Bullous Pemphigoid Antibodies Research Articles

P036 Pyoderma gangrenosum induced by Amgevita in a seropositive rheumatoid arthritis patient: a paradoxical effect of adalimumab?

Abstract Background/Aims In the recent years, the outlook of management of chronic inflammatory conditions have been promising with the introduction of biological therapies. With the patent protection of original biological drugs expires, biosimilar agents are introduced in replacement of the originators, though true clinical effectiveness remains debatable. As the rare side effect profiles of the originators are slowly emerging, these occurrences are yet to be observed in the biosimilars. We present the first case reported in the literature of pyoderma gangrenosum(PG) induced by Amgevita in a seropositive rheumatoid arthritis patient. Methods A 48-year-old gentleman had a long-standing history of seropositive rheumatoid arthritis, was commenced on Amgevita 40mg every other day in addition to his current regime of methotrexate and hydroxychloroquine in order to have better control of his disease acitivity. After 2 doses of Amgevita, he developed multiple blisters and ulcers on both of hands and the back of his neck. He denied any systemic illness and no new medication except Amgevita was initiated. He was referred urgently to the dermatologist which revealed on the dorsal aspect of hands several ulcers with fresh granulation and asymmetric border. Repeat immunology tests including vasculitis screen and bullous pemphigoid antibodies were negative. Blood and urine porphyrin screen came back negative. As skin biopsy’s findings were non-specific and inconclusive, treatment with Amgevita was reintroduced. Unfortunately, after a single dose of Amgevita, this patient developed similar ulcerations in the same distribution as previous. Amgevita was ceased immediately and patient was started a short course of steroid, which has led to complete resolution of the ulcers. Results Following his second presentation, it was concluded that the cause for his dermatological manifestation was Amgevita. Second opinion from the dermatologists and pathologist was sought and a diagnosis of pyoderma gangrenosum induced by Amgevita was finally made. Though patient’s skin lesions recovered after the withdrawal of Amgevita, he experienced severe flares of his joints. It was clear that he required another agent to help with his disease activity. The decision to initiate Benepali, another anti-TNF agent was made and the patient had managed to tolerate it well without any side effect. Conclusion Several recent studies have shown good therapeutic outcomes in patients with pyoderma gangrenosum treated with anti-TNF α antagonists, particularly in refractory cases. As our case proves that Amgevita can precipitate the manifestation of PG, this is suggestive that the anti-TNF α antagonist may have double-edged properties. There were very few reported cases of pyoderma gangrenosum linked to Humira, but no case of pyoderma gangrenosum induced by adalimumab-atto was known. This case highlights the paradoxical effect of anti-TNF α antagonists in PG and recognition of this association will allow the clinicians to withdraw the causative drugs promptly. Disclosure Y. Tan: None. S. Kavaklieva: None. F. Wood: None.

Bullous Pemphigoid With Nail Damage Associated With Kaposi Sarcoma: A Case Report

Introduction: Bullous pemphigoid is an acquired autoimmune bullous skin disease that occurs in the elderly, and nail involvement is relatively rare. Kaposi sarcoma(KS) is a rare opportunistic tumor in patients with iatrogenic immunosuppression and is rarely associated with autoimmune bullotic disease. Here we reported of a 73-year-old female with bullous pemphigoid with nail damage and KS. Case presentation: The patient was diagnosed with bullous pemphigoid based on her history, bullous pemphigoid antibody, and skin pathological examination. And nail involvement occurred 20 days prior to the recurrence of the disease. Moreover, KS occurred during high-dose glucocorticoid therapy in this patient. Discussion: The patient was admitted to hospital because of a five-month history of erythema and blisters and a one-month history of nodules and plaques. The diagnosis of bullous pemphigoid was followed by treatment with glucocorticoids and intravenous gamma globulin. During treatment, the disease recurred due to discontinuation of glucocorticoids. There were erythema and blisters all over the body, and purple nodules, plaques appeared in the base of some blisters. Pathological examination of nodules was consistent with KS. In addition, nail involvement occurred 20 days before recurrence. The particularity of this case lies in the simultaneous occurrence of nail involvement, bullous pemphigoid, and KS. Conclusion: Patients with autoimmune bullous skin disease may suddenly develop abnormal nails, and such patients should be vigilant about recurrence of the disease. Doctors should be aware of the possibility of KS when rapidly progressing purplish red plaques or nodules appear in patients receiving high doses of corticosteroids or immunosuppressants.

Demographics and Autoantibody Profiles of Pemphigoid Patients with Underlying Neurologic Diseases

Bullous pemphigoid (BP) is an autoantibody-mediated blistering disease that is often associated with neurologic disease. BP antibodies target two epidermal adhesion molecules, known as BP180 and BP230. Homologues to these proteins are found in the brain, and it is hypothesized that neurologic disease leads to the production of autoantibodies that can cross-react with their cutaneous forms. To better understand the link between BP and neurologic disease, we evaluated primary demographic features (age, sex, race, ethnicity, and elapsed time between onset of skin symptoms and BP diagnosis), severity of BP, and IgG and IgE autoantibody levels in BP control individuals and patients with BP with preceding Parkinson disease, dementia, and stroke. The main findings of this study are that patients with BP with preceding neurologic disease have a shorter elapsed time between onset of skin disease and BP diagnosis and that patients with preceding Parkinson disease or dementia, but not stroke, are significantly older than patients with BP without neurologic disease. However, no significant differences in clinical presentation, BP severity scores, or autoantibody (IgG and IgE) responses were observed among the groups. These findings suggest that, despite the age difference, the clinical phenotype of BP is not affected by preceding neurologic disease.

Eosinophils as putative therapeutic targets in bullous pemphigoid.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease and is characterized by the presence of autoantibodies directed against the hemidesmosomal proteins BP180 and BP230 that can be detected in the skin and serum of BP patients. Histologically, the dermal infiltration of eosinophils is obvious. The objective of this review was to present evidence that eosinophils play a key role in the pathogenesis of BP. Eosinophils, together with cytokines and chemokines regulating their production, recruitment and activation, are abundantly present in lesional skin, in blisters and in peripheral blood of patients with BP. Recently, using a cryosection model, eosinophils were demonstrated to induce dermal-epidermal separation in the presence of BP antibodies. Thus, eosinophils and their products, as well as mediators regulating their function, present promising targets for the treatment of BP.

The Value of BP230 Enzyme-Linked Immunosorbent Assay in the Diagnosis and Immunological Follow-Up of Bullous Pemphigoid

Background: The value of 230-kDa bullous pemphigoid antibody (BP230) enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bullous pemphigoid (BP) was investigated, but in the immunological follow-up of the disease remains unknown. Objective: Evaluation of BP230 ELISA for diagnosis, follow-up and prediction of relapse in BP. Methods: Monocenter retrospective and prospective study. Patients with typical BP. Detection of autoantibodies by indirect immunofluorescence (IIF), BP180 and BP230 ELISA tests at diagnosis, during the treatment (disease control or failure) and at treatment stop (relapse or not 3 months after). Results: 74 patients were included. At diagnosis, BP230 ELISA sensitivity was lower than IIF and BP180 ELISA. Combining both ELISA added a weak gain of sensitivity. Both tests paralleled the clinical evolution, especially in case of disease control. At the end of the treatment, BP230 ELISA was not different in patients with or without relapse. Conclusion: In routine practice, BP230 ELISA does not seem to be a useful additional test in typical BP.

Improvement of Intravenous Immunoglobulin Therapy for Bullous Pemphigoid by Adding Immunosuppressive Agents

Various antibody-mediated autoimmune disorders are treated with intravenous immunoglobulin (IVIg). While the exact action of IVIg is unknown, it likely acts to rapidly and selectively lower the level of pathogenic antibodies. The most effective use of IVIg, an expensive and potentially toxic treatment of autoimmune disorders, remains undetermined. We propose that the addition of immunosuppressive agents to the IVIg regimen may increase the ability of IVIg to lower the level of pathogenic antibodies. For 16 months, we observed a 78-year-old patient with autoantibody-mediated bullous pemphigoid who was treated with IVIg and an adjuvant therapy on 2 separate occasions as well as IVIg alone on 2 other occasions. We observed the greatest depression of bullous pemphigoid antibodies when IVIg was combined with an immunosuppressive agent. These results support the hypothesis that agents that suppress antibody synthesis can offset the rebound in the level of individual antibody that follows their depletion and thus can improve the effectiveness of IVIg treatment while reducing the cost and the potential toxic effects of therapy.

A new passive transfer animal model of bullous pemphigoid

Evaluation of: Nishie W, Sawamura D, Goto M et al. Humanuzation of autoantigen. Nat. Med. 13, 378–383 (2007).The development of a conventional passive transfer model of bullous pemphigoid (BP) is made impossible by the fact that BP antibodies that react with the immunodominant and potentially pathogenic epitope within the NC16A region fail to cross-react with the murine form of COL17. A recent paper by Nishie et al. describes one of the first successful murine models of organ-specific autoimmunity based on genetic modification and ‘humanization’ of the autoantigen (COL17). A COL17 knockout mouse (a murine equivalent of non-Herlitz junctional epidermolysis bullosa) was rescued by the human ortholog, which made it susceptible to a disease resembling BP on passive transfer of BP patients’ sera. The data presented provides in vivo evidence of the pathogenicity of circulating anti-NC16A autoantibodies from patients with BP.

Persistent Herpes Gestationis Treated with High-dose Intravenous Immunoglobulin

Sir, Pemphigoid gestationis (PG) is a rare autoimmune blistering disease specific to pregnancy that usually presents in the second or third trimester or during the immediate postpartum period (1, 2). PG may develop in association with other autoimmune diseases, as well as with trophoblastic tumours, hydatiform mole and choriocarcinoma (3). The pathogenesis of PG is not fully established. However, most patients develop antibodies against 2 hemidesmosomal proteins, BP180 (BPAG2, collagen XVII) and, less frequently, BP230. Historically known as the herpes gestationis factors, these circulating antibodies belong to the heat-stable immunoglobulin G1 subclass. Sera from PG can recognize 5 distinct epitopes within BP180 NC16A, 4 of which have been reported as major antigenic sites targeted by bullous pemphigoid antibodies (4). The PG has a strong association with human leukocyte antigens HLA-DR3 and HLA-DR4 or both, and virtually all patients with a history of PG have demonstrable anti-HLA antibodies (5). During pregnancy PG is often treated with topical corticosteroids and systemic antihistamines. However, in some patients with severe disease, systemic steroid therapy is needed. If the disease continues, treatment options are limited. Although plasmapheresis, cyclophosphamide, azathioprine, cyclosporine and dapsone, as monotherapy or in combined regimens all have been reported to be beneficial, they have potential side-effects that can be harmful to both the mother and the foetus (6, 7). There are only 2 case reports in the literature describing the use of intravenous immunoglobulin (IVIg) therapy in PG (6, 8). We report here a persistent case of PG, refractory to the conventional treatment with steroid plus azathioprine or dapsone, which responded positively to IVIg.

Granulocyte‐derived elastase and gelatinase B are required for dermal–epidermal separation induced by autoantibodies from patients with epidermolysis bullosa acquisita and bullous pemphigoid

Epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) are two clinically and immunologically distinct autoimmune subepidermal blistering skin diseases associated with IgG autoantibodies against the dermal-epidermal junction. BP antibodies are directed against the hemidesmosomal antigens BP180 and BP230, and those in patients with EBA target type VII collagen, a major component of anchoring fibrils. While the pathogenetic mechanisms of subepidermal blistering in BP have been previously studied using a passive transfer mouse model, the effector pathways of blister formation in EBA are largely unknown. Autoantibodies to type VII collagen and BP180 have recently been shown to induce leucocyte-mediated subepidermal cleavage in cryosections of human skin. The aim of the present study was to identify human leucocyte protease(s) instrumental in dermal-epidermal separation induced by autoantibodies to type VII collagen and BP180. When incubated with cryosections of human skin pretreated with IgG from patients with EBA or BP but not from patients with anti-laminin 5 mucous membrane pemphigoid or healthy controls, granulocytes were recruited to the dermal-epidermal junction and induced subepidermal splits. A combination of broad-range protease inhibitors as well as inhibitors of serine and matrix metalloproteases completely abolished dermal-epidermal separation induced by EBA or BP autoantibodies. When characterizing the proteases involved more specifically, selective inhibition of human leucocyte elastase or gelatinase B/MMP-9 was also found to result in suppression of blistering. These findings strongly suggest that elastase and gelatinase B are essential for granulocyte-mediated proteolysis resulting in dermal-epidermal separation in EBA and BP patients' skin.

An Antibody to BP 180 kDa Antigen is Able to Induce an Increase of Intracellular Ca 2+ Concentration in DJM-1 (Human Squamous Cell Carcinoma) Cells

To elucidate the induction of transmembrane signal transduction of keratinocytes by bullous pemphigoid (BP) antibodies, we assessed the intracellular Ca 2+ concentration ([Ca 2+ ]i) in DJM-1 cells (a squamous cell carcinoma cell line). The time course of fluo-3-fluorescence intensity was investigated, and transient increase of fluorescence intensity in DJM-1 cells was observed when the DJM-1 cells were incubated with rabbit IgG anti-human BP 180 kDa antigen (BP 180). No increase of fluorescence intensity was observed when the cells were incubated with rabbit IgG anti-human BP 230 kDa antigen (BP 230). This transient increase of fluorescence intensity was inhibited by U-73122, a specific inhibitor of phospholipase C. These results indicate the possibility that an antibody to BP 180 induces transmembrane signal transduction in keratinocytes.

Characterization of bullous pemphigoid antibody in clinically active and remission stage

Characterization of bullous pemphigoid antibody in clinically active and remission stage

Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.

To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.

Significance of IgG‐subclass antibody determinations in bullous pemphigoid

The presence of circulating basement membrane zone (BMZ) antibodies is characteristic of patients with bullous pemphigoid (BP) and are routinely employed in making the diagnosis. The positive tests, however, occur in 50–70% of patients with BP, thus necessitating consideration of other tests in a significant number of patients. The purpose of this study was to examine the efficacy of indirect immunofluorescence (IF) tests specific for various IgG subclasses antibodies to BMZ in BP, especially in patients who are seronegative on routine indirect IF tests with fluorescein-conjugated antibodies to IgG. BMZ antibodies primarily are of IgG4 subclass and are present in all BMZ antibody positive BP cases. Of BP patients negative for BMZ antibodies, 72% were found positive when tested for IgG4 subclass antibodies. In conclusion, testing for IgG4 subclass BMZ antibodies enhances the sensitivity of serum tests from 68.5% to 91%. This may be due in part to the inherent increased sensitivity of the assay and for detecting subclasses of IgG and in part, due to the subclass distribution of the BP antibodies. © 1996 Wiley-Liss, Inc.

Significance of IgG-subclass antibody determinations in bullous pemphigoid.

The presence of circulating basement membrane zone (BMZ) antibodies is characteristic of patients with bullous pemphigoid (BP) and are routinely employed in making the diagnosis. The positive tests, however, occur in 50-70% of patients with BP, thus necessitating consideration of other tests in a significant number of patients. The purpose of this study was to examine the efficacy of indirect immunofluorescence (IF) tests specific for various IgG subclasses antibodies to BMZ in BP, especially in patients who are seronegative on routine indirect IF tests with fluorescein-conjugated antibodies to IgG. BMZ antibodies primarily are of IgG4 subclass and are present in all BMZ antibody positive BP cases. Of BP patients negative for BMZ antibodies, 72% were found positive when tested for IgG4 subclass antibodies. In conclusion, testing for IgG4 subclass BMZ antibodies enhances the sensitivity of serum tests from 68.5% to 91%. This may be due in part to the inherent increased sensitivity of the assay and for detecting subclasses of IgG and in part, due to the subclass distribution of the BP antibodies.

In Situ-Bound Antibodies Eluted From the Skin of Patients with Bullous Pemphogoid Are Preferentially Directed Against the 230-kD Bullous Pemphigoid Antigen

Two bullous pemphigoid antigens (BPAs) have been identified: a 230-kD intracellular hemidesmosome-associated molecule and a 180-kD transmembrane hemidesmosome-associated molecule. Although patients with bullous pemphigoid (BP) have been shown to have circulating antibodies directed against one or both BPAs, the antigenic specificity of tissue-bound BP autoantibodies has not been studied. Because these skin-bound antibodies may play an important role in disease initiation, we sought to determine their antigenic specificity. In situ-bound BP antibodies were eluted with glycine from salt-split perilesional skin biopsy specimens and subjected to immunoprecipitation. Ten of 13 patients had glycine-eluted antibodies that recognized the 230-kD BPA, whereas four of 13 patients had glycine-eluted antibodies that recognized the 180-kD BPA. When these glycine-eluted BP skin specimens were sequentially eluted with urea, we found either antibodies of the same specificity or loss of reactivity to one of the BPAs, but no reactivity to any BPAs previously undetected with glycine elution. Sequential elution with sodium dodecylsulfate revealed no detectable BP antibodies. The heavy- and light-chain isotypes of the circulating, tissue-bound, and eluted BP antibodies were very similar, suggesting that we are not eluting certain subsets of BP antibodies. Sera from these patients contained circulating antibodies that recognized either one or both BPAs. Our observations demonstrate that in situ-bound antibodies eluted from the skin of patients with BP are preferentially directed against the 230-kD BPA.

Familial autoimmune enteropathy with circulating anti-bullous pemphigoid antibodies and chronic autoimmune hepatitis

In a family of four children (two boys and two girls), the two brothers had severe, protracted watery diarrhea beginning at 2 and 3 weeks of life, respectively. Duodenal mucosa in both patients showed total villous atrophy and severe inflammatory infiltration of the entire bowel. The first patient also had lymphoid cell infiltration of the pancreas and died at 6 weeks of age. The second boy is alive at 2 years of age and is immunocompetent, but still receives total parenteral nutrition. Indirect immunofluorescence studies revealed circulating antibodies to enterocytes, smooth muscle, thyroid, and islet cells. Bullous pemphigoid antibodies (230 and 180 kd), specific for hemidesmosomal proteins and usually associated with a subepidermal blistering skin disease, were detected by direct and indirect immunofluorescence studies and by Western immunoblot. A diagnosis of autoimmune hepatitis was made, based on evidence of chronic active hepatitis and circulating anti-smooth muscle antibody. Immunosuppressive treatments induced partial clinical remission of the diarrhea but no resolution of the small bowel injury. At 16 months of age, remission of the diarrhea occurred, but persistent autoimmune hepatitis led us to maintain treatment with prednisone and azathioprine, and later with cyclosporine. In this child, as in other patients with autoimmune disease, the link between autoantibodies and organ damage remains uncertain but immunosuppressive treatment is indicated. (J P EDIATR 1994;125:858-62)

Childhood bullous pemphigoid: Immunohistochemical, immunoelectron microscopic, and Western blot analysis

The most reliable diagnostic criteria of childhood bullous pemphigoid (BP) have been the linear deposition of IgG and C3 at the basement membrane zone (BMZ) and the frequent presence of circulating IgG BMZ antibodies. To characterize further the immunologic features of childhood BP, we performed direct and indirect immunoelectron microscopy (IEM) and Western immunoblotting on specimens from a 3-month-old boy with BP who had immunofluorescence evidence of linear BMZ IgG and C3 deposition and circulating IgG BMZ antibody. By direct IEM, autoantibody deposits were seen in the upper portion of the lamina lucida and on the undersurface of basal keratinocytes. Indirect IEM showed that the binding sites of BP antibody were closely related to the intracellular attachment plaques of hemidesmosomes. By Western blotting, the patient's serum recognized 180 kd epidermal protein.

Bullous pemphigoid sera induce bullous-pemphigoid-like lesions in neonatal mice pretreated with a limited dose of ultraviolet B irradiation.

The role of bullous pemphigoid (BP) autoantibodies (Abs) in the pathogenesis of BP is unclear. Lack of a confirmed experimental animal model prevents studies of the pathogenetic role of BP Abs. We hypothesized that some alterations of BP antigens (BP Ags) will be necessary for causative binding of BP Abs to the Ags. To cause an artificial and reproducible alteration, neonatal mice were irradiated with ultraviolet B (UVB). After the irradiation of 600 mJ/cm2 of UVB, BP serum was intraperitoneally injected. When the BP sera, which recognized only the 230-kD BP Ag, or both 230- and 180-kD BP Ags, were transferred into the UVB-treated mice, erosions and vesicles appeared in 14-78% of the animals. Histopathological examination revealed subepidermal blister formation in the mice treated with UVB and BP sera. Electron microscopy demonstrated that the separation occurred at the level of the lamina lucida. Human IgG and C3 were deposited at the dermal-epidermal junction. Control animals to which healthy sera were injected after the same dose of UVB irradiation showed degeneration of the upper epidermis but no apparent dermal-epidermal cleft formation histopathologically. These results suggest that BP antibodies play a pathogenetic role in vivo. This animal model can contribute to a study of the pathogenesis of BP.

Relationship Between Antigens and Igg Subclasses in Bullous Pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized in part by the presence of tissue-bound and circulating antibodies (mostly of IgG) to the basement membrane zone (BMZ). We previously reported that IgG subclasses of BP antibodies were IgG1, IgG2 and IgG4, and that only BP IgG1 fixed complements. In this study, we examined whether BP IgG sub-classes bound to the same epitope of BP antigen or a different epitope. In an inhibition immunofluorescence studies, the complement fixing capability of IgG1 was inhibited by the pretreatment with IgG4 and partially inhibited by IgG2. On immunoblot analysis, IgG1 and IgG4 were bound to the same MW of BP antigen. In enzyme-linked immunosorbent assay (ELISA), the binding capability of IgG subclass fractions from patients with BP to synthetic peptide P1-2, exceeding normal IgG subclass fractions was seen in five IgG1, one IgG2 and two IgG4, from eight BP patients. The binding capability of IgG subclass fractions from the patients with BP to P1-1, exceeding the normal IgG fractions was seen in two IgG1, three IgG2 and one IgG4 from ten BP patients. On inhibition ELISA, the binding activity to P1-2 of IgG4 was partially inhibited by the pretreatment of IgG1 and IgG2. These findings suggest that BP IgG1, IgG2 and IgG4 could bind to the same epitope though considerable variation occurred between patients.

Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.