IgA-mediated epidermolysis bullosa acquisita: two cases and review of the literature

We describe 2 adult patients with a subepidermal bullous dermatosis with exclusively linear IgA depositions along the epidermal basement membrane zone that were deposited in the sublamina densa zone as witnessed by direct immunoelectron microscopy. Indirect immunofluorescence microscopy of patients' sera revealed circulating IgA autoantibodies that bound exclusively to the dermal site of salt-split skin substrate. Immunoblot analysis using dermal and keratinocyte extracts were negative. Indirect immunofluorescence microscopy using type VII collagen-deficient skin ("knockout" substrate) showed no IgA binding, whereas linear IgA binding was seen at the epidermal basement membrane zone in normal human skin. The autoantigen in the patients was thus type VII collagen. A diagnosis of IgA-mediated epidermolysis bullosa acquisita (IgA-EBA) was made. We systematically reviewed the literature of this subset of patients with linear IgA dermatosis on the basis of the following criteria: exclusive binding of serum-IgA to the dermal side of salt-split skin or IgA depositions in the sublamina densa zone by indirect or direct immunoelectron microscopy. We learned that IgA-EBA is clinically indistinguishable from the classic "lamina-lucida type" linear IgA dermatosis or from the inflammatory type of IgG-mediated epidermolysis bullosa acquisita (IgG-EBA). Only a minority of the patients with IgA-EBA showed milia or scarring or had therapy-resistant ocular symptoms as in the mechanobullous type of IgG-EBA. Most patients with IgA-EBA responded to dapsone therapy.     

Bullous systemic lupus erythematosus

Two patients with established systemic lupus erythematosus developed bullous dermatoses clinically suggestive of bullous pemphigoid (BP). Direct immunofluorescence demonstrated linear staining at the dermoepidermal junction for IgG and C3 in a pattern typical for BP. However, immunoelectron microscopy demonstrated deposits of IgG largely below the basal lamina, with little if any deposit present within the lamina lucida. These findings are consistent with systemic lupus erythematosus (SLE) and effectively rule out BP. In addition, the leukocyte attachment assay, an in vitro assay of the functional activity of tissue-deposited immune complexes, demonstrated strong attachment of leukocytes to the dermoepidermal junction of patients' skin, suggesting that the immune reactants were functional immune complexes of possible importance to the pathogenesis of the bullous lesions. To our knowledge, this is the first time that immunoelectron microscopy has been performed on skin from patients suspected of manifesting concurrent SLE and BP. Our findings cast doubt on previous such reports by demonstrating the existence of a bullous form of SLE resembling BP both clinically and by direct immunofluorescence.     

Linear IgA Bullous Dermatosis with Circulating IgG Autoantibodies to the 230 kD Epidermal Antigen

The patient was a 54-year-old woman with wide-spread bullous lesions on her trunk and oral mucosa. Histologic examination revealed a subepidermal blister with infiltration of neutrophils and eosinophils. Direct immunofluorescence showed an exclusively IgA deposition at the basement membrane zone (BMZ). Indirect immunofluorescence showed that the blister fluid, but not the serum, contained IgG antibodies against the BMZ antigen on the epidermal side of salt-split skin. Using immunoblot analysis with normal human epidermal extracts, both serum and blister fluid reacted with the 230 kD epidermal antigen. Using colloidal gold and direct immunoelectron microscopy, IgA deposition was detected in the lamina lucida. Clinically, the skin lesions responded well to dapsone. We diagnosed this case as linear IgA bullous dermatosis (LABD) with IgG class circulating autoantibodies against the epidermal 230 kD antigen. These antibodies were considered to be secondary to the damage to the epidermal basal keratinocyte in this case.     

U-serrated immunodeposition pattern differentiates type VII collagen targeting bullous diseases from other subepidermal bullous autoimmune diseases

BACKGROUND: Epidermolysis bullosa acquisita (EBA) can be differentiated from other subepidermal bullous diseases by sophisticated techniques such as immunoelectron microscopy, salt-split skin antigen mapping, fluorescence overlay antigen mapping, immunoblot and enzyme-linked immunosorbent assay. OBJECTIVES: To determine whether the diagnosis can also be made by routine direct immunofluorescence microscopy. METHODS: We studied frozen skin biopsies from 157 patients with various subepidermal immunobullous diseases. RESULTS: We found three distinct 'linear' fluorescence patterns at the basement membrane zone: true linear, n-serrated and u-serrated. The true linear pattern, often seen in conjunction with either the n- or the u-serrated pattern, was found in any subepidermal immunobullous disease with nongranular depositions. In bullous pemphigoid, mucous membrane pemphigoid, antiepiligrin cicatricial pemphigoid, p200 pemphigoid and linear IgA disease the n-serrated pattern was found, corresponding with depositions located in hemidesmosomes, lamina lucida or lamina densa. However, in EBA and bullous systemic lupus erythematosus the u-serrated staining pattern was seen, corresponding with the ultralocalization of type VII collagen in the sublamina densa zone. The diagnosis of EBA with IgG or IgA autoantibodies directed against type VII collagen was confirmed by immunoelectron microscopy, salt-split skin antigen mapping, fluorescence overlay antigen mapping or immunoblotting. CONCLUSIONS: Using this pattern recognition by direct immunofluorescence microscopy we discovered several cases of EBA which would otherwise have been erroneously diagnosed as a form of pemphigoid or linear IgA disease.     

A bullous skin disease patient with autoantibodies against separate epitopes in 1 mol/L sodium chloride split skin.

BACKGROUND--We describe a patient with a subepidermal bullous skin disease associated with autoantibodies recognizing separate epitopes in 1 mol/L sodium chloride (NaCl) split skin. OBSERVATIONS--Direct immunofluorescence microscopy showed deposits of immunoglobulins and C3 in a continuous pattern in the patient's epidermal basement membrane zone. Direct immunoelectron microscopy demonstrated thick deposits of IgG overlying the lamina lucida and the lamina densa in a unique pattern. The patient had circulating IgG anti-basement membrane zone antibodies that bound both sides of 1 mol/L NaCl split skin, exhibited at least a fourfold-higher titer against the dermal side of this test substrate, and bound basal keratinocyte hemidesmosomes as well as focal sites along the superior portion of the lamina densa on indirect immunoelectron microscopy. Affinity purification of anti-basement membrane zone antibodies against epidermal or dermal strips of 1 mol/L NaCl split skin yielded IgG that only bound the side of split skin from which it was eluted. The patient's serum contained IgG that immunoprecipitated and immunoblotted the 230- and 170-kd bullous pemphigoid antigens. Affinity purification of patient antibody against bullous pemphigoid antigen immobilized on nitrocellulose paper yielded IgG that bound only the epidermal side of 1 mol/L NaCl split skin. The patient showed no evidence of reactivity against type VII collagen by direct immunoelectron microscopy, indirect immunoelectron microscopy, or immunoblot. CONCLUSIONS--This patient's bullous skin disease is associated with IgG anti-basement membrane zone antibodies with two specificities: one recognizing the bullous pemphigoid antigen in the epidermal side of 1 mol/L NaCl split skin, and another binding a distinct, yet presently unidentified, epitope in the superior aspect of the lamina densa.     

Restriction of cicatricial pemphigoid antigens to the lamina densa: confirmation by indirect immunoelectron microscopy.

Circulating anti-basement membrane zone (BMZ) antibodies in a patient with cicatricial pemphigoid (CP) were examined using an indirect immunofluorescence test, indirect immunoperoxidase electron microscopy, and Western blot analysis. An indirect immunofluorescence test on salt-split skin revealed that the anti-BMZ antibodies reacted solely to the dermal side at the separating epidermal-dermal interface, and indirect immunoelectron microscopy on intact skin indicated localization of the corresponding antigens (CP antigens) over the lamina densa and within the lower half of the lamina lucida; there were no CP antigens beneath a melanocyte. Indirect immunoelectron microscopy on salt-split skin demonstrated that the CP antigens were partly dissociated from, but restricted to, the lamina densa. Western blot analysis showed no differences in molecular weight between the CP antigens and bullous pemphigoid (BP) antigens. CP antigens, as detected by this patient's serum, appear to be constituted of molecules quite similar to BP antigens, but with different epitopes. CP antigens may be shed from basal cells and locate in the area of anchoring filaments, where they play a role in connecting basal cells to the underlying lamina densa.     

Immunomapping of EBA sera to multiple epitopes on collagen VII: further evidence that anchoring fibrils originate and terminate in the lamina densa

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease with circulating antibodies to type VII collagen, a major component of anchoring fibrils located at the dermal-epidermal junction. The purpose of this study was to further confirm the ultrastructural organisation of anchoring fibrils and to assess the relationship between the clinical phenotype of EBA and target site of their autoantibodies on anchoring fibrils. We studied the ultrastructural binding site of circulating autoantibodies from two patients with atypical clinical features who predominantly presented with oral lesions, and compared this with two patients with clinically typical forms of EBA. Immunoblotting of whole dermal extracts showed labelling of 290-kDa bands consistent with that of type VII collagen as well as the non-collagenous (NC-1) domain fusion protein in three out of four patients' sera. Postembedding immunoelectron microscopy (IEM) using Lowicryl K11M embedded normal human skin and patients' sera demonstrated the majority of labelling within the lamina densa, not below the lamina densa. We conclude that EBA autoantibodies in these patient's sera bind to the NC-1 domain of collagen VII situated in the lamina densa of the epidermal basement membrane, regardless of the EBA clinical phenotype. This confirms the previous notion that anchoring fibrils originate and terminate in the lamina densa.     

Heterogeneity of Brunsting-Perry type pemphigoid: a case showing blister formation at the lamina lucida, immune deposition beneath the lamina densa and autoantibodies against the 290-kD polypeptide along the lamina densa

An otherwise healthy 31-year-old man presented with multiple, vesicular, subepidermal blistering on the head, face, chest and oral cavity, leaving shallow scar formation, typical of Brunsting-Perry type pemphigoid. Direct immunofluorescence showed linear deposition of immunoglobulin (Ig)G and C3 along the basement membrane zone (BMZ), and indirect showed anti-BMZ autoantibodies (IgG, >40×) reacting with the dermal side under the salt-split study. Immunofluorescence staining for type IV collagen and laminins, as well as routine electron microscopy, demonstrated that the cleavage level of the blister was intra-lamina lucida. The immunoperoxidase method applied to lesional skin demonstrated IgG deposits along the lamina densa. The post-embedding immunogold method demonstrated that the autoantibodies against BMZ reacted with the lamina densa and the dermis just beneath it. Immunoblot studies demonstrated that the autoantibodies reacted with the 290-kD polypeptide (suggesting type VII collagen) when dermal extract was used as the substrate. The patient was treated with combination therapy consisting of 30 mg prednisolone, 900 mg nicotinamide and 750 mg tetracycline, and the number of newly forming blisters decreased. We concluded that Brunsting-Perry type pemphigoid, a rare autoimmune blistering disease, includes cases showing characteristics of epidermolysis bullosa acquisita as well as bullous pemphigoid. This case showed discrepancy between the blistering level (intra-lamina lucida) and location of antigen (lamina densa and sub-lamina densa areas).     

Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin

Sixty-one bullous disease sera containing IgG anti-BMZ antibodies were examined by indirect immunofluorescence on intact skin and skin separated through the lamina lucida by incubation in 1.0 M NaCl. All sera produced an indistinguishable pattern of linear immunofluorescence on intact skin at dilutions of 1:10 or higher. On separated skin, antibodies bound to either the epidermal (epidermal pattern), dermal (dermal pattern), or epidermal and dermal (combined pattern) sides of the separation. The binding patterns were consistent on separated skin from several donors and titers of anti-basement membrane zone antibodies on separated skin were comparable to those on intact skin. Sera from 3 patients with herpes gestationis (HG), 36 patients with bullous pemphigoid (BP), and 1 patient with clinical and histologic features of epidermolysis bullosa acquisita (EBA) showed an epidermal pattern. Sera from 9 patients with BP showed a combined pattern and sera from 6 patients with EBA and 6 patients with clinical and histologic features of BP showed a dermal pattern. Indirect immunoelectron microscopy of selected sera showed antibodies producing the epidermal and combined patterns were anti-lamina lucida antibodies and those producing the dermal pattern were anti-sublamina densa antibodies. These results show indirect immunofluorescence on separated skin is a dependable method for differentiating bullous disease anti-lamina lucida and anti-sublamina densa antibodies and that differentiating between the antibodies is essential for accurate diagnosis in some patients. The results also suggest BP anti-lamina lucida antibodies may have more than one antigenic specificity.     

Bullous systemic lupus erythematosus with autoantibodies recognizing multiple skin basement membrane components, bullous pemphigoid antigen 1, laminin-5, laminin-6, and type VII collagen.

BACKGROUND: Bullous systemic lupus erythematosus is a generalized subepidermal blistering skin eruption in patients suffering from systemic lupus erythematosus. Type VII collagen was initially identified as the target antigen. OBSERVATION: We studied an unusual patient who had bullous systemic lupus crythematosus. The patient fulfilled the criteria of systemic lupus with an antinuclear antibody titer of 1:5120. Immunopathological testing revealed in vivo deposition of all IgG subclasses, secretory IgA1, and both light chains at the patient's skin basement membrane. The in vivo-bound IgG and IgA were localized at the hemidesmosomes and lamina densa. The patient's IgG and IgA circulating autoantibodies labeled both the epidermal roof and the dermal floor of salt-split skin and recognized the hemidesmosomal protein BP230 as well as the full-length native form and the recombinant noncollagenous domain 1 of type VII collagen (anchoring fibril). In addition, the patient's IgG autoantibodies recognized the anchoring filament proteins laminin-5 and laminin-6 (alpha3 chain and gamma2 chain). CONCLUSIONS: We conclude that patients with bullous systemic lupus erythematosus may have autoantibodies to multiple basement membrane components critical for epidermal-dermal junctional adhesion. Possible pathogenic mechanisms in this patient's clinical diseases include provocation of organ-specific disease (bullous disease) by systemic autoimmunity (lupus) and the "epitope spreading" immune phenomenon.     

Studies of cicatricial pemphigoid autoantibodies using direct immunoelectron microscopy and immunoblot analysis

We studied 11 consecutive patients with classical cicatricial pemphigoid (CP) using direct immunoelectron microscopy (IEM) and Western immunoblotting analysis. Direct IEM performed in the skin or gingival mucosa revealed in all 11 CP patients that immunoglobulins and complement deposits were usually thick and discontinuous along the dermoepidermal junction, mostly localized on the lamina densa and occasionally in the lamina lucida. By direct IEM, the ultrastructural aspect in CP differs from the pattern observed in bullous pemphigoid (BP) and from that of chronic epidermolysis bullosa acquisita (EBA). Nine CP patients were studied by Western immunoblotting and, of these nine, only two had detectable anti-basement membrane zone (BMZ) antibodies by indirect immunofluorescence on salt-split skin. By immunoblotting performed on protein extracts of heat-separated epidermis, eight out of the nine CP sera specifically reacted with two protein bands of approximately 230-240 kD and 180 kD, similar to those recognized by BP sera in co-migration experiments. By immunoblotting on skin BMZ extracts, none of these nine CP sera recognized the 290-kD major polypeptide of EBA antigen. Taken together, these results suggest that, in CP, the target-antigen, as identified on immunoblots, is similar to BP antigen, but with an abnormal expression within the dermoepidermal junction of patients, which may in part explain the scarring course of the disease.     

The lupus band: do the autoantibodies target collagen VII?

BACKGROUND: Circulating autoantibodies directed against basement membrane zone (BMZ) components from patients with bullous pemphigoid and epidermolysis bullosa acquisita have been used to identify their target antigen in the skin and to confirm pathogenicity. Although the pattern of immunofluorescence in those diseases is similar to the lupus band, little is known about the origin and pathogenesis of the lupus band. Identifying the binding sites of the lupus band could provide a clue to the nature of the autoantigen that stimulates autoantibody formation in the skin of patients with systemic lupus erythematosus (SLE) and might provide valuable insight into the factors that influence the localization and pathogenicity of the lupus band. OBJECTIVES: To investigate the relation between the lupus band and the main BMZ components and to identify the target epitopes of autoantibodies deposited in the skin of patients with SLE. METHODS: Colocalization of the main components of the skin BMZ in nonlesional SLE skin with the lupus band was investigated using conventional immunofluorescence and confocal laser scanning microscopy. The effect of collagenase and pepsin on the expression of the lupus band was correlated with the differential sensitivity of these proteases on the collagenous and noncollagenous (NC) domains of collagen VII. Reactivity of sera from patients with SLE to a complete recombinant human NC1 domain of type VII collagen was then investigated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Near complete colocalization of the lupus band with collagen VII was found in this study, and chemical degradation of the skin attenuated the expression of the lupus band. Collectively, the NC1 domain of collagen VII was suggested as the target antigen of the lupus band, but none of the sera from patients with SLE reacted with recombinant NC1 domain-coated ELISA plates. Alternative explanations for the results of the colocalization of the lupus band with collagen VII are discussed. CONCLUSIONS: The lupus band colocalized with collagen type VII. The findings of this study ruled out the NC1 domain of collagen VII as a target antigen for circulating autoantibodies in SLE patients with no clinical evidence of blistering. Further studies are required to determine if other regions of collagen VII or another BMZ component is the target antigen for the immunoglobulins of the lupus band.     

大疱性系统性红斑狼疮的皮肤基底膜带相关抗原

免疫印迹和盐裂皮肤间接免疫荧光检测 ５例大疱性系统红斑狼疮（ＢＳＬＥ）血清，对照为 ５例获得性大疤性表皮松解症（ＥＢＡ）、２０例类天疱疮（ＢＰ）、２０例ＳＬＥ和１０例正常人血清。结果表明，３例（３／５）ＢＳＬＥ血清结合盐裂皮肤真皮侧和真皮提取物中２９０ ０００抗原，其中２例ＢＳＬＥ血清也结合表皮提取物中 １６５ ０００抗原，结果与 ＥＢＡ和部分ＢＰ血清相同。ＳＬＥ血清未结合 ２９０ ０００和 １６５ ０００抗原。提示ＢＳＬＥ血清中存在ＥＢＡ和ＢＰ抗体，推测ＥＢＡ和ＢＰ抗原可能是ＢＳＬＥ的皮肤基底膜带相关抗原。     

The localization of target antigens and autoantibodies in linear IgA disease is variable: correlation of immunogold electron microscopy and immunoblotting

Linear IgA disease (LAD) of adults and children is a dapsone-responsive, autoimmune subepidermal blistering disease characterized by linear IgA deposits at the basement membrane zone (BMZ) of the skin and mucosa. Circulating IgA antibodies to BMZ components are often present. In this study we investigated the ultrastructural localization of the antigens and autoantibodies in six patients with LAD (five adults and one child). Using a direct postembedding immunogold electron microscopy (EM) technique, three different patterns of IgA antibody deposition were seen in the skin of four patients with LAD. The IgA deposits localized within the uppermost part of the lamina lucida and to the basal surface of the hemidesmosome in two patients, to the lamina lucida in one, and to the lamina densa in the fourth patient. Using an indirect immunogold EM technique and serum or purified blister fluid from two additional LAD patients, we showed that the serum autoantibodies of one patient bound to the hemidesmosome of the BMZ, while the autoantibodies in the blister fluid of the other patient bound to the lamina densa and sublamina densa including the anchoring fibrils in a labelling pattern similar to that of the monoclonal antibody (LH7.2) to collagen VII. All the autoantibodies binding to the hemidesmosome or lamina lucida recognized a protein in epidermal extracts of molecular weight 180 kDa or its breakdown product of 97 kDa, 200 kDa or 230 kDa. The antibodies binding to the lamina densa recognized proteins of 180 and 285 kDa. The antibodies that bound to the lamina densa and anchoring fibrils recognized collagen VII. In this immunogold EM study we have shown four patterns of IgA labelling in six patients with LAD, associated with five different antigens as recognized by immunoblotting. These results, together with our previous immunofluorescence and immunoblotting findings add support to the contention that LAD is a heterogeneous disease as regards both the target antigens and epitopes.     

Ocular involvement in IgA-epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune bullous disease with frequent ocular involvement, but visual loss is rare. In contrast, EBA patients with predominant IgA autoantibodies more frequently develop severe ocular involvement, which tends to be refractory to therapy. We report two patients with 'IgA-EBA' with ocular involvement. Both initially presented with a generalized bullous disease, and direct immunofluorescence microscopy demonstrated IgA in the basement membrane zone of the skin, and in the conjunctiva and cornea of patient 1. On salt-split patient skin, IgA was found predominantly on the dermal side of the artificial split in both patients. Direct immunoelectron microscopy demonstrated IgA below the lamina densa in close association with the anchoring fibrils in both patients. In patient 1, who had a prolonged course of the disease, the skin disorder responded well to treatment with cyclosporin, but the ocular involvement ended in bilateral blindness despite repeated surgical treatment. In patient 2, the blister formation and scarring conjunctivitis was stopped by a combination of prednisolone and colchicine. These patients show that in subepithelial blistering diseases, early delineation of disease nosology is critical to detect subtypes with severe ocular involvement such as 'IgA-EBA'. In addition, colchicine may be a valuable alternative in the treatment of EBA with ocular involvement.     

IgA basement membrane zone autoantibodies in bullous pemphigoid detect epidermal antigens of 270–280 kDa, 230 kDa, and 180 kDa molecular weight by immunoblotting

Bullous pemphigoid (BP) is an acquired subepidermal blistering disease characterized by circulating IgG autoantibodies binding to the 230 and 180 kDa hemidesmosomal proteins. Associated basement membrane zone (BMZ) autoantibodies of the IgA class have been reported in few BP patients. The incidence and clinical relevance of these IgA antibodies, as well as their target antigens are unknown. Sera of 26 patients with BP were analysed for circulating IgG- and IgA-anti-BMZ autoantibodies by indirect immunofluorescence on salt-split human skin. All of the patients had circulating IgG autoantibodies and, in addition, nine (35%) also had circulating anti-BMZ IgA antibodies, that bound to the epidermal side of salt-split skin. By immunoblotting, IgA antibodies in seven of nine sera recognized either the 180 kDa, the 230 kDa, or both BP antigens. Moreover, IgA anti-BMZ antibodies in seven sera also detected an epidermal protein of 270-280 kDa. IgA antibodies did not identify specific bands on immunoblots of dermal extracts. There was no clinical difference between BP patients with or without circulating anti-BMZ-IgA.     

Evidence that anti-basement membrane zone antibodies in bullous eruption of systemic lupus erythematosus recognize epidermolysis bullosa acquisita autoantigen

Circulating and tissue-deposited IgG antibodies to the cutaneous basement membrane zone (BMZ) were detected in 3 patients with the clinical, pathologic, and immunologic features of bullous eruption of systemic lupus erythematosus (SLE). The antibodies were present in sera and IgG fractions in all cases and in eluates of cutaneous immune deposits from one of the cases. The antibodies were easily detected in sera by indirect immunofluorescence on adult human thigh skin separated through the lamina lucida by incubation in 1.0 M NaCl but were less easily detected on intact neonatal foreskin. The antibodies had features of epidermolysis bullosa acquisita (EBA) anti-BMZ antibodies including binding to the dermal side of the BMZ in separated skin, binding to the cutaneous but not vascular or glomerular basement membranes, binding to and just below the lamina densa, and binding to 290 or 290 and 145 kD dermal proteins previously identified as components of the EBA autoantigen. The antibodies were relatively specific for SLE patients with features of bullous eruption of SLE since they were detected in 3 of 4 of those cases and in only 1 of 20 SLE patients without blisters. These results show anti-BMZ antibodies with features of EBA antibodies are present in patients with bullous eruption of SLE and suggest there may be a close relationship between that disease and EBA. The results also suggest that EBA antibodies may be part of the autoantibody spectrum of SLE and that separated skin is more sensitive than intact skin for their detection.     

The localization of the target antigens and antibodies in linear IgA disease is heterogeneous, and dependent on the methods used

Fifty-nine patients with linear IgA disease, 24 with onset in childhood and 35 with adult onset, were studied. Sera from all patients were tested by indirect immunofluorescence, using as substrates intact normal skin and normal skin which had been split through the lamina lucida region of the basement membrane zone by suction and by prolonged incubation with molar NaCl. This enabled the site of the target antigen for the circulating IgA antibodies to be determined. The sites of deposition of the IgA antibodies in vivo were detected by raising a suction blister in eight patients, and splitting seven patients’ biopsies by prolonged incubation with molar NaCl. Eighteen sera were positive with intact skin, and 34 with split skin. Twenty-nine sera were positive with suction blisters as substrate; 14 bound to the epidermal aspect of the split skin, seven in a combined pattern (binding to the epidermis and dermis) and six to the dermal aspect. Thirty-one sera bound to salt-split skin, 24 to the epidermal side and seven on the dermal side. There was discordance between the two methods of skin splitting in 15 sera. Seven sera gave a combined pattern with suction but with salt-split skin, five of these bound epidermally, one was dermal, and one negative. Five sera showed epidermal binding on saltsplit skin and were negative on suction blisters, and the reverse was seen with one serum. Two sera gave variable results on suction blisters. Direct immunofluorescence studies showed dermal binding on all eight patients with suction blisters, and epidermal binding in four and dermal binding in three patients with salt splitting. These results demonstrate that the location of the target antigens and the sites of deposition of the antibodies are dependent on the methods used. They also suggest that there are at least two different antigens, an epidermal- and a dermal-associated antigen. The sera reacting in the combined pattern may represent antibodies reacting with a different epitope of the epidermal antigen, with a further epidermal antigen, or with two target antigens.     

Inflammatory epidermolysis bullosa acquisita with coexistent IgA antibodies to plectin

We present a case of inflammatory epidermolysis bullosa acquisita (EBA) with IgA antibodies to plectin. Analysis of lesional skin biopsies by electron microscopy revealed the split level to be in the sublamina densa zone, corresponding to the diagnosis of EBA. Direct immunofluorescence of perilesional skin demonstrated u-serrated depositions of IgG and IgA that under immunoelectron microscopy were shown to be located in the sublamina densa. In contrast, indirect immunofluorescence on salt-split skin revealed circulating IgA antibodies that stained the roof rather than the floor of the blister. Immunoblotting showed these serum antibodies to be directed to the cytoplasmic hemidesmosomal antigen plectin. The antiplectin specificity of these antibodies was confirmed by 'knockout' immunofluorescence analysis; the serum IgA did not bind to skin sections of a patient with plectin-deficient epidermolysis bullosa. To our knowledge, this case demonstrates for the first time the existence of IgA antibodies against plectin.     

A Case of Nonscarring Inflammatory Epidermolysis Bullosa Acquisita: Characterization of IgG Autoantibodies by Immunofluorescence,Immunoblotting and Immunogold Electron Microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.