction of fibroblasts with the extracellular matrix resulting in an altered collagen lattice contraction in vitro.     

Anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita: differentiation by use of indirect immunofluorescence microscopy

Binding of autoantibodies to laminin 5 and type VII collagen causes anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita, respectively. Differentiation between these two dermal-binding autoimmune bullous dermatoses is not yet possible by indirect immunofluorescence microscopy. In this study we tested whether two recently described immunofluorescence techniques, "knockout" skin substrate and fluorescent overlay antigen mapping, can differentiate between anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita. A total of 10 sera were tested: 4 with antilaminin 5, and 6 with antitype VII collagen autoantibodies, as characterized by either immunoblot or immunoprecipitation analysis. Differentiation between anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita was possible in all 10 sera by indirect immunofluorescence using either knockout skin substrate or fluorescent overlay antigen mapping technique.     

遗传性大疱性表皮松解症致病基因研究进展

遗传性大疱性表皮松解症(EB)是由皮肤中各种结构蛋白的遗传基因突变导致的一组机械性大疱病，临床主要表现为机械性脆性皮肤、张力性大疱和结痂等。根据电子显微镜下水疱的位置，大疱性表皮松解症分为4类不同的临床亚型，分别是单纯型EB、交界型EB、营养不良型EB以及Kindler综合征。迄今为止，已发现19个导致大疱性表皮松解症发生的基因。本文对大疱性表皮松解症的致病基因以及各基因型和临床表型之间的关系进行了综述。     

A comparative study of immunohistochemistry and electron microscopy used in the diagnosis of epidermolysis bullosa

Electron microscopic examination still is the gold standard for classifying epidermolysis bullosa, although it is relatively expensive, time consuming, and not readily available. Immunoreagents have been developed recently to map antigens in the basement membrane on routinely processed specimens. The current study was performed to examine the diagnostic usefulness of immunohistochemistry, as compared with electron microscopic examination, for analyzing routine formalin-fixed paraffin-embedded sections of epidermolysis bullosa. This study investigated 39 consecutively diagnosed cases of epidermolysis bullosa in which both electron microscopic examination and immunohistochemistry were used. In each case, three monoclonal antibodies were used to stain for laminin 1, collagen IV, and keratin. The immunohistochemical patterns were defined as follows: epidermolysis bullosa simplex (laminin, collagen IV, or both at the dermal floor of the blister and keratin at both the dermal floor and the epidermal roof), junctional epidermolysis bullosa (laminin, collagen IV, or both at the dermal floor of the blister and keratin only at the epidermal roof), and dystrophic epidermolysis bullosa (collagen IV, laminin, or both, and keratin all at the epidermal roof). Altogether, electron microscopic examination subclassified epidermolysis bullosa into its three major forms in 37 of the 39 cases (95%), and immunohistochemistry in 33 of the 39 cases (85%). All of the classifiable cases were concordant. Specifically, immunohistochemistry was diagnostic in 10 of 14 (71%) epidermolysis bullosa simplex cases, 14 of 14 (100%) junctional epidermolysis bullosa cases, and 9 of 11 (82%) dystrophic epidermolysis bullosa cases. The most frequent cause for inconclusive immunohistochemical results was failure in staining of the basement membrane with the antibodies to both laminin and collagen IV. In conclusion, the use of immunohistochemistry on routinely processed specimens may be useful for subclassifying epidermolysis bullosa into its major forms in the majority of the cases, although it still cannot fully replace electron microscopic examination or immunofluorescence mapping in the diagnosis of epidermolysis bullosa.     

KF-1 monoclonal antibody defines a specific basement membrane antigen defect in dystrophic forms of epidermolysis bullosa

The monoclonal antibody, KF-1, identifies a noncollagenous constituent of the lamina densa of the basement membrane zone (BMZ) of skin. In order to determine whether this BMZ constituent is affected in epidermolysis bullosa (EB), a mechanobullous skin disease often resulting in marked disfigurement, we have examined skin from patients with various forms of this disease for binding by KF-1 as well as for binding by polyclonal antibodies to laminin, type IV collagen, and bullous pemphigoid antigen, three other known BMZ components of normal skin. In all specimens from patients with simplex and junctional forms of EB, all four antibodies bound normally. In contrast, absent or diminished KF-1 binding was noted in all skin specimens from patients with dystrophic EB; antibodies directed against the other BMZ constituents, however, bound normally. This suggests that KF-1 may play a role in the structural integrity of normal skin and its absence or diminution may be important in the pathogenesis of lesion formation in dystrophic EB.     

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.     

Autoantibodies in anti-p200 pemphigoid stain skin lacking laminin 5 and type VII collagen

We report the case of a patient with a widespread bullous skin disease and linear deposits of IgG and C3 at the dermal-epidermal junction using direct immunofluorescence microscopy. Indirect immunofluorescence analysis demonstrated circulating IgG autoantibodies that stained, like autoantibodies to laminin 5 and type VII collagen, the dermal side of 1 mol L-1 NaCl-split human skin. By immunoblotting dermal extracts, the patient's serum, like serum samples from two control patients, reacted with a 200-kDa protein. Using immunoelectron microscopy, the serum labelled a component of the lower lamina lucida, but not the lamina densa/sublamina densa region, distinguishing this from the type VII collagen localization pattern. By immunofluorescence microscopy on skin sections from patients lacking either laminin 5 (Herlitz's epidermolysis bullosa) or type VII collagen (recessive dystrophic epidermolysis bullosa of Hallopeau-Siemens), the patient's serum retained reactivity with these test substrates. The patient's disease responded rapidly to the use of topical corticosteroids and lesions healed without scarring or milia formation. Our results provide strong evidence for the hypothesis that the 200 kDa autoantigen is different from laminin 5 and type VII collagen. For this new disease, we propose the designation 'anti-p200 pemphigoid'.     

Pathogenesis of mechanobullous disorders

Recent advances in the molecular biology of the dermo-epidermal basement membrane zone have contributed greatly to our understanding of the etiopathogenetic pathways underlying mechanobullous disorders. Genetic linkage was established between the keratin gene clusters and epidermolysis bullosa simplex, and keratin mutations were identified in several patients. Anchoring filaments and the alpha 6 beta 4 integrin are likely to be affected in junctional EB. Genetic linkage was established between the collagen VII gene and both dominant and recessive subtypes of dystrophic epidermolysis bullosa, and different molecular abnormalities of collagen VII leading to formation of non-functional, rudimentary anchoring fibrils were observed in several families. These discoveries that led to definition of mutations underlying EB also help us to understand the normal physiology and function of the affected structures. They may also point the way to new therapeutic strategies for common acquired blistering diseases and disturbances of epithelialization in general.     

Case of epidermolysis bullosa acquisita with concomitant anti‐laminin‐332 antibodies

Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti‐laminin‐γ1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin deposition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blistering disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti‐laminin‐332‐type mucous membrane pemphigoid has pathogenic autoantibodies against laminin‐332, which is a basement membrane heterotrimeric protein composed of α3, β3 and γ2 laminin chains. We describe a 73‐year‐old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully controlled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibodies to both type VII collagen and laminin‐332 α3 chain by indirect immunofluorescence of 1 mol NaCl‐split normal human skin, various immunoblot analyses and enzyme‐linked immunosorbent assays. This case was a rare case of EBA with concomitant anti‐laminin‐332 antibodies.     

Epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune subepidermal bullous disease with clinical features similar to the genetic form of dystrophic epidermolysis bullosa. EBA is characterized by the presence of autoantibodies against type VII collagen which is a major component of the anchoring fibrils at the dermal‐epidermal junction. EBA can be divided into two main clinical types; mechanobullous and inflammatory EBA. Mechanobullous EBA, referred to as classic EBA, presents with skin fragility, blisters and dystrophic changes on trauma‐prone areas. Inflammatory EBA resembles other autoimmune subepidermal bullous diseases. Compelling evidence from mouse models supports a pathogenic role of autoantibodies against type VII collagen in EBA. Treatment of EBA is often unsatisfactory. The most widely used systemic treatment is corticosteroids. Colchicine and dapsone have been reported to be good treatment modalities when combined with corticosteroids. Some intractable cases of EBA have successfully been treated with intravenous immunoglobulin or rituximab.     

Hemidesmosomal variants of epidermolysis bullosa

Abstract: Epidermolysis bullosa (EB), a heterogeneous group of genodermatoses, is characterized by fragility and blistering of the skin, associated with characteristic extracutaneous manifestations. Based on clinical severity, constellation of the phenotypic manifestations, and the level of tissue separation within the cutaneous basement membrane zone, EB has been divided into distinct subcategories. Traditionally, these include the simplex, junctional and dystrophic variants of EB. Recent attention has been drawn to variants of EB demonstrating tissue separation at the level of hemidesmosomes, ultrastructurally recognizable adhesion complexes within the cutaneous basement membrane zone. Clinically, these hemidesmosomal variants manifest either as generalized atrophic benign epidermolysis bullosa (GABEB), EB with pyloric atresia, or EB with late-onset muscular dystrophy, Elucidation of basement membrane zone components by molecular cloning and development of mutation detection strategies have revealed that the hemidesmosomal variants of EB result from mutations in the genes encoding the subunit polypeptides of the 180-kD bullous pemphigoid antigen/type XVII collagen, the α₆β₄ integrin, or plectin, respectively. Collectively, these data add to the understanding of the molecular complexity of the cutaneous basement membrane zone in EB, as attested by the fact that mutations in 10 different genes can underlie different variants of EB. Elucidation of mutations in different forms of EB has direct application to genetic counseling and DNA-based prenatal testing in families with EB.     

Immunofluorescence studies using skin sections of recessive dystrophic epidermolysis bullosa patients indicated that the antigen of anti-p200 pemphigoid is not a fragment of type VII collagen

BACKGROUND: There are a large number of autoimmune bullous diseases, which have distinct autoantibodies. Several reports on cases with IgG autoantibodies against a novel 200 kDa dermal protein have been published, for which we suggested the term, anti-p200 pemphigoid. However, the nature of this 200 kDa antigen has not been well characterized. OBJECTIVE: In this study, we examined the relationship between the 200 kDa protein and type VII collagen. METHODS: We collected sera from 12 cases of anti-p200 pemphigoid and skin sections from six cases of recessive dystrophic epidermolysis bullosa (RDEB). The reactivity of these sera was examined by indirect immunofluorescence using sections of the disease skin. RESULTS: we have shown that all the 12 anti-p200 pemphigoid sera could react with basement membrane zone of five cases of RDEB, while epidermolysis bullosa acquisita (EBA) sera were negative in these skins. In addition, in a case of RDEB, EBA sera reacted with intracytoplasmic deposition of type VII collagen, while no anti-p200 pemphigoid sera showed this reactivity. CONCLUSION: These results strongly suggested that the 200 kDa antigen is not a fragment of type VII collagen, but a specific autoantigen.     

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.     

Abnormal binding of an anti-amnion antibody to epidermal basement membrane provides a novel diagnostic probe for junctional epidermolysis bullosa

AA3 is a novel antibody raised against human amnion, which reacts with the basement membrane of various epithelia of ectodermal origin. We used AA3 to examine the epidermal basement membrane zone in normal skin and different genetically determined types of epidermolysis bullosa (EB), by indirect immunofluorescence. AA3 staining was normal in dystrophic and simplex EB, but was markedly reduced in lesional and non-blistered skin in severe forms of junctional EB. In non-lethal junctional EB, the intensity of staining was variable and appeared to be inversely associated with disease severity, but did not correlate with demonstrable abnormalities of hemidesmosomes. AA3 binding was not reduced in pemphigoid lesions or normal suction blisters. It appeared to localize to the lamina lucida, but with different characteristics compared with antibodies to laminin and bullous pemphigoid antigen. These finding suggest that AA3 recognizes an antigen (or antigens) which may be involved in a primary biochemical defect in junctional EB. Moreover, this antibody may act as a new probe for this potentially lethal mechano-bullous disease.     

Epidermolysis bullosa simplex generalisata: Importance of immunofluorescence studies in early diagnosis

Summary An unusual case of severe generalized epidermolysis bullosa (EB) simplex is described. Its severity, oral involvement and early milia formation suggested a dystrophic form of the disease, but early immunofluorescence studies on skin biopsy material using bullous pemphigoid (BP) serum clearly showed the level of cleavage to be superficial to the dermo-epidermal junction and microscopy confirmed this. The diagnosis of EB simplex was thus quickly established allowing conservative treatment to be pursued with confidence, and preventing unnecessary exposure of the child to systemic corticoid or phenytoin therapy.