TY - THES A1 - Gehrig, Andrea T1 - Untersuchungen zu den molekularen Ursachen der X-gebundenen juvenilen Retinoschisis - vom Gendefekt zum Mausmodell T1 - Molecular studies of X-linked juvenile Retinoschisis - from gene defect to the mouse model N2 - Hereditäre Netzhautdegenerationen betreffen weltweit etwa 15 Millionen Menschen. Sie sind klinisch und genetisch auffällig heterogen. Bisher wurden 139 verschiedene chromosomale Genorte mit Netzhautdystrophien assoziiert, wovon inzwischen 90 Gene identifiziert werden konnten. Mit Hilfe verschiedener Klonierungsstrategien konnte in der vorgelegten Arbeit ein Beitrag zur Aufklärung der genetischen Ursachen einiger ausgewählter Retinopathien geleistet werden. So konnte durch die Positionsklonierung das Gen, das mit der X-gebundenen juvenilen Retinoschisis (RS) assoziiert ist, identifiziert werden. Funktionelle Analysen des Genproduktes sowie die Generierung eines Mausmodells der RS geben einen Einblick in die Physiologie der Retina sowie den Pathomechanismus der Erkrankung. Die genomische Organisation des Interphotorezeptor-Matrixproteoglykans-1 (IMPG1) wurde aufgeklärt und die chromosomale Lokalisation auf 6q13-15 bestimmt. Damit kartierte das Gen in eine Region, in die die Genorte für 7 Retinopathien des Menschen kartiert wurden. Durch Kopplungs- und Mutationsanalysen konnten unsere Arbeiten ausschließen, daß IMPG1 mit North Carolina Makuladystrophie (MCDR1) oder der progressiven bifokalen chorioretinalen Atrophie (PBCRA) in Zusammenhang steht. Die Diacylglycerin Kinase-3 (DAGK3) konnte nach der Bestimmung der genomischen Organisation in die Region 3q27-28 kartiert werden. Dieser chromosomale Abschnitt deckt sich mit der chromosomalen Lokalisation der autosomal dominanten Optikusatrophie (OPA1). Auch hier konnte mit Hilfe von Mutationsanalysen ein Ausschluß des Gens erfolgen. Die X-gebundene juvenile Retinoschisis ist eine häufige Ursache juveniler Makula-degenerationen und betrifft etwa 300.000 junge Männer weltweit. Charakteristische Kennzeichen der Erkrankung sind Aufspaltungen in den inneren Netzhautschichten, die zu zystischen Veränderungen der zentralen Retina führen. Ungefähr 50 % der Patienten entwickeln auch periphere Manifestationen. Durch die Arbeit unserer und anderer Forschergruppen konnte der Krankheitslokus in einen etwa 900 kb großen Bereich auf dem kurzen Arm des X-Chromosoms (Xp22.2) kartiert werden. Durch einen Vergleich der genomischen DNA Sequenzen mit öffentlich zugänglichen ESTs (expressed sequence tags) konnte ein retinaspezifisches Transkript identifiziert werden. Es besteht aus 6 Exonen und kodiert für ein putatives 224 Aminosäuren großes Protein, das sekretiert wird und ein hochkonserviertes Discoidindomänen-Motiv enthält. Discoidindomänen sind in Zelladhäsion oder in Zell-Zell Interaktionen involviert. Mutationsanalysen in RS-Patienten bestätigten, daß es sich bei diesem Transkript um RS1, d.h. um das krankheitsassoziierte Gen der X-gebundenen juvenilen Retinoschisis handelte. Das RS1-Protein (Retinoschisin) kommt in homo-oligomeren Komplexen, die über Disulfidbrücken miteinander verbunden sind, auf der Zelloberfläche der Photorezeptoren und der Bipolaren sowie in den synaptischen Regionen der äußeren (OPL) und innere plexiformen Schicht (IPL) vor. Um die Funktion des normalen Retinoschisins zu untersuchen und um einen Einblick in die RS-Pathogenese zu bekommen, wurde nach der Charakterisierung des orthologen murinen Gens (Rs1h) eine Retinoschisin-defiziente knock-out Maus generiert. Ophthalmologische und histologische Untersuchungen der Rs1h-/Y-Maus zeigen signifikante Parallelen zu dem RS-Erkrankungsbild des Menschen. Damit stellt die Rs1h knock-out Maus ein ideales Tiermodell für die Untersuchung des zugrundeliegenden Krankheitsmechanismusses dar. So konnten wir inzwischen zeigen, daß apoptotische Prozesse zur Degeneration der Photorezeptoren führen. Gegenwärtig werden mit diesem Tiermodell erste gentherapeutische Versuche durchgeführt. Diese Arbeiten sollen Aufschluß darüber geben, ob ein Adeno-assoziierter Virus (AAV)-Transfer des RS1 Gens in die erkrankte Retina ein möglicher Therapieansatz für RS auch beim Menschen sein könnte. N2 - The World Health Organization (WHO) estimates that worldwide approximately 15 million people are affected with hereditary retinal degenerations. Retinal dystrophies are clinically and genetically heterogeneous. To date, 90 retinal disease genes have been identified and of 139 retinal disease genes the chromosomal location is known. In this study, different strategies of disease gene cloning were utilized to elucidate the underlying genetic defects of selected retinopathies. This has led to the identification of the gene associated with X-linked juvenile retinoschisis (RS) by positional cloning. Both, functional analysis of the gene product, named retinoschisin (RS1), and the generation of a mouse model for RS provide novel insight into retinal physiology and the pathomechanism of the disease. The genomic organization of the interphotoreceptor matrix proteoglycan-1 (IMPG1) was established and its chromosomal localization was identified (6q13-15). Seven different human retinal dystrophies have previously been mapped to this region on chromosome 6. A possible genetic association between IMPG1 and two retinal dystrophies, North Carolina macular dystrophy (MCDR1) and progressive bifocal chorioretinal atrophy (PBCRA), was investigated. By means of linkage studies and mutation analysis in affected patients the involvement of IMPG1 in these two different diseases was ruled out. The genomic organization of diacylglycerol kinase-3 (DAGK3) was determined and the gene locus was mapped to chromosome 3q27-28. The autosomal dominant optic atrophy (OPA1) was independently localized to the same chromosomal region. This has prompted us to investigate the role of DAGK3 in OPA1. By mutation analysis such a correlation could be excluded. X-linked juvenile retinoschisis is a common cause of juvenile macular degeneration affecting approximately 300.000 young males worldwide. The disease is characterized by a slitting of the inner retinal layers resulting in cystic degeneration of the central retina. Half of the patients also develop peripheral manifestations. Our laboratory and others localized the RS gene to a 900 kb interval on the short arm of chromosome Xp22.2. Comparison of genomic DNA sequences with publicly available expressed sequence tags (ESTs) have identified a retina-specific transcript. This novel transcript is composed of six exons that encode a 224-amino acid protein including a 23 amino acid signal peptide. Bioinformatical analysis revealed that the putative protein consists almost exclusively of a discoidin domain wich is highly conserved from slime mold to human. Discoidin domains are implicated in cell adhesion or cell-cell interactions. On the basis of mutation analysis in patients affected with RS, we confirmed that the gene indeed is responsible for RS pathology. The RS1 protein is found at the cell surfaces of photoreceptors and bipolar cells and within the synaptic regions of the outer (OPL) and the inner plexiform layers (IPL) most likely as a homo-oligomeric complex. To clarify the function of the normal RS1 and to gain insight into RS pathogenesis a mouse model deficient of the endogenous RS1 protein was generated. For these purposes the genomic organization of the murine orthologous RS1 gene (Rs1h) was identified. Ophthalmologic and histologic analysis of Rs1h-/Y mice revealed significant parallels to the human RS phenotyp. Therefore, the knock-out mouse represents an ideal model to further study the underlying disease mechanism. Recently, we showed that apoptosis is the final pathway of photoreceptor degeneration in Rs1h-/Y mice. Most importantly this mouse model can serve as proof-of-concept for gene therapy. Towards this end we are testing adeno-associated virus (AAV)-based gene transfer of RS1 into the defect murine retina. This may pave the way for a gene based future intervention in humans affected with this condition. KW - Maus KW - Retinoschisis KW - Erbkrankheit KW - Molekulargenetik KW - X-gebundene juvenile Retinoschisis KW - Gendefekt KW - Mausmodell KW - X-linked juvenile retinoschisis KW - gene defect KW - mouse model Y1 - 2003 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-7212 ER - TY - JOUR A1 - Hoesl, Christine A1 - Fröhlich, Thomas A1 - Posch, Christian A1 - Kneitz, Hermann A1 - Goebeler, Matthias A1 - Schneider, Marlon R. A1 - Dahlhoff, Maik T1 - The transmembrane protein LRIG1 triggers melanocytic tumor development following chemically induced skin carcinogenesis JF - Molecular Oncology N2 - The incidence of melanoma and nonmelanoma skin cancer has increased tremendously in recent years. Although novel treatment options have significantly improved patient outcomes, the prognosis for most patients with an advanced disease remains dismal. It is, thus, imperative to understand the molecular mechanisms involved in skin carcinogenesis in order to develop new targeted treatment strategies. Receptor tyrosine kinases (RTK) like the ERBB receptor family, including EGFR/ERBB1, ERBB2/NEU, ERBB3, and ERBB4, are important regulators of skin homeostasis and their dysregulation often results in cancer, which makes them attractive therapeutic targets. Members of the leucine‐rich repeats and immunoglobulin‐like domains protein family (LRIG1‐3) are ERBB regulators and thus potential therapeutic targets to manipulate ERBB receptors. Here, we analyzed the function of LRIG1 during chemically induced skin carcinogenesis in transgenic mice expressing LRIG1 in the skin under the control of the keratin 5 promoter (LRIG1‐TG mice). We observed a significant induction of melanocytic tumor formation in LRIG1‐TG mice and no difference in papilloma incidence between LRIG1‐TG and control mice. Our findings also revealed that LRIG1 affects ERBB signaling via decreased phosphorylation of EGFR and increased activation of the oncoprotein ERBB2 during skin carcinogenesis. The epidermal proliferation rate was significantly decreased during epidermal tumorigenesis under LRIG1 overexpression, and the apoptosis marker cleaved caspase 3 was significantly activated in the epidermis of transgenic LRIG1 mice. Additionally, we detected LRIG1 expression in human cutaneous squamous cell carcinoma and melanoma samples. Therefore, we depleted LRIG1 in human melanoma cells (A375) by CRISPR/Cas9 technology and found that this caused EGFR and ERBB3 downregulation in A375 LRIG1 knockout cells 6 h following stimulation with EGF. In conclusion, our study demonstrated that LRIG1‐TG mice develop melanocytic skin tumors during chemical skin carcinogenesis and a deletion of LRIG1 in human melanoma cells reduces EGFR and ERBB3 expression after EGF stimulation. KW - ERBB receptors KW - LRIG1 KW - melanoma KW - mouse model KW - skin carcinogenesis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238925 VL - 15 IS - 8 SP - 2140 EP - 2155 ER - TY - JOUR A1 - Leonhardt, Ines A1 - Spielberg, Steffi A1 - Weber, Michael A1 - Albrecht-Eckardt, Daniela A1 - Bläss, Markus A1 - Claus, Ralf A1 - Barz, Dagmar A1 - Scherlach, Kirstin A1 - Hertweck, Christian A1 - Löffler, Jürgen A1 - Hünniger, Kerstin A1 - Kurzai, Oliver T1 - The fungal quorum-sensing molecule farnesol activates innate immune cells but suppresses cellular adaptive immunity JF - mBio N2 - Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-\(\alpha\)] and macrophage inflammatory protein 1 alpha [MIP-1 \(\alpha\)]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. KW - human dendritic cells KW - Pseudomonas aeruginosa KW - induced apoptosis KW - cytokine production KW - biofilm formation KW - Candida albicans KW - mouse model KW - systemic candidiasis KW - oxidative stress KW - carcinoma cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143756 VL - 6 IS - 2 ER - TY - JOUR A1 - Dandekar, Thomas A1 - Fieselmann, Astrid A1 - Fischer, Eva A1 - Popp, Jasmin A1 - Hensel, Michael A1 - Noster, Janina T1 - Salmonella - how a metabolic generalist adopts an intracellular lifestyle during infection JF - Frontiers in Cellular and Infection Microbiology N2 - The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, "-omics" data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology. KW - enterica serovar Typhimurium KW - bacterial invasion KW - mouse model KW - defenses KW - regulation KW - "-omics" KW - virulence KW - Salmonella-containing vacuole (SCV) KW - metabolism KW - nitric oxide Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149029 VL - 4 IS - 191 ER - TY - JOUR A1 - Kim, Jae Ho A1 - Franck, Julien A1 - Kang, Taewook A1 - Heinsen, Helmut A1 - Ravid, Rivka A1 - Ferrer, Isidro A1 - Cheon, Mi Hee A1 - Lee, Joo-Yong A1 - Yoo, Jong Shin A1 - Steinbusch, Harry W. A1 - Salzet, Michel A1 - Fournier, Isabelle A1 - Park, Young Mok T1 - Proteome-wide characterization of signalling interactions in the hippocampal CA4/DG subfield of patients with Alzheimer's disease JF - Scientific Reports N2 - Alzheimer's disease (AD) is the most common form of dementia; however, mechanisms and biomarkers remain unclear. Here, we examined hippocampal CA4 and dentate gyrus subfields, which are less studied in the context of AD pathology, in post-mortem AD and control tissue to identify possible biomarkers. We performed mass spectrometry-based proteomic analysis combined with label-free quantification for identification of differentially expressed proteins. We identified 4,328 proteins, of which 113 showed more than 2-fold higher or lower expression in AD hippocampi than in control tissues. Five proteins were identified as putative AD biomarkers (MDH2, PCLO, TRRAP, YWHAZ, and MUC19 isoform 5) and were cross-validated by immunoblotting, selected reaction monitoring, and MALDI imaging. We also used a bioinformatics approach to examine upstream signalling interactions of the 113 regulated proteins. Five upstream signalling (IGF1, BDNF, ZAP70, MYC, and cyclosporin A) factors showed novel interactions in AD hippocampi. Taken together, these results demonstrate a novel platform that may provide new strategies for the early detection of AD and thus its diagnosis. KW - imaging mass spectrometry KW - neuron navigator 3 KW - dentate gyrus KW - growth factor KW - mouse model KW - neurotrophic factor KW - entorhinal cortex KW - factor expression KW - oxidative stress KW - memory deficits Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151727 VL - 5 IS - 11138 ER - TY - THES A1 - Krämer, Franziska T1 - Molecular and Biochemical Investigations into VMD2, the gene associated with Best Disease T1 - Molekulare und biochemische Untersuchungen zur Charakterisierung des Morbus Best Gens, VMD2 N2 - Best disease (OMIM 153700) is an early-onset, autosomal dominant maculopathy characterized by egg yolk-like lesions in the central retina. The disease gene, the vitelliform macular dystrophy gene type 2 (VMD2), encodes a 585-aa VMD2 transmembrane protein, termed bestrophin. The protein is predominantly expressed on the basolateral side of the retinal pigment epithelium (RPE) and is thought to be involved in the transport of chloride ions. Bestrophin as well as three closely related VMD2-like proteins (VMD2L1-L3) contain multiple putative transmembrane (TM) domains and an invariant tripeptide (RFP) motif in the N-terminal half of the protein. This and the tissue-restricted expression to polarized epithelial cells are typical features of the VMD2 RFP-TM family. Best disease is predominantly caused by missense mutations, clustering in four distinct „hotspots“ in the evolutionary highly conserved N-terminal region of the protein. To further augment the spectrum of mutations and to gain novel insights into the underlying molecular mechanisms, we screened VMD2 in a large cohort of affected patients. In total, nine novel VMD2 mutations were identified, raising the total number of known Best disease-related mutations from 83 to 92. Eight out of nine novel mutations are hotspot-specific missense mutations, underscoring their functional/structural significance and corroborating the dominant-negative nature of the mutations. Of special interest is a one-basepair deletion (Pro260fsX288) encoding a truncated protein with a deletion of an important functional domain (TM domain four) as well as the entire C-terminal half of bestrophin. For the first time, a nonsense mutation leading to a 50 % non-functional protein has been identified suggesting that on rare occassions Best disease may be caused by haploinsufficiency. Molecular diagnostics strongly requires a reliable classification of VMD2 sequence changes into pathogenic and non-pathogenic types. Since the molecular pathomechanism is unclear at present, the pathogenicity of novel sequence changes of VMD2 are currently assessed in light of known mutations. We therefore initiated a publicly accessible VMD2 mutation database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html) and are collecting and administrating the growing number of mutations, rare sequence variants and common polymorphisms. Missense mutations may disrupt the function of proteins in numerous ways. To evaluate the functional consequences of VMD2 mutations in respect to intracellular mislocalization and/or protein elimination, a set of molecular tools were generated. These included the establishment of an in vitro COS7 heterologous expression assay, the generation of numerous VMD2 mutations by site-directed mutagenesis as well as the development of bestrophin-specific antibodies. Surprisingly, membrane fractionation/Western blot experiments revealed no significant quantitative differences between intact and mutant bestrophin. Irrelevant of the type or location of mutation, incorporation of mutant bestrophin to the membraneous fraction was observed. Thus, impaired membrane integration may be ruled out as causative pathomechanism of Best disease consistent with a dominant-negative effect of the mutations. In a different approach, efforts were directed towards identifying and characterizing the VMD2 RFP-TM protein family in mouse. While clarification of the genomic organization of murine Vmd2 was required as basis to generate Vmd2-targeted animals (see below), the study of closely related proteins (Vmd2L1, Vmd2L2 and Vmd2L3) may provide further clues as to the function of bestrophin. For this, biocomputational as well as RT PCR analyses were performed. Moreover, the novel genes were analyzed by real time quantitative RT PCR, displaying predominant expression in testis, colon and skeletal muscle of Vmd2, Vmd2L1 and Vmd2L3 transcripts, respectively as well as in eye tissue. Interestingly, neither an ORF was determined for murine Vmd2L2 nor was the transcript present in a panel of 12 mouse tissues, suggesting that murine Vmd2L2 may represent a functionally inactive pseudogene. The murine Vmd2L3 gene, as its human counterpart, is a highly differentially spliced transcript. Finally, generating mouse models of Best disease will provide essential tools to investigate the pathophysiology of bestrophin in vivo. We have initiated the generation of two different mouse lineages, one deficient of Vmd2 (knock-out) and the other carrying a human disease-related mutation (Tyr227Asn) in the orthologous murine gene (knock-in). Genetic engineering of both constructs has been achieved and presently, four ES clones harboring the homologous recombination event (Vmd2+/-) have been isolated and are ready for the subsequent steps to generate chimeric animals. The resulting mouse lineages will represent two key models to elucidate the functional role of bestrophin in Best disease, in RPE development and physiology. N2 - Morbus Best (OMIM 153700) ist eine autosomal dominant vererbte Makulopathie mit juvenilem Beginn. Charakteristisch sind Eidotter-ähnliche Läsionen im zentralen Bereich der Retina. Das krankheitsverursachende Gen, das vitelliforme Makuladystrophie-Gen Typ 2 (VMD2), kodiert für ein 585 Aminosäuren langes Transmembranprotein. Das als Bestrophin bezeichnete Protein ist vorwiegend auf der basolateralen Seite des retinalen Pigmentepithels (RPE) exprimiert und wahrscheinlich am Transport von Chloridionen beteiligt. Bestrophin wie auch die drei eng-verwandten VMD2-ähnlichen Proteine (VMD2L1-L3) gehören zur Familie der VMD2 RFP-TM Proteine und sind durch putative Transmembrandomänen (TM) und ein invariantes Tripeptid (RFP) gekennzeichnet. Morbus Best wird hauptsächlich durch „missense“ Mutationen verursacht die in vier Bereichen („hotspots“) akkumulieren. Um das Mutationsspektrum zu erweitern und darüber hinaus den zugrundeliegenden molekularen Mechanismus weitergehend aufzuklären, wurde das VMD2 Gen in betroffenen Patienten untersucht. Insgesamt wurden neun bisher nicht beschriebene Mutationen identifiziert, wodurch sich die Anzahl der bekannten krankheitsassoziierten Mutationen auf 92 erhöhte. Wie die meisten der bisher bekannten Mutationen befinden sich acht „missense“ Mutationen in den sogenannten „hotspots“ des Gens. Dies unterstreicht die funktionelle bzw. strukturelle Bedeutung der betroffenen Regionen sowie den dominant-negativen Effekt der Mutationen. Bemerkenswert ist eine atypische 1-Basenpaar-Deletion (Pro260fsX288) in Exon 7. Denn erstmals wurde eine „nonsense“ Mutation im VMD2 Gen identifiziert, die 50 % nicht-funktionelles Protein zur Folge hat. In seltenen Fällen scheint daher die durch „nonsense“ Mutationen bedingte Haploinsuffizienz der Krankheit zugrunde liegen. Die molekulare Diagnostik verlangt eine zuverlässliche Einteilung der VMD2 Sequenzänderungen in pathogene und nicht-pathogene Gruppen. Da der molekulare Pathomechanismus zurzeit weitgehend ungeklärt ist, wird die Pathogenität neuer Sequenzänderungen aufgrund bereits bekannter Mutationen eingestuft. Es wurde daher eine öffentlich zugängliche VMD2 Mutationsdatenbank eingerichtet (http://www.uni-wuerzburg.de/humangenetics/vmd2.html), in der die wachsende Zahl an Mutationen, Sequenzvarianten und Polymorphismen gesammelt und verwaltet wird. „Missense“ Mutationen können die Proteinfunktion auf verschiedene Weise beeinträchtigen. Geeignete molekulare Assays wurden daher etabliert, um die funkionellen Auswirkungen der VMD2 Mutationen in Hinblick auf die intrazelluläre Lokalisation zu untersuchen. In der vorliegenden Arbeit wurde ein heterologes COS7 Expressionssystem entwickelt, es wurden verschiedene Mutationen mittels „site-directed mutagenesis“ generiert sowie Bestrophin-spezifische Antikörper hergestellt. Überraschenderweise konnte anhand von Membranfraktionierungs- und Westernblot-Analysen keine signifikanten quantitativen Unterschiede zwischen intakten und mutierten Bestrophin nachgewiesen werden. Unabhängig von Art oder Position der Mutation konnte der Einbau von mutiertem Bestrophin in die Membran gezeigt werden. Die Experimente deuten erstmalig darauf hin daß eine fehlerhafte Membranintegration als kausaler krankheitsverursachender Mechanismus ausgeschlossen werden kann. Dies liegt in Übereinstimmung mit dem dominant-negativen Effekt der Mutationen. In einem alternativen Ansatz wurde die VMD2 RFP-TM Proteinfamilie im Mausgenom identifiziert und charakterisiert. Während die Aufklärung der genomischen Struktur des Vmd2 Gens die Grundlage zur Herstellung Vmd2 transgener Mäuse darstellte (siehe unten), gewährte die Charakterisierung der eng verwandten Vmd2L1-L3 Mausgene weitere Einblicke in die Funktion des Bestrophins. Hierzu wurden bioinformatische sowie RT PCR Analysen durchgeführt. Darüber hinaus wurde die präferientielle Expression der jeweiligen Transkripte in Testis, Kolon und Skelettmuskel sowie Augengewebe anhand von „real-time quantitative“ RT PCR nachgewiesen. Interessanterweise stellt Vmd2L2 wahrscheinlich ein funktionell inaktives Pseudogen dar. Ähnlich wie sein humanes Gegenstück wird das Maus Vmd2L3- Transkript auf mRNA Ebene differentiell prozessiert. Mausmodelle für Morbus Best stellen grundlegende Hilfsmittel dar, die Pathophysiology von Bestrophin in vivo zu untersuchen. Es wurde die Herstellung zweier verschiedener Mauslinien initiiert: zum einen ein Vmd2-defizientes „knock-out“ Modell, zum anderen eine „knock-in“ Maus, die eine humane krankheitsassoziierte Mutation (Tyr227Asn) im mausorthologen Gen trägt. Die entsprechenden Konstrukte wurden hergestellt und in ES Zellen eingeschleust. Ausgehend von bislang vier isolierten ES Klonen, die den Vmd2+/- Genotyp tragen, können nun in nachfolgenden Schritten chimäre Tiere generiert werden. Die resultierenden Mauslinien repräsentieren neue experimentelle Ansätze, die funktionelle Rolle des Bestrophins in Morbus Best sowie in der Entwicklung und Physiologie des RPEs aufzukären. KW - Best-Krankheit KW - Genmutation KW - Morbus Best KW - BMD KW - VMD2 KW - Makuladegeneration KW - Mausmodell KW - Best Disease KW - BMD KW - VMD2 KW - macular degeneration KW - mouse model Y1 - 2003 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-5761 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - THES A1 - Bäuerlein, Carina T1 - Identification of new predictive markers for an early diagnosis of an imminent acute Graft-versus-Host Disease T1 - Bestimmung neuer prädiktiver Marker zur Früherkennung einer drohenden akuten Graft-versus-Host Disease N2 - Acute graft-versus-host disease (aGvHD) is an immune syndrome associated with allogeneic hematopoietic cell transplantation (allo-HCT) that is mediated by alloreactive donor T cells attacking the gastrointestinal tract, liver, and skin of the host. Early diagnosis remains problematic and to date mainly relies on clinical symptoms and histopathology. Previously, different groups demonstrated that in order to cause aGvHD, alloreactive T cells require the expression of appropriate homing receptors to efficiently migrate from their priming sites to their target tissues. Therefore, the development of a predictive test based on the homing receptor expression profile of peripheral blood T cells seems attractive to identify patients at risk before the onset of aGvHD. The aim of this study was to analyze migrating alloreactive donor T cell kinetics in the peripheral blood early after allo-HCT in a murine model across minor histocompatibility antigens (miHAg) followed by a precise characterization of the homing receptor expression profile of migrating donor lymphocytes in order to identify suitable predictive markers. Combining daily bioluminescence imaging (BLI) and flow cytometry (FC) allowed defining two weeks of massive alloreactive donor T cell migration before clinical aGvHD symptoms became apparent. Peripheral blood donor T lymphocytes highly up-regulated the homing markers α4β7 integrin, and P- and E-selectin-ligand at peak time points of cell migration. The combination with the activation markers CD25 and CD69 and low expression levels of L-selectin allowed alloreactive donor T cell definition. Based on this migration phase we postulated a potential diagnostic window to precisely identify alloreactive donor T cells upon their homing receptor expression profile. Consequently, targeted pre-emptive treatment with rapamycin starting at the earliest detection time point of alloreactive donor T cells in the peripheral blood (day+6) significantly prolonged survival of treated mice. Based on this data, we propose a potential diagnostic window for alloreactive cell detection based on their homing receptor expression profile for a timely and effective therapeutic intervention before the clinical manifestation of aGvHD. N2 - Die allogene hämatopoetische Stammzelltransplantation ist oft die einzig mögliche Behandlungsmethode für maligne und nicht-maligne hämatologische Erkrankungen. Die Graft-versus-Host Disease stellt den größten, limitierenden Faktor dieser Therapie dar. Bei diesem Immunsyndrom greifen alloreaktive Spender-T-Zellen gezielt Organe des Empfängers an, insbesondere den Gastrointestinaltrakt, die Leber und die Haut. Die frühe Diagnose einer bevorstehenden, akuten GvHD gestaltet sich nach wie vor schwierig und basiert heutzutage hauptsächlich auf dem Auftreten klinischer Symptome und histopathologischen Befunden. Die Entwicklung eines prädiktiven Tests zur Früherkennung gefährdeter Patienten hat daher hohe Priorität. Verschiedene Gruppen zeigten kürzlich, dass Spender-T-Zellen spezifische Rezeptoren, sogenannte Homing-Rezeptoren, exprimieren müssen, um in die Zielorgane einwandern zu können. Deshalb scheint die Entwicklung eines auf dem spezifischen Homing-Rezeptor-Expressionsmuster der T-Zellen im peripheren Blut basierenden Tests vielversprechend, um gezielt Patienten zu identifizieren, die möglicherweise eine aGvHD entwickeln werden. Das Ziel dieser Arbeit war die genaue Analyse der Migrationskinetik alloreaktiver Spender-T-Zellen im peripheren Blut in einem klinisch relevanten Mausmodell mit Unterschieden in minor Histokompatibilitätsantigenen. Es folgte eine präzise Charakterisierung des Homing-Rezeptor-Expressionsprofils der migrierenden Spenderlymphozyten zu ausgewählten Zeitpunkten nach Transplantation, um mögliche, geeignete Rezeptoren für einen prädiktiven Test zu identifizieren. Die Kombination von täglicher in vivo Bildgebung der transplantierten Mäuse mit durchflusszytometrischen Analysen des peripheren Blutes ermöglichte es, eine zweiwöchige Phase massiver Spenderzellmigration vor dem Auftreten klinischer aGvHD Symptome zu definieren. Die detektierten Spenderlymphozyten zeigten eine stark erhöhte Expression des für die Migration in den Gastrointestinaltrakt wichtigen Moleküls α4β7 Integrin sowie der Liganden für P- und E-Selektin, die in das Haut-Homing involviert sind. Die Kombination dieser Marker mit der stark reduzierten Expression von L-Selektin, einem Marker für naive T-Zellen, sowie der signifikant höheren Expression der Aktivierungsmarker CD25 und CD69 im Vergleich zu syngen transplantierten Kontrolltieren ermöglichte die Definition von alloreaktiven Spender-T-Zellen. Eine gezielte, vorbeugende Behandlung mit Rapamycin, beginnend am Tag der Detektion erster alloreaktiver T-Zellen (Tag+6), erhöhte die Überlebensrate der behandelten Mäuse. Aufgrund dieser Daten schlagen wir ein potentielles, diagnostisches Fenster zur Anwendung prädiktiver Tests vor, um Patienten, mit erhöhtem aGvHD-Risiko rechtzeitig zu identifizieren und vorbeugend behandeln zu können. KW - Transplantat-Wirt-Reaktion KW - Graft-versus-host disease KW - Mausmodell KW - T-Zellhoming KW - Graft-versus-host disease KW - mouse model KW - T cell homing KW - Maus KW - Blutstammzelle Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78489 ER - TY - JOUR A1 - Kraft, Peter A1 - Schuhmann, Michael K. A1 - Garz, Cornelia A1 - Jandke, Solveig A1 - Urlaub, Daniela A1 - Mencl, Stine A1 - Zernecke, Alma A1 - Heinze, Hans-Jochen A1 - Carare, Roxana O. A1 - Kleinschnitz, Christoph A1 - Schreiber, Stefanie T1 - Hypercholesterolemia induced cerebral small vessel disease JF - PLoS ONE N2 - Background While hypercholesterolemia plays a causative role for the development of ischemic stroke in large vessels, its significance for cerebral small vessel disease (CSVD) remains unclear. We thus aimed to understand the detailed relationship between hypercholesterolemia and CSVD using the well described Ldlr\(^{−/-}\) mouse model. Methods We used Ldlr\(^{−/-}\) mice (n = 16) and wild-type (WT) mice (n = 15) at the age of 6 and 12 months. Ldlr\(^{−/-}\) mice develop high plasma cholesterol levels following a high fat diet. We analyzed cerebral capillaries and arterioles for intravascular erythrocyte accumulations, thrombotic vessel occlusions, blood-brain barrier (BBB) dysfunction and microbleeds. Results We found a significant increase in the number of erythrocyte stases in 6 months old Ldlr\(^{−/-}\) mice compared to all other groups (P < 0.05). Ldlr\(^{−/-}\) animals aged 12 months showed the highest number of thrombotic occlusions while in WT animals hardly any occlusions could be observed (P < 0.001). Compared to WT mice, Ldlr\(^{−/-}\) mice did not display significant gray matter BBB breakdown. Microhemorrhages were observed in one Ldlr\(^{−/-}\) mouse that was 6 months old. Results did not differ when considering subcortical and cortical regions. Conclusions In Ldlr\(^{−/-}\) mice, hypercholesterolemia is related to a thrombotic CSVD phenotype, which is different from hypertension-related CSVD that associates with a hemorrhagic CSVD phenotype. Our data demonstrate a relationship between hypercholesterolemia and the development of CSVD. Ldlr\(^{−/-}\) mice appear to be an adequate animal model for research into CSVD. KW - hypercholesterolemia KW - cerebral small vessel disease KW - mouse model KW - histology Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170493 VL - 12 IS - 8 ER - TY - JOUR A1 - Tsoneva, Desislava A1 - Minev, Boris A1 - Frentzen, Alexa A1 - Zhang, Qian A1 - Wege, Anja K. A1 - Szalay, Aladar A. T1 - Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis JF - Molecular Therapy Oncolytics N2 - Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb’s in vitro binding ability and functionality in cell culture, beside the significant increase of CD56\(^{bright}\) NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our “patient-like” humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system. KW - humanized tumor KW - mouse model KW - subcutaneous human tumors KW - Oncolytic vaccinia virus Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170786 VL - 5 ER -