Einfluss unterschiedlicher Dosierungen einer Therapie mit dem Granulozyten Kolonie-stimulierenden Faktor auf die frühe Entzündungsreaktion im murinen Schlaganfallmodell

Die erfolgreiche Übertragung der vielversprechenden Ergebnisse aus zahlreichen Kleintierstudien zu den neuroprotektiven und -regenerativen Eigenschaften des Wachstumsfaktors G-CSF auf eine multizentrische randomisierte Phase-IIa-Studie misslang. Bei näherer Betrachtung fiel eine deutlich höhere Dosierung des Wachstumsfaktors in der humanen Studie im Vergleich zu den erfolgreichen präklinischen Studien auf, sodass Fragen nach einem schädigenden Einfluss möglicher immunologischer Veränderungen aufkamen.Um dies zu überprüfen, wurde in einem murinen Schlaganfallmodell eine quantitative FACS-Analyse des frühen entzündlichen Hirninfiltrats und der Immunzellpopulationen im peripheren Blut mit besonderem Augenmerk auf neutrophile Granulozyten 24 Stunden nach (Schein-)Schlaganfall durchgeführt. Ergänzend erfolgte eine immun-histochemische Beurteilung des Infiltrationsverhaltens neutrophiler Granulozyten, wobei neben der räumlichen Verteilung die Zellmorphologie untersucht wurde.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Der Schlaganfall 1 1.1.1 Epidemiologie und sozioökonomische Relevanz 1 1.1.2 Ätiologie und Pathogenese 1 1.1.3 Klinische Symptomatik 2 1.1.4 Rekanalisierende Therapie 2 1.2 Immunologie des Schlaganfalls 3 1.2.1 Das Gehirn als immunprivilegiertes Organ 3 1.2.2 Immunantwort nach Schlaganfall 3 1.2.3 Bedeutung neutrophiler Granulozyten für die Immunantwort 6 1.2.4 Rolle des neutrophilen Granulozyten im Schlaganfall 7 1.3 Der Granulozyten Kolonie-stimulierende Faktor 10 1.3.1 G-CSF im Schlaganfall 10 1.3.2 Einführung 12 1.3.3 Präklinische Schlaganfallstudien mit G-CSF 14 1.3.4 klinische Schlaganfallstudien mit G-CSF 14 1.4 Dosistranslation von experimentellen zu klinischen Studien 16 2 Zielstellung der Arbeit 17 3 Material und Methoden 18 3.1 Versuchstiere 18 3.2 Schlaganfallmodell 18 3.3 Ein- und Ausschlusskriterien 19 3.4 Versuchsplan 20 3.5 Behandlung mit G-CSF 21 3.6 Euthanasie und transkardiale Perfusion 22 3.7 Quantifizierung der Leukozytenzahl im Vollblut 23 3.8 Durchflusszytometrische Aufarbeitung 23 3.8.1 Lyse- und Färbeprotokoll Blut 23 3.8.2 Isolation von Immunzellen aus dem Maushirn und Färbeprotokoll 24 3.9 Durchflusszytometrische Analyse 25 3.10 Histopathologische Aufarbeitung 26 3.10.1 Anfertigung histologischer Schnitte im Kryostat 26 3.10.2 Hämatoxylin-Eosin-Färbung 27 3.10.3 Immunfluoreszenz 27 3.10.4 Differenzierung von Granulozyten und Monozyten 29 3.10.5 Immunhistochemische Auszählung 29 3.10.6 Darstellung der räumlichen Verteilung 31 3.11 Statistische Auswertung 33 4 Ergebnisse 34 4.1 Durchflusszytometrie 34 4.1.1 Veränderungen des Körper- und Milzgewichts 34 4.1.2 Immunzellpopulationen im peripheren Blut 35 4.1.3 Immunzellpopulationen im Hirnisolat 38 4.2 Immunhistochemie 42 4.2.1 Differenzierung von Monozyten und Granulozyten 42 4.2.2 Quantitative Analyse 43 4.2.2.1 Ipsilateral / kontralateral 44 4.2.2.2 Meningeal / intrazerebral 45 4.2.2.3 Rund / stäbchenförmig 46 4.2.3 Räumliche Verteilung der neutrophilen Granulozyten 47 5 Diskussion 51 5.1 Zusammenfassung der Ergebnisse 51 5.2 G-CSF und die Einwanderung neutrophiler Granulozyten 53 5.3 G-CSF und die Zusammensetzung des frühen entzündlichen Infiltrats im Gehirn 54 5.4 G-CSF und die räumliche Verteilung einwandernder neutrophiler Granulozyten 56 5.5 G-CSF und die Zellmorphologie einwandernder neutrophiler Granulozyten 58 5.6 G-CSF und die periphere bzw. systemische Immunantwort 60 5.7 Methodische Einflussfaktoren und Limitationen der Arbeit 63 5.7.1 Berechnung der murinen Äquivalenzdosis 63 5.7.2 Wahl des Schlaganfallmodells 64 5.7.3 Immunologische Unterschiede zwischen Maus und Mensch 65 5.7.4 Methoden und Versuchstieranzahl 66 5.7.4.1 Durchflusszytometrie 66 5.7.4.2 Immunhistochemie 67 5.8 Ausblick 68 6 Zusammenfassung 70 Literaturverzeichnis XI Erklärung über die eigenständige Abfassung der Arbeit XXVII Tabellarischer Lebenslauf XXVIII Publikationen XXIX Danksagung XX

Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions

Body satisfaction and physical appearance in gender dysphoria

Gender dysphoria (GD) is often accompanied by dissatisfaction with physical appearance and body image problems. The aim of this study was to compare body satisfaction with perceived appearance by others in various GD subgroups. Data collection was part of the European Network for the Investigation of Gender Incongruence. Between 2007 and 2012, 660 adults who fulfilled the criteria of the DSM-IV gender identity disorder diagnosis (1.31:1 male-to-female [MtF]:female-to-male [FtM] ratio) were included into the study. Data were collected before the start of clinical gender-confirming interventions. Sexual orientation was measured via a semi-structured interview whereas onset age was based on clinician report. Body satisfaction was assessed using the Body Image Scale. Congruence of appearance with the experienced gender was measured by means of a clinician rating. Overall, FtMs had a more positive body image than MtFs. Besides genital dissatisfaction, problem areas for MtFs included posture, face, and hair, whereas FtMs were mainly dissatisfied with hip and chest regions. Clinicians evaluated the physical appearance to be more congruent with the experienced gender in FtMs than in MtFs. Within the MtF group, those with early onset GD and an androphilic sexual orientation had appearances more in line with their gender identity. In conclusion, body image problems in GD go beyond sex characteristics only. An incongruent physical appearance may result in more difficult psychological adaptation and in more exposure to discrimination and stigmatization

Freshwater gobies 30 million years ago: New insights into character evolution and phylogenetic relationships of dagger Pirskeniidae (Gobioidei, Teleostei)

The modern Gobioidei (Teleostei) comprise eight families, but the extinct dagger Pirskeniidae from the lower Oligocene of the Czech Republic indicate that further families may have existed in the past. However, the validity of the dagger Pirskeniidae has been questioned and its single genus dagger Pirskeniushas been assigned to the extant family Eleotridae in previous works. The objective of this study is to clarify the status of the dagger Pirskeniidae. Whether or not the dagger Pirskeniidae should be synonymised with the Eleotridae is also interesting from a biogeographical point of view as Eleotridae is not present in Europe or the Mediterranean Sea today. We present new specimens and re-examine the material on which the two known species of dagger Pirskeniusare based (dagger P.diatomaceusObrhelova, 1961;dagger P.radoniPrikryl, 2014). To provide a context for phylogenetically informative characters related to the palatine and the branchiostegal rays, three early-branching gobioids (Rhyacichthys,Protogobius,Perccottus), an eleotrid (Eleotris) and a gobiid (Gobius) were subjected to micro-CT analysis. The new data justify revalidation of the family dagger Pirskeniidae, and a revised diagnosis is presented for both dagger Pirskeniusand dagger Pirskeniidae. Moreover, we provide for the first time an attempt to relate a fossil gobioid to extant taxa based on phylogenetic analysis. The results indicate a sister-group relationship of dagger Pirskeniidae to the Thalasseleotrididae + Gobiidae + Oxudercidae clade. Considering the fossil record, the arrival of gobioids in freshwater habitats in the early Oligocene apparently had generated new lineages that finally were not successful and became extinct shortly after they had diverged. There is currently no evidence that the Eleotridae was present in the European ichthyofauna in the past

New fossil cichlid from the middle Miocene of East Africa revealed as oldest known member of the Oreochromini

A new genus and species of fossil cichlid fishes of middle Miocene age (12.5 Ma) is described from the Ngorora fish Lagerstatte (Tugen Hills, Kenya) in the East African Rift Valley. Parsimony analysis of morphological characters using published phylogenetic frameworks for extant cichlids combined with the application of a comprehensive best-fit approach based on morphology was employed to place the new fossil taxon in the phylogenetic context of the African cichlids. The data reveal that the fossil specimens can be assigned to the tribe Oreochromini within the haplotilapiines. +Oreochromimos kabchorensis gen. et sp. nov. shows a mosaic set of characters bearing many similarities to the almost pan-African Oreochromis and the East African lake-endemic Alcolapia. As the striking diversity of present-day African cichlids, with 1100 recognised species, has remained largely invisible in the fossil record, the material described here adds significantly to our knowledge of the Miocene diversity of the group. It effectively doubles the age of a fossil calibration point, which has hitherto been used to calibrate divergence times of the East African cichlids in molecular phylogenetic investigations. Furthermore, the comparative dataset derived from extant cichlids presented here will greatly facilitate the classification of fossil cichlids in future studies

Adenovirus serotype 5 E1A sensitizes tumor cells to NKG2D-dependent NK cell lysis and tumor rejection

The expression of the Adenovirus serotype 5 (Ad5) E1A oncogene sensitizes tumor cells to natural killer (NK) cell–mediated killing and tumor rejection in vivo. These effects are dependent on the ability of E1A to bind the transcriptional coadaptor protein p300. To test the hypothesis that E1A up-regulates ligands recognized by the NKG2D-activating receptor, we stably transfected the highly tumorigenic mouse fibrosarcoma cell line MCA-205 with Ad5-E1A or a mutant form of E1A that does not interact with p300 (E1A-Δp300). Ad5-E1A, but not E1A-Δp300, up-regulated the expression of the NKG2D ligand retinoic acid early inducible (RAE)-1, but not murine ULBP-like transcript 1, another NKG2D ligand, in four independently derived MCA-205 transfectants. The up-regulation of RAE-1 by E1A targeted MCA-205 tumor cells to lysis by NK cells, resulting in NKG2D-dependent tumor rejection in vivo. Moreover, the up-regulation of NKG2D ligands by E1A was not limited to mouse tumor cells, as E1A also increased the expression of NKG2D ligands on primary baby mouse kidney cells, human MB435S breast cancer cells, and human H4 fibrosarcoma cells

Factors associated with non-use of condoms among heterosexually-active single people in Germany: Results from the first representative, population-based German health and sexuality survey (GeSiD)

BACKGROUND: Against the backdrop of rising STI incidence among the heterosexual population, sexually active single people are at particularly high STI transmission risk. Gaining insight into circumstances related to condoms non-use in this population is therefore important for developing effective health interventions. METHODS: The nationally-representative survey, GeSiD (German Health and Sexuality Survey) undertaken 2018–2019, interviewed 4,955 people aged 18–75 years. A total of 343 heterosexually-active single participants answered a question about condom use at last sex. Data on sociodemographic characteristics, sexual behaviours and circumstances of last sex were analysed to identify independently associated factors. RESULTS: Condom non-use at last sex was reported more commonly by participants aged >35 years than by younger participants (48.5 vs 33.7%, respectively) and more likely among longer relationships (adjusted odds ratio [AOR]: 2.43) or early loving relationships (AOR: 3.59) than in one-night-stands. It was also associated with not discussing using condoms before sex (AOR: 6.50) and with reporting non-use of condoms at sexual debut (AOR: 4.75). CONCLUSIONS: Non-use of condoms is a common STI risk behaviour among heterosexually-active single people in Germany and so needs promoting from sexual debut throughout the life course, regardless of relationship type and age, but particularly among middle-aged and older people

Identification of a polyI:C-inducible membrane protein that participates in dendritic cell–mediated natural killer cell activation

The novel polyI:C-inducible membrane protein INAM triggers dendritic cell–mediated natural killer cell activation