Marian Dmochowski

Immunoblotting studies of linear IgA disease

Patients with linear IgA deposits at the basement membrane zone (BMZ) detected by direct immunofluorescence (IF) may show diverse clinical and laboratory findings. The aim of this study was to investigate the issue of target antigens for linear IgA disease (LAD) antibodies. We examined sera from 46 adults and children with exclusive IgA deposits at the BMZ, by both indirect IF on 1 M NaCl split human skin and immunoblotting. IgA anti-BMZ antibodies binding to the epidermal side of the split were found in 31 LAD sera. IgA anti-BMZ antibodies binding to the dermal side of the split were detected only in 4 LAD sera. No sera contained IgA anti-BMZ antibodies binding to both sides of the split. Immunoblotting revealed that 12 epidermal side-positive LAD sera reacted with the 97 kDa protein in the human epidermal extracts. Moreover, we found that 2 dermal side-positive LAD sera reacted with a protein of approximately 255 kDa on immunoblotting of the dermal extract. We conclude that there are at least two types of LAD. However, the nature of target antigens for LAD antibodies remains to be determined.

Identification of desmoglein 1 as autoantigen in a patient with intraepidermal neutrophilic IgA dermatosis type of IgA pemphigus

Abstract: In a 51‐year‐old female patient with intraepidermal neutrophilic IgA dermatosis (IEN) type of IgA pemphigus, circulating IgA, but not IgG, autoantibodies were detected to bind to the cell surface of the whole epidermis, being much stronger in the upper epidermis. In the patients skin a heavy intraepidermal IgA staining was observed throughout the whole epidermis, accompanied by a weak IgG and a more prominent C3 staining. IgA from the patients serum showed no reactivity either with epidermal proteins by immunoblot analysis, or with COS 7 cells transiently transfected with mammalian cell expression constructs containing full length human Dsc1, Dsc2 and Dsc3. Our patients IgA specifically reacted with conformational epitopes of human desmoglein (Dsg) 1 but not Dsg 3, when studied in a previously established, here for IgA antibody detection modified enzyme‐linked immunoabsorbent assay (ELISA) of baculovirus expression system. The immunoreactivity against keratinocyte cell surface was completely removed from the serum of the patient by pre‐incubation with recombinant Dsg1 baculoprotein. This finding indicates that the sera possess only IgA antibodies against the extracellular domain of Dsg1 baculoprotein, but no antibodies against components of keratinocyte cell surface other than Dsg1. This is the first case of IgA pemphigus where Dsg1 has been identified as the autoantigen.

Demonstration of antibodies to bovine desmocollin isoforms in certain pemphigus sera

Summary We have shown previously that IgG antibodies in certain pemphigus sera. particularly endemic Brazilian pemphigus foliaceus (BPF) sera. react with bovine desmocollins (Dsc). which are transmembranous glycoproteins of desmosome junctions. Desmocollins occur as three different isoforms (Dsc 1, 2 and 3). all of which are represented in the epidermis. In this study. we examined sera of various pemphigus types by immunoblotting purified bovine desmosomes and bovine Dsc l, 2 and 3 fusion proteins. expressed in pGEX expression vectors. Six of l5 (40.0%) BPF sera. two of 18 (11.1%) non‐endemic pemphigus foliaceus sera. eight of 39 (20.5%) pemphigus vulgaris (PV) sera. and two of l l (18.2%) normal sera. showed reactivity with Dsc from desmosomes. Experiments with fusioni proteins showed that no Dsc isoform was specifically recognized by sera of any individual pemphigus type. Our results indicate that the pathogenesis of pemphigus might be more complex than previously believed.

Unusual acantholytic bullous dermatosis associated with neoplasia and IgG and IgA antibodies against bovine desmocollins I and II

There are unusual cases of pemphigus that have antibodies nonreactive with either pemphigus vulgaris or pemphigus foliaceus antigens. We describe a patient with an acantholytic bullous dermatosis and lung cancer with intercellular IgG and IgA antibodies that differed in specificity from those of pemphigus vulgaris and pemphigus foliaceus and reacted with bovine desmocollins I and II and recombinant desmocollin protein.

Monopathogenic vs multipathogenic explanations of pemphigus pathophysiology

This viewpoint highlights major, partly controversial concepts about the pathogenesis of pemphigus. The monopathogenic theory explains intra‐epidermal blistering through the “desmoglein (Dsg) compensation” hypothesis, according to which an antibody‐dependent disabling of Dsg 1‐ and/or Dsg 3‐mediated cell–cell attachments of keratinocytes (KCs) is sufficient to disrupt epidermal integrity and cause blistering. The multipathogenic theory explains intra‐epidermal blistering through the “multiple hit” hypothesis stating that a simultaneous and synchronized inactivation of the physiological mechanisms regulating and/or mediating intercellular adhesion of KCs is necessary to disrupt epidermal integrity. The major premise for a multipathogenic theory is that a single type of autoantibody induces only reversible changes, so that affected KCs can recover due to a self‐repair. The damage, however, becomes irreversible when the salvage pathway and/or other cell functions are altered by a partnering autoantibody and/or other pathogenic factors. Future studies are needed to (i) corroborate these findings, (ii) characterize in detail patient populations with non‐Dsg‐specific autoantibodies, and (iii) determine the extent of the contribution of non‐Dsg antibodies in disease pathophysiology.

The analysis of IgG subclasses of anti-intercellular antibodies in pemphigus by an immunoblot technique

Immunofluorescence (IF) studies seem to indicate an IgG1 and/or IgG4 restriction of antibody response in pemphigus [3, 6, 7, 10, 13]. Immunoprecipitation and immunoblot (IB) studies [4, 5] have revealed that pemphigus vulgaris (PV) sera contain pan-IgG antibodies to a 130-kDa protein, while pemphigus foliaceus (PF) sera contain pan-IgG antibodies to a 160or 150-kDa protein (most likely desmoglein). Recently, it has been shown that endemic PF IgG4 specifically recognizes three antigenic glycopeptides (45, 62 and 80 kDa) obtained by sonication of human epidermal envelopes [2], An IgG4restricted response to these molecules was found in 48% of endemic PF sera tested [1]. In the present study, the IgG subclasses of pemphigus antibodies were investigated by both indirect IF on normal human skin sections and IB. We examined nine PV sera, five PF sera and five normal control sera. The mouse monoclonal antibody 52-3D against desmocollins (DC) I/II (a generous gift from Dr. D. R. Garrod, Manchester, UK) diluted 1:100 was also used as a control. For the IgG subclass analysis we used 1:20 diluted sera. Indirect IF for pan-IgG detection and IgG subclass analysis was performed as described elsewhere [13]. Dermoepidermal separation with dispase, extraction of normal human epidermis and preparation of bovine desmosomes were performed according to procedures described previously [5, 9, 11]. Both dispase-treated normal human epidermal extract and bovine desmosome preparations were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis as described by Laemmli [8] using 7% separating gel, and electrophoretically transferred to nitrocellulose sheets (Schleicher and Schuell, Dassel, FRG) [12], IB for the pan-IgG study was performed according to a previously described method [5, 9J. IB for the IgG subclass analysis was

Prospective studies on the routine use of a novel multivariant enzyme-linked immunosorbent assay for the diagnosis of autoimmune bullous diseases

Background: Serologic diagnosis of autoimmune blistering disease (AIBD) usually follows a sophisticated multistep algorithm. Objective: We sought validation of a multivariant enzyme‐linked immunosorbent assay (ELISA) in the routine diagnosis of AIBD. Methods: The multivariant ELISA comprising 6 recombinant immunodominant forms of major AIBD target antigens, ie, desmoglein 1, desmoglein 3, envoplakin, BP180, BP230, and type VII collagen was applied in: (1) a cohort of well‐characterized AIBD (n = 173) and control sera (n = 130), (2) a prospective multicenter study with 204 sera from patients with newly diagnosed AIBD with positive direct immunofluorescence microscopy, and (3) a prospective monocenter study with 292 consecutive sera from patients with clinical suspicion of AIBD in comparison with the conventional multistep diagnostic algorithm. Results: Concordant results in the multivariant ELISA compared with direct immunofluorescence microscopy were seen in 94% of patients with pemphigus and 71% of patients with pemphigoid (Cohen &kgr; value, 0.95 and 0.66) and with the conventional multistep diagnostic approach in 91% of patients with pemphigus and 88% of patients with bullous pemphigoid and 93% of autoantibody‐negative sera (Cohen &kgr;, 0.95, 0.84, and 0.78). Limitations: IgA autoantibodies and less common target antigens were not analyzed. Conclusions: The multivariant ELISA is a practical, highly standardized, and widely available novel diagnostic tool for the routine diagnosis of AIBD.

IgA antikeratinocyte surface autoantibodies from two types of intercellular IgA vesiculopustular dermatosis recognize distinct isoforms of desmocollin

We previously proposed the term intercellular IgA vesiculopustular dermatosis (IAVPD) for cases showing IgA antikeratinocyte surface autoantibodies. This condition is divided into two subtypes: intraepidermal neutrophilic IgA dermatosis (IEN), showing pustule formation in the entire epidermis, and subcorneal pustular dermatosis (SPD) showing pustule formation in the uppermost epidermis. We have previously reported that serum from certain IAVPD patients reacts with bovine desmocollin (Dsc), a desmosomal cadherin. In this study we showed that two Dsc isoforms with a slightly different molecular weight were recognized by the serum from these patients. Further analysis revealed that serum from patients with the SPD type, which stained the cell surface in the uppermost epidermis with immunofluorescence, seem to react with Dsc1. By contrast, serum from patients with the IEN type, which stained the cell surface at all levels of the epidermis with immunofluorescence, seemed to react with Dsc3. This difference of distribution between the two distinct Dsc molecules may contribute to the different clinicopathological features between the IEN and SPD type of IAVPD.

Autoimmunity-driven enzymatic remodeling of the dermal-epidermal junction in bullous pemphigoid and dermatitis herpetiformis.

Pathogenesis of blister formation in bullous pemphigoid (BP) and dermatitis herpetiformis (DH) is associated with destruction of numerous components of the dermal–epidermal junction. Proteolytic enzymes (PE) are involved in a multitude of physiological reactions and may have impact on the epidermal–dermal integrity. Involvement of various PE in inflammation and blister formation in BP and DH is intensively investigated using both morphologic and functional approaches, particularly in BP. The development into the full-blown stage in BP and DH may be caused by an impairment of the human Fc receptor regulatory system that may cause the inefficiently controlled activation of inflammatory cells and subsequent secretion of various proteases.

Bullous pemphigoid and neurodegenerative diseases: a study in a setting of a Central European university dermatology department

Bullous pemphigoid (BP) is an autoimmune blistering dermatosis of the elderly mediated by IgG and IgE antibodies to skin hemidesmosomal proteins, BP180 and/or BP230, that occur physiologically also in neuronal tissue. It was reported that BP is associated with neurodegenerative diseases (ND). We performed a retrospective study in a setting of a Central European university dermatology department on prevalence of ND in 94 BP patients. 26 out of 94 BP patients had at least one ND. ND included: Parkinson’s disease, dementia, stroke, hear loss, tinnitus, blindness, vertigo, neurosyphilis, systemic sclerosis, and epilepsy. Since population aging is conceivably responsible for the rising number of BP cases as a result of immunosenescence-related phenomena, the plausible BP-specific immunopathogenetic relationship between BP and ND deserves to be further experimentally explored.