TY - THES A1 - Herb, Stefanie Maria T1 - Regulation of MCMV immediate early gene expression by virally encoded miRNAs T1 - Regulation der MCMV immediate early Genexpression durch viral kodierte miRNAs N2 - Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model. N2 - In eukaryotischen Zellen wird die Expression von Genen durch das Zusammenspiel vieler verschiedener biologischer Regulatoren, wie microRNAs (miRNAs), kontrolliert. MiRNAs sind einzelsträngige, kurze, nicht-kodierende RNA-Moleküle, die aus sogenannten primären miRNAs und Vorläufer-miRNAs entstehen und die Genexpression auf Ebene der Posttranskription beeinflussen. Um ihre Funktion ausüben zu können, werden reife miRNAs in RNA-induzierte Silencing-Komplexe (RISCs) eingebaut und zu ihren Ziel-mRNAs geführt. Durch Wechselwirkungen zwischen der miRNA "seed-Region , die die Nukleotide 2 bis 8 vom 5'-Ende überspannt und der 3'UTR (3' untranslatierte Region) der Ziel-mRNA, unterdrückt RISC die Translation der Ziel-mRNA und kann deren Abbau durch direkte sowie indirekte Mechanismen induzieren. Die Expression von miRNAs wurde nicht nur in multizellulären Organismen, sondern in bereits zahlreichen Viren, insbesondere in der Virusfamilie der Herpesviridae, nachgew- iesen. Viruskodierte miRNAs kontrollieren dabei zelluläre wie auch virale mRNA-Transkripte und verleihen dem Virus einen Selektionsvorteil bzgl. Wachstum und Persistenz. Das mur- ine Cytomegalievirus (MCMV) ist ein β-Herpesvirus, das nach aktuellem Wissensstand 29 reife miRNAs kodiert, die allesamt während der lytischen Infektion identifziert wurden. Bioinformatische Analysen eines vor dieser Arbeit durchgeführten PAR-iCLIP-Experiments (photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking and immunoprecipitation), zeigten einen PAR-iCLIP Peak in der 3'UTR (3' untranslatierte Region) des immediate early 3-Transkripts (IE3) von MCMV. Unter Verwendung von RNAhbybrid, einem miRNA target prediction tool, fanden sich zwei virale miRNAs, näm- lich miR-m01-2-3p und miR-M23-2-3p mit potentiellen Bindestellen innerhalb der 3'UTR des MCMV IE3 Transkripts. Unsere konsekutiv durchgeführten Luciferase-Assays be- stätigten, dass sowohl miR-m01-2 als auch miR-M23-2 an die 3'UTR von IE3 binden. Beide viralen miRNAs führten zu einer verminderten Luciferaseaktivität unter Verwendung von Reportern, in denen die 3'UTR des IE3-Gens mit dem Luciferase-Transkript fusioniert war. xxiv Summary Das IE3 Protein gilt während des lytischen Zykluses als einer der wichtigsten Transkrip- tionsfaktoren von MCMV. Ebenfalls wurde der Einfluss der beiden viralen miRNAs auf die virale Reproduktion von uns untersucht. Hierfür wurden murine Zelllinien vor Infektion mit miR-m01-2 und miR- M23-2 transziert. Das Transfektionsverfahren optimierten wir zunächst durch Testung verschiedener Reagenzien und experimenteller Bedingungen. Schließlich zeigten wir mittels Plaqueassays, dass eine vor Infektion durchgeführte Transfektion mit miR-m01-2 und miR- M23-2 die Replikation von MCMV signifikant verzögerte. Unter Verwendung einer MCMV- Mutante, die durch Punktmutationen in beiden miRNA-Bindungsstellen innerhalb der IE3- 3'UTR charakterisiert war, ließ sich dieser Effekt aufheben. Unsere Experimente weisen somit stark darauf hin, dass miR-m01-2 und miR-M23-2 die Expression des IE3 Proteins regulieren und damit indirekt Einfluss auf die Genexpression während der lytischen Phase des Replikationszykluses von MCMV nehmen. Die miRNA-mediierte Repression der immediate early Genexpression stellt ein evolutionär konserviertes Merkmal von Zytomegalieviren dar. Für eine weitere Einordnung der Rolle dieser Genexpressionskontrolle bedarf es zukünftige Untersuchungen im MCMV-Tiermodell KW - miRNS KW - Cytomegalie-Virus KW - Herpes KW - Frühe Gene KW - PAR-CLIP KW - MCMV KW - miRNA KW - immediate early genes KW - lytic infection KW - IE3 KW - miRNA target KW - luciferase assay KW - CMV KW - HCMV KW - viral miRNAs Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323314 ER - TY - JOUR A1 - Wagner-Drouet, Eva A1 - Teschner, Daniel A1 - Wolschke, Christine A1 - Schäfer-Eckart, Kerstin A1 - Gärtner, Johannes A1 - Mielke, Stephan A1 - Schreder, Martin A1 - Kobbe, Guido A1 - Hilgendorf, Inken A1 - Klein, Stefan A1 - Verbeek, Mareike A1 - Ditschkowski, Markus A1 - Koch, Martina A1 - Lindemann, Monika A1 - Schmidt, Traudel A1 - Rascle, Anne A1 - Barabas, Sascha A1 - Deml, Ludwig A1 - Wagner, Ralf A1 - Wolff, Daniel T1 - Comparison of cytomegalovirus-specific immune cell response to proteins versus peptides using an IFN-γ ELISpot assay after hematopoietic stem cell transplantation JF - Diagnostics N2 - Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8\(^+\) counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients. KW - CMV KW - CMV-specific cellular immunity KW - hematopoietic stem cell transplantation KW - recall antigen KW - peptide KW - immune monitoring KW - IFN-γ ELISpot KW - T cells KW - antigen processing and presentation KW - immunosuppression Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228843 SN - 2075-4418 VL - 11 IS - 2 ER - TY - THES A1 - Gasparyan [geb. Düver], Franziska T1 - Virale Reaktivierungen nach allogener Stammzelltransplantation bei Kindern T1 - Viral reactivations following hematopoietic stem cell transplantation in pediatric patients N2 - Virale Reaktivierungen treten im Rahmen der Immundefizienz und Immunsuppression nach allogener Stammzelltransplantation häufig auf und können zu schwerwiegenden Komplikationen führen. Ziel dieser retrospektiven Studie war die Charakterisierung von viralen Reaktivierungen im ersten Jahr nach allogener Stammzelltransplantation, die Identifikation von Risikofaktoren sowie die Untersuchung des Einflusses viraler Reaktivierungen auf das Transplantationsoutcome. 107 pädiatrische allogene Stammzelltransplantationen im Zeitrahmen von Januar 2005 bis Dezember 2015 wurden in diesem Zusammenhang auf Infektionen mit dem Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Humanen Herpesvirus 6 (HHV 6), Herpes simplex Virus (HSV), Varicella zoster Virus (VZV) und Adenovirus (ADV) untersucht. N2 - Viral reactivation occurs frequently in the context of immunodeficiency and immunosuppression after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and can cause severe complications. The aim of this single-center retrospective analysis was to characterize viral infections in the first year after HSCT, to investigate risk factors and to study the impact of viral infections on transplantation outcome. 107 pediatric allo-HSCT from January 2005 through December 2015 were analyzed for infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV 6), herpes simplex virus (HSV), varicella zoster virus (VZV) and adenovirus (ADV). KW - Stammzelltransplantation KW - Pädiatrie KW - Reaktivierung KW - Virusinfektion KW - EBV KW - CMV KW - HHV 6 KW - Adenoviren KW - Herpes simplex KW - Varicella zoster Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-243537 ER - TY - JOUR A1 - Seif, Michelle A1 - Einsele, Hermann A1 - Löffler, Jürgen T1 - CAR T cells beyond cancer: hope for immunomodulatory therapy of infectious diseases JF - Frontiers in Immunology N2 - Infectious diseases are still a significant cause of morbidity and mortality worldwide. Despite the progress in drug development, the occurrence of microbial resistance is still a significant concern. Alternative therapeutic strategies are required for non-responding or relapsing patients. Chimeric antigen receptor (CAR) T cells has revolutionized cancer immunotherapy, providing a potential therapeutic option for patients who are unresponsive to standard treatments. Recently two CAR T cell therapies, Yescarta® (Kite Pharma/Gilead) and Kymriah® (Novartis) were approved by the FDA for the treatments of certain types of non-Hodgkin lymphoma and B-cell precursor acute lymphoblastic leukemia, respectively. The success of adoptive CAR T cell therapy for cancer has inspired researchers to develop CARs for the treatment of infectious diseases. Here, we review the main achievements in CAR T cell therapy targeting viral infections, including Human Immunodeficiency Virus, Hepatitis C Virus, Hepatitis B Virus, Human Cytomegalovirus, and opportunistic fungal infections such as invasive aspergillosis. KW - infectious diseases KW - mAb engineering KW - CAR T cells KW - HIV KW - HCV KW - CMV KW - invasive aspergillosis KW - HBV Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195596 SN - 1664-3224 VL - 10 IS - 2711 ER - TY - JOUR A1 - Zeller, Daniel A1 - Heidemeier, Anke A1 - Grigoleit, Götz Ulrich A1 - Müllges, Wolfgang T1 - Case report: subacute tetraplegia in an immunocompromised patient JF - BMC Neurology N2 - Background: Clinical reasoning in Neurology is based on general associations which help to deduce the site of the lesion. However, even “golden principles” may occasionally be deceptive. Here, we describe the case of subacute flaccid tetraparesis due to motor cortical lesions. To our knowledge, this is the first report to include an impressive illustration of nearly symmetric motor cortical involvement of encephalitis on brain MRI. Case presentation: A 51 year old immunocompromized man developed a high-grade pure motor flaccid tetraparesis over few days. Based on clinical presentation, critical illness polyneuromyopathy was suspected. However, brain MRI revealed symmetrical hyperintensities strictly limited to the subcortical precentral gyrus. An encephalitis, possibly due to CMV infection, turned out to be the most likely cause. Conclusion: While recognition of basic clinical patterns is indispensable in neurological reasoning, awareness of central conditions mimicking peripheral nervous disease may be crucial to detect unsuspected, potentially treatable conditions. KW - tetraparesis KW - motor cortex KW - CMV KW - encephalitis KW - case report Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157576 VL - 17 IS - 31 ER - TY - JOUR A1 - Wunsch, Marie A1 - Hohmann, Christopher A1 - Milles, Bianca A1 - Rostermund, Christina A1 - Lehmann, Paul V. A1 - Schroeter, Michael A1 - Bayas, Antonios A1 - Ulzheimer, Jochen A1 - Mäurer, Mathias A1 - Ergün, Süleyman A1 - Kuerten, Stefanie T1 - The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis JF - Viruses N2 - There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy. KW - B cells KW - CMV KW - EBV KW - ELISPOT KW - MS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146946 VL - 8 IS - 4 ER - TY - JOUR A1 - Zlamy, Manuela A1 - Almanzar, Giovanni A1 - Parson, Walther A1 - Schmidt, Christian A1 - Leierer, Johannes A1 - Weinberger, Birgit A1 - Jeller, Verena A1 - Unsinn, Karin A1 - Eyrich, Matthias A1 - Würzner, Reinhard A1 - Prelog, Martina T1 - Efforts of the human immune system to maintain the peripheral CD8+ T cell compartment after childhood thymectomy JF - Immunity & Ageing N2 - Background Homeostatic mechanisms to maintain the T cell compartment diversity indicate an ongoing process of thymic activity and peripheral T cell renewal during human life. These processes are expected to be accelerated after childhood thymectomy and by the influence of cytomegalovirus (CMV) inducing a prematurely aged immune system. The study aimed to investigate proportional changes and replicative history of CD8+ T cells, of recent thymic emigrants (RTEs) and CD103+ T cells (mostly gut-experienced) and the role of Interleukin-(IL)-7 and IL-7 receptor (CD127)-expressing T cells in thymectomized patients compared to young and old healthy controls. Results Decreased proportions of naive and CD31 + CD8+ T cells were demonstrated after thymectomy, with higher proliferative activity of CD127-expressing T cells and significantly shorter relative telomere lengths (RTLs) and lower T cell receptor excision circles (TRECs). Increased circulating CD103+ T cells and a skewed T cell receptor (TCR) repertoire were found after thymectomy similar to elderly persons. Naive T cells were influenced by age at thymectomy and further decreased by CMV. Conclusions After childhood thymectomy, the immune system demonstrated constant efforts of the peripheral CD8+ T cell compartment to maintain homeostasis. Supposedly it tries to fill the void of RTEs by peripheral T cell proliferation, by at least partly IL-7-mediated mechanisms and by proportional increase of circulating CD103+ T cells, reminiscent of immune aging in elderly. Although other findings were less significant compared to healthy elderly, early thymectomy demonstrated immunological alterations of CD8+ T cells which mimic features of premature immunosenescence in humans. KW - thymectomy KW - naive T cells KW - TRECs KW - TCR diversity KW - CMV KW - CD8 KW - telomeres Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146497 VL - 13 IS - 3 ER -