The p.Arg435His Variation of IgG3 With High Affinity to FcRn Is Associated With Susceptibility for Pemphigus Vulgaris—Analysis of Four Different Ethnic Cohorts

IgG3 is the IgG subclass with the strongest effector functions among all four IgG subclasses and the highest degree of allelic variability among all constant immunoglobulin genes. Due to its genetic position, IgG3 is often the first isotype an antibody switches to before IgG1 or IgG4. Compared with the other IgG subclasses, it has a reduced half-life which is probably connected to a decreased affinity to the neonatal Fc receptor (FcRn). However, a few allelic variants harbor an amino acid replacement of His435 to Arg that reverts the half-life of the resulting IgG3 to the same level as the other IgG subclasses. Because of its functional impact, we hypothesized that the p.Arg435His variation could be associated with susceptibility to autoantibody-mediated diseases like pemphigus vulgaris (PV) and bullous pemphigoid (BP). Using a set of samples from German, Turkish, Egyptian, and Iranian patients and controls, we were able to demonstrate a genetic association of the p.Arg435His variation with PV risk, but not with BP risk. Our results suggest a hitherto unknown role for the function of IgG3 in the pathogenesis of PV

Der Wirkungsmechanismus von Neurosteroiden auf das Cytoskelett neuronaler Zellen

Neurosteroide können langsame genomische und schnelle nicht-genomische Effekte zeigen. Die Synthese und der Metabolismus von Neurosteroiden werden entwicklungsbedingt reguliert. In den letzten Jahren sind immer mehr schnelle Steroideffekte bekannt geworden, die sowohl über klassische als auch über nicht-klassische Rezeptoren laufen. Zum heutigen Stand der Forschung sind die morphologischen Effekte von Neurosteroiden auf das neuronale Cytoskelett und die involvierten Signalkaskaden noch weitgehend unerforscht. In diesem Zusammenhang stellen sich auch die Fragen nach den verantwortlichen Rezeptoren und dem Transportmechanismus sowie der subzellulären Lokalisation der Steroide. Die im Rahmen meiner Promotion erhaltenen Ergebnisse zeigen, dass die Steroide DHEA und Testosteron eine Reorganisation des Aktincytoskeletts in neuronalen Zellen induzieren und dass diese Effekte diesen Steroiden und nicht ihren Folgemetaboliten zuzuordnen sind. DHEA bewirkt die Kontraktion der Zellen, eine erhöhte Ausbildung von Stressfasern und fokalen Adhäsionskomplexen sowie die Bildung von Filopodien. Der diesen Effekten zu Grunde liegende Signalweg konnte eindeutig identifiziert werden. DHEA induziert in neuronalen Zellen die Aktivierung des Rho-Signalwegs. Diese Aktivierung führt zu einem erhöhten Phosphorylierungsstatus der regulatorischen leichten Kette von Myosin II (MRLC) an Serin 19 und der damit verbundenen erhöhten Myosin-Aktin-Interaktion. Die Ausbildung von Filopodien wird vermutlich über eine Aktivierung der GTPase Cdc42 vermittelt. Testosteron induziert das Auswachsen langer Neuriten sowie eine Verminderung von Stressfasern in neuronalen Zellen. Diese Effekte sind abhängig von der Aktivität der PI3-Kinase. Die im Rahmen dieser Arbeit gewonnenen Erkenntnisse deuten darauf hin, dass Testosteron über die PI3-Kinase und FAK den Rac-Signalweg induziert, da es zu einer Inhibierung des Rho-Signalwegs kommt. Zahlreiche Erkenntnisse weisen darauf hin, dass DHEA und Testosteron die Aktivierung der beteiligten Signalwege über einen G-Protein gekoppelten Rezeptor induzieren. DHEA und Testosteron beeinflussen auch die Expression und die Lokalisation der regulatorischen leichten Ketten von Myosin II. Im Gegensatz zu DHEA (Lokalisation der MRLC in der kortikalen Region der Zelle), induziert Testosteron eine Umlokalisation der MRLC in den Zellkern. Daher ist es denkbar, dass die MRLCs, wie auch Aktin, als Transkriptionsfaktoren wirken können. Die Synthese eines funktionalen, fluoreszierenden DHEA-Derivats (DHEA-Bodipy) ermöglichte erstmals, den Transport und die subzelluläre Lokalisation von DHEA in neuronalen Zellen zu beobachten. DHEA-Bodipy wird in neuronalen Zellen in den Mitochondrien lokalisiert. Diese Lokalisation ergibt völlig neue Ansätze im Verständnis zellulärer Wirkungsorte von Steroiden und beteiligter Rezeptoren. Das in meiner Arbeit vorgestellte Verfahren zur Fluoreszenzmarkierung von Steroiden bietet vielfältige Möglichkeiten im Einsatz zellbiologischer Methoden. Nach diesem Verfahren hergestellte, fluoreszierende Steroide eignen sich aufgrund ihrer Stabilität sehr gut für die Untersuchung des Transports und der subzellulären Lokalisation von Steroiden an fixierten und lebenden Zellen sowie für Colokalisationsexperimente. Diese Methode grenzt somit auch die Anzahl möglicher molekularer Interaktionspartner ein. Für Testosteron konnte ebenfalls ein fluoreszierendes Testosteron-Derivat (Testosteron-Bodipy) synthetisiert werden. Die Aufklärung der Effekte von Steroiden auf das neuronale Cytoskelett und der beteiligten Signalkaskaden sowie die Identifizierung der zellulären Wirkungsorte ermöglichen therapeutische Ansätze zur Behandlung neurodegenerativer Erkrankungen, deren Ursachen in Abnormitäten des Cytoskeletts oder fehlregulierter Neurosteroidogenese zu begründen sind.Neurosteroids may show slow genomic and fast non-genomic effects. Both the synthesis and the metabolism of neurosteroids are regulated depending on the developmental state of the brain. In recent years an increasing number of fast steroid effects have become known which work both via classical as well as non-classical receptors. The current state of research does not provide much insight with regard to the morphological effects of neurosteroids upon the neuronal cytoskeleton and the signaling cascades involved. In this context also questions arise covering the critical receptors, the transport mechanisms involved and finally the subcellular localization of steroids. The results of my research described in my thesis indicate that both the steroids DHEA (Dehydroepiandrosterone) and Testosterone initiate a reorganization of the actin cytoskeleton in neuronal cells. Obviously these structural effects are related to these steroids and not to their metabolites. DHEA causes the contraction of the cells, the increased formation of stress fibers, focal adhesion complexes and filopodia. The underlying signaling pathway which includes these effects has been clearly identified. Within the neuronal cell line SH-SY5Y DHEA activates the Rho signaling cascade. This activation causes an increased phosphorylation of Ser19 of the myosin II regulatory light chain and leads to an enhancement of the myosin-actin-interaction. It is assumed that the formation of filopodia is initiated via the activation of the GTPase Cdc42. Testosterone causes the growth of long neurites and also the reduction of stress fibers in neuronal cells. These results are dependent upon the activity of the PI3-kinase. The findings of my study indicate that Testosterone activates the Rac signaling pathway via PI3-kinase and FAK. Furthermore an inhibition of the Rho signaling pathway is caused. Numerous findings point out that DHEA and Testosterone initiate their effects through a G-protein coupled receptor. DHEA and Testosterone also impact the expression and the localization of the regulatory light chain of myosin II (MRLC). Contrary to DHEA (localization of the MRLC in the cortical region of the cell) Testosterone induces the relocation of the MRLC into the nucleus. Therefore it is conceivable that the MRLCs as well as actin act like transcription factors. The synthesis of a functional, fluorescent DHEA derivative (DHEA-Bodipy) makes it possible, for the first time, to observe the transport and the subcellular localization of DHEA in neuronal cells. DHEA-Bodipy is found within the mitochondria of neuronal cells. This location leads to completely new approaches in the understanding of cellular action places of steroids and involved receptors. In my thesis a new procedure is presented to label steroids via fluorescent dyes and thus offers a variety of cellbiological methods. Fluorescent steroids which have been labeled this way are very stable and are well suited for the analysis of the transport and the subcellular localization of steroids in both fixed and live cells. This also holds true for colocalization experiments. My method also limits the number of possible molecular interacting partners. It was also possible to synthesize the fluorescent Testosterone derivative (Testosterone-Bodipy). The determination of the effects of steroids on the cytoskeleton and the participating signaling cascades as well as the identification of the cellular action centers facilitate therapeutical approaches to the treatment of neurodegenerative diseases which may be caused by anomalies of the cytoskeleton or deficiencies in the metabolism of neurosteroids

Nerve conduction velocity is regulated by the inositol polyphosphate-4-phosphatase II gene

Impairment of nerve conduction is common in neurodegenerative and neuroinflammatory diseases such as multiple sclerosis (MS), and measurement of evoked potentials (visual, motor, or sensory) has been widely used for diagnosis and recently also as a prognostic marker for MS. We used a classical genetic approach to identify novel genes controlling nerve conduction. First, we used quantitative trait mapping in F2 progeny of B10/SJL mice to identify EAE31, a locus controlling latency of motor evoked potentials (MEPs) and clinical onset of experimental autoimmune encephalomyelitis. Then, by combining congenic mapping, in silico haplotype analyses, and comparative genomics we identified inositol polyphosphate-4-phosphatase, type II (Inpp4b) as the quantitative trait gene for EAE31. Sequence variants of Inpp4b (C/A, exon 13; A/C, exon 14) were identified as differing among multiple mouse strains and correlated with individual cortical MEP latency differences. To evaluate the functional relevance of the amino acid exchanges at positions S474R and H548P, we generated transgenic mice carrying the longer-latency allele (Inpp4b(474R/548P)) in the C57BL/6J background. Inpp4b(474R/548P) mice exhibited significantly longer cortical MEP latencies (4.5 ± 0.22 ms versus 3.7 ± 0.13 ms; P = 1.04 × 10(-9)), indicating that INPP4B regulates nerve conduction velocity. An association of an INPP4B polymorphism (rs13102150) with MS was observed in German and Spanish MS cohorts (3676 controls and 911 cases) (P = 8.8 × 10(-3))

A family with atypical Hailey Hailey disease--is there more to the underlying genetics than ATP2C1?

The autosomal dominant Hailey Hailey disease (HHD) is caused by mutations in the ATP2C1 gene encoding for human secretory pathway Ca2+/Mn2+ ATPase protein (hSPCA1) in the Golgi apparatus. Clinically, HHD presents with erosions and hyperkeratosis predominantly in the intertrigines. Here we report an exome next generation sequencing (NGS) based analysis of ATPase genes in a Greek family with 3 HHD patients presenting with clinically atypical lesions mainly localized on the neck and shoulders. By NGS of one HHD-patient and in silico SNP calling and SNP filtering we identified a SNP in the expected ATP2C1 gene and SNPs in further ATPase genes. Verification in all 3 affected family members revealed a heterozygous frameshift deletion at position 2355_2358 in exon 24 of ATP2C1 in all three patients. 7 additional SNPs in 4 ATPase genes (ATP9B, ATP11A, ATP2B3 and ATP13A5) were identified. The SNPs rs138177421 in the ATP9B gene and rs2280268 in the ATP13A5 gene were detected in all 3 affected, but not in 2 non affected family members. The SNPs in the ATP2B3 and ATP11A gene as well as further SNPs in the ATP13A5 gene could not be confirmed in all affected family members. One may speculate that besides the level of functional hSPCA1 protein, levels of other ATPase proteins may influence expressivity of the disease and might also contribute, as in this case, to atypical presentations

Conservation scores and frequencies of SNPs in ATPase genes found in a Greek Family with atypical Hailey Hailey disease.

<p>Gene = Gene name for variant, ExonicFunc = synonymous, non-synonymous, indel, etc., SNV = single nucleotide variant, Aminoacid Change = variant change in nucleotide and protein format, ESP5400_ALL = MAF in Exome Sequencing Project dataset (5,400 exomes) for all populations, 1000genomes_ALL = MAF in 1000Genomes February 2012 release, dbSNP135 = RS# from the dbSNP database, AVSIFT = SIFT Pathogenicity score: closer to 0 is more damaging, LJB_PhyloP = Pathogenicity score from dbNSFP: conserved > 0.95, not conserved < 0.95, LJB_SIFT = Pathogenicity score from dbNSFP: tolerated < 0.95, deleterious > 0.95, LJB_PolyPhen2 = Pathogenicity score from dbNSFP: probably damaging > 0.85, possibly damaging 0.85–0.15, benign < 0.15, NA = not available.</p><p>Conservation scores and frequencies of SNPs in ATPase genes found in a Greek Family with atypical Hailey Hailey disease.</p

Clinical picture, pedigree, and electropherogram of the Hailey-Hailey disease (HHD) family.

<p>Erythematous slightly scaling vesicles, papules, and plaques on the right breast, shoulder, and neck of HHD-affected 1 (<b>a</b>). Pedigree of the HHD family. Black squares represent affected males (aged 70, 39 and 35 years), white squares unaffected males (aged 4, 9 and 9 years), white circles represent unaffected women, icons with a crossing line represent family members that had passed away (<b>b</b>). ATP2C1 mutation of HHD-affected 2 shown by electropherogram at position 2355_2358 in exon 24 resulting in subsequent frameshift displayed by heterozygous mismatch (<b>c</b>) Electropherogram of the unaffected HHD- healthy 1displaying the reference allele (<b>d</b>).</p

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 κ 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 κ, 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

Carboxypeptidase E Modulates Intestinal Immune Homeostasis and Protects against Experimental Colitis in Mice

<div><p>Enteroendocrine cells (EEC) produce neuropeptides, which are crucially involved in the maintenance of the intestinal barrier. Hence, EEC dysfunction is suggested to be involved in the complex pathophysiology of inflammatory bowel disease (IBD), which is characterized by decreased intestinal barrier function. However, the underlying mechanisms for EEC dysfunction are not clear and suitable models for a better understanding are lacking. Here, we demonstrate that Carboxypeptidase E (CPE) is specifically expressed in EEC of the murine colon and ileum and that its deficiency is associated with reduced intestinal levels of Neuropeptide Y (NPY) and Peptide YY (PYY), which are both produced by EEC. Moreover, <i>cpe^−/−</i> mice exhibit an aggravated course of DSS-induced chronic colitis compared to wildtype littermates. In addition, we observed elevated mucosal IL-6 and KC transcript levels already at baseline conditions in <i>cpe^−/−</i> mice. Moreover, supernatants obtained from isolated intestinal crypts of <i>cpe^−/−</i> mice lead to increased IL-6 and KC expression in MODE-K cells in the presence of LPS. This effect was reversible by co-administration of recombinant NPY, suggesting a CPE mediated immunosuppressive effect in the intestines by influencing the processing of specific neuropeptides. In this context, the chemotaxis of bone marrow derived macrophages towards respective supernatants was enhanced. In conclusion, our data point to an anti-inflammatory role of CPE in the intestine by influencing local cytokine levels and thus regulating the migration of myeloid immune cells into the mucosa. These findings highlight the importance of EEC for intestinal homeostasis and propose EEC as potential therapeutic targets in IBD.</p></div

Routine detection of serum anti-desmocollin autoantibodies is only useful in patients with atypical pemphigus

Autoantibodies against the 3 desmocollin (Dsc; Dsc1-Dsc3) isoforms have been described in different pemphigus variants. Here, we developed state-of-the-art detection systems for serum anti-Dsc1, Dsc2 and Dsc1 IgG and IgA. These assays were applied in 5 different cohorts including pemphigus vulgaris (PV) patients with compatible direct immunofluorescence (IF) microscopy but no reactivity against desmogleins 1 and 3 (n = 24) and sera from patients with autoimmune blistering diseases with positive direct IF microscopy taken at the time of diagnosis (n = 749). We found that detection of anti-Dsc serum reactivity is not helpful in the routine diagnosis of PV, pemphigus foliaceus and paraneoplastic pemphigus but may be valuable in pemphigus vegetans