Epidermolysis Bullosa (EB) Acquisita in an Adult Patient with Previously Unrecognized Mild Dystrophic EB and Biallelic COL7A1 Mutations.

Circulating anti-type VII collagen autoantibodies are frequently detected in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, evidence supporting their pathogenic role in inducing epidermolysis bullosa acquisita (EBA) has been provided for only one individual with dominant dystrophic epidermolysis bullosa (DDEB). We describe here a patient who presented with dystrophic toenails since early childhood and developed trauma-induced skin blisters and oral erosions at age 26 years. Direct immunofluorescence showed IgG deposits with a u-serrated pattern along the cutaneous basement membrane zone, while no change in the expression of collagen VII could be detected by antigen mapping. High-titre anti-collagen VII antibodies were detected by enzyme-linked immunoassay (ELISA). In parallel, sequencing of epidermolysis bullosa (EB) genes identified compound heterozygous COL7A1 missense c.410G>A (p.Arg137Gln) and splicing c.3674C>T (p.Ala1225_Gln1241del) mutations, previously unrecognized in dystrophic epidermolysis bullosa (DEB). Thus, our patient had RDEB "nails-only" and developed mechanobullous EBA in adulthood. These data support a pathogenic role of circulating autoantibodies to collagen VII in inducing EBA in selected patients with DEB. Unforeseen worsening of skin symptoms in DEB should prompt laboratory investigations for EBA.

Autoantibody Profile of a Cohort of 78 Italian Patients with Mucous Membrane Pemphigoid: Correlation Between Reactivity Profile and Clinical Involvement.

Direct diagnosis of mucous membrane pemphigoid (MMP) is not easy. Circulating autoantibodies targeting bullous pemphigoid antigens of 180 kDa and 230 kDa (BP180 and BP230), α6ß4 integrin, laminin 332 and type VII collagen (Col VII) are not always present. The aims of this study were to characterize the humoral immune response of a cohort of Italian patients with MMP, its association with clinical involvement and severity, and to design an algorithm for efficient serological diagnosis. Seventy-eight MMP sera were studied retrospectively by indirect immunofluorescence on salt-split skin, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Indirect immunofluorescence on salt-split skin resulted in the most sensitive approach for diagnosis of MMP. BP180 was the major autoantigen in MMP patients with oral and cutaneous involvement. Significant associations were found between BP180 reactivity and oral and cutaneous localization of the lesions (p = 0.006), and between Col VII positivity and Setterfield severity score (p = 0.020).

Efficiency of translation termination in humans is highly dependent upon nucleotides in the neighbourhood of a (premature) termination codon.

BACKGROUND: Spontaneous read-through of a premature termination codon (PTC) has so far not been observed in patients carrying nonsense mutations. This report describes a patient with junctional epidermolysis bullosa who was expected to die because of compound heterozygous nonsense mutations in the gene LAMA3 (R943X/R1159X), but was rescued by spontaneous read-through of the R943X allele. RESULTS AND CONCLUSION: FACS analysis of cells carrying various PTCs surrounded by their natural neighbouring codons revealed significant reporter gene expression despite the PTC only for this patient's genetic context. Gene expression could be abolished by replacing the first or third nucleotide before, or one of the two nucleotides following the PTC. Site-directed mutagenesis was used to identify genotypes allowing PTC read-through. The genetic context of the LAMA3 mutation R943X is close to a hypothetical consensus sequence for maximum PTC read-through. Bioinformatic analysis showed that this consensus sequence is present in four sequences from the NCBI reference database, each of which contains another in-frame termination codon three or four codons apart. This indicates strong selective pressure against leaky termination codons in the human genome. This patient's mutated full length mRNA escaped nonsense-mediated decay, leading to LAMA3 mRNA levels similar to those of a healthy control, and full length laminin α3 could be detected in culture supernatant of the patient's keratinocytes. Immunofluorescence analyses of skin biopsies and continuous clinical improvement of the patient's condition suggested accumulation of intact laminin-332 in the epidermal basement membrane. These findings provide important clues for the prediction of PTC read-through in human genetic disease.

Anti-desmoplakin antibodies in erythema multiforme and Stevens-Johnson syndrome sera: pathogenic or epiphenomenon?

The pathophysiology of erythema multiforme (EM), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) is unclear. Whether autoantibodies against desmoplakin (Dp) I and II play a pathogenic role or result from an epitope spreading phenomenon is uncertain. Our aim was to characterize the keratinocyte antigens recognized in EM, TEN and SJS. Of 33 patients studied, 2 had TEN, 1 SJS, 9 EM major and 21 EM minor, according to Roujeau's criteria. All sera were studied by indirect immunofluorescence (IIF), immunoblotting and immunoprecipitation. Twenty normal sera were used as controls. 10/33 sera reacted with polypeptides of 215 and/or 250-kDa molecular mass, which co-migrate with Dp I and II as assessed by an anti-Dp I and II monoclonal antibody on IB. In IP, none of the anti-Dp I and -Dp II 10 patient sera immunoprecipitated Dp I and/or II from radiolabeled keratinocyte extracts. Two of 10 patient sera (SJS, EM minor) reacted with DpI and II when denaturated by the IB procedure. The reactivity against intracellular antigens DpI and II as denaturated proteins may result from the epidermal damage produced by aggressive autoreactive T cells, playing therefore only a secondary role in the pathogenesis of the disease.

Identification of a Novel Non-desmoglein Autoantigen in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is an autoimmune bullous disease of the skin and mucous membranes characterized by the presence of circulating and tissue-bound autoantibodies against keratinocyte cell surface antigens, specifically desmoglein (Dsg) 1 and 3. The pathogenic role of anti-Dsg antibodies is well-established, while the mechanism of blister formation is only partly defined. We have applied a previously developed method for the efficient immortalization of IgG+ memory B cells to identify novel target antigens in PV. A human monoclonal antibody reactive with a hitherto unreported non-Dsg antigen was isolated. Immunoprecipitation and immunoblotting studies with keratinocyte extracts indicated α-catenin as the putative antigen, then confirmed by immunoblotting on the recombinant protein. Four of ten PV sera reacted with recombinant α-catenin. Although the isolated human monoclonal antibody was per se unable to dissociate keratinocyte monolayers and also to synergize with a pathogenic antibody in vitro, further studies are warranted to assess its possible in vivo contribution in the multifactorial pathogenesis and heterogeneous manifestations of PV disease.

Multicenter prospective study of the humoral autoimmune response in bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune bullous disease, associated with autoantibodies directed against the hemidesmosomal components BP180 and BP230. In this study for the first time different laboratories have analyzed the autoantibody profile in the same group of 49 prospectively recruited BP patients. The results show that: 1) disease severity and activity correlated with levels of IgG against the BP180-NC16A domain, but also against a COOH-terminal epitope of BP180, 2) distinct epitopes of the BP180 ectodomain other than BP180-NC16A were recognized by 96% of the BP sera; and 3) the combined use of BP180 and BP230 ELISA led to the detection of IgG autoantibodies in all the BP sera. These results demonstrate the usefulness of the combined ELISAs based on various BP180 and BP230 fragments in establishing the diagnosis of BP and support the concept that BP180 is the major autoantigen of BP.

The pathogenic activity of anti-desmoglein autoantibodies parallels disease severity in rituximab-treated patients with pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune blistering disease mediated by IgG autoantibodies targeting desmogleins (Dsgs). The anti-CD20 monoclonal antibody rituximab is increasingly used in corticosteroid-resistant PV patients. In a subset of rituximab-treated patients in remission, high ELISA index values have been reported; however, their significance remains so far unclear. OBJECTIVE: To address the discrepancy between anti-Dsg3 serum antibody titers and disease severity. MATERIALS & METHODS: 6 rituximab-treated PV patients were prospectively followed-up for two years and anti-Dsg3 autoantibodies levels and pathogenic activity were measured. RESULTS: All patients achieved complete remission without any serious side effects. Both anti-Dsg3 autoantibodies (p = 0.031) and their pathogenic activity (p = 0.003) were significantly related to disease severity. However, in selected patients, the dissociation index was a more sensitive indicator for PV clinical activity than the ELISA index. CONCLUSION: Our findings have demonstrated the existence of non-pathogenic autoantibodies in PV patients in remission, establishing the basis for the design of a system able to precisely monitor the course of disease.

Are clinical phenotype and autoantibody profile always concordant in pemphigus? A study in a cohort of pemphigus patients.

BACKGROUND: The clinical phenotype of different forms of pemphigus is reportedly defined by the anti-desmoglein (Dsg) autoantibody profile. In routine practice, however, this is not always the case. OBJECTIVES: To verify the relationship between the anti-Dsg1 and -3 autoantibody profiles and titers on the one hand and the clinical phenotype and disease activity on the other. MATERIALS AND METHODS: we followed-up clinically and serologically 20 pemphigus patients, including 3 mucosal pemphigus (mPV), 9 mucocutaneous pemphigus (mcPV), and 8 cutaneous pemphigus (PF). RESULTS: We found that the cutaneous and/or mucosal involvement and the autoantibody profile were only concordant in mPV patients. On the contrary, in other clinical forms this correlation was often absent. CONCLUSIONS: 1) The discrepancy between autoantibody profile and the clinical phenotype, at least in PF patients, appears to be due to non-pathogenic anti-Dsg3 antibodies; 2) in a proportion of patients the relationship between the Dsg1 and Dsg3 ELISA titers and the disease severity was absent; 3) in some patients, the anti-Dsg1 and -3 autoantibodies were lacking at diagnosis, suggesting a role of other antigens in the pathogenesis of the disease and, lastly, 4) the pure cutaneous and mucosal forms tend to respond more efficiently to the therapy than the mucocutaneous forms and have a persistent response.

Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.

Pemphigus vulgaris (PV) is an autoimmune blistering disease of skin and mucous membranes caused by autoantibodies to the desmoglein (DSG) family proteins DSG3 and DSG1, leading to loss of keratinocyte cell adhesion. To learn more about pathogenic PV autoantibodies, we isolated 15 IgG antibodies specific for DSG3 from 2 PV patients. Three antibodies disrupted keratinocyte monolayers in vitro, and 2 were pathogenic in a passive transfer model in neonatal mice. The epitopes recognized by the pathogenic antibodies were mapped to the DSG3 extracellular 1 (EC1) and EC2 subdomains, regions involved in cis-adhesive interactions. Using a site-specific serological assay, we found that the cis-adhesive interface on EC1 recognized by the pathogenic antibody PVA224 is the primary target of the autoantibodies present in the serum of PV patients. The autoantibodies isolated used different heavy- and light-chain variable region genes and carried high levels of somatic mutations in complementary-determining regions, consistent with antigenic selection. Remarkably, binding to DSG3 was lost when somatic mutations were reverted to the germline sequence. These findings identify the cis-adhesive interface of DSG3 as the immunodominant region targeted by pathogenic antibodies in PV and indicate that autoreactivity relies on somatic mutations generated in the response to an antigen unrelated to DSG3.

Demonstration of epitope-spreading phenomena in bullous pemphigoid: results of a prospective multicenter study.

Bullous pemphigoid (BP), the most common autoimmune subepidermal bullous disease, is associated with an autoantibody response to BP180 and BP230, two components of junctional adhesion complexes in human skin promoting dermo-epidermal cohesion. Retrospective analyses demonstrated that these autoantigens harbor several epitopes targeted by autoaggressive B and T cells. The aim of this prospective multicenter study was to assess the evolution of IgG autoantibodies in 35 BP patients over a 12-month observation period. Epitope-spreading (ES) events were detected in 17 of 35 BP patients (49%). They preferentially occurred in an early stage of the disease and were significantly related to disease severity at diagnosis. Moreover, in three patients, spreading of IgG reactivity to intracellular epitopes of BP180 and BP230 was preceded by recognition of the BP180 ectodomain. Finally, IgG reactivity with extracellular epitopes of BP180 and intracellular epitopes of BP230 correlated with the severity of BP in disease course. These findings support the idea that IgG recognition of the BP180 ectodomain is an early and crucial event in BP disease, followed by variable intra- and intermolecular ES events, which likely shape the individual course of BP.

Paraneoplastic pemphigus presenting as mild cutaneous features of pemphigus foliaceus and lichenoid stomatitis with antidesmoglein 1 antibodies.

Herein, we report a case of paraneoplastic pemphigus with mild skin features of pemphigus foliaceus and lichenoid stomatitis associated with B-cell lymphoma. A 49-year-old man presented with scattered blisters and erosions on the trunk along with mucosal blisters and erosions. Skin biopsy showed subcorneal acantholytic bulla and oral mucosal biopsy demonstrated lichenoid dermatitis. Direct immunofluorescence showed cell surface deposits of IgG and C3. Indirect immunofluorescence identified circulating IgG autoantibodies to the cell surfaces of normal human skin and also on the transitional epithelium of rat bladder. Enzyme-linked immunosorbent assay using recombinant baculoproteins showed positive antidesmoglein 1 autoantibodies (index 46) but negative antidesmoglein 3 autoantibodies (index 8). Immunoblot analysis using normal human epidermal extract detected BP230 and the 190 kDa periplakin, while immunoprecipitation using radiolabeled cultured keratinocyte immunoprecipitated BP230 and the 210 kDa envoplakin. We consider that the skin lesion was produced by humoral immunity whereas the oral lesion was produced by cellular immunity.

Sequential intramolecular epitope spreading of humoral responses to human BPAG2 in a transgenic model.

Bullous pemphigoid (BP) is a subepidermal autoimmune disease characterized by a humoral response to an epidermal basement membrane (BM) component, BP antigen 2 (BPAG2). BP patients have IgG autoantibodies against an immunodominant BPAG2 extracellular domain termed NC16A as well as additional epitopes located both in the intracellular and extracellular domains (ICD and ECD, respectively) of this autoantigen. To study the evolution of humoral responses to BPAG2, sequential serum samples obtained from C57BL/6Ncr mice grafted with otherwise syngeneic skin from transgenic mice expressing human BPAG2 (hBPAG2) in epidermal BM were studied for IgG reactivity to seven ECD and ICD hBPAG2 epitopes. All grafted mice developed specific IgG against hBPAG2 ECD and ICD epitopes. In seven of eight mice, anti-hBPAG2 IgG was initially directed against ECD epitopes; in six mice, humoral responses subsequently targeted additional ECD and ICD BPAG2 epitopes. In contrast to IgG specific for ECD epitopes, IgG against ICD epitopes was present at lower levels, detectable for shorter periods, and non-complement fixing. Interestingly, the appearance of IgG directed against ICD epitopes correlated with the development of graft loss in this experimental model. These studies provide a comprehensive and prospective characterization of the evolution of humoral immune responses to hBPAG2 in vivo.

The intracellular and extracellular domains of BP180 antigen comprise novel epitopes targeted by pemphigoid gestationis autoantibodies.

Pemphigoid gestationis (PG) is an autoimmune sub-epidermal bullous dermatosis of pregnancy associated with circulating autoantibodies targeting the extracellular non-collagenous (NC) 16A domain of bullous pemphigoid (BP) 180 antigen. In order to determine whether BP180 regions other than NC16A are recognized by PG autoantibodies, we have analyzed the reactivity of 15 PG patient sera against several BP180 antigenic sites by sensitive methods such as immunological screening and ELISA. Most PG sera tested (13 of 15) reacted with an epitope (amino acid 508-541) mapped in the NC16A domain. Of note, nine of 15 PG patient sera reacted with at least one additional antigenic site other than NC16A. Specifically, two epitopes in the BP180 extracellular domain and five epitopes in the intracellular one were recognized by three and seven PG sera, respectively. In addition, a representative intracellular epitope was recognized by PG autoantibodies as a portion of BP180 antigen both in denaturating and native conditions. Finally, reactivity against epitopes additional to NC16A was also detected at an early stage of the disease. The identification and characterization of hitherto unrecognized epitopes targeted by PG patient autoantibodies provide novel insights into the pathophysiology of humoral immune response to BP180 in PG.

Oral pemphigoid autoantibodies preferentially target BP180 ectodomain.

Mucous membrane pemphigoid (MMP) comprises a heterogenous group of autoimmune subepithelial bullous diseases very frequently having oral involvement. Very few studies have investigated the immunological status of a subset of MMP, termed oral pemphigoid (OP), presenting with exclusive oral lesions. In this study we show that 75% of 20 OP patients without scarring phenotype possessed circulating autoantibodies against the BP180 molecule, indicating a prominent role of this protein as a target antigen in OP. Of note, the frequency of reactivity against BP180 ectodomain epitopes in OP was similar to that previously reported for MMP with cicatricial phenotype, while the lack of significant recognition of BP180 intracellular domain appears to characterize OP with respect to other diseases of the pemphigoid group. Finally, the combined use of sensitive techniques allowed the detection of circulating autoantibodies in 90% of OP patients, supporting the usefulness of this approach in the diagnosis of MMP disease.