Immunologische Erkrankungen und Immuntherapie in der dermatologischen Praxis

Die chronisch entzündlichen Immundermatosen (Psoriasis mit und ohne Arthritis, Atopische Dermatitis, Urtikaria) stellen einen wichtigen und prozentual großen Anteil an den Krankheitsbildern in einer dermatologischen Praxis dar. Zur Optimierung der Versorgung der Patienten mit immun-dermatologischen Erkrankungen führen wir seit 2019 eine Spezialimmunsprechstunde durch. Hier stellen sich Patienten mit den oben genannten Erkrankungen, aber auch mit Akne inversa, bullösen Dermatosen, mit Hauterscheinungen im Rahmen oder wegen Nebenwirkungen von Therapien bei Autoimmunkrankheiten anderer Organsysteme (zum Beispiel Myasthenia Gravis, SLE, Morbus Sjögren, Multiple Sklerose), mit Immunmangelerkrankungen oder Hyposensibilisierung-Initiierung vor. Immunmodulierende Therapien mit Biologika (monoklonale Antikörper) sowie mit klein-molekularen Arzneimitteln, wie zum Beispiel JAK-Inhibitoren, zeigen ein günstiges Wirkungs-Sicherheits-Verhältnis und werden in unserer Praxis als therapeutisches Prinzip breit eingesetzt. Neben der immunologischen Diagnostik und den Immuntherapien gehört auch der Einsatz von relevanten Impfstoffen zum Gebiet der Immundermatologie. Gerade im Zusammenhang mit der SARS-CoV-2-Infektion und der Covid-19 Erkrankung ergaben sich bei unseren Patienten zahlreiche, häufig immunologische Fragen. Das Management der Therapie entzündlicher Immundermatosen mit immunmodulierenden Biologika ist komplex und umfasst die saubere Indikationsstellung, das Befolgen eines Therapiealgorithmus, das Erkennen und Diagnostizieren von potenziellen Nebenwirkungen sowie die Foto- und schriftliche Dokumentation. Dies in den Alltag einer dermatologischen Praxis organisatorisch und vor allem auch wirtschaftlich einzubetten stellt eine große Herausforderung dar. Die Zusammenarbeit der Praxis mit der Klinik ist, besonders auf dem Gebiet der Immuntherapie und in Zeiten der Corona-Pandemie, von essenzieller Bedeutung. Ein Teil unserer Patienten wurde aus Studien rekrutiert, die an Kliniken durchgeführt wurden. Durch die immunmodulierende Behandlung von mehr als 200 Patienten unter Praxisbedingungen innerhalb von zwei Jahren, haben wir reichlich Erfahrungen gesammelt. Diese werden in dieser Arbeit zusammengefasst unter Nutzung von 8 eigenen Publikationen (2 Erstautorin, 6 Mitautorin). Die klinischen Daten wurden retrospektiv analysiert. Es werden Therapieverläufe vorgestellt und Schlussfolgerungen gezogen. Mit der vorgelegten Arbeit soll ein Beitrag geleistet werden zur Anwendung von modernen Immuntherapien in der dermatologischen Praxis

ESRRB Is a Pivotal Target of the Gsk3/Tcf3 Axis Regulating Embryonic Stem Cell Self-Renewal

博士（医学）神戸大

Complementary Activity of ETV5, RBPJ, and TCF3 Drives Formative Transition from Naive Pluripotency.

The gene regulatory network (GRN) of naive mouse embryonic stem cells (ESCs) must be reconfigured to enable lineage commitment. TCF3 sanctions rewiring by suppressing components of the ESC transcription factor circuitry. However, TCF3 depletion only delays and does not prevent transition to formative pluripotency. Here, we delineate additional contributions of the ETS-family transcription factor ETV5 and the repressor RBPJ. In response to ERK signaling, ETV5 switches activity from supporting self-renewal and undergoes genome relocation linked to commissioning of enhancers activated in formative epiblast. Independent upregulation of RBPJ prevents re-expression of potent naive factors, TBX3 and NANOG, to secure exit from the naive state. Triple deletion of Etv5, Rbpj, and Tcf3 disables ESCs, such that they remain largely undifferentiated and locked in self-renewal, even in the presence of differentiation stimuli. Thus, genetic elimination of three complementary drivers of network transition stalls developmental progression, emulating environmental insulation by small-molecule inhibitors.This research was funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, European Commission (contract no. 200720, EuroSyStem) and the Louis Jeantet Foundation. The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council. AS is a Medical Research Council Professor

Single‐cell molecular profiling provides a high‐resolution map of basophil and mast cell development

Funder: Karolinska InstitutetFunder: Magnus Bergvall FoundationFunder: Lars Hierta Memorial FoundationFunder: Swedish Cancer SocietyFunder: Åke Wiberg FoundationAbstract: Background: Basophils and mast cells contribute to the development of allergic reactions. Whereas these mature effector cells are extensively studied, the differentiation trajectories from hematopoietic progenitors to basophils and mast cells are largely uncharted at the single‐cell level. Methods: We performed multicolor flow cytometry, high‐coverage single‐cell RNA sequencing analyses, and cell fate assays to chart basophil and mast cell differentiation at single‐cell resolution in mouse. Results: Analysis of flow cytometry data reconstructed a detailed map of basophil and mast cell differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of peritoneal basophil‐mast cell progenitors. Conclusions: A combination of molecular and functional profiling of bone marrow and peritoneal cells provided a detailed road map of basophil and mast cell development. An interactive web resource was created to enable the wider research community to explore the expression dynamics for any gene of interest

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.This work was supported by grants from Bloodwise, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, the National Institute for Health Research Cambridge Biomedical Research Centre, and core support grants by Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. S.N. and F.K.H. are recipients of Medical Research Council PhD studentships. D.G.K. is the recipient of a Bennett Fellowship from Bloodwise, and E.L. is the recipient of a Sir Henry Dale Fellowship from the Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-05-71648

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.

Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.Work in the author’s laboratory is supported by grants from the Leukaemia and Lymphoma Research, the Medical Research Council, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, and the National Institute for Health Research Cambridge Biomedical Research Centre and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute. D.G.K. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship. F.B. and F.J.T. are funded by the European Research Council (starting grant “LatentCauses”). For funding for the open access charge, the core support grant was provided by the Wellcome Trust-MRC Cambridge Stem Cell Institute. We acknowledge the support of the University of Cambridge, Cancer Research UK Institute (core grant C14303/A17197), and Hutchison Whampoa Limited.This is the final published version. It first appeared at http://www.cell.com/cell-stem-cell/abstract/S1934-5909%2815%2900162-9

Gene set control analysis predicts hematopoietic control mechanisms from genome-wide transcription factor binding data

Transcription factors are key regulators of both normal and malignant hematopoiesis. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) has become the method of choice to interrogate the genome-wide effect of transcription factors. We have collected and integrated 142 publicly available ChIP-Seq datasets for both normal and leukemic murine blood cell types. In addition, we introduce the new bioinformatic tool Gene Set Control Analysis (GSCA). GSCA predicts likely upstream regulators for lists of genes based on statistical significance of binding event enrichment within the gene loci of a user-supplied gene set. We show that GSCA analysis of lineage-restricted gene sets reveals expected and previously unrecognized candidate upstream regulators. Moreover, application of GSCA to leukemic gene sets allowed us to predict the reactivation of blood stem cell control mechanisms as a likely contributor to LMO2 driven leukemia. It also allowed us to clarify the recent debate on the role of Myc in leukemia stem cell transcriptional programs. As a result, GSCA provides a valuable new addition to analyzing gene sets of interest, complementary to Gene Ontology and Gene Set Enrichment analyses. To facilitate access to the wider research community, we have implemented GSCA as a freely accessible web tool (http://bioinformatics.cscr.cam.ac.uk/GSCA/GSCA.html)

Decoding the regulatory network of early blood development from single-cell gene expression measurements.

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.We thank J. Downing (St. Jude Children's Research Hospital, Memphis, TN, USA) for the Runx1-ires-GFP mouse. Research in the authors' laboratory is supported by the Medical Research Council, Biotechnology and Biological Sciences Research Council, Leukaemia and Lymphoma Research, the Leukemia and Lymphoma Society, Microsoft Research and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute. V.M. is supported by a Medical Research Council Studentship and Centenary Award and S.W. by a Microsoft Research PhD Scholarship.This is the accepted manuscript for a paper published in Nature Biotechnology 33, 269–276 (2015) doi:10.1038/nbt.315

Dtudy of the expression of oxidative and endoplasmic reticulum stress proteins in the pulp and the dentin after exposure to caries, mechanical trauma and resinous monomers

The goal of this study was to investigate the role of oxidative and endoplasmic reticulum (ER) stress in the process of reparative dentinogenesis which characterizes the reaction of the dentin-pulp complex to stimuli such as caries, the mechanical trauma caused by a cavity preparation and the application of resinous restorative materials. To achieve this goal, the expression of thioredoxin (Trx1) and its reductase (TrxR1), members of an important antioxidant system, as well as the expression of ERdj5, a protein-marker of ER stress, was studied by immunohistochemistry in paraffin sections of human teeth with caries, a cavity preparation and a filling with a resinous material or fully developed impacted third molars (control teeth). The expression of the proteins was also studied in primary human pulp cell cultures before and after incubation with low concentrations (10, 50, 100μΜ) of HEMA, a monomer released from resinous materials, for 15 minutes, 1 hour and 4 hours. Finally, this study investigated under similar experimental conditions, the expression of dentin sialoprotein (DSP), a protein indispensable for the mineralization of reparative dentin, in combination with ERdj5, by Western Blot and immunofluorescence. The results suggest that mechanisms related to oxidative and ER stress are involved in the process of reparative dentinogenesis. In teeth filled with a resinous material, an increased expression of thioredoxins was observed in the odontoblasts in the reparative dentin area. The expression of ERdj5 was also intense in the same area, mainly in the odontoblastic processes. In teeth with caries, an intense ERdj5 expression was observed in the odontoblasts and the dentinal tubules under the lesion and also in the adjacent pulp cells. In primary human pulp cell cultures, incubation with HEMA did not seem to affect the thioredoxin expression. On the contrary, while under normal culture conditions, ERdj5 expression was covering a large area of the cytoplasm, after incubation with HEMA for one hour, its expression increased and the protein was accumulated in the ER, a finding confirmed by Western Blot. Under similar conditions, DSP expression remained stable but immunofluorescence revealed an obvious relocalization of the protein from the cytoplasm and its accumulation in the ER. These results suggest that a part of the cytotoxicity of low HEMA concentrations in primary human pulp cell cultures may be related to the induction of ER stress. Moreover, the inhibition of mineralization observed under the impact of monomers seems to be related to a possible inhibition of the secretion of DSP, a key protein in reparative dentinogenesis, because of its retainment into the ER.Στόχος της μελέτης αυτής ήταν να διερευνηθεί ο ρόλος του οξειδωτικού στρες και του στρες του ενδοπλασματικού δικτύου (ER) στη διαδικασία της επανορθωτικής οδοντινογένεσης που χαρακτηρίζει την αντίδραση του συμπλέγματος οδοντίνης-πολφού σε ερεθίσματα όπως η τερηδόνα, το μηχανικό τραύμα από την παρασκευή κοιλότητας και η εφαρμογή ρητινωδών επανορθωτικών υλικών. Για το σκοπό αυτό μελετήθηκε αρχικά με ανοσοϊστοχημεία η έκφραση της θειορεδοξίνης (Trx1) και της αναγωγάσης της (TrxR1), πρωτεϊνών ενός σημαντικού αντιοξειδωτικού συστήματος, καθώς και της ERdj5, πρωτεΐνης-δείκτη του ER στρες, σε τομές παραφίνης ανθρώπινων δοντιών με τερηδόνα, παρασκευή κοιλότητας και έμφραξη με ένα ρητινώδες υλικό ή ακέραιων έγκλειστων τρίτων γομφίων (δόντια ελέγχου). Η έκφραση των πρωτεϊνών μελετήθηκε επίσης σε πρωτογενή καλλιέργεια ανθρώπινων πολφικών κυττάρων πριν και μετά την έκθεσή τους σε χαμηλές συγκεντρώσεις (10, 50, 100μM) του μονομερούς HEMA που απελευθερώνεται από τα ρητινώδη υλικά, για χρονικά διαστήματα 15 λεπτών, 1 ώρας και 4 ωρών. Μελετήθηκε τέλος στις ίδιες συνθήκες η έκφραση της οδοντινικής σιαλοπρωτεΐνης (DSP), πρωτεΐνης με καθοριστικό ρόλο στην ενασβεστίωση της επανορθωτικής οδοντίνης, σε συνδυασμό με την ERdj5, με Western Blot και ανοσοφθορισμό. Τα αποτελέσματα της μελέτης έδειξαν ότι μηχανισμοί που σχετίζονται με το οξειδωτικό και το ER στρες εμπλέκονται πιθανά στη διαδικασία της επανορθωτικής οδοντινογένεσης. Σε δόντια που είχαν εμφραχθεί με ρητινώδες υλικό, παρατηρήθηκε αυξημένη έκφραση των θειορεδοξινών στους οδοντινοβλάστες στην περιοχή της επανορθωτικής οδοντίνης, ενώ έντονη ήταν στην ίδια περιοχή και η έκφραση της ERdj5, κυρίως στις οδοντινοβλαστικές αποφυάδες. Σε δόντια με τερηδόνα, η έκφραση της ERdj5 ήταν έντονη στα οδοντινοσωληνάρια και στους οδοντινοβλάστες στην περιοχή της βλάβης, καθώς και στα παρακείμενα πολφικά κύτταρα. Στις πρωτογενείς καλλιέργειες ανθρώπινων πολφικών κυττάρων, η επώαση με HEMA δεν είχε κάποια επίπτωση στην έκφραση των θειορεδοξινών. Αντίθετα, ενώ κάτω από φυσιολογικές συνθήκες η έκφραση της ERdj5 φαινόταν διάχυτη στο κυτταρόπλασμα, μία ώρα μετά την επώαση με HEMA, παρατηρήθηκε αύξηση στην έκφρασή της, η οποία επιβεβαιώθηκε με το Western Blot, και συγκέντρωσή της με τη μορφή συσσωματωμάτων στο ER. Στο ίδιο χρονικό διάστημα, τα επίπεδα της DSP παρέμειναν σταθερά αλλά παρατηρήθηκε σαφής μετατόπιση της έκφρασής της από το κυτταρόπλασμα στο ER. Ενδέχεται επομένως η κυτταροτοξικότητα του ΗΕΜΑ να οφείλεται κατά ένα μέρος στην πρόκληση ER στρες στα πολφικά κύτταρα. Ειδικά η αναστολή της ενασβεστίωσης που προκαλούν τα μονομερή μπορεί να σχετίζεται με πιθανή αναστολή της έκκρισης πρωτεϊνών που συμμετέχουν στη διαδικασία της επανορθωτικής οδοντινογένεσης όπως η DSP, λόγω κατακράτησής τους μέσα στο ER, σαν αποτέλεσμα της ακολουθίας των αντιδράσεων που προκαλείται από την ενεργοποίηση του ER στρες