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.     

A subepidermal blistering disease with histopathological features of dermatitis herpetiformis and immunofluorescence characteristcs of bullous pemphigoid: a novel subepidermal blistering disease or a variant of bullous pemphigoid?

A 64-year-old man presented with a bullous eruption which clinically and histopathologically resembled dermatitis herpetiformis. However, direct immunofluorescence analysis showed IgG deposits at the basement membrane zone, indicating a relationship with bullous pemphigoid or epidermolysis bullosa acquisita. Indirect immunofluorescence studies on salt-split skin showed binding of IgG mainly on the dermal side of the blister. Immunoblot analysis revealed a novel 200 kDa dermal antigen that could be associated with a major pathogen in this blistering a disease. The histopathological similarity to dermatitis herpetiformis and the immunofluorescence findings indicating bullous pemphigoid or epidermolysis bullosa acquisita seem typical of a distinct subepidermal blistering disease characterized by this 200 kDa antigen. However, the pathogenetic role of autoantibodies against this antigen should be further elucidated before confirming whether this case represents a novel subepidermal blistering disease or a special variant of bullous pemphigoid.     

Lichen planus pemphigoides: case report and results of immunofluorescence and immunoelectron microscopic study

A Japanese woman with lichen planus pemphigoides is reported. Immunologic characteristics of lichen planus pemphigoides antigen in the patient were investigated by indirect immunofluorescence and compared with those of bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen. Ultrastructural localization of lichen planus pemphigoides antigen was studied with the use of immunoelectron microscopic techniques. Lichen planus pemphigoides antigen showed localization similar to that of bullous pemphigoid antigen but different from that of epidermolysis bullosa acquisita antigen. The antigenic stability of lichen planus pemphigoides antigen was different from that of bullous pemphigoid antigen or epidermolysis bullosa acquisita antigen. Thus this study demonstrates that lichen planus pemphigoides antigen is different from bullous pemphigoid antigen.     

Human orf complicated by epidermolysis bullosa acquisita

Orf is a DNA parapoxvirus transmitted to humans by contact with infected goats and sheep. Many complications have been reported after orf infection, including erythema multiforme. A few cases of autoimmune bullous dermatosis complicating orf disease have been reported to date. They are usually characterized by tense blister eruptions with or without mucosal involvement; linear deposition of C3, IgG and/or IgA along the basement membrane; and negativity of indirect immunofluorescence analysis and enzyme‐linked immunosorbent assay (ELISA) (performed in four of 11 reported cases). These analyses have targeted antigens of bullous pemphigoid, mucous membrane pemphigoid or epidermolysis bullosa acquisita, except one case of mucosal pemphigoid with antilaminin‐332 antibodies. We describe the case of a patient who presented with an ulceration on his finger 10 days after direct contact with a lamb during Eid al‐Adha. Four weeks later he developed a severe tense blistering eruption associated with mucous membrane erosions. Indirect immunofluorescence analysis using the patient's serum revealed circulating antibasement membrane IgG that bound the dermal side of salt‐split skin. ELISA was positive for recombinant immunodominant NC1 domain of type VII collagen. We finally diagnosed epidermolysis bullosa acquisita complicating probable human orf infection.     

Epidermolysis bullosa acquisita. Incidence in patients with basement membrane zone antibodies

We examined the incidence of epidermolysis bullosa acquisita in patients with circulating basement membrane zone antibodies. Serum samples from 100 sequential patients with basement membrane zone antibodies were tested by indirect immunofluorescence against 1 mol/L sodium chloride split skin and by Western immunoblot against epidermal and dermal extracts of skin. Ninety-two (92%) serum samples stained only the epidermal side of split skin, 5 (5%) stained only the dermal side, and 3 (3%) stained both sides. Four of the 5 serum samples with dermal staining but none of the serum samples with epidermal or combined staining reacted with the 290-kd epidermolysis bullosa acquisita antigen by Western immunoblot. These results indicate that approximately 5% of unselected patients with basement membrane zone antibodies have epidermolysis bullosa acquisita or bullous lupus erythematosus rather than bullous pemphigoid.     

Epidermolysis bullosa acquisita: report of a case with comparison of immunogold electron microscopy using pre- and postembedding labelling

Summary A patient with epidermolysis bullosa acquisita (EBA). who has been diagnosed as having bullous pemphigoid for 7 years, is reported. By immunoblotting, both the latest serum and a 4-year-stored serum sample of the patient, were shown to react with the 290-kDa EBA antigen or type VII collagen, but not with bullous pemphigoid antigens. Pre-embedding immunogold electron microscopy demonstrated that the serum bound to the 'anchoring plaque' and to both ends of the anchoring fibrils in the fashion reported previously. In contrast, postembedding immunoelectron microscopy showed binding mainly to the lamina densa. These results indicate that EBA antigens are localized mainly at the lamina densa. Further studies are necessary for confirmation.     

A Case of Bullous Pemphigoid with Antibodies against Intercellular 130 KD Antigen

Pemphigus and bullous pemphigoid are two typical autoimmune bullous diseases that involve circulating autoantibodies directed against the epidermal cell surface and the epidermal basement membrane zone, respectively. The coexistence of pemphigus and bullous pemphigoid is rare. We describe a case of a 79-year-old man who had tense bullae and erythematous, erosive lesions on his trunk and four extremities. Histopathology revealed subepidermal blister formation without any evidence of intraepidermal acantholytic changes. Direct immunofluorescence study demonstrated deposition of IgG on the epidermal intercellular spaces, as well as along the basement membrane zone; C₃ was detected only on the latter. Indirect immunofluorescence study using monkey esophagus as a substrate demonstrated the presence of circulating antibodies against both junctional and intercellular antigens. In order to analyze the precise nature of this patient's antibodies, indirect immunofluorescence study using cultured human keratinocytes and immunoblot analyses were performed. Pemphigus vulgaris sera showed smooth and uniform staining on intercellular spaces. The patient's serum showed a granular and uneven staining pattern. Immunoblot analysis showed that the patient's serum reacted with the typical 230 kd (bullous pemphigoid) antigen and 130 kd antigen, which is close to the pemphigus vulgaris antigen.     

Psoriasis vulgaris coexistent with epidermolysis bullosa acquisita

Summary Autoimmune bullous diseases, such as bullous pemphigoid or pemphigus vulgaris. occasionally develop in psoriatic patients. In addition, a novel subepidermal bullous disease with autoantibodies against a lower lamina lucida antigen of 200kDa has recently been reported in association with psoriasis. We describe here a patient with psoriasis vulgaris who developed epidermolysis bullosa acquisita (EBA). Direct immunofluorescence revealed linear deposition of IgCl and C3 at the basement membrane zone. The patient's serum bound to the dermal side of salt-split normal human skin. However, immnumohlol analysis demonstrated that the patient's serum reacted with an EBA antigen of 290 kDa. EBA should be included in the list of autoimmune diseases associated with psoriasis vulgaris.     

Bullous pemphigoid of childhood: immunofluorescent investigation

A four-month-old Japanese girl with bullous pemphigoid of childhood (BPC) was reported. The characteristics of BPC antigen were studied by immunofluorescent technique using the serum of this patient with variously treated normal human skin as substrate. This study showed that her BPC antigen was quite similar to bullous pemphigoid antigen seen in adult patients and that this BPC antigen was differed from epidermolysis bullosa acquisita (EBA) antigen.     

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.     

A case of mixed bullous disease of epidermolysis bullosa acquisita and linear IgA bullous dermatosis

A 75-year-old Japanese male visited us with bullous eruptions on the extremities. Physical examination revealed large bullae on the hands, lower legs and feet. The oral mucosa was also involved. Histology disclosed subepidermal blister with inflammatory cell infiltrates in the dermis. Direct immunofluorescence showed deposits of IgG and IgA at the cutaneous basement membrane zone. Indirect immunofluorescence on 1 M NaCl-split human skin sections demonstrated that the patient's IgG antibodies reacted with the dermal side of the split, while IgA antibodies reacted with the epidermal side. Immunoblotting showed that the patient's serum reacted with the NC1 domain of type VII collagen (290-kDa epidermolysis bullosa acquisita antigen) as well as the 120-kDa linear IgA bullous dermatosis antigen, LAD-1. Systemic prednisolone resulted in a favorable response. From the clinicopathological findings, the present case is not consistent with either epidermolysis bullosa acquisita or IgA bullous dermatosis. Therefore, we regarded the case as mixed bullous disease of epidermolysis bullosa acquisita and linear IgA bullous dermatosis. Such a case has not been previously reported.     

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.     

Anti-p200 pemphigoid responding to dapsone

Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disease. Clinical presentation is similar to standard bullous pemphigoid (BP) but mucous membranes and cephalic lesions are more frequent. Histology and direct immunofluorescence (IF) are identical to BP but indirect IF discloses linear deposits of immunoglobulin G (IgG) on the dermal side of artificial salt-split skin. Specific diagnosis is based on western immunoblotting that shows circulating IgG recognizing a 200-kDa protein localized on the dermal extract. The 200-kDa antigen was recently identified as laminin γ1. Anti-p200 pemphigoid should be considered before all atypical or topical steroid-resistant bullous disease, as well as mucous membranes pemphigoid or epidermolysis bullosa acquisita. Dapsone appears to be the most effective treatment and should be used as the first option in combination with topical steroids. In this report, we describe the case of a patient with a typical clinical and immunopathological anti-p200 pemphigoid, responding to a combination of topical steroids and dapsone.     

DIFFERENTIATION OF BULLOUS PEMPHIGOID FROM EPIDERMOLYSIS BULLOSA ACQUISITA ON FROZEN SKIN BIOPSIES

Patients with bullous pemphigoid and epidermolysis bullosa acquisita may have similar clinical, histologic, and routine immunohistologic features. These two diseases can be distinguished by routine diagnostic studies either on a patient's serum tested by indirect immunofluorescence on salt-split normal skin or by obtaining a fresh perilesional skin biopsy, inducing a split at the lamina lucida, and testing for the site of IgG deposition by direct immunofluorescence. Often the serum studies are negative, while direct immunofluorescent studies yield the characteristic linear IgG staining of the basement membrane zone. To eliminate the need for a repeat biopsy to make a laboratory differential diagnosis, we studied the efficacy of salt-splitting perilesional skin biopsies that had been previously submitted and frozen for routine direct immunofluorescent studies. The biopsies were thawed, salt-split, and processed for direct immunofluorescence. Three epidermolysis bullosa acquisita biopsies and seven bullous pemphigoid biopsies examined demonstrated IgG staining at sites consistent with their respective diagnoses. The IgG appeared in the dermal side of the split biopsies in epidermolysis bullosa acquisita and predominantly, or exclusively, in the epidermal side in bullous pemphigoid. Thus the direct immunofluorescent study of previously frozen and subsequently salt-split skin biopsies may be used for the differential diagnosis of bullous pemphigoid from epidermolysis bullosa acquisita. In most cases, it may eliminate the need for a repeat biopsy.     

Epidermolysis Bullosa Acquisita Localized to the Face

A 39-year-old woman had a three-year history of recurrent bullous eruption localized to her left cheeck. The diagnosis of epidermolysis bullosa acquisita was confirmed by means of direct immunofluorescence and direct immunoelectron microscopic studies performed on the perilesional salt-split skin. Topical corticosteroid treatment reduced pruritus and bullae formation. This case of localized epidermolysis bullosa acquisita on the face is reminiscent of Brunsting-Perry cicatricial pemphigoid. We also review the previously reported cases of localized epidermolysis bullosa acquisita.     

A practical technique for differentiation of subepidermal bullous diseases: localization of in vivo-bound IgG by laser scanning confocal microscopy

OBJECTIVE: To develop a practical technique to distinguish autoimmune subepidermal bullous diseases. DESIGN: A prospective study. SETTING: Academic referral center-the Department of Dermatology, Medical University of Warsaw.Patients Forty-two patients fulfilling clinical, immunological, and/or immunoelectron microscopic criteria for bullous pemphigoid (n = 31), mucous membrane pemphigoid (n = 6), or epidermolysis bullosa acquisita (n = 5), diagnosed as having disease and treated from January 1, 1997, to December 31, 2002. MAIN OUTCOME MEASURES: We applied laser scanning confocal microscopy to determine the localization of in vivo-bound IgG at the basement membrane zone in biopsy specimens taken from patients' skin to compare the localization of basement membrane zone markers: antibody against beta4 integrin, antibody against laminin 5, and antibody against type IV collagen. In vivo-bound IgG was visualized by labeling with fluorescein isothiocyanate-conjugated anti-human IgG antibody, whereas basement membrane zone markers were labeled with anti-mouse Cy5-conjugated antibodies. RESULTS: In patients with bullous pemphigoid, in vivo-bound IgG was localized on the epidermal side of laminin 5 and co-localized with beta4 integrin. In patients with mucous membrane pemphigoid, IgG was in vivo bound to the dermal-epidermal junction between localization of laminin 5 and type IV collagen. In patients with epidermolysis bullosa acquisita, in vivo-bound IgG was present on the dermal side of type IV collagen. CONCLUSIONS: Laser scanning confocal microscopy allows precise localization of in vivo-bound IgG in patients' skin and, thus, it is a rapid method for the differentiation of mucous membrane pemphigoid from bullous pemphigoid and epidermolysis bullosa acquisita. This tool is suitable for the routine diagnosis of individual patients and for retrospective studies. This method is of special value in those patients in whom circulating autoantibodies are not detectable.     

Identification of cicatricial pemphigoid antigens.

The nature of skin antigens defined by antibodies in patients with cicatricial pemphigoid was studied with use of the 1 mol/L of sodium chloride split skin technique and Western immunoblot analysis. Antibodies in the serum samples of three of seven patients with cicatricial pemphigoid reacted to the epidermal side of 1 mol/L of sodium chloride split skin, and antibodies in the serum sample of one patient reacted to the dermal side. With Western immunoblot analysis, three patients had antibodies to antigens in the epidermal extracts of skin. The antibodies reacted in all patients to a 160-kd antigen and in one patient to an additional 230-kd antigen. These two antigens are similar in molecular weight to the 230-kd major bullous pemphigoid antigen and to the 160-kd minor bullous pemphigoid antigen. However, while the basement membrane zone antibodies present in cicatricial pemphigoid were most often directed to the 160-kd antigen, those present in 38 patients with bullous pemphigoid reacted most often (in 34 patients [89%]) to the 230-kd antigen. None of the serum samples reacted to antigens in dermal extracts that contained the epidermolysis bullosa acquisita antigen. These results indicate that the basement zone membrane antibodies present in cicatricial pemphigoid are directed in part to epidermal antigens that are similar in molecular weight to bullous pemphigoid antigens. However, the frequency of reactions to different basement membrane zone antigens differs in the two diseases, which may account for the clinical differences between the two conditions.     

Does class switching contribute to remission in bullous pemphigoid?

A correlation between the titre of circulating IgG autoantibodies and disease activity has been difficult to demonstrate in bullous pemphigoid. We postulate that isotype switching from "inflammatory" IgG1 to "blocking" IgG4 subclass antibodies might contribute to disease remission. We studied 16 patients with bullous pemphigoid, 3 patients with cicatricial pemphigoid and 2 patients with epidermolysis bullosa acquisita at different stages of the disease. The titres of IgG subclass and total IgG basement membrane zone autoantibodies were correlated with clinical activity. Ten of the 16 bullous pemphigoid patients went into remission. The total IgG autoantibody levels showed variable changes. IgG1 autoantibody decreased in 7 patients (3 were unchanged) and IgG4 autoantibody increased in 9 patients. The 6 patients with clinical activity did not show such changes in IgG1 and IgG4 autoantibodies. Similar results were observed with the other bullous diseases. Our data suggest that isotype switching from "inflammatory" IgG1 to "blocking" IgG4 antibody correlates with improvement in bullous pemphigoid.     

Serological diagnosis of bullous pemphigoid (BP): comparison of the sensitivity of indirect immunofluorescence on salt-split to immunoblotting

Specialized immunological assays are required for the accurate diagnosis of bullous dermatoses such as bullous pemphigoid (BP), epidermolysis bullosa acquisita and bullous lupus erythematosus. The aim of this study was to analyse and compare the sensitivity of indirect immunofluorescence (IF) on salt-split skin and immunoblotting for the detection of circulating autoantibodies in BP. Of the BP patients selected for the study, 74/79 (94%) had circulating autoantibodies detected by at least one of the two methods. Both methods had comparable sensitivity and detected BP-specific autoantibodies in 82-85% of the patients. Because 20% of the patients were found to be positive by only one of the methods, both methods should be used in the diagnosis of BP. Indirect IF on salt-split skin is easier to perform and is preferable in routine analysis, but Western blotting may be used as a complementary assay with sera showing no reactivity on salt-split skin.     

Epidermolysis bullosa acquisita: an autoimmune disease with distinctive immunoultrastructural features

Epidermolysis bullosa acquisita is a subepidermal bullous disease of the skin with distinctive clinical, histologic, immunologic, and ultrastructural features. A case of epidermolysis bullosa acquisita is presented in which the onset of the disease was heralded by inflammatory blisters similar to herpes simplex or herpes zoster. The immunofluorescent findings were indistinguishable from bullous pemphigoid. Although some inflammatory blisters persisted throughout the evolution of the disease, the patient eventually developed noninflammatory blisters that healed with milia formation and scarring, lesions typical of classic epidermolysis bullosa acquisita. The diagnosis of epidermolysis bullosa acquisita was definitely made by electron and immunoelectron microscopy which showed a sub-lamina-densa blister cleavage plane and immune deposits beneath the lamina densa. This case illustrates that some cases of epidermolysis bullosa acquisita may have an inflammatory stage and immunofluorescent findings that make it difficult to distinguish from inflammatory bullous diseases. The value of electron microscopy and immunoelectron microscopy in making a firm diagnosis of epidermolysis bullosa acquisita is emphasized.