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There is substantial evidence that blister formation in pemphigus patients is mediated by auto-antibodies (auto-Abs) targeted against certain desmosomal cadherins, namely desmoglein1 (Dsg1) and desmoglein3 (Dsg3). Several pathogenic epitopes of Dsg3 are located at the amino-(NH2)-terminal end of the Dsg3 ectodomain, namely the extracellular domain 1 (EC1). On the other hand a great number of pemphigus patients exhibit auto-Abs directed against the more carboxy-(COOH)-terminal epitopes of Dsg3, e.g. within the EC4- and EC5-domain and these domains may play an essential role in maintaining desmosomal adhesion. Some pemphigus patients exhibit additional or solely auto-Abs against other desmosomal cadherins, especially desmocollin 3 (Dsc3). However, the pathogenic relevance of Dsc3-reactive immunoglobulin G (IgG) has not been directly shown. This study aimed to first establish a method to specifically isolate Dsg3-reactive IgG from PV sera and to further investigate their pathogenic capacity using a keratinocyte based in vitro assay. This method was further applied to sera of four Japanese patients suffering from atypical pemphigus all of them exhibiting a positive IgG reactivity against Dsc3. Sepharose based affinity chromatochraphy columns coated with recombinant baculovirus produced proteins of the extracellular domains of Dsg3 and Dsc3, respectively, were used to specifically isolate auto-Abs from pemphigus sera. Affinity purified IgG fractions were subsequently tested for antigen specificity using enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB). Reactivity with native Dsg3- and Dsc3-protein, respectively, was proven by immunofluorescence (IF) on cultured human keratinocytes, monkey esophagus and frozen sections of normal human skin. Finally, a keratinocyte based so-called dissociation assay served to investigate the in vitro pathogenicity of the affinity purified IgG fractions. Eight Dsg3-reactive PV patients showed Dsg3-domain-specific auto-antibodies by ELISA. Four of these patients were selected for further investigation based on their antibody profile, i.e. their epitope specificity. Two patients (#1 and #8) exhibited IgG directed against Dsg3EC1 and Dsg3EC4. Patient #2 exclusively expressed auto-Abs directed against Dsg3EC1 whereas patient #6 showed IgG reactivity against Dsg3EC4, only. Serum IgG was then affinity purified using the respective recombinant Dsg3-subdomains. Antigen specificity of the eluted IgG-fractions was subsequently verified by IB and ELISA. Isolated IgG fractions showed a characteristic intercellular staining pattern by IF using cultured human keratinocytes indicating positive reactivity with native Dsg3-protein. Finally Dsg3-, Dsg3EC1- and Dsg3EC4-specific IgG caused keratinocyte dissociation which was comparable to the positive control, a monoclonal antibody (AK23) directed against the NH2-terminus of Dsg3. These techniques were then applied to the sera of four atypical, i.e. two pemphigus vegetans and two pemphigus herpetiformis patients, in order to isolate Dsc3-specific IgG. All but one of these patients, who showed additional Dsg1 reactivity, exhibited IgG reactivity exclusively against Dsc3 but no other desmosomal cadherin. From all sera IgG fractions were successfully isolated and antigen specificity to Dsc3 was verified. Dsc3-reacitve IgG showed a characteristic intercellular staining pattern by IF on cultured human keratinocytes, monkey esophagus and human skin. Finally all isolated IgG fractions were able to induce loss of keratinocyte adhesion in vitro.Taken together this data strongly suggests a significant acantholytic effect of IgG directed against COOH-terminal epitopes of Dsg3 in addition to the well known pathogenic epitopes at the NH2-terminus of this auto-antigen. Moreover Dsc3-reactive IgG isolated from patients with atypical pemphigus variants proved to be pathogenic in vitro. For the first time these results directly show the acantholytic effect of Dsc3-reactive IgG and provides evidence for the pathogenic relevance of Dsc3-IgG in pemphigus patients lacking reactivity against other desmosomal cadherins. Further investigations are needed to elucidate the mechanisms by which auto-Abs directed against COOH-terminal epitopes of Dsg3 induce acantholysis. The pathogenic relevance of other epitopes of Dsg3 needs to be addressed. Finally screening of pemphigus patients’ sera for Dsc3-reactive IgG should provide further knowledge about their correlation with atypical pemphigus variants. antibodies application/pdf Characterization of pathogenic auto-antibodies directed against desmoglein 3 and desmocollin 3 in sera of pemphigus patients. Medical sciences Medicine Medizin English desmoglein 3 Pemphigus pemphigus Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg pathogenic Desmoglein 3 2013 doctoralThesis Charakterisierung pathogener Auto-Antikörper gegenüber Desmoglein 3 und Desmocollin 3 in Seren von Pemphigus Patienten. Medizin Hautkrankheiten Pathogenität desmocollin 3 ths PD Dr. med. Eming Rüdiger Eming, Rüdiger (PD Dr. med.) ppn:327850612 2013-06-14 https://archiv.ub.uni-marburg.de/diss/z2013/0533/cover.png monograph urn:nbn:de:hebis:04-z2013-05334 Autoantikörper opus:5005 Pemphigus vulgaris (PV) repräsentiert die häufigste klinische Variante innerhalb der Pemphigus-Gruppe der autoimmunen bullösen Hautkrankheiten. Blasenbildung beim Pemphigus wird durch Auto-Antikörper (auto-Ak) gegen die desmosomalen Cadherine Desmoglein 1 (Dsg1) und Desmoglein 3 (Dsg3) ausgelöst. Einige pathogene Epitope des Dsg3 liegen im amino-(NH2)-terminalen Bereich dieses Moleküls, genau genommen in der extrazellulären Domäne 1 (EC1). Andererseits weist eine große Anzahl an Pemphigus Patienten auto-Ak gegen Carboxy-(COOH)-terminale Epitope des Dsg3, z.B. innerhalb der EC4- oder EC5-Domänen auf. Manche Pemphigus Patienten besitzen zusätzlich oder ausschließlich auto-Ak gegen andere desmosomale Cadherine, besonders Desmocollin 3 (Dsc3). Die pathogenetische Relevanz dieser Dsc3-reaktiven auto-Ak konnte bislang nicht direkt gezeigt werden. Zunächst wurde eine Methode etabliert, um Dsg3-reaktives Immunglobulin G (IgG) aus PV Seren spezifisch zu isolieren und die pathogenetische Wirkung mithilfe eines Zell-basierten in vitro Assays zu untersuchen. Diese Methode wurde anschließend auf Seren von vier japanischen Patienten mit atypischem Pemphigus, welche alle eine positive IgG-Reaktivität gegen Dsc3 aufwiesen, angewendet. Mithilfe von Affinitätschromatographie-Säulen, die rekombinante Proteine der extrazellulären Domänen von Dsg3 bzw. Dsc3 enthielten, wurden auto-Ak aus den Seren von Pemphigus Patienten spezifisch isoliert. Die isolierten IgG-Fraktionen wurden bezüglich ihrer Antigenspezifität mittels enzyme-linked immunosorbent assay (ELISA) und Immunoblotting (IB) getestet. Immunfluoreszenz (IF) Untersuchungen auf humanen Keratinozyten, Affenösophagus und Gefrierschnitten menschlicher Haut wiesen die Reaktivität mit nativem Dsg3 bzw. Dsc3 nach. Ein mit Keratinozyten durchgeführter, funktioneller „Dissoziations-Assay“ diente der Untersuchung der in vitro-Pathogenität der aufgereinigten IgG-Fraktionen. Acht PV Patienten zeigten im ELISA Dsg3-spezifische auto-Ak. Vier dieser Patienten wurden aufgrund ihres geeigneten Antikörperprofils, d.h. der Epitopspezifität, für die weiteren Untersuchungen ausgewählt. Zwei Patienten (#1 und #8) zeigten IgG-Reaktivität gegen Dsg3EC1 und Dsg3EC4. Das Serum von Patient #2 beinhaltete ausschließlich auto-Ak gegen Dsg3EC1, wohingegen Patient #6 nur IgG-Reaktivität gegen Dsg3EC4 zeigte. Serum-IgG wurde anschließend mithilfe der jeweils entsprechenden rekombinanten Dsg3-Ektodomäne spezifisch aufgereinigt. Die Antigenspezifität der eluierten IgG-Fraktionen konnte mittels IB- und ELISA-Analysen verifiziert werden. Alle isolierten IgG-Fraktionen zeigten ein charakteristisches interzelluläres Färbemuster in der IF auf kultivierten humanen Keratinozyten. Durch diese Experimente wurde die positive Reaktivität mit rekombinantem und nativem Dsg3 nachgewiesen. Dsg3-, Dsg3EC1- und Dsg3EC4-spezifisches IgG induzierte die Dissoziation von Keratinozyten in einem vergleichbaren Ausmaß wie die Positivkontrolle, ein monoklonaler Antikörper gegen den NH2-Terminus von Dsg3 (AK23). Diese Technik wurde anschließend auf die Seren von vier atypischen, d.h. zwei Pemphigus Vegetans und zwei Pemphigus Herpetiformis Patienten, angewendet, um Dsc3-spezifisches IgG zu isolieren. Alle außer einem dieser Patienten, welcher zusätzlich Dsg1-reaktives IgG aufwies, zeigten ausschließlich Serum-Reaktivität gegen Dsc3 aber gegen kein anderes desmosomales Cadherin. Von allen vier Seren konnte Dsc3-reaktives IgG erfolgreich isoliert und die Antigenspezifität bestätigt werden. Die IgG-Fraktionen zeigten ein charakteristisches interzelluläres Färbemuster in der IF auf kultivierten humanen Keratinozyten, Affenösophagus und humaner Haut und induzierten den Adhäsionsverlust epidermaler Keratinozyten in vitro. Zusammengefasst legen diese Daten nahe, dass auch IgG gegen COOH-terminale Epitope des Dsg3, zusätzlich zu den bereits bekannten pathogenen NH2-terminalen Epitopen, einen akantholytischen Effekt aufweist. Dsc3-reaktives IgG, isoliert aus Seren von atypischen Pemphigus Patienten, ist pathogen in vitro. Diese Untersuchungen zeigen erstmalig den akantholytischen Effekt von Dsc3-reaktivem IgG und geben Hinweise auf die pathogene Relevanz von Dsc3-IgG in Seren von Pemphigus Patienten die keine Reaktivität gegen andere desmosomale Cadherine aufweisen. Weitere Untersuchungen sind nötig, um die Mechanismen zu untersuchen, die dem akantholytischen Effekt von auto-Ak gegen COOH-terminale Dsg3-Epitope zugrunde liegen. Zudem sollte die pathogene Relevanz anderer Epitope des Dsg3 näher untersucht werden. Schließlich erscheint das Screening von Pemphigus Patienten hinsichtlich Dsc3-reaktiven IgG sehr vielversprechend, um weitere Erkenntnisse über mögliche Korrelationen mit atypischen Varianten des Pemphigus zu erhalten. Desmocollin 3 Philipps-Universität Marburg https://doi.org/10.17192/z2013.0533 Rafei-Shamsabadi, David Ali Rafei-Shamsabadi David Ali 2013-08-29