Conformational Epitopes of Pemphigus Antigens (Dsg1 and Dsg3) Are Calcium Dependent and Glycosylation Independent

The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the antigenicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculoproteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens

Synergistic Pathogenic Effects of Combined Mouse Monoclonal Anti-Desmoglein 3 IgG Antibodies on Pemphigus Vulgaris Blister Formation

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies. Previously, we generated an active mouse model for PV by adoptive transfer of splenocytes from immunized or naive Dsg3−/− mice. In this study, we isolated 10 anti-Dsg3 IgG mAbs (NAK-series) from PV model mice generated by transfer of naive Dsg3−/− splenocytes. We characterized their epitopes using domain-swapped and point-mutated Dsg1/Dsg3 molecules and examined their pathogenic activities in blister formation in three different assays. In a passive transfer model using neonatal mice, eight of 10 NAK mAbs showed pathogenic activity when injected together with half the minimum pathogenic dose of anti-Dsg1 IgG autoantibodies from pemphigus foliaceus (PF) patients. None of the mAbs could induce the PV phenotype when individual hybridoma clones were inoculated by peritoneal injection into adult Rag2−/− mice. NAK mAbs displayed a range of potency in an in vitro dissociation assay using primary cultured mouse keratinocytes. Interestingly, when multiple hybridoma clones recognizing different epitopes were inoculated in combination, recipient mice developed the PV phenotype. In vitro dissociation assays confirmed that combined NAK mAbs had synergistic pathogenic effects. These findings indicate that although an individual anti-Dsg3 IgG is not sufficient to cause blistering in adult mice, several together can induce the PV phenotype. These mAbs will provide a valuable tool to investigate the molecular mechanisms of blister formation, mimicking the effects of the polyclonal IgG antibodies found in patients

Pathogenic Epitopes of Autoantibodies in Pemphigus Reside in the Amino-Terminal Adhesive Region of Desmogleins Which Are Unmasked by Proteolytic Processing of Prosequence

Pemphigus targets desmogleins (Dsgs), which are thought to be synthesized as inactive precursor proteins with prosequences that are cleaved by substilisin-like proprotein convertases, such as furin, to yield mature adhesive molecules. We hypothesized that some pemphigus pathogenic antibodies (Abs), which presumably interfere with adhesion, only bind the mature form. A pathogenic and three non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) isolated from a pemphigus foliaceus (PF) patient, were used for immunoprecipitation and ELISA of recombinant precursor and mature Dsg1. The pathogenic Ab binds mature Dsg1, whereas non-pathogenic Abs bind either only the precursor or both the precursor and mature Dsg1. Competition ELISA showed that the majority of PF sera target the same or nearby epitopes defined by the pathogenic anti-Dsg1 mAb that blocked >20% binding of 29 out of 40 PF sera. Furthermore, the immunoreactivity of 45 PF sera against the mature Dsg1 was 3.2 fold stronger than that against the precursor Dsg1 by ELISA. Similar results were observed in anti-Dsg3 Abs in 47 pemphigus vulgaris sera, suggesting that most pemphigus sera target epitopes that are unmasked by proteolytic processing. These findings support the idea that at least some pathogenic pemphigus autoantibodies induce the loss of cell adhesion by directly binding the trans-interaction site of Dsgs

A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus

Human Desmocollin 1 (Dsc1) Is an Autoantigen for the Subcorneal Pustular Dermatosis Type of IgA Pemphigus

IgA pemphigus showing IgA anti-keratinocyte cell surface autoantibodies is divided into subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN) types. We previously showed by immunoblotting that IgA from some IgA pemphigus patients reacted with bovine desmocollins (Dsc), but not human Dsc. To determine the antigen for IgA pemphigus, we focused on conformation-dependent epitopes of Dsc, because sera of patients with classical pemphigus recognize conformation-sensitive epitopes of desmogleins. We constructed mammalian expression vectors containing the entire coding sequences of human Dsc1, Dsc2, and Dsc3 and transiently transfected them into COS7 cells by lipofection. Immunofluorescence of COS7 cells transfected with single human Dscs showed that IgA antibodies of all six SPD-type IgA pemphigus cases reacted with the surface of cells expressing Dsc1, but not with cells expressing Dsc2 or Dsc3. In contrast, none of seven IEN-type IgA pemphigus cases reacted with cells transfected with any Dscs. These results convincingly indicate that human Dsc1 is an autoantigen for SPD-type IgA pemphigus, suggesting the possibility of an important role for Dsc1 in the pathogenesis of this disease. This study shows that a Dsc can be an autoimmune target in human skin disease

Асинхронный электропривод агломерационной машины

В выпускной работе предложено заменить морально устаревший и выработавший ресурс электропривод постоянного тока на асинхронный частотно-регулируемый электропривод. По результатам расчета требуемой мощности двигателя выбран электродвигатель фирмы SIEMENS серии 1LG4313-8AB, а также преобразователь частоты типа MICROMACTER 440. В процессе выполнения работы была определена область работы, рассчитаны параметры схемы замещения двигателя и его характеристики. Выполнена оптимизация контуров регулирования электропривода. В ходе имитационных исследований установлено, что обеспечивается требуемый диапазон регулирования 1:10, необходимая точность поддержания скорости не менее 3%, что соответствует техническому заданию.In the final work, it was proposed to replace the obsolete and exhausted resource of a DC electric drive with an asynchronous frequency-controlled electric drive. Based on the results of calculating the required engine power, a SIEMENS electric motor of the 1LG4313-8AB series and a frequency converter of the MICROMACTER 440 type were chosen. During the work, the work area was determined, the parameters of the motor replacement circuit and its characteristics were calculated. Optimization of the control circuits of the electric drive is performed. During simulations it was found that the required control range of 1:10 is provided, the necessary accuracy of maintaining the speed is not less than 3%, which corresponds to the technical specification

Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome

Epidermal LCs but not dermal DCs take up skin surface protein through intact tight junctions and mediate IgG1 antibody responses to bacterial antigen, conferring protective immunization