Production of a human monoclonal anti-epithelial cell surface antibody derived from a patient with pemphigus vulgaris.

The production of monoclonal autoantibodies derived from individuals with autoimmune diseases constitutes a powerful tool to analyse an autoimmune process at both the antigen and antibody levels. We established a human anti-epithelial cell surface monoclonal antibody by applying hybridoma technology using peripheral blood lymphocytes from a patient with pemphigus vulgaris using a heteromyeloma as the fusion partner. The F12 monoclonal antibody displays four major characteristics: (1) it belongs to the IgM, kappa class; (2) it binds to the cell surface of stratified squamous and simple epithelia; (3) it recognizes an antigenic determinant associated with the desmosomal complex as demonstrated by indirect immunoelectron microscopy; (4) by immunoblotting analysis, it reacts with a 185 kDa polypeptide which was also recognized by a few pemphigus vulgaris sera. Although the F12 monoclonal antibody does not have the immunochemical properties of classical pemphigus vulgaris autoantibodies, several arguments suggest its relevance to the pemphigus vulgaris autoimmune response and, therefore, the heterogeneity of the antigen/antibody systems involved in this autoimmune disorder.     

A Perspective of Pemphigus from Bedside and Laboratory-Bench

Pemphigus represents a distinct organ-specific acquired autoimmune disease characterized by intra-epidermal blistering, which is induced by autoantibodies against desmosomal cadherins, desmoglein 1 (Dsg1), and Dsg3. Pemphigus is currently divided into three distinct varieties, i.e., pemphigus vulgaris (PV), pemphigus foliaceus (PF) and other variants of pemphigus (mostly associated with inflammation), depending on clinical features, the level of separation in the epidermis, and immunologic characteristics of auto-antigens. Blistering pathomechanisms differ for each of the types of pemphigus. Pemphigus, which results from autoantibodies against desmogleins and possibly to other proteins, binds to the cell surface antigens. This binding may cause steric hindrance to homophilic adhesion of desmogleins, and may, in turn, lead to internalization of desmogleins and inhibition of desmogleins' integration into desmosomes, resulting in the formation of Dsg3-depleted desmosomes in PV or Dsg1-depleted desmosomes in PF. Furthermore, PV-IgG activates an "outside-in" signaling pathway to induce disassembly of desmosomal components from the inside of the cells by phosphorylation of proteins, including Dsg3. On the other hand, Pemphigus-IgG-augmented signaling pathways may be linked to the secretion of cytokines such as in case of pemphigus herpetiformis and chemokines that initiate or activate inflammation. In this article, the classification of pemphigus and the characteristic pathomechanisms for acantholysis will be reviewed, with particular emphasis on the molecular and biochemical cell biology of these diseases.     

Complement and cutaneous autoimmune blistering diseases

Cutaneous autoimmune blistering diseases are associated with tissue injury and fluid accumulation within the skin. The initial trigger for the organ-specific damage is autoantibodies targeting skin autoantigens, which are involved in cell–cell or cell–matrix adhesion in the skin. Pemphigus autoantibodies bind to desmosomal antigens and cause intraepidermal blisters, while pemphigoid autoantibodies interact with hemidesmosomal or hemidesmosome-associated antigens and lead to dermal–epidermal junction separation. Local complement activation is a common feature for these skin blistering diseases and some complement components are readily detected in the lesional skin and blister fluids. This review summarizes the current knowledge on the role the complement system in skin blister formation. Characterization of the pathogenically relevant complement cascade and relative contribution of different pathways into complement activation provides new insights of disease pathology and may help develop better therapeutic strategies for these potentially fatal cutaneous blistering disorders.     

IgG autoantibodies against desmocollin 3 in pemphigus sera induce loss of keratinocyte adhesion

Pemphigus is considered an autoimmune bullous skin disorder associated with IgG against the desmosomal components, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1). This concept is supported by the in vitro and in vivo pathogenicity of anti-Dsg3/Dsg1 IgG and the mucosal blistering phenotype of mice with a genetic deficiency of Dsg3. Mice deficient for another desmosomal adhesion molecule, desmocollin 3 (Dsc3), show a similar pemphigus phenotype, and we investigated the pathogenicity of Dsc3-reactive IgG autoantibodies that were identified previously in a subset of patients with atypical pemphigus. We here demonstrate that IgG against Dsc3 causes loss of adhesion of epidermal keratinocytes. Specifically, IgG against Dsc3 was purified from Dsc3-reactive pemphigus sera by affinity column chromatography using recombinant human Dsc3. Affinity purified IgG was functionally active and did not only react with recombinant Dsc3 but also with epidermis and cultured human keratinocytes. Moreover, Dsc3-reactive IgG induced loss of adhesion of epidermal keratinocytes in a dispase-based keratinocyte dissociation assay that was reversed on pre-adsorption with human Dsc3 but not Dsg3. These findings demonstrate that IgG autoantibodies against an additional component of the desmosomes, Dsc3, induce loss of keratinocyte adhesion and thus may contribute to blister formation in pemphigus.     

Pathogenesis of epidermolysis bullosa acquisita, an autoimmune subepidermal bullous disease

Autoimmune bullous diseases (ABDs) are organ-specific autoimmune diseases, in which blisters on the skin and mucous membranes develop through binding of pathogenic autoantibodies to target antigens. There are two major ABD groups: the pemphigus group, showing autoantibodies to desmosomal components; and the subepidermal ABD group, showing autoantibodies to hemidesmosomal components in the epidermal basement membrane zone. Recent immunological, biochemical and molecular biological studies revealed many new autoantigens, including desmocollins, various plakin family proteins and integrins. A revised ABD classification includes new disease entities such as paraneoplastic pemphigus, IgA pemphigus and anti-laminin γ1 pemphigoid. In addition to systemic corticosteroids and various immunosuppressive agents, various adjuvant therapies for ABDs have developed. Among them, intravenous immunoglobulin (IVIG) is a promising therapy, although the therapeutic mechanisms are still unknown. Various disease models for ABDs have developed, particularly for pemphigus vulgaris, bullous pemphigoid and epidermolysis bullosa acquisita (EBA), and these have provided insights into the pathogenesis of various ADBs that suggest possible new treatment strategies. However, the fundamental mechanisms in disruption of immune-tolerance are still unknown. EBA shows autoimmunity to type VII collagen, the major component of anchoring fibrils, and EBA pathogenesis has been studied in various disease models. Previous studies suggested that, following binding of autoantibodies to type VII collagen, activation of complement, cytokine release, neutrophil migration, Fcγ receptors (FcgRs) and metalloproteinases play important roles in induction of subepidermal blisters. In this issue of the Journal of Pathology, Kasperkiewicz and colleagues reveal important roles of activating FcgRIV and inhibitory FcgRIIB in EBA pathogenesis that were recognized by conducting elegant studies using both genetic analysis and functional animal model methods. The expression equilibrium of the activating and inhibitory FcgRs can be modulated towards the inhibitory FcgRIIB by IVIG therapy, resulting in beneficial clinical effects of IVIG in EBA and other autoimmune skin-blistering diseases.     

Epidermal adhesion molecules and basement membrane components as target structures of autoimmunity

 Intraepidermal and dermal-epidermal cohesion are of paramount importance for the integrity of the skin. Some constituent molecules of keratinocyte adhesion complexes and basement membrane-associated structures are the targets of antibody-mediated autoimmune reactions that give rise to various (muco-)cutaneous blistering diseases. The current state of our knowledge about these molecules – along with the main clinical, histological, and immunohistochemical features of the corresponding autoimmune diseases and their pathogenetic mechanisms – comprise the subjects surveyed in this review. Among the desmosomal cadherins (desmogleins and desmocollins) that mediate epidermal cell–cell adhesion, it has been demonstrated that desmoglein 1 and desmoglein 3 are the autoantigens of pemphigus foliaceus and pemphigus vulgaris, respectively, both diseases that result in intraepidermal blistering. Further, desmocollin autoantibodies may be involved in IgA pemphigus. Paraneoplastic pemphigus is associated with autoantibodies directed against the desmosomal plaque protein, desmoplakin. Of the constituents of hemidesmosomes, the plaque protein, BP230 (BPAG1), and the collagen-like transmembrane protein, BP180 (BPAG2), are the autoantigens of bullous pemphigoid and pemphigoid gestationis, the manifestations of both of which include subepidermal blistering. Several diseases arise from autoimmune reactions against certain proteins associated with the basement membrane located beneath hemidesmosomes, for example laminin 5 (cicatricial pemphigoid), ladinin (LAD-1; linear IgA disease), uncein, and collagen VII (epidermolysis bullosa acquisita), the last of which is the constituent protein of the anchoring fibrils. Such recent advances in the elucidation of the molecular nature of autoantigens may serve as the basis for the development of novel molecule-based therapeutic strategies. Received: 12 September1997 / Accepted: 27 October 1997     

A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease, which is characterized by autoantibodies to a specific desmosomal constituent, i.e. desmoglein 3 (Dsg3). In this study, we analyzed phosphorylation of desmosomal proteins and their molecular interactions after PV-IgG binding to Dsg3 using DJM-1 cells, a squamous cell carcinoma cell line, and normal human keratinocytes. Cells were metabolically labeled with 32P inorganic phosphate, followed by stimulation with the IgG fractions from five PV patients or normal individuals for 20 min. Phosphorylation of specific desmosomal components and their molecular interactions were studied in immunoprecipitates using PV-IgG and anti-plakoglobin (PG) antibodies. PV-IgG binding alone induced the phosphorylation of Dsg3 at serine residues. Although Dsg3 and PG were coprecipitated by PV-IgG-immunoprecipitation when treated with normal IgG, PG was not coprecipitated with Dsg3 when stimulated with PV-IgG, suggesting that PV-IgG binding to Dsg3 caused the dissociation of Dsg3 from PG. These results demonstrate that the binding of pathogenic PV autoantibodies to the cell surface antigen Dsg3, which is an adhesion molecule categorized into desmosomal cadherins, caused particular phosphorylation of Dsg3 and its dissociation from PG.     

Pemphigus antibody induced phosphorylation of keratinocyte proteins

The pemphigus family of autoimmune blistering diseases is characterized by an autoantibody response to desmosomal cadherins in epithelia. Autoantibodies against desmogleins, desmosome cell adhesion molecules, induce loss of cell–cell adhesion that is characterized clinically by blister formation. The mechanism by which these autoantibodies induce loss of cell–cell adhesion is under active investigation, but appears to involve a coordinated intracellular response including activation of intracellular signaling and phosphorylation of a number of proteins in the target keratinocyte. Activation of p38 mitogen activated protein kinase may have a critical role in the acantholytic mechanism as inhibitors of p38MAPK block the ability of pemphigus IgG to induce blistering in pemphigus animal models.     

Antibodies to desmogleins 1 and 3, but not to BP180, induce blisters in human skin grafted onto SCID mice

Pemphigus and bullous pemphigoid (BP) are blistering skin diseases associated with IgG autoantibodies to desmosomal and hemidesmosomal components. When autoantibodies to desmogleins 1 and 3 from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) or rabbit antibodies against the murine hemidesmosomal component BP180 are passively transferred into neonatal mice, they induce blisters in the skin of the mice. To develop an animal model that would duplicate the findings in the skin of the patients more closely, full-thickness human skin from healthy volunteers was grafted onto SCID mice. Injection of the purified IgG fraction from the serum of PF and PV patients led to subcorneal and suprabasal splits in the human grafts and human IgG was deposited intercellularly in the upper and lower layers of the epidermis, respectively. Interestingly, anti-BP180 autoantibodies purified from the serum of BP patients and from a rabbit immunized with recombinant human BP180 strongly bound to the basement membrane zone of the grafts (n=32), fixed murine complement, led to the recruitment of neutrophils to the upper dermis of the graft, but did not induce subepidermal blisters. We report a novel experimental model for PF and PV which should greatly facilitate further studies to dissect the immunopathological mechanisms in these diseases. Specifically, this model can be used to identify pathogenically relevant epitopes on human desmogleins 1 and 3 and to develop novel strategies for the treatment of pemphigus.     

Autoimmunity to desmocollin 3 in pemphigus vulgaris

Pemphigus vulgaris is a blistering disease associated with autoantibodies to the desmosomal adhesion protein, desmoglein 3. Genetic deficiency of desmoglein 3 in mice mimics autoimmunity to desmoglein 3 in pemphigus vulgaris, with mucosal-dominant blistering in the suprabasal layer of the epidermis. Mice with an epidermal-specific deletion of desmocollin 3, the other major desmosomal cadherin isoform expressed in the basal epidermis, develop suprabasal blisters in skin that are histologically identical to those observed in pemphigus vulgaris, suggesting that desmocollin 3 might be a target of autoantibodies in some pemphigus vulgaris patients. We now demonstrate that desmocollin 3 is an autoantigen in pemphigus vulgaris, illustrated in a patient with mucosal-dominant blistering. Six of 38 pemphigus vulgaris and one of 85 normal serum samples immunoprecipitate desmocollin 3 (P = 0.003). Incubation of patient IgG with human keratinocytes causes loss of intercellular adhesion, and adsorption with recombinant desmocollin 3 specifically prevents this pathogenic effect. Additionally, anti-desmocollin 3 sera cause loss of keratinocyte cell surface desmocollin 3, but not desmoglein 3 by immunofluorescence, indicating distinct cellular pathogenic effects in anti-desmocollin and anti-desmoglein pemphigus, despite their identical clinical presentations. These data demonstrate that desmocollin 3 is a pathogenic autoantigen in pemphigus vulgaris and suggest that pemphigus vulgaris is a histological reaction pattern that may result from autoimmunity to desmoglein 3, desmocollin 3, or both desmosomal cadherins.     

Pemphigus antibody induced phosphorylation of keratinocyte proteins

The pemphigus family of autoimmune blistering diseases is characterized by an autoantibody response to desmosomal cadherins in epithelia. Autoantibodies against desmogleins, desmosome cell adhesion molecules, induce loss of cell-cell adhesion that is characterized clinically by blister formation. The mechanism by which these autoantibodies induce loss of cell-cell adhesion is under active investigation, but appears to involve a coordinated intracellular response including activation of intracellular signaling and phosphorylation of a number of proteins in the target keratinocyte. Activation of p38 mitogen activated protein kinase may have a critical role in the acantholytic mechanism as inhibitors of p38MAPK block the ability of pemphigus IgG to induce blistering in pemphigus animal models.     

Immunoadsorption in pemphigus

The principle of extracorporal immunoadsorption (IA) is based on affinity adsorption of pathogenic (auto-)antibodies and circulating immune complexes (CIC) which reversibly bind to an immobilized ligand of the adsorber. In pemphigus, a blistering autoimmune disease affecting skin and mucous membranes, autoantibodies, mainly of the IgG subclass are directed against desmosomal adhesion molecules and other non-desmosomal antigens on the surface of epidermal keratinocytes, such as acetylcholine receptors. The pathogenicity of these autoantibodies has been shown in various in vitro and in vivo systems. Recently, IA was applied in severe pemphigus demonstrating that a rapid and dramatic decline in desmoglein (Dsg)-reactive autoantibodies is accompanied by clinical remission of mucocutaneous blisters and erosions. As an adjuvant treatment, IA was combined with systemic immunosuppressive medication and current protocols initially apply treatment cycles of 3-4 IAs on consecutive days followed by immunoapheresis once a week or repeating the initial cycle in 4 week intervals depending on the disease activity. IA in pemphigus is generally safe and well tolerated.     

Immunoadsorption in pemphigus

The principle of extracorporal immunoadsorption (IA) is based on affinity adsorption of pathogenic (auto-)antibodies and circulating immune complexes (CIC) which reversibly bind to an immobilized ligand of the adsorber. In pemphigus, a blistering autoimmune disease affecting skin and mucous membranes, autoantibodies, mainly of the IgG subclass are directed against desmosomal adhesion molecules and other non-desmosomal antigens on the surface of epidermal keratinocytes, such as acetylcholine receptors. The pathogenicity of these autoantibodies has been shown in various in vitro and in vivo systems. Recently, IA was applied in severe pemphigus demonstrating that a rapid and dramatic decline in desmoglein (Dsg)-reactive autoantibodies is accompanied by clinical remission of mucocutaneous blisters and erosions. As an adjuvant treatment, IA was combined with systemic immunosuppressive medication and current protocols initially apply treatment cycles of 3–4 IAs on consecutive days followed by immunoapheresis once a week or repeating the initial cycle in 4 week intervals depending on the disease activity. IA in pemphigus is generally safe and well tolerated.     

Rituximab in Treatment-Resistant Autoimmune Blistering Skin Disorders

Autoimmune blistering diseases are associated with autoantibodies to desmosomal (pemphigus group) or hemidesmosomal proteins (autoimmune subepidermal blistering disorders) that are essential for the structural integrity of the epidermis and dermoepidermal junction. Treatment is usually based on systemic glucocorticosteroids, which are often combined with additional immunosuppressants such as azathioprine and mycophenolate mofetil or immunomodulators including dapsone, antibiotics, and intravenous immunoglobulins. These interventions are sometimes not sufficient to induce remission and/or may be associated with intolerable adverse events. In such situations, the anti-CD20 antibody rituximab has been successfully applied in recent years. Rituximab transitorily depletes CD20-positive B lymphocytes from the circulation. It has been employed in more than 1 million patients with CD20-positive non-Hodgkin’s lymphoma and severe side effects were only rarely observed. Subsequently, the B cell-modulating effect of rituximab has encouraged its use in a variety of autoimmune diseases, including more than 40 patients with pemphigus. In addition, a few patients with bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita have received rituximab. In the majority of these patients, clinical remission was induced; however, serious adverse events were considerable higher compared to both patients with non-Hodgkin’s lymphoma or nonbullous autoimmune disorders like lupus erythematosus, dermatomyositis, and rheumatoid arthritis.     

Paraneoplastic pemphigus is associated with the DRB1*03 allele

Pemphigus is a group of autoimmune blistering diseases caused by autoantibodies directed against keratinocyte adhesion molecules. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF), in which autoantibodies bind, respectively, to desmoglein 3 and desmoglein 1, are strongly associated with HLA-class II DR4 and DR14 alleles. In paraneoplastic pemphigus (PNP), a rare variant associated with neoplasia, autoantibodies target proteins of the plakin family in addition to desmogleins 1 and 3. The presence of anti-desmoglein antibodies in all types of pemphigus raises the question of common molecular mechanisms of susceptibility, particularly similar MHC-class II allele associations, in the different forms of the disease. HLA-DRB1 typing was performed in 13 PNP patients and results were compared to those obtained from 84 healthy controls, 37 PV and 31 PF patients. Our data demonstrate a significant association of PNP with HLA-DRB1*03 allele which was found in 61.5% of the patients, whereas DRB1*04 and DRB1*14 appear not to be involved in PNP susceptibility. Therefore, the HLA-genetic background of PNP differs from that of other types of pemphigus, which suggests that distinct mechanism(s) initiate(s) the immunological response in this form of pemphigus.     

Pemphigus: The promises of peptide immunotherapy

Pemphigus is caused by IgG autoantibodies directed against desmogleins (Dsg), keratinocyte desmosomal glycoproteins belonging to the cadherin family. Anti-Dsg IgG antibodies interfere with adhesive of desmogleins, causing the detachment of keratinocytes (acantholysis). In our laboratory, an alternative approach to pemphigus immunotherapy has been tested. The peptidic vaccine approach is based on the identification of peptidic epitodes within the sequence of known autoantigens and the use of such peptides to evoke tolerance (as in autoimmune disease) or to boost effector immune mechanisms (as in immunotherapy of tumors). This approach shows that in silico mapping of low-similarity sequences may add to the prediction of peptide immunogenicity. Thus peptide immunotherapy for pemphigus deserve further investigations before joining the therapeutic arsenal for pemphigus and related disorders.     

Advances in pemphigus and its endemic pemphigus foliaceus (Fogo Selvagem) phenotype: a paradigm of human autoimmunity

Pemphigus encompasses a group of organ specific, antibody mediated autoimmune diseases of the skin characterized by keratinocyte detachment that leads to the development of blisters and erosions, which can become life-threatening. The pathogenic autoantibodies recognize desmogleins, which are members of the desmosomal cadherin family of cell adhesion molecules. Desmoglein 3 is targeted in pemphigus vulgaris while desmoglein 1 is targeted in pemphigus foliaceus and its endemic form, Fogo Selvagem. This review will briefly define the salient features of pemphigus and the proposed steps in pathogenesis. We will then summarize the most recent advances in three important areas of investigation: (i) epidemiologic, genetic, and immunologic features of Fogo Selvagem, (ii) molecular mechanisms of injury to the epidermis, and (iii) novel therapeutic strategies targeting specific steps in disease pathogenesis. The advances in each of these three seemingly separate areas contribute to the overall understanding of the pemphigus disease model. These recent advancements also underscore the dynamic interplay between the treatment of patients in a clinical setting and basic science research and have led to an integrative understanding of disease pathogenesis and treatment, allowing pemphigus to serve as a paradigm of human autoimmunity.     

Humoral and cellular autoimmunity in autoimmune bullous skin disorders

Autoimmune bullous skin diseases, such as pemphigus vulgaris (PV) and bullous pemphigoid (BP), are severe, frequently life-threatening skin disorders. Immunologically, they are characterized by the presence of serum autoantibodies (auto-Ab) targeting distinct adhesion molecules of the epidermis or dermoepidermal basement membrane zone. Antibody (Ab) binding interferes with the adhesive function of these molecules, leading to detachment and subsequently blister formation. PV is the classical example of an Ab-mediated autoimmune disease affecting epidermal adhesion. Auto-Ab against the desmosomal adhesion molecule, desmoglein 3 (Dsg3), are critical in the pathogenesis of this disease, since the transfer of serum IgG Ab reactive with Dsg3 into newborn mice induces a bullous skin disease resembling PV. Autoreactive T cell responses to Dsg3 may be critical in the pathogenesis of PV because: (1) Ab production generally requires T cell help; (2) the involvement of CD4+ T lymphocytes in PV has been suggested by the strong association with distinct HLA class II alleles, and (3) T cell recognition of epitopes of Dsg3 may be crucial for the initiation and perpetuation of the production of Dsg3-specific auto-Ab by B cells. In PV and BP, autoreactive CD4+ T cells recognize distinct epitopes of the extracellular portions of Dsg3 and BP180 [BP antigen 2 (BPAG2) or type XVII collagen], respectively, and produce preferentially T helper type 2 (TH2) cytokines. Auto-Ab of the TH2-dependent IgG4 subtype are preferentially seen in the active stages of both PV and BP, while auto-Ab of the TH1-dependent IgG1 subclass are predominant during the chronic course of these disorders. These observations suggest that autoreactive TH2 cells may provide targets to specifically modulate the T cell-dependent production of pathogenic auto-Ab in these disorders.     

Evaluation of Recombinant Antigen-Based Assays for Diagnosis of Bullous Autoimmune Diseases

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.     

Evaluation of recombinant antigen-based assays for diagnosis of bullous autoimmune diseases

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.