TY - JOUR A1 - Walker, Brian A. A1 - Mavrommatis, Konstantinos A1 - Wardell, Christopher P. A1 - Ashby, T. Cody A1 - Bauer, Michael A1 - Davies, Faith A1 - Rosenthal, Adam A1 - Wang, Hongwei A1 - Qu, Pingping A1 - Hoering, Antje A1 - Samur, Mehmet A1 - Towfic, Fadi A1 - Ortiz, Maria A1 - Flynt, Erin A1 - Yu, Zhinuan A1 - Yang, Zhihong A1 - Rozelle, Dan A1 - Obenauer, John A1 - Trotter, Matthew A1 - Auclair, Daniel A1 - Keats, Jonathan A1 - Bolli, Niccolo A1 - Fulciniti, Mariateresa A1 - Szalat, Raphael A1 - Moreau, Phillipe A1 - Durie, Brian A1 - Stewart, A. Keith A1 - Goldschmidt, Hartmut A1 - Raab, Marc S. A1 - Einsele, Hermann A1 - Sonneveld, Pieter A1 - San Miguel, Jesus A1 - Lonial, Sagar A1 - Jackson, Graham H. A1 - Anderson, Kenneth C. A1 - Avet-Loiseau, Herve A1 - Munshi, Nikhil A1 - Thakurta, Anjan A1 - Morgan, Gareth T1 - A high-risk, Double-Hit, group of newly diagnosed myeloma identified by genomic analysis JF - Leukemia N2 - Patients with newly diagnosed multiple myeloma (NDMM) with high-risk disease are in need of new treatment strategies to improve the outcomes. Multiple clinical, cytogenetic, or gene expression features have been used to identify high-risk patients, each of which has significant weaknesses. Inclusion of molecular features into risk stratification could resolve the current challenges. In a genome-wide analysis of the largest set of molecular and clinical data established to date from NDMM, as part of the Myeloma Genome Project, we have defined DNA drivers of aggressive clinical behavior. Whole-genome and exome data from 1273 NDMM patients identified genetic factors that contribute significantly to progression free survival (PFS) and overall survival (OS) (cumulative R2 = 18.4% and 25.2%, respectively). Integrating DNA drivers and clinical data into a Cox model using 784 patients with ISS, age, PFS, OS, and genomic data, the model has a cumlative R2 of 34.3% for PFS and 46.5% for OS. A high-risk subgroup was defined by recursive partitioning using either a) bi-allelic TP53 inactivation or b) amplification (≥4 copies) of CKS1B (1q21) on the background of International Staging System III, comprising 6.1% of the population (median PFS = 15.4 months; OS = 20.7 months) that was validated in an independent dataset. Double-Hit patients have a dire prognosis despite modern therapies and should be considered for novel therapeutic approaches. KW - cancer genomics KW - risk factors Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233299 VL - 33 ER - TY - JOUR A1 - Roy, Denis Claude A1 - Lachance, Sylvie A1 - Cohen, Sandra A1 - Delisle, Jean-Sébastien A1 - Kiss, Thomas A1 - Sauvageau, Guy A1 - Busque, Lambert A1 - Ahmad, Imran A1 - Bernard, Lea A1 - Bambace, Nadia A1 - Boumédine, Radia S. A1 - Guertin, Marie-Claude A1 - Rezvani, Katayoun A1 - Mielke, Stephan A1 - Perreault, Claude A1 - Roy, Jean T1 - Allodepleted T-cell immunotherapy after haploidentical haematopoietic stem cell transplantation without severe acute graft-versus-host disease (GVHD) in the absence of GVHD prophylaxis JF - British Journal of Haematology N2 - Graft-versus-host disease (GVHD) is a major cause of transplant-related mortality (TRM) after allogeneic haematopoietic stem cell transplantation (HSCT) and presents a challenge in haploidentical HSCT. GVHD may be prevented by ex vivo graft T-cell depletion or in vivo depletion of proliferating lymphocytes. However, both approaches pose significant risks, particularly infections and relapse, compromising survival. A photodepletion strategy to eliminate alloreactive T cells from mismatched donor lymphocyte infusions (enabling administration without immunosuppression), was used to develop ATIR101, an adjunctive therapy for use after haploidentical HSCT. In this phase I dose-finding study, 19 adults (median age: 54 years) with high-risk haematological malignancies were treated with T-cell-depleted human leucocyte antigen-haploidentical myeloablative HSCT followed by ATIR101 at doses of 1 x 10(4)-5 x 10(6) CD3(+) cells/kg (median 31 days post-transplant). No patient received post-transplant immunosuppression or developed grade III/IV acute GVHD, demonstrating the feasibility of ATIR101 infusion for evaluation in two subsequent phase 2 studies. Additionally, we report long-term follow -up of patients treated with ATIR101 in this study. At 1 year, all 9 patients receiving doses of 0 center dot 3-2 x 10(6) CD3(+) cells/kg ATIR101 remained free of serious infections and after more than 8 years, TRM was 0%, relapse-related mortality was 33% and overall survival was 67% in these patients. KW - haematopoietic stem cell KW - stem cell transplantation KW - graft-versus-host-disease KW - cell therapy and immunotherapy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227075 VL - 186 IS - 5 ER - TY - JOUR A1 - Boch, Tobias A1 - Spiess, Birgit A1 - Heinz, Werner A1 - Cornely, Oliver A. A1 - Schwerdtfeger, Rainer A1 - Hahn, Joachim A1 - Krause, Stefan W. A1 - Duerken, Matthias A1 - Bertz, Hartmut A1 - Reuter, Stefan A1 - Kiehl, Michael A1 - Claus, Bernd A1 - Deckert, Peter Markus A1 - Hofmann, Wolf‐Karsten A1 - Buchheidt, Dieter A1 - Reinwald, Mark T1 - Aspergillus specific nested PCR from the site of infection is superior to testing concurrent blood samples in immunocompromised patients with suspected invasive aspergillosis JF - Mycoses N2 - Invasive aspergillosis (IA) is a severe complication in immunocompromised patients. Early diagnosis is crucial to decrease its high mortality, yet the diagnostic gold standard (histopathology and culture) is time‐consuming and cannot offer early confirmation of IA. Detection of IA by polymerase chain reaction (PCR) shows promising potential. Various studies have analysed its diagnostic performance in different clinical settings, especially addressing optimal specimen selection. However, direct comparison of different types of specimens in individual patients though essential, is rarely reported. We systematically assessed the diagnostic performance of an Aspergillus‐specific nested PCR by investigating specimens from the site of infection and comparing it with concurrent blood samples in individual patients (pts) with IA. In a retrospective multicenter analysis PCR was performed on clinical specimens (n = 138) of immunocompromised high‐risk pts (n = 133) from the site of infection together with concurrent blood samples. 38 pts were classified as proven/probable, 67 as possible and 28 as no IA according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions. A considerably superior performance of PCR from the site of infection was observed particularly in pts during antifungal prophylaxis (AFP)/antifungal therapy (AFT). Besides a specificity of 85%, sensitivity varied markedly in BAL (64%), CSF (100%), tissue samples (67%) as opposed to concurrent blood samples (8%). Our results further emphasise the need for investigating clinical samples from the site of infection in case of suspected IA to further establish or rule out the diagnosis. KW - antifungal KW - aspergillosis KW - BAL KW - blood KW - cerebrospinal fluid KW - comparison KW - PCR KW - Aspergillus Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-214065 VL - 62 IS - 11 SP - 1035 EP - 1042 ER - TY - JOUR A1 - Gernert, Michael A1 - Tony, Hans-Peter A1 - Schwaneck, Eva Christina A1 - Gadeholt, Ottar A1 - Schmalzing, Marc T1 - Autologous hematopoietic stem cell transplantation in systemic sclerosis induces long-lasting changes in B cell homeostasis toward an anti-inflammatory B cell cytokine pattern JF - Arthritis Research & Therapy N2 - Background Autologous hematopoietic stem cell transplantation (aHSCT) is performed in patients with aggressive forms of systemic sclerosis (SSc). The profile of B cell reconstitution after aHSCT is not fully understood. The aim of this study was to investigate changes of B cell subsets and cytokine production of B cells in patients with SSc after aHSCT. Methods Peripheral blood of six patients with SSc was collected at defined intervals up to 16 months after aHSCT. Immunophenotyping was performed, and B cell function was determined by measuring cytokine secretion in supernatants of stimulated B cell cultures. Results Within 1 month after aHSCT, a peak in the percentage of CD38\(^{++}\)/CD10\(^+\)/IgD\(^+\) transitional B cells and CD38\(^{++}\)/CD27\(^{++}\)/IgD\(^−\) plasmablasts was detected. Long-term changes persisted up to 14 months after aHSCT and showed an increased percentage of total B cells; the absolute B cell number did not change significantly. Within the B cell compartment, an increased CD27/IgD\(^+\) naïve B cell percentage was found whereas decreased percentages of CD27\(^+\)/IgD\(^+\) pre-switched memory, CD27\(^+\)/IgD\(^−\) post-switched memory, and CD27\(^−\) /IgD\(^−\) double-negative B cells were seen after aHSCT. Cytokine secretion in B cell cultures showed significantly increased IL-10 concentrations 13 to 16 months after aHSCT. Conclusion A changed composition of the B cell compartment is present for up to 14 months after aHSCT indicating positive persisting effects of aHSCT on B cell homeostasis. The cytokine secretion profile of B cells changes in the long term and shows an increased production of the immune regulatory cytokine IL-10 after aHSCT. These findings might promote the clinical improvements after aHSCT in SSc patients. KW - Systemic sclerosis KW - B cells KW - Memory B cells KW - naïve B cells KW - Autologous hematopoietic stem cell transplantation KW - Interleukin-10 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201004 VL - 21 ER - TY - JOUR A1 - Jahn, Daniel A1 - Dorbath, Donata A1 - Kircher, Stefan A1 - Nier, Anika A1 - Bergheim, Ina A1 - Lenaerts, Kaatje A1 - Hermanns, Heike M. A1 - Geier, Andreas T1 - Beneficial effects of vitamin D treatment in an obese mouse model of non-alcoholic steatohepatitis JF - Nutrients N2 - Serum vitamin D levels negatively correlate with obesity and associated disorders such as non-alcoholic steatohepatitis (NASH). However, the mechanisms linking low vitamin D (VD) status to disease progression are not completely understood. In this study, we analyzed the effect of VD treatment on NASH in mice. C57BL6/J mice were fed a high-fat/high-sugar diet (HFSD) containing low amounts of VD for 16 weeks to induce obesity, NASH and liver fibrosis. The effects of preventive and interventional VD treatment were studied on the level of liver histology and hepatic/intestinal gene expression. Interestingly, preventive and to a lesser extent also interventional VD treatment resulted in improvements of liver histology. This included a significant decrease of steatosis, a trend towards lower non-alcoholic fatty liver disease (NAFLD) activity score and a slight non-significant decrease of fibrosis in the preventive treatment group. In line with these changes, preventive VD treatment reduced the hepatic expression of lipogenic, inflammatory and pro-fibrotic genes. Notably, these beneficial effects occurred in conjunction with a reduction of intestinal inflammation. Together, our observations suggest that timely initiation of VD supplementation (preventive vs. interventional) is a critical determinant of treatment outcome in NASH. In the applied animal model, the improvements of liver histology occurred in conjunction with reduced inflammation in the gut, suggesting a potential relevance of vitamin D as a therapeutic agent acting on the gut–liver axis. KW - vitamin D KW - obesity KW - NAFLD KW - NASH KW - inflammation KW - intestine KW - gut–liver axis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177222 VL - 11 IS - 1 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 - Rydzek, Julian A1 - Nerreter, Thomas A1 - Peng, Haiyong A1 - Jutz, Sabrina A1 - Leitner, Judith A1 - Steinberger, Peter A1 - Einsele, Hermann A1 - Rader, Christoph A1 - Hudecek, Michael T1 - Chimeric Antigen Receptor Library Screening Using a Novel NF-kappa B/NFAT Reporter Cell Platform JF - Molecular Therapy N2 - Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the T cell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor kappa B (NF kappa B) and nuclear factor of activated T cells (NFAT) reporter genes that generate a duplex output of enhanced CFP (ECFP) and EGFP, respectively. As a proof of concept, we modified reporter cells with CD19-specific and ROR1-specific CARs, and we detected high-level reporter signals that allowed distinguishing functional from non-functional CAR constructs. The reporter data were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary CAR-T cells (21 days). We challenged the reporter platform to a large-scale screening campaign on a ROR1-CAR library, and we showed that reporter cells retrieved a functional CAR variant that was present with a frequency of only 6 in 1.05 x 10(6). The data illustrate the potential to implement this reporter platform into the preclinical development path of novel CAR-T cell products and to inform and accelerate the selection of lead CAR candidates for clinical translation. KW - NF-κB/NFAT reporter cells KW - chimeric antigen receptor KW - library screening KW - cancer immunotherapy KW - ROR1 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227193 VL - 27 IS - 2 ER - TY - JOUR A1 - Da Vià, Matteo Claudio A1 - Solimando, Antonio Giovanni A1 - Garitano-Trojaola, Andoni A1 - Barrio, Santiago A1 - Munawar, Umair A1 - Strifler, Susanne A1 - Haertle, Larissa A1 - Rhodes, Nadine A1 - Vogt, Cornelia A1 - Lapa, Constantin A1 - Beilhack, Andreas A1 - Rasche, Leo A1 - Einsele, Hermann A1 - Kortüm, K. Martin T1 - CIC Mutation as a Molecular Mechanism of Acquired Resistance to Combined BRAF‐MEK Inhibition in Extramedullary Multiple Myeloma with Central Nervous System Involvement JF - The Oncologist N2 - Combined MEK‐BRAF inhibition is a well‐established treatment strategy in BRAF‐mutated cancer, most prominently in malignant melanoma with durable responses being achieved through this targeted therapy. However, a subset of patients face primary unresponsiveness despite presence of the activating mutation at position V600E, and others acquire resistance under treatment. Underlying resistance mechanisms are largely unknown, and diagnostic tests to predict tumor response to BRAF‐MEK inhibitor treatment are unavailable. Multiple myeloma represents the second most common hematologic malignancy, and point mutations in BRAF are detectable in about 10% of patients. Targeted inhibition has been successfully applied, with mixed responses observed in a substantial subset of patients mirroring the widespread spatial heterogeneity in this genomically complex disease. Central nervous system (CNS) involvement is an extremely rare, extramedullary form of multiple myeloma that can be diagnosed in less than 1% of patients. It is considered an ultimate high‐risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. Here we not only describe the first patient with an extramedullary CNS relapse responding to targeted dabrafenib and trametinib treatment, we furthermore provide evidence that a point mutation within the capicua transcriptional repressor (CIC) gene mediated the acquired resistance in this patient. KW - Multiple myeloma KW - Extramedullary disease KW - Capicua transcriptional repressor KW - Drug resistance KW - BRAF mutation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219549 VL - 25 IS - 2 ER - TY - JOUR A1 - Belic, Stanislav A1 - Page, Lukas A1 - Lazariotou, Maria A1 - Waaga-Gasser, Ana Maria A1 - Dragan, Mariola A1 - Springer, Jan A1 - Loeffler, Juergen A1 - Morton, Charles Oliver A1 - Einsele, Hermann A1 - Ullmann, Andrew J. A1 - Wurster, Sebastian T1 - Comparative Analysis of Inflammatory Cytokine Release and Alveolar Epithelial Barrier Invasion in a Transwell® Bilayer Model of Mucormycosis JF - Frontiers in Microbiology N2 - Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis. KW - mucormycosis KW - alveolar epithelium KW - in vitro model KW - cytokines KW - dendritic cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-252477 VL - 9 ER - TY - JOUR A1 - Gotru, Sanjeev Kiran A1 - van Geffen, Johanna P. A1 - Nagy, Magdolna A1 - Mammadova-Bach, Elmina A1 - Eilenberger, Julia A1 - Volz, Julia A1 - Manukjan, Georgi A1 - Schulze, Harald A1 - Wagner, Leonard A1 - Eber, Stefan A1 - Schambeck, Christian A1 - Deppermann, Carsten A1 - Brouns, Sanne A1 - Nurden, Paquita A1 - Greinacher, Andreas A1 - Sachs, Ulrich A1 - Nieswandt, Bernhard A1 - Hermanns, Heike M. A1 - Heemskerk, Johan W. M. A1 - Braun, Attila T1 - Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases JF - Scientific Reports N2 - Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation. KW - coagulation system KW - metals Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227455 VL - 9 ER - TY - JOUR A1 - Springer, Jan A1 - Walther, Grit A1 - Rickerts, Volker A1 - Hamprecht, Axel A1 - Willinger, Birgit A1 - Teschner, Daniel A1 - Einsele, Hermann A1 - Kurzai, Oliver A1 - Loeffler, Juergen T1 - Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR JF - Journal of Fungi N2 - The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens. KW - probe-based real-time PCR KW - Fusarium KW - bronchoalveolar lavage fluid KW - fungal molecular diagnostics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193111 SN - 2309-608X VL - 5 IS - 4 ER - TY - JOUR A1 - Kraus, Amelie J. A1 - Brink, Benedikt G. A1 - Siegel, T. Nicolai T1 - Efficient and specific oligo-based depletion of rRNA JF - Scientific Reports N2 - In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available. KW - parasite biology KW - RNA sequencing KW - transcriptomics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224829 VL - 9 ER - TY - JOUR A1 - Raselli, Tina A1 - Hearn, Tom A1 - Wyss, Annika A1 - Atrott, Kirstin A1 - Peter, Alain A1 - Frey-Wagner, Isabelle A1 - Spalinger, Marianne R. A1 - Maggio, Ewerton M. A1 - Sailer, Andreas W. A1 - Schmitt, Johannes A1 - Schreiner, Philipp A1 - Moncsek, Anja A1 - Mertens, Joachim A1 - Scharl, Michael A1 - Griffiths, William J. A1 - Bueter, Marco A1 - Geier, Andreas A1 - Rogler, Gerhard A1 - Wang, Yuqin A1 - Misselwitz, Benjamin T1 - Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis JF - Journal of Lipid Research N2 - Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7 alpha,25-dihydroxycholesterol (7 alpha,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7 alpha,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2(-/-) mice and mice with defects in the 7 alpha,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7 alpha-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH. KW - nonalcoholic fatty liver disease KW - Epstein-Barr virus-induced gene 2 KW - cholesterol 25 hydroxylase KW - 25-hydroxycholesterol 7 alpha-hydroxylase KW - mouse feeding model Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225004 VL - 60 IS - 7 ER - TY - JOUR A1 - Graf, Christiana A1 - Mondorf, Antonia A1 - Knop, Viola A1 - Peiffer, Kai-Henrik A1 - Dietz, Julia A1 - Friess, Julia A1 - Wedemeyer, Heiner A1 - Buggisch, Peter A1 - Mauss, Stefan A1 - Berg, Thomas A1 - Rausch, Michael A1 - Sprinzl, Martin A1 - Klinker, Hartwig A1 - Hinrichsen, Holger A1 - Bronowicki, Jean-Pierre A1 - Haag, Sebastian A1 - Hüppe, Dietrich A1 - Lutz, Thomas A1 - Poynard, Thierry A1 - Zeuzem, Stefan A1 - Friedrich-Rust, Mireen A1 - Sarrazin, Christoph A1 - Vermehren, Johannes T1 - Evaluation of point shear wave elastography using acoustic radiation force impulse imaging for longitudinal fibrosis assessment in patients with HBeAg-Negative HBV infection JF - Journal of Clinical Medicine N2 - Background: Accurate assessment of hepatic fibrosis in patients with chronic HBeAg-negative Hepatitis B is of crucial importance not only to predict the long-term clinical course, but also to evaluate antiviral therapy indication. The aim of this study was to prospectively assess the utility of point shear wave elastography (pSWE) for longitudinal non-invasive fibrosis assessment in a large cohort of untreated patients with chronic HBeAg-negative hepatitis B virus (HBV) infection. Methods: 407 consecutive patients with HBeAg-negative HBV infection who underwent pSWE, transient elastography (TE) as well as laboratory fibrosis markers, including fibrosis index based on four factors (FIB-4), aspartate to platelet ratio index (APRI) and FibroTest, on the same day were prospectively followed up for six years. Patients were classified into one of the three groups: inactive carriers (IC; HBV-DNA <2000 IU/mL and ALT <40 U/L); grey zone group 1 (GZ-1; HBV DNA <2000 IU/mL and ALT >40 U/L); grey zone group 2 (GZ-2; HBV-DNA >2000 IU/mL and ALT <40 U/L). Results: pSWE results were significantly correlated with TE (r = 0.29, p < 0.001) and APRI (r = 0.17; p = 0.005). Median pSWE values did not differ between IC, GZ-1 and GZ-2 patients (p = 0.82, p = 0.17, p = 0.34). During six years of follow-up, median pSWE and TE values did not differ significantly over time (TE: p = 0.27; pSWE: p = 0.05). Conclusion: Our data indicate that pSWE could be useful for non-invasive fibrosis assessment and follow-up in patients with HBeAg-negative chronic HBV infection. KW - HBV KW - non-invasive fibrosis assessment KW - point shear wave elastography KW - acoustic radiation force impulse imaging KW - transient elastography KW - fibrotest KW - APRI KW - FIB-4 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193916 SN - 2077-0383 VL - 8 IS - 12 ER - TY - JOUR A1 - Luber, Verena A1 - Lutz, Mathias A1 - Abele-Horn, Marianne A1 - Einsele, Hermann A1 - Grigoleit, Götz Ulrich A1 - Mielke, Stephan T1 - Excretion of Ascaris lumbricoides following reduced‐intensity allogeneic hematopoietic stem cell transplantation and consecutive treatment with mebendazole JF - Transplant Infectious Disease N2 - Here, we present the unique case of a 51‐year‐old German patient with multiple myeloma excreting Ascaris lumbricoides in his stool five weeks after allogeneic hematopoietic stem cell transplantation. Stool analysis remained negative for the presence of eggs, and there was no eosinophilia in the peripheral blood at any time around stem cell transplantation. The patient was commenced on a three‐day treatment with mebendazole, which was well tolerated. No serious interactions with the concomitant post‐transplant medication or negative effects on the hematopoiesis were observed, and the myeloma still is in complete remission. To our knowledge, this is the first report on excretion of A lumbricoides in the context of allogeneic stem cell transplantation. The case is remarkable with view to the fact that the parasite has supposedly survived all courses of myeloma treatment including autologous and allogeneic conditioning. Parasitosis with A lumbricoides has a worldwide prevalence of about a billion and is extremely rare in northern Europe. Possibly the patient got infected during a trip to Egypt years before multiple myeloma was diagnosed. KW - sirolimus KW - mycophenolic acid KW - multiple myeloma KW - mebendazole KW - hematopoietic stem cell transplantation KW - Ascaris lumbricoides Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219608 SN - 1399-3062 VL - 22 IS - 1 ER - TY - JOUR A1 - Loda, Sophia A1 - Krebs, Jonathan A1 - Danhof, Sophia A1 - Schreder, Martin A1 - Solimando, Antonio G. A1 - Strifler, Susanne A1 - Rasche, Leo A1 - Kortüm, Martin A1 - Kerscher, Alexander A1 - Knop, Stefan A1 - Puppe, Frank A1 - Einsele, Hermann A1 - Bittrich, Max T1 - Exploration of artificial intelligence use with ARIES in multiple myeloma research JF - Journal of Clinical Medicine N2 - Background: Natural language processing (NLP) is a powerful tool supporting the generation of Real-World Evidence (RWE). There is no NLP system that enables the extensive querying of parameters specific to multiple myeloma (MM) out of unstructured medical reports. We therefore created a MM-specific ontology to accelerate the information extraction (IE) out of unstructured text. Methods: Our MM ontology consists of extensive MM-specific and hierarchically structured attributes and values. We implemented “A Rule-based Information Extraction System” (ARIES) that uses this ontology. We evaluated ARIES on 200 randomly selected medical reports of patients diagnosed with MM. Results: Our system achieved a high F1-Score of 0.92 on the evaluation dataset with a precision of 0.87 and recall of 0.98. Conclusions: Our rule-based IE system enables the comprehensive querying of medical reports. The IE accelerates the extraction of data and enables clinicians to faster generate RWE on hematological issues. RWE helps clinicians to make decisions in an evidence-based manner. Our tool easily accelerates the integration of research evidence into everyday clinical practice. KW - natural language processing KW - ontology KW - artificial intelligence KW - multiple myeloma KW - real world evidence Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197231 SN - 2077-0383 VL - 8 IS - 7 ER - TY - JOUR A1 - Saraceni, Francesco A1 - Labopin, Myriam A1 - Brecht, Arne A1 - Kröger, Nicolaus A1 - Eder, Matthias A1 - Tischer, Johanna A1 - Labussiere-Wallet, Helene A1 - Einsele, Hermann A1 - Beelen, Dietrich A1 - Bunjes, Donald A1 - Niederwieser, Dietger A1 - Bochtler, Tilman A1 - Savani, Bipin N. A1 - Mohty, Mohamad A1 - Nagler, Arnon T1 - Fludarabine-treosulfan compared to thiotepa-busulfan-fludarabine or FLAMSA as conditioning regimen for patients with primary refractory or relapsed acute myeloid leukemia: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT) JF - Journal of Hematology & Oncology N2 - Background Limited data is available to guide the choice of the conditioning regimen for patients with acute myeloid leukemia (AML) undergoing transplant with persistent disease. Methods We retrospectively compared outcome of fludarabine-treosulfan (FT), thiotepa-busulfan-fludarabine (TBF), and sequential fludarabine, intermediate dose Ara-C, amsacrine, total body irradiation/busulfan, cyclophosphamide (FLAMSA) conditioning in patients with refractory or relapsed AML. Results Complete remission rates at day 100 were 92%, 80%, and 88% for FT, TBF, and FLAMSA, respectively (p=0.13). Non-relapse mortality, incidence of relapse, acute (a) and chronic (c) graft-versus-host disease (GVHD) rates did not differ between the three groups. Overall survival at 2years was 37% for FT, 24% for TBF, and 34% for FLAMSA (p=0.10). Independent prognostic factors for survival were Karnofsky performance score and patient CMV serology (p=0.01; p=0.02), while survival was not affected by age at transplant. The use of anti-thymocyte globulin (ATG) was associated with reduced risk of grade III-IV aGVHD (p=0.02) and cGVHD (p=0.006), with no influence on relapse. Conclusions In conclusion, FT, TBF, and FLAMSA regimens provided similar outcome in patients undergoing transplant with active AML. Survival was determined by patient characteristics as Karnofsky performance score and CMV serology, however was not affected by age at transplant. ATG appears able to reduce the incidence of acute and chronic GVHD without influencing relapse risk. KW - Acute myeloid leukemia (AML) KW - Active disease KW - Allogeneic transplantation KW - Sibling donor (MSD) KW - Unrelated donor (UD) KW - Conditioning regimen KW - Fludarabine-treosulfan (FT) KW - Thiotepa-busulfan-fludarabine (TBF) KW - Fludarabine KW - intermediate dose Ara-C KW - amsacrine KW - total body irradiation/busulfan KW - cyclophosphamide (FLAMSA) Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227345 VL - 12 IS - 44 ER - TY - JOUR A1 - López, Cristina A1 - Kleinheinz, Kortine A1 - Aukema, Sietse M. A1 - Rohde, Marius A1 - Bernhart, Stephan H. A1 - Hübschmann, Daniel A1 - Wagener, Rabea A1 - Toprak, Umut H. A1 - Raimondi, Francesco A1 - Kreuz, Markus A1 - Waszak, Sebastian M. A1 - Huang, Zhiqin A1 - Sieverling, Lina A1 - Paramasivam, Nagarajan A1 - Seufert, Julian A1 - Sungalee, Stephanie A1 - Russell, Robert B. A1 - Bausinger, Julia A1 - Kretzmer, Helene A1 - Ammerpohl, Ole A1 - Bergmann, Anke K. A1 - Binder, Hans A1 - Borkhardt, Arndt A1 - Brors, Benedikt A1 - Claviez, Alexander A1 - Doose, Gero A1 - Feuerbach, Lars A1 - Haake, Andrea A1 - Hansmann, Martin-Leo A1 - Hoell, Jessica A1 - Hummel, Michael A1 - Korbel, Jan O. A1 - Lawerenz, Chris A1 - Lenze, Dido A1 - Radlwimmer, Bernhard A1 - Richter, Julia A1 - Rosenstiel, Philip A1 - Rosenwald, Andreas A1 - Schilhabel, Markus B. A1 - Stein, Harald A1 - Stilgenbauer, Stephan A1 - Stadler, Peter F. A1 - Szczepanowski, Monika A1 - Weniger, Marc A. A1 - Zapatka, Marc A1 - Eils, Roland A1 - Lichter, Peter A1 - Loeffler, Markus A1 - Möller, Peter A1 - Trümper, Lorenz A1 - Klapper, Wolfram A1 - Hoffmann, Steve A1 - Küppers, Ralf A1 - Burkhardt, Birgit A1 - Schlesner, Matthias A1 - Siebert, Reiner T1 - Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic Burkitt lymphoma JF - Nature Communications N2 - Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing. KW - cancer genomics KW - lymphocytes KW - lymphoid tissues KW - oncology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-237281 VL - 10 ER - TY - JOUR A1 - Ribechini, Eliana A1 - Eckert, Ina A1 - Beilhack, Andreas A1 - Du Plessis, Nelita A1 - Walzl, Gerhard A1 - Schleicher, Ulrike A1 - Ritter, Uwe A1 - Lutz, Manfred B. T1 - Heat-killed Mycobacterium tuberculosis prime-boost vaccination induces myeloid-derived suppressor cells with spleen dendritic cell–killing capability JF - JCI Insight N2 - Tuberculosis patients and mice infected with live Mycobacterium tuberculosis accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that dead M. tuberculosis vaccines also may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of M. tuberculosis vaccines (heat-killed M. tuberculosis in incomplete Freund’s adjuvant, such as Montanide) but not single or control vaccines without M. tuberculosis strongly expanded CD11b\(^+\) myeloid cells in the spleen, leading to T cell suppression of proliferation and killing ex vivo. Dead M. tuberculosis vaccination induced the generation of CD11b\(^+\)Ly6C\(^{hi}\)CD115\(^+\) iNOS/Nos2\(^+\) monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs were positioned strategically in the splenic bridging channels and then positioned in the white pulp areas. Notably, within 6–24 hours, in a Nos2-dependent fashion, they produced NO to rapidly kill conventional and plasmacytoid DCs while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that M. tuberculosis vaccine induced M-MDSCs do not directly suppress effector T cells in vivo but, instead, indirectly by killing DCs. Collectively, we demonstrate that M. tuberculosis booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs. Our data suggest that formation of MDSCs by M. tuberculosis vaccines should be investigated also in clinical trials. KW - Immunology KW - Infectious disease Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201973 VL - 13 IS - 4 ER - TY - JOUR A1 - Thiem, Alexander A1 - Hesbacher, Sonja A1 - Kneitz, Hermann A1 - di Primio, Teresa A1 - Heppt, Markus V. A1 - Hermanns, Heike M. A1 - Goebeler, Matthias A1 - Meierjohann, Svenja A1 - Houben, Roland A1 - Schrama, David T1 - IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression JF - Journal of Experimental & Clinical Cancer Research N2 - Background Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. Methods We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53\(^{L22Q,W23S}\), a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53\(^{L22Q,W23S}\) in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy. KW - Melanoma KW - PD-L1 KW - CD274 KW - p53 KW - TP53 KW - JAK2 Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201016 VL - 38 ER -