Yasuo Kitajima

Usefulness of enzyme-linked immunosorbent assay using recombinant desmogleins 1 and 3 for serodiagnosis of 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.

Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just ‘witnesses of disease'?

Abstract:u2002 Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ‐specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyones satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re‐examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti‐desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert ‘witnesses’ in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome ‘food for thought’ as well as very specific suggestions for important future research directions – within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting – and clinically very relevant! – immunopathogenesis of PV in all its complexity.

Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients

BACKGROUNDnEpidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain.nnnOBJECTIVEnThe aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA.nnnMETHODSnWe developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera.nnnRESULTSnWhen we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity.nnnCONCLUSIONnWe conclude that the NC1+NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients.

An extremely severe case of cutaneous calcinosis with juvenile dermatomyositis, and successful treatment with diltiazem

A case of cutaneous calcinosis associated with juvenile dermatomyositis is described. The patient was a 3‐year‐old girl who had been diagnosed as having dermatomyositis at age 1u2003year. She was treated with prednisolone, but developed multiple calcified nodules in the subcutaneous tissues and intermuscular fascia. These nodules gradually increased in size despite continual therapy with steroids and aluminium hydroxide. Treatment with diltiazem completely suppressed the development of calcinosis.

New insights into desmosome regulation and pemphigus blistering as a desmosome-remodeling disease

Desmosomes in keratinocytes are the most important intercellular adhering junctions that provide structural strength for the epidermis. These junctions are connected directly with desmosomal cadherin proteins. Desmosomal cadherins are divided into four desmogleins (Dsgs), Dsg1–4, and three desmocollins (Dscs), Dsc1–3, all of which are involved in desmosomal adhesion by homo‐ and/or heterophilic binding between Dsgs and Dscs in a Ca2+‐dependent manner. Cadherins are present on the cell surface and anchor keratin intermediate filaments (KIFs) to their inner cytoplasmic surface to generate an intracellular KIF‐skeletal scaffold through several associate proteins, including plakoglobin, plakophillin, and desmoplakins. As such, the desmosomal contacts between adjacent cells generate an intercellular KIF scaffold throughout the whole epidermal sheet. However, despite these critical roles in maintaining epidermal adhesion and integrity, desmosomes are not static structures. Rather, they are dynamic units that undergo regular remodeling, i.e., assembly and disassembly, to allow for cell migration within the epidermis in response to outside‐in signaling during epidermal differentiation. Recently, two cell–cell adhesion states controlled by desmosomes have been recognized, including “stable hyperadhesion (Ca2+‐independent)” and “dynamic weak‐adhesion (Ca2+‐dependent)” conditions. These conditions are mutually reversible through cell signaling events involving protein kinase C (PKC) and epidermal growth factor receptor. Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti‐Dsg3 antibodies. Binding of these antibodies to Dsg3 causes endocytosis of Dsg3 from the cell surface and results in the specific depletion of Dsg3 from desmosomes, an event linked to acantholysis in the epidermis. This binding of anti‐Dsg3 antibody to Dsg3 in epidermal keratinocytes activates PKC, to generate the “weak‐adhesion (Ca2+‐dependent)” state of desmosomes. The weak‐adhesion desmosomes appear to be the susceptible desmosomal state and a prerequisite for Dsg3 depletion from desmosomes, pivotal and specific events leading to PV blistering. These observations allow us to propose a concept for pemphigus blistering disorders as a “desmosome‐remodeling impairment disease” involving a mechanism of Dsg3 nonassembly and depletion from desmosomes through PV immunoglobulin G‐activated intracellular signaling events.

Binding of pemphigus vulgaris IgG to antigens in desmosome core domains excludes immune complexes rather than directly splitting desmosomes

Backgroundu2002 Pemphigus vulgaris (PV) is characterized by autoantibodies against desmoglein (Dsg) 3 or both Dsg1 and Dsg3, i.e. desmosomal adhesion molecules.

150(th) anniversary series: Desmosomes and autoimmune disease, perspective of dynamic desmosome remodeling and its impairments in pemphigus.

Abstract Desmosomes are the most important intercellular adhering junctions that adhere two adjacent keratinocytes directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell–cell adhesion states of desmosomes, that is, “stable hyper-adhesion” and “dynamic weak-adhesion” conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal growth factor receptor (EGFR) during Ca2+-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a “desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events”.

A randomized double-blind trial of intravenous immunoglobulin for bullous pemphigoid.

BACKGROUNDnPatients with steroid-resistant bullous pemphigoid (BP) require an appropriate treatment option.nnnOBJECTIVEnA multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of high-dose intravenous immunoglobulin (IVIG; 400mg/kg/day for 5days) in BP patients who showed no symptomatic improvement with prednisolone (≥0.4mg/kg/day) administered.nnnMETHODSnWe evaluated the efficacy using the disease activity score on day15 (DAS15) as a primary endpoint, and changes in the DAS over time, the anti-BP180 antibody titer, and safety for a period of 57days as secondary endpoints.nnnRESULTSnWe enrolled 56 patients in this study. The DAS15 was 12.5 points lower in the IVIG group than in the placebo group (p=0.089). The mean DAS of the IVIG group was constantly lower than that of the placebo group throughout the course of observation, and a post hoc analysis of covariance revealed a significant difference (p=0.041). Furthermore, when analyzed only in severe cases (DAS≥40), the DAS15 differed significantly (p=0.046). The anti-BP180 antibody titers showed no difference between the two groups.nnnCONCLUSIONnIVIG provides a beneficial therapeutic outcome for patients with BP who are resistant to steroid therapy.

Japanese guidelines for the management of pemphigus

The Committee for Guidelines for the Management of Pemphigus was organized as one element of the Japanese Dermatological Association (JDA) and the Ministry of Health, Labour, and Welfare (MHLW) Research Project on Measures for Research Committee for Intractable Skin Disease. Pemphigus has been defined as a group of intractable autoimmune blistering diseases caused by anti‐desmoglein 1 and/or anti‐desmoglein 3 IgG autoantibodies by the MHLW. The diagnosis of this condition and the criteria for assessing its severity are based on suggestions from the MHLW Research Group. The clinical practice guidelines presented here are those that are currently recommended in Japan. However, symptoms and complications can vary widely among individual pemphigus patients, so not all therapies will be required to be in complete agreement with these guidelines.

Bullous Pemphigoid IgG Induces BP180 Internalization via a Macropinocytic Pathway

Bullous pemphigoid (BP) is an autoimmune blistering skin disease induced by pathogenic autoantibodies against a type II transmembrane protein (BP180, collagen type XVII, or BPAG2). In animal models, BP180 autoantibody-antigen interaction appears insufficient to develop blisters, but involvement of complement and neutrophils is required. However, cultured keratinocytes treated with BP-IgG exhibit a reduction in the adhesive strength and a loss of expression of BP180, suggesting that the autoantibodies directly affect epidermal cell-extracellular matrix integrity. In this study, we explored the consequences of two distinct epithelial cells treated with BP-IgG, particularly the fate of BP180. First, we followed the distribution of green fluorescent protein-tagged BP180 in an epithelial cell line, 804G, and normal human epidermal keratinocytes after autoantibody clustering. After BP-IgG treatment, the adhesive strength of the cells to their substrate was decreased, and BP180 was internalized in both cell types, together with the early endosomal antigen-1. By using various endocytosis inhibitors and a fluid-uptake assay, we demonstrated that BP-IgG-induced BP180 internalization is mediated via a macropinocytic pathway. Moreover, a macropinocytosis inhibitor rescued a BP-IgG-induced reduction in the adhesive strength of the cells from their substrate. The results of this study suggest that BP180 internalization induced by BP-IgG plays an important role in the initiation of disease pathogenesis.