Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. Previously, we have generated an active mouse model for PV by adoptive transfer of Dsg3−/− splenocytes. In this study, we isolated eight AK series, anti-Dsg3 IgG mAbs from the PV mouse model, and examined their pathogenic activities in induction of blister formation. Intraperitoneal inoculation of the AK23 hybridoma, but not the other AK hybridomas, induced the virtually identical phenotype to that of PV model mice or Dsg3−/− mice with typical histology of PV. Epitope mapping with domain-swapped and point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognized a calcium-dependent conformational epitope on Dsg3, which consisted of the V3, K7, P8, and D59 Dsg3-specific residues that formed the adhesive interface between juxtaposed Dsg, as predicted by the crystal structure. The epitopes of the mAbs that failed to show apparent pathogenic activity were mapped in the middle to carboxyl-terminal extracellular region of Dsg3, where no direct intermolecular interaction was predicted. These findings demonstrate the pathogenic heterogeneity among anti-Dsg3 IgG Abs due to their epitopes, and suggest the direct inhibition of adhesive interaction of Dsg as an initial molecular event of blister formation in pemphigus.

Pemphigus is an autoimmune blistering disease with two major subtypes, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Patients with pemphigus have circulating antidesmoglein (Dsg)1 and/or anti-Dsg3 IgG autoantibodies. We have previously developed enzyme-linked immunosorbent assays (ELISAs) using recombinant Dsg1 and Dsg3 expressed by baculovirus as a diagnostic tool for pemphigus. The purpose of this study was to evaluate the practical application of these ELISAs for clinical use with a large number of serum samples. We used 81 PV sera, 48 PF sera, 114 bullous pemphigoid (BP) sera, 124 collagen disease sera, nine sera of other non-pemphigus bullous diseases and 179 normal control sera. A cut-off value was determined by receiver-operating-characteristic plots. Forty-seven of 48 PF sera (97.9%) were positive in the Dsg1 ELISA and 79 of 81 PV sera (97.5%) were positive in the Dsg3 ELISA, while only two (1. 1%) and four (2.2%) of 179 normal sera were positive in Dsg1 and Dsg3 ELISAs, respectively. However, some disease control sera of BP and collagen diseases exceeded the cut-off value. Introduction of a grey zone helped to decrease the number of these false-positive sera. Furthermore, in three patients studied, the respective Dsg1 and Dsg3 ELISA scores showed parallel fluctuation with the disease activity along the time course. We conclude that Dsg1 and Dsg3 ELISAs provide a simple, sensitive and highly specific assay for the diagnosis of patients with PV and PF and that these ELISAs may be a valuable tool to monitor the disease activity. We also propose diagnostic criteria for pemphigus based on ELISA reactivity: if a serum is positive against Dsg3 it indicates a diagnosis of PV, regardless of reactivity against Dsg1; if a serum is negative for Dsg3 and positive for Dsg1, it indicates a diagnosis of PF.

We present here the recommendations resulting from this Delphi process. This international consensus includes intravenous CD20 inhibitors as a first line therapy option for moderate to severe pemphigus.

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Jasmonates mediate various physiological events in plant cells such as defense responses, flowering, and senescence through intracellular and intercellular signaling pathways, and the expression of a large number of genes appears to be regulated by jasmonates. In order to obtain information on the regulatory network of jasmonate-responsive genes (JRGs) in Arabidopsis thaliana (Arabidopsis), we screened 2880 cDNA clones for jasmonate responsiveness by a cDNA macroarray procedure. Since many of the JRGs reported so far have been identified in leaf tissues, the cDNA clones used were chosen from a non-redundant EST library that was prepared from above-ground organs. Hybridization to the filters was achieved using α-33 P-labeled single-strand DNAs synthesized from mRNAs obtained from methyl jasmonate (MeJA)-treated and untreated Arabidopsis seedlings. Data analysis identified 41 JRGs whose mRNA levels were changed by more than three fold in response to MeJA. This was confirmed by Northern blot analysis by using eight representatives. Among the 41 JRGs identified, 5 genes were JA biosynthesis genes and 3 genes were involved in other signaling pathways (ethylene, auxin, and salicylic acid). These results suggest the existence of a positive feedback regulatory system for JA biosynthesis and the possibility of crosstalk between JA signaling and other signaling pathways.

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1–496) and Dsg3 (1–566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26–87 of Dsg1 and 25–88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His25, Cys28, Ala29) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

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