Major Bullous Pemphigoid Antigen Research Articles

Searching for foreign antigens as possible triggering factors of autoimmunity: Torque Teno virus DNA prevalence is elevated in sera of patients with bullous pemphigoid

The Torque Teno virus (TTV), a member of virus genus Anellovirus has been shown to be commonly present in humans, yet without detectable pathogenicity. Recent studies imply that TTV may contribute to provoke autoimmune progresses in systemic lupus erythematosus and idiopathic inflammatory myopathies. We aimed to study the presence of TTV in a group of patients with autoimmune bullous diseases with a further goal to identify long-lasting foreign antigen, such as TTV as possible triggers of skin-specific autoimmunity. We performed in silico research to study similarities between known TTV sequences and antigens of bullous pemphigoid (BP), pemphigus vulgaris (PV) and dermatitis herpetiformis (DH). Basic Local Alignment Search Tool results showed matching regions for the major BP antigens BP180 and BP230, PV antigen desmoglein 3 and DH antigen transglutaminase 3 and disclosed overlapping, antigen-predicted sequences only for BP180 regions. We also assessed the prevalence of TTV in these disorders and compared them with the results from two healthy blood donor groups (group 1: sex- and age-matched for the general bullous group, n = 95; group 2: sex- and age-matched for BP, n = 50). Furthermore, we assayed lymphocytes from four TTV DNA and BP180 NC16A blot-positive BP patients and three controls in a standard lymphocyte transformation test with a TTV peptide from the conserved ORF(Open Reading Frame)1/N22 region. We found that the detection rate of TTV was comparable with that in healthy controls in the group of PV (19/33); whereas detection rates in DH showed a slight, but not significant tendency for elevation (17/20). Contrary, the TTV prevalence in BP patients was significantly elevated (group 1: 36/40 vs group 2: 31/50, P < 0.032). Lymphocytes from all four virus-positive BP patients heavily reacted to TTV peptide while two of the three healthy controls have shown not to recognize the viral sequences. Only the TTV carrier healthy control had a minor reaction at lowest peptide concentration. The combined in silico, polymerse chain reaction and in vitro cell assay data of the present study indicate that a TTV persistence may contribute to the pathogenesis of BP.

Analysis of Antigens Targeted by Circulating IgG and IgA Antibodies in Patients With Mucous Membrane Pemphigoid Predominantly Affecting the Oral Cavity

Mucous membrane pemphigoid (MMP) includes a range of variants frequently having oral, particularly gingival, lesions. It has been suggested that MMP exclusively involving the oral cavity could be a distinct subset, but the immunological status of patients with predominantly oral lesions has received little attention. Indirect immunofluorescence (IIF) on normal human skin and normal human salt-split skin (SSS) as a substrate and immunoblotting on extracts of human keratinocyte cultures and normal human skin were carried out in 28 consecutive patients with MMP predominantly affecting the gingiva. There was no significant difference between frequency of positive SSS-IIF among patients with exclusive oral lesions (42%) and with MMP not restricted to the oral cavity (78%) (P = 0.114). By immunoblotting, IgG of 13 sera (46%) reacted with the 180 kD bullous pemphigoid antigen (BPAg2), six (21%) reacted with the 230 kD bullous pemphigoid antigen (BPAg1), and two (7%) reacted with an antigen of 200 kD. IgA of three sera (11%) reacted with BPAg2 and three (11%) reacted with BPAg1. There was no significant difference between frequency of positive immunoblotting among patients with exclusive oral lesions (68%) and with MMP not restricted to the oral cavity (67%) (P = 1.00). Most of the MMP sera contain IgG and IgA antibodies to the two major bullous pemphigoid antigens, independent of clinical presentation. Thus, in the majority of cases, patients with exclusively gingival pemphigoid lesions do not appear to have a distinct variety of mucous membrane pemphigoid.

IgE autoantibodies directed against the major bullous pemphigoid antigen in patients with a severe form of pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized in part by circulating and tissue-bound IgG autoantibodies directed against the basement membrane zone. In addition, most of the patients with BP have increased serum IgE levels which seem to be correlated with the disease activity, whereas the presence of circulating anti-basement membrane zone IgE Abs has been reported in some patients. To elucidate whether IgE-dependent mechanisms play a role in the physiopathology of BP, we looked for the presence of IgE Abs specifically directed against the major BP Ag (BPAg1) in sera of BP patients at the onset and after remission of the disease. A radioimmunoassay and a 55-kDa recombinant protein (rBP55) obtained from a cDNA sequence, encoding the C-terminal region of the BPAg1 and containing the BPAg1 immunodominant epitopes, were used. Anti-rBP55 IgE Abs were found in 12 of the 19 sera tested. When the patients were divided into two groups according to the disease severity, anti-rBP55 IgE Abs were found only in patients with a severe form of the disease. Cytophilic IgE was detected on approximately 20% of peripheral blood eosinophils purified from BP patients. Immunohistochemistry studies suggested that some of the IgE-bearing cells in the lesional skin of BP patients are eosinophils. Immunostaining experiments revealed the existence of FcepsilonRI on both peripheral blood and tissue eosinophils. Taken together, these results suggest that IgE-dependent mechanisms could participate in the constitution of the lesions in BP.

Clinical manifestations in 100 Japanese bullous pemphigoid cases in relation to autoantigen profiles

The relationship between clinical findings and antigen profiles in 100 bullous pemphigoid (BP) patients has been investigated. The patients were divided into four groups based upon the results of immunoblot analysis, namely patients whose sera detected the 230-kDa BP antigen (BP230) and the 180-kDa BP antigen (BP180), those recognizing either BP230 or BP180 alone, and those recognizing neither antigen. Analysis by the chi-squared test showed predominant occurrence of oral (P < 0.05) and facial lesions (P < 0.005) in patients whose sera detected BP180, and these patients also tended to have more extensive lesions (P < 0.005). Patients that were positive for BP180 alone needed treatment with higher doses of steroids than the patients positive for BP230 alone (P < 0.05). Furthermore, all five recalcitrant cases, which did not respond well to steroid treatment, were shown to possess autoantibodies against BP180 in their sera. Patients with antibodies to BP230 had a tendency to have a high titre of anti-BMZ antibodies (P < 0.005). These results suggest that anti-BP180 antibodies may be more related to the disease severity than anti-BP230 antibodies. Bullous pemphigoid (BP) is an autoimmune blistering disorder characterized clinically by tense blister formation and immunologically by the presence of tissue-bound and circulating antibasement membrane zone (BMZ) autoantibodies. A number of immunoblot analyses have indicated that two major antigenic proteins of epidermal extracts, the 230-kDa BP antigen (BP230 or BPAG1) and the 180-kDa BP antigen (BP180, BPAG2 or type XVII collagen), are detected by sera from patients with BP in various patterns. Some BP sera recognize both antigen proteins, while others detect only BP230 or BP180 or none. Recently, cDNAs for these antigens have been isolated, and the characteristics for these molecules have been investigated in more detail. The exact relationship between these two proteins is still unclear; however, more recent studies showed that BP180 has an extracellular domain and polyclonal rabbit antibodies raised against an extracellular non-collagenous domain of the murine BP180 antigen were pathogenic in a passive transfer model. Several investigators have suggested a relationship between clinical findings and laboratory data in BP. Arbesman et al. compared the extent and duration of the disease with age, serum levels of IgE, IgA and IgG and titre of anti-BMZ antibodies. The results indicated that serum level of IgE and IgA correlated significantly to the extent of the disease and that titre of anti-BMZ antibodies correlated significantly (only in males) to the duration. There has been only one report which has analysed the relationship between pemphigoid antigens and clinical findings or laboratory data; no correlation was found. In a previous study, the reactivities of BP sera by immunoblotting using human epidermal extracts and two recombinant polypeptides produced from a mouse cDNA clone BPM1 were examined. The results of immunoblotting using epidermal extracts revealed that the two major BP antigens, BP230 and BP180, were detected by 74% and 51% of BP sera, respectively. Comparison of the clinical data with BP antigens gave the impression that the cases whose sera reacted only with BP180 were clinically more severe and less respondent to steroid treatment than the cases which detected BP230 alone on immunoblots. In the study reported here we have examined further the relationship between clinical and immunoblot data by applying a statistical analysis to the results previously obtained.

Bull�ses Pemphigoid unter dem Bild einer Prurigo simplex subacuta

Bullous pemphigoid (BP) is a bullous autoimmune disease of the elderly; it is characterized by tense bullae on both erythematous and otherwise apparently normal skin. Several clinical variants of BP have been described, and we now add our observations of two BP cases mimicking subacute prurigo. Both patients had suffered from intensely pruritic excoriated papules for several months before presentation. Blisters had never developed and did not occur during follow up. Histology showed changes of chronic dermatitis. In the serum of both patients, indirect immunofluorescence on NaCl-separated human skin revealed the presence of circulating antibodies binding to the roof of the artificial blisters. Perilesional skin biopsies showed linear IgG or C3-deposits in the basement membrane zone. Immunoblotting of epidermal and dermal extracts demonstrated the binding of the antibodies to an epidermal 230-kD protein, which is the known major bullous pemphigoid antigen. These cases are described as a basis for discussion of the clinical spectrum of bullous pemphigoid.

Paraneoplastischer Pemphigus

Paraneoplastic pemphigus (PP) is a newly reported autoimmune disease, which is always associated with an underlying neoplasia. It is characterized by painful mucosal erosions and ulcerations, and by polymorphous skin lesions reminiscent of erythema multiforme, pemphigus vulgaris and lichen planus pemphigoides. These patients have autoantibodies that bind to a distinct complex of epidermal proteins, including desmoplakin I (250 kD), major bullous pemphigoid antigen (230 kD), desmoplakin II (210 kD) and a 190-kD and a 170-kD protein, neither of which has yet been further characterized. Histological findings include acantholysis, epidermal cell necrosis, vacuolar interface changes and, sometimes, lichenoid infiltrates of the upper dermis. Direct immunofluorescence shows an intercellular deposition of immunoreactants in the epidermis and granular deposits at the dermal-epidermal junction. Indirect immunofluorescence with rodent bladder as substrate shows an intercellular pattern. This method is the most convenient and cost-effective method of screening for PP, since antigens of both pemphigus vulgaris and pemphigus foliaceus are not expressed in this tissue.

Paraneoplastic Pemphigus Is a Distinct Neoplasia-Induced Autoimmune Disease

A relationship between pemphigus and internal malignancy has been recognized for a long time. Some authors have suggested that patients with pemphigus have a greater incidence of malignancy, perhaps related to immunosuppressive treatment, 1 while others have noted a strong statistical association with both thymic and nonthymic neoplasms. 2 Immunoglobulins isolated from tumors have been shown to have reactivity similar to pemphigus intercellular substance antibodies by indirect immunofluorescence. 3 The importance of these findings was unclear until Anhalt and colleagues 4 published their seminal observations regarding five patients with a distinct blistering disorder and underlying malignancy. Serum samples obtained from their patients all showed unique banding patterns by immunoprecipitation that corresponded to autoantibodies to desmoplakins I and II, the major bullous pemphigoid antigen, and an as yet unidentified 190-kd protein. Clinical findings included a blistering eruption with stomatitis and conjunctivitis. Treatment results were disappointing, as four of the five patients were known to die of their disease.

Pemphigoid vegetans represents a bullous pemphigoid variant: Patient's IgG autoantibodies identify the major bullous pemphigoid antigen

Pemphigoid vegetans is a rare disease; only four cases have been reported. The relationship of pemphigoid vegetans to bullous pemphigoid is not completely defined. We describe a case in a 77-year-old woman who had multiple well-circumscribed, erythematous, erosive, and vegetating plaques in the axillae and inframammary areas and in the neck. Microscopic examination revealed epidermal hyperplasia, dermoepidermal junctional separation, and prominent dermal eosinophilia. In the perilesional skin IgG was deposited linearly at the basement membrane zone. In addition, our patient's IgG autoantibodies, primarily the IgG4 subclass, reacted with the epidermal side of salt-split normal human skin and with the 230 kd major bullous pemphigoid antigen. We conclude that pemphigoid vegetans is best classified as a bullous pemphigoid variant.

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.

The clinical expression of bullous pemphigoid is not determined by the specificity of the target antigen

The major bullous pemphigoid (BP) antigen is a 220-240-kDa polypeptide, although some BP sera recognize bands of 180-200 kDa or lower molecular weight. We have investigated to what extent this heterogeneity of the target antigen accounts for the clinical diversity of BP. Immunoblotting studies against extracts of salt-separated epidermis were performed on sera from 39 patients with BP. The blotting patters obtained were correlated with the clinical findings, with particular reference to prodromal itching, lesion morphology and severity, mucosal involvement, presence of milia, dapsone responsiveness and disease duration. The results confirm that the major BP antigen is a 220-kDa polypeptide, and that the 180-kDa polypeptide is a second and sometimes the sole BP antigen identified in immunoblots. Rarely, multiple bands of lower molecular weight were found. There was no correlation between the pattern of BP antigens detected in immunoblots and the clinical presentation and course of BP. There was considerable clinical diversity even among the nine patients showing specificity for a single 220-kDa target antigen. Although two patients with a single 180-kDa antigen specificity had a disease of unusually long duration, factors other than antigen specificity must determine the clinical expression of BP.

Characterization of Bullous Pemphigoid Antibodies by Use of Recombinant Bullous Pemphigoid Antigen Proteins

The seroreactivity of patients with bullous pemphigoid (BP) to recombinant proteins representing sequences in the carboxyl domain of the murine 230-kD BP antigen (BPA) was determined. Sera from 133 patients with BP, 20 patients with pemphigus, and 21 normal subjects were examined by Western blotting by using two recombinant proteins: RP120 (MW = 120 kD), representing the C-terminal half of the 230-kD BPA, and RP60 (MW = 60 kD), representing the C-terminal quarter. These RP120 and RP60 were recognized by 84% and 61%, respectively, of the BP sera that reacted with the 230-kD BPA in epidermal extract, and not by any of pemphigus or normal sera. Furthermore, these RP120 and RP60 were not recognized by any BP sera that reacted only with the 170-kD BPA, which is known to be another major BPA. These findings indicate that one or more of the major antigenic regions localizes in the carboxyl-half domain of the 230-kD BPA, and also suggest that the 230-kD BPA may be distinct from the 170-kD BPA.

Molecular identification of major and minor bullous pemphigoid antigens

The skin antigens defined by basement membrane zone antibodies in 38 patients with bullous pemphigoid were analyzed by Western immunoblot. Thirty-four patients (89%) had antibodies to a major bullous pemphigoid antigen with a molecular weight of approximately 230 kD. Twelve patients (32%) had antibodies to a minor bullous pemphigoid antigen with a molecular weight of approximately 160 kD; this included four patients who did not have antibodies to the 230 kD antigen. Depending on the epidermal extract used, a variable number of patients (up to 75%) also had antibodies to minor bullous pemphigoid antigens with molecular weights of approximately 180 and 200 kD. The results of this study confirm that bullous pemphigoid antigens are heterogeneous at the molecular level. Approximately 10% of patients have antibodies directed solely to a minor bullous pemphigoid antigen.

Immunoblot analysis of IgG subclasses of circulating antibodies in bullous pemphigoid

Sera from 20 patients with bullous pemphigoid (BP) were analyzed for the subclass distribution of IgG autoantibodies by Western immunoblotting of normal human heat-separated epidermal extracts. Strips were sequentially probed with patient sera, monoclonal antibodies (Mab) anti-human IgG 1–4, and a labeled anti-mouse Ig. The relative dilutions of each subclass-specific Mab were determined in dot immunobinding experiments in order to give uniform reactivity to the corresponding human IgG subclass. Circulating BP autoantibodies reacting with the major BP antigen of 220 kDa and/or a 165-kDa band showed a stricking predominance of IgG 4. Overall, IgG 4 was the major subclass reactive with the 220-kDa band, whereas, in addition to IgG 4, IgG 1 and IgG 2 were frequently represented in the response to the 165-kDa band. Complement fixation of circulating BP antibodies was studied by complement indirect immunofluorescence using human serum as source of complement and FITC goat anti-human C3, C1q, and C4, on normal human skin substrate. When circulating BP antibodies contained IgG 1 or IgG 2 in addition to IgG 4, they fixed C3 and, in some cases, C1q and C4, suggesting complement activation by either the classical or the alternate pathway.

Comparative expression of bullous pemphigoid antigens in normal human epidermis and cultured keratinocytes

A heterogeneity of the bullous pemphigoid (BP) antigen initially described as a 220- to 240-kDa polypeptide doublet expressed in normal human skin was recently demonstrated. The aim of our study was to compare the heterogeneity of BP antigen by the immunoblotting technique in extracts of both human epidermis and keratinocyte cultures. Extracts of epidermal tissues obtained from plastic surgery skin samples and secondary keratinocyte cultures were analyzed for their immunologic content defined by 30 BP sera. Twenty-six out of 30 tested sera showed similar binding reactivities with one or several polypeptide bands in both extracts. These sera defined seven antigens of molecular weights ranging from 240 to 97 kDa. Three groups of sera reacting with three, two, or only one antigen could be distinguished in which 180, 200, and 220 kDa polypeptides represented the major BP antigens both in vivo and in vitro. The binding reactivity of most immunofluorescent negative sera demonstrated the sensitivity of the immunoblotting technique to evidence the heterogeneity of BP antigens and antibodies. Keratinocyte cultures represent a reproducible substrate for such analysis and offer a standardization attempt in comparative investigations in BP.

A 230-kD Basic Protein Is the Major Bullous Pemphigoid Antigen

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of BP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP sera by both immunoprecipitation of extracts of radiolabeled cultured human keratinocytes and immunoblotting of extracts of normal human epidermis. Thirty-six of 37 BP sera immunoprecipitated the 230-kD polypeptide, and 11 of 13 BP sera identified the 230-kD polypeptide by immunoblotting. Eighty-six disease and normal control sera did not bind the 230-kD BP antigen. Immunoprecipitation was a more sensitive assay than was immunoblotting to detect the 230-kD antigen; BP sera that were weak or negative by immunoblotting were strongly positive by immunoprecipitation. To demonstrate that this 230-kD polypeptide shared epitopes, defined by BP sera, with the epidermal basement membrane zone, we showed that BP IgG affinity purified on this polypeptide bound the epidermal basement membrane zone by immunofluorescence. To further characterize the BP antigen and to compare the antigen synthesized by cultured human keratinocytes to the antigen extracted from epidermis, we analyzed the 230-kD BP antigen by two-dimensional gel electrophoresis. BP antigen immunoprecipitated from culture extracts or BP antigen identified by immunoblots of epidermal extracts was a single spot on two-dimensional gels, with a pI of about 8. These data indicate that, although other polypeptides are sometimes identified (e.g., a 166 kD band by immunoprecipitation or a 180 kD band on immunoblots), the major BP antigen identified in cultured human keratinocytes is similar to the antigen found in normal human epidermis and is a basic 230-kD polypeptide.