Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice

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1. Verfasser: Schneeweiß, Anna Maria Birthe 1991- (VerfasserIn)
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Sprache:English
Veröffentlicht: Münster 2018
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adam_text TABLE OF CONTENTS 1 INTRODUCTION.............................................................................................................................. 1 1.1 PSORIASIS.............................................................................................................................. 1 1.1.1 CLINICAL PICTURE.........................................................................................................2 1.1.2 SKIN PATHOLOGY AND HISTOLOGICAL HALLMARKS OF PSORIATIC PLAQUES ........................ 2 1.1.3 GENETIC PREDISPOSITION & ENVIRONMENTAL FACTORS ................................................. 4 1.1.4 IMMUNOPATHOGENESIS O F PSORIASIS .......................................................................... 5 1.1.5 ROLE O F KERATINOCYTES FOR DISEASE INITIATION & PROGRESSION .................................. 8 1.1.6 ROLE O F NEUTROPHILS FOR DISEASE INITIATION & PROGRESSION .................................. 10 1.1.7 THERAPEUTIC OPTIONS............................................................................................... 11 1.1.8 MOUSE MODELS O F PSORIASIS....................................................................................14 1.1.8.1 IMIQUIMOD-INDUCED PSORIASIS-LIKE SKIN INFLAMMATION ............................... 14 1.2 BACTERIAL EFFECTOR PROTEINS.............................................................................................. 16 1.2.1 THE YERSINIA OUTER PROTEIN M ............................................................................... 16 1.3 OBJECTIVES........................................................................................................................ 20 2 MATERIALS & METHODS............................................................................................................ 22 2.1 MATERIALS...........................................................................................................................22 2.1.1 EQUIPMENT................................................................................................................22 2.1.2 DISPOSABLES............................................................................................................. 23 2.1.3 PURCHASED CHEMICALS & KITS.................................................................................. 24 2.1.4 MEDIA & BUFFERS..................................................................................................... 25 2.1.5 PRIMARY ANTIBODIES................................................................................................. 27 2.1.6 SECONDARY ANTIBODIES............................................................................................. 28 2.1.7 PRIMERS......................................................................................................... 28 2.1.8 MOUSE LINES............................................................................................................. 28 2.1.9 SOFTWARE...................................................................................................................29 2.2 METHODS............................................................................................................................30 2.2.1 EXPERIMENTAL ANIMALS & HOUSING.........................................................................30 2.2.2 IN VIVO IMIQUIMOD-INDUCED PSORIASIS-LIKE SKIN INFLAMMATION MODEL ............ 30 2.2.3 SUBCUTANEOUS TREATMENT WITH RYOPM AND ITS VARIANTS ..................................... 30 2.2.4 EPICUTANEOUS TREATMENT WITH RYOPM...................................................................32 2.2.5 EVALUATION O F DISEASE SEVERITY..............................................................................33 2.2.6 FLOW CYTOMETRIC ANALYSIS O F IMMUNE CELLS.........................................................34 2.2.6.1 IN SKIN DRAINING LYMPH NODES.........................................................................34 2.2.6.2 IN LESIONAL SK IN .................................... . ........................................................ 34 2.2.7 HISTOLOGICAL EXAMINATION......................................................................................35 2.2.7.1 HEMATOXYLIN & EOSIN STAINING ...................................................................... 35 2.2.7.2 IMMUNOFLUORESCENT STAINING........................................................................... 35 2.2.7.3 TUNEL STAINING.............................................................................................. 35 2.2.7.4 FAM-FLICA STAINING O F ACTIVE CASPASE-1...................................................36 2.2.8 RNA EXTRACTION.......................................................................................................36 2.2.9 QUALITY CONTROL O F RN A .........................................................................................36 2.2.10 CDNA SYNTHESIS AND QUANTITATIVE REAL-TIME PCR ............................................... 37 2.2.11 RNA-SEQ ANALYSIS.................................................................................................. 37 2.2.12 PROTEIN ANALYSIS.......................................................................................................38 2.2.12.1 EXTRACTION O F TOTAL SKIN PROTEIN.....................................................................38 2.2.12.2 TOTAL PROTEIN QUANTIFICATION BY BCA ASSAY ................................................ 38 2.2.12.3 SDS-PAGE & WESTERN B LO T ........................................................................ 39 2.2.12.4 LEGENDPLEX*.............................................................................................40 2.2.13 CELL CULTURE O F PRIMARY CELLS..................................................................................41 2.2.13.1 ISOLATION OF PRIMARY NEUTROPHILS FROM BONE MARROW.................................41 2.2.13.2 COATING O F CELL CULTURE PLATES FOR KERATINOCYTE CULTURE .............................. 42 2.2.13.3 ISOLATION OF PRIMARY KERATINOCYTES ................................................................ 42 2.2.13.4 TREATMENT O F PRIMARY CELLS WITH RYOPM ..................................................... 42 2.2.13.5 CYTOSPINS......................................................................................................... 43 2.2.14 HUMAN SKIN SAMPLES...............................................................................................43 2.2.15 STATISTICAL ANALYSIS.................................................................................................. 43 3 RESULTS.................................................................................................................................... 44 3.1 SUBCUTANEOUSLY INJECTED RYOPM REDUCES CLINICAL SYMPTOMS O F PSORIASIS-LIKE SKIN INFLAMMATION IN M ICE................................................................................................................ 44 3.2 EPICUTANEOUS TREATMENT WITH RYOPM.......................................................................... 46 3.2.1 TOPICAL RYOPM TREATMENT INFLUENCES KERATINOCYTE ACTIVATION .......................... 51 3.2.2 TREATMENT WITH TOPICAL RYOPM HAS A MINOR EFFECT ON T C E LLS ......................... 53 3.2.3 TOPICALLY APPLIED RYOPM REDUCES NEUTROPHIL INFILTRATION IN LESIONAL SKIN.... 55 3.3 TOPICALLY APPLIED RYOPM PENETRATES HUMAN SKIN .................................................... 59 3.4 THE FIRST THREE LRRS MEDIATE DISEASE AMELIORATION .................................................. 60 3.4.1 INFLUENCE OF RYOPM ON KERATINOCYTES...................................................................64 3.4.2 MARGINAL ROLE O F T CELLS DURING RYOPM TREATMENT.............................................69 3.4.3 REDUCED NUMBERS O F NEUTROPHILS IN LESIONAL SKIN IN WT MICE ........................ 75 3.4.4 RNA-SEQ ANALYSIS.................................................................................................. 79 4 DISCUSSION............................................................................................................................... 93 4.1 RECOMBINANT YOPM AND YOPMUUU -3 SIMILARLY AMELIORATE ONGOING SKIN- INFLAMMATION WHEN INJECTED SUBCUTANEOUSLY ......................................................................... 93 4.1.1 POTENTIAL MECHANISM O F THE ANTI-INFLAMMATORY CAPACITY .................................. 99 4.1.2 ROLE O F T CELLS DURING RY OPM TREATMENT...........................................................102 4.1.3 EFFECT O F RY OPM ON KERATINOCYTES ..................................................................... 104 4.1.4 EFFECT O F RYOPM ON NEUTROPHILS.........................................................................109 4.2 POSSIBLE TRANSFER TO THE HUMAN PATIENT...................................................................... 111 5 CONCLUSION & OUTLOOK.........................................................................................................117
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spelling Schneeweiß, Anna Maria Birthe 1991- Verfasser (DE-588)1178714942 aut
Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice vorgelegt von Anna Maria Birthe Schneeweiß, M. Sc.
Münster 2018
119, XXXVII Blätter Illustrationen 30 cm 1 CD-ROM
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Dissertation Westfälische-Wilhelms-Universität Münster 2019 Beschränkt für den Austausch
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spellingShingle Schneeweiß, Anna Maria Birthe 1991-
Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice
subject_GND (DE-588)4113937-9
title Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice
title_auth Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice
title_exact_search Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice
title_full Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice vorgelegt von Anna Maria Birthe Schneeweiß, M. Sc.
title_fullStr Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice vorgelegt von Anna Maria Birthe Schneeweiß, M. Sc.
title_full_unstemmed Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice vorgelegt von Anna Maria Birthe Schneeweiß, M. Sc.
title_short Truncated rYopM reduces keratinocyte activation and neutrophil infiltration in ongoing IMQ-induced psoriasiform dermatitis in mice
title_sort truncated ryopm reduces keratinocyte activation and neutrophil infiltration in ongoing imq induced psoriasiform dermatitis in mice
topic_facet Hochschulschrift
url http://d-nb.info/1178343456/04
http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031609979&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
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